Differential Inhibition of Secretagogue-Stimulated Sodium Uptake in Adrenal Chromaffin Cells by Activation of D4 and D5 Dopamine Receptors

Abstract: Recent studies have demonstrated that D1-selective and D2-selective dopamine receptor agonists inhibit catecholamine secretion and Ca²⁺ uptake into bovine adrenal chromaffin cells by receptor subtypes that we have identified by PCR as D5, a member of the D1-like dopamine receptor subfamily, and D4, a member of the D2-like dopamine receptor subfamily. The purpose of this study was to determine whether activation of D5 or D4 receptors inhibits influx of Na⁺, which could explain inhibition of secretion and Ca²⁺ uptake by dopamine agonists. D1-selective agonists preferentially inhibited both dimethylphenylpiperazinium- (DMPP) and veratridine-stimulated ²²Na⁺ influx into chromaffin cells. The D1-selective agonists chloro-APB hydrobromide (CI-APB; 100 µ M ) and SKF-38393 (100 µ M ) inhibited DMPP-stimulated Na⁺ uptake by 87.5 ± 2.3 and 59.7 ± 4.5%, respectively, whereas the D2-selective agonist bromocriptine (100 µ M ) inhibited Na⁺ uptake by only 22.9 ± 5.0%. Veratridine-stimulated Na⁺ uptake was inhibited 95.1 ± 3.2 and 25.7 ± 4.7% by 100 µ M CI-APB or bromocriptine, respectively. The effect of CI-APB was concentration dependent. A similar IC₅₀ (∼18 µ M ) for inhibition of both DMPP- and veratridine-stimulated Na⁺ uptake was obtained. The addition of 8-bromo-cyclic AMP (1 m M ) had no effect on either DMPP- or veratridine-stimulated Na⁺ uptake. These observations suggest that D1-selective agonists are inhibiting secretagogue-stimulated Na⁺ uptake in a cyclic AMP-independent manner.     

Regulation of Anterior Pituitary D2 Dopamine Receptors by Magnesium and Sodium Ions

At D2 3,4-dihydroxyphenylethylamine (dopamine) receptors in anterior pituitary tissue, magnesium ions shifted receptors to agonist high-affinity states, but decreased the affinity of the antagonist [3H]spiperone. Conversely, sodium ions shifted the receptors to agonist low-affinity states, but increased the affinity of [3H]spiperone. Magnesium is proposed to stabilize the hormone-receptor-guanine nucleotide regulatory protein complex, whereas sodium appears to destabilize this ternary complex. Thus, magnesium and sodium appear to mediate their regulatory effects via a common component at the D2 dopamine-receptor ternary complex.     

Distinctive and Synergistic Signaling of Human Adenosine A2a and Dopamine D2L Receptors in CHO Cells

Adenosine A_2a receptor (A_2aR) colocalizes with dopamine D₂ receptor (D₂R) in the basal ganglia and modulates D₂R-mediated dopaminergic activities. A_2aR and D₂R couple to stimulatory and inhibitory G proteins, respectively. Their opposing roles in regulating neuronal activities, such as locomotion and alcohol consumption, are mediated by their opposite actions on adenylate cyclase, which often serves as “co-incidence detector” of various activators. On the other hand, the neural actions of A_2aR and D₂R are also, at least partially, independent of each other, as indicated by studies using D₂R and A_2aR knock-out mice. Here we co-expressed human A_2aR and human D_2LR in CHO cells and examined their signaling characteristics. Human A_2aR desensitized rapidly upon agonist stimulation. A_2aR activity (80%) was diminished after 2 hr of pretreatment with its agonist CGS21680. In contrast, human D_2LR activity was sustained even after 2 hr and 18 hr pretreatment with its agonist quinpirole. Long-term (18 hr) stimulation of human D_2LR also increased basal cAMP levels in CHO cells, whereas long-term (18 hr) activation of human A_2aR did not affect basal cAMP levels. Furthermore, long-term (18 hr) activation of D_2LR dramatically sensitized A_2aR-induced stimulation of adenylate cyclase in a pertussis toxin-sensitive way. Forskolin-induced cAMP accumulation was significantly increased after short-term (2 hr) human D_2LR stimulation and further elevated after long-term (18 hr) D_2LR activation. However, neither short-term (2 hr) nor long-term (18 hr) stimulation of A_2aR affected the inhibitory effects of D_2LR on adenylate cyclase. Co-stimulation of A_2aR and D_2LR could not induce desensitization or sensitization of D_2LR either. In summary, signaling t hrough A_2aR and D_2LR is distinctive and synergistic, supporting their unique and yet integrative roles in regulating neuronal functions when both receptors are present.     

Visualization of D1 Dopamine Receptors on Living Nucleus Accumbens Neurons and Their Colocalization with D2 Receptors

Abstract: To examine the substrate for dopamine (DA) synaptic action in the nucleus accumbens (nAcc), we visualized the cellular and subcellular distribution of DA receptors on postnatal nAcc neurons in culture using fluoroprobe derivatives of DA receptor ligands. Previously, we have shown that rhodamine- N -( p -aminophenethyl)-spiperone (NAPS) (10 n M ), a derivative of the D2 antagonist spiperone, labels D2-like receptors on living nAcc neurons. We now show that rhodamine-Sch-23390 (30 n M ), a derivative of the D1 antagonist, labels D1-like receptors. Putative specific membrane labeling reached a plateau after about 20 min. Labeling was stereospecific, as it was unaffected by competition with (−)-butaclamol, but blocked with (+)-butaclamol. We found that 52 ± 7% of nAcc medium-sized neurons showed D1 labeling, which extended onto the dendrites. Labeling was also seen on presynaptic terminals, often abutting D1-positive and D1-negative cell bodies, consistent with a presynaptic modulatory role for D1 receptors. Larger neurons, which may be GABAergic or cholinergic interneurons, were also labeled. By sequential labeling first with rhodamine-Sch-23390 and then rhodamine-NAPS, we found that 38 ± 6% of medium-sized neurons express both D1- and D2-like receptors, indicating that D1–D2 interactions may occur at the level of single postsynaptic neurons.     

Drug-Induced Up-Regulation of Dopamine D2 Receptors on Cultured Cells

Abstract: Ligand-induced up-regulation of recombinant dopamine D2 receptors was assessed using C₆ glioma cells stably expressing the short (415-amino-acid; D2_S) and long (444-amino-acid; D2_L) forms of the receptor. Overnight treatment of C6-D2_L cells with N-propylnorapomorphine (NPA) caused a time- and concentration-dependent increase in the density of receptors, as assessed by the binding of radioligand to membranes prepared from the cells, with no change in the affinity of the receptors for the radioligand. The effect of 10 µM NPA was maximal after 10 h, at which time the density of D2_L receptors was more than doubled. The agonists dopamine and quinpirole also increased the density of D2_L receptors. The receptor up-regulation was not specific for agonists, because the antagonists epidepride, sulpiride, and domperidone caused smaller (30–60%) increases in receptor density. Prolonged treatment with 10 µM NPA desensitized D2_L receptors, as evidenced by a reduced ability of dopamine to inhibit adenylyl cyclase, whereas treatment with sulpiride was associated with an enhanced responsiveness to dopamine. The magnitude of NPA-induced receptor up-regulation in each of four clonal lines of C6-D2_L cells (mean increase, 80%) was greater than in all four lines of C6-D2_S cells (33%). Inactivation of pertussis toxin-sensitive G proteins had no effect on the basal density of D2_L receptors or on the NPA-induced receptor up-regulation. Treatment with 5 µg/ml of cycloheximide, on the other hand, decreased the basal density of receptors and attenuated, but did not prevent, the NPA-induced increase. Chimeric D1/D2 receptors were used to identify structural determinants of dopamine receptor regulation. Treatment with the D1/D2 agonist NPA decreased the density of D1 and chimeric CH4 and CH3 receptors. The latter two receptors have D1 sequence from the amino-terminus to the amino-terminal end of transmembrane region (TM) VII and VI, respectively. CH2, with D1 sequence up to the amino-terminal end of TM V, and thus the third cytoplasmic loop of the D2 receptor, was up-regulated by NPA or the D2-selective agonist quinpirole. Quinpirole treatment decreased the density of CH3 and had no effect on CH4 or D1 receptors. The different responses of CH2 and CH3 to agonist treatment suggest a role for TM V and the third cytoplasmic loop in the direction of receptor regulation.     

Oxygen-Induced Modifications of Benzodiazepine Receptors and D2 Dopamine Receptors in the Rat Under Hyperoxia

Peripheral-type benzodiazepine Receptors (PBR) in the kidney and Central-type Benzodiazepine Receptors (CBR) in the cerebral cortex were not affected in rats exposed to chronic hyperoxia (85% O₂, ATA, 6 days). Nevertheless, cortical CBR showed a significant decrease (29%) after hyperbaric hyperoxia (100% O₂, 3.5 ATA, 2h) in rats at a preconvulsive stage, with no concomitant alteration of kidney PBR. A similar down-regulation of striatal D₂ dopamine receptors was noticed (27%) - after hyperbaric hyperoxia— without any modification of cortical PBR. On the contrary, an up regulation of liver PBR was obtained in the same conditions (20%). It is likely that receptors implicated in neurotransmission are particularly down regulated or altered under hyperbaric hyperoxia.     

Evolution of the Cholecystokinin and Gastrin Peptides and Receptors

The intestinal hormone, cholecystokinin (CCK), and the stomachhormone, gastrin, form a simple two member family of peptideswith much to offer students of hormone and receptor evolution.They share a common carboxyl-terminal tetrapeptide sequence,which is the bioactive site of each peptide and is also antigenic,making heterologous biological and immunological assays feasible.Current evidence indicates that CCK evolved in chordate ancestorsand that gastrin-like peptides that separately regulate stomachfunctions evolved from an ancestral CCK at the level of thedivergence of tetrapods from fish. This tentative conclusionmay require modification when the two separate CCK- and gastrin-likepeptides recently identified in the dogfish shark are characterizedfurther. The CCK-X receptor appears to be ancestral to the CCK-Aand CCK-B receptors identified in amniotes. The evolution ofgastrin and of CCK-A and -B receptors may have played rolesin the evolution of the stomach and the evolution of endothermyin vertebrate phylogeny.     

Regulation of Aromatic l-Amino Acid Decarboxylase by Dopamine Receptors in the Rat Brain

Decarboxylation of phenylalanine by aromatic L-amino acid decarboxylase (AADC) is the rate-limiting step in the synthesis of 2-phenylethylamine (PE), a putative modulator of dopamine transmission. Because neuroleptics increase the rate of accumulation of striatal PE, these studies were performed to determine whether this effect may be mediated by a change in AADC activity. Administration of the D1 antagonist SCH 23390 at doses of 0.01-1 mg/kg significantly increased rat striatal AADC activity in an in vitro assay (by 16-33%). Pimozide, a D2-receptor antagonist, when given at doses of 0.01-3 mg/kg, also increased AADC activity in the rat striatum (by 25-41%). In addition, pimozide at doses of 0.3 and 1 mg/kg increased AADC activity in the nucleus accumbens (by 33% and 45%) and at doses of 0.1, 0.3, and 1 mg/kg increased AADC activity in the olfactory tubercles (by 23%, 30%, and 28%, respectively). Analysis of the enzyme kinetics indicated that the Vmax increased with little change in the Km with L-3,4-dihydroxyphenylalanine as substrate. The AADC activity in the striatum showed a time-dependent response after the administration of SCH 23390 and pimozide: the activity was increased within 30 min and the increases lasted 2-4 h. Inhibition of protein synthesis by cycloheximide (10 mg/kg, 0.5 h) had no effect on the striatal AADC activity or on the increases in striatal AADC activity produced by pimozide or SCH 23390. The results indicate that the increases in AADC activity induced by dopamine-receptor blockers are not due to de novo synthesis of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular Cloning,Stable Expression and Desensitization of the Human Dopamine D1B / D5 Receptor

Abstract

The sub-family of dopamine D1-like receptors is now known to be comprised of at least two members: the originally cloned D1 receptor (herein referred to as the D1a receptor) and a related receptor referred to as the D1b, D1β or D5 dopamine receptor (herein referred to as the D1b/D5 receptor). Here, we characterize the D1b/D5 receptor expressed transiently in COS-7 cells and permanently in Ltk^? cells.

Transiently expressed human D1b/D5 receptors bind the D1 specific ligand [¹²⁵I]SCH 23982 saturably and with high affinity (K_D = 500 _pM). Competition for [¹²⁵I]SCH 23982 binding to rat D1b/D5 and human D1a and D1b/D5 receptors supports the contention that the two D1b/D5 receptors are species homologues. Furthermore, in COS-7 cells, as previously observed, dopamine competes for the binding of [¹²⁵I]SCH 23982 to human D1b/D5 receptors with a higher affinity than that seen at the human D1a receptor. These results are similar to those seen in Ltk^? cells permanently transfected with the human D1b/D5 receptor. In these cells, dopamine competition for [¹²⁵I]SCH 23982 binding is complex, sensitive to guanine nucleotides and of a higher affinity than that observed for dopamine binding to the human D1a receptor expressed in these same cells. In both D1a and D1b/D5 expressing Ltk^? cells, dopamine stimulates adenylyl cyclase with an EC₅₀ of = 200 nM. Furthermore, preincubation of Ltk^? cells expressing the D1a and D1b/D5 receptors with dopamine results in desensitization of the response of adenylyl cyclase to subsequent agonist stimulation.     

Regulation of Dopamine Receptors by Bupropion: Comparison with Antidepressants and CNS Stimulants

Abstract

Acute treatment of rats with the antidepressant bupropion increased [³H]spiperone binding to D₂ receptors in vivo. This dose- and time-dependent effect was greatest in striatum and minimal in cerebellum and pituitary. A parallel behavioral stimulation occurred in the same rats. Among 21 antidepressants and CNS stimulants tested, only those that activate dopamine (DA) transmission had similar effects: nomifensine, amineptine, methylphenidate, D-amphetamine, amfonelic acid, cocaine, benztropine and GBR 12909. Decreasing DA transmission with reserpine plus α-methyl-p-tyrosine prevented the action of bupropion. Finally, bupropion was inactive in vitro and ex-vivo. Therefore, we propose that bupropion and other DA-enhancing agents modify the characteristics of [³H]spiperone binding through the intervention of a dynamic regulation of the D₂ receptors by the neurotransmitter itself.     

Regulation and Expression of D-1, but not D-5, Dopamine Receptors in Human SK-N-MC Neuroblastoma Cells

Abstract

Human SK-N-MC neuroblastoma cells endogenously express functional D-1-like dopamine receptors. The recent identification of at least two distinct type of D-1-like receptors, D-1 and D-5, lead us to investigate the precise molecular identity of the receptors expressed in these cells. By using specific primer sets and amplification of the mRNA by RT-PCR, we show that only D-1, but not D-5, dopamine receptors are expressed in these cells. Treatment of cells with 100 μM dopamine initially caused an upregulation in D-1 mRNA expression, followed by attenuation of the message compared to control, untreated cells. The D-1 receptors in SK-N-MC cells, whose expression is controlled by dopamine in a bimodal manner, may be important in understanding how these receptors are regulated at the molecular level.     

Renal Sodium and Potassium Excretion in Diseased Cattle Estimated Without Measurement of the Urine Volume

Since measurement of urine volume can disturb the electrolyte excretion, and since such measurement is difficult to carry out on diseased cattle, a method to estimate the renal electrolyte excretion without measurement of urine volume has been tried and discussed. Depending on a constant renal excretion of creatinine, the method allows substitution of measurement of urine cratinine concentration for measurement of urine volume. The largest source of error in the use of this method is alteration in the glomerular filtration rate, and the error of the method is therefore very high in case of cardiovascular collapse (136 and 300 % was found here). The errors found were only 4–18 % in normal or slightly affected animals, and 0–44 % in animals with severe fluid and electrolyte complications without collapse. Apart from cases complicated by cardio-vascular collapse and acute uremia the method seems to be acceptable for clinical estimation of the renal electrolyte excretion.     

Exciting Times: New Advances Towards Understanding the Regulation and Roles of Kainate Receptors

Kainate receptors (KARs) are glutamate-gated ion channels that play fundamental roles in regulating neuronal excitability and network function in the brain. After being cloned in the 1990s, important progress has been made in understanding the mechanisms controlling the molecular and cellular properties of KARs, and the nature and extent of their regulation of wider neuronal activity. However, there have been significant recent advances towards understanding KAR trafficking through the secretory pathway, their precise synaptic positioning, and their roles in synaptic plasticity and disease. Here we provide an overview highlighting these new findings about the mechanisms controlling KARs and how KARs, in turn, regulate other proteins and pathways to influence synaptic function.

     

Dopaminergic Inhibition of Catecholamine Secretion from Chromaffin Cells: Evidence that Inhibition Is Mediated by D4 and D5 Dopamine Receptors

Abstract: Previous studies have suggested that activation of D2-like dopamine receptors inhibits catecholamine secretion from adrenal chromaffin cells. The purpose of this study was to determine whether the activation of D1-like receptors on chromaffin cells affects either catecholamine release from the cells or the inhibition of secretion by D2-like dopamine receptors. Both D1- and D2-selective agonists inhibited secretion elicited by dimethylphenylpiperazinium (DMPP), veratridine, and high K⁺ levels. The D1-selective agonists 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (Cl-APB) and SKF-38393 inhibited DMPP-stimulated catecholamine secretion in a concentration-dependent manner; 50% inhibition was obtained with ～10 µM Cl-APB and ～100 µM SKF-38393. Of the D2-selective agonists, bromocriptine was a more potent inhibitor of DMPP-stimulated catecholamine release than was quinpirole. The inhibition of secretion caused by Cl-APB or SKF-38393 was additive with the inhibition caused by bromocriptine. Pertussis toxin treatment (50 ng/ml, 18 h) attenuated the inhibitory effect of D2-selective, but not D1-selective, dopamine agonists. In addition, forskolin-stimulated adenylyl cyclase activity was inhibited by D2-selective, but not D1-selective, agonists. Neither D1- nor D2-selective agonists stimulated adenylyl cyclase activity in the cells, although cyclase activity was stimulated by forskolin, carbachol, and vasoactive intestinal peptide. DMPP-stimulated Ca²⁺ uptake was inhibited by both D1- and D2-selective dopamine agonists. PCR analysis was used to determine which of the dopamine receptor subtypes within the D1-like and D2-like subfamilies was responsible for the observed inhibition. PCR analysis indicated that mRNA for only D4 and D5 dopamine receptor subtypes was present in chromaffin cells. These combined data suggest that D1- and D2-selective agonists inhibit Ca²⁺ uptake and catecholamine secretion by activating D4 and D5 dopamine receptors on chromaffin cells.     

Dopamine D1 Receptors Regulate the Light Dependent Development of Retinal Synaptic Responses

Retinal synaptic connections and function are developmentally regulated. Retinal synaptic activity plays critical roles in the development of retinal synaptic circuitry. Dopamine receptors have been thought to play important roles in the activity-dependent synaptic plasticity in central nervous system. The primary goal of this study is to determine whether dopamine D1 receptor regulates the activity-dependent development of retinal light responsiveness. Accordingly, we recorded electroretinogram from wild type mice and mice with genetic deletion of D1 dopamine receptor (D1−/− mice) raised under cyclic light conditions and constant darkness. Our results demonstrated that D1−/− mice have reduced amplitudes of all three major components of electroretinogram in adulthood. When the relative strength of the responses is considered, the D1−/− mice have selective reduction of the amplitudes of a-wave and oscillatory potentials evoked by low-intermediate intensities of lights. During postnatal development, D1−/− mice have increased amplitude of b-wave at the time of eye-opening but reduced developmental increase of the amplitude of b-wave after eye opening. Light deprivation from birth significantly reduced the amplitudes of b-wave and oscillatory potentials, increased the outer retinal light response gain and altered the light response kinetics of both a- and b-waves of wild type mice. In D1−/− mice, the effect of dark rearing on the amplitude of oscillatory potentials was diminished and dark rearing induced effects on the response gain of outer retina and the kinetics of a-wave were reversed. These results demonstrated roles of dopamine D1 receptor in the activity-dependent functional development of mouse retina.     

红霉素促动力作用的易感性与多巴胺受体D3和神经肽受体Y5

通过应用核素法测定糖尿病胃瘫大鼠的胃排空 ,建立了糖尿病胃瘫大鼠的模型 ;据此模型观察了小剂量红霉素 (3mg kg)对糖尿病胃瘫大鼠液相胃排空的影响。结果显示该药能够显著减小其胃排空的一小时残留率 (R1H) [(6 3%± 6 .8% )vs.(36 %± 3.8% ) ];但此作用具有显著个体差异 ,根据所设标准分成红霉素有效组 (E组 )和无效组 (F组 )后 ,发现两组疗效具有显著差异。应用受体相关基因芯片初步筛选了这两组大鼠胃窦组织的差异表达基因。结果筛选出 10条差异表达基因 ,其中对多巴胺受体D3和神经肽Y受体Y5基因进行了RT PCR半定量验证 ,结果与芯片一致 ,即D3和Y5基因在E组中的表达水平显著高于F组 [D3受体 :(0 .2 6± 0 .0 4 )vs.(0 .16± 0 .0 4 ) ;Y5受体 :(0 .94± 0 .10 )vs .(0 .6 8± 0 .0 9) ],而与正常对照组和阴性对照组间未见明显差异。结果表明 :小剂量红霉素可以改善糖尿病胃瘫大鼠的胃排空 ,其疗效有明显的个体差异 ;红霉素有效组多巴胺受体D3(DRD3)和神经肽受体Y5 (NPYY5 )表达水平显著高于红霉素无效组。提示这两种受体可能与红霉素促动力作用的易感性相关。     

多巴胺D5受体转基因小鼠的建立

目的建立人多巴胺D5受体突变基因F173 L（D5F173L）,S390G（D5S390G）及正常人D5基因（hD5 WT）的转基因小鼠,利用该转基因动物模型来研究D5受体在原发性高血压中的发病机制。方法利用显微注射的技术将插入CMV启动子下游的D5F173L,D5S390G及hD5 WT基因的转基因载体注射入C57BL/6小鼠体内,建立多巴胺D5F173L,D5S390G及hD5 WT的转基因小鼠。通过PCR鉴定转基因小鼠的基因型。利用Western Blotting方法鉴定该受体蛋白在肾脏的表达情况。使用智能无创血压测量仪测量转基因小鼠的血压值。结果分别建立了多巴胺D5F173L,D5S390G及hD5 WT转基因C57BL/6小鼠,Western Blotting方法鉴定结果显示,与非转基因C57BL/6小鼠比较,D5转基因小鼠D5受体在肾脏有较高的表达。3-6月龄D5 F173L转基因小鼠的收缩压、舒张压和平均动脉血压均明显高于多巴胺D5S390G及hD5 WT转基因小鼠（n=6-8,P〈0.05）。结论多巴胺D5受体在原发性高血压发病中具有重要作用,但作用机理还有待于进一步研究。     

Characterisation of Inhibitory 5-Hydroxytryptamine Receptors That Modulate Dopamine Release in the Striatum

Abstract: The effect of a series of indoleamines on the potassium-evoked tritium release of previously accumulated [³H]dopamine from rat striatal slices has been investigated. The indoleamines 5-hydroxytryptamine, 5-methoxy-tryptamine, 5-methoxy- N, N' -dimethyltryptamine and tryptamine (10⁻⁷ to 10⁻³ M) all reduced potassium-evoked release of tritium, to a maximum of 50%. The uptake of [³H]dopamine was unaffected by these compounds. A series of 5-hydroxytryptamine antagonists were examined for their ability to reduce the inhibition of potassium-evoked tritium release induced by 5-methoxytryptamine. The relative order of antagonist potency obtained was methysergide > metergoline > methiothepin > cinanserin > cyproheptadine > mianserin, and was consistent with an action on 5-hydroxytryptamine receptors. It is concluded that there are inhibitory 5-hydroxytryptamine receptors located on the terminals of dopaminergic neurones in the striatum.