PubMed Health⌕ Search

Biomedical subjects

D W Bonhaus

Publications and source records attributed to D W Bonhaus.

At least 19 recordsLinked to original sources
PubMed2001

High-affinity interactions of ligands at recombinant guinea pig 5HT7 receptors.

The serotonin 5HT7 receptor has been implicated in numerous physiological and pathological processes from circadian rhythms to depression and schizophrenia. Clonal cell lines heterologously expressing recombinant receptors offer good models for understanding drug-receptor interactions and development of quantitative structure-activity relationships (QSAR). Comparative Molecular Field Analysis (CoMFA) is an important modern QSAR procedure that relates the steric and electrostatic fields of a set of aligned compounds to affinity. Here, we utilized CoMFA to predict affinity for a number of high-affinity ligands at the recombinant guinea pig 5HT7 receptor. Using R-lisuride as the template, a final CoMFA model was derived using procedures similar to those of our recent papers. The final cross-validated model accounted for >85% of the variance in the compound affinity data, while the final non-cross validated model accounted for >99% of the variance. Model evaluation was done using cross-validation methods with groups of 5 ligands. Twenty cross-validation runs yielded an average predictive r2(q2) of 0.779 +/- 0.015 (range: 0.669-0.867). Furthermore, 3D-chemical database search queries derived from the model yielded hit lists of promising agents with high structural similarity to the template. Together, these results suggest a possible basis for high-affinity drug action at 5HT7 receptors.

Animals↗
PubMed2000

Allosteric modulation of [(3)H]gabapentin binding by ruthenium red.

Gabapentin is an anticonvulsant with an unknown mechanism of action. However, it has been proposed that gabapentin acts by binding to voltage-gated calcium channels. To further characterize the interaction of gabapentin with its endogenous binding site in cerebral cortex, we tested for competitive and allosteric interactions between [(3)H]gabapentin and a variety of calcium channel binding ligands. Most ligands for voltage- or ligand-gated calcium channels (verapamil, the omega-conotoxins MVIIC and GVIA, ryanodine, caffeine, capsaicin, MK-801) had no significant effect on [(3)H]gabapentin binding. However, ruthenium red, a relatively nonselective calcium channel ligand, was found to robustly modulate [(3)H]gabapentin binding. Ruthenium red slowed the association and dissociation kinetics of [(3)H]gabapentin while increasing the number of detectable binding sites. Spermine and MgCl(2), which also bind to calcium channels and modulate [(3)H]gabapentin binding, were found to act in a similar manner. These findings support the contention that the principal endogenous binding site for gabapentin is a calcium channel; they characterize the nature of the allosteric interaction of spermine, MgCl(2) and ruthenium red with this binding site; and they suggest possible mechanisms by which gabapentin may modulate calcium channel function and ultimately produce therapeutic actions.

Acetates↗
PubMed1999

RS-127445: a selective, high affinity, orally bioavailable 5-HT2B receptor antagonist.

Efforts to define precisely the role of 5-HT2B receptors in normal and disease processes have been hindered by the absence of selective antagonists. To address this deficiency, we developed a series of naphthylpyrimidines as potentially useful 5-HT2B receptor antagonists. RS-127445 (2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine) was found to have nanomolar affinity for the 5-HT2B receptor (pKi = 9.5+/-0.1) and 1,000 fold selectivity for this receptor as compared to numerous other receptor and ion channel binding sites. In cells expressing human recombinant 5-HT2B receptors, RS-127445 potently antagonized 5-HT-evoked formation of inositol phosphates (pK(B) = 9.5+/-0.1) and 5-HT-evoked increases in intracellular calcium (pIC50 = 10.4+/-0.1). RS-127445 also blocked 5-HT-evoked contraction of rat isolated stomach fundus (pA2 = 9.5+/-1.1) and (+/-)alpha-methyl-5-HT-mediated relaxation of the rat jugular vein (pA2 = 9.9+/-0.3). RS-127445 had no detectable intrinsic activity in these assays. In rats, the fraction of RS-127445 that was bioavailable via the oral or intraperitoneal routes was 14 and 60% respectively. Intraperitoneal administration of RS-127445 (5 mg kg(-1)) produced plasma concentrations predicted to fully saturate accessible 5-HT2B receptors for at least 4 h. In conclusion, RS-127445 is a selective, high affinity 5-HT2B receptor antagonist suitable for use is vivo. The therapeutic potential of this molecule is being further evaluated.

Animals↗
PubMed1998

Ligand efficacy and potency at recombinant alpha2 adrenergic receptors: agonist-mediated [35S]GTPgammaS binding.

Alpha-2 adrenergic receptors (alpha2 AR) mediate incorporation of guanosine 5'-O-(gamma-thio)triphosphate ([35S]GTPgammaS) into isolated membranes via receptor-catalyzed exchange of [35S]GTPgammaS for GDP. In the current study, we used [35S]GTPgammaS incorporation to characterize the intrinsic activity and potency of agonists and antagonists at the cloned mouse alpha2a/d and human alpha2a, alpha2b, and alpha2c ARs. Full agonists increased [35S]GTPgammaS binding to membranes by 2- to 3-fold. Antagonists did not increase [35S]GTPgammaS binding but competitively inhibited agonist-stimulated [35S]GTPgammaS binding. Compounds with intrinsic activities less than that of the full agonists norepinephrine (NE) or epinephrine (EPI) were capable of antagonizing agonist-stimulated [35S]GTPgammaS binding. The agonistic properties of a number of alpha2 AR ligands were characterized at each alpha2 AR subtype. The rank order of agonist potency for selected compounds at the human receptors (with intrinsic activity compared with NE, defined as 1.0) was: alpha2a: Dexmedetomidine (0.73) > guanabenz (0.38) > UK-14304 (1.02) > clonidine (0.32) > ST-91 (0.63) > NE (1.00). alpha2b: Dexmedetomidine (1.10) > clonidine (0.18) > guanabenz (0.71) > NE (1.00) > ST-91 (0.44) > UK-14304 (0.59). alpha2c: Dexmedetomidine (1.03) > NE (1.00) > UK-14304 (0.75) > ST-91 (0.32) > or = clonidine (0.23) >> guanabenz (0). This report provides a functional characterization of adrenergic receptor ligands at human and mouse alpha2a/d AR. It also illustrates the utility of [35S]GTPgammaS incorporation as a functional marker of receptor activation.

Adenylyl Cyclases↗
PubMed1998

Enhanced delayed matching performance in younger and older macaques administered the 5-HT4 receptor agonist, RS 17017.

Recent evidence indicates that the 5-HT4 subtype of serotonin receptor may modulate central cholinergic activity in regions of the mammalian CNS important to memory such as the frontal cortex, hippocampus and amygdala. These receptors could represent targets for drugs designed for the symptomatic therapy of Alzheimer's disease (AD) and other disorders of memory. In the present study, the binding activity of RS 17017 (previously described as a selective 5-HT4 agonist) was assessed across a number of neurotransmitter receptors and binding sites, pharmacokinetic data were obtained, and the compound was evaluated in macaques for mnemonic effects via a computer-assisted delayed matching-to-sample task (DMTS). Binding data confirmed the 5-HT4 selectivity of the compound, while pharmacokinetic results revealed low oral bioavailability, but a large volume of distribution of the compound. Significant and reproducible improvements in DMTS accuracy were observed after oral administration of the compound across a dose-effect series in both younger and older monkeys. The results suggest that RS 17017 offers a potential for memory enhancement in disorders involving cognitive decline, and are consistent with a role for central 5-HT4 receptors in memory. Improvements in DMTS performance in aged monkeys may have particular implications for neurodegenerative conditions such as AD, whereas positive results in the younger monkeys indicate that RS 17017 (or similar compounds) may have additional potential in the therapeutics of memory disorders not necessarily associated with advanced age.

Aging↗
PubMed1998

Evidence for inverse agonism of SR141716A at human recombinant cannabinoid CB1 and CB2 receptors.

The cannabinoid receptor antagonist SR141716A has been suggested to be an inverse agonist at CB1 receptors in some isolated intact tissues. We found that the basal incorporation of [35S]-GTPgammaS in Chinese hamster ovary cells expressing human recombinant CB1 and CB2 receptors was inhibited by SR141716A (mean pEC50s 8.26 and 6.00, respectively), whereas cannabinol (10 microM) had no significant effect at hCB1 receptors but inhibited the binding at hCB2 receptors. As cannabinol had no effect on basal [35S]-GTPmicroS binding at hCB1 at a concentration 100 fold higher than its binding affinity (K = 0.1 microM), we conclude that endogenous cannabinoid receptor agonists are not a confounding factor and suggest the actions of SR141716A at the hCB1 receptor, and the actions of SR141716A and cannabinol at the hCB2 receptor, are due to inverse agonism.

Animals↗
PubMed1998

Dual activation and inhibition of adenylyl cyclase by cannabinoid receptor agonists: evidence for agonist-specific trafficking of intracellular responses.

Cannabinoid receptors couple to both Gs and Gi proteins and can consequently stimulate or inhibit the formation of cAMP. To test whether there is specificity among cannabinoid receptor agonists in activating Gs- or Gi-coupled pathways, the potency and intrinsic activity of various cannabinoid receptor ligands in stimulating or inhibiting cAMP accumulation were quantified. The rank order of potencies of cannabinoid receptor agonists in increasing or inhibiting forskolin-stimulated cAMP accumulation, in CHO cells expressing hCB1 receptors, was identical (HU-210 > CP-55,940 > THC > WIN-55212-2 > anandamide). However, the activities of these agonists were different in the two assays with anandamide and CP-55,940 being markedly less efficacious in stimulating the accumulation of cAMP than in inhibiting its formation. Studies examining the effects of forskolin on cannabinoid receptor mediated stimulation of adenyly cyclase also revealed differences among agonists in as much as forskolin enhanced the potency of HU-210 and CP-55,940 by approximately 100-fold but, by contrast, had no effect on the potency of WIN-55212-2 or anandamide. Taken together these findings demonstrate marked differences among cannabinoid receptor agonists in their activation of intracellular transduction pathways. This provides support for the emerging concept of agonist-specific trafficking of cellular responses and further suggests strategies for developing receptor agonists with increased therapeutic utility.

Adenylyl Cyclase Inhibitors↗
PubMed1997

Catecholamine modulatory effects of nepicastat (RS-25560-197), a novel, potent and selective inhibitor of dopamine-beta-hydroxylase.

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.

Animals↗
PubMed1996

Comparison of 5-HT4 receptors in guinea-pig colon and rat oesophagus: effects of novel agonists and antagonists.

5-HT4 receptors in isolated distal colon myenteric plexus of guinea-pig, mediating contraction of longitudinal smooth muscle, have been further characterized by selective agonists and antagonists. The indole agonists, 5-HT and 5-methoxytryptamine (5-MeOT), were full agonists (relative to 5-HT) with potency values (pEC50) of 8.0 +/- 0.1 (n = 50) and 7.8 +/- 0.1 (n = 12), respectively. 5-HT4 receptor agonists of other structural classes, including benzimidazolones (BIMU 1 and BIMU 8), and benzamides ((S)-zacopride, (R)-zacopride, renzapride, SC 49518) were partial agonists with intrinsic activities less than that of 5-HT. In general, the potencies for these compounds at 5-HT4 receptors in guinea-pig colon were similar to the potencies seen in the rat isolated oesophagus, where 5-HT4 receptors mediate relaxation. GR 113808 ¿[1-[2-[(methylsulfonyl)amino]ethyl]-4-piperidinyl] methyl1-methyl-1H-indole-3-carboxylate¿, RS 39604 ¿1-[4-amino-5-chloro-2-(3, 5-dimethoxybenzyloxy)phenyl]-3[1-[2-[(methylsulfonyl)amino] ethyl]-4-piperidinyl]-1-propanone hydrochloride and SB 204070 ¿(1-n-butyl-4-piperidinyl)methyl 8-amino-7-chloro-1, 4-benzodioxane-5-carboxylate¿ antagonized 5-HT responses with pA2 values of 9.1 +/- 0.1, 9.0 +/- 0.2 and 11.0 +/- 0.1, respectively. These affinity values were similar to those obtained at 5-HT4 receptors in isolated rat oesophagus (9.0+/- 0.4, 9.3 +/- 0.1 and 10.6 +/- 0.1 respectively). Despite these operational similarities between 5-HT4 receptors in guinea-pig colon and rat oesophagus, several novel compounds have revealed important differences between 5-HT4 receptors in the two tissues. For example, the substituted benzoate, RS 23597 ¿3-(piperidine-1-yl) propyl-4-amino-5-chloro-2-methoxybenzoate hydrochloride, acted as a partial agonist (intrinsic activity 0.5) in guinea-pig colon with a potency of 7.6 +/-0.1 (n = 16). In isolated rat oesophagus, however, this compound was a surmountable antagonist (pA2 = 7.8 +/- 0.1) with no intrinsic activity. In contrast, the substituted naphthalimide (S)RS 56532 ¿(S)-6-amino-5-chloro-2-(1-azabicyclo[2, 2, 2]octan-3-yl) 2,3-dihydro-1H-benz[de] isoquinoline-1,3-dione hydrochloride¿, was a potent (pEC50 = 7.9 +/- 0.1), efficacious partial agonist (intrinsic activity = 0.8) in the rat oesophagus. However, in guinea-pig colon, it was a surmountable antagonist with an affinity (pKB) of 9.4 +/- 0.1. Furthermore, several novel, selective, 5-HT4 compounds also showed opposing patterns of intrinsic activities similar to those described for RS 23597 and (S)RS 56532. It is concluded that these differences are inconsistent with differences in 5-HT4 receptor reserves, and may suggest that 5-HT4 receptors in the guinea-pig colon and the rat oesophagus can be operationally distinguished.

Animals↗
PubMed1996

The beta 1 sodium channel subunit modifies the interactions of neurotoxins and local anesthetics with the rat brain IIA alpha sodium channel in isolated membranes but not in intact cells.

Mammalian brain sodium channels consist of an alpha subunit and two smaller beta subunits. The role of the beta 1 subunit in modulating ligand interactions at these channels was examined using a cell line stably expressing human beta1 and rat brain IIA alpha subunits. Coexpression of the beta 1 subunit had no effect on the potencies of sodium channel blockers in inhibiting whole cell [3H]batrachotoxinin A benzoate ([3H]BTX) binding or veratridine-stimulated [14C]guanidinium influx. Coexpression of the beta 1 subunit also had no effect on the potencies of alpha scorpion toxin, brevetoxin, or RU 39568 in stimulating [14C]guanidinium influx. By contrast, coexpression of the beta 1 subunit had dramatic effects on ligand interactions in isolated membranes. In isolated membranes of cells expressing only the alpha subunit, the neurotoxins had no stimulatory effect on [3H]BTX binding and the potencies of local anesthetic-like channel inhibitors were 10-100-fold lower than those at native sodium channels. Whereas in membranes of cells coexpressing the beta 1 subunit, the neurotoxins increased [3H]BTX binding 30-fold and the potencies of the sodium channel inhibitors closely matched those found at native sodium channels. These findings indicate that the beta 1 subunit is not required for the binding of sodium channel activators or inhibitors but rather, that the beta 1 subunit may stabilize the alpha subunit in a functional conformation thereby allowing detection of these interactions in disrupted membranes.

Anesthetics, Local↗
PubMed1996

Characterization of [3H]GR 113808 binding to 5-HT4 receptors in brain tissues from patients with neurodegenerative disorders.

[3H]GR 113808 binding studies in guinea-pig brain tissue revealed a temperature dependency and pharmacological specificity consistent with labelling of a 5-HT4 receptor. Detailed comparison of competition data between brain regions from human and guinea-pig suggest minor differences in the rank order of affinity. Studies in brain tissue from patients with Alzheimer's Huntington's and Parkinson's disease have revealed specific deficits in 5-HT4 receptor densities.

Aged↗
PubMed1996

RS-17053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha, alpha-dimethyl-1H-indole-3-ethanamine hydrochloride), a selective alpha 1A-adrenoceptor antagonist, displays low affinity for functional alpha 1-adrenoceptors in human prostate: implications for adrenoceptor classification.

Norepinephrine (NE) contracts smooth muscle cells within the human lower urinary tract (LUT) (bladder neck, prostate, and urethra). Receptor distribution and pharmacological evidence have implicated activation of alpha 1A-adrenoceptors. We disclose the pharmacological properties of the novel, selective alpha 1A-adrenoceptor antagonist N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro- alpha,alpha-dimethyl-1H-indole-3-ethanamine hydrochloride (RS-17053) and examine critically the pharmacological identity of the alpha 1-adrenoceptor mediating contractions to NE in human LUT tissues. In several tissues from rat and cloned adrenoceptors, RS-17053 displayed high affinity for the alpha 1A-adrenoceptor (pKi and pA2 estimates of 9.1-9.9) and a 30-100-fold selectivity over the alpha 1B- and the alpha 1D-adrenoceptor subtypes (pK1 and pA2 estimates of 7.7-7.8). However, in isolated smooth muscle preparations from human LUT tissues, RS-17053 antagonized responses to NE only at high concentrations. Estimates of affinity (pA2) at alpha 1-adrenoceptors mediating NE-induced contractions were 7.5 in prostatic periurethral longitudinal smooth muscle (compared with 8.6 for prazosin), 6.9 in anterior fibromuscular stroma (prazosin, 8.9), and 7.1 in bladder neck (prazosin, 8.5). These findings indicate that contractile responses to NE in human LUT tissues are mediated by a receptor displaying pharmacological properties that are clearly different from those of the defined alpha 1A-adrenoceptor and raise the possibility that multiple forms of the alpha 1A-adrenoceptor may exist in human LUT that are discriminated by RS-17053. In this regard, the affinity estimates obtained with RS-17053 and other alpha 1-adrenoceptor antagonists in human LUT tissues are identical to those described for the putative alpha 1L-adrenoceptor.

Adrenergic alpha-Antagonists↗
PubMed1995

Allosteric interactions among agonists and antagonists at 5-hydroxytryptamine3 receptors.

Cooperation in the action of agonists suggests that there are multiple binding sites on 5-hydroxytryptamine3 (5-HT3) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5-HT3 receptors. The affinities of competitive 5-HT3 receptor antagonists were similar regardless of whether the receptors were labeled with [3H]RS-42358, [3H]granisetron, or 1-(m-[3H]chlorophenyl)biguanide ([3H]mCPG). By contrast, the affinities of the agonists 5-HT, mCPG, and phenylbiguanide were approximately 10-fold higher when the receptors were labeled with [3H]mCPG. The dissociation of [3H]mCPG, [3H]RS-42358, and [3H]RS-25259, but not [3H]granisetron, from both cloned and native 5-HT3 receptors was markedly slower in the presence of 5-HT or 2-methyl-5-HT than in the presence of antagonists such as RS-42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5-HT3 receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor.

Allosteric Regulation↗
PubMed1995

The interaction of RS 25259-197, a potent and selective antagonist, with 5-HT3 receptors, in vitro.

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.

Animals↗
PubMed1995

The pharmacology and distribution of human 5-hydroxytryptamine2B (5-HT2B) receptor gene products: comparison with 5-HT2A and 5-HT2C receptors.

1. Full length clones of the human 5-HT2B receptor were isolated from human liver, kidney and pancreas. The cloned human 5-HT2B receptors had a high degree of homology (approximately 80%) with the rat and mouse 5-HT2B receptors. 2. PCR amplification was used to determine the tissue distribution of human 5-HT2B receptor mRNA. mRNA encoding the 5-HT2B receptor was expressed with greatest abundance in human liver and kidney. Lower levels of expression were detected in cerebral cortex, whole brain, pancreas and spleen. Expression was not detected in heart. 3. Northern blot analysis confirmed the presence of 5-HT2B receptor mRNA (a 2.4 kB sized band) in pancreas, liver and kidney. An additional 3.2 kB sized band of hybridization was detected in liver and kidney. This raises the possibility of a splice variant of the receptor or the presence of an additional homologous receptor. 4. The human 5-HT2B receptor was expressed in Cos-7 cells and its ligand binding characteristics were compared to similarly expressed human 5-HT2A and 5-HT2C receptors. The ligand specificity of the human 5-HT2B receptor (5-HT > ritanserin > SB 204741 > spiperone) was distinct from that of the human 5-HT2A (ritanserin > spiperone > 5-HT > SB 204741) and 5-HT2C (ritanserin > 5-HT > spiperone = SB 204741) receptors. On the basis of a higher affinity for ketanserin and a lower affinity for yohimbine the human 5-HT2B receptor also appeared to differ from the rat 5-HT2B receptor. 5. These findings confirm the sequence of the human 5-HT2B receptor and they demonstrate that the receptor has a widespread tissue distribution. In addition, these data suggest that there are differences in ligand affinities between different species homologues of the receptor. Finally, the finding of two distinct bands on the Northern blots of liver and kidney raises the possibility of splice variants or subtypes of 5-HT2B receptors, within these tissues.

3T3 Cells↗
PubMed1995

Functional evidence equating the pharmacologically-defined alpha 1A- and cloned alpha 1C-adrenoceptor: studies in the isolated perfused kidney of rat.

1. The present study characterizes and classifies alpha 1-adrenoceptor-mediated vasoconstriction in the isolated perfused kidney of rat using quantitative receptor pharmacology and compares the results to radioligand binding studies (made in cloned alpha 1-adrenoceptor subtypes, native alpha 1A-adrenoceptors in submaxillary gland of rat, and alpha 1A-adrenoceptors in several other tissues of rat). 2. Concentration-effect curves to noradrenaline in the presence of 5-methyl-urapidil were biphasic, indicating alpha 1-adrenoceptor heterogeneity. The alpha 1-adrenoceptor subtype mediating the first phase (low affinity for 5-methyl-urapidil) could not be 'isolated' for detailed pharmacological characterization but was defined by a sensitivity to inhibition by chloroethylclonidine and an inability of methoxamine to activate the site. Additionally, vasoconstriction mediated by this alpha 1-adrenoceptor subtype or subtypes was abolished by nitrendipine (1 microM), thereby allowing characterization of the second, high affinity site for 5-methyl-urapidil. 3. The following antagonists interacted competitively with noradrenaline at the alpha 1-adrenoceptor for which 5-methyl-urapidil exhibits high affinity (pKB value): WB 4101 (10.3) > prazosin (9.5) approximately HV 723 (9.3) approximately 5-methyl-urapidil (9.2) > phenotolamine (8.6) > spiperone (pA2 = 8.1) approximately oxymetazoline (7.9). In contrast, insurmountable antagonism was seen with S(+)- and R(-)-niguldipine, the S(+)-isomer being approximately 30 fold more potent than the R(-)-isomer. Receptor protection experiments indicated that S(+)-niguldipine interacted directly with alpha 1-adrenoceptors. Dehydroniguldipine acted as a competitive antagonist (pKB = 9.0). Thus, the results with antagonists define the alpha 1-adrenoceptor as an alpha 1A-adrenoceptor. 4. An agonist 'fingerprint' was constructed in the presence of nitrendipine to define further the alpha 1A-adrenoceptor. The following order and relativity of agonist potency was obtained: cirazoline (1) approximately adrenaline (2) > noradrenaline (5) > phenylephrine (23) approximately amidephrine (31) > methoxamine (71) >> isoprenaline (1456) approximately dopamine (2210). 5. A high correlative association was shown between the affinity of antagonists obtained functionally in the isolated perfused kidney of rat and pKi values obtained from binding experiments with the cloned bovine alpha 1C-adrenoceptor (R2 = 0.85), native alpha 1A-adrenoceptors in submaxillary gland of rat (R2 = 0.79), and alpha 1A-adrenoceptors from several other tissues of rat (values taken from the literature, R2 = 0.89). 6. The present study demonstrates that the alpha 1A-adrenoceptor is the predominant alpha 1-adrenoceptor subtype mediating vasoconstrictor responses to exogenously administered noradrenaline in the isolated perfused kidney of rat. More importantly, alpha 1A-adrenoceptors mediating vasoconstrictor responses to noradrenaline exhibited a pharmacological equivalency to the cloned bovine alpha 1 c-adrenoceptor. Thus,definitive functional pharmacological data are provided for equating the two receptors and support results derived recently from molecular and radioligand binding studies.

Adrenergic alpha-Agonists↗