Effects of a combination of 3,4-methylenedioxymeth amphetamine and caffeine on real time stimulated dopamine release in the rat striatum: Studies using fast cyclic voltammetry.

It is well documented that caffeine exacerbates the hyperthermia associated with acute exposure to 3,4-methylenedioxymethamphetamine (MDMA) in rats. Previous reports have also indicated that MDMA-related enhancement of dopamine release is exacerbated in the presence of caffeine. In the present study we have examined whether the effects of MDMA on real-time stimulated dopamine release, in the absence of uptake inhibition, are accentuated in the presence of caffeine. Isolated striatal slices from adult male Wistar rats were treated acutely with MDMA, caffeine, or a combination, and their effects on single and 5pulse stimulated dopamine release monitored using the technique of fast cyclic voltammetry. Caffeine at 10 or 100µM had no significant effect on single pulse stimulated dopamine release. However 100µM caffeine caused a significant peak increase in 5pulse stimulated dopamine release. Both 1 and 30µM MDMA gave rise to a significant increase in both single and 5-pulse dopamine release and reuptake. A combination of 100µM caffeine and 1 or 30µM MDMA did not significantly enhance the effects of MDMA on single or 5pulse dopamine release and reuptake when compared to that applied alone. Utilizing single action potential dependent dopamine release, these results do not demonstrate a caffeine-enhanced MDMA-induced dopamine release.

Straight to flexible sigmoidoscopy: rationalization of 2-week wait referrals in suspected colorectal cancer.

AIM: The 2-week wait pathway was designed to decrease the time from presentation to primary care of patients with 'red flag' symptoms of suspected cancer for review by a specialist for the diagnosis or exclusion of cancer. In our tertiary referral centre we have found that 968 colonoscopies per year are required to satisfy the demand for the 2-week wait, leading to limited colonoscopy availability for other services. We sought to determine the yield of colorectal cancer found at colonoscopy referred via the 2-week wait and referenced to the original red flag symptoms. This was in order to select the most efficacious alternative primary investigation based upon presenting symptoms. METHOD: Electronic records were retrospectively analysed. All patients who went through the 2-week wait for suspicion of colorectal cancer in 2013 and were found to have colorectal cancer on colonoscopy were included. Patients not undergoing colonoscopy as the first investigation were excluded. The splenic flexure was deemed to be within the range of a flexible sigmoidoscope. RESULTS: In all, 2950 referrals were made. 968 colonoscopies were performed as the primary investigation of which 35 were found to have colorectal cancer. No patients referred with rectal bleeding and another symptom had a tumour more proximal to the range of flexible sigmoidoscopy. 80% of tumours proximal to the splenic flexure were suitable for CT diagnosis alone. CONCLUSION: Our data support the use of flexible sigmoidoscopy alone as an initial investigation for patients presenting with rectal bleeding with or without additional colorectal symptoms. Patients with anaemia (without bleeding) or change in bowel habit (without bleeding) may be investigated with CT colonography alone; colonoscopy may then be used selectively prior to surgery.

An in vitro approach to assessing a potential drug interaction between MDMA (ecstasy) and caffeine.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a popular recreational drug which causes long-term neurotoxicity and increased risk of fatality. In rats, MDMA toxicity is exacerbated by co-administration of caffeine. The aim of this study was to investigate whether caffeine altered the effects of MDMA in a battery of in vitro tests selected to model some of the known actions of MDMA in vivo. In cytotoxicity studies, caffeine modestly enhanced the effect of MDMA on neuronal N2a cell viability but not that of liver, intestinal or kidney cells. MDMA inhibited the formation of fluorescent metabolites by CYP2D6≫CYP3A4>CYP1A2 but this was not altered by caffeine. Similarly, the inhibition of synaptosomal [(3)H] 5-HT uptake by MDMA was not affected by the presence of caffeine. Thus, these in vitro tests failed to detect any substantial interaction between caffeine and MDMA, highlighting the difficulty of modelling in vivo drug interactions using in vitro tests. However, the results show that the inhibition of synaptosomal [(3)H] 5-HT uptake by MDMA was greater at 41°C and 25°C than at 37°C which raises the possibility that MDMA's effect on SERT in vivo may be increased as body temperature increases, contributing to its harmful effects in users.

Rectal cancer: prognostic indicators of long-term outcome in patients considered for surgery.

PURPOSE: Patients and clinicians seek an accurate prognosis after resectional surgery for rectal cancer. The aim of this study was to determine long-term outcomes after potentially curative surgery for rectal cancer with particular focus on factors associated with longer-term survival that are available to surgeons in the early post-operative setting. METHODS: We conducted a retrospective review of a prospectively gathered database of all primary rectal adenocarcinomas considered for surgery in the University Hospitals of Leicester National Health Service (NHS) Trust between 1998 and 2007. Survival was calculated using a Kaplan-Meier method. Factors thought to be associated with survival were subjected to univariate analysis followed by Cox proportion regression. RESULTS: One thousand and twelve patients with primary rectal adenocarcinoma diagnosed between 1998 and 2007 were identified. Eight hundred and fifty three patients did not have metastases at the time of presentation and 726 patients underwent major resectional surgery. Five-year survival was 66 %. Patients' age, Dukes' stage, UICC stage, nodal involvement and circumferential resection margin status were independently associated with long-term survival on multivariate analysis. CONCLUSION: This is one of the largest series of rectal cancers from a single NHS trust. We have demonstrated that age, Dukes' stage and CRM status are associated with long-term survival. These clinical factors are readily available to the surgeon at the time of first post-operative review and can provide a good clinical guide to prognosis.

Adenosine A1 receptor-mediated inhibition of dopamine release from rat striatal slices is modulated by D1 dopamine receptors.

Dopamine release is regulated by presynaptic dopamine receptors and interactions between adenosine and dopamine receptors have been well documented. In the present study, dopamine release from isolated striatal slices from Wistar rats was measured using fast cyclic voltammetry. Single-pulse stimulation (0.1 ms, 10 V) was applied every 5 min over a 2-h period. Superfusion with the adenosine (A)(1) receptor agonist N(6)-cyclopentyladenosine (CPA), but not the A(2) receptor agonist 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl] phenyl]propanoic acid (CGS 21680), inhibited dopamine release in a concentration-dependent manner (IC(50) 3.80 x 10(-7) m; n = 10). The dose-response curve to CPA was shifted to the right (IC(50) 6.57 x 10(-6) m; n = 6, P < 0.05 vs. control) by the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Neither the D(1) agonist 6-chloro-APB nor the D(1) antagonist R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3- benzazepine-7-ol (SCH 23390) altered dopamine release on their own. However, SCH 23390 (3 microm) significantly attenuated the response to CPA (IC(50) 1.44 x 10(-5) m; n = 6, P < 0.01 vs. control). Furthermore, the inhibitory effect of CPA was significantly increased in the presence of 6-chloro-APB (1 microm). In radioligand binding experiments, CPA interacted with high- and low-affinity states of [(3)H]DPCPX-lableled A(1) receptors. The high-affinity agonist binding to A(1) receptors was inhibited by the stable guanosine triphosphate analogue Gpp(NH)p. In contrast, neither the proportion nor the affinity of high-affinity A(1) receptors was altered by dopamine or SCH 23390. These results provide evidence that the inhibition of dopamine release by adenosine A(1) receptors is dependent, at least in part, on the simultaneous activation of D(1) dopamine receptors. While the mechanism underlying this interaction remains to be determined, it does not appear to involve an intramembrane interaction between A(1) and D(1) receptors.

MRI assessment of the bony pelvis may help predict resectability of rectal cancer.

OBJECTIVE: The outcome after surgical treatment of rectal cancer may be influenced by the technical difficulty of the operation, which is thought to be affected by pelvic size. The aim of this study was to examine the association between bony pelvic dimensions and CRM involvement. PATIENTS AND METHODS: All patients with primary rectal cancer between December 1999 and January 2002 were studied. Staging was performed by pelvic MRI. Nine pelvic dimensions were measured from the MR images on a workstation. Pathology reports were obtained for all patients and the mesorectal specimen was examined. Technical difficulty was assessed by circumferential resection margin (CRM) involvement. RESULTS: Of 126 patients with primary rectal cancer, 88 had staging MRI and rectal excision; there were significant differences between the sexes in all 9 pelvic dimensions (P < 0.05). In females, the interspinous diameter was significantly shorter in patients with CRM involvement compared with patients with a negative CRM. In female patients predicted to have a negative CRM, the anteroposterior diameter of the inlet, the anteroposterior diameter of the midplane and the transverse diameter of the midplane (interspinous distance) were significantly shorter in patients who actually had a positive CRM compared with those in whom the CRM was negative. In male patients, there was no correlation between pelvic dimensions and CRM status. CONCLUSIONS: In certain patients with rectal cancer, CRM positivity may be predicted from pre-operative MRI pelvic measurements. This may influence the choice of adjuvant therapy.

Dopamine D2 receptor modulation of carotid body type 1 cell intracellular calcium in developing rats.

Carotid chemoreceptor type 1 cells release dopamine, which inhibits carotid chemoreceptor activity via dopamine D2 autoreceptors on type 1 cells. Postnatal changes in dopaminergic modulation may be involved in postnatal chemoreceptor development. The present study explores dopaminergic modulation of the intracellular calcium ([Ca(2+)](i)) response to hypoxia in type 1 cells from 1, 3, and 11- to 16-day-old rats. Using fura-2, we studied the effects of quinpirole, a D2 receptor agonist, on type 1 cell [Ca(2+)](i) response to 90-s hypoxia challenges (Po(2) approximately 1-2 mmHg). Cells were sequentially exposed to the following challenges: 1) hypoxia control, 2) hypoxia plus quinpirole, and 3) hypoxia plus quinpirole plus sulpiride (D2 receptor antagonist). In the 11- to 16-day-old group, type 1 cell [Ca(2+)](i) increased approximately 3 to 4-fold over resting [Ca(2+)](i) in response to hypoxia. Quinpirole (10 microM) significantly blunted the peak [Ca(2+)](i) response to hypoxia. Repeat challenge with hypoxia plus 10 microM quinpirole in the presence of 10 microM sulpiride partially restored the hypoxia [Ca(2+)](i) response. In sharp contrast to the older aged group, 10 microM quinpirole had minimal effect on hypoxia response of type 1 cells from 1-day-olds and a small but significant effect at 3 days of age. We conclude that stimulation of dopamine D2 receptors inhibits type 1 cell [Ca(2+)](i) response to hypoxia, consistent with an inhibitory autoreceptor role. These findings suggest dopamine-mediated inhibition and oxygen sensitivity increase with age on a similar time course and do not support a role for dopamine as a major mediator of carotid chemoreceptor resetting.

Behavioural, hyperthermic and neurotoxic effects of 3,4-methylenedioxymethamphetamine analogues in the Wistar rat.

1. The ability of N-ethyl (MDEA) and N-butyl (MDBA) analogues of 3,4-methylenedioxymethamphetamine (MDMA, 'Ecstasy') to induce acute behavioural changes and increases in body temperature, and to cause serotonergic neurotoxicity, was assessed in young adult male Wistar rats. The in vitro ability of MDMA analogues to evoke presynaptic monoamine release from crude rat forebrain synaptosomal preparations pre-labelled with [3H]5-HT or [3H]DA was also measured. 2. In behavioural experiments, acute MDMA and MDEA (20 mg/kg, i.p.) significantly increased rat open-field locomotion scores, decreased open-field rearing, and induced stereotypy, Straub tail and head weaving. MDBA did not produce any of these behaviours. 3. After repeated dosing (8 x 20 mg/kg, i.p., twice daily for 4 days), MDMA > MDEA >> MDBA > or = saline at decreasing forebrain [3H]paroxetine binding levels and concentrations of 5-HT and 5-HIAA at 14 days post-treatment. None of the analogues caused any long-term changes in dopamine or noradrenaline concentrations in the forebrain. 4. Acute MDMA and MDEA (20 mg/kg, i.p.) produced significant acute increases in rat aural temperature compared with saline-treated animals, while 20 mg/kg MDBA caused no significant effects. 5. MDA, MDMA and MDEA were equipotent at inducing [3H]5-HT release from frontal cortex/hippocampal synaptosomes, while MDBA only evoked a significant release at 100 microM concentrations. The potency order for inducing [3H]DA release from striatal synaptosomes was MDA > MDMA > MDEA = MDBA. 6. This study shows that large N-alkyl substitution decreases the ability of MDMA analogues to evoke presynaptic 5-HT and DA release, induce acute hyperthermia, hyperlocomotion and behavioural changes, and cause long-term serotonergic neurotoxicity. 7. The structure-activity relationship data presented here indicate that the neurotoxic damage caused by substituted amphetamines requires a combination of acute hyperthermia and increased neurotransmitter release. Induction of one of these effects in isolation is not sufficient to cause serotonergic nerve terminal degradation.

Synthesis of D-hexos-5-uloses by novel in situ hydrolysis of epoxides derived from 6-deoxyhex-5-enopyranosides.

[reaction: see text] Epoxides derived from 2,3, 4-tri-O-protected-6-deoxyhex-5-enopyranosides are hydrolyzed in situ to ultimately give novel protected-D-hexos-5-ulose derivatives (sugar 1,5-dicarbonyls, 5-ketohexoses) in moderate to high yields. The products adopt a bicyclic structure (1,6-anhydropyranos-5-ulose) in solution with the pyranose ring in (4)C(1) conformation. The methodology has been used to prepare D-xylo-hexos-5-ulose (5-ketoglucose), a synthetic precursor to 1-deoxynojirimycin and a possible intermediate in the biosynthesis of inositols.

Functional coupling of a recombinant human 5-HT5A receptor to G-proteins in HEK-293 cells.

1. We have cloned, expressed and pharmacologically characterized the Human 5-HT5A receptor. 2. We have shown that ligand activation of the Human 5-HT5A receptor results in functional coupling to G-proteins in HEK-293 cells. 3. Stimulation of the receptor with 5-CT (5-carboxamidotryptamine) resulted in a dose-dependent increase in the % [35S]-GTPgammaS binding over the basal level. This is the first study to describe such G-protein activation for the Human 5-HT5A receptor in any cell. 4. A dose-dependent inhibition of cyclic AMP accumulation was observed in the recombinant Human 5-HT5A receptor cell line, suggesting a functional coupling to a G alpha i, G-protein in the HEK-293 cell line. 5. A ligand-stimulated reduction in the detectable level of the catalytic domain of protein kinase A (PKA) in nuclear extracts isolated from Human 5-HT5A expressing cells was observed. This observation was consistent with the reduction in the level of cyclic AMP accumulation, in response to receptor activation.

Apoptosis in C3H-10T1/2 cells: roles of intracellular pH, protein kinase C, and the Na+/H+ antiporter.

Changes in intracellular ion concentrations have been correlated with the activation of an endogenous endonuclease and thus internucleosomal DNA cleavage during apoptosis in many cell types. We investigated whether intracellular pH could play a significant role in apoptotic initiation and progression in C3H-10T1/2 cells, a cell strain that does not exhibit double-stranded DNA cleavage during apoptosis. Protein kinase C and the Na+/H+ antiporter, known regulators of intracellular pH, also were assessed for their involvement in apoptosis of C3H-10T1/2 cells. When a H+ ionophore was used to clamp intracellular pH to 6.0 or below, a significant level of apoptosis was induced in these cells within 6 h, whereas clamping at pH 6.75 did not induce significant amounts of apoptosis until 36 h after acidification. The acidified cells exhibited classic apoptotic morphology and chromatin condensation, similar to serum withdrawn cells, but failed to show internucleosomal DNA cleavage with electrophoresis of genomic DNA. Our results also suggest that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated inhibition of apoptosis in serum withdrawn C3H-10T1/2 cells functions through a sequential activation of protein kinase C and the Na+/H+ antiporter; thus, an alkalinization or an inhibition of acidification is involved in this apoptotic block. Serum withdrawal itself does not appear to act through a negative effect on either protein kinase C or the Na+/H+ antiporter. TPA was also capable of inhibiting the apoptosis induced by specific inhibitors of protein kinase C and the Na+/H+ antiporter, but the inhibition was successful only if the TPA was administered at least 20 min prior to the addition of the enzyme inhibitor. These results indicate that apoptosis in C3H-10T1/2 cells follows a pathway that involves intracellular acidification, but is independent of detectable endonuclease activity.

Nefiracetam (DM-9384) reverses apomorphine-induced amnesia of a passive avoidance response: delayed emergence of the memory retention effects.

Nefiracetam is a novel pyrrolidone derivative which attenuates scopolamine-induced learning and post-training consolidation deficits. Given that apomorphine inhibits passive avoidance retention when given during training or in a defined 10-12h post-training period, we evaluated the ability of nefiracetam to attenuate amnesia induced by dopaminergic agonism. A step-down passive avoidance paradigm was employed and nefiracetam (3 mg/kg) and apomorphine (0.5 mg/kg) were given alone or in combination during training and at the 10-12h post-training period of consolidation. Co-administration of nefiracetam and apomorphine during training or 10h thereafter produced no significant anti-amnesic effect. However, administration of nefiracetam during training completely reversed the amnesia induced by apomorphine at the 10h post-training time and the converse was also true. These effects were not mediated by a dopaminergic mechanism as nefiracetam, at millimolar concentrations, failed to displace either [3H]SCH 23390 or [3H]spiperone binding from D1 or D2 dopamine receptor subtypes, respectively. It is suggested that nefiracetam augments molecular processes in the early stages of events which ultimately lead to consolidation of memory.

D2 receptor antagonists do not modify guanine nucleotide-sensitive interactions between dopamine and D1 dopamine receptors under in vitro conditions.

This study investigated possible D1/D2 interactions in rat and bovine striatal tissue by examining the effects of D2 antagonists on the action of dopamine at D1 dopamine receptors. In addition, the extent to which D2 antagonists may induce an agonist low-affinity state of the D1 receptor was evaluated in comparison with the effects of the guanine nucleotide analogue 5'-guanylyl-imidodiphosphate [Gpp(NH)p]. In saturation experiments dopamine caused a dose-dependent decrease in rat striatal and bovine caudate D1 receptor density. This effect of dopamine, which has been shown to be sensitive to Gpp (NH)p, was not altered by pretreatment with either of the selective D2 antagonists eticlopride (200 nM) or domperidone (200 nM). Results from displacement experiments show that the affinity of dopamine for D1 receptors, and the proportion of receptors in an agonist high-affinity state, are reduced by Gpp(NH)p (100 microM) but not by eticlopride. A molar excess of dopamine (100 microM) promotes the dissociation of (+/-)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-o l ([3H]SCH 23390) from rat striatal D1 receptors at a rate that is significantly slower than when dissociation is initiated using 1 microM piflutixol. After pretreament with Gpp(NH)p, [3H]SCH 23390 dissociation induced by dopamine occurred at an even slower rate. Pretreatment with eticlopride had no effect on the dopamine-induced rate of [3H]SCH 23390 dissociation. These results indicate that all experimental approaches detected dopamine effects at D1 receptors that are Gpp(NH)p sensitive and D2 antagonist insensitive and provide no evidence to support a D1/D2 link operating at the receptor level.

Behavioural pharmacology of 'D-1-like' dopamine receptors: further subtyping, new pharmacological probes and interactions with 'D-2-like' receptors.

1. D-1 receptors are now recognised to play a critical psychopharmacological role in the regulation of unconditioned motor and numerous other aspects of behaviour. 2. There appears to exist a broad family of 'D-1-like' receptors in terms both of differential coupling to distinct messenger/transduction mechanisms and of gene cloning, whose behavioural roles remain to be clarified. 3. The adenylyl cyclase-inhibiting benzazepine SK&F 83959 induces behavioural responses in rats that are similar to those induced by the full efficacy cyclase-stimulating isochroman A 68930 but not to those induced by its high efficacy partial agonist benzazepine congener R-6-Br-APB; these data indicate roles for individual 'D-1-like' receptors in mediating distinct elements of dopaminergic behaviour. 4. The putative D-1 autoreceptor agonist B-HT 920 and the putative D-3 agonist 7-OH-DPAT demonstrate different behavioural profiles when given both alone and in combination with the selective 'D-1-like' antagonist BW 737C; D-3 receptors may participate in cooperative/synergistic but not in oppositional 'D-1-like': 'D-2-like' interactions. 5. Such interactions apparent at the level of behaviour are complemented by evidence for similar interactions at numerous alternative levels of function, though these may differ between rodent and primate species. 6. A broader range of more selective agonists and antagonists, able to distinguish between individual members of the 'D-1-like' and of the 'D-2-like' receptor families are needed to clarify these issues.

Dopamine-induced reduction in the density of guanine nucleotide-sensitive D1 receptors in human postmortem brain in the absence of apparent D1: D2 interactions.

The effects of dopamine and guanine nucleotides on the binding of the D1 dopamine receptor antagonist ligand [3H]SCH 23390 were examined in membranes prepared from putamen, caudate and nucleus accumbens of human postmortem brain. Dopamine induced a concentration-dependent decrease in the apparent maximum number of binding sites (Bmax) in each brain region studied, and displaced binding in a biphasic manner consistent with the presence of both high and low affinity states of the D1 receptor; the GTP analogue Gpp(NH)p transformed this biphasic displacement to a monophasic pattern consistent with a shift of high affinity sites to a low affinity state. However, the selective D2 antagonist eticlopride did not reverse the action of dopamine to decrease Bmax. These data suggest that dopamine decreases Bmax for D1 receptors through a high affinity, guanine nucleotide-sensitive agonist binding site, but fail to reveal D1:D2 interactions at this synaptic level.

Cholinergic activation of Cl- secretion in rat colonic epithelia.

Acetylcholine receptor agonists and antagonists were used in a pharmacological analysis to identify which muscarinic receptor(s) may be involved in cholinergic regulation of Cl- secretion across rat colonic mucosa in vitro. A comparative ligand binding analysis for each of the antagonists was carried out in parallel. Both studies elicited identical rank order potencies (atropine > or = 4-diphenyl-acetoxy-N-piperidine methiodide (4-DAMP) > pirenzepine > 11-[[2[(diethylamino)methyl]-1-pipiridinyl]acetyl[5,11- dihydro-6H-pyrido[2,3-b]]1,4]benzodiazepine-6-one (AF-DX 116). Cholinomimetic-induced Cl- secretion was predominantly mediated by activation of muscarinic receptors in rat isolated colonic mucosa, with only a modest contribution from nicotinic receptors. Short circuit current responses evoked by the selective muscarinic M1 receptor agonist 4-[[(3-chlorophenyl)amino]carbonyl]-N,N,N-trimethyl-2-butyn-1-a minium chloride (McN-A-343) suggest that this receptor subtype, which is thought to be neuronally sited, also plays a minor role in regulation of intestinal ion transport. The principal epithelial cell receptors responsible for acetylcholine receptor-mediated Cl- secretion appear to belong to the M3 class.

The kinetics of [3H]SCH 23390 dissociation from rat striatal dopamine D1 receptors: effect of dopamine.

The present study investigated possible allosteric interactions between dopamine and [3H]SCH 23390 ((R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepi n-7-ol)- labelled dopamine D1 receptors in rat striatum. As previously described, dopamine prevented [3H]SCH 23390 binding in a mixed competitive/non-competitive manner, causing both a loss of ligand affinity and a decrease in Bmax. The effect of dopamine was largely reversed following pretreatment of the membranes with 100 microM Gpp(NH)p (5'-guanylylimidodiphosphate) and was significantly enhanced by omission of Na+ from the incubation buffer. In dissociation kinetic studies, two methods of initiating ligand dissociation were used: dilution into 100-fold volume excess of buffer or addition of a molar excess of drug. Both methods yielded similar rates of [3H]SCH 23390 dissociation. Inclusion of dopamine in the volume excess of buffer did not alter the k-1 for [3H]SCH 23390 dissociation. However, when 100 microM dopamine was used instead of 1 microM piflutixol to initiate dissociation, a significant slowing of the rate of dissociation of [3H]SCH 23390 occurred. This effect of dopamine on k-1 was Na(+)-dependent since in the absence of Na+ the dopamine-induced rate of dissociation was only slightly slower than control values. Under neither condition did dopamine accelerate the rate of ligand dissociation, indicating that dopamine does not interact allosterically with [3H]SCH 23390 binding sites. These data, therefore, preclude an allosteric mechanism to explain the dopamine-induced decrease in dopamine D1 receptor density and provide direct evidence that dopamine masks ligand binding by binding to a high affinity site which can be modulated by Gpp(NH)p and Na+.

Chronic antagonist treatment does not alter the mode of interaction of dopamine with rat striatal dopamine receptors.

Chronic treatment with the D1 and D2 dopamine receptor antagonists SCH 23390 (0.5 mg/kg) and haloperidol decanoate (25 mg/kg) caused an up-regulation in D1 and D2 receptor densities, respectively, with no change in KD. Dopamine (20 microM) interacted with both receptor subtypes in a mixed competitive/non-competitive manner, causing a reduction in ligand binding affinity and an apparent decrease in receptor density. In the presence of dopamine, both vehicle-treated and SCH 23390-treated striatal preparations showed a significant loss in affinity for 3H-SCH 23390 binding to D1 receptors and a decrease in D1 receptor density of approximately 26%. Similarly, dopamine caused a substantial loss in 3H-spiperone binding affinity to D2 receptors and a 46% decrease in Bmax in both vehicle-treated and haloperidol-treated membranes. Thus, receptor up-regulation does not appear to alter the mode of interaction of dopamine with rat striatal dopamine receptors.

Agonist and antagonist interactions with D1 dopamine receptors: agonist-induced masking of D1 receptors depends on intrinsic activity.

The effects of agonist and antagonist compounds on the equilibrium binding of the D1 antagonist ligand [3H]SCH 23390 were examined in membranes from the striatum of the rat. The antagonist SK&F 83566 interacted with D1 receptors in the manner of a competitive antagonist, causing a decrease in the affinity of the binding of [3H]SCH 23390, without altering the maximum number of binding sites (Bmax). The interaction of agonist compounds with the D1 receptor appeared to be more complex. The drug SK&F 75670, a weak partial agonist, also acted competitively at D1 sites. However, agonists with moderate (SK&F 38393, CY 208-243) or full (dopamine) intrinsic activity to stimulate adenylate cyclase, interacted with D1 binding sites in a mixed competitive/non-competitive manner, causing both a decrease in ligand affinity and a decrease in Bmax. The benzazepine analogue, which also has full agonist activity, SK&F 82958, only caused a reduction in Bmax. Furthermore, there was a positive relationship between the intrinsic activity of agonists and the magnitude of the reductions in Bmax which they induced. In the presence of the GTP analogue, Gpp(NH)p, CY 208-243 no longer caused an apparent reduction in the number of receptors. These data suggests that the apparent loss of D1 receptors, induced by agonists, may result from an interaction with a guanine-nucleotide sensitive, high affinity agonist binding site and that the degree of interaction with this site depends on the intrinsic D1 activity of the agonist.