Atypical SCH23390 Binding Sites Are Present on Bovine Adrenal Medullary Membranes

D1-selective dopamine receptor agonists inhibit secretagogue-stimulated catecholamine secretion from bovine adrenal chromaffin cells. The purpose of the studies reported here was to use the radiolabeled D1-selective dopamine receptor antagonist, SCH23390, to characterize putative D1-like dopamine receptors responsible for this effect. Characterization of SCH23390 binding sites demonstrated an unusual pharmacological profile inconsistent with classical D1-like receptors. [¹²⁵I]SCH23390 bound to adrenal medullary membranes was competed for by non-radioactive iodo-SCH23390 (Kd = 490 ± 50 nM), but not by (+)butaclamol. Other classical D1 antagonists had little, if any, effect. Competition with dopamine receptor agonists demonstrated a relative rank order of potency profile characteristic of D1-like dopamine receptors, however, K_is were higher than those found in other tissues. The K_is for competition of [¹²⁵I]SCH23390 binding by C1-APB and SKF38393 (16 and 118 M, respectively) are nearly identical to the IC₅₀s previously observed for inhibition of secretion (9 and 100 M, respectively). Combined these data suggest that adrenal medullary membranes contain a novel SCH23390 binding site involved in the inhibition of secretion by D1-selective agonists.     

Specific [3H]SCH23390 Binding to Dopamine D1 Receptors in Cerebral Cortex and Neostriatum: Role of Disulfide and Sulfhydryl Groups

Receptor binding studies were performed in cerebral cortex (CTX) and neostriatum (CPU; caudate-putamen) using the dopamine D1 antagonist [3H]SCH23390. Because receptors are of protein nature, we examined the role of disulfide bonds (--SS--) and sulfhydryl groups (--SH) in the specific binding of [3H]SCH23390. Furthermore, membrane preparations contain a certain amount of lipid, so that treatments with --SH and --SS-- reagents could determine whether the fixation of the radioligand was to protein or to the lipid moiety. Pretreatment of CTX and CPU membranes with dithioerythritol, L-dithiothreitol, or 5,5'-dithiobis(2-nitrobenzoic acid), as well as with the alkylating agent N-ethylmaleimide, produced dose-dependent decreases of specific [3H]SCH23390 binding in membrane preparations from both tissues. These changes were not reversible after up to two washes, but could be prevented in part if the treatments were performed in the presence of dopamine. Additional protection experiments were conducted with (+)- and (-)-butaclamol, as well as with (+)- and (-)-SKF38393. A series of saturation experiments (with pretreated membranes in the absence of reactives) demonstrated that the alkylation of --SS-- groups reduced specific [3H]SCH23390 binding mainly through an affinity change, but L-dithiothreitol and 5,5-dithiobis(2-nitrobenzoic acid) decreased the number of binding sites. The affinity of the receptor to agonists was examined with the two enantiomers of SKF38393; the inhibition curves showed that residual binding was not affected and stereospecificity was conserved. The present results provide evidence for the participation of both --SS-- and --SH groups in the recognition site of the dopamine D1 receptor in both the CTX and the CPU.     

Dependence of the coupling of dopamine receptors to G proteins on the protein redox state in the neural plasma membranes of pond snail

Binding analysis using [3H]dopamine has shown that reduction of protein thiol groups with dithiothreitol (DTT) led to a dual effect on the receptors. First, the amount of dopamine-binding sites on the membranes and their affinity to the ligand were decreased. Second, the affinity of the receptors to [3H]dopamine was enhanced in the presence of GDP. Binding of D(1) antagonist [3H]SCH23390 to dopamine receptors increased following DTT treatment, opposite to the case with D(1) agonist [3H]SKF38393. The displacement of [3H]GDP by GTPgammaS was depressed by dopamine. Stimulation of [3H]GDP binding by dopamine was potentiated after incubation with DTT. Membrane nitrosylation eliminated the reciprocal dependence of GDP and dopamine binding to the membranes. It is suggested that binding of dopamine to the receptors can lead to both stimulation and inhibition of G protein activity, and the ratio of these effects depends on the reduction and oxidation of sulfhydryl groups of membrane proteins. Thiol reduction potentiated inhibitory action of dopamine receptors on coupled G proteins, and nitrosylation led to their uncoupling.     

Binding of [3H]SCH23390 in Rat Brain: Regional Distribution and Effects of Assay Conditions and GTP Suggest Interactions at a D1-Like Dopamine Receptor

The compound [9-3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7- ol] was synthesized, and the binding of this purportedly selective antagonist of D1 3,4-dihydroxyphenylethylamine (dopamine) receptors was characterized. The regional distribution of high-affinity, specific [3H]SCH23390 binding sites in the rat brain correlated well with levels of endogenous dopamine. Receptor densities were greatest in corpus striatum, nucleus accumbens, and olfactory tubercle; intermediate levels were found in several limbic and cortical areas, whereas few sites were detectable in cerebellum, brainstem, and ol-factory bulb. Specific binding in caudate-putamen was found to be both temperature- and pH-dependent, with optima at 25-30 degrees C and pH 7.8-8.0. Scatchard or Woolf analyses of binding in caudate-putamen suggest that most of the sites are either of a single class or of classes with similar characteristics (KD = 0.7 +/- 0.1 nM; Bmax = 347 +/- 35 fmol/mg of protein). Both dopamine and cis-flupenthixol altered the slope but not the intercept of lines generated by Scatchard analysis, suggesting a competitive mode of inhibition of [3H]SCH23390 binding. Competition for binding by dopamine or the D1 agonist SKF38393 was inhibited by guanine nucleotides, whereas GTP had little effect on the competition for binding by the antagonist cis-flupenthixol. The competition for [3H]SCH23390 binding sites by dopamine was much more sensitive to GTP than was competition for [3H]spiperone binding. These data support the hypotheses that [3H]SCH23390 binds to recognition sites that differ from those previously described using other radiolabeled dopamine antagonists and that these sites have the characteristics expected of dopamine receptors.     

Binding of [3H]SKF 38393 to Dopamine D-l Receptors in Rat Striatum In Vitro; Estimation of Receptor Molecular Size by Radiation Inactivation

[3H]SKF 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine) binds with high affinity to 3,4-dihydroxyphenylethylamine (dopamine) D-1 receptors in rat striatum in vitro (KD = 7 and 14 nM in nonfrozen and frozen striatum, respectively). The number of binding sites (Bmax) was approximately 80.0 pmol/g of original tissue, a value similar to the Bmax for the dopamine D-1 antagonist SCH 23390. Nondisplaceable [3H]SKF 38393 binding was approximately 45% of total binding. Irradiation (0-4 Mrad) of frozen whole striata decreased the number of [3H]SKF 38393 binding sites monoexponentially without changing the binding affinity. The functional molecular mass for the agonist dopamine D-1 binding site was 132,800 daltons, which is higher than the functional molecular mass of the antagonist dopamine D-1 binding site (approximately 80,000 daltons).     

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.     

Identification and characterization of functional D1 dopamine receptors in a human neuroblastoma cell line

Dopamine stimulated human neuroblastoma SK-N-MC cells to accumulated cyclic AMP. The D1 agonist SKF (R)-38393 also stimulated cyclic AMP production whereas the response to dopamine was inhibited by the D1 antagonist SCH (R)-23390. Membranes from SK-N-MC cells bound the D1 ligand [125I]SCH 23982 with a Kd of 2.1 nM and a Bmax of 102 fmol/mg protein. Binding was displaced by dopamine, SKF 38393, and SCH 23390. Up to 40% of the receptors were in an agonist high affinity, guanine nucleotide-sensitive state, compared to only 6% in rat striatum. A D1 photoaffinity probe labeled a 72 kDa protein in both SK-N-MC and rat striatal membranes. Thus, SK-N-MC human neuroblastoma cells contain D1 dopamine receptors which are similar to those found in mammalian striatum, but which are more tightly coupled to adenylate cyclase. SK-N-MC cells may be a useful model to investigate the properties and regulation of D1 dopamine receptors.     

D1 and D2 Dopamine Receptors in Caudate-Putamen of Nonhuman Primates (Macaca fascicularis)

D1 and D2 dopamine receptors were characterized in the caudate-putamen region of nonhuman primate brains (Macaca fascicularis). D1 dopamine receptors were identified with [3H]SCH 23390 and D2 receptors with [3H]-spiperone. Scatchard analysis of [3H]SCH 23390 saturation data using washed membranes revealed a single high-affinity binding site (KD, 0.352 +/- 0.027 nM) with a density (Bmax) of 35.7 +/- 2.68 pmol/g original wet tissue weight (n = 10). The affinity of [3H]spiperone for the D2 site was 0.039 +/- 0.007 nM and the density was 25.7 +/- 1.97 pmol/g original wet tissue weight (n = 10). D1 and D2 receptors in nonhuman primates may be differentiated on the basis of drug affinities and stereoselectivity. In competition experiments, RS-SKF 38393 was the most selective D1 agonist, whereas (+)-4-propyl-9-hydroxynaphthoxazine [(+)-PHNO] was the most selective D2 agonist. Apomorphine was essentially nonselective for D1 or D2 binding sites. Of the antagonists, R-SKF 83566 and SCH 23390 were the most selective for the D1 site, whereas YM-09151-2 was the most selective for the D2 site. cis-Flupentixol and (S)-butaclamol were the least selective dopamine antagonists. D1 receptors bound benzazepine antagonists (SCH 23390/SCH 23388, R-SKF 83692/RS-SKF 83692) stereoselectively whereas D2 receptors did not. Conversely D2 receptors bound (S)-sulpiride and (+)-PHNO more potently than their enantiomers whereas D1 receptors showed little stereoselectively for each of these isomeric pairs. These binding characteristics may be utilized for evaluation of individual receptor function in vivo.     

The alpha1-adrenergic receptor not the DA(1)-dopaminergic receptor mediates cyclosporine-SKF38393 renovascular interaction

In this study, we investigated the effect of acute exposure to cyclosporine A (CyA) on renal vasodilations evoked by the DA(1) dopaminergic agonist SKF38393 and whether dopamine DA(1) receptors are directly involved in the interaction. Changes evoked by CyA in SKF38393 vasodilations were evaluated in phenylephrine-preconstricted isolated perfused rat kidneys in the absence and presence of SCH23390, a DA(1) receptor antagonist. SKF38393 (3 x 10(-8) to 3 x 10(-6) mol) produced dose-dependent reductions in the renal perfusion pressure that were significantly attenuated in tissues pretreated with SCH23390 or CyA. Unlike SKF38393, the vasodilatory action of sodium nitroprusside, a nitrovasodilator, was not altered by CyA. The attenuating effect of CyA on SKF38393 vasodilations was preserved in preparations pretreated with SCH23390, suggesting that sites other than DA(1) receptors may be involved in CyA-SKF38393 interaction. The study was then extended to investigate the possible involvement of renal alpha1-adrenoceptors in the interaction. Blockade of alpha(1)-adrenoceptors by prazosin (30 nmol/L) significantly reduced the vasodilatory effect of SKF38393 and virtually abolished the CyA-induced attenuation of SKF38393 responses. Further, CyA failed to alter SKF38393 vasodilations when the renal tone was raised with prostaglandin F2alpha (PGF2alpha), a vasoconstrictor whose effect is independent of alpha(1)-adenoceptors. Together, these findings support earlier reports that both DA(1) and alpha(1)-receptors mediate the renal vasodilatory action of SKF38393 and suggest that CyA interacts selectively with the alpha(1)-receptor component to compromise SKF38393 responses.     

Lack or Inhibition of Dopaminergic Stimulation Induces a Development Increase of Striatal Tyrosine Hydroxylase-Positive Interneurons

We examined the role of endogenous dopamine (DA) in regulating the number of intrinsic tyrosine hydroxylase-positive (TH⁺) striatal neurons using mice at postnatal day (PND) 4 to 8, a period that corresponds to the developmental peak in the number of these neurons. We adopted the strategy of depleting endogenous DA by a 2-day treatment with α-methyl-p-tyrosine (αMpT, 150 mg/kg, i.p.). This treatment markedly increased the number of striatal TH⁺ neurons, assessed by stereological counting, and the increase was highly correlated to the extent of DA loss. Interestingly, TH⁺ neurons were found closer to the clusters of DA fibers after DA depletion, indicating that the concentration gradient of extracellular DA critically regulates the distribution of striatal TH⁺ neurons. A single i.p. injection of the D1 receptor antagonist, {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 (0.1 mg/kg), the D2/D3 receptor antagonist, raclopride (0.1 mg/kg), or the D4 receptor antagonist, L-745,870 (5 mg/kg) in mice at PND4 also increased the number of TH⁺ neurons after 4 days. Treatment with the D1-like receptor agonist {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 (10 mg/kg) or with the D2-like receptor agonist, quinpirole (1 mg/kg) did not change the number of TH⁺ neurons. At least the effects of {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 were prevented by a combined treatment with {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393. Immunohistochemical analysis indicated that striatal TH⁺ neurons expressed D2 and D4 receptors, but not D1 receptors. Moreover, treatment with the α4β2 receptor antagonist dihydro-β-erythroidine (DHβE) (3.2 mg/kg) also increased the number of TH⁺ neurons. The evidence that DHβE mimicked the action of {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 in increasing the number of TH⁺ neurons supports the hypothesis that activation of D1 receptors controls the number of striatal TH⁺ neurons by enhancing the release of acetylcholine. These data demonstrate for the first time that endogenous DA negatively regulates the number of striatal TH⁺ neurons by direct and indirect mechanisms mediated by multiple DA receptor subtypes.     

Dopamine D1 receptors characterized with [3H]SCH 23390. Solubilization of a guanine nucleotide-sensitive form of the receptor

Dopamine D1 receptors were solubilized from canine and bovine striatal membranes with the detergent digitonin. The receptors retained the pharmacological characteristics of membrane-bound D1 receptors, as assessed by the binding of the selective antagonist [3H]SCH 23390. The binding of [3H]SCH 23390 to solubilized receptor preparations was specific, saturable, and reversible, with a dissociation constant of 5 nM. Dopaminergic antagonists and agonists inhibited [3H]SCH 23390 binding in a stereoselective and concentration-dependent manner with an appropriate rank order of potency for D1 receptors. Moreover, agonist high affinity binding to D1 receptors and its sensitivity to guanine nucleotides was preserved following solubilization, with agonist dissociation constants virtually identical to those observed with membrane-bound receptors. To ascertain the molecular basis for the existence of an agonist-high affinity receptor complex, D1 receptors labeled with [3H] dopamine (agonist) or [3H]SCH 23390 (antagonist) prior to, or following, solubilization were subjected to high pressure liquid steric-exclusion chromatography. All agonist- and antagonist-labeled receptor species elute as the same apparent molecular size. Treatment of brain membranes with the guanine nucleotide guanyl-5'-yl imidodiphosphate prior to solubilization prevented the retention of [3H]dopamine but not [3H]SCH 23390-labeled soluble receptors. This suggests that the same guanine nucleotide-dopamine D1 receptor complex formed in membranes is stable to solubilization and confers agonist high affinity binding in soluble preparations. These results contrast with those reported on the digitonin-solubilized dopamine D2 receptor, and the molecular mechanism responsible for this difference remains to be elucidated.     

Regulation of DARPP-32 phosphorylation by three distinct dopamine D1-like receptor signaling pathways in the neostriatum

Dopamine D(1)-like receptors play a key role in dopaminergic signaling. In addition to G(s/olf)/adenylyl cyclase (AC)-coupled D(1) receptors, the presence of D(1)-like receptors coupled to G(q)/phospholipase C (PLC) has been proposed. Benzazepine D(1) receptor agonists are known to differentially activate G(s/olf)/AC and G(q)/PLC signaling. By utilizing SKF83959 and SKF83822, we investigated the D(1)-like receptor signaling cascades, which regulate DARPP-32 phosphorylation at Thr34 (the PKA-site) in mouse neostriatal slices. Treatment with SKF83959 or SKF83822 increased DARPP-32 phosphorylation. The SKF83959- and SKF83822-induced increase in DARPP-32 phosphorylation was largely, but partially, antagonized by a D(1) receptor antagonist, SCH23390, and the residual SCH23390-insensitive increase was abolished by an adenosine A(2A) receptor antagonist. In addition, the SKF83959-induced, SCH23390-sensitive increase in DARPP-32 phosphorylation was enhanced by a PLC inhibitor. Analysis in slices from D(1)R/D(2)R-DARPP-32 mice revealed that both D(1) receptor agonists regulate DARPP-32 phosphorylation in striatonigral, but not in striatopallidal, neurons. Thus, dopamine D(1)-like receptors are coupled to three signaling cascades in striatonigral neurons: (i) SCH23390-sensitive G(s/olf)/AC/PKA, (ii) adenosine A(2A) receptor-dependent G(s/olf)/AC/PKA, and (iii) G(q)/PLC signaling. Interestingly, G(q)/PLC signaling interacts with SCH23390-sensitive G(s/olf)/AC/PKA signaling, resulting in its inhibition. Three signaling cascades activated by D(1)-like receptors likely play a distinct role in dopaminergic regulation of psychomotor functions.     

In Situ Molecular Size of Agonist Dopamine D-2 Binding Sites in Rat Striatum

Specific binding of [3H]N-propylnorapomorphine [( 3H]NPA) to 3,4-dihydroxyphenylethylamine (dopamine) D-2 receptors was investigated in rat striatum in vitro. For various dopamine receptor substances, the rank order of potency to inhibit [3H]NPA binding was spiroperidol greater than or equal to NPA greater than LY 171555 greater than SCH 23390 greater than SKF 38393. A single high-affinity binding site was found in membranes prepared in either Tris-citrate buffer or imidazole buffer; the affinity constants were 0.11 and 0.76 nM, respectively. The number of receptors (33 pmol/g wet weight) was independent of whether the membranes were prepared in Tris-citrate buffer or imidazole buffer and was similar to the number of receptors estimated by [3H]spiroperidol binding to dopamine receptors. Irradiation inactivation of frozen whole rat striata showed a monoexponential loss of [3H]NPA binding sites without a change in the binding affinity. The target size of the [3H]NPA binding site was 81,000 daltons, which shows that the functional molecular entity to bind the dopamine D-2 agonist was smaller than the molecular entity to bind the dopamine D-2 antagonist [3H]spiroperidol (target size, 137,000 daltons).     

Chronic stimulation of D1 dopamine receptors in human SK-N-MC neuroblastoma cells induces nitric-oxide synthase activation and cytotoxicity

Elevated synaptic levels of dopamine may induce striatal neurodegeneration in l-DOPA-unresponsive parkinsonism subtype of multiple system atrophy (MSA-P subtype), multiple system atrophy, and methamphetamine addiction. We examined the participation of dopamine and D1 dopamine receptors in the genesis of postsynaptic neurodegeneration. Chronic treatment of human SK-N-MC neuroblastoma cells with dopamine or H2O2 increased NO production and accelerated cytotoxicity, as indexed by enhanced nitrite levels and cell death. The antioxidant sodium metabisulfite or SCH 23390, a D1 dopamine receptor-selective antagonist, partially blocked dopamine effects but together ablated dopamine-mediated cytotoxicity, indicating the participation of both autoxidation and D1 receptor stimulation. Direct activation of D1 dopamine receptors with SKF R-38393 caused cytotoxicity, which was refractory to sodium metabisulfite. Dopamine and SKF R-38393 induced overexpression of the nitric-oxide synthase (NOS) isoforms neuronal NOS, inducible NOS (iNOS), and endothelial NOS in a protein kinase A-dependent manner. Functional studies showed that approximately 60% of total NOS activity was due to activation of iNOS. The NOS inhibitor N(G)-nitro-l-arginine methyl ester and genistein, wortmannin, or NF-kappaB SN50, inhibitors of protein tyrosine kinases phosphatidylinositol 3-kinase and NF-kappaB, respectively, reduced nitrite production by dopamine and SKF R-38393 but were less effective in attenuating H2O2-mediated effects. In rat striatal neurons, dopamine and SKF R-38393, but not H2O2, accelerated cell death through increased expression of neuronal NOS and iNOS but not endothelial NOS. These data demonstrate a novel pathway of dopamine-mediated postsynaptic oxidative stress and cell death through direct activation of NOS enzymes by D1 dopamine receptors and its associated signaling pathways.     

Aromatic L-Amino Acid Decarboxylase Is Modulated by D1 Dopamine Receptors in Rat Retina

Aromatic L-amino acid decarboxylase (AAAD) activity of rat retina increases when animals are placed in a lighted environment from the dark. The increase of activity can be inhibited by administering the selective dopamine D1 receptor agonist SKF 38393, but not the selective D2 agonist quinpirole, or apomorphine. Conversely, in the dark, enzyme activity can be enhanced by administering the selective D1 antagonist SCH 23390 or haloperidol, but not the selective D2 antagonist (-)-sulpiride. Furthermore, in animals exposed to room light for 3 h, the D1 agonist SKF 38393 reduced retinal AAAD activity, and this effect was prevented by prior administration of SCH 23390. In contrast, quinpirole had little or no effect when administered to animals in the light. Kinetic analysis indicated that the apparent Vmax for the enzyme increases with little change in the apparent Km for the substrate 3,4-dihydroxyphenylalanine or the cofactor pyridoxal-5'-phosphate. We suggest that dopamine released in the dark tonically occupies D1 receptors and suppresses AAAD activity. When the room light is turned on, D1 receptors are vacated and selective D1 agonists can either prevent the rise of AAAD or reverse light-enhanced AAAD activity.     

Hydrogen peroxide causes uncoupling of dopamine D1-like receptors from G proteins via a mechanism involving protein kinase C and G-protein-coupled receptor kinase 2

Dopamine, via activation of D1-like receptors, inhibits Na,K-ATPase and Na,H-exchanger in renal proximal tubules and promotes sodium excretion. This effect of dopamine is not seen in conditions associated with oxidative stress such as hypertension, diabetes, and aging due to uncoupling of D1-like receptors from G proteins. To identify the role of oxidative stress in uncoupling of the D1-like receptors, we utilized primary cultures from rat renal proximal tubules. Hydrogen peroxide (H2O2), an oxidant, treatment to the cell cultures increased the level of malondialdehyde, a marker of oxidative damage. Further, H2O2 decreased membranous D1-like receptor numbers and proteins, D1-like agonist (SKF 38393)-mediated [35S]GTPgammaS binding and SKF 38393-mediated inhibition of Na,K-ATPase. Moreover, H2O2 treatment to the cultures caused membranous translocation of G-protein-coupled receptor kinase 2 (GRK 2) and increased serine phosphorylation of D1A receptors accompanied by an increase in protein kinase C (PKC) activity. Interestingly, PKC inhibitors blocked the H2O2-mediated stimulation of GRK 2 and serine phosphorylation of D1A receptors. Further, GRK 2 antisense but not scrambled oligonucleotides attenuated the effect of H2O2 on membranous expression of GRK 2. Moreover, direct activation of PKC with phorbol ester (PMA) resulted in reduction of SKF 38393-mediated [35S]GTPgammaS binding. We conclude that H2O2 stimulates PKC leading to the activation of GRK 2, which causes serine phopshorylation of D1A receptors and receptor G-protein uncoupling in these cells, resulting in impairment in D1-like receptor function.     

Pharmacological characterization of mammalian D1 and D2 dopamine receptors expressed in Drosophila Schneider-2 cells

Mammalian D1 and D2 dopamine receptors were stably expressed in Drosophila Schneider-2 (S2) cells and screened for their pharmacological properties. Saturable, dose-dependent, high affinity binding of the D1-selective antagonist [3H]SCH-23390 was detected only in membranes from S2 cells induced to express rat dopamine D1 receptors, while saturable, dose-dependent, high affinity binding of the D2-selective antagonist [3H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D2 receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D1 and D2 receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D1 receptor bound the (+)-enantiomer of the D1-selective agonist SKF38393 with higher affinity than the (-)-enantiomer, while the dopamine D2 receptor bound the (-)-enantiomer of the D2-selective agonist norpropylapomorphine with higher affinity than the (+)-enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPgammaS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D1 and D2 receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D1 and D2 dopamine receptors free of promiscuous G protein contaminants.     

D1-D2 dopamine receptor interaction within the nucleus accumbens mediates long-loop negative feedback to the ventral tegmental area (VTA)

The objective of the present study was to examine the effects of perfusion of dopamine (DA) D1- and D2-like receptor agonists in the nucleus accumbens (ACB) on the long-loop negative feedback regulation of mesolimbic somatodendritic DA release in the ventral tegmental area (VTA) of Wistar rats employing ipsilateral dual probe in vivo microdialysis. Perfusion of the ACB for 60 min with the D1-like receptor agonist SKF 38393 (SKF, 1-100 microM) dose-dependently reduced the extracellular levels of DA in the ACB, whereas the extracellular levels of DA in the VTA were not changed. Similarly, application of the D2-like receptor agonist quinpirole (Quin, 1-100 microM) through the microdialysis probe in the ACB reduced the extracellular levels of DA in the ACB in a concentration-dependent manner, whereas extracellular levels of DA in the VTA were not altered. Co-application of SKF (100 microM) and Quin (100 microM) produced concomitant reductions in the extracellular levels of DA in the ACB and VTA. The reduction in extracellular levels of DA in the ACB and VTA produced by co-infusion of SKF and Quin was reversed in the presence of either 100 microM SCH 23390 (D1-like antagonist) or 100 microM sulpiride (D2-like antagonist). Overall, the results suggest that (a) activation of dopamine D1- or D2-like receptors can independently regulate local terminal DA release in the ACB, whereas stimulation of both subtypes is required for activation of the negative feedback pathway to the VTA.     

Physicochemical modulation of agonist-induced [35s]GTPgammaS binding: implications for coexistence of multiple functional conformations of dopamine D1-like receptors

Dopamine agonist-stimulated [35S]GTPgammaS binding to membrane G proteins was studied in select brain regions under experimental conditions that permit the activation of receptor coupling to the G proteins Gi, Gs, or Gq. Agents studied were agonists known to be effective at various dopamine receptor/effector systems and included quinelorane (D2-like/Gi), SKF38393 (D1-like/Gq, D1-like/Gs), SKF85174 (D1-like/Gs), and SKF83959 (D1-like/Gq). Dopamine and SKF38393 significantly stimulated [35S]GTPgammaS binding to normal striatal membranes by 161% and 67% above controls. Deoxycholate, which enhances agonist-induced phospholipase C (PLC) stimulation, markedly enhanced the agonistic effects of dopamine and SKF38393 to 530% and 637% above controls, respectively. The enhancing effects of deoxycholate were reversed if it was washed off the membranes before agonist addition. The thiol-reducing agent, dithiothreitol, completely abolished the effects of SKF38393 and SKF83959, whereas SKF85174 effects were augmented. Agonist responses were concentration-related, and highest efficacies were obtained in the hippocampus, thus paralleling both the brain regional distribution and agonist efficacies previously observed in phosphoinositide hydrolysis assays. These findings suggest that D1-like receptor conformations that mediate agonist stimulation of Gs/adenylylcyclase may be structurally different from those that mediate Gq/PLC activation. Although the exact mechanism of deoxycholate's effect awaits elucidation, the results are consistent with the emerging concept of functional selectivity whereby deoxycholate could create a membrane environment that facilitates the transformation of the receptor from a conformation that activates Gs/adenylylcyclase to one that favors Gq/PLC signaling.     

Characterization of Dopamine and β-Adrenergic Receptors Linked to Cyclic AMP Formation in Intact Cells of the Clone D384 Derived from a Human Astrocytoma

3,4-Dihydroxyphenylethylamine (dopamine) and beta-adrenergic receptor agonists and antagonists were assessed for their effects on cyclic AMP accumulation in human astrocytoma derived clone D384 cells. Dopamine, SKF 38393, and 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene increased cyclic AMP content with Ka values of 2.0, 0.2, and 1.6 microM. The D1-selective antagonists SCH 23390 (Ki, 1.2 nM) and SKF 83566 (Ki, 0.8 nM) were over 5,000-fold more potent than the D2-selective antagonist domperidone (Ki, 6.7 microM) at inhibiting dopamine stimulation of cyclic AMP formation. SCH 23388 (Ki, 560 nM; the S-enantiomer of SCH 23390) was 400-fold less potent than SCH 23390. Isoprenaline, adrenaline, salbutamol, and noradrenaline increased cyclic AMP content with Ka values of 0.13, 0.12, 0.22, and 7.60 microM. The beta 2-selective antagonist ICI 118,551 (Ki,0.8 nM) was almost 8,000-fold more potent than the beta 1-selective antagonist practolol (Ki, 5.9 microM) at inhibiting isoprenaline stimulated cyclic AMP accumulation. These results demonstrate that D384 cells express D1-dopamine and beta 2-adrenergic receptors linked to adenylate cyclase. Furthermore, the dopamine receptor expressed by D384 cells exhibits a pharmacological profile typical of a mammalian striatal D1-receptor and therefore the use of this clone represents another approach to studying central D1-receptors.