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Current therapies for gastroparesis metoclopramide and domperidone carry risks of extrapyramidal symptoms and life-threatening cardiac arrhythmias. Trazpiroben, a novel, potent dopamine D 2 /D 3 receptor antagonist, has low brain permeation and very low affinity for human ether-à-go-go-related gene (hERG) channel inhibition, potentially improving on safety profiles of existing therapies. Trazpiroben demonstrated the following receptor affinities: high for D 2 and D 3 , moderate for D 4 , and minimal for D 1 and D 5 It demonstrated moderate affinity for adrenergic α 1B ( α 1B ) and 5-hydroxytryptamine (5HT) 2A receptors and low potential for off-target adverse events (AEs). Trazpiroben potently inhibited dopamine-activated D 2L receptor activation of cognate G-proteins in human embryonic kidney 293 cell membranes and was a neutral D 2L receptor antagonist. In vivo, trazpiroben dose-dependently increased prolactin release in orally dosed rat (0.1-1 mg/kg). Additionally, multiple oral doses in the rat (100 mg/kg) and dog (50 mg/kg) for 3 days produced robust plasma exposures and prolactin increases in both species. Trazpiroben inhibited retching/vomiting in the dog with apomorphine-induced emesis with a potency (0.1-1 mg/kg) like that of trazpiroben-mediated prolactin increases in rat. Oral trazpiroben (1, 10, and 30 mg/kg) did not affect rat rotarod performance, suggesting low brain penetration. Trazpiroben concentrations were low in cerebrospinal fluid versus plasma after multiple oral doses for 4 days in rat and dog. Trazpiroben weakly inhibited the hERG channel current (concentration causing half-maximal inhibition of control-specific binding of 15.6 µM), indicating little potential for disrupting cardiac rhythm. Overall, trazpiroben is a potent D 2 /D 3 receptor antagonist designed to avoid the serious potential AEs associated with current gastroparesis therapies. SIGNIFICANCE STATEMENT: Trazpiroben is a novel, potent dopamine D 2 /D 3 selective receptor antagonist designed to avoid adverse effects associated with the current pharmacological therapies metoclopramide and domperidone. Preclinical studies have demonstrated low brain penetration and weak affinity for the hERG channel, indicating that trazpiroben is not expected to be associated with central nervous system or cardiovascular safety issues. With these pharmacological properties, trazpiroben may represent a viable new treatment option for gastroparesis because of a potentially improved safety profile relative to existing therapies., (Copyright © 2021 The Author(s).)

Gastroparesis is a chronic neuromuscular disorder of the upper gastrointestinal tract in which episodic exacerbation can lead to frequent hospitalizations and severe disability. Dopamine D 2 /D 3 receptor antagonists have been used to treat patients with gastroparesis with some efficacy; however, their chronic use is limited owing to associated central nervous system (CNS) or cardiovascular safety concerns. Trazpiroben (TAK-906) is a dopamine D 2 /D 3 receptor antagonist under development for the long-term treatment of gastroparesis. Preclinical studies in rat and dog have shown trazpiroben to have minimal brain penetration and low affinity for the human ether-à-go-go-related gene (hERG) potassium channel (IC 50 ,  15.6 µM), thereby reducing the risk of the CNS and cardiovascular adverse effects seen with other dopamine D 2 /D 3 receptor antagonists. This phase 1 trial evaluated the safety, pharmacokinetics, and pharmacodynamics of trazpiroben in healthy participants. Trazpiroben was rapidly absorbed and eliminated (T max , ∼1.1 hours; t 1/2 , 4-11 hours) after administration of single (5-300 mg) and multiple (50 or 100 mg) doses. Receptor target engagement was confirmed for all doses, as indicated by an increase in serum prolactin levels compared with placebo (mean prolactin C max , 134.3 ng/mL after administration of trazpiroben 10 mg vs 16.1 ng/mL with placebo). Therapeutically relevant single and multiple doses of trazpiroben were well tolerated in healthy participants, and no clinically meaningful cardiovascular adverse effects were observed across the whole dose range. These data support the further development of trazpiroben for the treatment of gastroparesis., (© 2021 Altos Therapeutics, LLC. Clinical Pharmacology in Drug Development published by Wiley Periodicals LLC on behalf of American College of Clinical Pharmacology.)

Background and Purpose: The current study examined the utility of the recently described prostacyclin (prostanoid IP) receptor antagonist RO1138452 (2-(4-(4-isopropoxybenzyl)-phenylamino) imidazoline) as a tool for classifying prostanoid receptors., Experimental Approach: pA(2) values were determined on isolated smooth muscle and platelet preparations., Key Results: RO1138452 antagonized relaxation of human pulmonary artery, guinea-pig aorta and rabbit mesenteric artery induced by the selective IP agonist cicaprost. Schild plots had slopes close to unity, generating pA(2) values of 8.20, 8.39 and 8.12 respectively. Non-surmountable antagonism was sometimes found with the higher concentrations of RO1138452, attributable to the EP(3) contractile action of cicaprost. RO1138452 did not block relaxation of guinea-pig trachea induced by the EP(2)-selective agonist butaprost. In contrast, there was a modest inhibition of butaprost-induced relaxation of human pulmonary artery by RO1138452, implying activation of both EP(2) and IP receptors by butaprost. RO1138452 did not affect relaxation induced by PGE(2) (EP(4) agonist) and substance P (NK(1)/endothelium-dependent agonist) in rabbit mesenteric artery. In human and rat platelet-rich plasmas, RO1138452 antagonized cicaprost-induced inhibition of platelet aggregation in a surmountable manner; pA(2) values may have been affected by binding of RO1138452 to plasma protein. RO1138452 did not affect the inhibitory actions of PGD(2) (DP(1) agonist) and NECA (adenosine A(2A) agonist) in human platelets., Conclusions and Implications: The data indicate that RO1138452 is a potent and selective IP receptor antagonist. RO1138452 represents an important addition to our armoury of prostanoid receptor antagonists and a potential clinical agent in situations where prostacyclin has a pathophysiological function.

Nepicastat (RS-25560-197) is a novel, selective, and potent inhibitor of dopamine beta-hydroxylase, which modulates catecholamine levels (reduces norepinephrine and elevates dopamine) in cardiovascular tissues. This study was designed to evaluate the cardiovascular effects of nepicastat. Acute oral administration of nepicastat (0.3, 1, 3, 10, and 30 mg/kg) produced attenuation of the pressor and positive chronotropic responses to preganglionic sympathetic nerve stimulation (about twofold to sixfold shift in the frequency-response curve) in pithed spontaneously hypertensive rats (SHRs). In inactin-anesthetized SHRs, the antihypertensive effects of nepicastat (3 mg/kg, i.v.) were accompanied by a significant decrease in renal vascular resistance (38%), a tendency toward an increase in renal blood flow (22%), and no adverse effects on urine output and Na/K excretion. In conscious, unrestrained, telemetry-implanted SHRs, nepicastat (30 and 100 mg/kg/day for 30 days) produced dose-dependent decreases in mean arterial blood pressure (peak decrease of 20 and 42 mm Hg, respectively) without evoking reflex tachycardia. Long-term, concurrent administration of nepicastat (30 mg/kg/day, p.o.) and a subthreshold dose of enalapril (1 mg/kg/day, p.o.) produced greater antihypertensive effects than those produced by nepicastat alone. In normal dogs, nepicastat (5.0 mg/kg, p.o., b.i.d., for 4.5 days) blunted the positive chronotropic and pressor response to tyramine. These findings suggest that nepicastat functionally modulates sympathetic drive to cardiovascular tissues and may be of value in the treatment of cardiovascular disorders associated with overactivation of the sympathetic nervous system such as hypertension and congestive heart failure.

1. Inhibitory modulation of sympathetic nerve function may have a favourable impact on the progression of congestive heart failure. Nepicastat is a novel inhibitor of dopamine-beta-hydroxylase, the enzyme which catalyses the conversion of dopamine to noradrenaline in sympathetic nerves. The in vitro pharmacology and in vivo catecholamine modulatory effects of nepicastat were investigated in the present study. 2. Nepicastat produced concentration-dependent inhibition of bovine (IC50 = 8.5 +/- 0.8 nM) and human (IC50 = 9.0 +/- 0.8 nM) dopamine-beta-hydroxylase. The corresponding R-enantiomer (RS-25560-198) was approximately 2-3 fold less potent than nepicastat. Nepicastat had negligible affinity (> 10 microM) for twelve other enzymes and thirteen neurotransmitter receptors. 3. Administration of nepicastat to spontaneously hypertensive rats (SHRs) (three consecutive doses of either 3, 10, 30 or 100 mg kg-1, p.o.; 12 h apart) or beagle dogs (0.05, 0.5, 1.5 or 5 mg kg-1, p.o.; b.i.d., for 5 days) produced dose-dependent decreases in noradrenaline content, increases in dopamine content and increases in dopamine/noradrenaline ratio in the artery (mesenteric or renal), left ventricle and cerebral cortex. At the highest dose studied, the decreases in tissue noadrenaline were 47%, 35% and 42% (in SHRs) and 88%, 91% and 96% (in dogs) in the artery, left ventricle and cerebral cortex, respectively. When tested at 30 mg kg-1, p.o., in SHRs, nepicastat produced significantly greater changes in noradrenaline and dopamine content, as compared to the R-enantiomer (RS-25560-198), in the mesenteric artery and left ventricle. 4. Administration of nepicastat (2 mg kg-1, b.i.d, p.o.) to beagle dogs for 15 days produced significant decreases in plasma concentrations of noradrenaline and increases in plasma concentrations of dopamine and dopamine/noradrenaline ratio. The peak reduction (52%) in plasma concentration of noradrenaline and the peak increase (646%) in plasma concentration of dopamine were observed on day-6 and day-7 of dosing, respectively. 5. The findings of this study suggest that nepicastat is a potent, selective and orally active inhibitor of dopamine-beta-hydroxylase which produces gradual modulation of the sympathetic nervous system by inhibiting the biosynthesis of noradrenaline. This drug may, therefore, be of value in the treatment of cardiovascular disorders associated with over-activation of the sympathetic nervous system, such as congestive heart failure.

Substantial evidence indicates that serotonin receptors are involved in the regulation of acetylcholine release in CNS regions important to mnemonic processes, and may thus be exploited pharmacologically as targets for memory improvement. In the present study, the (R) and (S) isomers of a potent serotonin (5-HT3) receptor ligand, RS-56812 were evaluated for potential memory effects in five macaques trained to perform a delayed matching-to-sample (DMTS) task. While both isomers enhanced certain aspects of task performance, the (R) isomer produced more systematic improvements. This differential sensitivity to the isomers in regard to DMTS performance appears to parallel the higher 5-HT3 receptor affinity of the R enanantiomer. The results are consistent with a potential therapeutic role for RS-56812 in disorders involving cognitive decline.

1. Compared to rats maintained on the normal NaCl (0.33%) diet, animals maintained on the low NaCl (0%) diet for 4 weeks exhibited increased plasma aldosterone and chloride and decreased urinary sodium excretion. 2. Rats maintained on the high NaCl (8%) diet for 4 weeks showed increased systolic blood pressure, water intake, urine volume, sodium and dopamine excretion and decreased plasma aldosterone and glomerular filtration rate. 3. Administration of SCH 23390 (10 mg/kg, po), but not domperidone to the high salt diet rats attenuated the diuretic effect, indicating the involvement of DA1 rather than DA2 receptors. The dopamine decarboxylase inhibitor, carbidopa (30 mg/kg, i.p.), also reduced the high salt-induced diuresis. 4. Kidney sections from rats fed the low NaCl diet showed a 63-100% decrease (P < 0.001-0.02) in cortical and medullary DA1 and DA2 binding sites, while rats fed the high NaCl diet demonstrated only a 70% decrease (P < 0.01-0.02) in cortical DA1 binding, without affecting DA2 binding. 5. These data indicate that chronic modification of dietary salt profoundly affects the sodium, water and dopamine excretion and leads to selective modulation of renal dopamine receptor subtypes.

The effects of chronic dietary sodium chloride (NaCl) consumption on renal function and brain dopamine receptors were studied in adult, male normotensive rats. Compared to rats maintained on the normal NaCl (0.33%) diet, animals maintained on the low NaCl (0%) diet for 4 weeks exhibited significant increases in plasma aldosterone, chloride and changes in urinary electrolyte excretion. In contrast, rats maintained on the high NaCl (8%) diet for 4 weeks demonstrated significant increases in urine volume and urinary sodium, chloride and dopamine excretions and water intake. Rats fed the high NaCl diet displayed a 42-59% decrease (p < 0.001-0.05) in D1 binding in the nucleus accumbens (NA), olfactory tubercle (OT) and the striatum (STM), without any effects on D2 binding in these brain regions. Rats maintained on the low NaCl diet also demonstrated decreased D1 binding in the ventral (24%, p < 0.02) and lateral (29%, p < 0.01) STM, but not in the OT, NA, entopeduncular nucleus and substantia nigra. Rats fed low or high NaCl diets exhibited a 35-180% increase (p < 0.01-0.05) in D2 binding in several mid-brain areas (e.g. hypothalamus, thalamus and hippocampus) and hindbrain regions (e.g. superior colliculus and nucleus tractus solitarius) without affecting the D1 binding. These data indicate that chronic modification of dietary salt intake profoundly affects the renal handling of sodium/water excretion and leads to selective up- and/or down-regulation of DA receptor subtypes in different areas of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

1. A series of isoquinolines have been identified as 5-HT3 receptor antagonists. One of these, RS 25259-197 [(3aS)-2-[(S)-1-azabicyclo[2.2.2]oct-3-yl]-2,3,3a,4,5,6-hexahydro- 1- oxo-1H-benzo[de]isoquinoline-hydrochloride], has two chiral centres. The remaining three enantiomers are denoted as RS 25259-198 (R,R), RS 25233-197 (S,R) and RS 25233-198 (R,S). 2. At 5-HT3 receptors mediating contraction of guinea-pig isolated ileum, RS 25259-197 antagonized contractile responses to 5-HT in an unsurmountable fashion and the apparent affinity (pKB), estimated at 10 nM, was 8.8 +/- 0.2. In this tissue, the -log KB values for the other three enantiomers were 6.7 +/- 0.3 (R,R), 6.7 +/- 0.1 (S,R) and 7.4 +/- 0.1 (R,S), respectively. The apparent affinities of RS 25259-197 and RS 25259-198, RS 25233-197 and RS 25233-198 at 5-HT3 receptors in membranes from NG-108-15 cells were evaluated by a [3H]-quipazine binding assay. The -log Ki values were 10.5 +/- 0.2, 8.4 +/- 0.1, 8.6 +/- 0.1 and 9.5 +/- 0.1, respectively, with Hill coefficients not significantly different from unity. Thus, at these 5-HT3 receptors, the rank order of apparent affinities was (S,S) > (R,S) > (S,R) = (R,R). 3. RS 25259-197 displaced the binding of the selective 5-HT3 receptor ligand, [3H]-RS 42358-197, in membranes from NG-108-15 cells, rat cerebral cortex, rabbit ileal myenteric plexus and guinea-pig ileal myenteric plexus, with affinity (pKi) values of 10.1 +/- 0.1, 10.2 +/- 0.1, 10.1 +/- 0.1 and 8.3 +/- 0.2, respectively. In contrast, it exhibited low affinity (pKi <6.0) at 28 other receptors in binding assays, including adrenoceptors (alpha1A, alpha 1B, alpha2A, alpha 2B ,beta1, beta2), muscarinic (M1-M4), dopamine (D1, D2), opioid and other 5-HT(5-HTlA, 5-HTlD, 5-HT2C, 5-HT4) receptors.4. RS 25259-197 was tritium labelled (specific activity: 70 Ci mmol-1) and evaluated in pharmacological studies. Saturation studies with [3H]-RS 25259-197 in membranes from NG-108-15 and cloned homomeric a subunits of the 5-HT3 receptor from N1E-1 15 cells expressed in human kidney 293E1 cells,revealed an equilibrium dissociation constant (Kd) of 0.05 +/- 0.02 and 0.07 +/- 0.01 nM, and Bmax of610 +/- 60 and 1068 +/- 88 fmol mg-1, respectively. Competition studies in NG-108-15 cells indicated a pharmacological specificity entirely consistent with labelling a 5-HT3 receptor, i.e. RS 25259-197> granisetron> (S)-zacopride> tropisetron> (R)-zacopride> ondansetron> MDL 72222.5. In contrast to the majority of radioligands available to label 5-HT3 receptors, [3H]-RS 25259-197 labelled a high affinity site in hippocampus from human post-mortem tissue with an equilibrium dissociation constant (Kd) of 0.15 +/- 0.07 nM and density (BmaX) of 6.8 +/- 2.4 fmol mg-1 protein. Competition studies in this tissue indicated a pharmacological specificity consistent with labelling of a 5-HT3receptor.6. Quantitative autoradiographic studies in rat brain indicated a differential distribution of 5-HT3receptor sites by [3H]-RS 25259-197. High densities of sites were seen in nuclear tractus solitaris and area postrema, a medium density in spinal trigeminal tract, ventral dentate gyrus and basal medial amygdala,and a low density of sites in hippocampal CAl, parietal cortex, medium raphe and cerebellum.7 In conclusion, the functional, binding and distribution studies undertaken with the radiolabelled and non-radiolabelled RS 25259-197 (S,S enantiomer) established the profile of a highly potent and selective5-HT3 receptor antagonist.

1. The pharmacological effects in vivo, of RS 25259-197, a selective 5-HT3 receptor antagonist, have been investigated. 2. In anaesthetized rats, RS 25259-197, administered by the intravenous, intraduodenal or transdermal route, dose-dependently inhibited the von Bezold-Jarisch reflex induced by 2-methyl 5-HT (ID50 = 0.04 micrograms kg-1, i.v., 3.2 micrograms kg-1, i.d. and 32.8 micrograms per chamber, respectively). In this regard, when administered intraduodenally, RS 25259-197 was more potent and exhibited a longer duration of action than either ondansetron or granisetron. 3. In conscious ferrets, RS 25259-197, administered intravenously or orally, dose-dependently inhibited emesis induced by cisplatin. The ID50 estimates of RS 25259-197 were 1.1 micrograms kg-1, i.v. and 3.2 micrograms kg-1, p.o. In this respect, RS 25259-197 was more potent than ondansetron and equipotent with granisetron. 4. In conscious dogs, RS 25259-197, administered intravenously or orally, dose-dependently inhibited emesis induced by cisplatin (ID50 = 1.9 micrograms kg-1, i.v. and 8.5 micrograms kg-1, p.o.), dacarbazine (ID50 = 4.1 micrograms kg-1, i.v. and 9.7 micrograms kg-1, p.o.), actinomycin D (ID50 = 4.9 micrograms kg-1, i.v. and 2.5 micrograms kg-1, p.o.) and mechlorethamine (ID50 = 4.4 micrograms kg-1, i.v. and 3.0 micrograms kg-1, p.o.). Against each of the emetogenic agents, RS 25259-197 was very much more potent than ondansetron. When tested at equi-effective intravenous doses against cisplatin-induced emesis in dogs, RS 25259-197 had a longer duration of anti-emetic activity (7 h) than ondansetron (4 h). At doses up to and including 1000 microg kg-1, p.o., neither RS25259-197 nor ondansetron was capable of inhibiting apomorphine-induced emesis.5. At doses up to 1000 microg kg-1, i.v., RS 25259-197 produced no meaningful haemodynamic changes in anaesthetized dogs.6. In summary, RS 25259-197 is a novel, highly potent and orally active 5-HT3 receptor antagonist in vivo. With respect to its anti-emetic activity, RS 25259-197 appears to be a significant improvement over ondansetron in terms of potency and duration of action.

In the present studies, the pharmacology and regulation of the functional muscarinic receptors on HSDM1C1 cells were probed using phosphoinositide (PI) turnover assays. In addition, the receptor binding of the putative M3-selective radioligand, [3H]4-DAMP, to cell homogenates was characterized. Carbachol (EC50 = 9 microM), (+)muscarine (EC50 = 4.5 microM) and cis-dioxolane (EC50 = 0.72 microM) were full agonists which stimulated PI turnover by 13.3 +/- 1.0 fold above basal values. The potencies of numerous agonists in this assay system were relatively similar to their affinities in receptor binding assays. Exposure of HSDM1C1 cells to 10 nM-10 microM muscarine during the last 24h of [3H]myo-inositol-labeling resulted in a concentration-dependent reduction in the cis-dioxolane affinity and maximal PI response induced by subsequent treatment with cis-dioxolane. Pertussis toxin (5-2000 ng/ml) caused a partial reduction in the cis-dioxolane-induced PI turnover. Likewise, exposure of the HSDM1C1 cells to an active phorbol ester (TPA) resulted in a partial inhibition of the cis-dioxolane-induced (100 microM) PI turnover. The half-maximal effect of TPA was produced at 1.8 +/- 0.3 nM. [3H]4-DAMP binding to cell homogenates was of high affinity (Kd = 0.19 +/- 0.04 nM) and moderate capacity (Bmax = 201 +/- 22 fmol/mg protein). The pharmacological specificity (4-DAMP > p-FHHSiD > dicyclomine > pirenzepine > methoctramine > AFDX-116 > gallamine) resembled that for [3H]NMS binding and correlated well with that observed for inhibition of PI turnover. These studies further support the identification of M3 receptors on HSDM1C1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The pharmacological properties of the (R) and (S) enantiomers of RS 56532 have been studied in vitro and in vivo. In radioligand binding studies at 5-HT4 receptors in guinea-pig striatum, (S) RS 56532 exhibited a higher affinity than (R) RS 56532 (-log Ki = 7.6 and 6.5, respectively). (S) RS 56532 acted as a potent agonist at 5-HT4 receptors mediating relaxation of rat oesophageal muscularis mucosae (-log EC50 = 7.9) while (R) RS 56532 acted as a weaker agonist at this receptor (-log EC50 < 6.0). These data suggest that at 5-HT4 receptors, the enantiomeric selectivity of RS 56532 was (S) > (R). In binding studies at 5-HT3 receptors in rat cortex, (R) RS 56532, conversely, exhibited a higher affinity than (R) RS 56532 (-log Ki = 9.1 and 8.0, respectively). At 5-HT3 receptors in guinea-pig isolated ileum, (R) RS 56532 exhibited an affinity (-log KB) of 7.9, whereas (S) RS 56532 (1 nM-1 microM) was inactive. No agonism was observed at ileal 5-HT3 receptors with either enantiomers. These data suggest that at 5-HT3 receptors in rat and guinea-pig, both enantiomers acted as antagonists, with (R) > (S) RS 56532. At the non-5-HT3, high affinity '(R) zacopride' site, (R) RS 56532 exhibited a higher affinity than (S) RS 56532 (-log Ki = 6.1 and 4.9). This site was insensitive to potent 5-HT3 antagonists such as (R) YM 060 or ondansetron. However, it was recognized with relatively high affinity (-log Ki = 7.5) by the (R), but not (S) enantiomer, of RS 42358 (-log Ki = 4.7). Since (S) RS 42358 is a high affinity 5-HT3 receptor antagonist, these data further highlight the dissimilarity between the 5-HT3 receptor and the '(R) zacopride' site. The '(R) zacopride' site also appeared to be pharmacologically distinct from the 5-HT4 receptor, since 5-HT4 ligands such as renzapride, SDZ 205,557 or RS 23597-190 exhibited low affinities. The enantiomeric selectivity of (R) and (S) RS 56532 in vivo was consistent with findings in vitro. At 5-HT4 receptors mediating tachycardia in the pig, 5-HT induced a dose-dependent tachycardia (ED50 = 3 micrograms kg-1, i.v.; maximum response = 90-100 beats min-1). (S) RS 56532 increased heart rate by 88 min-1 with a potency of (ED50) of 3 micrograms kg-1, i.v.(ABSTRACT TRUNCATED AT 400 WORDS)

Bradykinin (BK) and its analogs (1 nM-100 microM) stimulated phosphoinositide (PI) turnover in murine fibrosarcoma (HSDM1C1) cells in a concentration-dependent manner. The relative potencies (EC50) were: BK = 48 +/- 4 nM; Lys-BK = 39 +/- 3 nM; Met-Lys-BK = 158 +/- 33 nM, Des-Arg9-BK = 2617 +/- 598 nM (means +/- SEM, n = 3-14). All these analogs were full agonists and they produced up to 5.4 +/- 0.4-fold stimulation of PI turnover at the highest concentration (10-100 microM) of the peptides. In contrast, the analogs [D-Arg0-HYP3-Thienyl5,8-D-Phe7]-BK (HYP3-antagonist), [D-Arg0-HYP3-Thienyl,5,8-D-Phe7]-BK (Thienyl antagonist) and Des-Arg9-Leu8-BK were inactive, as agonists, at 0.1 nM-1 microM in this system. These data suggested that BK-induced PI turnover in these cells was mediated via B2-type of BK receptors. This was confirmed further by the fact that both the B2-selective Hyp3- and Thienyl-antagonists inhibited BK-induced PI turnover with KBS of 369 +/- 51 nM and 368 +/- 118 nM respectively while the B1-selective antagonist, Des-Arg9-Leu8-BK, was inactive at 1 microM. [3H]BK receptor binding studies revealed two binding sites, one with high affinity (Kd = 0.24 +/- 0.06 nM; Bmax = 1.4 +/- 0.4 pmol/g tissue) and the other with low affinity (Kd = 18.5 +/- 0.95 nM; Bmax = 25.1 +/- 0.52 pmol/g tissue), on HSDM1C1 cell homogenates. The rank order of affinity of BK analogs at inhibiting specific [3H]BK binding was similar to that found for PI turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

The pharmacological activity of RS 42358-197, a novel 5-HT3 receptor antagonist has been evaluated in vitro and in vivo. In functional experiments in vitro, RS 42358-197 behaved as a competitive antagonist against 5-HT-induced contractions in the guinea pig ileum (low-potency phase), yielding a pA2 estimate of 8.1. RS 42358-197 was devoid of any agonistic or antagonistic activity at 5-HT1-like receptors (contraction of canine saphenous vein), 5-HT2 receptors (contraction of rabbit aorta) or 5-HT4 receptors (contraction of guinea pig ileum, high-potency phase). RS 42358-197 failed to affect the concentration-effect curve to substance P in guinea pig ileum. In anesthetized rats. RS 42358-197, administered by the intravenous, intraduodenal or transdermal route, dose-dependently inhibited the Bezold-Jarisch reflex induced by 2-methyl 5-HT (ID50:0.05 micrograms/kg; i.v., 5.7 micrograms/kg; i.d., and 11.6 micrograms/chamber, respectively). In this regard, when administered intraduodenally, RS 42358-197 was more potent and exhibited a longer duration of action than either ondansetron or granisetron. In dogs, RS 42358-197, administered either intravenously or orally, dose-dependently inhibited the emesis induced by cisplatin, actinomycin and cyclophosphamide, but not that induced by apomorphine. When tested at maximally effective doses against cisplatin-induced emesis in dogs, RS 42358-197 had a longer duration of antiemetic activity (> 6 h) than ondansetron (2 h). RS 42358-197, administered orally, also afforded protection against cisplatin-induced emesis in ferrets. At doses that showed marked anti-emetic activity in dogs (10-100 micrograms/kg; i.v. and 100-1000 micrograms/kg; i.d.), RS 42358-197 did not produce any hemodynamic changes.(ABSTRACT TRUNCATED AT 250 WORDS)

The S-isomer of the novel 5-HT3 receptor antagonist RS-42358 ((S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1-H- benzo[de]isoquinolin-1-one, RS-42358-197) disinhibited behaviour in the mouse suppressed by the aversive situation of the light/dark test box. RS-42358-197 was effective at sub-ng/kg dose levels and the efficacy was maintained over a 100 million-fold dose range. In contrast, the R-isomer was ineffective at all doses studied. The S-isomer also disinhibited a suppressed behaviour in social interaction and elevated X-maze tests in the rat and reduced anxiety-related behaviours in a marmoset human threat test. RS-42358-197 prevented the exacerbation of the suppression of behaviour in the mouse light/dark test following withdrawal from treatment with alcohol, nicotine, cocaine and diazepam. Thus, the S-isomer of RS-42358 has a consistent non-sedating anxiolytic profile in rodent and primate models. It is exceptionally potent and a maintained efficacy at high doses distinguishes its actions from many other 5-HT3 receptor antagonists.

1. The potency of indole analogues has been studied, in vitro, at 5-hydroxytryptamine4 (5-HT4) receptors mediating contractions of guinea-pig ileum and relaxation of rat oesophagus. These have been compared to other 5-HT receptors in canine saphenous vein (5-HT1-like), rabbit aorta (5-HT2), and guinea-pig ileum (5-HT3). 2. At receptors mediating 5-HT4 responses in ileum and oesophagus, the rank orders of potency were similar. These rank orders differed from those observed at 5-HT1-like, 5-HT2, and 5-HT3 receptors. In particular, 5-hydroxy N,N, dimethyltryptamine but not 5-methoxy N,N, dimethyltryptamine acted as agonists at 5-HT4 receptors. At 5-HT1-like, 5-HT2 and 5-HT3 receptors these compounds were both active. 3. The 5-HT receptors mediating contractions of canine cephalic vein exhibited a rank order profile similar to that observed at receptors mediating contractions of canine saphenous vein, suggesting stimulation of a 5-HT1-like receptor. 4. The rank order of potency of the substituted indoles differed at 5-HT receptors mediating responses in canine saphenous vein, rabbit aorta and guinea-pig ileum (determined in the presence of 5-methoxytryptamine to desensitize 5-HT4 receptors), suggesting the presence of three distinct receptors. Indeed, at 5-HT3 receptors in the ileum, only three agonists (5-HT, 2-methyl-5-HT and 5-hydroxy N,N, dimethyltryptamine) elicited a response, while all remaining compounds were inactive. 5. It is concluded that rank orders of indole potency can prove useful in the delineation of 5-HT subtypes and together with differential antagonist affinities support the existence of four 5-HT receptor subtypes.

The action of the cervane alkaloid, imperialine, has been assessed at M1, M2 and M3 receptors in functional assays and at M1, M2, M3 and putative M4 sites in binding studies. In functional studies, imperialine acted as a selective surmountable antagonist at M2 receptors in guinea-pig isolated atria and uterus (-log KB = 7.7 and 7.4, respectively), in comparison to M1 receptors in canine isolated saphenous vein (-log KB = 6.9) or M3 receptors in a range of guinea-pig isolated smooth muscles including ileum, trachea, fundus, seminal vesicle or oesophagus (-log KB = 6.6-6.8). In rat aorta, the -log KB value at the M3 receptor (5.9) was slightly, but significantly, lower. In competition radioligand binding studies, imperialine was also selective toward to M2 sites in rat myocardium (-log Ki = 7.2) with respect to M1 and M3 sites (rat cerebral cortex, rat submaxillary gland; -log Ki = 6.1 and 5.7, respectively). However, it did not significantly discriminate between rat cardiac M2 sites and putative M4 sites in rabbit lung (-log Ki = 6.9). Imperialine resembles the alkaloid himbacine in terms of its pharmacological profile at muscarinic receptor subtypes in that it acts as an M2 selective antagonist with respect to M1 or M3 sites. It may also provide a second, commercially available, antagonist with which to discriminate between M1 and M4 receptors.

Binding of [3H]GBR12935 to homogenates of mouse and rat striatum and kidney was studied. [3H]GBR12935 bound to both tissue preparations with high affinity (mouse striatum Kd = 2.4 +/- 0.4 nM, n = 4; mouse kidney Kd = 3.8 +/- 0.9 nM, n = 4), in a saturable (striatal Bmax = 1.5 +/- 0.4 pmol/mg protein; kidney Bmax = 4.9 +/- 0.5 pmol/mg protein) and reversible manner. Saturation experiments revealed the presence of a single class of high affinity binding sites in both tissues of both species. Mouse kidney appeared to possess a greater density of [3H]GBR12935 binding sites than the striatum while the reverse situation prevailed for the rat. Although two dopamine uptake inhibitors, namely GBR12909 and benztropine, displaced [3H]GBR12935 binding from striatal and kidney homogenates with a similar affinity in both tissues of these species, unlabelled mazindol, (+/-)cocaine, nomifensine and amfonelic acid were significantly (P < 0.001-0.02) more potent inhibitors of [3H]GBR12935 binding in the striatum than in the kidney. While the pharmacological profile of [3H]GBR12935 binding in the rodent striatum compared well with that of the dopamine transporter reported previously, the pharmacology in the kidney was considerably different to that in the striatum. GBR12909 (1-30 mg/kg, i.p.), a close analog of GBR12935, induced significant antidiuretic and antinatriuretic effects in spontaneously hypertensive rats. These data suggest that while [3H]GBR12935 labels the dopamine uptake sites in the brain, it does not appear to label similar sites in the kidney. The mechanism of action of GBR12909 on sodium and water excretion remains to be determined.

1. The effects of acute desensitization of muscarinic receptors mediating contractile responses of the guinea-pig ileal longitudinal muscle were studied in vitro, using similar conditions for both functional and radioligand binding studies. 2. The pA2 values for a number of muscarinic antagonists (pirenzepine, methoctramine, (+/-)para-fluoro-hexahydro-siladifenidol and 4-diphenylacetoxy-N-methyl piperidine-methiodide) indicated that the contractile response to carbachol was mediated through an M3 muscarinic receptor. In binding experiments the muscarinic receptor subtype population in ileal longitudinal muscle was found to be heterogeneous, consisting of approximately 77% M2 and 23% M3 receptors. 3. Pre-exposure of ileal longitudinal muscle to 10 microM carbachol for 30 min produced a reduction (28 +/- % of control maximum) in the maximum contractile response and a dextral shift in the concentration-effect curve to carbachol. Prior equilibration (60 min) with (+/-)p-F-HHSiD (1 microM), but not with methoctramine (1 microM) or pirenzepine (0.3 microM), prevented the desensitization. Desensitization under these conditions did not alter either the apparent affinity, the total number of binding sites or the relative, proportions of M2 and M3 muscarinic receptors, as determined in radioligand binding studies. Desensitization did not cause any meaningful change in either the apparent affinity of carbachol or the proportion of the high and low affinity binding sites. 4. It is concluded that desensitization of the contractile responses of the guinea-pig ileal longitudinal muscle is a result of M3 but not M2 muscarinic receptor desensitization. Acute desensitization, therefore, is not accompanied by meaningful changes in the total number of both M2 and M3 receptors or by alterations in the affinity of the receptor to ligands.

1. The muscarinic pharmacology of a novel oxadiazole muscarinic agonist, (+/-) L-660,863, [+/-3-(3-amino-1,2,4-oxadiazole-5-yl)-quinuclidine] has been studied using pharmacological, radioligand binding and biochemical techniques, in vitro. 2. In isolated tissue experiments, (+/-)L-660,863 was a more potent agonist than carbachol in all preparations studied, being most potent at muscarinic receptors mediating negative chronotropy in guinea-pig right, spontaneously beating atria and least potent at receptors mediating contractions in canine saphenous vein and endothelial denuded rabbit aorta (-log EC50 values were 8.8, 6.6 and 6.3, respectively. The apparent affinities (-log KA) of (+/-)L-660,863) estimated by receptor inactivation, showed some selectivity toward the atrial M2 muscarinic receptor (-log KA = 7.6) in comparison to the M1 or M3 muscarinic receptors (-log KA = 5.4 and 6.2) respectively. This degree of selectivity was also observed in competition radioligand binding studies. 3. At M3 muscarinic receptors mediating inositol phosphates (IPs) accumulation in longitudinal muscle of guinea-pig ileum, the potency of (+/-)L-660,863 (-log EC50 value = 6.2) was similar to the apparent affinity calculated at M3 muscarinic receptors in the functional studies (see above). In contrast, at muscarinic receptors mediating IPs accumulation in guinea-pig atria and ventricles, the potency for (+/-)L-660,863 (-log EC50 = 6.2 and 6.4, respectively) was lower than the apparent affinity calculated at M2 muscarinic receptors from inotropic and binding studies in cardiac tissue (see above).(ABSTRACT TRUNCATED AT 250 WORDS)

The muscarinic receptor mediating stimulation of PI hydrolysis in guinea pig atria and ventricles has been studied. The non-selective muscarinic agonist (+)-cis-dioxolane elicited this response, concentration-dependently, with a potency indicative of a low receptor reserve. The potency of a novel, M2-selective agonist, L-660,863 (-log EC50 = 6.3, atria; 6.0, ventricles) was observed to be lower than its apparent affinity (-log KA = 7.6) for M2 receptors, indicating an action probably mediated by a population distinct from that producing negative inotropy in the same tissue. The inhibition of the response to (+)-cis-dioxolane by several muscarinic antagonists (atropine, pirenzepine, AF-DX 116, methoctramine, HHSiD and pFHHSiD) generated an affinity profile for this receptor also dissimilar to that described for the receptor mediating the classical cardiac 'M2' response. Although no other muscarinic receptor mRNA has been detected in this tissue, these data suggest the presence of a second population of muscarinic sites, which may signify an M2 receptor diversity.

The effect of centrally and peripherally administered dopamine D1 and D2 specific compounds on core body temperature in mice was investigated. Quinpirole (LY-17155), a D2 agonist, induced a dose-dependent fall in body temperature (2.4-11.6%; p less than 0.003) when injected intraperitoneally (ip, 0.3-3.0 mg/kg) and intracerebroventricularly (icv, 0.1 mg/kg). This quinpirole-induced (1.0 mg/kg, ip) hypothermia was reversed by the central and peripheral administration of the D2 antagonists S-(-)-sulpiride (3.0-30.0 mg/kg, ip; 0.1-3.0 mg/kg, icv) and spiperone (0.03 and 0.1 mg/kg, ip; 0.03-3.0 mg/kg, icv). Domperidone, a D2 antagonist which does not cross the blood brain barrier, had no effect on quinpirole-induced hypothermia (1.0-10.0 mg/kg, ip). Domperidone partially reversed quinpirole-induced hypothermia at 0.1-30.0 mg/kg, icv. The D1 agonist, SKF-38393 at a high dose of 10.0 mg/kg, ip mildly attenuated quinpirole-induced hypothermia (a 1.8% increase in temperature). SKF-38393 at 10.0 mg/kg, icv potentiated quinpirole-induced hypothermia. SCH-23390 (0.1-3.0 mg/kg, ip), a D1 antagonist, had no effect on quinpirole-induced hypothermia and potentiated the hypothermia when administered icv. An ineffective icv dose of spiperone (0.01 mg/kg) in reversing quinpirole-induced hypothermia was rendered effective by prior administration of SCH-23390 (0.1-3.0 mg/kg, icv) but not by SKF-38393 (1.0-10.0 mg/kg, icv). These data suggest a central D2 receptor mechanism mediating hypothermia in mice which is capable of being modulated by the D1 receptor.

1. Apparent affinity constants (KD) for prenalterol, an agonist of low intrinsic efficacy at beta 1-adrenoceptors in rat left atria, have been determined by use of receptor desensitization and functional antagonism induced by isoprenaline and carbachol, respectively. The values obtained have been compared to those values estimated with the irreversible beta-adrenoceptor antagonist, bromoacetylalprenololmenthane (BAAM). 2. The -log KD values for prenalterol estimated by desensitization or irreversible antagonism ranged from 6.8-7.1 and 6.2-7.1, respectively. 3. Carbachol produced functional antagonism of the response to prenalterol even though it was removed before addition of prenalterol. This effect was mediated by M2-muscarinic receptors. Pretreatment of animals with pertussis toxin did not affect the functional antagonism elicited by carbachol. The apparent KD value obtained after pre-exposure to carbachol (6.8) was similar to those estimated by use of either alkylation with BAAM or desensitization with isoprenaline (see above). 4. It is concluded that acute desensitization or functional antagonism of responses to agonists of low intrinsic efficacy provides a means to estimate apparent KD constants. This approach could be useful to characterize receptors for which an irreversible antagonist may not be available.

RS-93427, a novel analog of prostacyclin, increased adenylate cyclase activity in human platelet membranes (EC50 = 42 nM) to approximately the same maximum level as that produced by prostacyclin (EC50 = 87 nM). The concentration-response curve for RS-93427 appeared to be monophasic. However, a selective prostaglandin D2 antagonist (BW A868C) significantly reduced the stimulation of adenylate cyclase produced by low concentrations of RS-93427 (3.2 to 32 nM). RS-93520, a stereoisomer of RS-93427, also stimulated adenylate cyclase activity but in a biphasic pattern. BW A868C reduced the activation produced by low concentrations of RS-93520 with a 100-fold shift in the response curve. Maximum stimulation by RS-93520 (4.5-fold) was less than that obtained with prostaglandin D2 (7.3-fold). Thus, the stimulation of adenylate cyclase activity by low concentrations of RS-93520 is due to an interaction with prostaglandin D2 receptors while the activation by RS-93427 is mediated by both prostacyclin and prostaglandin D2 receptors. Additional data in support of these conclusions was obtained when these prostaglandins were tested as inhibitors of ADP-induced platelet aggregation in the presence or absence of BW A868C. The potent stimulation of prostaglandin receptors with chimeric molecules provides some insight into the structural features required for receptor activation.

Muscarinic receptors mediating the release of epithelial derived relaxant factor (EpDRF) have been studied by using both contractions of the guinea-pig tracheal strip (with epithelium intact or denuded) or a coaxial bioassay assembly (rat anococcygeus-recipient; guinea-pig trachea-donor tissue). Indomethacin (1 microM/1) and physostigmine (0.1 microM/1) were both present throughout the study. In the tracheal strip studies, the potencies and maximal effects of all agonists studied (acetylcholine, arecoline, bethanechol, carbachol, (+)cis-dioxolane, ethoxyethyltrimethylammonium, L-660,863, (+/-)methacholine and OXA-22) were not affected or were only slightly (but significantly) reduced by removal of the epithelium. The -log KB for the muscarinic antagonists, atropine, pirenzepine, methoctramine and 4-DAMP (4-diphenyl-acetoxy-N-methylpiperidine) were also not affected and the -log KB values were consistent with M3 muscarinic receptor function. However, the -log KB value of para-fluoro-hexahydro-siladifendol (p-F-HHSiD) was significantly (P less than 0.05) increased upon epithelial denudation (epithelium intact, 7.1; epithelium removed, 7.6). The coaxial bioassay assembly provided more convincing evidence for release of EpDRF in that all muscarinic agonists studied caused relaxations of a precontracted anococcygeus tissue. These relaxations were observed only in the presence of a tracheal tube possessing an intact epithelium. The rank order of potencies for agonists at receptors mediating EpDRF dependent relaxation were similar to those estimated at receptors causing contraction. These data suggested that a substantial receptor reserve was associated with the receptors mediating both EpDRF release and contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

The biochemical and pharmacological properties of [3H]MK-801 binding to the N-methyl-D-aspartate (NMDA) receptor-channel in homogenates of mouse, guinea pig and dog brain, dog cerebral cortex and rat spinal cord were determined using radioligand binding techniques. Specific [3H]MK-801 binding increased linearly with increasing tissue concentration and in general represented 80-93% of the total binding at 6-8 nM radioligand concentration. [3H]MK-801 interacted with brain and spinal homogenates with high affinity. The dissociation constants (KD) for all tissues studied were similar ranging between 7.9 and 11.9 nM, whereas the maximum number of binding sites (Bmax) showed a wide, tissue-dependent range (0.1-6.75 pmol/mg protein). The rank order of tissue enrichment was found to be as follows: mouse brain much greater than dog cerebral cortex much greater than dog brain much greater than guinea pig brain much greater than rat spinal cord. Specific [3H]MK-801 binding in rodent and dog brain, dog cerebral cortex and rat spinal cord exhibited a similar pharmacological profile (correlation coefficients = 0.93-0.99). The rank order of potency of unlabelled compounds competing for [3H]MK-801 binding was: (+)MK-801 greater than (-)MK-801 greater than phencyclidine greater than (-)cyclazocine much greater than (+)cyclazocine greater than or equal to ketamine greater than (+)N-allyl-N-normetazocine greater than (-)N-allyl-N-normetazocine greater than (-)pentazocine greater than (+)pentazocine. NMDA, Kainate, quisqualate and several other compounds failed to inhibit [3H]MK-801 binding at 100 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The biochemical and pharmacological properties of 5-HT3 receptors in homogenates of NG108-15 and NCB-20 neuroblastoma cells and rat cerebral cortex have been ascertained by the use of [3H]-quipazine and [3H]-GR65630 binding. 2. In NG108-15 and NCB-20 cell homogenates, [3H]-quipazine bound to a single class of high affinity (NG108-15: Kd = 6.2 +/- 1.1 nM, n = 4; NCB-20: Kd = 3.0 +/- 0.9 nM, n = 4; means +/- s.e.means) saturable (NG108-15: Bmax = 1340 +/- 220 fmol mg-1 protein; NCB-20: Bmax = 2300 +/- 200 fmol mg-1 protein) binding sites. In rat cortical homogenates, [3H]-quipazine bound to two populations of binding sites in the absence of the 5-hydroxytryptamine (5-HT) uptake inhibitor, paroxetine (Kd1 = 1.6 +/- 0.5 nM, Bmax1 = 75 +/- 14 fmol mg-1 protein; Kd2 = 500 +/- 300 nM, Bmax2 = 1840 +/- 1040 fmol mg-1 protein, n = 3), and to a single class of high affinity binding sites (Kd = 2.0 +/- 0.5 nM, n = 3; Bmax = 73 +/- 6 fmol mg-1 protein) in the presence of paroxetine. The high affinity (nanomolar) component probably represented 5-HT3 binding sites and the low affinity component represented 5-HT uptake sites. 3. [3H]-paroxetine bound with high affinity (Kd = 0.02 +/- 0.003 nM, n = 3) to a site in rat cortical homogenates in a saturable (Bmax = 323 +/- 45 fmol mg-1 protein, n = 3) and reversible manner. Binding to this site was potently inhibited by 5-HT uptake blockers such as paroxetine and fluoxetine (pKi s = 8.6-9.9), while 5-HT3 receptor ligands exhibited only low affinity (pK; < 7). No detectable specific [3H]-paroxetine binding was observed in NG108-15 or NCB-20 cell homogenates. 4. [3H]-quipazine binding to homogenates of NG108-15, NCB-20 cells and rat cortex (in the presence of 0.1 microM paroxetine) exhibited similar pharmacological characteristics. 5-HT3 receptor antagonists competed for [3H]-quipazine binding with high nanomolar affinities in the three preparations and the rank order of affinity was: (S)-zacopride > quarternized ICS 205-930 2 granisetron > ondansetron > ICS 205-209 (R)-zacopride > quipazine > renzapride > MDL-72222 > butanopride > metoclopramide. 5. [3H]-GR65630 labelled a site in NCB-20 cell homogenates with an affinity (Kd = 0.7 + 0.1 nms n = 4) and density (B&#95;&#95; = 1800 + 1000 fmol mg- protein) comparable to that observed with [3H]-quipazine. Competition studies also indicated a good correlation between the pharmacology of 5-HT3 binding sites when [3H]-GR65630 and [3H]-quipazine were used in these cells. 6. In conclusion, [3H]-quipazine labelled 5-HT3 receptor sites in homogenates of NG108-15 cells, NCB-20 cells and rat cerebral cortex. In rat cortical homogenates, [3H]-quipazine also bound to 5-HT uptake sites, which could be blocked by 0.1 microM paroxetine. The pharmacological specificity of the 5-HT3 receptor labelled by [3H]-quipazine was similar in the neuroblastoma cells and rat cortex and was substantiated in NCB-20 cells by the binding profile of the selective 5-HT3 receptor antagonist, [3H]-GR65630.

[3H](3-Me-His2) thyrotropin-releasing hormone ([3H]MeTRH) bound to TRH receptors in rodent, rabbit and dog brain and spinal cord (SC), and in rat, sheep, bovine and dog anterior pituitary (PIT) glands, with high affinity (dissociation constants, KdS = 5-9 nM; n = 3-4) but to different densities of these sites (Bmax range 6-145 fmol/mg protein) (rabbit SC greater than sheep PIT much greater than G.pig brain greater than dog brain greater than rat brain greater than bovine and dog PIT). Various TRH analogs competitively inhibited [3H]MeTRH binding in these tissues with a similar rank order of potency: MeTRH greater than TRH greater than CG3703 greater than or equal to RX77368 greater than or equal to MK-771 greater than TRH Glycinamide greater than Glu1-TRH much greater than CG3509 greater than or equal to NVal2-TRH much much greater than TRH free acid much much greater than and cyclo-His-Pro, indicating a pharmacological similarity of CNS and pituitary TRH receptors. While most TRH analogs displaced [3H]MeTRH binding with a similar potency in the different species, TRH exhibited a 2-fold lower affinity in the rat and G.pig brain than in other tissues of other species. Similarly, CG3703 was 2.4-4.5 times more active in the rabbit brain than in the rodent and dog brain, and also more potent in the rabbit brain as compared to the sheep PIT.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this study was to characterize the biochemical and pharmacological properties of bradykinin receptors in the guinea pig central and peripheral tissues using radioligand binding techniques. Specific [(3)H]bradykinin ([(3)H]BK) receptor binding to homogenates of guinea pig cerebral cortex, hippocampus, spinal cord, ileum, kidney, heart, vas deferens and uterus was of high affinity, saturable and reversible. Scatchard analysis of saturation (0.005-1 nM) data revealed the presence of a single population of non-interacting nanomolar affinity (generally 0.14-0.38 nM) binding sites in all these tissues, with the ileum having the highest affinity (K(D) = 0.02 nM) and the greatest density of sites (B(max) = 5.8 +/- 1.8 pmol/g tissue). The rank order of tissue enrichment in terms of [(3)H]BK binding sites was: ileum > uterus > kidney > heart > vas deferens > spinal cord > cerebral cortex > hippocampus. Unlabelled BK and its analogs inhibited [(3)H]BK binding in the above tissues in a concentration-dependent manner and with the same rank order of potency: BK > Lys-BK > Met-Lys-BK > [ d -Arg (0)-Hyp (3)- d -Phe (7)]BK ? [ d -Arg (0)-Hyp (3)-Thi (5,8)- d -Phe (7)]BK ? Des-Arg (9)-BK . A similar rank order of potency of agonists was observed for their ability to contract guinea pig uterine and ileal smooth muscle strips. The pharmacological profile of [(3)H]BK receptor binding, using BK agonists and antagonists, and that of functional responses was consistent with the identification of BK receptors of the B(2)-type in the guinea pig central nervous system and peripheral tissues.

1. Neuronal 5-hydroxytryptamine (5-HT) receptors mediating contraction of guinea-pig ileal segments have been characterized in vitro by the use of methysergide to block 5-HT1-like and 5-HT2 receptors. Concentration-response curves to 5-HT were biphasic (first phase, defined as those responses occurring between 1 nM and 0.32 microM 5-HT, -log EC50 = 7.15 +/- 0.08; second phase, defined as these responses occurring between 0.32 microM and 32 microM 5-HT, -log EC50 = 5.32 +/- 0.03) but monophasic to 5-methoxytryptamine (-log EC50 = 7.0 +/- 0.08) and 2 methyl 5-HT (-log EC50 = 5.2 +/- 0.13). The maximal response of the first phase to 5-HT and the maximal response to 5-methoxytryptamine were 30 +/- 4% and 35 +/- 5% respectively of the maximum response to the second phase of the 5-HT concentration-effect curve (set at 100%). In contrast, the maximal response to 2-methyl-5-HT equalled that obtained with 5-HT (second phase). 2. The responses comprising the second phase of the concentration-effect curve to 5-HT were antagonized by 1 microM ICS 205-930, ondansetron, granisetron, quipazine, N-methyl-quipazine and (R,S)-zacopride and the following pKB values, with 5-HT as the agonist, were obtained at the 5-HT3 receptor: ICS 205-930 7.61 +/- 0.05, ondansetron 6.90 +/- 0.04, granisetron 7.90 +/- 0.04, (S)-zacopride 8.11 +/- 0.06, (R,S)-zacopride 7.64 +/- 0.11, and (R)-zacopride 7.27 +/- 0.06. 3. Under conditions of 5-HT1-like, 5-HT2 and 5-HT3 receptor blockade, the following rank order of agonism was observed: 5-HT > 5-methoxytryptamine = renzapride > (S)-zacopride > (R,S-zacopride > 5-carboxamidotryptamine > BRL 24682 > (R-zacopride > metoclopramide > 2-methyl-5-HT > sulpiride. 8-Dihydroxydiphenylaminotetralin (8-OHDPAT), GR 43175, N,N-dipropyl-5-carboxamidotryptamine, ondansetron, ICS 205-930, granisetron, quipazine and N-methyl-quipazine were inactive as agonists and antagonists. Relative to 5-HT, (R,S)-zacopride acted as a partial agonist (intrinsic activity, alpha = 0.80; -log EC50 = 6.3 + 0.12; -log KA = 6.1 + 0.03) as did (R)-zacopride (alpha = 0.4, -log EC,0 5.7 + 0.08, -log KA = 5.5 + 0.11). (S)-zacopride acted as a full agonist (-log EC,0 = 6.9 + 0.03). ICS 205-930 (3 microM) antagonized competitively responses to 5-HT, 5 methoxytryptamine, (RS)- and (S)- zacopride and 5-carboxamidotryptamine yielding -log KB estimates ranging from 6.1-6.5. 4. It is concluded that two different 5-HT receptors mediate excitatory neuronal responses in the guineapig ileum. 5-HT3 receptors mediate the second phase of the biphasic concentration-response curve, whereas a receptor with properties distinct from the 5-HT1-like, 5-HT2 and 5-HT3 subtypes mediates the initial phase of the concentration-response curve. This receptor, which exhibits a close similarity to the 5-HT4 subtype is: (1) stimulated by 5-methoxytryptamine but not 2-methyl-5-HT; (2) stimulated selectively by certain substituted benzamides; (3) recognizes the optical isomers of zacopride and (4) is blocked by relatively high concentrations ICS 205-930 (pKB = 6.0-6.5) but not ondansetron, granisetron, quipazine or N-methyl-quipazine.

1. para-Fluoro-hexahydrosila-difenidol (p-F-HHSiD) has been proposed as an M3 selective antagonist. However, the M3 selectivity is variable in that it exhibits a high pA2 value for M3 muscarinic receptors in guinea-pig ileum but a low value at muscarinic receptors in guinea-pig trachea. 2. The pA2 value in the trachea was found to be agonist independent since similar pA2 values were found when acetylcholine, carbachol, (+)-cis-dioxolane or OXA-22 were used (7.13, 7.03, 6.85 and 6.97, respectively). The pA2 value was not meaningfully increased when the equilibrium period was increased from 60 to 180 min. The pA2 value was unaffected by blockade of M1 or M2 receptors, using 0.1 microM pirenzepine or methoctramine (7.03 and 7.14, respectively). p-F-HHSiD and atropine appeared to act at the same site, as adjudged by combination concentration-ratio studies. 3. The pA2 values for p-F-HHSiD vary by 10 fold between ileal (8.0) and tracheal M3 receptors (7.0). The precise reason for this is unknown, but appears to be unrelated to conditions of disequilibrium that could be detected. The antagonist should therefore only be employed to distinguish M3 or M1 from M2 receptors. In this respect, although the M1/M3 vs M2 discrimination is relatively large (68 fold), p-F-HHSiD exhibits similar properties to other putative M3 selective antagonists such as 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP) or the parent compound, hexahydrosiladifenidol (HHSiD).

Muscarinic receptors mediate diverse effects on the vasculature. Recently, a consensus has been arrived at with regard to muscarinic receptor classification (Levine & Birdsall, 1989). As a result, it may now be possible to clarify the role of each subtype in the responses of vascular tissues to muscarinic agonists. It is apparent that vascular muscarinic receptors form a heterogeneous population. M1 receptors contract canine venous tissue, whilst M3 receptors contract porcine and bovine coronary arteries. M3 receptors also mediate EDRF-dependent relaxant responses in the majority of tissues studied to date. M2 receptors elicit relaxations by a decrease in sympathetic outflow in canine femoral vein, rabbit ear artery and rat portal vein. These conclusions are primarily derived from functional estimations of equilibrium dissociation constants, since comparable radioligand binding data are both scarce and contradictory. It is concluded that all three major subtypes of receptors are present in the vasculature. However, the limited selectivity of the available antagonists, the lack of extensive use of such compounds and the unavailability of selective agonists clearly indicate the need for more definitive studies to be undertaken.

The interaction of gallamine with muscarinic receptor subtypes was examined using radioligand binding studies. In competition studies using [3H]N-methylscopolamine [( 3H]NMS), gallamine displayed high affinity for the rat cardiac and guinea-pig uterine M2 muscarinic receptors and for the atypical muscarinic receptor present in chicken heart. Gallamine displayed low affinity for rat glandular and human 1321 N1 astrocytoma cell M3 receptors and also for the M4 receptors of NG108-15 and PC12 cells. The compound displayed intermediate affinity for M1 receptors of rat cortex labeled using [3H]pirenzepine. The interaction of gallamine with the M1 and M2 receptors appeared to be competitive at the low concentrations required to determine affinity estimates. Thus, gallamine inhibited the binding of [3H]pirenzepine to M1 receptors and [3H]NMS to M2 receptors at concentrations that were 263- and 23-fold lower, respectively, than those required to decrease radioligand dissociation kinetics. Furthermore, gallamine, at a concentration that inhibited between 63 and 71% of specific radioligand binding, had no effect on the observed rate of association of the radioligand with either the M1 or the M2 receptor. At the M3 glandular receptor, there was little separation between the concentrations of gallamine that produced inhibition of binding and those that decreased the association and dissociation rates of [3H]NMS. It is therefore difficult to determine if the inhibition of binding seen in competition studies on the M3 receptor was produced through a competitive or an allosteric mechanism. Despite its possible allosteric properties at the M3 receptor, gallamine can be used to detect heterogeneity of muscarinic receptor subtypes in several tissues and therefore represents a useful tool for defining muscarinic receptor subtypes.

Agonist-induced desensitization has been utilized to discriminate and independently "isolate" the neuronal excitatory receptors to 5-hydroxytryptamine (5-HT) in the guinea pig ileum (5-HT3 and putative 5-HT4 receptors). Electrically stimulated longitudinal muscle myenteric plexus preparations, and non-stimulated segments of whole ileum were used. Exposure to 5-methoxytryptamine (10 mumol/l) inhibited completely responses to 5-HT at the putative 5-HT4 receptor without affecting 5-HT3-mediated responses. Conversely, exposure to 2-methyl-5-HT (10 mumol/l) inhibited completely responses to 5-HT at the 5-HT3 receptor without affecting putative 5-HT4-mediated responses. The inhibition with 5-methoxytryptamine and 2-methyl-5-HT, either alone or in combination, appeared selective as responses to KCl, DMPP, carbachol, histamine, and substance P were unaffected or only very slightly modified. Furthermore, the pA2 values for ICS 205-930 at the putative 5-HT4 (pA2 = 6.2 to 6.5) and 5-HT3 (pA2 = 7.6 to 8.1) receptors (estimated in the presence of 2-methyl-5-HT and 5-methoxytryptamine, respectively) were consistent with those estimated in the absence of desensitization. 5-Methoxytryptamine, but not 2-methyl-5-HT, suppressed completely but reversibly the concentration-effect curve to renzapride, suggesting that responses to this agent are mediated exclusively via agonism at the putative 5-HT4 receptor. It is concluded that 5-methoxytryptamine and 2-methyl-5-HT can be utilized as selective probes to discriminate the putative 5-HT4 receptor from the 5-HT3 receptor in guinea pig ileum. This finding is of importance as no selective antagonist exists for the putative 5-HT4 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The antagonistic actions of parafluorohexahydrosiladiphenidol (pFHHSiD) at muscarinic receptors has been studied in cardiac muscle, smooth muscle and cell culture preparations. In this paper, the classification scheme of Doods et al. (1987) is employed. This scheme is based upon differential affinities of muscarinic antagonists. pFHHSiD exhibited high pA2 values at M3 receptors mediating contractions of guinea-pig ileum and oesophageal muscularis mucosae (7.8 and 8.2 respectively) whereas low values were determined at M2 receptors mediating negative inotropic responses in guinea-pig atria (6.0). Intermediate pA2 values were determined at M1 receptors mediating contractions of the canine femoral and saphenous veins. 2. The pA2 values of pFHHSiD at receptors mediating endothelial-dependent relaxation of rat aortic rings, rabbit jugular vein and canine femoral artery (7.6-7.9) were similar to those determined on the ileum. However, the pA2 values of pFHHSiD at receptors mediating contractions of the guinea-pig trachea (7.1), which has been previously shown to possess M3 receptors, were different from those determined in the ileum. 3. The similarity in pA2 values of pFHHSiD between the M3 receptors in guinea-pig ileum and the receptors mediating endothelial-dependent relaxations provide further evidence for the role of M3 receptors in this vascular response. Taken together, pA2 values for pFHHSiD range from 7.1 to 8.2, depending upon the M3 preparation used. The selectivity of the compound therefore for the M3 versus the M2 muscarinic receptor ranged from 13 to 163 fold. 4. At muscarinic receptors mediating stimulation of phosphatidylinositol hydrolysis, pFHHSiD paradoxically displayed a high affinity for the M1 receptor in the SH-SY5Y cell line (pA2 = 7.9) as well as for the M3 receptor in the human astrocytoma (1321 NI cell line (pA2 = 7.6). The value at the M1 receptor in SH-SY5Y cells was greater than was observed at M1 receptors mediating contractions of both the canine saphenous and femoral veins (7.1). 5. pFHHSiD, therefore, clearly delineated M3 from M2 muscarinic receptors, whilst the separation between M1 and M3 receptors was variable. The reason for the anomalous affinity estimates in some functional studies remains unclear. These data indicate that the pA2 values for pFHHSiD appear to be tissue-dependent since the M3 selectivity varies according to the preparations studied. As a result the utility of pFHHSiD in muscarinic receptor classification is limited.

The present study was undertaken to further characterize the atypical beta adrenoceptor in guinea pig ileum. Tension was developed in isolated segments of ileum using transmural electrical stimulation of enteric cholingeric nerves. The ability of isoproterenol to relax the ileum, via beta-1 adrenoceptor and atypical beta adrenoceptor agonism, was measured. Propranolol (5 x 10(-6) M) and bromoacetylaprenololmetane blocked beta-1 adrenoceptors but, at the concentrations tested, were without affinity at atypical beta adrenoceptors. (-)-Alprenolol and (-)-dihydroalprenolol, however, acted as competitive antagonists at both sites (pA2 values of 8.2 and 8.81 at beta-1 adrenoceptors and 6.47 and 6.43 at atypical beta adrenoceptors, respectively). (-)-Alprenolol also exerted agonistic activity at the atypical beta adrenoceptor. [3H](-)-Dihydroproalprenolol failed to identify beta-1 adrenoceptors or atypical beta adrenoceptors but, instead, bound to a putative lipophilic site unrelated to ileal adrenoceptors. Before this study, nadolol (pA2 = 4.7) was the only documented antagonist at the atypical beta adrenoceptor in guinea pig ileum. Thus, the present results detail two additional pharmacological probes which exhibit about a 100-fold greater affinity than nadolol for the atypical site.

1. The alpha 2-adrenoceptor binding sites of rabbit spleen and rat kidney, labelled with [3H]-rauwolscine, were characterized using a range of subtype selective ligands. 2. In rabbit spleen, the alpha-2-adrenoceptor binding sites displayed high affinity for oxymetazoline and WB 4101 and low affinity for prazosin and chlorpromazine suggesting the presence of an alpha 2A subtype. 3. There was evidence for heterogeneity of the alpha 2-adrenoceptor binding sites present in rabbit spleen. The results obtained with oxymetazoline and WB 4101 indicated that at least 75% of the [3H]-rauwolscine binding sites in this preparation displayed a pharmacology consistent with the presence of an alpha 2A subtype. 4. In rat kidney, the alpha 2-adrenoceptor binding sites displayed high affinity for prazosin and chlorpromazine and low affinity for oxymetazoline and WB 4101 suggesting the presence of an alpha 2B subtype. 5. The inclusion of guanylylimidodiphosphate (Gpp(NH)p, 0.1 mM) did not modify the pharmacology of the alpha 2-adrenoceptor binding sites present in the two preparations. Furthermore, when the two membrane preparations were combined, the resultant pharmacology was still consistent with the presence of two receptors that retained the characteristics of the alpha 2A and alpha 2B subtypes. 6. Imiloxan was identified as a selective alpha 2B ligand while benoxathian displayed a high degree of selectivity for the alpha 2A-adrenoceptor binding site. The selectivity of imiloxan for the alpha 2B-adrenoceptor binding site, coupled with its specificity for alpha 2-adrenoceptors, should make it a valuable tool in the classification of alpha 2-adrenoceptor subtypes.

The biochemical and pharmacological properties of mu (mu), kappa (kappa) and delta (delta) opioid receptors were ascertained in dog cerebral cortex homogenates. The selective peptides, [3H]D-Pen2-D-Pen5enkephalin [( 3H]DPDPE) and [3H]D-Ala2-MePhe4-Glyol5-enkephalin [3H]Glyol; [3H]DAMGO), bound to delta- and mu-opioid receptors with high affinity (dissociation constants, Kd values = 4.7 and 1.6 nM) but to different densities of binding sites (Bmax values of 49.2 and 6.6 fmol/mg protein, respectively) in washed homogenates of dog cerebral cortex. In contrast, the non-peptides, [3H]U69593 [( 3H]U69) and [3H]etorphine [( 3H]ET), labeled a high concentration of kappa-opioid receptors (respective Bmax values of 67.2 and 76.6 fmol/mg protein) of high affinity (respective Kds of 1.4 and 0.47 nM) in the same tissue homogenates. Thus, the relative rank order of opioid receptor densities was: kappa greater than delta much greater than mu. The selective labeling of the kappa-receptors with two different drugs [( 3H]U69 and [3H]ET) failed to reveal the possible existence of multiple kappa-sites based on the relative Bmax values of the two radioligands. This conclusion was further supported by the similarity of the pharmacological specificity of both [3H]U69 and [3H]ET binding, where all the opioids tested produced 100% inhibition of these labels and where the rank order of potency of opioids at inhibiting the binding of these probes was: U50488 greater than U69593 greater than dynorphin-(1-8) greater than naloxone much greater than morphine much greater than Glyol (DAMGO) greater than DPDPE.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic receptors present in longitudinal ileum were characterized using the non-selective radioligand [3H]N-methylscopolamine [( 3H]NMS) and the M3 selective radioligand [3H]4-diphenylacetoxy-N-methylpiperidine methiodide [( 3H]4DAMP). In saturation studies, [3H]4DAMP, but not [3H]NMS, identified two populations of binding sites with 17% of the sites (155 fmol/mg protein) displaying high affinity (Kd = 0.39 nM) for [3H]4DAMP and the remaining sites displaying low affinity for the radioligand (Kd = 4.43 nM). In competition studies gallamine and methoctramine, but not AF-DX 116, identified two populations of [3H]NMS binding sites. Affinity estimates for gallamine and methoctramine indicated that 80% of the [3H]NMS binding sites were of the M2 subtype. The minor population of [3H]NMS binding sites could not be readily characterized, due partly to the low selectivity of the competing ligands and also to the relatively low density of the sites. In studies using the M3 muscarinic receptor selective radioligand [3H]4DAMP, the minor population of sites could be preferentially labeled by using a low concentration (0.4 nM) of [3H]4DAMP. Under these conditions, [3H]4DAMP labeled approximately equal levels of the two muscarinic receptor binding sites present in the ileum. Competition studies with AF-DX 116, gallamine and methoctramine indicated that the two [3H]4DAMP binding sites displayed the pharmacology expected of the M2 and M3 receptors, respectively. These results provide additional evidence that longitudinal ileal smooth muscle membranes contain both M2 and M3 muscarinic receptors and indicate that [3H]4DAMP is a useful ligand for identifying heterogeneity of muscarinic receptor subtypes.

1. Oral doses of 10-100 mg/kg of BRL 13776 lowered the blood pressure of both deoxycorticosterone acetate (DOCA)/NaCl-treated hypertensive rats and untreated normotensive rats. 2. BRL 13776 (100 mg/kg, orally) also reduced the blood pressure of renal hypertensive cats (cellophane perinephritis model). 3. No tolerance developed to the blood-pressure lowering action of BRL 13776 when an oral daily dose of 100 mg/kg was administered repeatedly for up to 15 days to hypertensive rats and cats. 4. The fall in blood pressure to BRL 13776 in rats was associated with a reduction of tissue catecholamines. 5. The catecholamine depletion occurred in all the peripheral tissues examined but in the brain was restricted to certain regions, these being the hind-brain on single dosing and the hind-brain, hypothalamus and mid-brain on repeated dosing. Catecholamine levels in the cerebral hemispheres were not affected by either single or repeated doses of BRL 13776. 6. BRL 13776 caused some reduction of the 5-hydroxytryptamine content of the heart but not of whole brain or any brain region. 7. Neither single doses (up to 900 mg/kg orally) nor repeated doses (100-300 mg/kg orally) of BRL 13776 produced any significant behavioural effects in animals. 8. BRL 13776 is a new type of agent to display both antihypertensive and monoamine-depleting properties. The reduction of noradrenaline in certain brain regions may be a cause of the antihypertensive response but depletion in the periphery could contribute in a major or minor way. The differential action on noradrenaline in the brain together with the lack of effect on 5-hydroxytryptamine might also explain the apparent absence of behavioural effects.

The synthesis and activity in the spontaneously hypertensive rat of several 4-(1,2,3,4-tetrahydronaphthyl-2-amino)-1-(4-fluorophenyl)-1-but anones is reported. Maximal antihypertensive activity was associated with 5,6-dimethoxy substitution in the aminotetralin moiety.

1. The alpha 1-adrenoceptors present in membranes of rat liver, cortex and submaxillary gland were labelled with [3H]-prazosin and the affinity of 15 ligands for these receptors was determined. 2. In saturation studies, [3H]-prazosin bound with high affinity (Kd = 30-39 pM) to a single population of sites in all three preparations. 3. In competition studies using rat cortex, evidence for heterogeneity of the alpha 1-adrenoceptor binding sites was obtained. Displacement isotherms for amidephrine, benoxathian, oxymetazoline, phentolamine and WB 4101 were biphasic and were consistent with the presence of both alpha 1A- and alpha 1B-adrenoceptor subtypes as described by Morrow & Creese (1986) and Han et al. (1987). 4. The rat liver and submaxillary gland membrane preparations both possessed homogeneous populations of alpha 1-adrenoceptors. However, there were pharmacological differences between the receptors in these two preparations. Rat submaxillary gland alpha 1-adrenoceptors displayed high affinity for amidephrine, benoxathian, oxymetazoline, phentolamine and WB 4101 and therefore appeared to represent alpha 1A-adrenoceptors. Rat liver alpha 1-adrenoceptors possessed lower affinity for these ligands (6-65 fold) suggesting that these receptors were of the alpha 1B-subtype. 5. Spiperone exhibited 12.9 fold higher affinity for rat liver alpha 1B-adrenoceptors than for rat submaxillary gland alpha 1A-adrenoceptor and may therefore represent the first alpha 1B-adrenoceptor selective ligand.

The muscarinic receptor present in chicken cardiac membranes was characterised using a ligand binding approach and compared to the M1, M2 and M3 receptors that can be identified in ligand binding studies at present. [3H]N-methylscopolamine and [3H]pirenzepine appeared to label the same population of muscarinic receptors in chicken cardiac membranes since the density of sites labeled by the two radioligands was similar. Furthermore, affinity estimates of 8 muscarinic receptor antagonists for chicken cardiac muscarinic receptors were the same irrespective of whether [3H]N-methylscopolamine or [3H]pirenzepine was used as the radioligand. The chicken cardiac muscarinic receptor displayed high affinity for pirenzepine (pKi = 7.9) and so did not appear to represent an M2 receptor. Despite the high affinity of chicken cardiac muscarinic receptors for pirenzepine, affinity estimates for dicyclomine (pKi = 8.0), CPPS (pKi = 8.4) and 4DAMP (pKi = 8.6) in chicken heart were not consistent with the presence of M1 receptors. The chicken cardiac muscarinic receptor also displayed significant differences to the M3 receptor since it displayed high affinity for AF-DX 116 (pKi = 7.1) and methoctramine (pKi = 8.4). Finally, chicken heart muscarinic receptors displayed high affinity for gallamine (pKi = 7.0) and pirenzepine suggesting that the receptor was different to the M4 muscarinic receptor of the NG108-15 cell line. These findings suggest that chicken heart expresses a novel muscarinic receptor subtype distinct from the M1, M2, M3, and M4 subtypes already described.