Littérature scientifique sur le sujet « DOPAMINE D2-LIKE RECEPTORS »

Three-dimensional computer models of the rat D2, D3 and D4 dopamine receptor subtypes have been constructed based on the diffraction co-ordinates for bacteriorhodopsin, another membrane-bound protein containing seven transmembrane domains presumed to be arranged in a similar spatial orientation. Models were assembled by aligning the putative transmembrane domains of the dopamine receptors with those of bacteriorhodopsin using sequence similarities, and then superimposing these modelled alpha-helices on to the bacteriorhodopsin-derived co-ordinates. These models explore the potential hydrogen bonding, electrostatic and stacking interactions within the receptor which may be important for maintaining the conformation of these receptors, and thereby provide target sites for agonist binding. Proposed interactions between the catecholamine ligands and these receptors appear to account for the affinity, although not the specificity, of these agonist ligands for the different dopamine receptor subtypes. Such models will be useful for establishing structure-function relationships between ligands and the dopamine receptors, and may ultimately provide a template for the design of receptor-specific drugs.

The activity patterns of subthalamic nucleus (STN) neurons are intimately related to motor function/dysfunction and modulated directly by dopaminergic neurons that degenerate in Parkinson's disease (PD). To understand how dopamine and dopamine depletion influence the activity of the STN, the functions/signaling pathways/substrates of D2-like dopamine receptors were studied using patch-clamp recording. In rat brain slices, D2-like dopamine receptor activation depolarized STN neurons, increased the frequency/irregularity of their autonomous activity, and linearized/enhanced their firing in response to current injection. Activation of D2-like receptors in acutely isolated neurons reduced transient outward currents evoked by suprathreshold voltage steps. Modulation was inhibited by a D2-like receptor antagonist and occluded by voltage-dependent Ca2+ (Cav) channel or small-conductance Ca2+-dependent K+ (SKCa) channel blockers or Ca2+-free media. Because Cav channels are targets of Gi/o-linked receptors, actions on step- and action potential waveform-evoked Cav channel currents were studied. D2-like receptor activation reduced the conductance of Cav2.2 but not Cav1 channels. Modulation was mediated, in part, by direct binding of Gβγ subunits because it was attenuated by brief depolarization. D2 and/or D3 dopamine receptors may mediate modulation because a D4-selective agonist was ineffective and mRNA encoding D2 and D3 but not D4 dopamine receptors was detectable. Brain slice recordings confirmed that SKCa channel-mediated action potential afterhyperpolarization was attenuated by D2-like dopamine receptor activation. Together, these data suggest that D2-like dopamine receptors potently modulate the negative feedback control of firing that is mediated by the functional coupling of Cav2.2 and SKCa channels in STN neurons.

Dopamine receptors are G protein-coupled receptors that are divided into two subgroups, “D1-like” receptors (D1 and D5) that couple to the Gs protein and “D2-like” receptors (D2, D3, and D4) that couple to Gi. Although inhaled dopamine has been reported to induce bronchodilation in patients with asthma, functional expression of dopamine receptor subtypes has never been described on airway smooth muscle (ASM) cells. Acute activation of Gi-coupled receptors inhibits adenylyl cyclase activity and cAMP synthesis, which classically impairs ASM relaxation. In contrast, chronic activation of Gi-coupled receptors produces a paradoxical enhancement of adenylyl cyclase activity referred to as heterologous sensitization. We questioned whether the dopamine D2-like receptor is expressed on ASM, whether it exhibits classical Gi-coupling, and whether it modulates ASM function. We detected the mRNA encoding the dopamine D2 receptor in total RNA isolated from native human ASM and from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the dopamine D2 receptor protein in both native human and guinea pig ASM and cultured HASM cells. The dopamine D2 receptor protein was immunohistochemically localized to both human and guinea pig ASM. Acute activation of the dopamine D2 receptor by quinpirole inhibited forskolin-stimulated adenylyl cyclase activity in HASM cells, which was blocked by the dopamine D2 receptor antagonist L-741626. In contrast, the chronic pretreatment (1 h) with quinpirole potentiated forskolin-stimulated adenylyl cyclase activity, which was inhibited by L-741626, the phospholipase C inhibitor U73122, or the protein kinase C inhibitor GF109203X. Quinpirole also stimulated inositol phosphate synthesis, which was inhibited by L-741626 or U73122. Chronic pretreatment (1 h) of the guinea pig tracheal rings with quinpirole significantly potentiated forskolin-induced airway relaxation, which was inhibited by L-741626. These results demonstrate that functional dopamine D2 receptors are expressed on ASM and could be a novel therapeutic target for the relaxation of ASM.

Recent studies have revealed peculiar functional and genetic features of dopamine receptors in migraine. As peripheral blood lymphocytes (PBL) may represent a tool for peripheral detection of neuroreceptors, we compared the expression of dopamine D3 (DRD3) and D4 (DRD4) receptors on PBL in migraine patients and in healthy controls using radioligand binding assay techniques in the presence of antidopamine D2-like receptor antibodies. The dopamine D2-like receptor agonist [3H]7-OH-DPAT was used as a radioligand. An increased density of both DRD3 ( P = 0.0006) and DRD4 ( P = 0.002) on PBL was observed in migraineurs compared with controls. This up-regulation might reflect central and/or peripheral dopamine receptor hypersensitivity due to hypofunction of the dopaminergic system. These findings support the view that dopamine D2-like receptors are involved in the determination of the so-called migraine trait, which may help to elucidate several clinical features of the disease.

D1-like (D1, D5) and D2-like (D2, D3, D4) dopamine receptors interact in the kidney to produce a natriuresis and a diuresis. Disruption of D1 or D3 receptors in mice results in hypertension that is caused, in part, by a decreased ability to excrete an acute saline load. We studied D1-like and D2-like receptor interaction in anesthetized spontaneously hypertensive rats (SHR) by the intrarenal infusion of Z-1046 (a novel dopamine receptor agonist with rank order potency of D3≥D4>D2>D5>D1). Z-1046 increased glomerular filtration rate (GFR), urine flow, and sodium excretion in normotensive Wistar-Kyoto rats but not in SHRs. The lack of responsiveness to Z-1046 in SHRs was not an epiphenomenon, because intrarenal cholecystokinin infusion increased GFR, urine flow, and sodium excretion to a similar extent in the two rat strains. We conclude that renal D1-like and D2-like receptor interaction is impaired in SHRs. The impaired D1-like and D2-like receptor interaction in SHRs is not caused by alterations in the coding sequence of the D3 receptor, the D2-like receptor expressed in rat renal tubules that has been shown to be involved in sodium transport. Because the diuretic and natriuretic effects of D1-like receptors are, in part, caused by an interaction with D2-like receptors, it is possible that the decreased Z-1046 action in SHRs is secondary to the renal D1-like receptor dysfunction in this rat strain.

The localization of dopamine stores and the expression and localization of dopamine (DAT) and vesicular monoamine transporters (VMAT) type-1 and -2 and of dopamine D1-like and D2-like receptor subtypes were investigated in rat submandibular, sublingual, and parotid salivary glands by HPLC with electrochemical detection, as well as immunochemical and immunohistochemical techniques. Male Wistar rats of 2 mo of age were used. The highest dopamine levels were measured in the parotid gland, followed by the submandibular and sublingual glands. Western blot analysis revealed DAT, VMAT-1, VMAT-2, and dopamine receptors immunoreactivity in membrane preparations obtained from the three glands investigated. Immunostaining for dopamine and transporters was developed within striated ducts. Salivary glands processed for dopamine receptors immunohistochemistry developed an immunoreaction primarily in striated and excretory ducts. In the submandibular gland, acinar cells displayed strong immunoreactivity for the D2 receptor, while cells of the convoluted granular tubules were negative for both D1-like and D2-like receptors. Parotid glands acinar cells displayed the highest immunoreactivity for both D1 and D2 receptors compared with other salivary glands. The above localization of dopamine and dopaminergic markers investigated did not correspond closely with neuron-specific enolase (NSE) localization. This indicates that at least in part, catecholamine stores and dopaminergic markers are independent from glandular innervation. These findings suggest that rat major salivary glands express a dopaminergic system probably involved in salivary secretion. The stronger immunoreactivity for dopamine transporters and receptors in striated duct cells suggests that the dopaminergic system could regulate not only quality, but also volume and ionic concentration of saliva.

Dopamine is an endogenous catecholamine that modulates many functions including behavior, movement, nerve conduction, hormone synthesis and release, blood pressure, and ion fluxes. Dopamine receptors in the brain have been classically divided into D1 and D2 subtypes, based on pharmacological data. However, molecular biology techniques have identified many more dopamine receptor subtypes. Several of the receptors cloned from the brain correspond to the classically described D1 and D2 receptors. Several D1 receptor subtypes have been cloned (D1A, D1B, and D5) and are each coupled to the stimulation of adenylyl cyclase. The D2 receptor has two isoforms, a shorter form, composed of 415 amino acids, is termed the D2short receptor. The long form, called the D2long receptor, is composed of 444 amino acids; both are coupled to the inhibition of adenylyl cyclase. The D3 and D4 receptors are closely related to, but clearly distinct from, the D2 receptor. They have not yet been linked to adenylyl cyclase activity. Outside of the central nervous system, the peripheral dopamine receptors have been classified into the DA1 and DA2 subtypes, on the basis of synaptic localization. The pharmacological properties of DA1 receptors roughly approximate those of D1 and D5 receptors, whereas those of DA2 receptors approximate those of D2 receptors. A renal dopamine receptor with some pharmacological features of the D2 receptor but not linked to adenylyl cyclase has been described in the renal cortex and inner medulla. In the inner medulla, this D2-like receptor, termed DA2k, is linked to stimulation of prostaglandin E2 production, apparently due to stimulation of phospholipase A2. Of the cloned dopamine receptors, only the mRNA of the D3 receptor has been reported in the kidney. The DA1 receptor in the kidney is associated with renal vasodilation and an increase in electrolyte excretion. The DA1-related vasodilation and inhibition of electrolyte transport is mediated by cAMP. The role of renal DA2 receptors remains to be clarified. Although DA1 and DA2 receptors may act in concert to decrease transport in the renal proximal convoluted tubule, the overall function of DA2 receptors may be actually the opposite of those noted for DA1 receptors. Dopamine has been postulated to act as an intrarenal natriuretic hormone. Moreover, an aberrant renal dopaminergic system may play a role in the pathogenesis of some forms of hypertension. A decreased renal production of dopamine and/or a defective transduction of the dopamine signal is/are present in some animal models of experimental hypertension as well as in some forms of human essential hypertension.

Dopamine receptors have been divided into two major types – D1 and D2 – based primarily on pharmacological and biochemical criteria. Recent advances in the molecular biology of the dopamine receptor system have allowed the identification and characterisation of at least five distinct neuronal dopamine receptor genes (D1 to D5). These genes encode dopamine receptors belonging to the D1 receptor family, termed D1 and D5, and three D2-like receptors, termed D2, D3 and D4. These receptors are distinguished on the basis of their primary structure, chromosomal location, mRNA size and tissue distribution, and biochemical and pharmacological differences. Although individually these receptor subtypes may not be directly and exclusively involved in the maintenance or expression of schizophrenia, alterations of any of the receptors may contribute to the perturbation or instability of dopaminergic homeostasis in the brain. What was once thought to be a simple two-receptor system seems to have emerged as an intricate and interactive entity. This review summarises what is currently understood about dopamine receptors, their role in antipsychotic drug action, and their association with psychosis.

Dopamine plays an important role in the pathogenesis of hypertension by regulating epithelial sodium transport, vascular smooth muscle contractility and production of reactive oxygen species and by interacting with the renin–angiotensin and sympathetic nervous systems. Dopamine receptors are classified into D1-like (D1 and D5) and D2-like (D2, D3 and D4) subtypes based on their structure and pharmacology. Each of the dopamine receptor subtypes participates in the regulation of blood pressure by mechanisms specific for the subtype. Some receptors regulate blood pressure by influencing the central and/or peripheral nervous system; others influence epithelial transport and regulate the secretion and receptors of several humoral agents. This review summarizes the physiology of the different dopamine receptors in the regulation of blood pressure, and the relationship between dopamine receptor subtypes and hypertension.

The ability of G protein coupled receptors to heterooligomerize and create novel signaling complexes has demonstrated the tremendous potential of these receptors to access diverse signaling cascades, as well as to modulate the nature of the transduced signal. In the dopamine receptor field, the existence of a D1-like receptor in brain that activated phospatidylinositol turnover has been shown, but definition of the molecular entity remained elusive. We discovered that the D1 and D2 receptors form a heterooligomer, which on activation of both receptors, coupled to Gq to activate phospholipase C and generate intracellular calcium release. The activation of Gq by the D1-D2 heterooligomer has been shown to occur in cells expressing both receptors, as well as in striatum, distinct from Gs/olf or Gi/o activation by the D1 and D2 receptor homooligomers, respectively. The activation of the D1-D2 receptor heterooligomer in brain led to a calcium signal–mediated increase in phosphorylation of calmodulin kinase lla. The calcium signal rapidly desensitized and the receptors cointernalized after occupancy of either the D1 or D2 binding pocket. Thus, the D1-D2 heterooligomer directly links the action of dopamine to rapid calcium signaling and likely has important effects on dopamine-mediated synaptic plasticity and its functional correlates in brain.

The G-protein coupling specificity of D2-like dopamine receptors was investigated using both receptor-G protein fusions and membranes of cells in which pertussis toxin-resistant mutants of individual Gi-family G proteins could be expressed in an inducible fashion. A range of ligands displayed agonism at the long isoform of the human dopamine D2 receptor. However, varying degrees of efficacy were observed for individual ligands as monitored by their capacity to load [35S] GTPγS onto each of Gi1, Gi2, Gi3 and Go1. By contrast, S-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine (S-(-)-3PPP) was a partial agonist when Go1 was the target G protein but an antagonist/inverse agonist at Gi1, Gi2, Gi3. In ligand binding assays dopamine identified both high and low affinity states at each of the dopamine D2 receptor-G protein fusion proteins and the high affinity state was eliminated by guanine nucleotide. S-(-)-3PPP bound to an apparent single state of the constructs where the D2 receptor was fused to Gi1, Gi2 or Gi3. However, it bound to distinct high and low affinity states of the D2 receptor-Go1 fusion with the high affinity state being eliminated by guanine nucleotide. Similarly, although dopamine identified guanine nucleotide-sensitive high affinity states of the D2 receptor when expression of pertussis toxin-resistant forms of either Gi2 or Go1 was induced, S-(-)-3PPP identified a high affinity site only in the presence of Go1. These results demonstrate S-(-)-3PPP to be a protean agonist at the D2l receptor and may explain in vivo actions of this ligand. Furthermore, in agreement with previous studies, the ability of the dopamine D2l receptor to couple promiscuously to Gαi1-3, and Gαo1 was demonstrated. However, despite high homology between dopamine D2l and D3 receptors, the G protein-coupling specificity of the D3 receptor has not been well characterised. Again using both receptor-G protein fusions and membranes of cells in which pertussis toxin-resistant mutants of individual Gi-family G proteins could be expressed in an inducible fashion, we confirmed the selective coupling of the D3 receptor to Gαo1. A range of ligands displayed agonism at the D2l receptor and the D3 receptor when coupled to Gαo1. As a general trend, agonists, including dopamine, displayed a higher potency at the D3 receptor. This perhaps reflects the role of D3 as an autoreceptor. Of particular interest was the demonstration that S-(-)-3PPP has both a higher efficacy and potency at the D3 receptor when coupled to Gαo1. The investigations into dopamine receptor-G protein coupling highlighted the utility of the [35S]GTPγS binding assay as a method of directly measuring receptor catalysed nucleotide exchange on the α subunit of G proteins. However, the expense associated with the use of radiolabels makes this assay less attractive, particularly for high-throughput screening programmes. In an attempt to develop a non-radioactive assay equivalent to the [35S] GTPγS assay an immunisation programme was initiated to generate antibodies selective against the active (GTP bound) conformation of G proteins. 4 way primary screening of 1632 hybridomas generated from mice immunized with GTPS-loaded Gi1 and isolated using an automated robotic colony picker, identified 3 antibodies that interacted with the constitutively active Q204L but neither the constitutively inactive G203A nor wild type form of Gi1. This profile extended to other closely related Gi-family G proteins but not to the less closely related Gs and Gq/G11 families. Each of these antibodies was, however, also able to identify wild type, GDP-bound Gi- family G proteins in the presence of AlF4- which mimics the presence of the terminal phosphate of GTP and hence generates an active conformation of the G protein. Stimulation of cells co-expressing a wild type Gisubunit and the dopamine D2 receptor with the agonist ligand nor-apomorphine also allowed these conformation selective antibodies to bind the G protein. Such reagents allow the development of label- free assays for G protein-coupled receptor-mediated activation of Gi- family G proteins.

La regulació dopaminèrgica presinàptica és important per mantenir un equilibri homeostàtic dels nivells emmagatzemats de dopamina i el seu alliberament. Els canvis en la neurotransmissió de dopamina contribueixen a trastorns neurològics i psiquiàtrics. Treballs recents del nostre grup (Ma et al., 2015; González-Sepúlveda et al.,-presentada) van descriure importants efectes de diverses classes de fàrmacs dopaminèrgics sobre la síntesi de dopamina, inclosa la L-DOPA (emprada en Parkinson), la tetrabenazina (Huntington) i aripiprazol (esquizofrènia). En aquest estudi, vam confirmar i ampliar aquestes troballes i vam comparar els efectes dels antipsicòtics agonistes parcials D2R cariprazina i brexpiprazol, els pricostimulants amfetamina i metilfenidat i diversos altres compostos selectius i experimentals. L’estriat cerebral de rata va ser trocejat i incubat ex-vivo en presència o absència d’aquests fàrmacs a diferents concentracions. De manera espontània, la dopamina i la serotonina es van acumular al llarg del temps i van assolir nivells d’emmagatzematge gairebé màxims. Aquest enfocament experimental ens va permetre avaluar la seva síntesi i dinàmica d’emmagatzematge sota la influència d’agents farmacològics escollits. Els nostres resultats podrien ser útils per comprendre els mecanismes d’acció dels antipsicòtics, i podrien facilitar més investigacions amb models animals i assajos clínics mitjançant nous agents dopaminèrgics.
La regulación dopaminérgica presináptica es importante para mantener un equilibrio homeostático de los niveles almacenados y liberación de dopamina. Los cambios en la neurotransmisión de dopamina contribuyen a los trastornos neurológicos y psiquiátricos. Hallazgos recientes de nuestro grupo (Ma et al., 2015; González-Sepúlveda et al., presentado) describieron los fuertes efectos de varias clases de medicamentos dopaminérgicos en la síntesis de dopamina, incluida L-DOPA (utilizada en Parkinson), tetrabenazina (Huntington) y aripiprazol (esquizofrenia). En este estudio, confirmamos y ampliamos esos hallazgos y comparamos los efectos de los antipsicóticos agonistas parciales D2R cariprazina y brexpiprazol, las psicoestimulantes anfetamina y metilfenidato varios otros compuestos selectivos y experimentales. El estriado cerebral de rata fue troceado e incubado ex-vivo en presencia o ausencia de estos fármacos a diferentes concentraciones. Espontáneamente, la dopamina y la serotonina se acumularon con el tiempo alcanzando niveles de almacenamiento casi máximos. Este enfoque experimental nos permitió evaluar su dinámica de síntesis y almacenamiento bajo la influencia de los agentes farmacológicos elegidos. Nuestros resultados podrían ser útiles para comprender los mecanismos de acción de los antipsicóticos, y podrían facilitar la investigación futura con modelos animales y ensayos clínicos utilizando nuevos agentes dopaminérgicos.
Presynaptic dopaminergic regulation is important to maintain a homeostatic balance of dopamine stored levels and release. Changes in dopamine neurotransmission contribute to neurological and psychiatric disorders. Recent findings from our group (Ma et al., 2015; González-Sepúlveda et al.,-submitted) describe strong effects of several classes of dopaminergic drugs on dopamine synthesis, including L-DOPA (used in Parkinson), tetrabenazine (Huntington) and aripiprazole (schizophrenia). In this study, I confirm and extend those findings and compare the effects of D2R partial agonist antipsychotics cariprazine and brexpiprazole, the psychostimulants amphetamine and methylphenidate, and several other selective and experimental compounds. Rat brain striatum was minced and incubated ex-vivo in the presence or absence of these drugs at different concentrations. Spontaneously, dopamine and serotonin accumulated over time reaching near-maximal storage levels. This experimental approach allowed me to evaluate their synthesis and storage dynamics under the influence of chosen pharmacological agents. My results could be useful to understand the mechanisms of action of antipsychotics, and they could facilitate further research with animal models and clinical trials using new dopaminergic agents.

Among several neurodegenerative disorders, Parkinson’s is one of the utmost widespread diseases, occur due to the degeneration of the dopaminergic neurons in the region of midbrain which is identified by motor signs like tremor & bradykinesia; non-motor features (like impaired vision, depression, sleeping disorders). Currently, there is no treatment available that can cure PD. However, there is an imbalance between signaling pathway in the basal ganglia circuit. The direct pathway is downregulated and mediated via dopamine D1-like receptors. In contrast, the indirect pathway is upregulated and this is mediated via dopamine D2-like receptors. Using dopamine D2-like receptors antagonists, PD symptoms can be ameliorated. Increased histaminergic signaling has been observed in PD. Herein, we want to analyze the interaction between approved antihistamine drugs with dopamine D2-like receptors. We curated a list of 58 approved antihistamine drugs to analyze their affinity with dopamine D2-like receptors as an inhibitor. Literature indicates a substantial correlation between the antihistamines and dopamine D2-like receptors. Thus, targeting these drugs might regulate the disrupted indirect pathway in the basal ganglia circuit. Through molecular docking analysis, we identified that bilastine, a highly potent and selective inhibitor of histamine H1 receptor shows high affinity with dopamine D2-like receptors and might inhibit the aberrant signaling cascade of indirect pathway and upregulated histaminergic signaling simultaneously. Further studies are required to determine the action.

博士
臺灣大學
臨床醫學研究所
95
Previous studies have shown that dopamine inhibited angiotensin II (AII)- or low salt diet-induced increase of plasma aldosterone concentration (PAC) through the D2-like dopamine receptors. Our previous work showed that belong to the D2-like dopamine receptors, both D2 and D4 dopamine receptor (D2R and D4R) expressed on human adrenal cortex and aldosterone producing adenoma (APA) and their physiologic function seemed different. Therefore, my main subject was to explore the role of D2-like dopamine receptors in the regulation of aldosterone secretion of human adrenal cortical cells. By the way of understanding the cell molecular change in APA, we wished to discover the role of D2-like receptors in the pathogenesis of this subtype of human adrenal hypertension. The molecular mechanisms of D2R and D4R were studied by series of experiments. Aldosterone is the most important mineralocorticoid, which regulates sodium and potassium concentration and maintains the adequacy of body fluid. Consequently, the secretion of aldosterone must be under a precise and complicated control. Among the many regulators, AII, plasma potassium concentration, and the adrenal corticotrophin hormone are the most important stimulators. There have been many literatures discussing their role and the regulatory mechanisms of aldosterone secretion. Plasma AII concentration may rapidly respond to body fluid deficiency and salt depletion, therefore it plays the main role in regulation of aldosterone secretion and blood pressure regulation. On the other hands, the inhibitory regulators of aldosterone secretion were much less discussed and far from being understood. Among the inhibitory regulators, atrial natriuretic peptide and dopamine are relatively more reported. The inhibitory role of dopamine in the aldosterone secretion was first reported about 30 years ago. Dopamine did not alter the basal PAC, but it inhibited the increase of the PAC under volume depletion or salt depletion. On the other hand, dopamine antagonist, metoclopramide induced the increase of the PAC. These reports suggested that the dopamine system has a tonic inhibitory effect on aldosterone secretion in the usual physical condition. Dopamine and its antagonist have similar effects on cultured bovine or rat adrenal cortical cells. This finding showed that dopamine inhibited aldosterone secretion could be directly acting on the adrenal cortical cells rather than by the way of indirectly modulating the other regulators involving the aldosterone secretion. The earlier studied have demonstrated that dopamine has its inhibitory effect on aldosterone secretion through D2-like dopamine receptors. Our previous study revealed that the increase of the APA patients’ PAC by metoclopramide was inversely correlated to the expression of CYP11B2 mRNA of these adenomas. This result hinted that the more D2-like dopamine activity, the less CYP11B2 expression in the APA. There are five dopamine receptors discovered. Among the D2-like dopamine receptors, except D3 dopamine receptor, both D2 and D4 dopamine receptors’ mRNA expressed in human adrenal cortex and aldosterone producing adenoma. By different pharmacological inhibitors, we have shown that these two D2-like dopamine receptors seemed to play opposite regulatory roles in aldosterone secretion. In this project, we analyzed the surgical specimen of APA patients to compare the expression of CYP11B2, angiotensin II type 1 receptor, D2R and D4R. We found that the APA had less D2R and D4R than the non-tumor adrenal cortex. The amount of AR1R of the tumor portions was similar to that of the non-tumor adrenal cortex. As expected, the tumor portions had much more CYP11B2 mRNA than the non-tumor adrenal cortex. In consistence with the protein analysis, both D2R and D4R mRNA of the tumor portions were less than those of the non-tumor adrenal cortex, and the mRNA of AT1R of the tumor portions and non-tumor adrenal cortex were similar. By linear regression analysis, we found that the patients’ PAC was positively correlated to the CYP11B2 mRNA expression and negatively correlated to the D2R mRNA expression. On the other hand, the patients’ PAC did not have significant correlation with AT1R and D4R mRNA. The expression of D2R mRNA was more abundance than D4R mRNA in both the tumor portions and the non-tumor adrenal cortex. In order to understanding the cause-result relationship between the D2R decrease and the PAC increase, we used the human adrenal cortical carcinoma cell line, NCI-H295R (H295R), as a cell model to test the role of D2R in regulation of aldosterone secretion. D2R agonist, bromocriptine, did not alter the basal aldosterone secretion. But it significantly inhibited the AII (10-8 mol/L)-stimulated acute (30 min) and chronic (24 hr) aldosterone secretion. Bromocriptine also attenuated the AII-stimulated CYP11B2 mRNA expression. The effect of bromocriptine could be revered by simultaneously giving D2R antagonist, raclopride. In order to mimic the down-regulation of D2R in APA, we used shRNA of D2R to generate a D2R-depleted clone of H295R cells. The D2R-depleted H295R cells have similar basal 24 hr aldosterone secretion and CYP11B2 mRNA expression. Under AII treatment, the D2R-depleted H295R cells have more aldosterone secretion and CYP11B2 mRNA expression than the wild type H295R cells. Dopamine did not alter aldosterone secretion and CYP11B2 mRNA expression in wild type H295R cells. However, dopamine significantly enhanced AII-stimulated aldosterone secretion and CYP11B2 mRNA expression in D2R-depleted H295R cells. Giving the D2R antagonist, raclopride, to block the residual D2R, the enhancing effect of dopamine was further augmented. To understanding the mechanism of the D2R modulation of the aldosterone secretion, we examined the AII-induced PKC and calcium signaling pathway. AII induced phosphorylation of PKC α/β、μ、ε as wells as their translocation to cell membrane. Bromocriptine significantly attenuated AII-stimulated PKCμ (Ser916) phosphorylation and its translocation to membrane. We also observed the reciprocal change of cytoplasmic PKCμ. The effect of bromocriptine on PKCμ activation could be reversed by raclopride. Depleting 60% PKCμ by PKCμ-specific shRNA attenuated AII-stimulated CYP11B2 mRNA expression and aldosterone secretion. We also demonstrated that the APA expressed more abundant phospho-PKCμ than the non-tumor adrenal cortex. In consistence with previous reports, AI-stimulated aldosterone secretion and CYP11B2 mRNA expression were both calcium dependent. Bromocriptine attenuated AII-stimulated increase of cytoplasmic inositol 1,4,5 triphosphate and [Ca2+]。 We demonstrated both D4R and AT1R expression in APA, human normal adrenal cortex, primary cultured human adrenal cortical cells, and H295R cells. AII stimulated aldosterone secretion and CYP11B2 mRNA expression in primary cultured human adrenal cortical cells as well as in H295R cells. But the former responded more to AII stimulation. D4R agonist, PD168,077 enhanced AII-stimulated aldosterone secretion and CYP11B2 mRNA expression in both cultured cells. D4R antagonist, L745,870 reversed the effect of PD168,077. AII stimulated PKC α/β、μ、ε phosphorylation and translocation to cell membrane in primary cultured human adrenal cortical cells as well as in H295R cells. PD168,077 selectively enhanced PKC ε activation. Transferring PKC ε-selective inhibitory peptide to prevent PKCε translocation to cell membrane attenuated AII-stimulated aldosterone secretion and CYP11B2 mRNA expression. PD168,077 also enhanced AII-stimulated increase of cytoplasmic IP3 and [Ca2+]. L745,870 could reverse this effect of PD168,077. Intracellular [Ca2+] chelator, BAPATA, did not inhibit AII-stimulated PKCε phosphorylation. On the other hand, transferring PKC ε-selective inhibitory peptide attenuated AII-stimulated increase of cytoplasmic IP3 and [Ca2+]. AII induced the increase of cytoplasmic [Ca2+] within few seconds, but PD167,077 took several minutes to enhance AII-stimulated PKCε phosphorylation. This result suggested that D4R could augment AII-stimulated cytoplasmic [Ca2+] increase directly by increasing cytoplasmic IP3 or indirectly by enhancing PKCε phosphorylation. Finally, we tried to understand the role of D2R on the tumorigenesis of APA. In consistence with previous reports, AII stimulated proliferation of primary cultured human adrenal cortical cells. Bromocriptine inhibited this cell proliferation, and raclopride reversed it. Bromocriptine did not induce H295R cells apoptosis, but it significantly inhibited the DNA synthesis H295R cells. Bromocriptine attenuated AII-stimulated ERK1/2 phosphorylation and thereafter ERK1/2 translocation from the cytosol to the nuclear in H295R cells. PD98059 which inhibited ERK1/2 phosphorylation also inhibited the proliferation of H295R cells. Analyzing the APA surgical specimen, we found that APA expressed much more phosphorylated ERK1/2 than the non-tumor adrenal cortex, though the total ERK1/2 amounts were similar in APA and the non-tumor adrenal cortex, Bromocriptine did not alter the expression of p21, p27, and p53 of H295R cells, Bromocriptine attenuated AII-stimulated cyclin D1 expression in primary cultured human adrenal cortical cells. Here, we demonstrated the inhibitory effect of D2R on the proliferation of adrenal cortical cells by attenuating the ERK1/2 phosphorylation. Consequently, down-regulation of D2R at least partially contributed to the increase of the ERK1/2 phosphorylation in APA and its tumorigenesis. In conclusion, we focused on APA, a relative homogenous subgroup of the hypertensive patients, to discuss the role of D2-like dopamine receptors in the human adrenal hypertension. The decreased D2R expression in APA negatively correlated to CYP11B2 mRNA expression in APA as well as the patients’ PAC. We further showed the opposite functions of D2R and D4R in the cultured cell models. We demonstrated their effects on the different AII signaling molecules, and the role of these signaling molecules in AII-stimulated aldosterone secretion were proved by the molecular biology techniques. We also provided evidence that D2R inhibited the proliferation of the adrenal cortical cells. Finally, we showed the difference of these signaling molecules between APA and the non-tumor adrenal cortex that confirmed the significance of the signaling molecular modification in the clinical disease, APA.

Tese de mestrado em Biologia Evolutiva e do Desenvolvimento, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016
Este estudo tem como objectivo compreender o papel de um neurotransmissor, a dopamina, na regulação de comportamentos sociais e cooperativos. Para uma melhor compreensão de cooperação, é necessário compreender primeiramente o que é sociabilidade. Sociabilidade pode ser definida pela vivência de dois ou mais indivíduos como parte do mesmo grupo. Para a sociabilidade ocorrer, é ainda necessário, existir interacção entre os membros do grupo, tendo sempre em conta o contexto social em que a interacção ocorre, assim como o comportamento do indivíduo com o qual a interacção está a ocorrer. Após a percepção destes dois componentes cada membro do grupo terá que modelar o seu próprio comportamento para que a que a sua acção se enquadre ao que lhe é exigido pelo contexto social em que se encontra. O contexto social em que um animal se encontra é definido pelo conjunto de factores ambientais (eg. taxa de predação, recurso de alimento e a forma como os seus pares se estão a comportar). Apenas após a consideração dos aspectos referidos acima, é que os comportamentos cooperativos podem emergir. Cooperação pode ser definida como uma acção que é realizada por um indivíduo A, que por sua vez beneficia um individuo B. Dentro de comportamentos cooperativos podemos definir vários tipos de interacções dependendo do receptor do benefício resultante dessa interacção. Esta troca de benefícios é designada de reciprocidade. A reciprocidade pode ser directa, quando dois indivíduos se entreajudam, ou indirecta quando numa interacção cooperativa o indivíduo beneficiado ajuda um terceiro indivíduo. A investigação em cooperação e sociabilidade que tem sido desenvolvida têm-se focado maioritariamente na ecologia mas também nos aspectos evolutivos de como cooperação e sociabilidade poderá ter surgido e mantida ao longo do tempo. Contudo, pouco se conhece sobre os seus mecanismos regulatórios. O presente estudo pretende desvendar o papel de um neurotransmissor na regulação de comportamentos cooperativos e sociais, a dopamina. Dopamina é uma catecolamina que está envolvida em várias funções centrais de um organismo, tal como na locomoção, na cognição, na aprendizagem e no sistema mesolímbico de recompensa. Em 2011 foi descrita a “Social decision-making network” que consiste num conjunto de núcleos cerebrais que estão envolvidos na regulação de sociabilidade, tal como o núcleo accumbens e a área pré-óptica, juntamente com o sistema mesolímbico de recompensa também está incluído na “Social decison-making network”. Admite-se desta forma, que um comportamento social para ser repetido, é porque despoletou alguma recompensa num “helper” (ajudante). Tendo isto em conta o presente estudo tem como principal objectivo tentar uma melhor compreensão do papel da dopamina na regulação comportamentos sociais e cooperativos. Compreender de que forma a dopamina regula comportamentos cooperativos e sociais de ”helpers”. Foi usado como objecto de estudo, o ciclídeo Neolamprolugus pulcher, sendo que esta espécie vive em famílias com uma estrutura social robusta. Estas famílias são constituídas por um casal dominante e um conjunto de “helpers” que varia de um a trinta, em que apenas o par dominante se reproduz. Os ajudantes podem ser indivíduos sexualmente maturos ou não, o seu papel na família consiste em defesa do território contra predadores e intrusos, manutenção do território e a ajudar a criar a ninhada dos dominantes. Os predadores podem ser predadores de ovos ou de adultos. Para este efeito foram realizadas injecções intramusculares em indivíduos ajudantes, com agonistas e antagonistas específicos para receptores D1 e D2. Após a manipulação da actividade de cada um destes receptores realizou-se observações de forma a serem detectadas diferenças comportamentais (eg. número de comportamentos agressivos, submissos e de afilação).Para melhor compreensão da função dos receptores dopaminérgicos na regulação de comportamentos cooperativos e sociais em ajudantes, foram formadas 8 famílias com um casal dominante e dois ajudantes, um grande e um pequeno. Começou-se por realizar um estudo de calibração para as dosagens das drogas em estudo, agonista de D1-like (SKF-38393), antagonista de D1-like (SCH-23390), agonista de D2-like (Quinpirole) e antagonista de D2-like (Metoclopramida). Para a execução do estudo de calibração foram escolhidas 3 doses para cada uma das drogas, um dose alta, uma dose média e uma dose baixa, estas doses foram escolhidas tendo em conta estudos anteriores realizados noutros organismos. Como controlo foi injectada uma solução salina. Para este estudo foram criados dois grupos com 4 famílias cada, em que cada grupo apenas foi injectado com drogas para uma das classes de receptores, 4 famílias estiveram sujeitas ao tratamento para os receptores D1-like (8 ajudantes no total) e 4 famílias estiveram sujeitas ao tratamento para os receptores D2-like (8 ajudantes no total). O desenho experimental consistiu em realizar várias sessões de observações de 15 minutos em diferentes tempos, uma observação antes da injecção, uma sessão de observação 15 minutos após a injecção, outra observação 30 minutos após a injecção e outra observação 60 minutos após a injecção. Com este estudo foi possível concluir que os receptores D1-like e D2-like estão de facto a modular a agressividade, submissão e comportamento aflitivo dos ajudantes. Após o estudo de calibração testou-se o papel dos receptores dopaminérgicos na regulação comportamental dos ajudantes, quando estes são sujeitos a diferentes contextos sociais. Para tal, foram usadas 10 famílias constituídas por um casal dominante e 2 ajudantes (um grande e um pequeno ajudante), a duas tarefas distintas mais uma tarefa de controlo. Estas tarefas consistiram em estimular certos comportamentos por parte dos ajudantes, tal como comportamentos de manutenção do território tal como escavar e limpar o abrigo e comportamentos de defesa contra intrusos. Para induzirmos comportamentos de ajuda na manutenção do território preenchemos o abrigo do casal com areia para induzir o comportamento de escavar por parte dos ajudantes para que estes desobstruíssem o abrigo. Para a indução de comportamentos de defesa do território foi apresentada à família um predador de ovos num tubo de apresentação, para induzir comportamentos agressivos contra o intruso por parte da família mas em especial por parte dos ajudantes. Como controlo para as tarefas foi elaborada uma observação sem manipulação do ambiente antes e depois da injecção. Semelhante ao que foi feito na primeira experiência foram realizados períodos de observação de 15min para cada tarefa, antes e depois da injecção. Neste caso cada ajudante foi injectado intramuscular com uma dose de cada uma das drogas, agonista do D1-like (SKF-38393), antagonista de D1-like (SCH-23390), agonista de D2-like (Quinpirole), antagonista de D2-like (Metoclopramida), e controle (solução de 0.9% NaCl). Esta experiência permitiu demonstrar que os receptores D2-like estão de facto a regular a agressividade, submissão e afilação em ajudantes de N.pulcher, no entanto foi observado que esta depende do contexto social a que o ajudante está sujeito. Isto porque não foi constatado um aumento no número de comportamentos agressivos em todas as tarefas apresentadas, apenas nas tarefas em que tal comportamento era contextualmente exigido (eg. Na presença de um intruso). É assim evidenciado que apesar da dopamina regular a sociabilidade de N.pulcher, especialmente os receptores D2-like, estes parecem estar a ter em conta o contexto social a que os ajudantes estão sujeitos. Após a manipulação farmacológica dos receptores dopaminérgicos no cérebro dos ajudantes averiguou-se também de que forma a actividade dopaminérgica estava distribuída no cérebro de um ajudante. Para isso foram executadas microdissecções das macro-areas de cérebros de indivíduos ajudantes (que pertencem a uma família), e a indivíduos que não eram ajudantes (que se encontravam em tanques de agregação). Indivíduos de tanques de agregação também se encontravam dentro de uma estrutura hierárquica forte, mas no entanto esta não era uma família. Dissecou-se as seguintes macro-areas: “forebrain”, tecto óptico, cerebelo, diencéfalo e tronco cerebral. Após as dissecções foram medidas as concentrações de dopamina e dos seus metabolitos (HVA e DOPAC), nas várias macro-areas em estudo. Ao analisar a contracção total de dopamina e dos seus metabolitos no cérebro de ajudantes versus não-ajudantes, constatou-se a não existência de diferenças significativas entre estes. No entanto, quando analisadas as diferentes macro-areas separadamente, foi evidenciado que os ajudantes têm significativamente mais dopamina e HVA no “forebrain” comparativamente com os não-ajudantes. Considerando a concentração de DOPAC, observou-se ainda que os ajudantes têm significativamente mais DOPAC no diencéfalo e no tronco cerebral. Estes resultados vão de encontro com estudos anteriores, que demonstram que áreas como o hipotálamo anterior estão envolvidas na regulação de comportamentos sociais, e que a elevada concentração de dopamina no “forebrain” está correlacionada com a percepção de uma recompensa. Os resultados deste estudo sugerem que os ajudantes de N.pulcher sentem uma recompensa por pertenceram a uma família. Sugerindo que, ao contrário do que se pensava anteriormente, ser um ajudante de uma família de N.pulcher é um caso de reciprocidade directa e não de um comportamento altruísta.
Cooperation is an evolutionary enigma that has intrigued biologists ever since Darwin. Much has been researched on the functional mechanisms of cooperation however; the physiological framework has only recently become a focus. Here we report on three experiments focussing the role of dopamine in social behaviour of a notorious cooperatively breeding teleost fish species. Dopamine is involved in the modulation of animals’ reward system and social decision network, suggesting that it might be involved in sociability. We studied Neolamprologus pulcher, a cooperative cichlid fish from Lake Tanganyika, East Africa. These fish live in families with a dominant pair and a variable number of subordinates helping the dominant breeders in territory maintenance and defence, showing altruistic behaviour by engaging in alloparental care. We aimed at dopaminergic receptors D1 and D2, blocking or stimulating their activity with injections of agonists or antagonists (SKF-3893, SCH-23390, Quinpirole and Metoclopramide). Our data suggest that the two dopaminergic receptors have different regulatory roles for the social behaviour of these fish. The major focus seems to be on D2 receptor, which is influencing the aggressive, submissive and affiliative behaviour. Specifically, the D2 receptor is stimulated there was an increase of aggression, while when blocked it increases submission and affiliative behaviour. Interestingly, social context is the switch in which D2 influence is observe, helpers have into account the social context and they will not behave in discordance with the environment. Finally, when analysing the concentration of dopamine and its metabolites we found that helpers have a higher dopaminergic activity in the diencephalon and a higher concentration of dopamine the forebrain (e.g. Telecephalon). Higher levels of dopamine in helpers’ telencephalon points out towards the direct existence of reward from living in a stabilized family. These data provide the first insight into the role of dopamine for the social behaviour of a cooperative fish species.

abstract: Patients with schizophrenia have deficits in sensorimotor gating, the ability to gate out irrelevant stimuli in order to attend to relevant stimuli. Prepulse inhibition (PPI) of the startle response is a reliable and valid model of sensorimotor gating across species. Repeated D2-like agonist treatment alleviates prior PPI deficits in rats, termed a PPI recovery, and is observable 28 days after treatment. The aim of the current project is to illuminate the underlying mechanism for this persistent change of behavior and determine the clinical relevance of repeated D2-like agonist treatment. Our results revealed a significant increase in Delta FosB, a transcription factor, in the nucleus accumbens (NAc) 10 days after repeated D2-like agonist treatment. Additionally, we investigated if Delta FosB was necessary for long-lasting PPI recovery and discovered a bilateral infusion of dominant-negative Delta JunD prevented PPI recovery after repeated D2-like agonist treatment. To further develop the underlying mechanism of PPI recovery, we observed that dominant negative mutant cyclic adenosine monophosphate (cAMP) response biding element protein (CREB) prevented repeated D2-like agonist-induced Delta FosB expression in the NAc. We then compared our previous behavioral and intracellular findings to the results of repeated aripiprazole, a novel D2-like partial agonist antipsychotic, to determine if repeated D2-like receptor agonist action is a clinically relevant pharmacological approach. As compared to previous PPI recovery and Delta FosB expression after repeated D2-like agonist treatment, we found similar PPI recovery and Delta FosB expression after repeated aripiprazole treatment in rats. We can conclude that repeated D2-like agonist treatment produces persistent PPI recovery through CREB phosphorylation and Delta FosB, which is necessary for PPI recovery. Furthermore, this pharmacological approach produces behavioral and intracellular changes similar to an effective novel antipsychotic. These findings suggest the underlying intracellular mechanism for sustained PPI recovery is clinically relevant and may be a potential target of therapeutic intervention to alleviate sensorimotor gating deficits, which are associated with cognitive symptoms of schizophrenia.
Dissertation/Thesis
Ph.D. Psychology 2013

The behavioral effects of drugs that act on the brain’s dopamine (DA) system change with repeated exposure to the drug. Antipsychotic drugs, that block DA receptors, produce progressively greater effects on behavior with repeated testing. For example, rats repeatedly treated with a low dose of the D2 receptor-preferring antagonist haloperidol do not initially exhibit catalepsy, a response quantified by time spent on a horizontal bar without active movement. However, with repeated drug-environment pairings animals show a reduction in exploration and increases in catalepsy. The current thesis examined the drug-environment relationship in catalepsy sensitization, and how different DA receptor subtypes control this phenomenon. Treatment with a D2, but not D3 or D1-like receptor-preferring antagonist produced catalepsy sensitization. Catalepsy sensitization developed in one test environment did not transfer to another environment. Similarly, rats with a history of haloperidol treatments outside of the test environment (unpaired group) did not exhibit significant catalepsy when given haloperidol for the first time prior to catalepsy testing. Previous exposure to the catalepsy test environment led to a more rapid development of catalepsy sensitization. Thus, drug-environment interaction is critical for the development and expression of catalepsy sensitization. Rats previously given haloperidol and tested with saline in the drug paired environment exhibited conditioned catalepsy. The acquisition of conditioned catalepsy is dependent on D1-like receptors, while its expression is dependent on D3 receptors. Conditioned catalepsy showed gradual day-to-day extinction with repeated saline treatment in the previously haloperidol-paired environment. Following extinction, the response to haloperidol in previously sensitized rats shifted from environment-specific to environment-independent suggesting that a putative haloperidol drug cue alone can elicit conditioned catalepsy. In summary, treatment with a D2, but not D1-like or D3 receptor-preferring antagonist in a particular test environment produces catalepsy sensitization, while acquisition of conditioned catalepsy is dependent on D1-like receptors, and its expression is dependent on D3 receptors. Importantly, the acquisition and expression of sensitization to haloperidol is conditional on the presence of drug-associated environmental stimuli. Our findings provide further insight into the current understanding of learning processes involved in the action of antipsychotic drugs and the dissociable effects of D1-like, D2 and D3 receptors controlling this phenomenon.
Thesis (Ph.D, Neuroscience Studies) -- Queen's University, 2012-08-12 15:51:00.467

碩士
國立陽明大學
藥理學研究所
96
Methamphetamine (MA) is a widely abused psychostimulant. In animal models, the pharmacological effects of MA or amphetamine depend largely on the dose and pattern of administration. Earlier studies reported that acute doses of MA administration to the human produce improvements in cognitive processing. However, more recent studies showed that long-term MA use is associated with impaired performance on a number of cognitive tasks. MA has been shown to activate central dopaminergic system, but long-term use of MA may induce oxidative stress and neurotoxicity by multiple mechanisms. The aim of the present study is to examine the effects of acute, acute binge and binge MA on the memory performance of step-though passive avoidance tasks in ICR mice and the roles of dopamine D2-like receptors and oxidative stress. The results showed that acute (1 injection), acute binge (4 injections at 2 h interval) and binge (total 8 injections, b.i.d for four days) MA (3 mg/kg, i.p.) administration induced memory impairment in mice. Pretreatment with various selective D2-like receptor antagonists (D2: eticlopride 0.1 mg/kg; D4: L745,870 0.03 mg/kg) attenuated MA-induced amnesia in acute administration protocol, but had no significant effects on those in acute binge and binge administration protocols. In addition, pretreatment with eticlopride, but not L745,870, also decreased MA-induced increase in the locomotor activity. In contrast, pre- or post-treatment with an antioxidant, baicalein (1 mg/kg, i.p.), had no effect on acute MA-induced memory impairment. However, pretreatment with baicalein attenuated MA-induced memory impairment in acute binge and binge administration protocols with no effect on MA-induced increase in the locomotor activity. In conclusion, the present study demonstrated that MA can induce amnesia in the passive avoidance test in ICR mice after acute, acute binge and binge administrations. Acute MA-induced memory impairment may attribute to the overactivity of D2-like receptors by increased release of dopamine, whereas acute binge or binge MA-induced memory impairment could be due to MA-induced oxidative damage to the memory-associated nervous system. Moreover, MA-induced memory impairment in the passive avoidance test and change of locomotor activity in mice may be independent, probably involving different nervous pathways.

Dopamine receptor subtypes and incentive learning explains that dopamine receptors are G protein-coupled and form two families: D1-like receptors, including D1 and D5, stimulate adenylyl cyclase and cyclic adenosine monophosphate (cAMP); D2-like receptors, including D2, D3, and D4, inhibit cAMP. Antipsychotic medications are dopamine receptor antagonists and their clinical potency is strongly correlated with blockade of D2 receptors, implicating overactivity of D2 receptors in psychosis in schizophrenia. D1- and D2-like receptors appear to be involved in unconditioned locomotor activity and incentive learning. D1-like receptors are implicated more strongly in incentive learning and D2-like receptors more strongly in locomotion. D3 receptors may play a relatively greater role in expression than acquisition of incentive learning. Dopamine receptor subtypes form heteromers with each other and with the receptors of other neurotransmitters (e.g., glutamate, adenosine, ghrelin) and the signaling properties of these heteromers can differ from those of either receptor in isolation.