Adenosine and dopamine receptor antagonist binding in the rat ventral and dorsal striatum: lack of changes after a neonatal bilateral lesion of the ventral hippocampus.

There is experimental evidence from radioligand binding experiments for the existence of strong antagonistic interactions between different subtypes of adenosine and dopamine receptors in the striatum, mainly between adenosine A1 and dopamine D1 and between adenosine A2A and dopamine D2 receptors. These interactions seem to be more powerful in the ventral compared to the dorsal striatum, which might have some implications for the treatment of schizophrenia. The binding characteristics of different dopamine and adenosine receptor subtypes were analysed in the different striatal compartments (dorsolateral striatum and shell and core of the nucleus accumbens), by performing saturation experiments with the dopamine D1 receptor antagonist [125I]SCH-23982, the dopamine D2-3 receptor antagonist [3H]raclopride, the adenosine A1 receptor antagonist [3H]DPCPX and the adenosine A2A receptor antagonist [3H]SCH 58261. The experiments were also performed in rats with a neonatal bilateral lesion of the ventral hippocampus (VH), a possible animal model of schizophrenia. Both dopamine D2-3 and adenosine A2A receptors follow a similar pattern, with a lower density of receptors (40%) in the shell of the nucleus accumbens compared with the dorsolateral caudate-putamen. A lower density of adenosine A1 receptors (20%) was also found in the shell of the nucleus accumbens compared with the caudate-putamen. On the other hand, dopamine D1 receptors showed a similar density in the different striatal compartments. Therefore, differences in receptor densities cannot explain the stronger interactions between adenosine and dopamine receptors found in the ventral, compared to the dorsal striatum. No statistical differences in the binding characteristics of any of the different adenosine and dopamine receptor antagonists used were found between sham-operated and VH-lesioned rats.     

Protective effects of N-acetylserotonin against 6-hydroxydopamine-induced neurotoxicity

The present work studied in vivo neuroprotective effects of n-acetylserotonin (NAS), the immediate precursor of melatonin, on the dopaminergic system, in rats lesioned with the unilateral intrastriatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA). Two weeks after the lesion, the dopamine receptor agonist, apomorphine, produced rotational asymmetry, and the NAS treatment significantly reduced the motor deficit following the apomorphine challenge. The apomorphine-induced rotational behavior was blocked by 84, 86 and 53% after NAS, at doses of 2, 5 and 10 mg/kg, i.p., respectively. The injection of 6-OHDA significantly decreased DA, DOPAC and HVA levels in the rat striatum. In contrast, the NAS (2, 5 and 10 mg/kg, i.p., daily for 7 days) treatment partially reversed the decreases caused by 6-OHDA, and the neurotransmitter levels were brought to approximately 50% of that observed in the contralateral sides. NAS was more efficient at the smaller doses. NAS (5 mg/kg) produced an up-regulation of D1 (37%) and D2 (37%) receptors associated with a decrease in Kd values.     

Antipeptide antibodies that recognize a lymphocyte substance P receptor

In an effort to investigate the presence of substance P (SP) receptors on lymphocytes, polyclonal antibodies against SP receptors were developed. The immunogen used to generate these antibodies was a peptide encoded by an RNA complementary to the mRNA for SP. The rationale for using this SP complementary peptide (termed SP CP) as an immunogen resulted from the observation that 3H-SP bound to microtiter wells coated with SP CP in a dose dependent and saturable fashion. Furthermore, binding was blocked with excess unlabeled SP or SP antagonist, D-Pro2-D-Phe7-D-Trp9-SP. Inasmuch as the peptide, SP CP, specifically bound 3H-SP, we hypothesized that antibodies against this peptide might recognize a SP receptor binding site. Using the SP receptor positive lymphoblast cell line, IM-9, affinity-purified antibodies against SP CP but not antibodies against keyhole limpet hemocyanin recognized a molecule on the surface of IM-9 cells. Anti-SP CP binding to IM-9 cells was blocked with excess SP antagonist, suggesting that the antibody and the SP antagonist were competing for the same binding site. In support of this possibility, anti-SP CP antibodies blocked 3H-SP binding to IM-9 cells. An immunoaffinity column coupled with antibodies against SP CP bound protein from solubilized IM-9 cells. This isolated protein bound 125I-Tyr8-SP and binding was specifically blocked with SP as well as by SP antagonist, neurokinin A, and eledoisin. Passthrough material did not bind SP suggesting that a SP receptor had been purified. Western blot analysis of solubilized IM-9 cell proteins using anti-SP CP antibodies but not preimmune IgG recognized a single protein of 58,000 D. Taken together, these results demonstrate that antibodies against SP CP recognize a SP receptor present on the lymphocyte cell line, IM-9.     

Regulation of D1A and D2 dopamine receptor mRNA during ontogenesis, lesion and chronic antagonist treatment.

The developmental characteristics of D1A and D2 dopamine receptor mRNA levels were determined by Northern blot analyses. Striatal D1A and D2 dopamine receptor mRNAs of male Fischer 344 rats were about 60% of adult (day 120) levels at postnatal day 1 and reached their highest levels at day 30 (126 and 139% adult levels) and then decreased by day 120 (100%). D1 and D2 dopamine receptors showed much greater quantitative changes with densities at day 30 about 6- and 14-fold higher than at day 1, respectively, while mRNA levels showed only a 2-fold increase. The highest level of D2 dopamine receptor mRNA in the midbrain was reached at day 14 (195% of adult levels) while the level at day 1 was 31% higher than that at day 120. Striatal beta-actin mRNA levels decreased gradually as the rats developed with the level at postnatal day 1 almost twice that at day 120 postpartum. Treatment of adult rats with the selective D2 dopamine receptor antagonist, haloperidol (0.5 mg/kg/day, s.c., for 2 h, 7, 14, 21 days or 21 days + 3 days withdrawal) had no effect on striatal D2 dopamine receptor mRNA levels in spite of significant increases in dopamine receptor density at the later time points. However, 21 days following a 6-hydroxydopamine lesion of the nigrostriatal pathway, striatal D2 dopamine receptor mRNA levels were increased by 53%.     

Pharmacological evaluation of (+)-2- [123I]A-69024: a radioligand for in vivo studies of dopamine D1 receptors

(+)-2-[123I] A-69024, [(+)-1-(2-[123I] iodo-4,5-dimethoxy-benzyl)-7-hydroxy-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline], is a specific and enantioselective dopamine D1 receptor radioligand. The in vivo biodistribution of this radioligand in rats showed high brain uptake and a distribution consistent with the density of dopamine D1 receptors. Highest uptake was observed in the striatum (0.65 %ID/g) at 5 min followed by clearance. As a measure of specificity the striatum/cerebellar ratio reached a maximum of 3.9 at 30 min post-injection. Radioactivity in the striatum was reduced by 68% by pre-administration of the D1 antagonist SCH 23390. Pre-administration of other dopamine binding drugs, spiperone (D2), 7-OH-DPAT (D3), and clozapine (D4) displayed no inhibitory effect on (+)-2-[123I]A-69024 accumulation in any brain region. Ketanserin (5-HT2/5-HT2C) and haloperidol (D2 receptor antagonist/sigma receptor ligand) also displayed no inhibitory effect in any brain region studied. With the pharmacologically inactive enantiomer, (-)-2-[123I]A-69024, the brain uptake was determined to be non-specific since a striatum/cerebellar ratio of near 1 was observed throughout the time course of the experiment. (+)-2-[123I]A-69024 displays enantioselectivity for dopamine D1 receptors and may deserve further investigation as a possible SPECT radioligand.     

On the specificity of vitamin D compounds binding to chick pig intestinal 1,25-dihydroxyvitamin D3 receptor

The binding activity of four vitamin D metabolites and/or analogs for the intestinal 1,25-dihydroxyvitamin D3 receptor was evaluated after incubation at 25 degrees C for 1 h or at 0-4 degrees C for 18 h. The incubation conditions, which had no effect on the binding of 1,25-dihydroxyvitamin D3, had a dramatic effect on the binding of 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 and a small but reproducible effect on 24,25-dihydroxyvitamin D3 binding to receptor. Affinities 10- to 20-fold higher were obtained for 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, and affinities 3-fold higher were obtained for 24,25-dihydroxyvitamin D3 at the 0-4 degrees C/18-h incubation. A comparison of intestinal receptor from chick and pig with nine vitamin D compounds showed no major differences between the two species. The relative affinity of the vitamin D analogs to compete with tritiated 1,25-dihydroxyvitamin D3 for the receptor in pig nuclear extract, expressed as ratios of the molar concentration required for 50% binding of the tritiated 1,25-dihydroxyvitamin D3 compared to nonradioactive 1,25-dihydroxyvitamin D3, are as follows: 1,25-dihydroxyvitamin D3 (1) = 1,25-dihydroxyvitamin D2 = 24-homo-1,25-dihydroxyvitamin D3 greater than 1,24,25-trihydroxyvitamin D3 (4) greater than 25-hydroxyvitamin D3 (21) = 10-oxo-19-nor-25-hydroxyvitamin D3 = 1 alpha-hydroxyvitamin D3 (37) greater than 24,25-dihydroxyvitamin D2 (257) much much greater than vitamin D3 (greater than 10(6)).     

D2-dopamine receptor and alpha2-adrenoreceptor-mediated analgesic response of quercetin

Quercetin, a bioflavonoid (100-300 mg/kg) produced dose dependent increase in tail-flick latency, the analgesic effect being sensitive to reversal by naloxone (1 mg/kg). Prior treatment with haloperidol (1 mg/kg), D1/D2 receptor antagonist haloperidol, sulpiride (50 mg/kg), a selective D2 receptor antagonist, yohimbine (5 mg/kg), a alpha2-adrenoreceptor antagonist but not by SCH 23390 a, selective D1 receptor antagonist blocked this response. Apomorphine (1 mg/kg) a mixed D1/D2 dopamine receptor agonist, and quinpirole (0.5 mg/kg), a selective D2 receptor agonist also produced antinociception, that was reversed by haloperidol (1 mg/kg), sulpiride (50 mg/kg), but not by yohimbine (5 mg/kg). The antinociceptive action of quercetin (200 mg/kg) was potentiated by D2 agonist quinpirole (0.2 mg/kg). Dopamine D1 receptor agonist SKF38393 (10 and 15 mg/kg) failed to alter the antinociceptive effect of quercetin (200 mg/kg). Quercetin (200 mg/kg) reversed reserpine (2 mg/kg-4 hr) induced hyperalgesia, which was reversed by sulpiride but not by yohimbine. Thus, a role of dopamine D2 and alpha2-adrenoreceptors is postulated in the antinociceptive action of quercetin.     

High-and Low-Affinity States of Striatal D2 Receptors Are Not Affected by 6-Hydroxydopamine or Chronic Haloperidol Treatment

Specific D2 binding in rat striatum was characterized and then the effects of chronic disruption of dopaminergic activity on antagonist and agonist binding to these sites were studied. D2 receptors were defined as those sites capable of binding [3H]spiperone in the presence of cinanserin, a 5-HT2 antagonist, but not in the presence of (+)-butaclamol, a D2 and 5-HT2 blocker. Saturation, competition, and kinetic analyses suggested that D2 receptors are a homogeneous population exhibiting more complex interactions with agonists than antagonists. Antagonist binding was monophasic and guanine nucleotide-insensitive whereas agonist binding was biphasic and guanine nucleotide-sensitive. D2 receptor density was elevated by more than 40% following dopamine depletion by 6-hydroxydopamine or chronic receptor blockade by haloperidol. However neither treatment altered the affinities or magnitudes of the high- and low-affinity components associated with agonist binding to the D2 receptor.     

D1 Dopamine Receptors of NS20Y Neuroblastoma Cells Are Functionally Similar to Rat Striatal D1 Receptors

Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two- to three-fold over baseline. The pseudo-Km for dopamine was approximately 2 microM, whereas for dihydrexidine it was approximately 30 nM. The effects of both drugs were blocked by either the D1-selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)-butaclamol (Ki, 5 nM). Both (-)-butaclamol and the D2-selective antagonist (-)-sulpiride were ineffective (Ki greater than 3 microM). Forskolin (10 microM), prostaglandin E1 (1 microM), and adenosine (10 microM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 microM). Finally, muscarinic receptor stimulation (100 microM carbachol) inhibited both D1- and forskolin-stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum.     

Effect of angiotensin-(1-7) on jejunal absorption of water in rats

The effect of angiotensin-(1-7) on jejunal water absorption in rats was investigated. The jejunal sac of anesthetized rats was filled with two ml of tyrode solution containing 3.7 MBq of tritiated water. A femoral vein was cannulated for administration of peptides and drugs. Infusion of Ang-(1-7) at the dose of 0.7 ng/kg.min produced a significant increase in jejunal water absorption compared to control (32% increase). The Ang-(1-7) antagonist A-779 abolished the effect of Ang-(1-7) on water absorption. A reduction of the Ang-(1-7) effect was also produced by treatment with the AT(1) receptor antagonist, losartan or the AT(2) receptor antagonist, PD123.177. The increase in jejunal water absorption produced by Ang-(1-7) was blocked by the nitric oxide synthase inhibitor, L-NAME and by indomethacin. These data suggest that the effect of Ang-(1-7) on the jejunal loop is mediated by activation of a multiple angiotensin receptors and/or by an atypical angiotensin receptor. Furthermore, the effect of Ang-(1-7) on jejunal water absorption is mediated by nitric oxide and by a cyclooxygenase-dependent mechanism.     

Specific [3H]SCH23390 Binding to Dopamine D1 Receptors in Cerebral Cortex and Neostriatum: Evidence for Heterogeneities in Affinity States and Cortical Distribution

The tritiated antagonist SCH23390 was used to identify dopamine D1 receptors in the cerebral cortex and neostriatum. The kinetic properties of binding were investigated in parallel experiments with membrane preparations from both tissues. The densities of receptors (Bmax) and the dissociation constants (KD) were determined from saturation curves, and the specificity of binding verified in competition experiments using agonists and antagonists. The cortical D1 receptor displays the same pharmacological selectivity (including stereospecificity) and kinetic properties as the neostriatal D1 receptor. From both the dissociation kinetics by dilution and the competition curves, it could be established that there is an heterogeneity of binding probably due to high- and low-affinity states. Endogenous dopamine, 4-hydroxy-3-methoxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and 3-methoxytyramine contents, as well as D1 receptor distribution, were measured for the neostriatum and four localized cortical areas: anterior cingulate, primary somatosensory, primary visual, and piriform-entorhinal. For the regions examined, the distribution of D1 receptors is heterogeneous, but correlates very well (r greater than 0.98) with the endogenous levels of dopamine and its major metabolites.     

Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes

A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.     

Effect of age on duodenal 1,25-dihydroxyvitamin D-3 receptors in Wistar rats

We confirmed our previous observation that duodenal Ca2+ absorption and serum 1,25-dihydroxyvitamin D (1,25-(OH)2D) levels declined concurrently in old (24 months old) rats as compared to young (6 months old) rats. It is well known that 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) expresses its action after binding to specific receptor molecules. In this paper, we compared certain properties of rat duodenal 1,25-(OH)2D3 receptors from old and young animals. Receptor preparations were incubated with [3H]1,25-(OH)2D3 to quantitate the number of unoccupied and total receptor sites and showed that total and unoccupied receptor sites decreased by 22 and 16%, respectively in old rats. Endogenously occupied sites were reduced by 43% in duodenum of the old rat and, consequently, the percentage of receptor occupancy also declined. Age did not affect the dissociation constant (KD) of 1,25-(OH)2D3 from the receptor; the sedimentation coefficient (3.3 S) of the tritiated 1,25-(OH)2D3-receptor complex in sucrose density centrifugation; or its affinity for DNA. The data are consistent with the hypothesis that the age-related decline in Ca2+ absorption in the intestine may be due, in part, to the decrement in the circulating level of 1,25-(OH)2D and a reduction of intestinal 1,25-(OH)2D3 receptor occupancy status.     

Effects of the cannabinoid receptor agonist CP 55,940 and the cannabinoid receptor antagonist SR 141716 on intracranial self-stimulation in Lewis rats.

Lewis rats were trained to self-stimulate the medial forebrain bundle (MFB) using a rate-frequency paradigm. They were then tested for the effects of the cannabinoid receptor agonist CP 55,940, the selective cannabinoid receptor antagonist SR 141716 and the dopamine D1 receptor antagonist SCH 23390. CP 55,940 (0, 10, 25 and 50 microg/kg i.p.) had no effect on MFB self-stimulation behaviour as assessed by the M50, the stimulation frequency at which half-maximal response rates were obtained. With SR 141716, only a very high dose (20 mg/kg i.p.) caused a significant inhibition of the rewarding efficacy of the stimulation. This was seen as an increase in the M50. All other doses of SR 141716 (0, 1, 3, 10 mg/kg i.p.) were ineffective in modulating the M50. By comparison, a relatively low dose (0.06 mg/kg i.p.) of SCH 23390 caused a large increase in M50. These results indicate a relatively modest influence, if any at all, of exogenous or endogenous cannabinoids on reward-relevant neurotransmission.     

Lipidation and glycosylation of a T cell antigen receptor (TCR) transmembrane hydrophobic peptide dramatically enhances in vitro and in vivo function

A T cell antigen receptor (TCR) transmembrane sequence derived peptide (CP) has been shown to inhibit T cell activation both in vitro and in vivo at the membrane level of the receptor signal transduction. To examine the effect of sugar or lipid conjugations on CP function, we linked CP to 1-aminoglucosesuccinate (GS), N-myristate (MYR), mono-di-tripalmitate (LP1, LP2, or LP3), and a lipoamino acid (LA) and examined the effects of these compounds on T cell activation in vitro and by using a rat model of adjuvant-induced arthritis, in vivo. In vitro, antigen presentation results demonstrated that lipid conjugation enhanced CP's ability to lower IL-2 production from 56.99%+/-15.69 S.D. observed with CP, to 12.08%+/-3.34 S.D. observed with LA. The sugar conjugate GS resulted in only a mild loss of in vitro activity compared to CP (82.95%+/-14.96 S.D.). In vivo, lipid conjugation retarded the progression of adjuvant-induced arthritis by approximately 50%, whereas the sugar conjugated CP, GS, almost completely inhibited the progression of arthritis. This study demonstrates that hydrophobic peptide activity is markedly enhanced in vitro and in vivo by conjugation to lipids or sugars. This may have practical applications in drug delivery and bioavailability of hydrophobic peptides.     

Synthesis and binding characteristics of [3H] H-Tyr-Ticpsi[CH2-NH] Cha-Phe-OH, a highly specific and stable delta-opioid antagonist.

Substitution of the Phe3 aromatic ring in H-Tyr-Ticpsi[CH2-NH]Phe-Phe-OH with cyclohexylalanine (Cha) has been reported to result in a compound, H-Tyr-Ticpsi[CH2-NH]Cha-Phe-OH (TICP[psi]), showing substantially increased delta-opioid antagonist potency and high delta selectivity. TICP[psi] was radiolabeled by catalytic tritiation of its precursor Tyr(3',5'-I2)1TICP[psi]. Binding characteristics of the new tritiated pseudopeptide were determined using the radioligand binding assay in rat brain membranes. On the basis of the results of saturation binding studies performed at 25 degrees C, an equilibrium dissociation constant (Kd) of 0.35 nM and a receptor density (Bmax) of 112 fmol/mg protein were calculated. This new tritiated ligand exhibits high affinity for delta-opioid receptors, whereas its binding to mu and kappa receptors is weak. A study of [H3]TICP[psi] binding displacement by various receptor-selective opioids showed the following rank order of potency: delta > kappa = mu. These receptor binding characteristics of the ligand, together with its high specific radioactivity (41.3 Ci/mmol) and stability, makes it a useful tool for labeling delta-opioid receptors, both in vitro and in vivo.     

Cross-talk between M2 muscarinic and D1 dopamine receptors in the cat adrenal medulla.

In the study reported here we have reached two conclusions. First, the cat adrenal medulla chromaffin cell possesses a dopamine D1 receptor that seems to be coupled to an adenylyl cyclase. Second, this receptor regulates the muscarinic-mediated catecholamine release response through a negative feed-back loop which uses cyclic AMP as a second messenger. These conclusions are supported by the following findings: (i) SKF38393 (a selective D1 receptor agonist), but not quinpirole (a selective D2 agonist), inhibits the methacholine-mediated catecholamine release responses in a concentration-dependent manner (IC50 of around 1-2 microM). (ii) SCH23390 (a selective D1 antagonist), but not sulpiride (a selective D2 antagonist), reversed by 70% the inhibitory effects of SKF38393. (iii) Dibutyril cyclic AMP (500 microM) inhibited by 80% the secretory effects of methacholine.     

Mechanisms regulating the cell surface residence time of the alpha 2A-adrenergic receptor

Despite considerable insights concerning the mechanisms regulating short-term agonist-mediated G protein-coupled receptor (GPCR) internalization, little is known about the mechanisms regulating GPCR surface residence over long periods of time. Herein, we experimentally evaluated mechanisms regulating the surface t(1/2) of various alpha(2A)-adrenergic receptor (alpha(2A)AR) structures. The Delta 3i alpha(2A)AR (lacking the third intracellular loop), D79N alpha(2A)AR (impaired G protein coupling), and CAM alpha(2A)AR (enhanced G protein coupling) all exhibited a cell surface alpha(2A)AR turnover in Chinese hamster ovary cells that was faster than that of the wild type (WT). Cell surface receptor turnover could be slowed with ligand occupancy of D79N alpha(2A)AR (agonist or antagonist) and CAM alpha(2A)AR (antagonist only) but not the Delta 3i- or WT alpha(2A)AR. This selective ligand-induced surface stabilization was paralleled by a dramatic ligand-dependent receptor density upregulation for D79N- and CAM alpha(2A)AR structures. Receptors which exhibited surface turnover and density that could be modulated by ligand (D79N and CAM) also demonstrated structural instability, measured by a loss of radioligand binding capacity in detergent solution over time without parallel changes in receptor protein content. In contrast, the shorter surface t(1/2) of the Delta 3i alpha(2A)AR, whose cell surface t(1/2) and steady state density were not altered by ligand occupancy, occurred in the context of a structurally stable receptor in detergent solution. These results demonstrate that changes in receptor structure which alter receptor-G protein coupling (either an increase or decrease) are paralleled by structural instability and ligand-induced surface stabilization. These studies also provide criteria for assessing the structural instability of the alpha(2A)AR that can likely be generalized to all GPCRs.     

Chronic administration of N-methyl-D-aspartate (NMDA) receptor antagonists induced in rats, a facilitation of striatal dopaminergic type D2 transmission: behavioral and biochemical study]

Compared with control rats, rats treated with ketamine (15 mg/kg/day, p.o.) or MK-801 (0.1 to 0.4 mg/kg/day, p.o.) for 6 weeks showed significantly increased: 1) behavioural responses to D2 dopaminergic agents, either agonist or antagonist, 2) striatal D2 receptor mRNA expression, 3) striatal D2 receptor density, without any presynaptic change in dopaminergic or serotoninergic neurotransmission. These results suggest that the functional expression of striatal D2 receptor is postsynaptically regulated by glutamate-triggered events through the NMDA receptor subtype.     

Dopamine receptor-mediated regulation of striatal cholinergic activity: positron emission tomography studies with norchloro[18F]fluoroepibatidine

Large numbers of in vitro studies and microdialysis studies suggest that dopaminergic regulation of striatal acetylcholine (ACh) output is via inhibitory dopamine D2 receptors and stimulatory dopamine D1 receptors. Questions remain as to the relative predominance of dopamine D2 versus D1 receptor modulation of striatal ACh output under physiological conditions. Using positron emission tomography, we first demonstrate that norchloro[18F]fluoroepibatidine ([18F]NFEP), a selective nicotinic ACh receptor (nAChR) ligand, was sensitive to changes of striatal ACh concentration. We then examined the effect of quinpirole (D2 agonist), raclopride (D2 antagonist), SKF38393 (D1 agonist), and SCH23390 (D1 antagonist) on striatal binding of [18F]NFEP in the baboon. Pretreatment with quinpirole increased the striatum (ST) to cerebellum (CB) ratio by 26+/-6%, whereas pretreatment with raclopride decreased the ST/CB ratio by 22+/-2%. The ratio of the distribution volume of [18F]NFEP in striatum to that in cerebellum, which corresponds to (Bmax/K(D)) + 1 (index for nAChR availability), also showed a significant increase (29 and 20%; n = 2) and decrease (20+/-3%; n = 3) after pretreatment with quinpirole and raclopride, respectively. However, both the D1 agonist and antagonist had no significant effect. This suggests that under physiological conditions the predominant influence of endogenous dopamine on striatal ACh output is dopamine D2, not D1, receptor-mediated.