Pharmacological characterization of purinergic receptors in the rat vas deferens

Using the isolated rat vas deferens, we have confirmed the existence of P1 purinergic receptors whose activation results in an inhibition of the neurogenic twitch of the vas deferens. The observed order of potency for agonists (adenosine ethyl carboxamide greater than 2-chloroadenosine greater than adenosine greater than 5'-AMP greater than 5'-ADP greater than ATP) and antagonism of these effects by theophylline supports a P1-mediated response. Metabolically stable analogs of ATP elicited dose-dependent contractile responses which were quantitatively greater than, but qualitatively comparable to, ATP-induced responses. The order of potency for the eliciting contraction was the following: adenylyl-5-imidodiphosphate = beta-gamma-methylene ATP greater than adenosine tetraphosphate much greater than ATP greater than ADP. Interestingly, these compounds also produced an inhibition of the neurogenic twitch with a similar rank order of potency. This response was not due to the activation of P1 receptors insofar as high concentrations of theophylline failed to attenuate either the inhibition of the neurogenic twitch or the contractile response induced by these agonists. Thus, these data demonstrate the presence of both P1 and P2 purinergic receptors in the rat vas deferens. In addition, the data are consistent with the idea that two distinct classes of P2 receptors exist in this tissue. Furthermore, these data suggest that the rat vas deferens provides a useful tissue for studying compounds which interact with both major subtypes of purinergic receptors.     

Pharmacological characterization and autoradiographic localization of dopamine receptors in human epicardial arteries.

The pharmacological properties and the anatomical localization of dopamine (DA) D1 and D2 receptor sites were studied in normal samples of the human right coronary and anterior interventricular arteries by assessing the effect of DA on the cyclic AMP generating system and by using combined radioreceptor binding and autoradiographic techniques. DA caused a concentration-dependent accumulation of cyclic AMP in membranes of right and anterior interventricular coronary arteries. This effect was antagonized by the selective D1 receptor antagonist SCH 23390 and by other DA receptor antagonists. D2 receptor responses negatively coupled to cyclic AMP generation were obtained by incubating membranes of coronary arteries with DA together with SCH 23390 or with D2 receptor agonists. This D2 effect was abolished by the selective D2 receptor antagonist (-)-sulpiride. [3H]SCH 23390 was bound to sections of the coronary arteries in a manner consistent with the labeling of D1 sites. Light microscope autoradiography revealed the localization of D1 sites in the medial layer of the coronary arteries. [3H]Spiroperidol, in the presence of ketanserin, was bound to sections of the coronary arteries in a manner consistent with the labeling of D2 sites. D2 receptor sites were located within the adventitia and the adventitial-medial border of the two arteries, and are probably prejunctional in nature. These findings indicate the existence of both D1 and D2 receptor sites in human right and anterior interventricular arteries. Moreover, they suggest that coronary vasodilation induced by DA or DA receptor agonists may be the result of a direct coronary vasodilatory activity.     

Modulation of mesoprefrontal dopamine neurons by central benzodiazepine receptors. I. Pharmacological characterization

The benzodiazepine (BZ) recognition sites on the gamma-aminobutyric acid receptor/chloride ionophore complex have been suggested to be involved in the modulation of mesoprefrontal dopamine (DA) neurons. We have examined further the effects of different classes of BZ receptor ligands on DA metabolism in the prefrontal cortex. The anxiogenic inverse agonist FG 7142 elevated selectively 3,4-dihydroxyphenylacetic acid (DOPAC) levels and DO-PAC/DA ratio in the prefrontal cortex in a dose- and time-dependent manner. The activating effect was not, however, observed in any other mesocortical, mesolimbic or nigrostriatal DA terminal fields examined. Pretreatments with BZ agonists such as diazepam, flurazepam, lorazepam and CGS 9896 and BZ antagonists such as Ro15-1788 and CGS 8216 and barbiturates such as pentobarbital, significantly antagonized the beta-carboline-induced elevation of prefrontal DOPAC levels. Furthermore, a significant correlation was found between the pharmacological profile of different BZ receptor ligands on prefrontal DA metabolism and their profiles in behavioral, electrophysiological and receptor binding studies. Agonists increased DA levels and consequently decreased DOPAC/DA ratio in the prefrontal cortex. Inverse agonists, on the other hand, significantly elevated prefrontal DOPAC levels and DOPAC/DA ratio in a dose-dependent manner. Antagonists such as Ro15-1788 and CGS 8216, at low doses, did not alter mesoprefrontal DA metabolism, but at higher doses did elevate DOPAC/DA ratio in the prefrontal cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of the muscarinic receptors mediating phosphoinositide hydrolysis in rat myometrium

Activation of rat uterine myometrial muscarinic receptors with a variety of agonists results in increased phosphatidylinositol metabolism. Activation with carbachol is concentration- and time-dependent and is most apparent by following the accumulation of inositol monophosphate although there are small but significant increases of inositol bisphosphate and inositol trisphosphate. Carbachol stimulation of phospholipid turnover is greatest in the upper third of the uterus. The carbachol-induced increase of inositol monophosphate is antagonized by atropine and by the selective M-3 muscarinic receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methobromide. Pirenzepine, a selective M-1 receptor antagonist is less active, whereas gallamine and 11-2[[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one, selective M-2 receptor antagonists, are minimally effective suggesting that muscarinic M-3 receptors modulate phospholipid turnover in the rat myometrium. Displacement of tritium-quinuclidinyl benzilate binding by muscarinic antagonists also supports the presence of M-3 receptors in the uterus. Incubation with phorbol 12, 13-dibutyrate significantly reduced the accumulation of inositol monophosphate induced by carbachol implying that protein kinase C might modulate the responsiveness of the M-3 receptors in the rat uterus. Our results suggest that the intracellular concentration of calcium required for the contraction of the rat myometrium may be modulated, in part, through M-3 muscarinic receptors coupled to phospholipase C-activated turnover of phosphoinositides.     

Pharmacological characterization of dopamine sulfoconjugation by human platelets

Human platelets accumulate dopamine (DA) from the incubation medium in vitro and convert a significant percentage of the accumulated DA to dopamine sulfate (DAS). Platelet content of both DA and DAS increased linearly with incubation time and tissue concentration. Increasing the concentration of DA in the incubation medium also increased platelet content of both DA and DAS. However, after prolonged incubation times or in the presence of high concentrations of DA, the percentage conversion of DA to DAS decreased; this decrease was reversed at least partially by incubation in the presence of sodium sulfate, suggesting that sulfate availability may limit sulfoconjugation under some conditions. Incubation with fluoxetine produced a concentration-dependent decrease in both DA and DAS content of the platelets. In contrast, the agent 2,6-dichloro-4-nitrophenol, an inhibitor of the enzyme phenol sulfotransferase, did not interfere with DA uptake, but did inhibit DAS formation in a concentration-dependent manner. These studies suggest that the human platelet may represent a useful model system for the study of factors regulating DA sulfoconjugation.     

Pharmacological characterization of muscarinic receptors mediating inhibition of adenylate cyclase activity in the rat retina

Incubation of the rat retina with acetylcholine resulted in about a 20 to 30% decrease of basal cyclic AMP accumulation. Oxotremorine, arecoline, [4-hydroxy-2-butynyl]trimethylammonium chloride, m-chlorocarbanilate and carbachol also inhibited cyclic AMP accumulation. Nicotine had no effect. The response was blocked by atropine and pirenzepine but not gallamine. Intraocular injection of pertussis toxin 72 hr before testing also blocked the response to acetylcholine. The presence of forskolin exaggerated the response to acetylcholine. Intraocular injection of the cholinotoxin AF64A resulted in apparent supersensitivity of the response to acetylcholine. Our results suggest that rat retina contains muscarinic M1 receptors that are coupled negatively to adenylate cyclase.     

Pharmacological analysis of the actions of SKF 82526 on cardiovascular dopamine receptors

We have performed experiments with SKF 82526, a selective dopamine (DA1) receptor agonist to determine whether this compound would activate either ganglionic and/or central DA receptors. Bilateral hindlimb perfusion was carried out at controlled flow rates and changes in hindlimb perfusion pressure were recorded to evaluate the action of SKF 82526 on vascular resistance in anesthetized dogs. Intracisternal administration of SKF 82526 (10 and 40 micrograms/kg) did not produce any changes in blood pressure, heart rate or hindlimb vascular resistance. When the same doses were administered i.v., SKF 82526 produced hypotension and a decrease in perfusion pressure in the innervated limb, whereas perfusion pressure in the denervated limb was not altered. Intravenous SKF 82526 did not produce any changes in heart rate. When given into the lower abdominal aorta, SKF 82526 caused a dose-dependent decrease in perfusion pressure only in the innervated hindlimb, no significant changes in perfusion pressure occurred in the denervated limb. The hypotensive and the hindlimb vasodilatory actions of SKF 82526 could be antagonized by RS-sulpiride. It was discovered that SCH 23390, a selective DA1 receptor antagonist was most potent in blocking the hypotensive action of i.v. SKF 82526; however, it did not influence the neurogenic hindlimb vasodilation produced by intra-aortic SKF 82526. On the other hand, R-sulpiride, another selective DA1 receptor antagonist significantly antagonized the hypotensive as well as hindlimb vasodilatory actions of SKF 82526. S-sulpiride, a selective DA2 receptor antagonist, was least effective in blocking hypotension and did not influence the hindlimb vasodilatory action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of dopamine receptors in the stellate ganglia with selective DA1 and DA2 receptor agonists and antagonists

Experiments were performed with fenoldopam (SKF-82526), a selective DA1 receptor agonist, and quinpirole (LY-171555), a selective DA2 receptor agonist, to determine their actions on ganglionic transmission. Fenoldopam caused significant inhibition of the tachycardia elicited during preganglionic stellate stimulation; however, it did not alter the positive chronotropic responses to postganglionic stellate stimulation, suggesting that the compound exerts its inhibitory action at the ganglia. Electrophysiological experiments in the isolated stellate ganglia showed that fenoldopam produced inhibition of ganglionic transmission as indicated by a significant reduction in the magnitude of the compound postganglionic action potential elicited during preganglionic nerve stimulation. The inhibition of ganglionic transmission produced by fenoldopam both under in vivo and in vitro conditions was antagonized by R-sulpiride and metoclopramide, but not by SCH 23390, S-sulpiride or phentolamine. Quinpirole produced significant inhibition of the tachycardia elicited during both preganglionic as well as postganglionic cardiac sympathetic nerve stimulation. This action of quinpirole was antagonized by RS-sulpiride. In electrophysiological experiments it was discovered that quinpirole caused a significant reduction in the magnitude of the compound action potential elicited during stimulation of preganglionic stellate nerve fibers. This inhibition of ganglionic transmission produced by quinpirole was antagonized by S- but not by R-sulpiride. Although phentolamine antagonized the inhibitory action of quinpirole, it was much less effective than S-sulpiride. Norepinephrine also produced inhibition of ganglionic transmission in the isolated stellate ganglia which was antagonized by phentolamine but not by S-sulpiride. These results demonstrate the presence of two subtypes of specific dopamine receptors in the stellate ganglia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of 5-hydroxytryptamine2 and 5-hydroxytryptamine3 receptors in rat dorsal root ganglion cells

5-Hydroxytryptamine (5-HT) depolarized 87% of the rat dorsal root ganglion cells recorded. 5-HT increased the input resistance (Rin) in 50%, decreased Rin in 41% and produced both responses in 9% of the responding cells. When 5-HT increased the Rin, the response was mimicked by the 5-HT2 agonists alpha-methyl-5-HT, (+/-)-1-(2,5-dimethoxy-4 iodophenyl)-2-aminopropane HCl, quipazine and MK 212 (6-chloro-1-[1-piperazinyl]-pyrazine), but not by 2-methyl-5-HT or carboxamidotryptamine. The response to 5-HT was antagonized by ketanserin, spiperone and methiothepin. The unsurmountable blockade induced by higher concentrations of ketanserin was not explained by pseudo-irreversible antagonism or multiple receptor subtypes, but could result from a two-state receptor model or multiple subtypes of the 5-HT2 receptor. This conclusion is supported by the partial agonist action of DOI. Cells responding to 5-HT with depolarization and decreased Rin responded similarly to 2-methyl-5-HT and phenylbiguanide, but not to alpha-methyl-5-HT or carboxyamidotryptamine. This response was surmountably blocked by ICS 205-930 (3-tropanyl-indole-3-carboxylate) (pA2 = 10.3) and MDL 72222 (3-tropanyl-3,5-dichlorobenzoate)(pA2 = 7.8). The arylpiperazines, quipazine and MK 212, antagonized the action of 2-methyl-5-HT with IC50 values of 8 and 4 nM, respectively. These data indicate that 5-HT2 receptors mediate the increased Rin and 5-HT3 receptors mediate the decreased Rin.     

Pharmacological characterization of lithium reabsorption in the rat.

The effects of furosemide, chlorothiazide and acetazolamide upon fractional lithium excretion (FELi) were studied in acutely lithium-loaded rats. Changes in FELi were compared with the fractional excretions of sodium FENa and calcium FECa. Furosemide and acetazolamide, given separately, produced large and substantially equivalent increases in FELi. Responses of FENa and FECa to acetazolamide were much less than to furosemide. Chlorothiazide, alone or in combination with the other agents, produced moderate increases in FENa, modest increases in FECa and essentially no change in FELi. When furosemide was superimposed upon a previously established acetazolamide diuresis, the response in FELi was additive to that from acetazolamide alone. These results suggest that lithium reabsorption occurs at nephron sites primarily affected by acetazolamide and furosemide in the rat and that a significant amount of lithium reabsorption may take place in the loop of Henle. Acetazolamide, in combination with furosemide, appeared to depress renal tissue lithium accumulation.     

Pharmacological characterization of the receptor mediating electrophysiological responses to dopamine in the rat medial prefrontal cortex: a microiontophoretic study

The pharmacological profile of the receptor mediating inhibitory effects of dopamine (DA) in the rat medial prefrontal cortex (PFC) was characterized using extracellular single unit recording and microiontophoretic techniques. Iontophoretic application of DA inhibited 65% of spontaneously active cells in the deep layers of the PFC, while producing little effect on cells in superficial laminae. The D2 selective antagonist, sulpiride, specifically attenuated DA-induced inhibition of deep layer PFC neurons, without blocking the inhibitory effects of gamma-aminobutyric acid (GABA) or serotonin (5-HT). Surprisingly, sulpiride antagonism did not appear to be stereospecific, as both its (-)- and (+)-isomers proved equally effective at blocking the inhibitory effects of DA. In contrast to sulpiride, the D1 selective antagonist, SCH23390, was much less effective at attenuating inhibitory responses to DA. The effects of selective agonists also were examined on DA-sensitive PFC neurons. The D2 selective agonist, LY171555, and the D1 selective agonist, SKF38393, produced inhibitory effects on a small number of DA-sensitive PFC neurons. However, the majority of cells tested were inhibited only by DA and not by LY171555 or SKF38393. In addition, coiontophoresis of LY171555 and SKF38393. In addition, coiontophoresis of LY171555 and SKF38393 failed to inhibit the majority of DA-sensitive PFC cells tested. LY171555, but not SKF38393, significantly attenuated DA-induced inhibition when applied simultaneously, suggesting that the D2 selective agonist might possess partial agonist/weak antagonist activity at this receptor. These results indicate that the receptor mediating the inhibitory effects of DA in the medial PFC has the pharmacological characteristics of a D2 subtype. However, this receptor may not be identical to D2 sites in other brain regions.     

Pharmacological characterization of alpha adrenoceptors in the rat gastric fundus

Alpha receptors on the intramural cholinergic neurons and on the smooth muscle cells are involved in the inhibitory effect of catecholamines on rat gastric fundus motility. The pharmacological characteristics of these alpha receptors were assessed using longitudinal muscle strips of the rat gastric fundus, contracted to a similar degree by electrical stimulation and by methacholine. All alpha agonists studied (norepinephrine, phenylephrine, methoxamine, clonidine, UK-14,304 and B-HT 920) concentration-dependently inhibited the stimulation-induced contractions. Norepinephrine, phenylephrine, methoxamine and clonidine also inhibited the methacholine-induced contractions, but for the same concentration of agonist, the inhibition was less pronounced than during electrical stimulation-induced contractions; UK-14,304 and B-HT 920 inhibited the methacholine-induced contractions only in a concentration of 10(-4) M. The effect of clonidine and UK-14,304 on electrical stimulation-induced contractions was antagonized competitively by the alpha antagonists rauwolscine and yohimbine (slope in the Schild plot not different from 1). The effect of norepinephrine and phenylephrine on methacholine-induced contractions was antagonized by the alpha antagonists prazosin, corynanthine and yohimbine; against phenylephrine, the antagonism was competitive (slope in the Schild plot not different from 1). It is concluded that the muscular alpha receptors in the rat gastric fundus are of the alpha-1-type. On the postganglionic cholinergic neurons, alpha-2-like receptors are present; it is not yet clear whether the pronounced effect of alpha-1 agonists on the cholinergic neuron activity is due to interaction with these receptors.     

Pharmacological characterization of opioid effects in the rat hippocampal slice

The potencies of several opiates and opioid peptides for potentiating the synaptic activation of CA1 pyramidal cells were compared in the rat hippocampal slice preparation. Morphine and the opioid peptides [D-Ala2, D-Leu5]-enkephalin (DADL), beta-endorphin and Tyr-D-Ser-Gly-Phe-Leu-Thr (a delta agonist) caused a concentration-dependent shift to the left in the input-output curve constructed by plotting population spike amplitude (a measure of evoked firing) as a function of the dendritic field excitatory postsynaptic potential. The concentration-response curves for DADL and morphine had similar slopes and maxima, although the curve for morphine was biphasic due to the addition of a nonopiate effect that became apparent at higher concentrations (greater than or equal to 20 microM). The EC50 values were 68 nM for DADL and 3000 nM for morphine. The IC50 values of naloxone against equieffective concentrations of DADL and morphine were not significantly different. Perfusion of slices with a combination of nearly maximally effective concentrations of DADL and morphine resulted in an effect that was no greater than the maximum effect obtained by either drug alone. The results suggest that these opioids produce their actions through a common pathway. The rank order of potency to produce identical effects was DADL greater than Tyr-D-Ser-Gly-Phe-Leu-Thr greater than beta-endorphin greater than morphine. The kappa agonist ethylketocyclazocine was inactive at concentrations up to 10 microM. The data suggest that delta opioid receptors play a key role in the epileptiform action of these opiates in the CA1 region of the rat hippocampus. However, this opioid response may be different from those characterized in peripheral preparations because ethylketocyclazocine appears to be inactive in the hippocampal CA1 region.     

Pharmacological characterization of PD 118717, a putative piperazinyl benzopyranone dopamine autoreceptor agonist.

PD 118717 (7-[3-[4-(2-pyrimidinyl)-1-piperazinyl]-propoxy]-2H-1- benzopyran-2-one sulfate) proved to be a dopamine (DA) D-2 autoreceptor agonist in biochemical and electrophysiological studies in rats and to exhibit an antipsychotic-like profile in behavioral tests in rodents and monkeys. In vitro binding studies indicated that PD 118717 bound selectively to DA D-2 vs. D-1 receptors and exhibited agonist binding properties (biphasic inhibitory curves and GTP shift) similar to DA. It also had significant affinity for serotonin-(5-HT)1A but not 5-HT1B and 5-HT2 receptors. PD 118717 was active in antagonizing the tau-butyrolactone-induced accumulation of dopa in rat striatum and mesolimbic regions. PD 118717 also depressed the firing of DA neurons in substantia nigra pars compacta of rats. In both of the latter tests the effects of PD 118717 were reversed by haloperidol. PD 118717 decreased brain DA metabolism, decreased DA utilization, decreased accumulation of dopa after inhibition of L-aromatic amino acid decarboxylase, stimulated serum corticosterone and inhibited stimulated serum prolactin levels. PD 118717 did not alter striatal acetylcholine levels; nor did it induce locomotor stimulation or stereotypy in normal animals, suggesting a lack of postsynaptic DA stimulation of normosensitive DA receptors. In tests designed to reveal even weak postsynaptic DA agonist effects, PD 118717 stimulated locomotor activity in 6-hydroxydopamine-lesioned animals and relatively higher doses induced a low degree of stereotyped behavior when combined with the DA D-1 agonist SKF 38393. PD 118717 decreased the accumulation of 5-hydroxytryptophan in brain, an effect probably due to an agonist action at 5-HT1A receptors. PD 118717 decreased spontaneous locomotor activity in rodents, antagonized amphetamine-stimulated hyperactivity in mice and inhibited Sidman avoidance in monkeys, effects seen with antipsychotic agents. Unlike DA antagonist antipsychotics, PD 118717 did not induce extrapyramidal dysfunction in monkeys. PD 118717 displayed behavioral activity after p.o. dosing and its effects did not show tolerance on repeated dosing. In conclusion, PD 118717 has the profile of a DA autoreceptor agonist in neurochemical and neurophysiological tests and produces effects suggestive of antipsychotic efficacy without neurological side effect liability in preclinical behavioral tests.     

Pharmacological characterization of adrenergic receptors of a rabbit cerebral artery in vitro.

In the light of controversy over the functional significance of the abundant sympathetic innervation of large cerebral blood vessels, an in vitro analysis of the adrenergic receptors mediating contractile effects in the rabbit basilar artery was undertaken. Beta adrenergic stimulation had no effect, and agents that block norepinephrine (NE) uptake did not alter contractile responses to l-NE. The l-NE dose-response curve could be resolved into two components S-shaped curves: the first had an ED50 OF 10(-5) M and reached a plateau at 10(-4) M; the second component continued above 10(-3) M without reaching a plateau. Phenylephrine and epinephrine dose-response curves were also biphasic; d-NE responses corresponded to the second phase of the l-NE curve. Relative potencies for the two components were different. For the first component, these were 1:0.23:0.18:0.03 for l-NE, epinephrine, phenylephrine and d-NE, respectively; relative potencies for the second component of the curve were 1:1:0.11:0.23. Phentolamine dissociation constants were analyzed separately for each component. The value for low l-NE concentrations was 5 X 10 (-8) M and, for higher concentrations, it was 3 X 10(-6) . The insensitivity of the alpha adrenergic receptor and the poor responsiveness of the muscle to its activation with agonist concentrations below 10(-4) M can probably account for the small contractile responses to nerve stimulation of large pial arteries in spite of their abundant innervation.     

Pharmacological studies on stress-induced increase in frontal cortical dopamine metabolism in the rat

The effects of a variety of minor tranquilizers and of benzodiazepine inverse agonists on the stress-induced increase in frontal cortical dopamine metabolism have been studied in the rat. Electric footshock stress increased 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the frontal (but not parietal) cortex and in the nucleus accumbens but not in the striatum or ventral tegmental area. Similar stress-induced alterations of frontal cortical DOPAC levels were observed after DSP4-induced noradrenergic denervation or after adrenalectomy. Other types of stress, e.g. conditioned fear (exposure to an environment paired previously with footshock) or swim stress also provoked an elevation of DOPAC levels in the prefrontal cortex. When administered systemically, the anxiolytic agents meprobamate, CL 218,872, CGS 9896, suriclone and the hypnotic/anxiolytic drugs zolpidem and zopiclone all prevented the electric footshock stress-induced augmentation of cortical DOPAC levels whereas the gamma-aminobutyric acid receptor agonists progabide, muscimol and depamide or the sedative alpha-1 adrenoceptor antagonist prazosin were ineffective. The preventive effect of diazepam and zolpidem on the stress-induced biochemical response was antagonized by the benzodiazepine antagonist CGS 8216 but not by the gamma-aminobutyric acid receptor antagonist bicuculline. In nonstressed rats, systemic administration of the anxiogenic benzodiazepine inverse agonists beta-CCM (methyl-beta-carboline-3-carboxylate) and beta-CCE (ethyl-beta-carboline-3-carboxylate), but not of the benzodiazepine antagonists Ro 15-1788 or CGS 8216, caused an increase in frontal cortical DOPAC similar to that provoked by stress and which was antagonized by zolpidem.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of 5-hydroxytryptamine receptors in the canine terminal ileum and ileocolonic junction

The effects of 5-hydroxytryptamine (5-HT), the 5-HT1-like receptor agonist 5-carboxamidotryptamine and the 5-HT3 receptor agonist 2-methyl-5-hydroxytryptamine were studied on circular muscle strips of the canine terminal ileum and ileocolonic junction. Serial administration of 5-HT or of 5-carboxamidotryptamine induced slow tonic contractions that at higher concentrations of 5-HT (10(-4)-3 x 10(-4] were preceded by an initial relaxation and a fast phasic contraction. The concentration-response curves to both agonists were competitively shifted to the right by the mixed 5-HT1/5-HT2 receptor antagonist methysergide. The initial relaxation and fast phasic contraction were inhibited by the 5-HT3 receptor antagonist ICS 205-930 and tetrodotoxin. Atropine blocked the fast phasic contraction, but enhanced the relaxation. During acetylcholine-induced contractions, 5-HT and 2-methyl-5-hydroxytryptamine (greater than or equal to 10(-5) M), but not 5-carboxamidotryptamine, evoked relaxations that were blocked by ICS 205-930 and tetrodotoxin, but not by adrenoceptor antagonists. Thus, in the canine terminal ileum and ileocolonic junction, 5-HT stimulates neuronal 5-HT3 receptors and excitatory 5-HT1-like receptors located on smooth muscle. Stimulation of the 5-HT3 receptors results in an acetylcholine-mediated contraction and a relaxation mediated by an as yet unknown nonadrenergic noncholinergic neurotransmitter.     

Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent [3H]acetylcholine release from rabbit retina labeled in vitro with [3H]choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of [3H]acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of [3H]acetylcholine with the following order of potency: apomorphine greater than or equal to SKF(R)82526 greater than SKF 85174 greater than SKF(R)38393 greater than or equal to pergolide greater than or equal to dopamine (EC50 = 4.5 microM) greater than SKF(S)82526 greater than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of [3H]acetylcholine: SCH 23390 (IC50 = 1 nM) greater than (+)-butaclamol greater than or equal to cis-flupenthixol greater than fluphenazine greater than perphenazine greater than trans-flupenthixol greater than R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating [3H]acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by [3H]SCH 23390, or as determined by adenylate cyclase activity. [3H]SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of [3H]SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate [3H]acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at [3H]SCH 23390 binding sites (r = 0.755, P greater than .05, n = 8). The potencies of antagonists to inhibit dopamine-evoked [3H]acetylcholine release were correlated with their potencies to inhibit the dopamine-stimulated adenylate cyclase (r = 0.759, P less than .05, n = 5) and with their affinities at [3H]SCH 23390 binding sites (r = 0.998, P less than .01, n = 7). We conclude that in rabbit retina dopamine evokes calcium-dependent [3H]acetylcholine release through activation of a site with the pharmacological characteristics of a D-1 dopamine receptor.     

Pharmacological characterization of rat renal medulla dopamine-sensitive cyclic adenosine monophosphate generating system

The dopamine (DA) DA-1 and DA-2 receptors coupled to 3'-5'-cyclic adenosine monophosphate (cAMP) generating system were characterized in membrane particles of the rat kidney medulla. In confirmation of reports using central and other peripheral tissues, activation of DA-1 receptors with DA, apomorphine or SKF 82526 induced accumulation of cAMP. This effect was blocked by the DA-1 receptors antagonist SCH 23390 and by the other DA-2 receptor antagonists fluphenazine and haloperidol. DA-2 receptor responses coupled negatively to the cAMP generating system were obtained by incubating renal medulla membrane particles with DA or SKF 82526 together with SCH 23390. DA-2 receptor responses were also elicited with the receptor agonists quinpirole and bromocriptine in the absence of SCH 23390. These inhibitory effects on cAMP generation were abolished by the DA-2 receptor antagonist l-sulpiride. Our findings suggest that rat renal medulla contains DA DA-1 and DA-2 receptors similar to those found in brain and in other peripheral tissues. The physiological significance of these receptors, if any, should be established in future studies.