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.

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+.