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Pugh, Edward N., Teresa Duda, Ari Sitaramayya, and Rameshwar K. Sharma. "Photoreceptor Guanylate Cyclases: A Review." Bioscience Reports 17, no. 5 (October 1, 1997): 429–73. http://dx.doi.org/10.1023/a:1027365520442.
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Almost three decades of research in the field of photoreceptor guanylate cyclases are discussed in this review. Primarily, it focuses on the members of membrane-bound guanylate cyclases found in the outer segments of vertebrate rods. These cyclases represent a new guanylate cyclase subfamily, termed ROS-GC, which distinguishes itself from the peptide receptor guanylate cyclase family that it is not extracellularly regulated. It is regulated, instead, by the intracellularly-generated Ca2+ signals. A remarkable feature of this regulation is that ROS-GC is a transduction switch for both the low and high Ca2+ signals. The low Ca2+ signal transduction pathway is linked to phototransduction, but the physiological relevance of the high Ca2+ signal transduction pathway is not yet clear; it may be linked to neuronal synaptic activity. The review is divided into eight sections. In Section I, the field of guanylate cyclase is introduced and the scope of the review is briefly explained; Section II covers a brief history of the investigations and ideas surrounding the discovery of rod guanylate cyclase. The first five subsections of Section III review the experimental efforts to quantify the guanylate cyclase activity of rods, including in vitro and in situ biochemistry, and also the work done since 1988 in which guanylate cyclase activity has been determined. In the remaining three subsections an analytical evaluation of the Ca2+ modulation of the rod guanylate cyclase activity related to phototransduction is presented. Section IV deals with the issues of a biochemical nature: isolation and purification, subcellular localization and functional properties of rod guanylate cyclase. Section V summarizes work on the cloning of the guanylate cyclases, analysis of their primary structures, and determination of their location with in situ hybridization. Section VI summarizes studies on the regulation of guanylate cyclases, with a focus on guanylate cyclases activating proteins. In Section VII, the evidence about the localization and functional role of guanylate cyclases in other retinal cells, especially in “on-bipolar” cells, in which guanylate cyclase most likely plays a critical role in electrical signaling, is discussed. The review concludes with Section VIII, with remarks about the future directions of research on retinal guanylate cyclases.
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Yamazaki, Akio, Matsuyo Yamazaki, Russell K. Yamazaki, and Jiro Usukura. "Illuminated Rhodopsin Is Required for Strong Activation of Retinal Guanylate Cyclase by Guanylate Cyclase-Activating Proteins†." Biochemistry 45, no. 6 (February 2006): 1899–909. http://dx.doi.org/10.1021/bi0519396.
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HONKAWA, Hanayo, Osamu HISATOMI, Yoshihiro KISHIDA, and Fumio TOKUNAGA. "Two Guanylate Cyclase Activating Proteins in Medaka Retina." Interdisciplinary Information Sciences 8, no. 1 (2002): 25–32. http://dx.doi.org/10.4036/iis.2002.25.
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Baehr, Wolfgang, and Krzysztof Palczewski. "Focus on Molecules: Guanylate cyclase-activating proteins (GCAPs)." Experimental Eye Research 89, no. 1 (June 2009): 2–3. http://dx.doi.org/10.1016/j.exer.2008.12.016.
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Palczewski, Krzysztof, Izabela Sokal, and Wolfgang Baehr. "Guanylate cyclase-activating proteins: structure, function, and diversity." Biochemical and Biophysical Research Communications 322, no. 4 (October 2004): 1123–30. http://dx.doi.org/10.1016/j.bbrc.2004.07.122.
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Sokal, Izabela, Andrei Alekseev, and Krzysztof Palczewski. "Photoreceptor guanylate cyclase variants: cGMP production under control." Acta Biochimica Polonica 50, no. 4 (December 31, 2003): 1075–95. http://dx.doi.org/10.18388/abp.2003_3633.
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Changes in the Ca2+ concentration are thought to affect many processes, including signal transduction in a vast number of biological systems. However, only in few cases the molecular mechanisms by which Ca2+ mediates its action are as well understood as in phototransduction. In dark-adapted photoreceptor cells, the equilibrium level of cGMP is maintained by two opposing activities, such as phosphodiesterase (PDE) and guanylate cyclase (GC). Upon absorption of photons, rhodopsin-G-protein-mediated activation of PDE leads to a transient decrease in [cGMP] and subsequently to lowering of [Ca2+]. In turn, lower [Ca2+] increases net production of cGMP by stimulation of GC until dark conditions are re-established. This activation of GC is mediated by Ca2+ -free forms of Ca2+ -binding proteins termed GC-activating proteins (GCAPs). The last decade brought the molecular identification of GCs and GCAPs in the visual system. Recent efforts have been directed toward understanding the properties of GC at the physiological and structural levels. Here, we summarize the recent progress and present a list of topics of ongoing research.
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Rätscho, Nina, Alexander Scholten, and Karl-Wilhelm Koch. "Expression profiles of three novel sensory guanylate cyclases and guanylate cyclase-activating proteins in the zebrafish retina." Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1793, no. 6 (June 2009): 1110–14. http://dx.doi.org/10.1016/j.bbamcr.2008.12.021.
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Shahu, Manisha Kumari, Fabian Schuhmann, Alexander Scholten, Ilia A. Solov’yov, and Karl-Wilhelm Koch. "The Transition of Photoreceptor Guanylate Cyclase Type 1 to the Active State." International Journal of Molecular Sciences 23, no. 7 (April 5, 2022): 4030. http://dx.doi.org/10.3390/ijms23074030.
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Membrane-bound guanylate cyclases (GCs), which synthesize the second messenger guanosine-3′, 5′-cyclic monophosphate, differ in their activation modes to reach the active state. Hormone peptides bind to the extracellular domain in hormone-receptor-type GCs and trigger a conformational change in the intracellular, cytoplasmic part of the enzyme. Sensory GCs that are present in rod and cone photoreceptor cells have intracellular binding sites for regulatory Ca2+-sensor proteins, named guanylate-cyclase-activating proteins. A rotation model of activation involving an α-helix rotation was described as a common activation motif among hormone-receptor GCs. We tested whether the photoreceptor GC-E underwent an α-helix rotation when reaching the active state. We experimentally simulated such a transitory switch by integrating alanine residues close to the transmembrane region, and compared the effects of alanine integration with the point mutation V902L in GC-E. The V902L mutation is found in patients suffering from retinal cone–rod dystrophies, and leads to a constitutively active state of GC-E. We analyzed the enzymatic catalytic parameters of wild-type and mutant GC-E. Our data showed no involvement of an α-helix rotation when reaching the active state, indicating a difference in hormone receptor GCs. To characterize the protein conformations that represent the transition to the active state, we investigated the protein dynamics by using a computational approach based on all-atom molecular dynamics simulations. We detected a swinging movement of the dimerization domain in the V902L mutant as the critical conformational switch in the cyclase going from the low to high activity state.
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Li, Ning, Robert N. Fariss, Kai Zhang, Annie Otto-Bruc, Francoise Haeseleer, Darin Bronson, Ning Qin, et al. "Guanylate-cyclase-inhibitory protein is a frog retinal Ca2+-binding protein related to mammalian guanylate-cyclase-activating proteins." European Journal of Biochemistry 252, no. 3 (March 15, 1998): 591–99. http://dx.doi.org/10.1046/j.1432-1327.1998.2520591.x.
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Marino, Valerio, Giuditta Dal Cortivo, Paolo Enrico Maltese, Giorgio Placidi, Elisa De Siena, Benedetto Falsini, Matteo Bertelli, and Daniele Dell’Orco. "Impaired Ca2+ Sensitivity of a Novel GCAP1 Variant Causes Cone Dystrophy and Leads to Abnormal Synaptic Transmission Between Photoreceptors and Bipolar Cells." International Journal of Molecular Sciences 22, no. 8 (April 14, 2021): 4030. http://dx.doi.org/10.3390/ijms22084030.
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Guanylate cyclase-activating protein 1 (GCAP1) is involved in the shutdown of the phototransduction cascade by regulating the enzymatic activity of retinal guanylate cyclase via a Ca2+/cGMP negative feedback. While the phototransduction-associated role of GCAP1 in the photoreceptor outer segment is widely established, its implication in synaptic transmission to downstream neurons remains to be clarified. Here, we present clinical and biochemical data on a novel isolate GCAP1 variant leading to a double amino acid substitution (p.N104K and p.G105R) and associated with cone dystrophy (COD) with an unusual phenotype. Severe alterations of the electroretinogram were observed under both scotopic and photopic conditions, with a negative pattern and abnormally attenuated b-wave component. The biochemical and biophysical analysis of the heterologously expressed N104K-G105R variant corroborated by molecular dynamics simulations highlighted a severely compromised Ca2+-sensitivity, accompanied by minor structural and stability alterations. Such differences reflected on the dysregulation of both guanylate cyclase isoforms (RetGC1 and RetGC2), resulting in the constitutive activation of both enzymes at physiological levels of Ca2+. As observed with other GCAP1-associated COD, perturbation of the homeostasis of Ca2+ and cGMP may lead to the toxic accumulation of second messengers, ultimately triggering cell death. However, the abnormal electroretinogram recorded in this patient also suggested that the dysregulation of the GCAP1–cyclase complex further propagates to the synaptic terminal, thereby altering the ON-pathway related to the b-wave generation. In conclusion, the pathological phenotype may rise from a combination of second messengers’ accumulation and dysfunctional synaptic communication with bipolar cells, whose molecular mechanisms remain to be clarified.
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Ikuta, Tohru, Diana Gutsaeva, James Parkerson, Shobha Yerigenahally, and C. Alvin Head. "Hydroxyurea Induces Fetal Hemoglobin Expression by Activating cAMP Signaling Pathways In a cAMP- and cGMP-Dependent Manner; New Hypothesis to Account for a Role of Non-Erythroid Cells In Fetal Hemoglobin Induction." Blood 116, no. 21 (November 19, 2010): 1622. http://dx.doi.org/10.1182/blood.v116.21.1622.1622.
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Abstract Abstract 1622 Despite considerable concerns and efforts, the mechanism of action of hydroxyurea (HU) for the induction of fetal hemoglobin (HbF) remains elusive. For example, clinical studies with HU suggest that bone marrow reserve is critical for HbF response to HU, but the underlying mechanism remains unknown. We and others have demonstrated that HU activates the cGMP signaling pathway in erythroid cells, which plays a role in HbF induction. However, the mechanisms by which intracellular signals are transduced to downstream cascades of cyclic nucleotide-dependent pathways in erythroid cells treated with HU remain to be established. Here we present evidence that HU induces HbF expression by activating the cAMP signaling pathway through two independent mechanisms: cAMP and cGMP. To study signal transduction by HU in cyclic nucleotide-dependent pathways, we initially focused on identifying substrates for cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) that are expressed in erythroid-lineage cells. We found that vasodilator-stimulated phosphoprotein (VASP), which is a 46/50-kDa phosphoprotein expressed in platelets at high levels, is also expressed in erythroid-lineage cells. VASP can be phosphorylated by cyclic nucleotide-elevating agents such as forskolin (activator of adenylate cyclase) and nitric oxide donors (activator of soluble guanylate cyclase). Interestingly, cAMP and cGMP phosphorylate distinct serine residues of VASP; Ser157 is phosphorylated by cAMP-elevating agents, while cGMP-elevating agents phosphorylated Ser239. Although HU increased both intracellular cAMP and cGMP levels in CD34+-derived erythroblasts, we found that Ser157, but not Ser239, is phosphorylated in adult erythroid cells treated with HU, suggesting activation of the cAMP signaling pathway. However, HU-induced HbF expression was down-regulated by inhibiting the activity of adenylate cyclase or soluble guanylate cyclase, suggesting that both enzymes are involved in HU-induced HbF expression. Our studies found that HU decreased the expression of cGMP-inhibitable phosphodiesterase 3B in a manner dependent on soluble guanylate cyclase, resulting in activation of the cAMP signaling pathway. Although a recent study showed that HU directly activates soluble guanylate cyclase, our studies showed that HU is unable to directly stimulate the enzyme activity of adenylate cyclase. Furthermore, HU induced the expression of cyclooxygenase-1 (COX-1) and increased the production of prostaglandin E2 (PGE2) that activates adenylate cyclase through G protein-coupled E-prostanoid receptors. Plasma PGE2 levels were also elevated in sickle cell patients upon HU therapy. These results demonstrate that HU induces HbF expression by activating the cAMP pathway by cAMP- and cGMP-dependent mechanisms, producing redundancy in the response of HbF to HU. Both cAMP and cGMP may represent major molecules that transduce signals from HU to the fetal globin gene. It is known that non-erythroid cells such as leukocytes and monocytes produce a large amount of PGE2. Thus, the involvement of PGE2 in HU-induced HbF expression may suggest an important role of non-erythroid cells as well as bone marrow reserve in the induction of HbF expression. More interestingly, several single nucleotide polymorphisms with amino acid changes have been demonstrated for COX-1; some genetic variants exhibit reduced COX activities. If SCD patients have some mutations in the COX-1 gene, such patients might be resistant to HU therapy. Disclosures: No relevant conflicts of interest to declare.
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Xiao, Sijia, Qianbin Li, Liqing Hu, Zutao Yu, Jie Yang, Qi Chang, Zhuo Chen, and Gaoyun Hu. "Soluble Guanylate Cyclase Stimulators and Activators: Where are We and Where to Go?" Mini-Reviews in Medicinal Chemistry 19, no. 18 (November 29, 2019): 1544–57. http://dx.doi.org/10.2174/1389557519666190730110600.
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Soluble Guanylate Cyclase (sGC) is the intracellular receptor of Nitric Oxide (NO). The activation of sGC results in the conversion of Guanosine Triphosphate (GTP) to the secondary messenger cyclic Guanosine Monophosphate (cGMP). cGMP modulates a series of downstream cascades through activating a variety of effectors, such as Phosphodiesterase (PDE), Protein Kinase G (PKG) and Cyclic Nucleotide-Gated Ion Channels (CNG). NO-sGC-cGMP pathway plays significant roles in various physiological processes, including platelet aggregation, smooth muscle relaxation and neurotransmitter delivery. With the approval of an sGC stimulator Riociguat for the treatment of Pulmonary Arterial Hypertension (PAH), the enthusiasm in the discovery of sGC modulators continues for broad clinical applications. Notably, through activating the NO-sGC-cGMP pathway, sGC stimulator and activator potentiate for the treatment of various diseases, such as PAH, Heart Failure (HF), Diabetic Nephropathy (DN), Systemic Sclerosis (SS), fibrosis as well as other diseases including Sickle Cell Disease (SCD) and Central Nervous System (CNS) disease. Here, we review the preclinical and clinical studies of sGC stimulator and activator in recent years and prospect for the development of sGC modulators in the near future.
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Avesani, Anna, Laura Bielefeld, Nicole Weisschuh, Valerio Marino, Pascale Mazzola, Katarina Stingl, Tobias B. Haack, Karl-Wilhelm Koch, and Daniele Dell’Orco. "Molecular Properties of Human Guanylate Cyclase-Activating Protein 3 (GCAP3) and Its Possible Association with Retinitis Pigmentosa." International Journal of Molecular Sciences 23, no. 6 (March 17, 2022): 3240. http://dx.doi.org/10.3390/ijms23063240.
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The cone-specific guanylate cyclase-activating protein 3 (GCAP3), encoded by the GUCA1C gene, has been shown to regulate the enzymatic activity of membrane-bound guanylate cyclases (GCs) in bovine and teleost fish photoreceptors, to an extent comparable to that of the paralog protein GCAP1. To date, the molecular mechanisms underlying GCAP3 function remain largely unexplored. In this work, we report a thorough characterization of the biochemical and biophysical properties of human GCAP3, moreover, we identified an isolated case of retinitis pigmentosa, in which a patient carried the c.301G>C mutation in GUCA1C, resulting in the substitution of a highly conserved aspartate residue by a histidine (p.(D101H)). We found that myristoylated GCAP3 can activate GC1 with a similar Ca2+-dependent profile, but significantly less efficiently than GCAP1. The non-myristoylated form did not induce appreciable regulation of GC1, nor did the p.D101H variant. GCAP3 forms dimers under physiological conditions, but at odds with its paralogs, it tends to form temperature-dependent aggregates driven by hydrophobic interactions. The peculiar properties of GCAP3 were confirmed by 2 ms molecular dynamics simulations, which for the p.D101H variant highlighted a very high structural flexibility and a clear tendency to lose the binding of a Ca2+ ion to EF3. Overall, our data show that GCAP3 has unusual biochemical properties, which make the protein significantly different from GCAP1 and GCAP2. Moreover, the newly identified point mutation resulting in a substantially unfunctional protein could trigger retinitis pigmentosa through a currently unknown mechanism.
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Scholten, Alexander, and Karl-Wilhelm Koch. "Differential Calcium Signaling by Cone Specific Guanylate Cyclase-Activating Proteins from the Zebrafish Retina." PLoS ONE 6, no. 8 (August 2, 2011): e23117. http://dx.doi.org/10.1371/journal.pone.0023117.
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Hilfiker, S. "Neuronal calcium sensor-1: a multifunctional regulator of secretion." Biochemical Society Transactions 31, no. 4 (August 1, 2003): 828–32. http://dx.doi.org/10.1042/bst0310828.
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Ca2+ ions play a crucial role not only as the trigger for neurotransmitter release, but also in other aspects of brain function, such as short-term and long-term modulation of synaptic efficacy, which may underlie certain forms of learning and memory. The actions of Ca2+ are mediated by Ca2+-binding proteins, including a group of proteins known as neuronal calcium sensor (NCS) proteins. The NCS family includes NCS-1, visinin-like proteins, recoverins, guanylate cyclase-activating proteins and potassium channel-interacting proteins. Some members of this family, such as recoverin and guanylate cyclase-activating protein, are only expressed in photoreceptor cells and have been implicated in the control of visual transduction pathways, while the functional roles of the other members are largely unknown. NCS-1 was originally identified in Drosophila in a screen for neuronal hyperexcitability mutants. NCS-1 is an N-terminally myristoylated protein that contains four EF-hand motifs, three of which are able to bind Ca2+ in the submicromolar range. Overexpression of NCS-1 has been shown to enhance evoked neurotransmitter release, paired-pulse facilitation and exocytosis in several neuronal and neuroendocrine cell types. Recent experiments suggest that NCS-1 interacts directly with phosphatidylinositol 4-hydroxykinase in yeast as well as mammalian cells, suggesting that it may enhance neuronal secretion by modulating cellular trafficking steps in a phosphoinositide-dependent manner. In contrast, an involvement of NCS-1 in the expression and regulation of voltage-gated Ca2+ channels and K+ channels has also been proposed, which may be attributed, at least in part, to the effects of NCS-1 on vesicular trafficking pathways. The present review describes current knowledge about the cellular functions and molecular mechanisms by which NCS-1 may regulate neurotransmitter release.
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Bonì, Francesco, Valerio Marino, Carlo Bidoia, Eloise Mastrangelo, Alberto Barbiroli, Daniele Dell’Orco, and Mario Milani. "Modulation of Guanylate Cyclase Activating Protein 1 (GCAP1) Dimeric Assembly by Ca2+ or Mg2+: Hints to Understand Protein Activity." Biomolecules 10, no. 10 (October 5, 2020): 1408. http://dx.doi.org/10.3390/biom10101408.
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The guanylyl cyclase-activating protein 1, GCAP1, activates or inhibits retinal guanylyl cyclase (retGC) depending on cellular Ca2+ concentrations. Several point mutations of GCAP1 have been associated with impaired calcium sensitivity that eventually triggers progressive retinal degeneration. In this work, we demonstrate that the recombinant human protein presents a highly dynamic monomer-dimer equilibrium, whose dissociation constant is influenced by salt concentration and, more importantly, by protein binding to Ca2+ or Mg2+. Based on small-angle X-ray scattering data, protein-protein docking, and molecular dynamics simulations we propose two novel three-dimensional models of Ca2+-bound GCAP1 dimer. The different propensity of human GCAP1 to dimerize suggests structural differences induced by cation binding potentially involved in the regulation of retGC activity.
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Mendez, A., M. E. Burns, I. Sokal, A. M. Dizhoor, W. Baehr, K. Palczewski, D. A. Baylor, and J. Chen. "Role of guanylate cyclase-activating proteins (GCAPs) in setting the flash sensitivity of rod photoreceptors." Proceedings of the National Academy of Sciences 98, no. 17 (August 7, 2001): 9948–53. http://dx.doi.org/10.1073/pnas.171308998.
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Venema, Richard C., Virginia J. Venema, Hong Ju, M. Brennan Harris, Connie Snead, Tamas Jilling, Christiana Dimitropoulou, Michael E. Maragoudakis, and John D. Catravas. "Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase." American Journal of Physiology-Heart and Circulatory Physiology 285, no. 2 (August 2003): H669—H678. http://dx.doi.org/10.1152/ajpheart.01025.2002.
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Soluble guanylate cyclase (sGC) is an important downstream intracellular target of nitric oxide (NO) that is produced by endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). In this study, we demonstrate that sGC exists in a complex with eNOS and heat shock protein 90 (HSP90) in aortic endothelial cells. In addition, we show that in aortic smooth muscle cells, sGC forms a complex with HSP90. Formation of the sGC/eNOS/HSP90 complex is increased in response to eNOS-activating agonists in a manner that depends on HSP90 activity. In vitro binding assays with glutathione S-transferase fusion proteins that contain the α- or β-subunit of sGC show that the sGC β-subunit interacts directly with HSP90 and indirectly with eNOS. Confocal immunofluorescent studies confirm the subcellular colocalization of sGC and HSP90 in both endothelial and smooth muscle cells. Complex formation of sGC with HSP90 facilitates responses to NO donors in cultured cells (cGMP accumulation) as well as in anesthetized rats (hypotension). These complexes likely function to stabilize sGC as well as to provide directed intracellular transfer of NO from NOS to sGC, thus preventing inactivation of NO by superoxide anion and formation of peroxynitrite, which is a toxic molecule that has been implicated in the pathology of several vascular diseases.
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Abbas, Seher, Valerio Marino, Laura Bielefeld, Karl-Wilhelm Koch, and Daniele Dell’Orco. "Constitutive Activation of Guanylate Cyclase by the G86R GCAP1 Variant Is Due to “Locking” Cation-π Interactions that Impair the Activator-to-Inhibitor Structural Transition." International Journal of Molecular Sciences 21, no. 3 (January 23, 2020): 752. http://dx.doi.org/10.3390/ijms21030752.
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Guanylate Cyclase activating protein 1 (GCAP1) mediates the Ca2+-dependent regulation of the retinal Guanylate Cyclase (GC) in photoreceptors, acting as a target inhibitor at high [Ca2+] and as an activator at low [Ca2+]. Recently, a novel missense mutation (G86R) was found in GUCA1A, the gene encoding for GCAP1, in patients diagnosed with cone-rod dystrophy. The G86R substitution was found to affect the flexibility of the hinge region connecting the N- and C-domains of GCAP1, resulting in decreased Ca2+-sensitivity and abnormally enhanced affinity for GC. Based on a structural model of GCAP1, here, we tested the hypothesis of a cation-π interaction between the positively charged R86 and the aromatic W94 as the main mechanism underlying the impaired activator-to-inhibitor conformational change. W94 was mutated to F or L, thus, resulting in the double mutants G86R+W94L/F. The double mutants showed minor structural and stability changes with respect to the single G86R mutant, as well as lower affinity for both Mg2+ and Ca2+, moreover, substitutions of W94 abolished “phase II” in Ca2+-titrations followed by intrinsic fluorescence. Interestingly, the presence of an aromatic residue in position 94 significantly increased the aggregation propensity of Ca2+-loaded GCAP1 variants. Finally, atomistic simulations of all GCAP1 variants in the presence of Ca2+ supported the presence of two cation-π interactions involving R86, which was found to act as a bridge between W94 and W21, thus, locking the hinge region in an activator-like conformation and resulting in the constitutive activation of the target under physiological conditions.
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Solessio, Eduardo, Shobana S. Mani, Nicolas Cuenca, Gustav A. Engbretson, Robert B. Barlow, and Barry E. Knox. "Developmental regulation of calcium-dependent feedback in Xenopus rods." Journal of General Physiology 124, no. 5 (October 25, 2004): 569–85. http://dx.doi.org/10.1085/jgp.200409162.
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The kinetics of activation and inactivation in the phototransduction pathway of developing Xenopus rods were studied. The gain of the activation steps in transduction (amplification) increased and photoresponses became more rapid as the rods matured from the larval to the adult stage. The time to peak was significantly shorter in adults (1.3 s) than tadpoles (2 s). Moreover, adult rods recovered twice as fast from saturating flashes than did larval rods without changes of the dominant time constant (2.5 s). Guanylate cyclase (GC) activity, determined using IBMX steps, increased in adult rods from ∼1.1 s−1 to 3.7 s−1 5 s after a saturating flash delivering 6,000 photoisomerizations. In larval rods, it increased from 1.8 s−1 to 4.0 s−1 9 s after an equivalent flash. However, the ratio of amplification to the measured dark phosphodiesterase activity was constant. Guanylate cyclase–activating protein (GCAP1) levels and normalized Na+/Ca2+, K+ exchanger currents were increased in adults compared with tadpoles. Together, these results are consistent with the acceleration of the recovery phase in adult rods via developmental regulation of calcium homeostasis. Despite these large changes, the single photon response amplitude was ∼0.6 pA throughout development. Reduction of calcium feedback with BAPTA increased adult single photon response amplitudes threefold and reduced its cutoff frequency to that observed with tadpole rods. Linear mathematical modeling suggests that calcium-dependent feedback can account for the observed differences in the power spectra of larval and adult rods. We conclude that larval Xenopus maximize sensitivity at the expense of slower response kinetics while adults maximize response kinetics at the expense of sensitivity.
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Codega, Paolo, Luca Della Santina, Claudia Gargini, Diana E. Bedolla, Tatiana Subkhankulova, Frederick J. Livesey, Luigi Cervetto, and Vincent Torre. "Prolonged illumination up-regulates arrestin and two guanylate cyclase activating proteins: a novel mechanism for light adaptation." Journal of Physiology 587, no. 11 (June 1, 2009): 2457–72. http://dx.doi.org/10.1113/jphysiol.2009.168609.
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Rosenzweig, Derek H., K. Saidas Nair, Konstantin Levay, Igor V. Peshenko, John W. Crabb, Alexander M. Dizhoor, and Vladlen Z. Slepak. "Interaction of retinal guanylate cyclase with the α subunit of transducin: potential role in transducin localization." Biochemical Journal 417, no. 3 (January 16, 2009): 803–12. http://dx.doi.org/10.1042/bj20081513.
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Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between Gαt (the transducin α subunit) and retGC. Gαt co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC–Gαt complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with both Gαt and retGC. The Gαt–retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound Gαt stronger than the GTP[S] (GTPγS; guanosine 5′-[γ-thio]triphosphate) form. Neither Gαt nor Gβγ affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein–protein interaction between Gαt and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors.
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Biasi, Amedeo, Valerio Marino, Giuditta Dal Cortivo, Paolo Enrico Maltese, Antonio Mattia Modarelli, Matteo Bertelli, Leonardo Colombo, and Daniele Dell’Orco. "A Novel GUCA1A Variant Associated with Cone Dystrophy Alters cGMP Signaling in Photoreceptors by Strongly Interacting with and Hyperactivating Retinal Guanylate Cyclase." International Journal of Molecular Sciences 22, no. 19 (October 6, 2021): 10809. http://dx.doi.org/10.3390/ijms221910809.
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Guanylate cyclase-activating protein 1 (GCAP1), encoded by the GUCA1A gene, is a neuronal calcium sensor protein involved in shaping the photoresponse kinetics in cones and rods. GCAP1 accelerates or slows the cGMP synthesis operated by retinal guanylate cyclase (GC) based on the light-dependent levels of intracellular Ca2+, thereby ensuring a timely regulation of the phototransduction cascade. We found a novel variant of GUCA1A in a patient affected by autosomal dominant cone dystrophy (adCOD), leading to the Asn104His (N104H) amino acid substitution at the protein level. While biochemical analysis of the recombinant protein showed impaired Ca2+ sensitivity of the variant, structural properties investigated by circular dichroism and limited proteolysis excluded major structural rearrangements induced by the mutation. Analytical gel filtration profiles and dynamic light scattering were compatible with a dimeric protein both in the presence of Mg2+ alone and Mg2+ and Ca2+. Enzymatic assays showed that N104H-GCAP1 strongly interacts with the GC, with an affinity that doubles that of the WT. The doubled IC50 value of the novel variant (520 nM for N104H vs. 260 nM for the WT) is compatible with a constitutive activity of GC at physiological levels of Ca2+. The structural region at the interface with the GC may acquire enhanced flexibility under high Ca2+ conditions, as suggested by 2 μs molecular dynamics simulations. The altered interaction with GC would cause hyper-activity of the enzyme at both low and high Ca2+ levels, which would ultimately lead to toxic accumulation of cGMP and Ca2+ in the photoreceptor outer segment, thus triggering cell death.
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ROELOFS, Jeroen, Helena SNIPPE, Reinhard G. KLEINEIDAM, and Peter J. M. Van HAASTERT. "Guanylate cyclase in Dictyostelium discoideum with the topology of mammalian adenylate cyclase." Biochemical Journal 354, no. 3 (March 8, 2001): 697–706. http://dx.doi.org/10.1042/bj3540697.
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The core of adenylate and guanylate cyclases is formed by an intramolecular or intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a heterodimer and are activated by G-proteins. In contrast, membrane-bound guanylate cyclases have only one transmembrane spanning region and one cyclase domain, and are activated by extracellular ligands to form a homodimer. In the cellular slime mould, Dictyosteliumdiscoideum, membrane-bound guanylate cyclase activity is induced after cAMP stimulation; a G-protein-coupled cAMP receptor and G-proteins are essential for this activation. We have cloned a Dictyostelium gene, DdGCA, encoding a protein with 12 transmembrane spanning regions and two cyclase domains. Sequence alignment demonstrates that the two cyclase domains are transposed, relative to these domains in adenylate cyclases. DdGCA expressed in Dictyostelium exhibits high guanylate cyclase activity and no detectable adenylate cyclase activity. Deletion of the gene indicates that DdGCA is not essential for chemotaxis or osmo-regulation. The knock-out strain still exhibits substantial guanylate cyclase activity, demonstrating that Dictyostelium contains at least one other guanylate cyclase.
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Tsuruda, Toshihiro, Kinta Hatakeyama, Hiroyuki Masuyama, Yoko Sekita, Takuroh Imamura, Yujiro Asada, and Kazuo Kitamura. "Pharmacological stimulation of soluble guanylate cyclase modulates hypoxia-inducible factor-1α in rat heart." American Journal of Physiology-Heart and Circulatory Physiology 297, no. 4 (October 2009): H1274—H1280. http://dx.doi.org/10.1152/ajpheart.00503.2009.
Full textAbstract:
Mechanical load and ischemia induce a series of adaptive physiological responses by activating the expression of O2-regulated genes, such as hypoxia inducible factor-1α (HIF-1α). The aim of this study was to explore the interaction between HIF-1α and soluble guanylate cyclase (sGC) and its second messenger cGMP in cultured cardiomyocytes exposed to hypoxia and in pressure-overloaded heart. In cultured cardiomyocytes of neonatal rats, either sGC stimulator BAY 41-2272 or cGMP analog 8-bromo-cGMP decreased the hypoxia (1% O2/5% CO2)-induced HIF-1α expression, whereas the inhibition of protein kinase G by KT-5823 reversed the effect of BAY 41-2272 on the expression under hypoxic conditions. In pressure-overloaded heart induced by suprarenal aortic constriction (AC) in 7-wk-old male Wistar rats, the administration of BAY 41-2272 (2 mg·kg−1·day−1) for 14 days significantly suppressed the protein expression of HIF-1α ( P < 0.05), vascular endothelial growth factor ( P < 0.01), and the number of capillary vessels ( P < 0.01) induced by pressure overload. This study suggests that the pharmacological sGC-cGMP stimulation modulates the HIF-1α expression in response to hypoxia or mechanical load in the heart.
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Li, Ji, Xiaoyue Hu, Pradeepa Selvakumar, Raymond R. Russell, Samuel W. Cushman, Geoffrey D. Holman, and Lawrence H. Young. "Role of the nitric oxide pathway in AMPK-mediated glucose uptake and GLUT4 translocation in heart muscle." American Journal of Physiology-Endocrinology and Metabolism 287, no. 5 (November 2004): E834—E841. http://dx.doi.org/10.1152/ajpendo.00234.2004.
Full textAbstract:
AMP-activated protein kinase (AMPK) is a serine-threonine kinase that regulates cellular metabolism and has an essential role in activating glucose transport during hypoxia and ischemia. The mechanisms responsible for AMPK stimulation of glucose transport are uncertain, but may involve interaction with other signaling pathways or direct effects on GLUT vesicular trafficking. One potential downstream mediator of AMPK signaling is the nitric oxide pathway. The aim of this study was to examine the extent to which AMPK mediates glucose transport through activation of the nitric oxide (NO)-signaling pathway in isolated heart muscles. Incubation with 1 mM 5-amino-4-imidazole-1-β-carboxamide ribofuranoside (AICAR) activated AMPK ( P < 0.01) and stimulated glucose uptake ( P < 0.05) and translocation of the cardiomyocyte glucose transporter GLUT4 to the cell surface ( P < 0.05). AICAR treatment increased phosphorylation of endothelial NO synthase (eNOS) ∼1.8-fold ( P < 0.05). eNOS, but not neuronal NOS, coimmunoprecipitated with both the α2 and α1 AMPK catalytic subunits in heart muscle. NO donors also increased glucose uptake and GLUT4 translocation ( P < 0.05). Inhibition of NOS with Nω-nitro-l-arginine and Nω-methyl-l-arginine reduced AICAR-stimulated glucose uptake by 21 ± 3% ( P < 0.05) and 25 ± 4% ( P < 0.05), respectively. Inhibition of guanylate cyclase with ODQ and LY-83583 reduced AICAR-stimulated glucose uptake by 31 ± 4% ( P < 0.05) and 22 ± 3% ( P < 0.05), respectively, as well as GLUT4 translocation to the cell surface ( P < 0.05). Taken together, these results indicate that activation of the NO-guanylate cyclase pathway contributes to, but is not the sole mediator of, AMPK stimulation of glucose uptake and GLUT4 translocation in heart muscle.
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Nozik-Grayck, Eva, Timothy J. McMahon, Yuh-Chin T. Huang, Christine S. Dieterle, Jonathan S. Stamler, and Claude A. Piantadosi. "Pulmonary vasoconstriction by serotonin is inhibited byS-nitrosoglutathione." American Journal of Physiology-Lung Cellular and Molecular Physiology 282, no. 5 (May 1, 2002): L1057—L1065. http://dx.doi.org/10.1152/ajplung.00081.2001.
Full textAbstract:
Nitric oxide (NO) functions as an endothelium-derived relaxing factor by activating guanylate cyclase to increase cGMP levels. However, NO and related species may also regulate vascular tone by cGMP-independent mechanisms. We hypothesized that naturally occurring NO donors could decrease the pulmonary vascular response to serotonin (5-HT) in the intact lung through chemical interactions with 5-HT2receptors. In isolated rabbit lung preparations and isolated pulmonary artery (PA) rings, 50–250 μM S-nitrosoglutathione (GSNO) inhibited the response to 0.01–10 μM 5-HT. The vasoconstrictor response to 5-HT was mediated by 5-HT2receptors in the lung, since it could be blocked completely by the selective inhibitor ketanserin (10 μM). GSNO inhibited the response to 5-HT by 77% in intact lung and 82% in PA rings. In PA rings, inhibition by GSNO could be reversed by treatment with the thiol reductant dithiothreitol (10 mM). 3-Morpholinosydnonimine (100–500 μM), which releases NO and O[Formula: see text] simultaneously, also blocked the response to 5-HT. Its chemical effects, however, were distinct from those of GSNO, because 5-HT-mediated vasoconstriction was not restored in isolated rings by dithiothreitol. In the intact lung, neither NO donor altered the vascular response to endothelin, which activates the same second-messenger vasoconstrictor system as 5-HT. These findings, which did not depend on guanylate cyclase, are consistent with chemical modification by NO of the 5-HT2G protein-coupled receptor system to inhibit vasoconstriction, possibly by S-nitrosylation of the receptor or a related protein. This study demonstrates that GSNO can regulate vascular tone in the intact lung by a reversible mechanism involving inhibition of the response to 5-HT.
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Karna, Keshab Kumar, Bo-Ram Choi, Chul-Young Kim, Hye-Kyung Kim, and Jong-Kwan Park. "Rubus occidentalis and Ellagic Acid Affect the Contractility of Penile Corpus Cavernosum Smooth Muscle through the Nitric Oxide-Cyclic Guanosine Monophosphate and Cyclic Adenosine 3′,5′-Monophosphate Signaling Pathway." Journal of Clinical Medicine 11, no. 10 (May 23, 2022): 2947. http://dx.doi.org/10.3390/jcm11102947.
Full textAbstract:
The present study was designed to evaluate the relaxation effect of Rubus occidentalis (RO) and ellagic acid (EA) on rabbit penile corpus cavernosum smooth muscle (PCCSM). Rabbit PCCSM was treated with ROE or EA after preincubation with nitric oxide synthase (NOS), guanylate cyclase (GC), adenylyl cyclase (AC) or protein kinase A (PKA) blocker. Cyclic nucleotides in the perfusate were analyzed using radioimmunoassay (RIA). Subsequently, perfused PCCSMs were subjected to analysis to evaluate the expression level of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS). The interaction of ROE or EA with phosphodiesterase (PDE) 5 and PDE4 inhibitors, such as udenafil (UDE) and rolipram (ROL), were also evaluated. Both ROE and EA relaxed the PCCSM in a concentration-dependent manner. Coincubation of ROE or EA with NOS, GC, AC, or PKA blocker significantly decreased the ROE- and EA-induced relaxation. Pretreatment of ROE and EA significantly upregulated the cyclic guanosine monophosphate (cGMP), cyclic adenosine 3′,5′-monophosphate (cAMP), and eNOS levels in the perfused PCCSM. Furthermore, the treatment of ROE and EA markedly increased the UDE- and ROL-induced relaxation of the PCCSM. In conclusion, ROE and EA induced PCCSM relaxation by activating the nitric oxide (NO)-cGMp and cAMp signaling pathways and may have a synergistic action to improve erectile function.
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Liu, Xiao-Ming, Gary B. Chapman, Kelly J. Peyton, Andrew I. Schafer, and William Durante. "Antiapoptotic Action of Carbon Monoxide on Cultured Vascular Smooth Muscle Cells." Experimental Biology and Medicine 228, no. 5 (May 2003): 572–75. http://dx.doi.org/10.1177/15353702-0322805-30.
Full textAbstract:
Vascular smooth muscle cells (SMCs) generate carbon monoxide (CO) from the degradation of heme by the enzyme heme oxygenase. Because recent studies indicate that CO influences the properties of vascular SMCs, we examined whether this diatomic gas regulates apoptosis in vascular SMCs. Treatment of cultured rat aortic SMCs with a cytokine cocktail consisting of interleukin-1β (5 ng/ml), tumor necrosis factor-α (20 ng/ml), and interferon-γ (200 U/ml) for 48 hr stimulated apoptosis, as demonstrated by DNA laddering, caspase-3 activation, and annexin V staining. However, the exogenous addition of CO (200 ppm) completely blocked cytokine-mediated apoptosis. The antiapoptotic action of CO was partially reversed by the soluble guanylate cyclase inhibitor, H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 μM). In contrast, the p38 mitogen-activated protein kinase inhibitor, SB203580 (10 μM), had no effect on SMC apoptosis. These findings indicate that CO is a potent inhibitor of vascular SMC apoptosis and that it blocks apoptosis, in part, by activating the cGMP signaling pathway. The ability of CO to inhibit vascular SMC apoptosis may play a critical role in attenuating lesion formation at sites of arterial damage.
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BURGOYNE, Robert D., and Jamie L. WEISS. "The neuronal calcium sensor family of Ca2+-binding proteins." Biochemical Journal 353, no. 1 (December 18, 2000): 1–12. http://dx.doi.org/10.1042/bj3530001.
Full textAbstract:
Ca2+ plays a central role in the function of neurons as the trigger for neurotransmitter release, and many aspects of neuronal activity, from rapid modulation to changes in gene expression, are controlled by Ca2+. These actions of Ca2+ must be mediated by Ca2+-binding proteins, including calmodulin, which is involved in Ca2+ regulation, not only in neurons, but in most other cell types. A large number of other EF-hand-containing Ca2+-binding proteins are known. One family of these, the neuronal calcium sensor (NCS) proteins, has a restricted expression in retinal photoreceptors or neurons and neuroendocrine cells, suggesting that they have specialized roles in these cell types. Two members of the family (recoverin and guanylate cyclase-activating protein) have established roles in the regulation of phototransduction. Despite close sequence similarities, the NCS proteins have distinct neuronal distributions, suggesting that they have different functions. Recent work has begun to demonstrate the physiological roles of members of this protein family. These include roles in the modulation of neurotransmitter release, control of cyclic nucleotide metabolism, biosynthesis of polyphosphoinositides, regulation of gene expression and in the direct regulation of ion channels. In the present review we describe the known sequences and structures of the NCS proteins, information on their interactions with target proteins and current knowledge about their cellular and physiological functions.
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Dhandapani, Krishnan M., and Darrell W. Brann. "The role of glutamate and nitric oxide in the reproductive neuroendocrine system." Biochemistry and Cell Biology 78, no. 3 (April 2, 2000): 165–79. http://dx.doi.org/10.1139/o00-015.
Full textAbstract:
The preovulatory surge of gonadotropin releasing hormone (GnRH) is essential for mammalian reproduction. Recent work has implicated the neurotransmitters glutamate and nitric oxide as having a key role in this process. Large concentrations of glutamate are found in several hypothalamic nuclei known to be important for GnRH release and glutamate receptors are also located in these key hypothalamic nuclei. Administration of glutamate agonists stimulate GnRH and LH release, while glutamate receptor antagonists attenuate the steroid-induced and preovulatory LH surge. Glutamate has also been implicated in the critical processes of puberty, hormone pulsatility, and sexual behavior. Glutamate is believed to elicit many of these effects by activating the release of the gaseous neurotransmitter, nitric oxide (NO). NO potently stimulates GnRH by activating a heme containing enzyme, guanylate cyclase, which in turn leads to increased production of cGMP and GnRH release. Recent work has focused on identifying anchoring and (or) clustering proteins that target glutamate receptors to the synapse and couple the glutamate-NO neurotransmission system. The present review will discuss these new findings, as well as the role of glutamate and nitric oxide in important mammalian reproductive events, with a focus on the hypothalamic control of preovulatory GnRH release. Key words: glutamate, nitric oxide, GnRH, postsynaptic density, hypothalamus.
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Marsden, P. A., and B. J. Ballermann. "Tumor necrosis factor alpha activates soluble guanylate cyclase in bovine glomerular mesangial cells via an L-arginine-dependent mechanism." Journal of Experimental Medicine 172, no. 6 (December 1, 1990): 1843–52. http://dx.doi.org/10.1084/jem.172.6.1843.
Full textAbstract:
Endothelium-derived nitric oxide (NO) causes vasodilatation by activating soluble guanylate cyclase, and glomerular mesangial cells respond to NO with elevations of intracellular guanosine 3',5'-cyclic monophosphate (cGMP). We explored whether mesangial cells can be stimulated to produce NO and whether NO modulates mesangial cell function in an autocrine or paracrine fashion. Tumor necrosis factor alpha (TNF-alpha) raised mesangial cell cGMP levels in a time- and concentration-dependent manner (threshold dose 1 ng/ml, IC50 13.8 ng/ml, maximal response 100 ng/ml). TNF-alpha-induced increases in mesangial cGMP content were evident at 8 h and maximal at 18-24 h. The TNF-alpha-induced stimulation of mesangial cell cGMP production was abrogated by actinomycin D or cycloheximide suggesting dependence on new RNA or protein synthesis. Hemoglobin and methylene blue, both known to inhibit NO action, dramatically reduced TNF-alpha-induced mesangial cell cGMP production. Superoxide dismutase, known to potentiate NO action, augmented the TNF-alpha-induced effect. Ng-monomethyl-L-arginine (L-NMMA) decreased cGMP levels in TNF-alpha-treated, but not vehicle-treated mesangial cells in a concentration-dependent manner (IC50 53 microM). L-arginine had no effect on cGMP levels in control or TNF-alpha-treated mesangial cells but reversed L-NMMA-induced inhibition. Interleukin 1 beta and lipopolysaccharide (LPS), but not interferon gamma, also increased mesangial cell cGMP content. Transforming growth factor beta 1 blunted the mesangial cell response to TNF-alpha. TNF-alpha-induced L-arginine-dependent increases in cGMP were also evident in bovine renal artery vascular smooth muscle cells, COS-1 cells, and 1502 human fibroblasts. These findings suggest that TNF-alpha induces expression in mesangial cell of an enzyme(s) involved in the formation of L-arginine-derived NO. Moreover, the data indicate that NO acts in an autocrine and paracrine fashion to activate mesangial cell soluble guanylate cyclase. Cytokine-induced formation of NO in mesangial and vascular smooth muscle cells may be implicated in the pathogenesis of septic shock.
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Jin, J. G., K. S. Murthy, J. R. Grider, and G. M. Makhlouf. "Activation of distinct cAMP- and cGMP-dependent pathways by relaxant agents in isolated gastric muscle cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 264, no. 3 (March 1, 1993): G470—G477. http://dx.doi.org/10.1152/ajpgi.1993.264.3.g470.
Full textAbstract:
The mechanism of action of vasoactive intestinal peptide (VIP) was examined in isolated gastric and taenia coli muscle cells and compared with that of nitric oxide (NO), sodium nitroprusside (SNP), and isoproterenol. In gastric muscle cells, VIP stimulated NO production, increased adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) levels, and induced relaxation in a concentration-dependent fashion. The NO synthase inhibitor NG-nitro-L-arginine abolished NO and cGMP production and partly inhibited relaxation. The soluble guanylate cyclase inhibitor LY 83583 abolished cGMP production and partly inhibited relaxation. (R)-p-adenosine 3',5'-cyclic phosphorothioate [(R)-p-cAMPS], a preferential inhibitor of cAMP-dependent protein kinase (cAK), and KT5823, a preferential inhibitor of cGMP-dependent protein kinase (cGK), partly inhibited relaxation separately and abolished relaxation in combination. The pattern implied that VIP induced relaxation by activation of cAK and by NO-mediated stimulation of cGMP and activation of cGK. In taenia coli muscle cells, VIP did not increase NO production or cGMP levels: relaxation was accompanied by an increase in cAMP and was partly inhibited by (R)-p-cAMPS and KT5823 and abolished by a combination of both inhibitors. Isoproterenol increased only cAMP levels in both cell types, which induced relaxation by activating cAK at low concentrations of agonist and both cAK and cGK at high concentrations in a pattern identical to that observed with VIP in taenia coli muscle cells. SNP and NO increased only cGMP levels in both cell types, which induced relaxation by activating cGK only. We conclude that cAK and cGK can be activated separately and mediate relaxation independently.(ABSTRACT TRUNCATED AT 250 WORDS)
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Steinbrecher, Kris A., Jeffrey A. Rudolph, Guangju Luo, and Mitchell B. Cohen. "Coordinate upregulation of guanylin and uroguanylin expression by hypertonicity in HT29-18-N2 cells." American Journal of Physiology-Cell Physiology 283, no. 6 (December 1, 2002): C1729—C1737. http://dx.doi.org/10.1152/ajpcell.00010.2002.
Full textAbstract:
Guanylin and uroguanylin are particulate guanylate cyclase-activating peptides that are secreted from the epithelia of the intestine, kidney, pancreas, and salivary gland. These peptides elicit chloride and bicarbonate secretion via the cystic fibrosis transmembrane conductance regulator. To test the hypothesis that hypertonicity mediates an increase in guanylin and uroguanylin mRNA, we subjected HT29-18-N2 to osmotic stress. Guanylin and uroguanylin RNA were increased substantially in the presence of hypertonicity but only with solutes that were relatively impermeable to the cell membrane. This hypertonicity-mediated increase was transcriptional and did not require protein synthesis. Herbimycin A and mitogen-activated protein kinase inhibitors SB-203580 and PD-98059 had no effect on basal or induced levels of guanylin or uroguanylin. Both staurosporine and prolonged exposure to phorbol ester reduced basal levels and completely blocked hypertonicity-related increases in guanylin or uroguanylin RNA. These data suggest that serine/theonine protein kinases, possibly protein kinase C (PKC), mediate the hypertonicity-associated increase in guanylin and uroguanylin RNA. We conclude that guanylin and uroguanylin are released in response to hypertonic stress and that regulation of these genes may be mediated by PKC isoforms.
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Morales-Cámara, Samuel, Susana Alexandre-Moreno, Juan-Manuel Bonet-Fernández, Raquel Atienzar-Aroca, José-Daniel Aroca-Aguilar, Jesús-José Ferre-Fernández, Carmen-Dora Méndez, et al. "Role of GUCA1C in Primary Congenital Glaucoma and in the Retina: Functional Evaluation in Zebrafish." Genes 11, no. 5 (May 14, 2020): 550. http://dx.doi.org/10.3390/genes11050550.
Full textAbstract:
Primary congenital glaucoma (PCG) is a heterogeneous, inherited, and severe optical neuropathy caused by apoptotic degeneration of the retinal ganglion cell layer. Whole-exome sequencing analysis of one PCG family identified two affected siblings who carried a low-frequency homozygous nonsense GUCA1C variant (c.52G > T/p.Glu18Ter/rs143174402). This gene encodes GCAP3, a member of the guanylate cyclase activating protein family, involved in phototransduction and with a potential role in intraocular pressure regulation. Segregation analysis supported the notion that the variant was coinherited with the disease in an autosomal recessive fashion. GCAP3 was detected immunohistochemically in the adult human ocular ciliary epithelium and retina. To evaluate the ocular effect of GUCA1C loss-of-function, a guca1c knockout zebrafish line was generated by CRISPR/Cas9 genome editing. Immunohistochemistry demonstrated the presence of GCAP3 in the non-pigmented ciliary epithelium and retina of adult wild-type fishes. Knockout animals presented up-regulation of the glial fibrillary acidic protein in Müller cells and evidence of retinal ganglion cell apoptosis, indicating the existence of gliosis and glaucoma-like retinal damage. In summary, our data provide evidence for the role of GUCA1C as a candidate gene in PCG and offer new insights into the function of this gene in the ocular anterior segment and the retina.
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Payne, Annette M., Susan M. Downes, David A. R. Bessant, Catherine Plant, Tony Moore, Alan C. Bird, and Shomi S. Bhattacharya. "Genetic analysis of the guanylate cyclase activator 1B (GUCA1B) gene in patients with autosomal dominant retinal dystrophies: Table 1." Journal of Medical Genetics 36, no. 9 (September 1, 1999): 691–93. http://dx.doi.org/10.1136/jmg.36.9.691.
Full textAbstract:
The guanylate cyclase activator proteins (GCAP1 and GCAP2) are calcium binding proteins which by activating Ret-GC1 play a key role in the recovery phase of phototransduction. Recently a mutation in theGUCA1A gene (coding for GCAP1) mapping to the 6p21.1 region was described as causing cone dystrophy in a British family. In addition mutations in Ret-GC1have been shown to cause Leber congenital amaurosis and cone-rod dystrophy. To determine whether GCAP2 is involved in dominant retinal degenerative diseases, the GCAP2 gene was screened in 400 unrelated subjects with autosomal dominant central and peripheral retinal dystrophies.A number of changes involving the intronic as well as the coding sequence were observed. In exon 1 a T to C nucleotide change was observed leaving the tyrosine residue 57 unchanged. In exon 3 a 1 bp intronic insertion, a single nucleotide substitution G to A in the intron 3′ of this exon, and a GAG to GAT change at codon 155 were observed. This latter change results in a conservative change of glutamic acid to aspartic acid. In exon 4 a 7 bp intronic insertion, a single nucleotide A to G substitution in the intron 5′ of this exon, and a single base pair change C to G in the intron 3′ of exon 4 were seen. None of these changes would be expected to affect correct splicing of this gene. All these changes were observed in controls. The results of this study do not show any evidence so far that GCAP2 is involved in the pathogenesis of autosomal dominant retinal degeneration in this group of patients. All the changes detected were found to be sequence variations or polymorphisms and not disease causing.
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Kamenarova, Kunka, Marta Corton, Blanca García-Sandoval, Patricia Fernández-San Jose, Valentin Panchev, Almudena Ávila-Fernández, Maria Isabel López-Molina, Christina Chakarova, Carmen Ayuso, and Shomi S. Bhattacharya. "NovelGUCA1AMutations Suggesting Possible Mechanisms of Pathogenesis in Cone, Cone-Rod, and Macular Dystrophy Patients." BioMed Research International 2013 (2013): 1–15. http://dx.doi.org/10.1155/2013/517570.
Full textAbstract:
Here, we report two novelGUCA1A(the gene for guanylate cyclase activating protein 1) mutations identified in unrelated Spanish families affected by autosomal dominant retinal degeneration (adRD) with cone and rod involvement. All patients from a three-generation adRD pedigree underwent detailed ophthalmic evaluation. Total genome scan using single-nucleotide polymorphisms and then the linkage analysis were undertaken on the pedigree. Haplotype analysis revealed a 55.37 Mb genomic interval cosegregating with the disease phenotype on chromosome 6p21.31-q15. Mutation screening of positional candidate genes found a heterozygous transition c.250C>T in exon 4 ofGUCA1A, corresponding to a novel mutation p.L84F. A second missense mutation, c.320T>C (p.I107T), was detected by screening of the gene in a Spanish patients cohort. Using bioinformatics approach, we predicted that either haploinsufficiency or dominant-negative effect accompanied by creation of a novel function for the mutant protein is a possible mechanism of the disease due to c.250C>T and c.320T>C. Although additional functional studies are required, our data in relation to the c.250C>T mutation open the possibility thattransacting factors binding to de novo created recognition site resulting in formation of aberrant splicing variant is a disease model which may be more widespread than previously recognized as a mechanism causing inherited RD.
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Ames, James B. "Structural Insights into Retinal Guanylate Cyclase Activator Proteins (GCAPs)." International Journal of Molecular Sciences 22, no. 16 (August 13, 2021): 8731. http://dx.doi.org/10.3390/ijms22168731.
Full textAbstract:
Retinal guanylate cyclases (RetGCs) promote the Ca2+-dependent synthesis of cGMP that coordinates the recovery phase of visual phototransduction in retinal rods and cones. The Ca2+-sensitive activation of RetGCs is controlled by a family of photoreceptor Ca2+ binding proteins known as guanylate cyclase activator proteins (GCAPs). The Mg2+-bound/Ca2+-free GCAPs bind to RetGCs and activate cGMP synthesis (cyclase activity) at low cytosolic Ca2+ levels in light-activated photoreceptors. By contrast, Ca2+-bound GCAPs bind to RetGCs and inactivate cyclase activity at high cytosolic Ca2+ levels found in dark-adapted photoreceptors. Mutations in both RetGCs and GCAPs that disrupt the Ca2+-dependent cyclase activity are genetically linked to various retinal diseases known as cone-rod dystrophies. In this review, I will provide an overview of the known atomic-level structures of various GCAP proteins to understand how protein dimerization and Ca2+-dependent conformational changes in GCAPs control the cyclase activity of RetGCs. This review will also summarize recent structural studies on a GCAP homolog from zebrafish (GCAP5) that binds to Fe2+ and may serve as a Fe2+ sensor in photoreceptors. The GCAP structures reveal an exposed hydrophobic surface that controls both GCAP1 dimerization and RetGC binding. This exposed site could be targeted by therapeutics designed to inhibit the GCAP1 disease mutants, which may serve to mitigate the onset of retinal cone-rod dystrophies.
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Perri, Roman E., Daniel A. Langer, Suvro Chatterjee, Simon J. Gibbons, Jay Gadgil, Sheng Cao, Gianrico Farrugia, and Vijay H. Shah. "Defects in cGMP-PKG pathway contribute to impaired NO-dependent responses in hepatic stellate cells upon activation." American Journal of Physiology-Gastrointestinal and Liver Physiology 290, no. 3 (March 2006): G535—G542. http://dx.doi.org/10.1152/ajpgi.00297.2005.
Full textAbstract:
NO antagonizes hepatic stellate cell (HSC) contraction, although activated HSC in cirrhosis demonstrate impaired responses to NO. Decreased NO responses in activated HSC and mechanisms by which NO affects activated HSC remain incompletely understood. In normal rat HSC, the NO donor diethylamine NONOate (DEAN) significantly increased cGMP production and reduced serum-induced contraction by 25%. The guanylate cyclase (sGC) inhibitor 1 H-[1,2,4]oxadiazolo-[4,3- a]quinoxalin-1-one (ODQ) abolished 50% of DEAN effects, whereas the cGMP analog 8-bromoguanosine 3′,5′-cyclic monophosphate (8-BrcGMP) reiterated half the observed DEAN response, suggesting both cGMP-dependent protein kinase G (PKG)-dependent and -independent mechanisms of NO-mediated antagonism of normal HSC contraction. However, NO donors did not increase cGMP production from in vivo activated HSC from bile duct-ligated rats and showed alterations in intracellular Ca2+ accumulation suggesting defective cGMP-dependent effector pathways. The LX-2 cell line also demonstrated lack of cGMP generation in response to NO and a lack of effect of ODQ and 8-BrcGMP in modulating the NO response. However, cGMP-independent effects in response to NO were maintained in LX-2 and were associated with S-nitrosylation of proteins, an effect reiterated in primary HSC. Adenovirus-based overexpression of PKG significantly attenuated contraction of LX-2 by 25% in response to 8-BrcGMP. In summary, these studies demonstrate that NO affects HSC through cGMP-dependent and -independent pathways. The HSC activation process is associated with maintenance of cGMP-independent actions of NO but defects in cGMP-PKG-dependent NO signaling that are improved by PKG gene delivery in LX-2 cells. Activating targets downstream from NO-cGMP in activated HSC may represent a novel therapeutic target for portal hypertension.
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Plano, Santiago Andrés, Fernando Martín Baidanoff, Laura Lucía Trebucq, Sebastián Ángel Suarez, Fabio Doctorovich, Diego Andrés Golombek, and Juan José Chiesa. "Redox and Antioxidant Modulation of Circadian Rhythms: Effects of Nitroxyl, N-Acetylcysteine and Glutathione." Molecules 26, no. 9 (April 26, 2021): 2514. http://dx.doi.org/10.3390/molecules26092514.
Full textAbstract:
The circadian clock at the hypothalamic suprachiasmatic nucleus (SCN) entrains output rhythms to 24-h light cycles. To entrain by phase-advances, light signaling at the end of subjective night (circadian time 18, CT18) requires free radical nitric oxide (NO•) binding to soluble guanylate cyclase (sGC) heme group, activating the cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG). Phase-delays at CT14 seem to be independent of NO•, whose redox-related species were yet to be investigated. Here, the one-electron reduction of NO• nitroxyl was pharmacologically delivered by Angeli’s salt (AS) donor to assess its modulation on phase-resetting of locomotor rhythms in hamsters. Intracerebroventricular AS generated nitroxyl at the SCN, promoting phase-delays at CT14, but potentiated light-induced phase-advances at CT18. Glutathione/glutathione disulfide (GSH/GSSG) couple measured in SCN homogenates showed higher values at CT14 (i.e., more reduced) than at CT18 (oxidized). In addition, administration of antioxidants N-acetylcysteine (NAC) and GSH induced delays per se at CT14 but did not affect light-induced advances at CT18. Thus, the relative of NO• nitroxyl generates phase-delays in a reductive SCN environment, while an oxidative favors photic-advances. These data suggest that circadian phase-locking mechanisms should include redox SCN environment, generating relatives of NO•, as well as coupling with the molecular oscillator.
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Bassot, Arthur, Marie-Agnès Chauvin, Nadia Bendridi, Jingwei Ji-Cao, Guillaume Vial, Léa Monnier, Birke Bartosch, et al. "Regulation of Mitochondria-Associated Membranes (MAMs) by NO/sGC/PKG Participates in the Control of Hepatic Insulin Response." Cells 8, no. 11 (October 25, 2019): 1319. http://dx.doi.org/10.3390/cells8111319.
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Under physiological conditions, nitric oxide (NO) produced by the endothelial NO synthase (eNOS) upregulates hepatic insulin sensitivity. Recently, contact sites between the endoplasmic reticulum and mitochondria named mitochondria-associated membranes (MAMs) emerged as a crucial hub for insulin signaling in the liver. As mitochondria are targets of NO, we explored whether NO regulates hepatic insulin sensitivity by targeting MAMs. In Huh7 cells, primary rat hepatocytes and mouse livers, enhancing NO concentration increased MAMs, whereas inhibiting eNOS decreased them. In vitro, those effects were prevented by inhibiting protein kinase G (PKG) and mimicked by activating soluble guanylate cyclase (sGC) and PKG. In agreement with the regulation of MAMs, increasing NO concentration improved insulin signaling, both in vitro and in vivo, while eNOS inhibition disrupted this response. Finally, inhibition of insulin signaling by wortmannin did not affect the impact of NO on MAMs, while experimental MAM disruption, using either targeted silencing of cyclophilin D or the overexpression of the organelle spacer fetal and adult testis-expressed 1 (FATE-1), significantly blunted the effects of NO on both MAMs and insulin response. Therefore, under physiological conditions, NO participates to the regulation of MAM integrity through the sGC/PKG pathway and concomitantly improves hepatic insulin sensitivity. Altogether, our data suggest that the induction of MAMs participate in the impact of NO on hepatocyte insulin response.
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Ducrocq, Claire, Claudine Servy, Mare Cudic, and Béatrice Blanchard. "Intervention du monoxyde d'azote, NO, et de ses dérivés oxydés, particulièrement chez les mammifères." Canadian Journal of Physiology and Pharmacology 79, no. 2 (February 1, 2001): 95–102. http://dx.doi.org/10.1139/y00-077.
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Nitric oxide (NO) is a natural and stable free radical produced in soil and water by the bacteriological reduction of nitrites and nitrates and in animals by the enzyme oxidation of L-arginine. NO is biosynthesised by finely regulated enzymatic systems called NO-synthases and readily diffuses through tissues. It reacts rapidly with hemoproteins and iron-sulphur centers to form nitrosylated compounds. It oxidises more slowly to form nitrogen oxides that nitrosate thiols into thionitrite. NO is transported in these various forms and released spontaneously or through yet unclear mechanisms into most cells; it also regulates oxygen consumption at the mitochondrial respiratory chain level through interaction with cytochrome oxidase. In the cardiovascular system, NO lowers blood pressure by activating a hemoprotein, the guanylate cyclase present in muscle cells; through such interaction it acts also as a neuromediator and neuromodulator in the nervous system. However, many of NO's roles result from rapid coupling to other radicals; for example, it reacts with the superoxide anion (O2) to form oxoperoxinitrate (ONOO, also known as peroxynitrite). This strong oxidant of metallic centers, thiols, and antioxidants is also able to convert tyrosine to 3-nitrotyrosine and to act upon tyrosine residues contained in proteins. The biological aspects of the roles of NO are presented with particular respect to the rapid interactions of NO with hemoproteins' iron and other radicals. Concurrently, NO oxidation enables nitrosation reactions primarily of thiols but ultimately of nucleic bases. The thionitrite function (R-S-NO) thus formed and the dimerisation and nitration of tyrosine residues are protein post-translational modifications that are being investigated in animals.Key words: nitric oxide, peroxynitrite, nitration, nitrosation, nitrosylation. [Translated by the editors.]
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Bondarenko, Vladimir A., Fumio Hayashi, Jiro Usukura, and Akio Yamazaki. "Involvement of rhodopsin and ATP in the activation of membranous guanylate cyclase in retinal photoreceptor outer segments (ROS-GC) by GC-activating proteins (GCAPs): a new model for ROS-GC activation and its link to retinal diseases." Molecular and Cellular Biochemistry 334, no. 1-2 (November 26, 2009): 125–39. http://dx.doi.org/10.1007/s11010-009-0323-y.
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Bominaar, A. A., F. Kesbeke, B. E. Snaar-Jagalska, D. J. Peters, P. Schaap, and P. J. Van Haastert. "Abberant chemotaxis and differentiation in Dictyostelium mutant fgdC with a defective regulation of receptor-stimulated phosphoinositidase C." Journal of Cell Science 100, no. 4 (December 1, 1991): 825–31. http://dx.doi.org/10.1242/jcs.100.4.825.
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Dictyostelium cells use extracellular cyclic AMP both as a chemoattractant and as a morphogen inducing cell-type-specific gene expression. Cyclic AMP binds to surface receptors, activates one or more G-proteins, and stimulates adenylate cyclase, guanylate cyclase and phosphoinositidase C. Mutant fgdC showed aberrant chemotaxis, and was devoid of cyclic AMP-induced gene expression and differentiation. Both the receptor- and G-protein-mediated stimulation of adenylate cyclase and guanylate cyclase were unaltered in mutant fgdC as compared to wild-type cells. In wild-type cells phosphoinositidase C was activated about twofold by the cyclic AMP receptor. In mutant fgdC cells, however, the enzyme was inhibited by about 60%. These results suggest that phosphoinositidase C is regulated by a receptor-operated activation/inhibition switch that is defective in mutant fgdC. We conclude that activation of phosphoinositidase C is essential for Dictyostelium development.
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Simon, Christoph, Nora Mirza, Wolfgang A. Bethge, Wichard Vogel, Christoph Faul, Hans-Georg Rammensee, Lothar Kanz, and Sebastian P. Haen. "Central Nervous System Complications After Allogeneic Hematopoietic Cell Transplantation – Treatment Related Morbidity, Toxicity, Graft Versus Host Disease Or New Autoimmunity." Blood 122, no. 21 (November 15, 2013): 2069. http://dx.doi.org/10.1182/blood.v122.21.2069.2069.
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Abstract Graft versus host disease (GvHD) is still the leading complication of allogeneic hematopoietic cell transplantation (HCT). While the skin, liver and gut are the main sites of GvHD manifestation, evidence is emerging that the central nervous system (CNS) may also be involved. Vascular and infectious complications after allogeneic HCT are rare and represent a diagnostic and therapeutic challenge, as the differentiation between inflammatory, vascular, infectious and malignant causes of CNS symptoms can be difficult. Most data available so far are based on animal models or case reports. We recently reported the development of new-autoinflammatory T cell responses against autoantigens of the retina as part of the CNS (ASH Annual Meeting 2012, Abstract #3060). In this study, we evaluated CNS manifestations of GvHD and other CNS complications not directly associable with inflammatory CNS changes after allogeneic HCT. Data of 854 patients that underwent allogeneic HCT at our institution between 2003 and 2012 were analyzed. We identified 25 patients (3%) suffering from 39 different CNS manifestations. The cohort comprised 12 women and 13 men with a median age of 38 years at transplantation (range 20-69 years) and of 39 years at the onset of CNS symptoms (range 21-69 years). Patients had been treated for ALL (n=10, 40%), AML (n=13, 52%), PMF (n=1, 4%) and PNH (n=1, 4%). Median onset of CNS symptoms was 3 months after allogeneic HCT (range 0-24 months). By that time, 19 patients (76%) were in complete remission, 6 patients (24%) had experienced relapse. Patients had been transplanted 1-3 times (total 37 HCT) with myeloablative (n=14, 38%) or dose-reduced intensity conditioning regimen protocols (n=23, 62%). 13 HCT were performed with grafts from related (35%), 24 with grafts from unrelated donors (65%). HLA-identical, mismatched and haploidentical grafts were given in 18, 11 and 8 cases, respectively. Patients with ALL undergoing prophylactic CNS irradiation tended to be more at risk for inflammatory CNS complications. Ten of all investigated patients suffered from visual loss due to retinal affection. Six of them experienced inflammation of the optic nerve and the retina (5 without detection of viruses or other causes, 1 due to CMV), and 3 patients experienced severe optical nerve atrophy. Infections of the CNS were seen in 3 cases and were due to VZV, HHV-6 and toxoplasmosis. Cerebral vasculitis was present in 6 further patients, while 8 patients had severe leukencephalopathy. Unspecific CNS symptoms including sedation and seizures were observed in 4 patients. Here, the underlying cause could not be further discriminated. Five further patients suffered from bleeding (n=4) or ischemic (n=1) CNS complications. The symptoms of 2 patients could be attributed to isolated meningeal relapse of the disease. Of 4 patients with ocular symptoms (cone degeneration and CNS vascultitis, n=1; anemic retinopathy, n=1; optic neuritis, n=2) we obtained peripheral mononuclear cells (PBMC) which were evaluated in ELISPOT and intracellular cytokine staining assays. Before testing, PBMC were prestimulated for 12 days with T cell epitopes derived from retinal proteins (membrane-bound retinal guanylate cyclase 1 protein (retGC), guanylate cyclase activating proteins 1 and 2 (GCAP1 and GCAP2) and retinoid binding protein 3 (RBP3)) that had been identified by polymorphic protein sequence based prediction of epitopes using the internet based databases EpiToolKit and SYFPEITHI. In one patient with cone degeneration and optic nerve atrophy, antigen specific T cells against retGC derived epitopes could be detected. Our data indicate that CNS complications of allogeneic HCT may differ with regard to the affected organ (eye, CNS vascular system, brain) and the pathophysiology of the symptoms (inflammation, toxicity). Most of the complications were associated with inflammatory changes of the eye and the brain representing either GvHD or new autoimmunity. Without immunohistochemic examination, differentiation between GvHD and new autoimmunity is difficult, since inflammatory reactions of allogeneic T cells may be due to recognition of neo-autoantigens. Disclosures: No relevant conflicts of interest to declare.
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McKeown, Alex S., and Timothy W. Kraft. "Adaptive potentiation in rod photoreceptors after light exposure." Journal of General Physiology 143, no. 6 (May 12, 2014): 733–43. http://dx.doi.org/10.1085/jgp.201411163.
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Photoreceptors adapt to changes in illumination by altering transduction kinetics and sensitivity, thereby extending their working range. We describe a previously unknown form of rod photoreceptor adaptation in wild-type (WT) mice that manifests as a potentiation of the light response after periods of conditioning light exposure. We characterize the stimulus conditions that evoke this graded hypersensitivity and examine the molecular mechanisms of adaptation underlying the phenomenon. After exposure to periods of saturating illumination, rods show a 10–35% increase in circulating dark current, an adaptive potentiation (AP) to light exposure. This potentiation grows as exposure to light is extended up to 3 min and decreases with longer exposures. Cells return to their initial dark-adapted sensitivity with a time constant of recovery of ∼7 s. Halving the extracellular Mg concentration prolongs the adaptation, increasing the time constant of recovery to 13.3 s, but does not affect the magnitude of potentiation. In rods lacking guanylate cyclase activating proteins 1 and 2 (GCAP−/−), AP is more than doubled compared with WT rods, and halving the extracellular Mg concentration does not affect the recovery time constant. Rods from a mouse expressing cyclic nucleotide–gated channels incapable of binding calmodulin also showed a marked increase in the amplitude of AP. Application of an insulin-like growth factor-1 receptor (IGF-1R) kinase inhibitor (Tyrphostin AG1024) blocked AP, whereas application of an insulin receptor kinase inhibitor (HNMPA(AM)3) failed to do so. A broad-acting tyrosine phosphatase inhibitor (orthovanadate) also blocked AP. Our findings identify a unique form of adaptation in photoreceptors, so that they show transient hypersensitivity to light, and are consistent with a model in which light history, acting via the IGF-1R, can increase the sensitivity of rod photoreceptors, whereas the photocurrent overshoot is regulated by Ca-calmodulin and Ca2+/Mg2+-sensitive GCAPs.
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Hasebe, Masaharu, and Masami Yoshino. "Nitric oxide/cGMP/PKG signaling pathway activated by M1-type muscarinic acetylcholine receptor cascade inhibits Na+-activated K+ currents in Kenyon cells." Journal of Neurophysiology 115, no. 6 (June 1, 2016): 3174–85. http://dx.doi.org/10.1152/jn.00036.2015.
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The interneurons of the mushroom body, known as Kenyon cells, are essential for the long-term memory of olfactory associative learning in some insects. Some studies have reported that nitric oxide (NO) is strongly related to this long-term memory in Kenyon cells. However, the target molecules and upstream and downstream NO signaling cascades are not completely understood. Here we analyzed the effect of the NO signaling cascade on Na+-activated K+ (KNa) channel activity in Kenyon cells of crickets ( Gryllus bimaculatus). We found that two different NO donors, S-nitrosoglutathione (GSNO) and S-nitroso- N-acetyl-dl-penicillamine (SNAP), strongly suppressed KNa channel currents. Additionally, this inhibitory effect of GSNO on KNa channel activity was diminished by 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), and KT5823, an inhibitor of protein kinase G (PKG). Next, we analyzed the role of ACh in the NO signaling cascade. ACh strongly suppressed KNa channel currents, similar to NO donors. Furthermore, this inhibitory effect of ACh was blocked by pirenzepine, an M1 muscarinic ACh receptor antagonist, but not by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) and mecamylamine, an M3 muscarinic ACh receptor antagonist and a nicotinic ACh receptor antagonist, respectively. The ACh-induced inhibition of KNa channel currents was also diminished by the PLC inhibitor U73122 and the calmodulin antagonist W-7. Finally, we found that ACh inhibition was blocked by the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME). These results suggested that the ACh signaling cascade promotes NO production by activating NOS and NO inhibits KNa channel currents via the sGC/cGMP/PKG signaling cascade in Kenyon cells.
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Imanishi, Yoshikazu, Lili Yang, Izabela Sokal, S?awomir Filipek, Krzysztof Palczewski, and Wolfgang Baehr. "Diversity of Guanylate Cyclase-Activating Proteins (GCAPs) in Teleost Fish: Characterization of Three Novel GCAPs (GCAP4, GCAP5, GCAP7) from Zebrafish (Danio rerio) and Prediction of Eight GCAPs (GCAP1-8) in Pufferfish (Fugu rubripes)." Journal of Molecular Evolution 59, no. 2 (August 2004): 204–17. http://dx.doi.org/10.1007/s00239-004-2614-y.
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Roth, Nora, Christoph Faul, Christiane Dorn, Wichard Vogel, Wolfgang A. Bethge, Lothar Kanz, Hans-Georg Rammensee, and Sebastian P. Haen. "Development of New Autoimmunity Against T Cell Antigens Derived From Retinal Proteins After Allogeneic Hematopoietic Cell Transplantation." Blood 120, no. 21 (November 16, 2012): 3060. http://dx.doi.org/10.1182/blood.v120.21.3060.3060.
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Abstract Abstract 3060 Introduction: Graft versus host disease (GvHD) is mainly mediated by T cells recognizing major (MHC) and minor (miHAG) histocompatibility antigens (human leukocyte antigens and MHC-restricted epitopes, respectively). The clinical appearance of a GvHD affecting the central nervous system (CNS) and the retina as part of the CNS is rare and evidence is limited to single case reports. Some publications describe the development of new autoimmunity after hematopoietic cell transplantation (HCT) manifested as hemolytic anemia (AIHA), immune thrombocytopenia (ITP) or myasthenia gravis. Of note, new autoinflammatory diseases affecting the retina have not been reported. In this study we investigated the GvHD of the retina and examined the development of new autoimmune T cell responses against epitopes derived from proteins exclusively expressed in the retina. Patients and Methods: We analyzed T cells from 8 women and 12 men with a median age of 55 years (range 29 – 69 years) that had underwent HCT. Underlying diseases were acute lymphoblastic leukemia (n = 1), acute myeloid leukemia, (n = 6), chronic myeloid leukemia (n = 1), myelodysplastic syndrome (n = 3), myeloproliferative syndromes (primary myelofibrosis, n = 2; essential thrombocytemia with secondary myelofibrosis, n = 2; polycythemia vera with secondary myelofibrosis, n = 1), B-cell non-Hodgkin lymphoma (gray zone lymphoma, n = 1; follicular lymphoma, n = 1; peripheral T cell lymphoma, n = 1) and Hodgkin's lymphoma (n = 1). Potential T cell epitopes from four unique highly polymorphic retinal proteins (membrane-bound retinal guanylate cyclase 1 protein (retGC), the guanylate cyclase activating proteins 1 and 2 (GCAP1 and GCAP2) and the retinoid binding protein 3 (RBP3)) were identified using 2 approaches. First, genomic DNA derived from both donor and recipient coding for these proteins was sequenced by Sanger sequencing in search of single nucleotide polymorphisms (SNP). Second, alternate peptide expression based on known SNP was predicted using internet based databases (EpiToolKit). The predicted epitopes were synthesized and used in T cell assays. Peripheral blood mononuclear cells (PBMCs) from patients after hematopoietic regeneration (neutrophils > 500/μl) were stimulated with SNP peptide pairs (peptides pairs differing in one amino acid) and analyzed by IFNg-ELISPOT and flow cytometry. Results: In 5 out of 20 patients (25%), strong T cell responses against peptides derived from retGC as well as from GCAP1 and GCAP2 were observed which were not detectable before HCT and not reflected by a difference in the DNA sequence between donor and recipient. Two patients of the cohort presented with visual loss which was due to cone dystrophy (n = 1) and retrobulbar optic neuritis (n = 1). In the patient with cone dystrophy, we observed circulating antigen specific T cells against peptides derived from retGC. The patient with retrobulbar optic neuritis did not have antigen specific T cell responses. In 2 clinically silent patients, we found IFNg producing CD4+ T cells that recognized a predicted GCAP1-derived self-peptide. One patient also had a strong T cell response against a GCAP2-derived self-peptide. The T cells specifically recognized the peptide represented in the autologous DNA sequence; no reactivity was seen after stimulation with the SNP peptide. Furthermore, the T cell reactions persisted over time and were still detectable one year after HCT. In another patient, T cell responses against the pair of GCAP2 peptides were detected. Here, the reactivity against one peptide could not be discriminated due to limited availability of patient T cells. One further patient displayed T cell responses against GCAP and retGC peptides, which were directed against both self- and SNP peptides. As controls we stimulated T cells from 5 HLA-matched healthy individuals with all respective peptides and observed no T cell reaction. Conclusions: 25% of the patients revealed strong T cell responses against retinal autoantigens after HCT. T cell responses detected late after HCT as observed in 3 patients might indicate a chronic antigen exposure. Clinical manifestations were cone dystrophy (here, antigen-specific T cells against cone protein-derived peptides could be detected) and retrobulbar optic neuritis. To our knowledge, this is the first report on antigen-specificity of neoautoinflammatory cells after allogeneic HCT. Disclosures: No relevant conflicts of interest to declare.
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Robinson, Phyllis R., and Richard H. Cote. "Characterization of guanylate cyclase in squid photoreceptors." Visual Neuroscience 3, no. 1 (July 1989): 1–7. http://dx.doi.org/10.1017/s0952523800012451.
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AbstractLight causes a rapid, 1.7-fold increase in cyclic GMP concentration in intact squid retinas (Johnson et al. (1986)). To determine whether light-induced changes in cyclic GMP concentration result from activation of guanylate cyclase, we have studied the regulation of guanylate cyclase activity in squid (Loligo pealei) photoreceptors. The enzyme is membrane-associated and activity is enhanced by the detergents Triton X-100 or digitonin. The enzyme requires divalent cations, Mn2+ being preferred over Mg2+. The dependence of enzyme activity on the MnGTP concentration deviates from simple Michaelis-Menten kinetics. Under conditions where a light-induced binding of GTP to the guanine nucleotide regulatory protein can be observed, no light-induced change in guanylate cyclase could be detected.