Journal articles on the topic 'PKC inhibitors'
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1
Saberi, Behnam, Mie Shinohara, Maria D. Ybanez, Naoko Hanawa, William A. Gaarde, Neil Kaplowitz, and Derick Han. "Regulation of H2O2-induced necrosis by PKC and AMP-activated kinase signaling in primary cultured hepatocytes." American Journal of Physiology-Cell Physiology 295, no. 1 (July 2008): C50—C63. http://dx.doi.org/10.1152/ajpcell.90654.2007.
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Recent studies have suggested that, in certain cases, necrosis, like apoptosis, may be programmed, involving the activation and inhibition of many signaling pathways. In this study, we examined whether necrosis induced by H2O2 is regulated by signaling pathways in primary hepatocytes. A detailed time course revealed that H2O2 treated to hepatocytes is consumed within minutes, but hepatocytes undergo necrosis several hours later. Thus, H2O2 treatment induces a “lag phase” where signaling changes occur, including PKC activation, Akt (PKB) downregulation, activation of JNK, and downregulation of AMP-activated kinase (AMPK). Investigation of various inhibitors demonstrated that PKC inhibitors were effective in reducing necrosis caused by H2O2 (∼80%). PKC inhibitor treatment decreased PKC activity but, surprisingly, also upregulated Akt and AMPK, suggesting that various PKC isoforms negatively regulate Akt and AMPK. Akt did not appear to play a significant role in H2O2-induced necrosis, since PKC inhibitor treatment protected hepatocytes from H2O2 even when Akt was inhibited. On the other hand, compound C, a selective AMPK inhibitor, abrogated the protective effect of PKC inhibitors against necrosis induced by H2O2. Furthermore, AMPK activators protected against H2O2-induced necrosis, suggesting that much of the protective effect of PKC inhibition was mediated through the upregulation of AMPK. Work with PKC inhibitors suggested that atypical PKC downregulates AMPK in response to H2O2. Knockdown of PKC-α using antisense oligonucleotides also slightly protected (∼22%) against H2O2. Taken together, our data demonstrate that the modulation of signaling pathways involving PKC and AMPK can alter H2O2-induced necrosis, suggesting that a signaling “program” is important in mediating H2O2-induced necrosis in primary hepatocytes.
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Silva, J. M., M. Hamel, M. Sahmi, and C. A. Price. "Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro." Reproduction 132, no. 6 (December 2006): 909–17. http://dx.doi.org/10.1530/rep-06-0058.
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The objective of this study was to determine the major intracellular signalling pathways used by FSH and insulin to stimulate cytochrome P450 aromatase (Cyp19) mRNA and oestradiol accumulation in oestrogenic bovine granulosa cellsin vitro. Bovine granulosa cells from small follicles (2–4 mm diameter) were cultured for 6 days under non-luteinizing conditions in the presence of insulin at 100 ng/ml, or insulin (10 ng/ml) and FSH (1 ng/ml). On day 4 of culture, specific inhibitors of phosphatidylinositol 3-kinase (PI3K; LY-294002), protein kinase C (PKC; GF-109203X), protein kinase A (PKA; H-89) or mitogen-activated protein (MAP) kinase activation (PD-98059) were added. The addition of PI3K and PKC inhibitors, but not of PKA inhibitor, significantly decreased insulin-stimulated Cyp19 mRNA levelsand oestradiol accumulation (P< 0.001). The PKA inhibitor significantly decreased FSH-stimulated Cyp19 mRNA abundance and oestradiol secretion, whereas PI3K and PKC inhibitors decreased oestradiol secretion without affecting Cyp19 mRNA accumulation. Inhibition of MAP kinase pathway significantly increased Cyp19 mRNA abundance ininsulin- and FSH-stimulated cells.P450scc mRNA levels and progesterone secretion were not affected by any inhibitor in either experiment. Although FSH stimulates Cyp19 expression predominantly through PKA, oestradiol secretion is altered by PI3K and PKC pathways independently of Cyp19 mRNA levels. In addition, we suggest that Cyp19 is under tonic inhibition mediated through a MAP kinase pathway.
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Liedtke, Carole M., and Thomas Cole. "Antisense oligodeoxynucleotide to PKC-δ blocks α1-adrenergic activation of Na-K-2Cl cotransport." American Journal of Physiology-Cell Physiology 273, no. 5 (November 1, 1997): C1632—C1640. http://dx.doi.org/10.1152/ajpcell.1997.273.5.c1632.
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A role for protein kinase C (PKC)-δ and -ζ isotypes in α1-adrenergic regulation of human tracheal epithelial Na-K-2Cl cotransport was studied with the use of isotype-specific PKC inhibitors and antisense oligodeoxynucleotides to PKC-δ or -ζ mRNA. Rottlerin, a PKC-δ inhibitor, blocked 72% of basolateral-to-apical, bumetanide-sensitive36Cl flux in nystatin-permeabilized cell monolayers stimulated with methoxamine, an α1-adrenergic agonist, with a 50% inhibitory concentration of 2.3 μM. Methoxamine increased PKC activity in cytosol and a particulate fraction; the response was insensitive to PKC-α and -βIIisotype-specific inhibitors, but was blocked by general PKC inhibitors and rottlerin. Rottlerin also inhibited methoxamine-induced PKC activity in immune complexes of PKC-δ, but not PKC-ζ. At the subcellular level, methoxamine selectively elevated cytosolic PKC-δ activity and particulate PKC-ζ activity. Pretreatment of cell monolayers with antisense oligodeoxynucleotide to PKC-δ for 48 h reduced the amount of whole cell and cytosolic PKC-δ, diminished whole cell and cytosolic PKC-δ activity, and blocked methoxamine-stimulated Na-K-2Cl cotransport. Sense oligodeoxynucleotide to PKC-δ and antisense oligodeoxynucleotide to PKC-ζ did not alter methoxamine-induced cotransport activity. These results demonstrate the selective activation of Na-K-2Cl cotransport by cytosolic PKC-δ.
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Hu, Hui-Juan, and Robert W. Gereau. "ERK Integrates PKA and PKC Signaling in Superficial Dorsal Horn Neurons. II. Modulation of Neuronal Excitability." Journal of Neurophysiology 90, no. 3 (September 2003): 1680–88. http://dx.doi.org/10.1152/jn.00341.2003.
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Protein kinases belonging to the protein kinase A (PKA), protein kinase C (PKC), and extracellular signal-related kinase (ERK) families have been identified as key players in modulating nociception at the level of the spinal cord dorsal horn, yet little is known about the effects of these kinases on membrane properties of the dorsal horn neurons. PKA, PKC, and ERK exert inhibitory effects on transient potassium currents (A-type currents or IA) in mouse superficial dorsal horn neurons ( Hu et al. 2003 ). Here we aimed to determine the effects of these kinases on action potential firing and membrane properties of these neurons to evaluate the impact of the modulation of IA (and other conductances) in these neurons. We found that activating PKC and PKA has dramatic effects on action potential firing, reflecting an increase in the excitability of superficial dorsal horn neurons. In addition, we found that inhibitors of both PKC and ERK signaling decrease the excitability of dorsal horn neurons, suggesting that these kinases exert a tonic excitation of these cells. Consistent with our findings that these kinases inhibit A-type currents, we found that PKA, PKC, and ERK act to shorten the first-spike latency after depolarization induced by current injection. In addition, activation of these kinases increases spike frequency and action potential amplitude of dorsal horn neurons. Interestingly, we found that the effects of PKA and PKC activators are blocked by inhibitors of ERK signaling, suggesting that PKA and PKC may exert their actions by activation of ERKs.
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Reilein, Amy R., Irina S. Tint, Natalia I. Peunova, Grigori N. Enikolopov, and Vladimir I. Gelfand. "Regulation of Organelle Movement in Melanophores by Protein Kinase A (PKA), Protein Kinase C (PKC), and Protein Phosphatase 2A (PP2A)." Journal of Cell Biology 142, no. 3 (August 10, 1998): 803–13. http://dx.doi.org/10.1083/jcb.142.3.803.
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We used melanophores, cells specialized for regulated organelle transport, to study signaling pathways involved in the regulation of transport. We transfected immortalized Xenopus melanophores with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogues of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. α-Melanocyte-stimulating hormone (MSH), a stimulus which increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. We conclude that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A.
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Itoh, Hiroyuki, Shinji Yamamura, J. Anthony Ware, Shaobin Zhuang, Shinsuke Mii, Bo Liu, and K. Craig Kent. "Differential effects of protein kinase C on human vascular smooth muscle cell proliferation and migration." American Journal of Physiology-Heart and Circulatory Physiology 281, no. 1 (July 1, 2001): H359—H370. http://dx.doi.org/10.1152/ajpheart.2001.281.1.h359.
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Vascular smooth muscle cell (SMC) migration and proliferation contribute to intimal hyperplasia, and protein kinase C (PKC) may be required for both events. In this report, we investigated the role of PKC in proliferation and migration of SMC derived from the human saphenous vein. Activation of PKC by phorbol-12,13-dibutyrate (PDBu) or (−)-indolactam [(−)-ILV] increases SMC proliferation. Downregulation of PKC activity by prolonged incubation with phorbol ester or inhibition of PKC with chelerythrine in SMC diminished agonist-stimulated proliferation. In contrast, stimulation of PKC with PDBu or (−)-ILV inhibited basal and agonist-induced SMC chemotaxis. Moreover, downregulation of PKC or inhibition with chelerythrine accentuated migration. We postulated that the inhibitory effect of PKC on SMC chemotaxis was mediated through cAMP-dependent protein kinase (protein kinase A, PKA). In support of this hypothesis, we found that activation of PKC in SMC stimulated PKA activity. The cAMP agonist forskolin significantly inhibited SMC chemotaxis. Furthermore, the inhibitory effect of PKC on SMC chemotaxis was completely reversed by cAMP or PKA inhibitors. In search of the PKC isotype(s) underlying these differential effects of PKC in SMC, we identified eight isotypes expressed in human SMC. Only PKC-α, -βI, -δ, and -ε were eliminated by downregulation, suggesting that one or more of these four enzymes facilitate the observed phorbol ester-dependent effects of PKC in SMC. In summary, we found that PKC activation enhances proliferation but inhibits migration of human vascular SMC. These differential effect of PKC on vascular cells appears to be mediated through PKC-α, -βI, -δ, and/or -ε.
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Wilkinson, S. E., and J. S. Nixon. "PKC Inhibitors in the Therapy of Autoimmune Diseases." Current Pharmaceutical Design 2, no. 6 (December 1996): 596–609. http://dx.doi.org/10.2174/1381612802666221004184418.
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The protein kinase C ( PKC) family of isoenzymes was thought to mediate a wide range of signal transduction processes in cells. However, it is now widely accepted that its role may have been overstated. The advent of selective PKC inhibitors has led to a re-appraisal of the role this enzyme plays in these processes. This review shows how the structural lead provided by staurosporine, a non-selective protein kinase inhibitor, was used as a basis for the design of substituted bisindolylmaleimides with significantly improved selectivity for protein kinase C over cAMP-dependent protein kinase and phophorylase kinase. Evidence from studies with these inhibitors implicates PKC in inflammatory responses such as the neutrophil respiratory burst and antigen-driven T cell proliferation. Potent, orally bioavailable bisindolylmaleimide PKC inhibito_rs such as Ro 32-0432 inhibit phorbol ester -induced inflammation in rodents. These agents also selectively inhibit T cell mediated responses in animal models of arthritis and encephalomyelitis. Taken together, these results suggest that selective inhibitors of PKC may be useful in the therapy of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and psoriasis.
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Liu, Wen, Yuan Wei, Peng Sun, Wen-Hui Wang, Thomas R. Kleyman, and Lisa M. Satlin. "Mechanoregulation of BK channel activity in the mammalian cortical collecting duct: role of protein kinases A and C." American Journal of Physiology-Renal Physiology 297, no. 4 (October 2009): F904—F915. http://dx.doi.org/10.1152/ajprenal.90685.2008.
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Flow-stimulated net K secretion ( JK) in the cortical collecting duct (CCD) is mediated by an iberiotoxin (IBX)-sensitive BK channel, and requires an increase in intracellular Ca2+ concentration ([Ca2+]i). The α-subunit of the reconstituted BK channel is phosphorylated by PKA and PKC. To test whether the BK channel in the native CCD is regulated by these kinases, JK and net Na absorption ( JNa) were measured at slow (∼1) and fast (∼5 nl·min−1·mm−1) flow rates in rabbit CCDs microperfused in the presence of mPKI, an inhibitor of PKA; calphostin C, which inhibits diacylglycerol binding proteins, including PKC; or bisindolylmaleimide (BIM) and Gö6976, inhibitors of classic and novel PKC isoforms, added to luminal (L) and/or basolateral (B) solutions. L but not B mPKI increased JK in CCDs perfused at a slow flow rate; a subsequent increase in flow rate augmented JK modestly. B mPKI alone or with L inhibitor abolished flow stimulation of JK. Similarly, L calphostin C increased JK in CCDs perfused at slow flow rates, as did calphostin C in both L and B solutions. The observation that IBX inhibited the L mPKI- and calphostin C-mediated increases in JK at slow flow rates implicated the BK channel in this K flux, a notion suggested by patch-clamp analysis of principal cells. The kinase inhibited by calphostin C was not PKC as L and/or B BIM and Gö6976 failed to enhance JK at the slow flow rate. However, addition of these PKC inhibitors to the B solution alone or with L inhibitor blocked flow stimulation of JK. Interpretation of these results in light of the effects of these inhibitors on the flow-induced elevation of [Ca2+]i suggests that the principal cell apical BK channel is tonically inhibited by PKA and that flow stimulation of JK in the CCD is PKA and PKC dependent. The specific targets of the kinases remain to be identified.
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Wu, D., I. J. Clarke, and C. Chen. "The role of protein kinase C in GH secretion induced by GH-releasing factor and GH-releasing peptides in cultured ovine somatotrophs." Journal of Endocrinology 154, no. 2 (August 1997): 219–30. http://dx.doi.org/10.1677/joe.0.1540219.
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Abstract The involvement of protein kinase C (PKC) in the action of GH-releasing factor (GRF) and synthetic GH-releasing peptides (GHRP-2 and GHRP-6) was investigated in ovine somatotrophs in primary culture. In partially purified sheep somatotrophs, GRF and GHRP-2 caused translocation of PKC activity from the cytosol to the cell membranes and caused GH release in a dose- and time-dependent manner. GHRP-6 did not cause PKC translocation. The PKC inhibitors, calphostin C, staurosporine and chelerythrine, partially reduced GH release in response to GRF and GHRP-2 at doses which selectively inhibit PKC activity. These inhibitors totally abolished GH release caused by phorbol 12-myristate 13-acetate (PMA). Down-regulation of PKC by the treatment of cells with phorbol 12,13-dibutyrate for 16 h caused a significant (P<0·001) reduction in total PKC activity and totally abolished PKC translocation in response to a challenge with GRF, GHRP-2 or PMA. In addition, down-regulation abolished GH release in response to GRF, GHRP-2 or GHRP-6. Treatment of cells with H89, a selective PKA inhibitor, totally blocked GH release caused by either GRF or GHRP-2 and partially reduced PMA-induced GH release. H89 had no effect on PKC translocation caused by GRF, GHRP-2 or PMA and did not affect GH release caused by GHRP-6. These data suggest that GHRP-2 and GRF activate PKC in addition to stimulating adenylyl cyclase activity. Although the cAMP–protein kinase A (PKA) pathway is the major signalling pathway employed by GRF and GHRP-2, the activation of PKC may potentiate signalling via the cAMP–PKA pathway in ovine GH secretion. Importantly, the effect of PMA in increasing the secretion of GH from ovine somatotrophs is effected, in part, by up-regulation of the cAMP–PKA pathway. We conclude that there is cross-talk between the PKC pathway and the cAMP–PKA pathway in ovine somatotrophs during the action of GRF or GHRP. Journal of Endocrinology (1997) 154, 219–230
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Takahashi, Hideyuki, and Hideo Namiki. "Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors." Biochemical Journal 405, no. 2 (June 27, 2007): 331–40. http://dx.doi.org/10.1042/bj20070299.
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ATP-competitive inhibitors of PKC (protein kinase C) such as the bisindolylmaleimide GF 109203X, which interact with the ATP-binding site in the PKC molecule, have also been shown to affect several redistribution events of PKC. However, the reason why these inhibitors affect the redistribution is still controversial. In the present study, using immunoblot analysis and GFP (green fluorescent protein)-tagged PKC, we showed that, at commonly used concentrations, these ATP-competitive inhibitors alone induced redistribution of DAG (diacylglycerol)-sensitive PKCα, PKCβII, PKCδ and PKCϵ, but not atypical PKCζ, to the endomembrane or the plasma membrane. Studies with deletion and point mutants showed that the DAG-sensitive C1 domain of PKC was required for membrane redistribution by these inhibitors. Furthermore, membrane redistribution was prevented by the aminosteroid PLC (phospholipase C) inhibitor U-73122, although an ATP-competitive inhibitor had no significant effect on acute DAG generation. Immunoblot analysis showed that an ATP-competitive inhibitor enhanced cell-permeable DAG analogue- or phorbol-ester-induced translocation of endogenous PKC. Furthermore, these inhibitors also enhanced [3H]phorbol 12,13-dibutyrate binding to the cytosolic fractions from PKCα–GFP-overexpressing cells. These results clearly demonstrate that ATP-competitive inhibitors cause redistribution of DAG-sensitive PKCs to membranes containing endogenous DAG by altering the DAG sensitivity of PKC and support the idea that the inhibitors destabilize the closed conformation of PKC and make the C1 domain accessible to DAG. Most importantly, our findings provide novel insights for the interpretation of studies using ATP-competitive inhibitors, and, especially, suggest caution about the interpretation of the relationship between the redistribution and kinase activity of PKC.
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Hou, Lili, Lei Zhu, Min Zhang, Xingyi Zhang, Guoqing Zhang, Zhenwei Liu, Qiang Li, and Xin Zhou. "Participation of Antidiuretic Hormone (ADH) in Asthma Exacerbations Induced by Psychological Stress via PKA/PKC Signal Pathway in Airway-Related Vagal Preganglionic Neurons (AVPNs)." Cellular Physiology and Biochemistry 41, no. 6 (2017): 2230–41. http://dx.doi.org/10.1159/000475638.
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Aims: Present study was performed to examine whether ADH was implicated in psychological stress asthma and to explore the underlying molecular mechanism. Methods: We not only examined ADH levels in the cerebrospinal fluid (CSF) via radioimmunoassay, but also measured ADH receptor (ADHR) expression in airway-related vagal preganglionic neurons (AVPNs) through real-time PCR in all experimental mice. Western blotting was performed to evaluate the relationship between ADH and PKA/PKC in psychological stress asthma. Finally, the role of PKA/PKC in psychological stress asthma was analyzed. Results: Marked asthma exacerbations were noted owing to significantly elevated levels of ADH and ADHR after psychological stress induction as compared to OVA alone (asthma group). ADHR antagonists (SR-49095 or SR-121463A) dramatically lowered higher protein levels of PKAα and PKCα induced by psychological stress as compared to OVA alone, suggesting the correlation between ADH and PKA/PKC in psychological stress asthma. KT-5720 (PKA inhibitor) and Go-7874 (PKC inhibitor) further directly revealed the involvement of PKA/PKC in psychological stress asthma. Some notable changes were also noted after employing PKA and PKC inhibitors in psychological stress asthma, including reduced asthmatic inflammation (lower eosinophil peroxidase (EPO) activity, myeloperoxidase (MPO) activity, immunoglobulin E (IgE) level, and histamine release), substantial decrements in inflammatory cell counts (eosinophils and lymphocytes), and decreased cytokine secretion (IL-6, IL-10, and IFN-γ), indicating the involvement of PKA/PKC in asthma exacerbations induced by psychological stress. Conclusion: Our results strongly suggested that ADH participated in psychological stress-induced asthma exacerbations via PKA/PKC signal pathway in AVPNs.
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Haddad, Georges E., Bernell R. Coleman, Aiqiu Zhao, and Krista N. Blackwell. "Regulation of atrial contraction by PKA and PKC during development and regression of eccentric cardiac hypertrophy." American Journal of Physiology-Heart and Circulatory Physiology 288, no. 2 (February 2005): H695—H704. http://dx.doi.org/10.1152/ajpheart.00783.2004.
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ANG II plays a major role in development of cardiac hypertrophy through its AT1 receptor subtype, whereas angiotensin-converting enzyme (ACE) inhibitors are effective in reversing effects of ANG II on the heart. The objective of this study was to investigate the role of PKA and PKC in the contractile response of atrial tissue during development and ACE inhibitor-induced regression of eccentric hypertrophy induced by aortocaval shunt. At 1 wk after surgery, sham and shunt rats were divided into captopril-treated and untreated groups for 2 wk. Then isometric contraction was assessed by electrical stimulation of isolated rat left atrial preparations superfused with Tyrode solution in the presence or absence of specific inhibitors KT-5720 (for PKA) and Ro-32-0432 (for PKC) and high Ca2+. Peak tension developed was greater in shunt than in sham hearts. However, when expressed relative to tissue mass, hypertrophied muscle showed weaker contraction than muscle from sham rats. In sham rats, peak tension developed was more affected by PKC than by PKA inhibition, whereas this differential effect was reduced in the hypertrophied heart. Treatment of shunt rats with captopril regressed left atrial hypertrophy by 67% and restored PKC-PKA differential responsiveness toward sham levels. In the hypertrophied left atria, there was an increase in the velocity of contraction and relaxation that was not evident when expressed in specific relative terms. Treatment with ACE inhibitor increased the specific velocity of contraction, as well as its PKC sensitivity, in shunt rats. We conclude that ACE inhibition during eccentric cardiac hypertrophy produces a negative trophic and a positive inotropic effect, mainly through a PKC-dependent mechanism.
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Murthy, K. S., J. R. Grider, J. F. Kuemmerle, and G. M. Makhlouf. "Sustained muscle contraction induced by agonists, growth factors, and Ca2+ mediated by distinct PKC isozymes." American Journal of Physiology-Gastrointestinal and Liver Physiology 279, no. 1 (July 1, 2000): G201—G210. http://dx.doi.org/10.1152/ajpgi.2000.279.1.g201.
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The role of protein kinase C (PKC) in sustained contraction was examined in intestinal circular and longitudinal muscle cells. Initial contraction induced by agonists (CCK-8 and neuromedin C) was abolished by 1) inhibitors of Ca2+ mobilization (neomycin and dimethyleicosadienoic acid), 2) calmidazolium, and 3) myosin light chain (MLC) kinase (MLCK) inhibitor KT-5926. In contrast, sustained contraction was not affected by these inhibitors but was abolished by 1) the PKC inhibitors chelerythrine and calphostin C, 2) PKC-ε antibody, and 3) a pseudosubstrate PKC-ε inhibitor. GDPβS abolished both initial and sustained contraction, whereas a Gαq/11 antibody inhibited only initial contraction, implying that sustained contraction was dependent on activation of a distinct G protein. Sustained contraction induced by epidermal growth factor was inhibited by calphostin C, PKC-α,β,γ antibody, and a pseudosubstrate PKC-α inhibitor. Ca2+ (0.4 μM) induced an initial contraction in permeabilized muscle cells that was blocked by calmodulin and MLCK inhibitors and a sustained contraction that was blocked by calphostin C and a PKC-α,β,γ antibody. Thus initial contraction induced by Ca2+, agonists, and growth factors is mediated by MLCK, whereas sustained contraction is mediated by specific Ca2+-dependent and -independent PKC isozymes. G protein-coupled receptors are linked to PKC activation via distinct G proteins.
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Gomes, Pedro, and P. Soares-da-Silva. "Role of cAMP-PKA-PLC signaling cascade on dopamine-induced PKC-mediated inhibition of renal Na+-K+-ATPase activity." American Journal of Physiology-Renal Physiology 282, no. 6 (June 1, 2002): F1084—F1096. http://dx.doi.org/10.1152/ajprenal.00318.2001.
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We studied the molecular events set into motion by stimulation of D1-like receptors downstream of Na+-K+-ATPase, while measuring apical-to-basal ouabain-sensitive, amphotericin B-induced increases in short-circuit current in opossum kidney (OK) cells. The D1-like receptor agonist SKF-38393 decreased Na+-K+-ATPase activity (IC50, 130 nM). This effect was prevented by the D1-like receptor antagonist SKF-83566, overnight cholera toxin treatment, the protein kinase A (PKA) antagonist H-89, or the PKC antagonist chelerythrine, but not the mitogen-activated PK inhibitor PD-098059 or phosphatidylinositol 3-kinase inhibitors wortmannin and LY-294002. Dibutyryl cAMP (DBcAMP) and phorbol 12,13-dibutyrate (PDBu) both effectively reduced Na+-K+-ATPase activity. PKA downregulation abolished the inhibitory effects of SKF-38393 and DBcAMP but not those of PDBu. PKC downregulation abolished inhibition by PDBu, SKF-38393, and DBcAMP. The phospholipase C (PLC) inhibitor U-73122 prevented inhibition by SKF-38393 and DBcAMP. However, DBcAMP increased PLC activity. Although OK cells express both Gsα and Gq/11α proteins, D1-like receptors are coupled to Gsα proteins only, as evidenced by studies in cells treated overnight with specific antibodies raised against Gsα and Gq/11α proteins. We conclude that PLC and Na+-K+-ATPase are effector proteins for PKA and PKC, respectively, after stimulation of D1-like receptors coupled to Gsα proteins, in a sequence of events that begins with adenylyl cyclase-PKA system activation followed by PLC-PKC system activation.
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Rashid, Gloria, Eleanora Plotkin, Osnat Klein, Janice Green, Jacques Bernheim, and Sydney Benchetrit. "Parathyroid hormone decreases endothelial osteoprotegerin secretion: role of protein kinase A and C." American Journal of Physiology-Renal Physiology 296, no. 1 (January 2009): F60—F66. http://dx.doi.org/10.1152/ajprenal.00622.2007.
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Parathyroid hormone (PTH), which is elevated in patients with chronic renal failure, has been shown to participate in the development of vascular calcification. Previous studies have demonstrated that PTH may promote endothelial expressions of proinflammatory parameters. On the basis of these data, we evaluated whether PTH may have an impact on endothelial osteoprotegerin (OPG), a vascular-protective factor which may control vascular calcification. Endothelial cells were stimulated with 10−12 to 10−10 mol/l PTH. PKC and PKA are the main cellular pathways of PTH. Inhibitors and activators of PKC or PKA were used to determine whether these signaling pathways are involved in the control of endothelial OPG. PTH induced a decrease in OPG secretion and mRNA expression. Treatment of PTH-stimulated cells by calphostin C (PKC inhibitor) induced a further decrease in OPG secretion, while Rp-cAMP (PKA inhibitor) had no additional effect. In nonstimulated cells, a PKC activator significantly stimulated OPG secretion, while a PKA activator was associated with a decline. These effects were blunted in the presence of calphostin C and Rp-cAMP, respectively. An increase in OPG secretion induced by a PKC activator indicates that the basal OPG secretion is mediated through PKC. The decrease induced by a PKA activator, which is similar to the decrease observed with PTH, suggests that the action of PTH on OPG secretion and mRNA expression may be due to the PKA pathway.
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Liu, M., J. Xu, J. Liu, M. E. Kraw, A. K. Tanswell, and M. Post. "Mechanical strain-enhanced fetal lung cell proliferation is mediated by phospholipase C and D and protein kinase C." American Journal of Physiology-Lung Cellular and Molecular Physiology 268, no. 5 (May 1, 1995): L729—L738. http://dx.doi.org/10.1152/ajplung.1995.268.5.l729.
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The signaling pathways by which intermittent strain (60 cycles/min, 15 min/h) regulates proliferation of mixed fetal rat lung cell in vitro have been investigated. Adenosine 3',5'-cyclic monophosphate (cAMP) content and cAMP-dependent protein kinase (PKA) activity were not affected by strain. The stimulatory effect of strain on DNA synthesis was also not influenced by the cyclic nucleotide-dependent protein kinase inhibitors H-8 or HA-1004, the adenylate cyclase inhibitor SQ-22536, or a PKA inhibitor and cAMP antagonist, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). In contrast, intracellular concentrations of two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), were dramatically increased after a short period of strain. This increase in second messengers was accompanied by an increased tyrosine phosphorylation of phospholipase C-gamma 1. Phospholipase D activity was also increased by strain. Mechanical strain elicited a shift in the subcellular distribution of PKC activity from cytosol to membranes shortly after the onset of strain. The specific activity of PKC in the membranes increased 6- to 10-fold within 5-15 min and remained increased throughout a 48-h period of intermittent strain. Strain-induced PKC activation and DNA synthesis were blocked by the PKC inhibitors H-7, staurosporine, and calphostin C, as well as by the phospholipase C inhibitor U-73,122. We conclude that mechanical strain of mixed fetal rat lung cells activates phospholipid turnover via phospholipases, followed by PKC activation, which then triggers the downstream events that lead to cell proliferation.
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Murai, Y., Y. Okabe, and E. Tanaka. "Activation of protein kinase A and C prevents recovery from persistent depolarization produced by oxygen and glucose deprivation in rat hippocampal neurons." Journal of Neurophysiology 107, no. 9 (May 1, 2012): 2517–25. http://dx.doi.org/10.1152/jn.00537.2011.
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Intracellular recordings were made from rat hippocampal CA1 neurons in rat brain slice preparations to investigate whether cAMP-dependent protein kinase (PKA) and calcium/phospholipid-dependent protein kinase C (PKC) contribute to the membrane dysfunction induced by oxygen and glucose deprivation (OGD). Superfusion of oxygen- and glucose-deprived medium produced a rapid depolarization ∼5 min after the onset of the superfusion. When oxygen and glucose were reintroduced immediately after the rapid depolarization, the membrane depolarized further (persistent depolarization) and reached 0 mV after 5 min from the reintroduction. The pretreatment of the slice preparation with PKA inhibitors, H-89 and Rp-cAMPS, and an adenylate cyclase inhibitor, SQ 22, 536, significantly restored the membrane toward the preexposure potential level after the reintroduction of oxygen and glucose in a concentration-dependent manner. On the other hand, a phospholipase C inhibitor, U73122, a PKC inhibitor, GF109203X, and a nonselective protein kinase inhibitor, staurosporine, also significantly restored the membrane after the reintroduction. Moreover, an inositol-1,4,5-triphosphate receptor antagonist, 2-aminoethyl diphenylborinate, and calmodulin inhibitors, trifluoperazine and W-7, significantly restored the membrane after the reintroduction, while neither an α-subunit-selective antagonist for stimulatory G protein, NF449, a Ca2+/calmodulin-dependent kinase II inhibitor, KN-62, nor a myosin light chain kinase inhibitor, ML-7, significantly restored the membrane after the reintroduction. These results suggest that the activation of PKA and/or PKC prevents the recovery from the persistent depolarization produced by OGD. The Ca2+/calmodulin-stimulated adenylate cyclase may contribute to the activation of PKA.
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Sriwai, Wimolpak, Huiping Zhou, and Karnam S. Murthy. "Gq-dependent signalling by the lysophosphatidic acid receptor LPA3 in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA." Biochemical Journal 411, no. 3 (April 14, 2008): 543–51. http://dx.doi.org/10.1042/bj20071299.
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The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA3 receptors, but not LPA1 and LPA2, receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the Gαq, but not the Gαi, minigene. Initial contraction and MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-β (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC20 and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IκBα were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr696 was masked by phosphorylation of MYPT1 at Ser695 by PKA derived from IκB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-β and MLCK and phosphorylation of MLC20, and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC20. Although Rho kinase was activated, phosphorylation of MYPT1 at Thr696 by Rho kinase was masked by phosphorylation of MYPT1 at Ser695 via cAMP-independent PKA derived from the NF-κB pathway.
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Hartzell, H. C., Y. Hirayama, and J. Petit-Jacques. "Effects of protein phosphatase and kinase inhibitors on the cardiac L-type Ca current suggest two sites are phosphorylated by protein kinase A and another protein kinase." Journal of General Physiology 106, no. 3 (September 1, 1995): 393–414. http://dx.doi.org/10.1085/jgp.106.3.393.
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We previously showed (Frace, A.M. and H.C. Hartzell. 1993. Journal of Physiology. 472:305-326) that internal perfusion of frog atrial myocytes with the nonselective protein phosphatase inhibitors microcystin or okadaic acid produced an increase in the L-type Ca current (ICa) and a decrease in the delayed rectifier K current (IK). We hypothesized that microcystin revealed the activity of a protein kinase (PKX) that was basally active in the cardiac myocyte that could phosphorylate the Ca and K channels or regulators of the channels. The present studies were aimed at determining the nature of PKX and its phosphorylation target. The effect of internal perfusion with microcystin on ICa or IK was not attenuated by inhibitors of protein kinase A (PKA). However, the effect of microcystin on ICa was largely blocked by the nonselective protein kinase inhibitors staurosporine (10-30 nM), K252a (250 nM), and H-7 (10 microM). Staurosporine and H-7 also decreased the stimulation of ICa by isoproterenol, but K252a was more selective and blocked the ability of microcystin to stimulate ICa without significantly reducing isoproterenol-stimulated current. Internal perfusion with selective inhibitors of protein kinase C (PKC), including the autoinhibitory pseudosubstrate PKC peptide (PKC(19-31)) and a myristoylated derivative of this peptide had no effect. External application of several PKC inhibitors had negative side effects that prevented their use as selective PKC inhibitors. Nevertheless, we conclude that PKX is not PKC. PKA and PKX phosphorylate sites with different sensitivities to the phosphatase inhibitors calyculin A and microcystin. In contrast to the results with ICa, the effect of microcystin on IK was not blocked by any of the kinase inhibitors tested, suggesting that the effect of microcystin on IK may not be mediated by a protein kinase but may be due to a direct effect of microcystin on the IK channel.
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LIU, Qing-Rong, Ping-Wu ZHANG, Zhicheng LIN, Qi-Fu LI, Amina S. WOODS, Juan TRONCOSO, and George R. UHL. "GBPI, a novel gastrointestinal- and brain-specific PP1-inhibitory protein, is activated by PKC and inactivated by PKA." Biochemical Journal 377, no. 1 (January 1, 2004): 171–81. http://dx.doi.org/10.1042/bj20030128.
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The activities of PP1 (protein phosphatase 1), a principal cellular phosphatase that reverses serine/threonine protein phosphorylation, can be altered by inhibitors whose activities are themselves regulated by phosphorylation. We now describe a novel PKC (protein kinase C)-dependent PP1 inhibitor, namely GBPI (gut and brain phosphatase inhibitor). The shorter mRNA that encodes this protein, GBPI-1, is expressed in brain, stomach, small intestine, colon and kidney, whereas a longer GBPI-2 splice variant mRNA is found in testis. Human GBPI-1 mRNA encodes a 145-amino-acid, 16.5 kDa protein with pI 7.92. GBPI contains a consensus PP1-binding motif at residues 21–25 and consensus sites for phosphorylation by enzymes, including PKC, PKA (protein kinase A or cAMP-dependent protein kinase) and casein kinase II. Recombinant GBPI-1-fusion protein inhibits PP1 activity with IC50=3 nM after phosphorylation by PKC. Phospho-GBPI can even enhance PP2A activity by >50% at submicromolar concentrations. Non-phosphorylated GBPI-1 is inactive in both assays. Each of the mutations in amino acids located in potential PP1-binding sequences, K21E+K22E and W25A, decrease the ability of GBPI-1 to inhibit PP1. Mutations in the potential PKC phosphoacceptor site T58E also dramatically decrease the ability of GBPI-1 to inhibit PP1. Interestingly, when PKC-phosphorylated GBPI-1 is further phosphorylated by PKA, it no longer inhibits PP1. Thus, GBPI-1 is well positioned to integrate PKC and PKA modulation of PP1 to regulate differentially protein phosphorylation patterns in brain and gut. GBPI, its closest family member CPI (PKC-potentiated PP1 inhibitor) and two other family members, kinase-enhanced phosphatase inhibitor and phosphatase holoenzyme inhibitor, probably modulate integrated control of protein phosphorylation states in these and other tissues.
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Volk, Kenneth A., Russell F. Husted, Peter M. Snyder, and John B. Stokes. "Kinase regulation of hENaC mediated through a region in the COOH-terminal portion of the α-subunit." American Journal of Physiology-Cell Physiology 278, no. 5 (May 1, 2000): C1047—C1054. http://dx.doi.org/10.1152/ajpcell.2000.278.5.c1047.
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In an effort to gain insight into how kinases might regulate epithelial Na+ channel (ENaC) activity, we expressed human ENaC (hENaC) in Xenopus oocytes and examined the effect of agents that modulate the activity of some kinases. Activation of protein kinase C (PKC) by phorbol ester increased the activity of ENaC, but only in oocytes with a baseline current of <2,000 nA. Inhibitors of protein kinases produced varying effects. Chelerythrine, an inhibitor of PKC, produced a significant inhibition of ENaC current, but calphostin C, another PKC inhibitor, had no effect. The PKA/protein kinase G inhibitor H-8 had no effect, whereas the p38 mitogen-activated protein kinase inhibitor, SB-203580 had a significant inhibitory effect. Staurosporine, a nonspecific kinase inhibitor, was the most potent tested. It inhibited ENaC currents in both oocytes and in M-1 cells, a model for the collecting duct. Site-directed mutagenesis revealed that the staurosporine effect did not require an intact COOH terminus of either the β- or γ-hENaC subunit. However, an intact COOH terminus of the α-subunit was required for this effect. These results suggest that an integrated kinase network regulates ENaC activity through an action that requires a portion of the α-subunit.
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Sajan, Mini P., Michael Leitges, Colin Park, David M. Diamond, Jin Wu, Barbara C. Hansen, Mildred A. Duncan, et al. "Control of β-Site Amyloid Precursor Protein-Cleaving Enzyme-1 Expression by Protein Kinase C-λ/ι and Nuclear Factor κ-B." Current Alzheimer Research 18, no. 12 (December 27, 2021): 941–55. http://dx.doi.org/10.2174/1567205019666211222120448.
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Βackground: β-Amyloid precursor protein-cleaving enzyme-1 (BACE1) initiates the production of Aβ-peptides that form Aβ-plaque in Alzheimer’s disease. Methods: Reportedly, acute insulin treatment in normal mice, and hyperinsulinemia in high-fat-fed (HFF) obese/diabetic mice, increase BACE1 activity and levels of Aβ-peptides and phospho- -thr-231-tau in the brain; moreover, these effects are blocked by PKC-λ/ι inhibitors. However, as chemical inhibitors may affect unsuspected targets, we presently used knockout methodology to further examine PKC-λ/ι requirements. We found that total-body heterozygous PKC-λ knockout reduced acute stimulatory effects of insulin and chronic effects of hyperinsulinemia in HFF/obese/diabetic mice, on brain PKC-λ activity and production of Aβ1-40/42 and phospho-thr-231-tau. This protection in HFF mice may reflect that hepatic PKC-λ haploinsufficiency prevents the development of glucose intolerance and hyperinsulinemia. Results: On the other hand, heterozygous knockout of PKC-λ markedly reduced brain levels of BACE1 protein and mRNA, and this may reflect diminished activation of nuclear factor kappa-B (NFκB), which is activated by PKC-λ and increases BACE1 and proinflammatory cytokine transcription. Accordingly, whereas intravenous administration of aPKC inhibitor diminished aPKC activity and BACE1 levels by 50% in the brain and 90% in the liver, nasally-administered inhibitor reduced aPKC activity and BACE1 mRNA and protein levels by 50-70% in the brain while sparing the liver. Additionally, 24-hour insulin treatment in cultured human-derived neurons increased NFκB activity and BACE1 levels, and these effects were blocked by various PKC-λ/ι inhibitors. Conclusion: PKC-λ/ι controls NFκB activity and BACE1 expression; PKC-λ/ι inhibitors may be used nasally to target brain PKC-λ/ι or systemically to block both liver and brain PKC-λ/ι, to regulate NFκB-dependent BACE1 and proinflammatory cytokine expression.
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Wrede, CE, LM Dickson, MK Lingohr, I. Briaud, and CJ Rhodes. "Fatty acid and phorbol ester-mediated interference of mitogenic signaling via novel protein kinase C isoforms in pancreatic beta-cells (INS-1)." Journal of Molecular Endocrinology 30, no. 3 (June 1, 2003): 271–86. http://dx.doi.org/10.1677/jme.0.0300271.
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It is possible that activation of protein kinase C (PKC) isoforms by free fatty acids (FFA) plays a role in the failure of pancreatic beta-cell mass expansion to compensate for peripheral insulin resistance in the pathogenesis of type-2 diabetes. The effect of lipid moieties on activation of conventional (PKC-alpha and -beta1), novel (PKC-delta) and atypical (PKC-zeta) PKC isoforms was evaluated in an in vitro assay, using biotinylated neurogranin as a substrate. Oleoyl-Coenzyme A (CoA) and palmitoyl-CoA, but not unesterified FFA, significantly increased the activity of all PKC isoforms (P< or =0.05), particularly that for PKC-delta. It was found that FFA (0.4 mM oleate/complexed to 0.5% bovine serum albumin) inhibited IGF-I-induced activation of protein kinase B (PKB) in the pancreatic beta-cell line (INS-1), but this was alleviated in the presence of the general PKC inhibitor (Go6850; 1 microM). To further investigate whether conventional or novel PKC isoforms adversely affect beta-cell proliferation, the effect of phorbol ester (phorbol 12-myristate 13-acetate; PMA)-mediated activation of these PKC isoforms on glucose/IGF-I-induced INS-1 cell mitogenesis, and insulin receptor substrate (IRS)-mediated signal transduction was investigated. PMA-mediated activation of PKC (100 nM; 4 h) reduced glucose/IGF-I mediated beta-cell mitogenesis (>50%; P< or =0.05), which was reversible by the general PKC inhibitor Go6850 (1 microM), indicating an effect of PKC and not due to a non-specific PMA toxicity. PMA inhibited IGF-I-induced activation of PKB, correlating with inhibition of IGF-I-induced association of IRS-2 with the p85 regulatory subunit of phosphatidylinositol-3 kinase. However, in contrast, PMA activated the mitogen-activated protein kinases, Erk1/2. Titration inhibition analysis using PKC isoform inhibitors indicated that these PMA-induced effects were via novel PKC isoforms. Thus, FFA/PMA-induced activation of novel PKC isoforms can inhibit glucose/IGF-I-mediated beta-cell mitogenesis, in part by decreasing PKB activation, despite an upregulation of Erk1/2. Thus, activation of novel PKC isoforms by long-chain acyl-CoA may well contribute to decreasing beta-cell mass in the pathogenesis of type-2 diabetes, similar to their inhibition of insulin signal transduction which causes insulin resistance.
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Dow, Robert L. "Progress Towards Selective Inhibition of Protein Kinase C." Current Medicinal Chemistry 1, no. 3 (October 1994): 192–203. http://dx.doi.org/10.2174/092986730103220214163307.
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Abstract: Protein kinase C (PKC), a member of the serine/threonine family of protein kinases, is an integral part of cellular signal transduction. There is mounting evidence that inappropriate activation of PKC may be responsible for a number of disease states. Potent, selective inhibitors of PKC could serve as valuable tools for def,lning these signaling pathways, as well as, for the treatment of diseases mediated through aberrant PKC activity. Several mechanistically-distinct classes of inhibitors have been identified which potently and selectively suppress PKC activity. The most advanced and promising of these agents are Go-6976 and Ro-32-0432, which arose from optimization studies on staurosporine, an indolecarbazole-based natural product. Though these compounds inhibit PKC competitively with respect to nucleotide cofactor, they exhibit little inhibitory activity against a range of other nucleotide-dependent enzymes. More modest advances have begun to be made with respect to selective inhibition of the various PKC isozymes. This review will update efforts directed towards the discovery of inhibitors of PKC, with a focus on the associated selectivity parameters.
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Klausen, Christian, David L. Severson, John P. Chang, and Hamid R. Habibi. "Role of PKC in the regulation of gonadotropin subunit mRNA levels: interaction with two native forms of gonadotropin-releasing hormone." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 289, no. 6 (December 2005): R1634—R1643. http://dx.doi.org/10.1152/ajpregu.00186.2005.
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Gonadotropin-releasing hormone (GnRH) is an important regulator of reproduction in all vertebrates through its actions on the production and secretion of pituitary gonadotropin hormones (GtHs). Most vertebrate species express at least two GnRHs, including one form, designated chicken (c)GnRH-II or type II GnRH, which has been well conserved throughout evolution. The goldfish brain and pituitary contain salmon GnRH and cGnRH-II. In goldfish, GnRH-induced luteinizing hormone (LH) secretion involves PKC; however, whether PKC mediates GnRH stimulation of GtH subunit mRNA levels is unknown. In this study, we used inhibitors and activators of PKC to examine its possible involvement in GnRH-induced increases in GtH-α, follicle-stimulating hormone (FSH)-β and LH-β mRNA levels in primary cultures of dispersed goldfish pituitary cells. Treatment with PKC inhibitors calphostin C and GF109203X unmasked a basal repression of GtH subunit mRNA levels by PKC; both inhibitors increased GtH subunit mRNA levels in a dose-dependent manner. PKC activators, 12- O-tetradecanoylphorbol 13-acetate (TPA), and 1,2-dioctanoyl- sn-glycerol, stimulated GtH subunit mRNA levels, whereas an inactive phorbol ester (4-α-TPA) was without effect. Thus, a dual, inhibitory and stimulatory, influence for PKC in the regulation of GtH subunit mRNA levels is suggested. In contrast, PKC inhibitor- and activator-induced effects were, for the most part, additive to those of GnRH, suggesting that conventional and novel PKCs are unlikely to be involved in GnRH-stimulated increases in GtH subunit mRNA levels. Our data illustrate major differences in the signal transduction of GnRH effects on GtH secretion and gene expression in the goldfish pituitary.
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Miakotina, Olga L., and Jeanne M. Snyder. "Signal transduction events involved in TPA downregulation of SP-A gene expression." American Journal of Physiology-Lung Cellular and Molecular Physiology 286, no. 6 (June 2004): L1210—L1219. http://dx.doi.org/10.1152/ajplung.00416.2003.
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Surfactant protein A (SP-A), the most abundant pulmonary surfactant protein, plays a role in innate host defense and blocks the inhibitory effects of serum proteins on surfactant surface tension-lowering properties. SP-A mRNA and protein are downregulated by phorbol esters (TPA) via inhibition of gene transcription. We evaluated the TPA signaling pathways involved in SP-A inhibition in a lung cell line, H441 cells. TPA caused sustained phosphorylation of p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK, and c-Jun-NH2-terminal kinase. An inhibitor of conventional and novel isoforms of protein kinase C (PKC) and two inhibitors of p44/42 MAPK kinase partially or completely blocked the inhibitory effects of TPA on SP-A mRNA levels. In contrast, inhibitors of conventional PKC-α and -β, stress-activated protein kinases, protein phosphatases, protein kinase A, and the phosphatidylinositol 3-kinase pathway had no effect on the TPA-mediated inhibition of SP-A mRNA. TPA also stimulated the synthesis of c-Jun mRNA and protein in a time-dependent manner. Inhibitors of the p44/42 MAPK signaling pathway and PKC blocked the TPA-mediated phosphorylation of p44/42 MAPK and the increase in c-Jun mRNA. We conclude that TPA inhibits SP-A gene expression via novel isoforms of PKC, the p44/42 MAPK pathway, and the activator protein-1 complex.
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Lin, Hung-Yun, Paul M. Yen, Faith B. Davis, and Paul J. Davis. "Protein synthesis-dependent potentiation by thyroxine of antiviral activity of interferon-γ." American Journal of Physiology-Cell Physiology 273, no. 4 (October 1, 1997): C1225—C1232. http://dx.doi.org/10.1152/ajpcell.1997.273.4.c1225.
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We have studied the prenuclear signal transduction pathway by which thyroid hormone potentiates the antiviral activity of human interferon-γ (IFN-γ) in HeLa cells, which are deficient in thyroid hormone receptor (TR). The action of thyroid hormone was compared with that of milrinone, which has structural homologies with thyroid hormone.l-Thyroxine (T4), 3,5,3′-l-triiodothyronine (T3), and milrinone enhanced the antiviral activity of IFN-γ up to 100-fold, a potentiation blocked by cycloheximide. The 5′-deiodinase inhibitor 6- n-propyl-2-thiouracil did not block the T4 effect. 3,3′,5,5′-Tetraiodothyroacetic acid prevented the effect of T4 but not of milrinone. The effects of T4 and milrinone were blocked by inhibitors of protein kinases C (PKC) and A (PKA) and restored by PKC and PKA agonists; only the effect of T4 was blocked by genistein, a tyrosine kinase inhibitor. In separate models, milrinone was shown not to interact with nuclear TR-β. T4 potentiation of the antiviral activity of IFN-γ requires PKC, PKA, and tyrosine kinase activities but not traditional TR.
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Ali, A., A. W. Ford-Hutchinson, and D. W. Nicholson. "Activation of protein kinase C down-regulates leukotriene C4 synthase activity and attenuates cysteinyl leukotriene production in an eosinophilic substrain of HL-60 cells." Journal of Immunology 153, no. 2 (July 15, 1994): 776–88. http://dx.doi.org/10.4049/jimmunol.153.2.776.
Full textAbstract:
Abstract An eosinophilic substrain of HL-60 cells (HL-60#7) predominantly synthesized cysteinyl leukotrienes after stimulation with the calcium ionophore A23187. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) specifically attenuated cysteinyl leukotriene production without affecting the biosynthesis of non-cysteinyl leukotrienes. The inhibition of cysteinyl leukotriene biosynthesis was prevented only by specific PKC inhibitors (staurosporine and bisindolylmaleimide) but not by inhibitors of tyrosine kinases (genistein, tyrphostin 47, and herbimycin A), protein kinase A (KT5720), or the oxidative burst (apocynin). Similar results were obtained when LTC4 synthase enzymatic activity was measured directly in the presence of saturating concentrations of exogenously added substrates. Therefore, the inhibitory effects of PKC activation on cysteinyl leukotriene formation in intact cells was attributable to effects on the LTC4 synthase enzyme. The mechanism of inhibition of LTC4 synthase by PKC activation was determined by kinetic analysis to be noncompetitive in both eosinophil-like HL-60#7 cells and monocytic THP-1 cells. Contrary to the effect of PKC activation on cysteinyl leukotriene biosynthesis, the formation of prostaglandin E2 and thromboxane B2 was elevated twofold to threefold after PMA treatment, which was prevented by the PKC inhibitor, staurosporine. We propose a regulatory model in which PKC activation shifts the profile of eicosanoid mediators produced by eosinophils from cysteinyl leukotrienes to prostanoids.
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Oriji, G. K., and H. R. Keiser. "Action of protein kinase C in endothelin-induced contractions in rat aortic rings." American Journal of Physiology-Cell Physiology 271, no. 1 (July 1, 1996): C398—C404. http://dx.doi.org/10.1152/ajpcell.1996.271.1.c398.
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Endothelin (ET) is a potent vasoconstrictor peptide that induces characteristically long-lasting contractions. We used both intact and endothelium-denuded rat aortic rings to investigate the role of protein kinase C (PKC) in ET-induced contractions. ET (10(-9) M) and phorbol 12,13-dibutyrate (PDBu), a PKC activator, produced a gradual and sustained contraction of greater magnitude in denuded aortic rings than in intact rings. When aortic rings were pretreated with graded concentrations of different PKC inhibitors, inhibition of ET-induced contractions began at 10(-9)M and was nearly complete at 10(-3)M, and the reduction was greater in intact than in denuded rings. Pretreatment of aortic rings with PDBu or NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, potentiated ET-induced contractions. PKC enzyme assay showed activation of PKC in aortic rings that were treated with either ET or PDBu, inhibition after pretreatment with PKC inhibitors, and no change with 4 alpha-phorbol 12,13-didecanoate (PDD), an inactive phorbol ester. ET significantly increased nitrate and nitrite production, which was further increased by pretreatment with PKC inhibitors. PDBu prevented ET-induced nitrate/nitrite production, and PDD had no effect. These results strongly suggest that PKC mediates, in part, ET-induced contractions in rat aortic rings and that an intact endothelium is required for maximum inhibition by PKC inhibitors because PKC stimulated by ET inhibits nitric oxide release.
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Matsumoto, Shigeji, Shinki Yoshida, Mizuho Ikeda, Chikako Saiki, and Mamoru Takeda. "Effects of PKC and PKA Inhibitors on the cAMP-Stimulant-Induced Enhancement of Tetrodotoxin-Resistant Na+ (Nav1.8) Currents." Open Pharmacology Journal 2, no. 1 (February 28, 2008): 17–19. http://dx.doi.org/10.2174/1874143600802010017.
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The protein kinase C (PKC) inhibitor bisindolymaleimide Ro-31-8425 (Ro-31-8425) decreases the peak tetrodotoxin- resistant (TTX-R) Na+ (Nav1.8) current in nodose ganglion (NG) neurons, and this decrease is not altered by simultaneous application of 8-bromo-cAMP (8-Br-cAMP), phorbol 12-myristate 13-acetate (PMA, a PKC activator) or forskolin (a cAMP analogue). Intracellular application of the endogenous protein kinase A (PKA) inhibitor, protein kinase inhibitor (PKI) abolishes the increase in the peak Nav1.8 current that occurs in response to the applications of 8-BrcAMP, PMA, forskolin, or prostaglandin E2 (PGE2, an adenyl cyclase activator). At a higher concentration (0.5 mM) compared with a sufficient concentration (0.01 mM) to block the cAMP-stimulant Nav1.8 current, PKI still attenuated the Ro-31-8425-induced decrease in peak Nav1.8 current. When we considered these results together, cAMP-stimulantinduced modification of Nav1.8 currents is mediated by the activation of both PKA and PKC, and PKC may be located upstream of PKA.
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Getz, Todd M., Kamala Bhavaraju, and Satya P. Kunapuli. "CPI-17 Contributes to Calcium-Independent ADP-Induced Platelet Shape Change through P2Y1-Gq-PKC Pathways." Blood 110, no. 11 (November 16, 2007): 3644. http://dx.doi.org/10.1182/blood.v110.11.3644.3644.
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Abstract The initial event in platelet activation is the reorganization of the cytoskeleton causing the platelets to change from a discoid to a spiculated spheroid shape. Platelet shape change is primarily regulated by the phosphorylation of myosin light chain kinase. We have shown that this process is mediated through both calcium-dependent and calcium-independent Rho kinase pathways. CPI-17, a Protein kinase C (PKC) phosphorylated inhibitory protein of myosin light chain phosphatase, has been shown to have a role in platelet shape change downstream of thrombin-induced platelet activation. CPI-17 is a 17 kDa protein expressed in human platelets shown to inhibit myosin light chain phosphotase activity via PKCs. In this study we examined the role of CPI-17 in ADP-induced shape change and phosphorylation of CPI-17, downstream of the Gq coupled, P2Y1, and the Gi coupled, P2Y12 receptors. CPI-17 phosphorylation occurred upon activation of platelets with 2MeSADP. This phosphorylation was abolished in the presence of the P2Y1 receptor antagonist, MRS-2179. These results indicated that Gq signaling is important for platelet shape change and phosphorylation of CPI-17. In the presence of the calcium chelator, BAPTA, platelets changed shape in response to 2MeSADP; CPI-17 phosphorylation, however, was unaffected by BAPTA treatment under these conditions. However, CPI-17 phosphorylation was inhibited in the presence of the pan PKC inhibitors. These results indicate that CPI-17 phosphorylation occurs downstream of PKC activation. In the presence of BAPTA, treatment with PKC inhibitors decreased platelet shape change possibly due to reduced CPI-17 phosphorylation. The shape change caused by p160ROCK downstream of G12/13 pathways was unaffected by pan PKC inhibitors, but abolished by p160ROCK inhibitors H1152 or Y27632. Platelets incubated with BAPTA, pan PKC inhibitors, and p160ROCK inhibitor H1152, abolished ADP-induced platelet shape change and CPI-17 phosphorylation. In conclusion, ADP-induced platelet shape change occurs through a Gq-mediated, calcium-independent signaling pathway regulated by CPI-17 phosphorylation via PKC activation.
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Adderley, Shaquria P., Meera Sridharan, Elizabeth A. Bowles, Alan H. Stephenson, Mary L. Ellsworth, and Randy S. Sprague. "Protein kinases A and C regulate receptor-mediated increases in cAMP in rabbit erythrocytes." American Journal of Physiology-Heart and Circulatory Physiology 298, no. 2 (February 2010): H587—H593. http://dx.doi.org/10.1152/ajpheart.00975.2009.
Full textAbstract:
Activation of the β-adrenergic receptor (β-AR) or the prostacyclin receptor (IPR) results in increases in cAMP and ATP release from erythrocytes. cAMP levels depend on a balance between synthesis via adenylyl cyclase and hydrolysis by phosphodiesterases (PDEs). Previously, we reported that cAMP increases associated with activation of the β-AR and IPR in rabbit and human erythrocytes are tightly regulated by distinct PDEs ( 1 ). Importantly, inhibitors of these PDEs potentiated both increases in cAMP and ATP release. It has been shown that increases in protein kinase (PK) activity can activate PDE3 and PDE4. Both PKA and PKC are present in the erythrocyte and can phosphorylate and activate these PDEs. Here we investigate the hypothesis that PKA regulates PDE activity associated with the β-AR and both PKA and PKC regulate the PDE activity associated with the IPR in rabbit erythrocytes. Pretreatment of erythrocytes with the PKA inhibitor, H89 (10 μM), in the presence of the PDE4 inhibitor, rolipram (10 μM), augmented isoproterenol (1 μM)-induced cAMP increases. In contrast, in the presence of the PDE3 inhibitor, cilostazol (10 μM), pretreatment of erythrocytes with either H89 (1 μM) or two chemically dissimilar inhibitors of PKC, calphostin C (1 μM) or GFX109203X (1 μM), potentiated iloprost (1 μM)-induced cAMP increases. Furthermore, pretreatment of erythrocytes with both H89 and GFX109203X in the presence of cilostazol augmented the iloprost-induced increases in cAMP to a greater extent than either PK inhibitor individually. These results support the hypothesis that PDEs associated with receptor-mediated increases in cAMP in rabbit erythrocytes are regulated by kinases specific to the receptor's signaling pathway.
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Sugimoto, T., M. Kanatani, H. Kaji, T. Yamaguchi, M. Fukase, and K. Chihara. "Second messenger signaling of PTH- and PTHRP-stimulated osteoclast-like cell formation from hemopoietic blast cells." American Journal of Physiology-Endocrinology and Metabolism 265, no. 3 (September 1, 1993): E367—E373. http://dx.doi.org/10.1152/ajpendo.1993.265.3.e367.
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The second messenger signaling mechanisms of parathyroid hormone (PTH)- and PTH-related peptide (PTHRP)-stimulated osteoclast-like cell formation were investigated in mouse hemopoietic blast cells that possessed PTH binding sites. Human (h) PTH-(1-34) or hPTHRP-(1-34) resulted in a dose-dependent stimulation of tartrate-resistant acid phosphatase-positive multinucleated cells (MNC) formation. Pretreatment with [Nle8,18Tyr34]hPTH-(3-34) significantly blocked hPTH-(1-34)- and hPTHRP-(1-34)-stimulated MNC formation. Dibutyryladenosine 3',5'-cyclic monophosphate (10(-4) M) and forskolin (10(-5) M) as well as the stimulatory diastereoisomer of adenosine 3',5'-cyclic phosphorothioate (Sp-cAMPS), a direct activator of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) (10(-4) M), stimulated MNC formation, and Rp-cAMPS, an inhibitor of PKA activation (10(-4) M), almost completely inhibited MNC formation stimulated by the aforementioned agents but not by 1,25-dihydroxyvitamin D3. Moreover, Rp-cAMPS significantly blocked PTH- and PTHRP-stimulated MNC formation. Treatment with calcium ionophores (10(-8) and 10(-7) M) and phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator (10(-8) to 10(-6) M), but not 4 alpha-phorbol 12,13-didecanoate, a phorbol incapable of activating PKC, stimulated MNC formation. Two PKC inhibitors [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride and staurosporine] equally blocked PTH- and PTHRP-stimulated MNC formation. The combined pretreatment with Rp-cAMPS and PKC inhibitors completely blocked PTH- and PTHRP-stimulated MNC formation. Present findings indicate that the activation of PKA and PKC is directly linked to PTH- and PTHRP-stimulated osteoclast-like cell formation from hemopoietic blast cells.
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Lee, Min Young, Soo Hyun Park, Yun Jung Lee, Jung Sun Heo, Jang Hern Lee, and Ho Jae Han. "EGF-induced inhibition of glucose transport is mediated by PKC and MAPK signal pathways in primary cultured chicken hepatocytes." American Journal of Physiology-Gastrointestinal and Liver Physiology 291, no. 4 (October 2006): G744—G750. http://dx.doi.org/10.1152/ajpgi.00551.2005.
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EGF is a regulator of a wide variety of processes in various cell systems. Hepatocytes are important sites in the body's metabolism and function. Glucose transporter 2 (GLUT2) is a major transporter that is expressed strongly in hepatocytes. Therefore, this study examined the effect of EGF on GLUT2 and its related signal cascades in primary cultured chicken hepatocytes. EGF decreased [3H]deoxyglucose uptake in a dose- and time-dependent manner (>10 ng/ml, 2 h). AG-1478 (an EGF receptor antagonist) and genistein and herbimycin A (tyrosine kinase inhibitors) blocked the EGF-induced decrease in [3H]deoxyglucose uptake, which correlated with the GLUT2 expression level. In addition, the EGF-induced decrease in GLUT2 protein expression was inhibited by staurosporine, H-7, or bisindolylmaleimide I (PKC inhibitors), PD-98059 (a MEK inhibitor), SB-203580 (a p38 MAPK inhibitor), and SP-600125 (a JNK inhibitor), suggesting a role of both PKC and MAPKs (p44/42 MAPK, p38 MAPK, and JNK). In particular, EGF increased the translocation of PKC isoforms (PKC-α, -β1, -γ, -δ, and -ζ) from the cytosol to the membrane fraction and increased the activation of p44/42 MAPK, p38 MAPK, and JNK. Moreover, PKC inhibitors blocked the EGF-induced phosphorylation of three MAPKs. In conclusion, EGF decreases the GLUT2 expression level via the PKC-MAPK signal cascade in chicken hepatocytes.
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Liang, Mingyu, and Franklyn G. Knox. "Nitric oxide activates PKCα and inhibits Na+-K+-ATPase in opossum kidney cells." American Journal of Physiology-Renal Physiology 277, no. 6 (December 1, 1999): F859—F865. http://dx.doi.org/10.1152/ajprenal.1999.277.6.f859.
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Nitric oxide (NO) reduces the molecular activity of Na+-K+-ATPase in opossum kidney (OK) cells, a proximal tubule cell line. In the present study, we investigated the cellular mechanisms for the inhibitory effect of NO on Na+-K+-ATPase. Sodium nitroprusside (SNP), a NO donor, inhibited Na+-K+-ATPase in OK cells, but not in LLC-PK1cells, another proximal tubule cell line. Similarly, phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, inhibited Na+-K+-ATPase in OK, but not in LLC-PK1, cells. PKC inhibitors staurosporine or calphostin C, but not the protein kinase G inhibitor KT-5823, abolished the inhibitory effect of NO on Na+-K+-ATPase in OK cells. Immunoblotting demonstrated that treatment with NO donors caused significant translocation of PKCα from cytosolic to particulate fractions in OK, but not in LLC-PK1, cells. Furthermore, the translocation of PKCα in OK cells was attenuated by either the phospholipase C inhibitor U-73122 or the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. U-73122 also blunted the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. The phospholipase A2inhibitor AACOCF3 did not blunt the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. AACOCF3 alone, however, also decreased Na+-K+-ATPase activity in OK cells. In conclusion, our results demonstrate that NO activates PKCα in OK, but not in LLC-PK1, cells. The activation of PKCα in OK cells by NO is associated with inhibition of Na+-K+-ATPase.
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Kiang, Juliann G., Sharon C. Kiang, Yuang-Taung Juang, and George C. Tsokos. "N ω-nitro-l-arginine inhibits inducible HSP-70 via Ca2+, PKC, and PKA in human intestinal epithelial T84 cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 282, no. 3 (March 1, 2002): G415—G423. http://dx.doi.org/10.1152/ajpgi.00138.2001.
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The nitric oxide (NO) synthase inhibitor N ω-nitro-l-arginine (l-NNA) inhibits heat stress (HS)-induced NO production and the inducible 70-kDa heat shock protein (HSP-70i) in many rodent organs. We used human intestinal epithelial T84 cells to characterize the inhibitory effect of l-NNA on HS-induced HSP-70i expression. Intracellular Ca2+ concentration ([Ca2+]i) was measured using fura-2, and protein kinase C (PKC), and PKA activities were determined. HS increased HSP-70i mRNA and protein in T84 cells exposed to 45°C for 10 min and allowed to recover for 6 h. l-NNA treatment for 1 h before HS inhibited the induction of HSP-70i mRNA and protein, with an IC50 of 0.0471 ± 0.0007 μM. Because the HS-induced increase in HSP-70i mRNA and protein is Ca2+ dependent, we measured [Ca2+]i after treating cells withl-NNA. l-NNA at 100 μM significantly decreased resting [Ca2+]i. Likewise, treatment with 1 μM GF-109203X or H-89 (inhibitors of PKC and PKA, respectively) for 30 min also significantly decreased [Ca2+]i and inhibited HS-induced increase in HSP-70i. GF-109203X- or H-89-treated cells failed to respond tol-NNA by further decreasing [Ca2+]i and HSP-70i. l-NNA effectively blocked heat shock factor-1 (HSF1) translocation from the cytosol to the nucleus, a process requiring PKC phosphorylation. These results suggest that l-NNA inhibits HSP-70i by reducing [Ca2+]i and decreasing PKC and PKA activity, thereby blocking HSF1 translocation from the cytosol to the nucleus.
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Hermann-Kleiter, Natascha, Nikolaus Thuille, Christa Pfeifhofer, Thomas Gruber, Michaela Schäfer, Christof Zitt, Armin Hatzelmann, Christian Schudt, Michael Leitges, and Gottfried Baier. "PKCθ and PKA are antagonistic partners in the NF-AT transactivation pathway of primary mouse CD3+ T lymphocytes." Blood 107, no. 12 (June 15, 2006): 4841–48. http://dx.doi.org/10.1182/blood-2005-10-4044.
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AbstractWe here investigate the crosstalk of PKC and PKA signaling during primary CD3+ T-lymphocyte activation using pharmacologic inhibitors and activators in combination with our established panel of PKC isotype–deficient mouse T cells in vitro. PKCθ and PKA inversely affect the CD3/CD28-induced IL-2 expression, whereas other PKC isotypes are dispensable in this signaling pathway. Gene ablation of PKCθ selectively results in a profound reduction of IL-2 production; however, complete abrogation of IL-2 production in these PKCθ–/– T cells was achieved only by simultaneous coactivation of the cAMP/PKA pathway in CD3+ T cells. Conversely, the reduced IL-2 production in PKC inhibitor–treated T cells can be rescued by inhibition of the cAMP/PKA pathway in wild-type but not in PKCθ–/– T cells. Mechanistically, the cAMP/PKA and PKCθ pathways converge at the level of NF-AT, as shown by DNA binding analysis. The combined increase in PKA and decrease in PKCθ activity leads to an enhanced inhibition of nuclear NF-AT translocation. This PKCθ/PKA crosstalk significantly affects neither the NF-κB, the AP-1, nor the CREB pathways. Taken together, this opposite effect between the positive PKCθ and the negative cAMP/PKA signaling pathways appears rate limiting for NF-AT transactivation and IL-2 secretion responses of CD3+ T lymphocytes.
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Wong, Lid B., C. Lucy Park, and Donovan B. Yeates. "Neuropeptide Y inhibits ciliary beat frequency in human ciliated cells via nPKC, independently of PKA." American Journal of Physiology-Cell Physiology 275, no. 2 (August 1, 1998): C440—C448. http://dx.doi.org/10.1152/ajpcell.1998.275.2.c440.
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The intracellular mechanisms whereby the inhibitory neurotransmitter neuropeptide Y (NPY) decreases ciliary beat frequency (CBF) were investigated in cultured human tracheal and bronchial ciliated cells. CBF was measured by nonstationary analysis laser light scattering. NPY at 1 and 10 μM decreased CBF from a baseline of 6.7 ± 0.5 ( n = 12) to 6.1 ± 0.5 ( P < 0.05) and 5.8 ± 0.4 ( P < 0.01) Hz, respectively. Prior application of PYX-1, an NPY antagonist, prevented the decreases of CBF induced by both doses of NPY. Two broad protein kinase C (PKC) kinase inhibitors, staurosporine and calphostin C, also abolished the NPY-induced decrease in CBF. The NPY-induced decrease in CBF was abolished by GF 109203X, a novel PKC (nPKC) isoform inhibitor, whereas this decrease in CBF was not attenuated by Gö-6976, a specific inhibitor of conventional PKC isoforms. Because pretreatment with NPY did not block the stimulation of CBF by forskolin and pretreatment with forskolin did not abolish the NPY-induced inhibition of CBF, this NPY receptor-mediated signal transduction mechanism appears to be independent of the adenylate cyclase-protein kinase A (PKA) pathway. Inhibition of Ca2+-ATPase by thapsigargin also prevented the suppression of CBF induced by subsequent application of NPY. These novel data indicate that, in cultured human epithelia, NPY decreases CBF below its basal level via the activation of an nPKC isoform and Ca2+-ATPase, independent of the activity of PKA. This is consistent with the proposition that NPY is an autonomic efferent inhibitory neurotransmitter regulating mucociliary transport.
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Ghobrial, Irene, Ujjal Singha, Yazan Alsayed, Alissa Huston, Lanie Francis, Ganwei Lu, Judy Anderson, Suzanne Lentzsch, and G. David Roodman. "Molecular Mechanisms Regulating Migration and Adhesion in Response to the Chemokine SDF-1 in Multiple Myeloma (MM)." Blood 106, no. 11 (November 16, 2005): 2491. http://dx.doi.org/10.1182/blood.v106.11.2491.2491.
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Abstract Malignant plasma cells home to the bone marrow (BM). However, the mechanisms by which cells are recruited into and confined to the BM are not well understood. The G-protein coupled receptor CXCR4 and its chemokine SDF-1 regulate migration in lymphocytes. In this study, we explore the molecular mechanisms involved in migration and adhesion of plasma cells in response to SDF-1. The following inhibitors were used: the Gi protein inhibitor pertussis toxin, PTX (Sigma, Aldrich, MO) 50–100ng/ml for 16 hrs; the PI3K inhibitor LY294002 (EMD Biosciences, CA) 25–50uM for 20 minutes; the mTOR inhibitor rapamycin (LC Laboratories, MA) 20–50nM for 16 hrs; the MEK inhibitor PD098059 (Alexis Biochemicals, CA) 25–50uM for 90 minutes; the p38MAPKinase inhibitor SB203580 (Calbiochem, CA) 10uM for 16 hrs; the PKC isosyme inhibitors (Biomol International LP, PA), PKC beta 5–20uM, PKC epsilon 5–20uM, PKC zeta 5–20uM; and the PLC inhibitors D609 and U73122 (EMD Biosciences, San Diego, CA) 5–20uM for 1 hr. The concentrations and durations of these inhibitors did not induce apoptosis. The transwell migration assay (Costar, Corning, NY) was used with MM cell lines (MM.1S, OPM2 and Kas6/1). Serial concentrations of SDF-1 were used in the lower chambers. The adhesion assay (EMD Biosciences, San Diego, CA) was used with MM cells treated with the inhibitors and in the presence or absence of serial concentrations of SDF-1. Student-t test was used for statistical analysis. SDF-1 induced a bell-shaped migration curve of MM cells, with 10–30nM inducing maximum migration (324% compared to untreated control) while higher doses of SDF-1 (100nM) did not induce migration in all MM cells tested. All inhibitors were tested in the presence or absence of 30nM SDF-1. PTX 50ng/ml significantly inhibited migration to 30% as compared to control in the presence of 30nM SDF-1 (p=0.004). The PI3K inhibitor and MEK inhibitors inhibited migration by 57% and 58% respectively indicating that the PI3K and ERK MAPkinase play a role in MM migration. The combination of the two agents was not additive (59%) indicating signaling through the same pathway. Immunoblotting confirmed that ERK1/2 are downstream of PI3K. The p38MAPkinase inhibitor did not affect migration. All PKC inhibitors induced a dose dependent inhibition of migration as compared to untreated control, PKC-epsilon (75%–33%), PKC-beta (63–30%), PKC-zeta (49–40%) indicating that all isoenzymes play a role in migration of MM cells. The PLC inhibitors inhibited migration in a similar fashion (52–24%). Serial concentrations of SDF-1 increased adhesion in MM cells in a dose dependent fashion with 10nM inducing 180% increase in adhesion, 30nM inducing 296% and 100nM inducing 365% increase in adhesion as compared to control. PTX 50ng/ml in the presence of 30nM SDF-1 inhibited migration to 64% as compared to control. Similar results were observed with LY294002 and PD098059. In summary, we delineate molecular mechanisms that regulate MM migration and adhesion in response to SDF-1. We demonstrate that MM cells migrate in response to SDF-1 through the Gi protein, PI3K, PKC, PLC and ERK MAPkinase pathways, but not through the p38MAPkinase pathway, and that ERK MAPkinase is downstream of PI3K. Studies to delineate the role of these inhibitors in MM homing in vivo are underway so that future clinical trials designed to regulate MM homing and migration can be performed. Supported in part by an ASH Scholar Award and an MMRF grant.
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Cunningham, Rochelle, Rajatsubhra Biswas, Marc Brazie, Deborah Steplock, Shirish Shenolikar, and Edward J. Weinman. "Signaling pathways utilized by PTH and dopamine to inhibit phosphate transport in mouse renal proximal tubule cells." American Journal of Physiology-Renal Physiology 296, no. 2 (February 2009): F355—F361. http://dx.doi.org/10.1152/ajprenal.90426.2008.
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The present experiments were designed to detail factors regulating phosphate transport in cultured mouse proximal tubule cells by determining the response to parathyroid hormone (PTH), dopamine, and second messenger agonists and inhibitors. Both PTH and dopamine inhibited phosphate transport by over 30%. The inhibitory effect of PTH was completely abolished in the presence of chelerythrine, a PKC inhibitor, but not by Rp-cAMP, a PKA inhibitor. By contrast, both chelerythrine and Rp-cAMP blocked the inhibitory effect of dopamine. Chelerythrine inhibited PTH-mediated cAMP accumulation but also blocked the inhibitory effect of 8-bromo-cAMP on phosphate transport. On the other hand, Rp-cAMP had no effect on the ability of DOG, a PKC activator, to inhibit phosphate transport. PD98059, an inhibitor of MAPK, had no effect on PTH- or dopamine-mediated inhibition of sodium-phosphate cotransport. Finally, compared with 8-bromo-cAMP, 8-pCPT-2′- O-Me-cAMP, an activator of EPAC, had no effect on phosphate transport. These results outline significant differences in the signaling pathways utilized by PTH and dopamine to inhibit renal phosphate transport. Our results also suggest that activation of MAPK is not critically involved in PTH- or dopamine-mediated inhibition of phosphate transport in mouse renal proximal tubule cells in culture.
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Ali, Atef, and Marc-André Sirard. "Protein kinases influence bovine oocyte competence during short-term treatment with recombinant human follicle stimulating hormone." Reproduction 130, no. 3 (September 2005): 303–10. http://dx.doi.org/10.1530/rep.1.00387.
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The aim of this study was to investigate the effect of short-term treatment (first 2 or 6 h) with recombinant human follicle-stimulating hormone (r-hFSH) during in vitro maturation (IVM) on the developmental competence of bovine oocytes. The roles of protein kinase A (PKA) and protein kinase C (PKC) (possibly involved in FSH response), were investigated using activators (Sp-cAMPS, PMA) or inhibitors (Rp-cAMPS, sphingosine) of these two protein kinases, respectively. The developmental competence of bovine oocytes was measured by the rate of blastocyst formation after in vitro fertilization (IVF). Our results showed that when cumulus–oocyte complexes (COCs) were cultured with r-hFSH for the first 6 h, a highly significant (P < 0.0001) improvement is seen in blastocyst development rate as a proportion of oocytes in culture compared with those matured with r-hFSH for the first 2 or 24 h. A transient exposure (6 h) to the highest dose (100 μM) of forskolin (an activator of adenylate cyclase) increased (P < 0.05) the rate of blastocyst formation. But the PKA inhibitors (Rp-cAMPS) did not affect the stimulatory effects of r-hFSH on the blastocyst yield. However, stimulation of PKC by low doses of PMA (0.1–0.5 μM) during short-term treatment, enhanced (P < 0.0001) the developmental capacity of oocytes, while sphingosine (a specific inhibitor of PKC) inhibited (P < 0.05) the stimulatory effects of r-hFSH on the rate of blastocyst formation. Our results indicate that although the developmental capacity of bovine oocytes in vitro can be modulated by both the PKA, and the PKC pathways, the activation of PKC during short-term treatment can mimic the effect of r-hFSH on the cytoplasmic maturation in bovine oocytes in vitro.
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KAPADIA, RINA, PETER D. YURCHENCO, and KURT AMSLER. "Binding of the Renal Epithelial Cell Line LLC-PK1 to Laminin Is Regulated by Protein Kinase C." Journal of the American Society of Nephrology 10, no. 6 (June 1999): 1214–23. http://dx.doi.org/10.1681/asn.v1061214.
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Abstract. The α6β1 integrin heterodimer has been implicated in the mediation of renal epithelial cell binding to laminin, and it has been suggested that this binding is important for renal morphogenesis and development. Studies of nonrenal cells have suggested that the functional activity of α6β1 integrin is regulated by protein kinase C (PKC) activity. In this study, the binding of a renal epithelial cell line, LLC-PK1, to laminin was characterized and the role of PKC activity in the modulation of binding was investigated. LLC-PK1 cells bound to laminin-coated surfaces in a time- and laminin concentration-dependent manner. Binding was strongly inhibited by anti-β1 integrin antibodies and by anti-α6 integrin antibodies. Antibodies against α2 integrin and α3 integrin had little inhibitory effect. Cells bound to both whole laminin and laminin fragment E8, i.e., the fragment to which the α6β1 integrin heterodimer binds. Exposure of cells to PKC activators for as little as 2 h enhanced cell binding to laminin approximately twofold, in a protein synthesis-dependent manner. PKC inhibitors antagonized this effect. PKC-stimulated binding was also inhibited by anti-β1 integrin and anti-α6 integrin antibodies. PKC activation did not alter expression of β1 integrin subunits at the cell surface after short time periods (2 to 4 h), but expression was increased after longer time periods (24 h). These results indicate that the renal epithelial cell line LLC-PK1 binds to laminin via the α6β1 integrin heterodimer and binding is enhanced by PKC activation. The PKC-mediated enhancement of binding requires protein synthesis and is mediated in part by activation of surface α6β1 integrin.
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Fang, Xianjun, Shuangxing Yu, Janos L. Tanyi, Yiling Lu, James R. Woodgett, and Gordon B. Mills. "Convergence of Multiple Signaling Cascades at Glycogen Synthase Kinase 3: Edg Receptor-Mediated Phosphorylation and Inactivation by Lysophosphatidic Acid through a Protein Kinase C-Dependent Intracellular Pathway." Molecular and Cellular Biology 22, no. 7 (April 1, 2002): 2099–110. http://dx.doi.org/10.1128/mcb.22.7.2099-2110.2002.
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ABSTRACT Lysophosphatidic acid (LPA) is a natural phospholipid with multiple biological functions. We show here that LPA induces phosphorylation and inactivation of glycogen synthase kinase 3 (GSK-3), a multifunctional serine/threonine kinase. The effect of LPA can be reconstituted by expression of Edg-4 or Edg-7 in cells lacking LPA responses. Compared to insulin, LPA stimulates only modest phosphatidylinositol 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt) that does not correlate with the magnitude of GSK-3 phosphorylation induced by LPA. PI3K inhibitors block insulin- but not LPA-induced GSK-3 phosphorylation. In contrast, the effect of LPA, but not that of insulin or platelet-derived growth factor (PDGF), is sensitive to protein kinase C (PKC) inhibitors. Downregulation of endogenous PKC activity selectively reduces LPA-mediated GSK-3 phosphorylation. Furthermore, several PKC isotypes phosphorylate GSK-3 in vitro and in vivo. To confirm a specific role for PKC in regulation of GSK-3, we further studied signaling properties of PDGF receptor β subunit (PDGFRβ) in HEK293 cells lacking endogenous PDGF receptors. In clones expressing a PDGFRβ mutant wherein the residues that couple to PI3K and other signaling functions are mutated with the link to phospholipase Cγ (PLCγ) left intact, PDGF is fully capable of stimulating GSK-3 phosphorylation. The process is sensitive to PKC inhibitors in contrast to the response through the wild-type PDGFRβ. Therefore, growth factors, such as PDGF, which control GSK-3 mainly through the PI3K-PKB/Akt module, possess the ability to regulate GSK-3 through an alternative, redundant PLCγ-PKC pathway. LPA and potentially other natural ligands primarily utilize a PKC-dependent pathway to modulate GSK-3.
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Al, Z., and C. M. Cohen. "Phorbol 12-myristate 13-acetate-stimulated phosphorylation of erythrocyte membrane skeletal proteins is blocked by calpain inhibitors: possible role of protein kinase M." Biochemical Journal 296, no. 3 (December 15, 1993): 675–83. http://dx.doi.org/10.1042/bj2960675.
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Human erythrocytes contain cytosolic protein kinase C (PKC) which, when activated by phorbol 12-myristate 13-acetate (PMA), induces the phosphorylation of the membrane skeletal proteins band 4.1, band 4.9 and adducin. We found that brief treatments of erythrocytes with PMA resulted in a decrease in cytosolic PKC content and in the transient appearance in the cytosol of a Ca(2+)- and phospholipid-independent 55 kDa fragment of PKC, called PKM. Prolonged treatment with PMA resulted in the complete and irreversible loss of erythrocyte PKC. To investigate the possible role of calpain in this process, the calpain inhibitors leupeptin and E-64 were sealed inside erythrocytes by reversible haemolysis. Both inhibitors prolonged the lifetime of PKC in PMA-treated cells, and leupeptin was shown to block the PMA-stimulated appearance of PKM in the cytosol. Significantly, leupeptin also completely blocked PMA-stimulated phosphorylation of membrane and cytosolic substrates. This effect was mimicked by other calpain inhibitors (MDL-28170 and calpain inhibitor I), but did not occur when other protease inhibitors such as phenylmethanesulphonyl fluoride, pepstatin A or chymostatin were used. In addition, the phosphorylation of exogenous histone sealed inside erythrocytes was also blocked by leupeptin. Immunoblotting showed that leupeptin did not prevent the PMA-induced translocation of PKC to the erythrocyte membrane. Thus inhibition of PKC phosphorylation of membrane skeletal proteins by calpain inhibitors was not due to inhibition of PKC translocation to the membrane. Our results suggest that PMA treatment of erythrocytes results in the translocation of PKC to the plasma membrane, followed by calpain-mediated cleavage of PKC to PKM. This cleavage, or some other leupeptin-inhibitable process, is a necessary step for the phosphorylation of membrane skeletal substrates, suggesting that the short-lived PKM may be responsible for membrane skeletal phosphorylation. Our results suggest a potential mechanism whereby erythrocyte PKC may be subject to continual down-regulation during the lifespan of the erythrocyte due to repeated activation events, possibly related to transient Ca2+ influx. Such down-regulation may play an important role in erythrocyte survival or pathophysiology.
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Talior, Ilana, Tamar Tennenbaum, Toshio Kuroki, and Hagit Eldar-Finkelman. "PKC-δ-dependent activation of oxidative stress in adipocytes of obese and insulin-resistant mice: role for NADPH oxidase." American Journal of Physiology-Endocrinology and Metabolism 288, no. 2 (February 2005): E405—E411. http://dx.doi.org/10.1152/ajpendo.00378.2004.
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Oxidative stress is thought to be one of the causative factors contributing to insulin resistance and type 2 diabetes. Previously, we showed that reactive oxygen species (ROS) production is significantly increased in adipocytes from high-fat diet-induced obese and insulin-resistant mice (HF). ROS production was also associated with the increased activity of PKC-δ. In the present studies, we hypothesized that PKC-δ contributes to ROS generation and determined their intracellular source. NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) reduced ROS levels by 50% in HF adipocytes, and inhibitors of NO synthase (l-NAME, 1 mM), xanthine oxidase (allopurinol, 100 μM), AGE formation (aminoguanidine, 10 μM), or the mitochondrial uncoupler (FCCP, 10 μM) had no effect. Rottlerin, a selective PKC-δ inhibitor, suppressed ROS levels by ∼50%. However, neither GÖ-6976 nor LY-333531, effective inhibitors toward conventional PKC or PKC-β, respectively, significantly altered ROS levels in HF adipocytes. Subsequently, adenoviral-mediated expression of wild-type PKC-δ or its dominant negative mutant (DN-PKC-δ) in HF adipocytes resulted in either a twofold increase in ROS levels or their suppression by 20%, respectively. In addition, both ROS levels and PKC-δ activity were sharply reduced by glucose depletion. Taken together, these results suggest that PKC-δ is responsible for elevated intracellular ROS production in HF adipocytes, and this is mediated by high glucose and NADPH oxidase.
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Yan, Rong, Zhicheng Wang, Weilin Zhang, Suping Li, Changgeng Ruan, and Kesheng Dai. "Calpain Regulates ADAM17-Mediated Platelet Glycoprotein Ibalpha Ectodomain Shedding." Blood 114, no. 22 (November 20, 2009): 2996. http://dx.doi.org/10.1182/blood.v114.22.2996.2996.
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Abstract Abstract 2996 Poster Board II-974 Introduction: The interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet adhesion and thrombus formation. Thus GPIbalpha ectodomain shedding has important implications for thrombosis and hemostasis. A disintegrin and metalloproteinase 17 (ADAM17) was identified recently to play an essential role in agonist induced GPIbalpha shedding. Calpain, the calcium-dependent neutral protease, has been thought to play a key role in GPIbalpha shedding, whereas it still remains unclear the role of calpain in ADAM17-mediated GPIbalpha shedding. Methods: Washed platelets isolated from vein blood of healthy donors were pre-incubated with various reagents. GPIbalpha fragments were detected by Western blot with anti- GPIbalpha C- or N-terminal antibodies. GPIbalpha surface expression was determined by flow cytometry with anti-GPIbalpha N-terminal antibody. Results: Western blot and flow cytometry results showed that cell permeable calpain inhibitors, MDL 28170, calpain inhibit I and calpain inhibitor II, significantly reduced calmodulin inhibitor W7- and calcium ionophore A23187-induced GPIbalpha shedding. Dibucaine induced GPIbalpha shedding was potently inhibited by calpain inhibitors. W7 and A23187 induced talin cleavage was abolished by calpain inhibitors. Calpain inhibitors had no obvious effect on N-ethylmaleimide-induced GPIbalpha shedding. In order to investigate the role of protein kinase C (PKC) in calpain-mediated GPIbalpha shedding, calpain inhibitors or PKC inhibitor (bisindolylmaleimide I hydrochloride, BIM) were incubated with platelets respectively, and then platelets were further incubated with PKC activator phorbol-ester (PMA) or dibucaine. PMA-induced or dibucaine-mediated GPIbalpha shedding did not inhibit by calpain inhibitors or PKC inhibitor respectively, indicating that PKC is not involved in calpain-mediated GPIbalpha shedding. In addition, two CHO cell lines expressing wild-type GPIb-IX and GPIb-IX mutant with truncated GPIbalpha lacking filamin A binding site were used to test the role of filamin A in calpain-regulated GPIbalpha shedding. Dibucaine-induced GPIbalpha shedding was inhibited by MDL28170 in both of the cell lines. Conclusion: The data indicate that calpain plays key roles in ADAM17-mediated GPIbalpha ectodomain shedding and the calpain-regualted GPIbalpha shedding is independent of PKC activity or filamin A cleavage. Disclosures: No relevant conflicts of interest to declare.
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Vijayaragavan, Kausalia, Mohamed Boutjdir, and Mohamed Chahine. "Modulation of Nav1.7 and Nav1.8 Peripheral Nerve Sodium Channels by Protein Kinase A and Protein Kinase C." Journal of Neurophysiology 91, no. 4 (April 2004): 1556–69. http://dx.doi.org/10.1152/jn.00676.2003.
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Voltage-gated Na+ channels (VGSC) are transmembrane proteins that are essential for the initiation and propagation of action potentials in neuronal excitability. Because neurons express a mixture of Na+ channel isoforms and protein kinase C (PKC) isozymes, the nature of which channel is being regulated by which PKC isozyme is not known. We showed that DRG VGSC Nav1.7 (TTX-sensitive) and Nav1.8 (TTX-resistant), expressed in Xenopus oocytes were differentially regulated by protein kinase A (PKA) and PKC isozymes using the two-electrode voltage-clamp method. PKA activation resulted in a dose-dependent potentiation of Nav1.8 currents and an attenuation of Nav1.7 currents. PKA-induced increases (Nav1.8) and decreases (Nav1.7) in peak currents were not associated with shifts in voltage-dependent activation or inactivation. The PKA-mediated increase in Nav1.8 current amplitude was prevented by chloroquine, suggesting that cell trafficking may contribute to the changes in Nav1.8 current amplitudes. A dose-dependent decrease in Nav1.7 and Nav1.8 currents was observed with the PKC activators phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate. PMA induced shifts in the steady-state activation of Nav1.7 and Nav1.8 channels by 6.5 and 14 mV, respectively, in the depolarizing direction. The role of individual PKC isozymes in the regulation of Nav1.7 and Nav1.8 was determined using PKC-isozyme-specific peptide activators and inhibitors. The decrease in the Nav1.8 peak current induced by PMA was prevented by a specific ϵPKC isozyme peptide antagonist, whereas the PMA effect on Nav1.7 was prevented by ϵPKC and βIIPKC peptide inhibitors. The data showed that Nav1.7 and Nav1.8 were differentially modulated by PKA and PKC. This is the first report demonstrating a functional role for ϵPKC and βIIPKC in the regulation of Nav1.7 and Nav1.8 Na+ channels. Identification of the particular PKC isozymes(s) that mediate the regulation of Na+ channels is essential for understanding the molecular mechanism involved in neuronal ion channel regulation in normal and pathological conditions.
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Wei, Xiaoyu, Ting Lan, Yuanqun Zhou, Jun Cheng, Pengyun Li, Xiaorong Zeng, and Yan Yang. "Mechanism of α1-Adrenergic Receptor-Induced Increased Contraction of Rat Mesenteric Artery in Aging Hypertension Rats." Gerontology 67, no. 3 (2021): 323–37. http://dx.doi.org/10.1159/000511911.
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<b><i>Introduction:</i></b> Vasoconstriction is triggered by an increase in intracellular-free calcium concentration. Growing evidence indicates that contraction is also regulated by calcium-independent mechanisms involving RhoA-Rho kinase (ROCK), protein kinase C (PKC), and so on. In this study, we studied the changes of vascular reactivity as well as the underlying signaling pathways in aging spontaneously hypertensive rats (SHRs). <b><i>Methods:</i></b> The artery tension induced by α1-adrenergic receptor activator (α1-AR) phenylephrine (PE) was measured in the absence or presence of myosin light chain kinase (MLCK), PKC, and ROCK inhibitors. The α1-AR, PKC, ROCK, phosphorylation of myosin light chain (MLC), and PKC-potentiated phosphatase inhibitors of 17 kDa (CPI-17) of rat mesenteric arteries were analyzed at the mRNA level or protein level. <b><i>Results:</i></b> The vascular tension measurements showed that there was a significant increase in the mesenteric artery contraction induced by PE in old SHR. MLCK inhibitor ML-7 can similarly inhibit PE-induced vasoconstriction. PKC inhibitor GF109203X has the weakest inhibitory effect on PE-induced contraction in old SHR. At the presence of ROCK inhibitor H1152, PE-induced contraction was significantly reduced in young Wistar-Kyoto (WKY) rats, but this phenomenon disappeared in other rats. Furthermore, in old SHR the protein expression of α1-AR decreased and phosphorylation of MLC and CPI-17 were upregulated and MLC phosphatase (MLCP) activity was significantly lower. The expressions of PKC were upregulated in SHR and old rats. In addition, the expression of ROCK-1 was decreased and ROCK-2 was significantly upregulated with age in SHR. <b><i>Conclusion:</i></b> In aging hypertension, the expression/activity of PKC or ROCK-2/CPI-17 excessively increased, MLCP activity decreased and MLC phosphorylation enhanced, leading to increased α1-AR-induced vasoconstriction.
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Kawano, Takahito, Junichi Inokuchi, Masatoshi Eto, Masaharu Murata, and Jeong-Hun Kang. "Activators and Inhibitors of Protein Kinase C (PKC): Their Applications in Clinical Trials." Pharmaceutics 13, no. 11 (October 20, 2021): 1748. http://dx.doi.org/10.3390/pharmaceutics13111748.
Full textAbstract:
Protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinase, is classed into three subfamilies based on their structural and activation characteristics: conventional or classic PKC isozymes (cPKCs; α, βI, βII, and γ), novel or non-classic PKC isozymes (nPKCs; δ, ε, η, and θ), and atypical PKC isozymes (aPKCs; ζ, ι, and λ). PKC inhibitors and activators are used to understand PKC-mediated intracellular signaling pathways and for the diagnosis and treatment of various PKC-associated diseases, such as cancers, neurological diseases, cardiovascular diseases, and infections. Many clinical trials of PKC inhibitors in cancers showed no significant clinical benefits, meaning that there is a limitation to design a cancer therapeutic strategy targeting PKC alone. This review will focus on the activators and inhibitors of PKC and their applications in clinical trials.
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Niedbala, MJ, and M. Stein-Picarella. "Role of protein kinase C in tumor necrosis factor induction of endothelial cell urokinase-type plasminogen activator." Blood 81, no. 10 (May 15, 1993): 2608–17. http://dx.doi.org/10.1182/blood.v81.10.2608.2608.
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Abstract Tumor necrosis factor (TNF) can promote endothelial cell transcription, synthesis, and secretion of urokinase plasminogen activator (uPA) augmenting extracellular matrix remodeling and influencing cellular differentiation. In this report, the role of the protein kinase C (PKC) pathway in mediating TNF induction of uPA in human umbilical vein endothelial cells is described. The PKC inhibitors (H-7, staurosporine, and calphostin C), but not HA-1004, inhibited TNF-induced uPA expression, synthesis, and secretion in a dose-dependent manner. Analysis of cell-free conditioned medium obtained from PKC inhibitor- treated cultures by micro-enzyme-linked immunosorbent assay methodologies using uPA- and plasminogen activator inhibitor type 1 (PAI-1)-specific monoclonal antibodies indicate that the decrease in uPA activity observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography was a direct result of decreased extracellular uPA antigen and not a consequence of increased PAI-1 antigen. The effect of PKC inhibitors was specific for TNF-mediated increased uPA expression because cytokine induction of PAI-1 was not influenced by these agents. Northern blot analyses also showed that PKC inhibitor treatment of endothelial cells resulted in a decreased steady- state level of uPA mRNA with no measurable change in PAI-1 mRNA in cultures incubated with TNF. Downregulation of cellular PKC by 18 hours of phorbol myristate acetate (PMA) pretreatment of endothelial cell cultures abolished TNF-mediated extracellular uPA induction. This effect was specific for PMA because 4-alpha PMA pretreatment of cells, which does not stimulate PKC, was ineffective in altering TNF induction of endothelial cell uPA. Induction of PKC directly with PMA, mezerein, and (-)-octylindolactam V increased endothelial cell levels of extracellular uPA in a time- and dose-dependent manner. In addition, this increase in endothelial cell extracellular uPA activity mediated by PKC agonists could be inhibited with PKC inhibitors. Endothelial cells treated with TNF acquire the ability to invade extracellular matrix and reorganize into tube-like structures when grown on Matrigel- coated culture dishes, a behavior blocked by H-7, but not by HA 1004. In summary, these data implicate a role for the PKC pathway in the TNF- mediated induction of uPA expression, subsequent matrix remodeling, and the formation of tube-like structures, a process important in neovascularization, wound healing, and leukocyte extravasation.