Pharmacological characterisation of human 5-HT2 receptor subtypes.

Prompted by conflicting literature, this study compared the pharmacology of human 5-hydroxytryptamine2 (5-HT2) receptors expressed in SH-SY5Y cells using a fluorometric imaging plate reader (FLIPR) based Ca2+ assay. 5-Hydroxytryptamine (5-HT) increased intracellular calcium concentration ([Ca2+]i) at 5-HT2A, 5-HT2B and 5-HT2C receptors (pEC(50)=7.73+/-0.03, 8.86+/-0.04 and 7.99+/-0.04, respectively) and these responses were inhibited by mesulergine (pKB=7.42+/-0.06, 8.77+/-0.10 and 9.52+/-0.11). A range of selective agonists and antagonists displayed the expected pharmacology at each receptor subtype. Sodium butyrate pretreatment increased receptor expression in SH-SY5Y/5-HT2B (15-fold) and SH-SY5Y/5-HT2C cells (7-fold) and increased agonist potencies and relative efficacies. In contrast, sodium butyrate pretreatment of SH-SY5Y/5-HT(2A) cells did not affect receptor expression. The present study provides a direct comparison of agonist and antagonist pharmacology at 5-HT(2) receptor subtypes in a homogenous system and confirms that agonist potency and efficacy varies with the level of receptor expression.

Pharmacological actions of a novel, high-affinity, and selective human dopamine D(3) receptor antagonist, SB-277011-A.

SB-277011-A (trans-N-[4-[2-(6-cyano-1,2,3, 4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolininecarboxamide), is a brain-penetrant, high-affinity, and selective dopamine D(3) receptor antagonist. Radioligand-binding experiments in Chinese hamster ovary (CHO) cells transfected with human dopamine D(3) or D(2 long) (hD(3), hD(2)) receptors showed SB-277011-A to have high affinity for the hD(3) receptor (pK(i) = 7.95) with 100-fold selectivity over the hD(2) receptor and over 66 other receptors, enzymes, and ion channels. Similar radioligand-binding data for SB-277011-A were obtained from CHO cells transfected with rat dopamine D(3) or D(2). In the microphysiometer functional assay, SB-277011-A antagonized quinpirole-induced increases in acidification in CHO cells overexpressing the hD(3) receptor (pK(b) = 8.3) and was 80-fold selective over hD(2) receptors. Central nervous system penetration studies showed that SB-277011-A readily entered the brain. In in vivo microdialysis studies, SB-277011-A (2. 8 mg/kg p.o.) reversed the quinelorane-induced reduction of dopamine efflux in the nucleus accumbens but not striatum, a regional selectivity consistent with the distribution of the dopamine D(3) receptor in rat brain. SB-277011-A (2-42.3 mg/kg p.o.) did not affect spontaneous locomotion, or stimulant-induced hyperlocomotion. SB-277011-A (4.1-42.2 mg/kg p.o.) did not reverse prepulse inhibition deficits in apomorphine- or quinpirole-treated rats, but did significantly reverse the prepulse inhibition deficit in isolation-reared rats at a dose of 3 mg/kg p.o. SB-277011-A (2.5-78. 8 mg/kg p.o.) was noncataleptogenic and did not raise plasma prolactin levels. Thus, dopamine D(3) receptor blockade produces few of the behavioral effects characteristic of nonselective dopamine receptor antagonists. The effect of SB-277011-A on isolation-induced prepulse inhibition deficit suggests that blockade of dopamine D(3) receptors may benefit the treatment of schizophrenia.

Regulation of human D(1), d(2(long)), d(2(short)), D(3) and D(4) dopamine receptors by amiloride and amiloride analogues.

1. The modulatory effects of the allosteric effectors methylisobutylamiloride (MIA), benzamil and amiloride have been examined at human D(1), D(2), D(3) and D(4) dopamine receptors. The subtype selectivity and the mechanism of action of this allosteric regulation was examined. 2. In radioligand dissociation experiments each modulator accelerated dissociation from all four receptor subtypes indicating allosteric regulation. MIA displayed selectivity for the D(3) subtype for acceleration of radioligand dissociation. 3. In equilibrium binding (pseudo-competition) experiments the three compounds inhibited radioligand binding at the four receptor subtypes. Inhibition curves for D(1), D(2(short)), D(2(long)) and D(3) receptors were described by Hill coefficients exceeding unity and data were fitted best by a model that assumes binding of modulator to both the primary and allosteric binding sites of the receptor (the allosteric/competitive model). 4. At the D(4) subtype, Hill coefficients of unity described the binding data for amiloride and benzamil, consistent with competitive inhibition. The Hill coefficient for MIA at the D(4) subtype was less than unity and data could be fitted well by the allosteric/competitive model, but it was not possible to define unambiguously the modulatory mechanism. For this effect a better definition of the mechanism could be obtained by simultaneous analysis of data obtained in the presence of a range of concentrations of a purely competitive ligand. 5. MIA reduced the potency with which dopamine stimulated [(35)S]-GTPgammaS binding at the D(2) receptor. The effects of MIA could be described by the allosteric/competitive model with effects of MIA to inhibit the binding of dopamine but not its ability to induce a response.

Pharmacological characterization of extracellular acidification rate responses in human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells.

This study characterized pharmacologically the functional responses to agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) receptors separately expressed in cloned cells using the cytosensor microphysiometer. Dopaminergic receptor agonists caused increases in extracellular acidification rate in adherent Chinese hamster ovary (CHO) clones expressing hD2, hD3 or hD4 receptors. Acidification rate responses to agonists in other cell lines expressing these receptors were smaller than those in adherent CHO cells. The time courses and maximum increases in acidification rate of the agonist responses in adherent CHO cells were different between the three dopamine receptor clones. Responses were blocked by pretreatment of cells with pertussis toxin or amiloride analogues. Most agonists had full intrinsic activity at each of the dopamine receptor subtypes, as compared to quinpirole, however both enantiomers of UH-232 and (-)3-PPP were partial agonists in this assay system. The functional potency of full agonists at each of the three receptors expressed in CHO cells was either higher than, or similar to, the apparent inhibition constants (Ki) determined in [125I]-iodosulpride competition binding studies. Functional selectivities of the agonists were less than radioligand binding selectivities. The rank orders of agonist potencies and selectivities were similar, but not identical, to the rank orders of radioligand binding affinities and selectivities. The dopamine receptor antagonists, iodosulpride and clozapine, had no effect on basal acidification rates but inhibited acidification responses in CHO cells to quinpirole in an apparently competitive manner. Antagonist potencies closely matched their radioligand binding affinities in these cells.

Comparison of the functional potencies of ropinirole and other dopamine receptor agonists at human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells.

1. The aim of the present study was to characterize functional responses to ropinirole, its major metabolites in man (SKF-104557 (4-[2-(propylamino)ethyl]-2-(3H) indolone), SKF-97930 (4-carboxy-2-(3H) indolone)) and other dopamine receptor agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) receptors separately expressed in Chinese hamster ovary cells using microphysiometry. 2. All the receptor agonists tested (ropinirole, SKF-104557, SKF-97930, bromocriptine, lisuride, pergolide, pramipexole, talipexole, dopamine) increased extracellular acidification rate in Chinese hamster ovary clones expressing the human D2, D3 or D4 receptor. The pEC50s of ropinirole at hD2, hD3 and hD4 receptors were 7.4, 8.4 and 6.8, respectively. Ropinirole is therefore at least 10 fold selective for the human dopamine D3 receptor over the other D2 receptor family members. 3. At the hD2 and hD3 dopamine receptors all the compounds tested were full agonists as compared to quinpirole. Talipexole and the ropinirole metabolite, SKF-104557, were partial agonists at the hD4 receptor. 4. Bromocriptine and lisuride had a slow onset of agonist action which precluded determination of EC50s. 5. The rank order of agonist potencies was dissimilar to the rank order of radioligand binding affinities at each of the dopamine receptor subtypes. Functional selectivities of the dopamine receptor agonists, as measured in the microphysiometer, were less than radioligand binding selectivities. 6. The results show that ropinirole is a full agonist at human D2, D3 and D4 dopamine receptors. SKF-104557 the major human metabolite of ropinirole, had similar radioligand binding affinities to, but lower functional potencies than, the parent compound.

Novel 1,2,3,4-tetrahydroisoquinolines with high affinity and selectivity for the dopamine D3 receptor.

Using clearance and brain penetration studies as a screen, tetrahydroisoquinoline 3 was identified as a lead having low clearance in rats (CLb 20 ml/min/kg). Introduction of a 7-CF3SO2O- substituent into the tetrahydroisoquinoline, followed by replacement of the biphenylamido group of 3 by a 3-indolylpropenamido group gave 31, having high D3 receptor affinity (pKi 8.4) and 150 fold selectivity over the D2 receptor.

Gamma-aminobutyric acidA receptor function is desensitised in rat cultured cerebellar granule cells following chronic flunitrazepam treatment.

This study examined gamma-aminobutyric acidA (GABA(A)) receptor function in cultured rat cerebellar granule cells by using microphysiometry following chronic flunitrazepam exposure, and correlated the findings with the alpha1 and beta2/3 subunit protein expression and [3H]muscimol binding after the same treatment paradigm. Flunitrazepam treatment reduced (p < 0.05) the maximal GABA-stimulated increase in extracellular acidification rate (Emax) (16.5 +/- 1.2% and 11.3 +/- 1.0%, 2-day control and treated cells, respectively; 17.4 +/- 1.0% and 9.9 +/- 0.7%, 7-day control and treated cells, respectively; best-fit Emax +/- SEM, n = 7), without affecting the GABA concentration required to elicit 50% of maximal response (EC50) (1.2 +/- 1.7 and 2.3 +/- 1.8 microM, 2-day control and treated cells, respectively; 1.7 +/- 1.5 and 1.5 +/- 1.5 microM, 7-day control and treated cells, respectively; best-fit EC50 +/- SEM, n = 7). Flunitrazepam exposure also abolished the flunitrazepam potentiation of the GABA response, caused a transient reduction of the GABA(A) receptor alpha1 and beta2/3 subunit proteins over the initial 2 days, but did not alter [3H]muscimol binding compared with vehicle-treated cells. The results suggest that changes in GABA(A) receptor subunit protein expression, rather than loss of [3H]muscimol binding sites, underlie the chronic flunitrazepam-mediated desensitisation of GABA(A) receptor function.

Fused aminotetralins: novel antagonists with high selectivity for the dopamine D3 receptor.

Starting from a series of 2-aminotetralins 1, a novel series of N-[4-(4-phenylbenzoylamino)butyl]-octahydrobenzoquinolines and hexahydrobenzoindoles with high potency and selectivity for the dopamine D3 receptor has been designed. The effect of ligand chirality on binding affinity has been established. Selected derivatives (e.g. 2o, 2p) show high functional selectivity and enhanced in vivo properties compared to 1.

Functional effects of the muscarinic receptor agonist, xanomeline, at 5-HT1 and 5-HT2 receptors.

Xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-me thylpyridine] has been reported to act as a functionally selective muscarinic partial agonist with potential use in the treatment of Alzheimer's disease. This study examined the functional activity of xanomeline at 5-HT1 and 5-HT2 receptors in native tissue and/or human cloned receptors. Xanomeline had affinity for muscarinic receptors in rat cortical membranes where the ratio of the displacement affinity of [3H]-Quinuclidinyl benzilate vs that of [3H]-Oxotremorine-M was 16, indicative of partial agonist activity. Radioligand binding studies on human cloned receptors confirmed that xanomeline had substantial affinity for M1, M2, M3, M4, M5 receptors and also for 5-HT1 and 5-HT2 receptor subtypes. Carbachol and xanomeline stimulated basal [35S]-GTPgammaS binding in rat cortical membranes with micromolar affinity. The response to carbachol was attenuated by himbacine and pirenzepine with pA2 of 8.2, 6.9 respectively consistent with the response being mediated, predominantly, via M2 and M4 receptors. Xanomeline-induced stimulation of [35S]-GTPgammaS binding was inhibited by himbacine with an apparent pKb of 6.3, was not attenuated by pirenzepine up to 3 microM and was inhibited by the selective 5-HT1A antagonist WAY100635 with an apparent pKb of 9.4. These data suggest the agonist effect of xanomeline in this tissue is, in part, via 5-HT1A receptors. Similar studies on human cloned receptors confirmed that xanomeline is an agonist at human cloned 5-HT1A and 5-HT1B receptors. In studies using the fluorescent cytoplasmic Ca2+ indicator FLUO-3AM, xanomeline induced an increase in cytoplasmic Ca2+ concentration in SH-SY5Y cells expressing recombinant human 5-HT2C receptors. Atropine antagonized this response, consistent with mediation via endogenously-expressed muscarinic receptors. In the presence of atropine, xanomeline antagonized 5-HT-induced cytoplasmic changes in Ca2+ concentration in cells expressing h5-HT2A, h5-HT2B and h5-HT2c receptors with potencies similar to its affinity at these receptors. These studies indicate that xanomeline is a potent agonist at 5-HT1A and 5-HT1B receptors and an antagonist at 5-HT2 receptor subtypes.

Measurement of GABAA receptor function in rat cultured cerebellar granule cells by the Cytosensor microphysiometer.

1. gamma-Aminobutyric acid (GABA), acting via the GABAA receptor, increased the extracellular acidification rate of rat primary cultured cerebellar granule cells, measured by the Cytosensor microphysiometer. 2. The optimal conditions for the measurement of GABAA receptor function in cerebellar granule cells by microphysiometry were: cells seeded at 9-12 x 10(5) cells/transwell cup and maintained in vitro for 8 days, GABA stimulation performed at 25 degrees C, with a stimulation time of 33 s. 3. GABA stimulated a concentration-dependent increase in the extracellular acidification rate with an EC50 of 2.0 +/- 0.2 microM (mean +/- s.e.mean, n = 7 experiments) and maximal increase (Emax) over basal response of 15.4 +/- 1.2%. 4. The sub-maximal GABA-stimulated increase in acidification rate could be potentiated by the 1,4-benzodiazepine, flunitrazepam (100 nM). The 10 nM GABA response showed the maximal benzodiazepine facilitation (GABA alone, 1.4 microV s-1, GABA + flunitrazepam, 3.8 microV s-1, mean increment over basal, n = 7). 5. The GABA-stimulated increase in acidification rate was inhibited by the GABAA antagonist, bicuculline (100 microM) (90% inhibition at 1 mM GABA). 6. The results of this study show that activation of GABAA receptors in rat cerebellar granule cells caused an increase in the extracellular acidification rate; an effect which was potentiated by benzodiazepines and inhibited by a GABAA receptor antagonist. This paper defines the conditions and confirms the feasibility of using microphysiometry to investigate GABAA receptor function in primary cultured CNS neurones. The microphysiometer provides a rapid and sensitive technique to investigate the regulation of the GABAA receptor in populations of neurones.

Expression and functional characterisation of a synthetic version of the human D4 dopamine receptor in a stable human cell line.

A synthetic version of the human D4 (hD4) dopamine receptor was prepared. The G/C content of the natural gene was reduced by 14% without altering the amino acid composition of the corresponding protein sequence. HEK293 cells were transfected with the synthetic hD4 gene and stable clones resistant to G418 selected. The hD4 receptor expressed from the synthetic gene had identical pharmacological characteristics to the native hD4 receptor [(1991) Nature 350, 610-619; (1992) Nature 358, 149-152]. Functional studies with cells expressing the synthetic hD4 gene indicated negative coupling of this receptor to adenylate cyclase.

Synthesis and antihypertensive activity of 4-(substituted-carbonylamino)-2H-1-benzopyrans.

The synthesis and antihypertensive activity of a series of novel 4-(substituted-carbonylamino)-2H-1-benzopyran-3-ols, administered orally to conscious spontaneously hypertensive rats, are described. Optimum activity was observed for compounds with alkyl, amino, or aryl groups flanking the carbonyl group. Of the alkyl and amino series the most potent compounds contained the methyl and methylamino groups, respectively. Several analogues have been compared with cromakalim (1) for their effects on potassium ion efflux in the rabbit mesenteric artery using rubidium-86 as a marker. The ability of each compound to enhance rubidium-86 efflux is approximately parallelled by its blood pressure lowering activity, and thus these analogues, like compound (1), belong to the series of drugs which have been classified as potassium-channel activators.

Potassium efflux enhancement by cromakalim (BRL 34915) in rabbit mesenteric artery: an indirect effect independent of calcium?

Experiments have been performed in order to investigate the calcium and exposure time dependency of cromakalim (BRL 34915)-stimulated rubidium efflux in rabbit isolated mesenteric artery. Removal of calcium from the bathing medium prolonged the effects of cromakalim on rubidium efflux. Lanthanum was without effect on cromakalim-induced efflux whilst high concentrations of nifedipine were required to produce a significant inhibitory effect. Decreasing the exposure time to cromakalim, either in the presence or absence of calcium, led to a progressive loss of the response. However, significant increases in rubidium efflux rate were observed after very short exposures (15 sec) to the drug. In normal medium, exposure to cromakalim resulted in an inhibition of a second response when the drug was reapplied. Blockade by tetraethylammonium of the initial rubidium efflux response to cromakalim did not reverse the inhibition of the second response. These results suggest that the stimulation by cromakalim of rubidium efflux in rabbit isolated mesenteric artery is independent of calcium influx and requires only a short initial exposure to the drug in order to develop a response. The development and maintenance of the response after the removal of the drug suggest that cromakalim does not directly interact with the potassium channel through which rubidium efflux enhancement is observed.

Comparative effects of K+ channel blockade on the vasorelaxant activity of cromakalim, pinacidil and nicorandil.

Three agents with K+ channel blocking activity, procaine, 4-aminopyridine (4-AP) and tetraethylammonium (TEA), were tested for inhibition of vasorelaxation and 86Rb+ efflux induced by cromakalim (BRL 34915), pinacidil and nicorandil in rabbit isolated mesenteric artery. The potency order for inhibition of vasorelaxation was procaine greater than 4-AP greater than TEA and for inhibition of efflux was procaine = 4-AP greater than TEA. The K+ channel blockers did not discriminate between cromakalim, pinacidil or nicorandil on efflux but demonstrated preferential inhibition of vasorelaxation to cromakalim greater than pinacidil greater than nicorandil. In addition, the maximum response to cromakalim was depressed but that to pinacidil and nicorandil was not. The results confirm the role of K+ channel activation in vasorelaxation to cromakalim, pinacidil and nicorandil, but suggest that additional mechanisms may be involved for pinacidil and, in particular, for nicorandil.

Specificity of action of the novel antihypertensive agent, BRL 34915, as a potassium channel activator. Comparison with nicorandil.

Experiments have been performed to investigate the specificity of the mechanism of action of the novel antihypertensive agent, BRL 34915. BRL 34915 (0.5-100 microM) and nicorandil (10-500 microM) stimulated the efflux of rubidium from preloaded rabbit isolated mesenteric arteries. BRL 34915 also caused an increase in the rubidium efflux rate constant in other vascular smooth muscles. Tetraethylammonium (0.1-30 mM) inhibited BRL 34915 (10 microM), nicorandil (100 microM) and potassium (30 mM) induced stimulations of rubidium efflux, but had no effect on noradrenaline (30 microM) induced efflux. Only noradrenaline induced efflux was inhibited by apamin (3-100 nM). Examination of other second messenger systems demonstrated that BRL 34915 (at concentrations up to 100 microM) did not have any appreciable effect on cGMP accumulation in rabbit mesenteric artery, cAMP or cGMP phosphodiesterase in rat heart, or cAMP and inositol phosphate accumulation in rat brain slices. Nicorandil (100 microM) caused a small increase in cGMP accumulation in rabbit mesenteric artery. Radioligand binding studies showed that BRL 34915 did not interact with dihydropyridine, 5-hydroxytryptamine, dopamine, alpha 1, alpha 2 or beta adrenoceptor binding sites. [3H]-BRL 34915 did not bind specifically to any site in any tissue studied, either in vitro or ex vivo. Thus we have been unable to demonstrate an effect of BRL 34915 other than of increasing potassium efflux in rabbit vascular smooth muscle. This lends support to other evidence suggesting that BRL 34915 relaxes vascular smooth muscle (and hence lowers blood pressure) by a novel, and specific, mechanism involving hyperpolarisation of the smooth muscle cell membrane.