In Vivo Microdialysis Evidence for Transient Dopamine Release by Benzazepines in Rat Striatum

Abstract: The effects of benzazepine derivatives on extracellular levels of dopamine (DA) and l -3,4-dihydroxyphenylacetic acid (DOPAC) in the dorsal striatum of freely moving rats were studied using in vivo microdialysis. Direct injection of SKF-38393 (0.5 or 1.5 µg/0.5 µl), a selective D₁ receptor agonist, into the striatum through a cannula secured alongside a microdialysis probe produced a rapid dose-dependent transient increase in striatal DA efflux and a more gradual reduction in efflux of DOPAC. The rapid increase in DA efflux was not affected by infusion of tetrodotoxin (TTX; 2 µ M ) or Ca²⁺-free Ringer's solution and occurred after either enantiomer of SKF-38393. A TTX-insensitive increase in DA level similar to that induced by SKF-38393 was also seen after other benzazepines acting as agonists (SKF-75670 and SKF-82958, each 1.5 µg in 0.5 µl) and antagonists (SCH-23390, 1.5 µg in 0.5 µl) at the D₁ receptor and after (+)-amphetamine. These effects were inhibited by infusion of nomifensine (100 µ M ). It is concluded that the transient increases in striatal DA efflux seen after intrastriatal injection of SKF-38393 and other benzazepines are not mediated by presynaptic D₁ receptors but by an amphetamine-like action on the dopamine transporter.     

SKF38393抑制大鼠DRG分离神经元GABA-激活电流

在大鼠新鲜分离ＤＲＧ神经元标本上应用全细胞膜片箝记录,观察了多巴胺Ｄ１受体的选择性激动剂ＳＫＦ３８３９３ＨＣＩ对ＧＡＢＡ－激活电流的作用。大部分受检细胞对ＧＡＢＡ敏感,１０＾－６－１０＾－３－ｍｏｌ／Ｌ ＧＡＢＡ可于引起呈剂量依赖性的明显去敏感作用的内向电流。     

In vivo CREB phosphorylation mediated by dopamine and NMDA receptor activation in mouse hippocampus and caudate nucleus

The pattern of CREB phosphorylation was investigated in the caudate nucleus and hippocampus 10 min or 3 h after i.p. injection of dopamine or NMDA receptor agonists alone, or in combination with antagonists. Ten minutes after C57BL/6 J mice were injected with either the dopamine D1 receptor agonist SKF-38393 hydrobromide or NMDA, immunoreactivity of phosphorylated CREB (pCREB) was significantly increased in all parts of the caudate nucleus but not in hippocampal regions. However, 3 h after the injection of SKF-38393, pCREB levels in the caudate nucleus did not differ significantly from the pCREB levels in control animals, whereas pCREB levels were still elevated 3 h after NMDA injection. Except for the D1 receptor antagonist SCH-23390, which induced CREB phosphorylation in the caudate nucleus, dopamine and NMDA receptor antagonists had little effect on pCREB levels by themselves. However, the NMDA receptor antagonist CGS-19755 injected i.p. blocked both the NMDA- and SKF-38393-induced rise of pCREB levels in the caudate nucleus. Similarly, the D1 receptor antagonist SCH-23390 inhibited the effects produced by SKF-38393 or NMDA. Interestingly, the D2 receptor antagonist sulpiride also blocked the SKF-38393-triggered rise of pCREB. The results demonstrated that NMDA and dopamine receptors modulate pCREB levels in the caudate nucleus and suggest mutual permissive roles for both receptors.     

The Selective Inhibition of the D1 Dopamine Receptor Results in an Increase of Metabolized Dopamine in the Rat Striatum

Our aim was to study the specific role of the postsynaptic D(1) receptors on dopaminergic response and analyze the metabolized dopamine (DA) in the rat striatum. We used male Wistar rats to evaluate the effects of different doses of a D(1) agonist (SKF-38393) and a D(1) antagonist (SCH-23390), and their co-administration. The levels of DA and L-3, 4-dihydroxyphenylacetic acid (DOPAC) were measured using high performance liquid chromatography. The systemic injection of SKF-38393 alone at 1, 5 and 10 mg/kg did not alter the DA and DOPAC levels or the DOPAC/DA ratio. In contrast, injection of SCH-23390 alone at 0.25, 0.5 and 1 mg/kg significantly increased the DA and DOPAC levels, as well as the DOPAC/DA ratio, compared with the respective control groups. The co-administration of SCH-23390+SKF-38393 did not alter the DA or DOPAC levels, but it did significantly inhibit the SCH-23390-induced increase of the DA and DOPAC levels. The SCH-23390+SKF-38393 and the SCH-23390-only groups showed an increase in the DOPAC/DA ratio. The co-administration of SCH-23390+PARGYLINE significantly decreased the DOPAC levels and the DOPAC/DA ratio compared with the control and SCH-23390 groups. Taken together, our results showed that selective inhibition with SCH-23390 produced an increase in metabolized DA via striatal monoamine oxidase. These findings also contribute to the understanding of the role of postsynaptic D(1) receptors in the long-loop negative feedback system in the rat striatum.     

大麻素抑制大鼠三叉神经节神经元ATP激活电流

本文在培养的大鼠三叉神经节(trigeminal ganglion,TG)神经元上采用全细胞膜片钳技术,探讨大麻素对大鼠TG神经元ATP激活电流(ATP-activated currents,IATP)的影响.结果显示(1)胞外给予ATP,大部分受检细胞(67/75,89.33%)可记录到一个内向电流,且具有剂量依赖性.该电流可被P2X嘌呤受体特异性拮抗剂PPADS所阻断.(2)预加WIN55212-2[大麻素受体1(cannabinoid receptor 1,CB1受体)激动剂]可对IATP产生抑制作用,此作用呈剂量依赖性,并可被CB1受体特异性拮抗剂AM281阻断.预加不同浓度的WIN55212-2(1x10-13、1x10-12、1x10-11、1x10-10、1x10-9和1x10-8mol/L)对IATP(1x10-4mol/L ATP)的抑制作用分别为(8.14±3.14)%、(20.11±2.72)%、(46.62±3.51)%、(72.16±5.64)%、(80.21±2.80)%和(80.59±3.55)%.(3)预加WIN55212-2后IATP的浓度-反应曲线明显下移;最大反应浓度时的IATP幅值减小了(58.02±4.21)%,而阈值基本不变;预加WIN55212.2前后曲线的EC50值非常接近(1.15x10-4mol/L vs 1.27x10-4 mol/L).(4)预加forskolin[腺苷酸环化酶(adenylyl cyclase,AC)激动剂]或8-Br-cAMP可以逆转WIN55212-2对IATP的抑制作用.以上结果表明,大麻素可能作用于CB1受体,通过抑制AC-cAMP-PKA途径发挥对IATP的抑制作用.     

Involvement of G protein-coupled receptor kinase 4 and 6 in rapid desensitization of dopamine D1 receptor in rat IEC-6 intestinal epithelial cells

Dopamine-induced inhibition of Na(+)-K(+)-ATPase has been suggested to play a role in the regulation of Na(+) absorption at the intestinal level, and these effects were mediated by dopamine D(1)-like receptors. The aim of this work was to evaluate the effect of the activation of the D(1)-like receptors on the activity of the Na(+)/H(+) exchanger (NHE) in the rat intestinal epithelial cell line IEC-6. The presence of D(1) receptors was confirmed by immunoblotting. The dopamine D(1)-like receptor agonist SKF-38393 produced a concentration-dependent inhibition of NHE activity and stimulation of adenylyl cyclase (AC), this being antagonized by the D(1) selective antagonist SKF-83566. Effects of SKF-38393 on NHE and AC activities were maximal at 5 min of exposure to the agonist and rapidly diminished with no effect at 25 min. Exposure of cells for 25 min to dibutyryl-cAMP (0.5 mM) or to the AC activator forskolin (3 microM) effectively inhibited NHE activity. Pretreatment of cells with heparin (1 microM), a nonselective G protein-coupled receptor kinase (GRK) inhibitor, prevented the loss of effects on NHE activity after 25 min exposure to SKF-38393. The presence of GRK4, GRK6A, and GRK6B was confirmed by immunoblotting. Overnight treatment with the anti-GRK4-6 antibody complexed with Lipofectin was also effective in preventing loss of the effects of SKF-38393 on NHE and AC activities. It is concluded that dopamine D(1) receptors in IEC-6 rapidly desensitize to D(1)-like agonist stimulation and GRK4 and 6 appear to be involved in agonist-mediated responsiveness and desensitization.     

Dopamine Stimulates [3H]Phorbol 12,13-Dibutyrate Binding in Cultured Striatal Cells

The effect of dopamine (DA) on the binding of [3H]phorbol 12,13-dibutyrate ([3H]PdBu) in cultured rat striatal cells was examined. DA maximally increased specific [3H]PdBu binding by 70 +/- 10%, an increase comparable to that observed with norepinephrine (NE). This finding suggests that DA activates protein kinase C in cultured striatal cells, because increases in [3H]PdBu binding reflect translocation of protein kinase C. Half-maximal stimulation was observed with 10(-6) M DA. The peak response was observed at 2-3 min after addition of 10(-4) M DA, but [3H]PdBu binding was still increased above basal at 30 min. DA was not acting via an adrenergic receptor. Prazosin (10(-6) M) blocked the response to NE, suggesting mediation by an alpha 1-adrenergic receptor, but had little effect on the response to DA. Conversely, the D1 receptor antagonist SCH-23390 (10(-6) M) blocked the response to DA, but only partially inhibited the response to NE. Morphine (10(-6) M) inhibited the response to DA by 46 +/- 14%, but did not affect significantly the response to NE. The DA effect on [3H]PdBu binding is apparently independent of the increase in cyclic AMP seen on D1 receptor activation. Forskolin, apomorphine, and the D1 agonist SKF-38393 all increased cyclic AMP in striatal cells, but were less effective than DA in stimulating [3H]PdBu binding. The D2 agonist quinpirole was ineffective in stimulating either cyclic AMP or [3H]PdBu binding.     

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.     

The discriminative stimulus properties of the D-1 agonist SK&F 38393 and the D-2 agonist (-)-NPA are mediated by separate mechanisms

The dopamine D-1 agonist SK&F 38393 (10 mg/kg) the D-2 agonist (-)-NPA (0.04 mg/kg) and d-amphetamine (1.0 mg/kg) were established as discriminative stimuli versus saline in rats. The stimulus induced by SK&F 38393 was stereoselective, since the R-(+)-, but not the S-(-)-enantiomer was effective. It was mimicked by two partial D-1 agonists with central effects, SK&F 75670 and Lu 24-040, but not by the peripheral agonist fenoldopam. D-2 agonists and d-amphetamine were ineffective. The effect of SK&F 38393 was antagonized by SCH 23390, but not by its inactive enantiomer SCH 23388 or by the D-2 antagonist YM 09151-2. The (-)-NPA stimulus was dependent on postsynaptic D-2 receptors: It was mimicked by quinpirole and pergolide in stimulant dosages, whereas the partial D-2 agonist (-)-3-PPP inhibited the effect of (-)-NPA. The dopamine synthesis inhibitor alpha-methyl-p-tyrosine did not antagonize the effect of (-)-NPA. Likewise, the above-mentioned D-1 agonists produced saline responding. D-amphetamine produced partial substitution to (-)-NPA. The (-)-NPA stimulus was blocked by YM 09151-2, but not by SCH 23390. In d-amphetamine-trained rats, quinpirole, (-)-NPA and pergolide produced generalization, whereas SK&F 38393 was ineffective. Both SCH 23390 and YM 09151-2 antagonized the effect of d-amphetamine. It is concluded that the cues induced by SK&F 38393 and (-)-NPA are mediated by separate D-1 and D-2 sites, whereas both sites contribute to the effect of d-amphetamine.     

Characteristics of the brain dopamine system in mice carrying the quaking neurological mutation

Dopamine (DA) metabolism and the response to dopaminergic drugs were studied in quaking (QK) mice with neurological mutation expressed in demyelinization of the brain neurons and constant shaking. It has been shown that apomorphine in a low dose (0.25 mg/kg) produced a more significant decrease in locomotor activity in Qk than in control mice. Qk mice appeared to be less sensitive to the blockade by haloperidol of apomorphine (2.5 mg/kg)-induced climbing. DA1 receptor agonist, SKF-38393 caused less pronounced climbing in Qk mice than in the control. There were no changes in DA level in striatum and n. accumbens, whereas 3,4-dihydroxyphenylacetic acid in n. accumbens and homovanillic acid level in striatum were elevated. It was suggested that the increased DA metabolism and the altered sensitivity of pre- and postsynaptic DA receptors are involved in the shaking behaviour of Qk mice.     

Dopamine effects on identified rat vagal motoneurons

Catecholaminergic neurons of the A2 area play a prominent role in brain stem vagal circuits. It is not clear, however, whether these neurons are noradrenergic or adrenergic, i.e., display tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbetaH) immunoreactivity (-IR) or dopaminergic (i.e., TH- but not DbetaH-IR). Our aims were to investigate whether a subpopulation of neurons in the A2 area was dopaminergic and, if so, to investigate the effects of dopamine (DA) on the membrane of gastric-projecting vagal motoneurons. We observed that although the majority of A2 neurons were both TH- and DbetaH-IR, a small percentage of nucleus tractus solitarius neurons were TH-IR only, suggesting that DA itself may play role in these circuits. Whole cell recordings from thin brain stem slices showed that 71% of identified gastric-projecting motoneurons responded to DA (1-300 microM) with either an excitation (28%) or an inhibition (43%) of the membrane; the remaining 29% of the neurons were unresponsive. The DA-induced depolarization was mimicked by SK 38393 and prevented by pretreatment with SCH 23390. Conversely, the DA-induced inhibition was mimicked by bromoergocryptine and prevented by pretreatment with L741626. When tested on the same neuron, the effects of DA and NE were not always similar. In fact, in neurons in which DA induced a membrane depolarization, 77% were inhibited by NE, whereas 75% of neurons unresponsive to DA were inhibited by NE. Our data suggest that DA modulates the membrane properties of gastric-projecting motoneurons via D1- and D2-like receptors, and DA may play different roles than norepinephrine in brain stem vagal circuits.     

Involvement of D1 receptors in depression-like behavior of ovariectomized rats

The aim of the present study was to explore the mood effects of D1 receptor agonist, SKF-38393 and D1 receptor antagonist, SCH-23390 alone or in combination with a low dose of 17β-estradiol (17β-E2) in the adult ovariectomized female rats (OVX). OVX rats of Wistar strain were used in all experiments. Two weeks after surgery rats were chronically treated with vehicle, a low dose of 17β-E2 (5.0 μg/rat), SKF-38393 (0.1 mg/kg), SCH-23390 (0.1 mg/kg), SKF-38393 plus 17β-E2 or SCH-23390 plus 17β-E2 for 14 days before the forced swimming test. We found that SCH-23390 significantly decreased immobility time in the OVX females. A combination of SCH-23390 with a low dose of 17β-E2 induced more profound decrease of immobility time in the OVX rats compared to the rats treated with SCH-23390 alone. On the contrary, SKF-38393 failed to modify depression-like behavior in the OVX rats. In addition, SKF-38393 significantly blocked the antidepressant-like effect of 17β-E2 in OVX rats. Thus, the D1 receptor antagonist SCH-23390 alone or in combination with a low dose of 17β-E2 exerted antidepressant-like effect in OVX female rats, while the D1 receptor agonist SKF-38393 produced depressant-like profile on OVX rats.     

The effect of the deprivation of paradoxical sleep on the rotational and stereotyped behavior induced by selective agonists of the dopamine receptors

The ability of selective D1 and D2 agonists of dopamine (DA) receptors SKE-38393 and Ly-171555 to induce rotational and stereotypes behaviour were studied in rats with unilateral striatal kainic acid lesion before and after procedure of REM sleep deprivation (REMSD) lasting for 5 days. It was found that REMSD SKF-38393 given along induced the ipsilateral rotation. REMSD increased the circling and decreased an oral stereotype simultaneously when SKF-38393 and Ly-171555 were given together. The depletion of brain DA in part with alpha-methyl-p-tyrosine (AMPT) in nondeprived rats fails to influence the rotational behaviour and stereotype when SKF 38393 was injected 30 min following Ly-171555. After REMSD the AMPT pretreatment prevents the rotational behaviour caused by Ly-171555 which restores SKF-38393. The results suggest that prolonged REMSD may induce nonidentical changes in the sensitivity of the postsynaptic D1 and D2 receptors.     

NMDA Receptor Involvement in Toxicity to Dopamine Neurons In Vitro Caused by the Succinate Dehydrogenase Inhibitor 3-Nitropropionic Acid

Abstract: Exposure of mesencephalic dopamine neurons to an irreversible inhibitor of succinate dehydrogenase (SDH), 3-nitropropionic acid (3-NPA), for 24 h on day 12 in vitro, produced a dose-dependent loss of high-affinity dopamine uptake when measured 48 h following 3-NPA removal. ATP concentrations in the cultures were reduced by 57% after 3 h of treatment with the highest concentration of 3-NPA tested (500 µ M ). To determine whether glutamate receptors mediated the dopamine toxicity by 3-NPA, cultures were examined for their sensitivity to excitatory amino acid-induced toxicity. Mesencephalic cultures exposed to either 100 µ M NMDA or kainate, on day 12 for 24 h, showed complete loss of dopamine uptake following 48 h of recovery. The NMDA and non-NMDA antagonists, MK-801 (1 µ M ) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 15 µ M ), completely prevented the effects of NMDA or kainate, respectively, when present at the time of toxin exposure. In cultures treated with 3-NPA, MK-801, but not CNQX, significantly attenuated the loss of dopamine uptake. Direct measurement of the effect of 3-NPA on SDH activity showed that 3-NPA dose-dependently inhibited SDH in vitro in a manner commensurate with the loss of dopamine uptake by 3-NPA. MK-801 had no effect on basal SDH activity or on 3-NPA inhibition of SDH. These data are consistent with the interpretation that metabolic inhibition in dopamine neurons can trigger a secondary excitotoxicity that is mediated by NMDA receptors.     

Subunit-specific modulation of [(3)H]MK-801 binding to NMDA receptors mediated by dopamine receptor ligands in rodent brain

Dopamine D1 receptor (D1R) ligands may directly interact with the NMDA receptor (NMDAR), but detailed knowledge about this effect is lacking. Here we identify D1R ligands that directly modulate NMDARs and examine the contributions of NR2A and NR2B subunits to these interactions. Binding of the open channel blocker [(3)H]MK-801 in membrane preparations from rat- and mouse brain was used as a biochemical measure of the functional state of the NMDAR channel. We show that both D1R agonist A-68930 and dopamine receptor D2 antagonist haloperidol can decrease [(3)H]MK-801 binding with increased potency in membranes from the NR2A(-/-) mice (i.e. in membranes containing NR2B only), as compared to the inhibition obtained in wild-type membranes. Further, a wide range of D1R agonists such as A-68930, SKF-83959, SKF-83822, SKF-38393 and dihydrexidine were able to decrease [(3)H]MK-801 binding, all showing half maximal inhibitory concentrations ~20 μM, and with significant effects occurring at or above 1 μM. With membranes from D1R(-/-) mice, we demonstrate that these effects occurred through a D1R-independent mechanism. Our results demonstrate that dopamine receptor ligands can selectively influence NR2B containing NMDARs, and we characterize direct inhibitory NMDAR effects by different D1R ligands.     

The effect and mechanism of dopamine D1 receptors on the proliferation of osteosarcoma cells

The physiological and pathological roles of dopamine D1 receptors (DR1) in the regulation of functions in tissues and organs have been recognized. However, whether DR1 are expressed in the osteosarcoma cells and inhibit the proliferation of these cells is unknown. In the present study, we found that DR1 were expressed in the osteosarcoma cells (OS732 cells). SKF-38393 (DR1 agonist) and the overexpression of DR1 decreased the proliferation of OS732 cells; SCH-23390 (DR1 antagonist) and the knockdown of DR1 increased the proliferation of OS732 cells, and both SCH-23390 and the knockdown of DR1 abolished the effect of SKF-38393 on the proliferation of OS732 cells. In addition, SKF-38393 down-regulated the phosphorylation of ERK1/2, PI3K, and Akt; SCH-23390 up-regulated the phosphorylation of ERK1/2, PI3K, and Akt, and SCH-23390 cancelled the effect of SKF-38393. The effect of SKF-38393 on the phosphorylation of ERK1/2, PI3K, and Akt and the proliferation of OS732 cells was similar to PD98059 (an ERK inhibitor) or LY294002 (a PI3K inhibitor), respectively. In conclusion, our results suggest that DR1 are expressed in the osteosarcoma cells and inhibit the proliferation of osteosarcoma cells by the down-regulation of the ERK1/2 and PI3K-Akt pathways. These findings provide a novel target for the treatment of the osteosarcoma.     

Neurotensin triggers dopamine D2 receptor desensitization through a protein kinase C and beta-arrestin1-dependent mechanism

The peptide neurotensin (NT) is known to exert a potent excitatory effect on the dopaminergic system by inhibiting D2 dopamine (DA) receptor (D2R) function. This regulation is dependent on activation of PKC, a well known effector of the type 1 NT receptor (NTR1). Because PKC phosphorylation of the D2R has recently been shown to induce its internalization, we hypothesized that NT acts to reduce D2R function through heterologous desensitization of the D2R. In the present study, we first used HEK-293 cells to demonstrate that NT induces PKC-dependent D2R internalization. Furthermore, internalization displayed faster kinetics in cells expressing the D2R short isoform, known to act as an autoreceptor in DA neurons, than in cells expressing the long isoform, known to act as a postsynaptic D2R. In patch clamp experiments on cultured DA neurons, overexpression of a mutant D2S lacking three key PKC phosphorylation sites abrogated the ability of NT to reduce D2R-mediated cell firing inhibition. Short interfering RNA-mediated inhibition of β-arrestin1 and dynamin2, proteins important for receptor desensitization, reduced agonist-induced desensitization of D2R function, but only the inhibition of β-arrestin1 reduced the effect of NT on D2R function. Taken together, our data suggest that NT acutely regulates D2 autoreceptor function and DA neuron excitability through PKC-mediated phosphorylation of the D2R, leading to heterologous receptor desensitization.     

Dopamine Receptors Modulate Cytotoxicity of Natural Killer Cells via cAMP-PKA-CREB Signaling Pathway

Dopamine (DA), a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK) cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R) with agonist {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R) with agonist quinpirole attenuated NK cells. Simultaneously, {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB) level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 were blocked by {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA), prevented the {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC), counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The results may provide more targets of therapeutic strategy for neuroimmune diseases.     

左旋千金藤啶碱D1激动—D2阻滞作用引起伏核双相放电活动的电生理研究

为确定左旋千金藤啶碱（ＳＰＤ）对中脑边缘ＤＡ神经系统的作用特性,本研究采用细胞外记录的电生理学方法,观察微电泳和尾静脉给药对６－ＯＨＤＡ损毁及未损毁大鼠的伏核（ＮＡｃ）单位放电的影响。结果显示：ＳＰＤ累积给药（０．０２－２ｍｇ／ｋｇ,ｉｖ）可诱发ＮＡｃ神经元双相放电特征,即小剂量抑制、大剂量兴奋。预先给予Ｄ２受体拮抗剂ｓｐｅｐｅｒｏｎｅ,ＳＰＤ则仅产生兴奋效应,并被Ｄ１拮抗剂ＳＣＨ－２３３９０所翻     

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.