Histamine H3-receptor agonists and imidazole-based H3-receptor antagonists can be thermodynamically discriminated.

BACKGROUND AND PURPOSE: Studies suggest that measurement of thermodynamic parameters can allow discrimination of agonists and antagonists. Here we investigate whether agonists and antagonists can be thermodynamically discriminated at histamine H(3) receptors. EXPERIMENTAL APPROACH: The pK(L) of the antagonist radioligand, [(3)H]-clobenpropit, in guinea-pig cortex membranes was estimated at 4, 12, 21 and 30 degrees C in 20 mM HEPES-NaOH buffer (buffer A), or buffer A containing 300 mM CaCl(2), (buffer A(Ca)). pK(I)' values for ligands with varying intrinsic activity were determined in buffer A and A(Ca) at 4, 12, 21 and 30 degrees C. KEY RESULTS: In buffer A, the pK(L) of [(3)H]-clobenpropit increased with decreasing temperature while it did not change in buffer A(Ca). The Bmax was not affected by temperature or buffer and n (H) values were not different from unity. In buffer A, pK(I)' values for agonists remained unchanged or decreased with decreasing temperature, while antagonist pK(I) values increased with decreasing temperature; agonist binding was entropy-driven while antagonist binding was enthalpy and entropy-driven. In buffer A(Ca), temperature had no effect on antagonist and agonist pK(I) values; both agonist and antagonist binding were enthalpy and entropy-driven. CONCLUSIONS AND IMPLICATIONS: The binding of H(3)-receptor agonists and antagonists can be thermodynamically discriminated under conditions where agonist pK(I)' values are over-estimated (pK(I)' not = pK(app)). However, under conditions when agonist pK(I) approximately pK(app), the thermodynamics underlying the binding of agonists are not different to those of antagonists.

Correlation of apparent affinity values from H3-receptor binding assays with apparent affinity (pKapp) and intrinsic activity (alpha) from functional bioassays.

BACKGROUND AND PURPOSE: Agonist apparent affinities (pK(I)') in histamine H(3)-receptor binding assays were higher than expected from apparent affinity values (pK(app)) estimated in bioassay. Here, we investigate whether the degree of pK(I)' overestimation is related to agonist intrinsic efficacy, by studying the effect of buffer composition on the pK(I)' of ligands with varying intrinsic activity. EXPERIMENTAL APPROACH: In the guinea-pig ileum bioassay, intrinsic activity (alpha) was determined from the maximal inhibition of the contraction produced by increasing agonist concentration. pK(app) values were estimated using the method of Furchgott. The pK(L) of [(3)H]clobenpropit in guinea-pig cerebral cortex was estimated by saturation analysis in 20 mM HEPES-NaOH buffer (buffer B(0,0,0)), or buffer B(0,0,0) containing 70 mM CaCl(2), 100 mM NaCl and 100 mM KCl (buffer B(0.07,0.1,0.1)). PK(I) values were determined in competition studies in both buffers. KEY RESULTS: [(3)H]clobenpropit saturation isotherms had n (H) values of unity in both buffers. In buffer B(0.07,0.1,0.1), agonist pK(I)' values were closer to pK(app) values than in buffer B(0,0,0) but were associated with n (H) values <1. A two-site analysis of agonist data in buffer B(0.07, 0.1, 0.1) provided a better fit than a one-site fit and low affinity values (pK(IL)) were comparable to pK(app). Differences between the pK(I)' in buffer B(0,0,0) and pK(IL) values in buffer B(0.07,0.1,0.1) (DeltapK) were correlated with alpha. CONCLUSIONS AND IMPLICATIONS: H(3)-receptor binding assays conducted in buffer B(0,0,0) and buffer B(0.07,0.1,0.1) can provide a measure of ligand affinity (pK(app)) and intrinsic efficacy. The assay predicts that some ligands previously classified as H(3)-receptor antagonists may possess residual intrinsic efficacy.

A comparison of the effects of gastrin, somatostatin and dopamine receptor ligands on rat gastric enterochromaffin-like cell secretion and proliferation.

Gastrin regulates ECL cell histamine release and is a critical determinant of acid secretion. ECL cell secretion and proliferation is inhibited by gastrin antagonists and somatostatin but little is known about the role of dopamine agonists in this process. Since the ECL cell exhibits all three classes of receptor we evaluated and compared the effects of the gastrin receptor antagonist, (YF476), lanreotide (SST agonist) and novel dopaminergic agents (BIM53061 and BIM27A760) on ECL cell histamine secretion and proliferation. Highly enriched (>98%) ECL cell preparations prepared from rat gastric mucosa using a FACS approach were studied. Real-time PCR confirmed presence of the CCK2, SS2 and SS5 and D1 receptors on ECL cells. YF476 inhibited histamine secretion and proliferation with IC(50)s of 1.25 nM and 1.3 x 10(-11) M respectively, values 10-1000x more potent than L365,260. Lanreotide inhibited secretion and proliferation (2.2 nM, 1.9 x 10(-10) M) and increased YF476-inhibited proliferation a further 5-fold. The dopamine agonist, BIM53061, inhibited gastrin-mediated ECL cell secretion and proliferation (17 nM, 6 x 10(-10) M) as did the novel dopamine/somatostatin chimera BIM23A760 (22 nM, 4.9 x 10(-10) M). Our studies demonstrate that the gastrin receptor antagonist, YF476, is the most potent inhibitor of ECL cell histamine secretion and proliferation. Lanreotide, a dopamine agonist and a dopamine/somatostatin chimera inhibited ECL cell function but were 10-1000x less potent than YF476. Agents that selectively target the CCK2 receptor may provide alternative therapeutic strategies for gastrin-mediated gastrointestinal cell secretion and proliferation such as evident in the hypergastrinemic gastric carcinoids associated with low acid states.

Isolation of BVDV from bovine serum by EMEA/CVMP and 9 CFR: a comparison.

Data derived from both experience and validation exercises on the behaviour and properties of BVDV is presented as applied to recovery of the virus from bovine serum used in the industrial production of animal biologicals. Licensed products both in the EU and the U.S. must use ingredients shown to be free of this agent and both regulatory agencies have published regulations governing this topic (EU: EMEA/CVMP and US: 9 CFR). The two systems differ in some specifics covering cell cultures used, timing of testing within the assays and the use of multiple approaches to the isolation of this virus. It was found that selected cell lines instead of primary cells could be used for BVDV isolation. FA tests, however, needed to be performed weekly for maximum sensitivity instead of once at the end of the test. BVDV interference assay (comparative titration) was found to be an unreliable test for the detection of non-CPE BVDV in cell culture. BVDV neutralization assays, while desirable from an informational standpoint, were determined to be unnecessary in serum that was to be adequately irradiated. Ultracentrifuge concentration of BVDV from serum was found to be a satisfactory method of increasing the sensitivity of assays for this agent, although quantification of the virus was found unnecessary in serum to be irradiated. Recommendations for the harmonization of 9 CFR and EMEA/CVMP into one assay are given.

Potential histamine H2-receptor antagonists. 2. N-alpha-Guanylhistamine.

The agonist molecule, histamine, has been used as a starting point for the design of potential H2-receptor antagonists. Converting the side-chain amino group into a guanidine yielded the first histamine H2-receptor antagonist, Nalpha-guanylhistamine. Antagonism of H2 receptors was demonstrated by the inhibition of histamine-stimulated gastric acid secretion in the rat at high dose levels (approximate ID50 800 MUmol/kg, iv) and by the inhibition of histamine-stimulated tachycardia of guinea-pig right atrium (pA2 equals 3.9). Guanylhistamine behaves as a partial agonist at histamine H2 receptors.

Inotropic "A" ring substituted sulmazole and isomazole analogues.

A series of "A" ring substituted sulmazole and isomazole analogues have been prepared and evaluated as inotropic agents. pKA's, protonation sites, and log P values were measured for selected compounds and their electronic properties were calculated. No simple correlation between inotropic activity and pKA, protonation site, or log P value was observed. However, in vitro inotropism did correlate with the calculated charge density of the "B" ring imidazo nitrogen atom. The 6-position of sulmazole appeared to be the most tolerant toward substituents, the 6-amino derivative 7 being a more potent inotrope than sulmazole itself. 4-Methoxyisomazole 13 had comparable in vivo inotropic properties to those of isomazole.

Development of peptide 3D structure mimetics: rational design of novel peptoid cholecystokinin receptor antagonists.

The two hormones cholecystokinin and gastrin share the same C-terminal sequence of amino acids, namely Gly(29)-Trp(30)-Met(31)-Asp(32)-Phe(33)-NH(2). Nevertheless, this congruence has not precluded using this structure to develop selective ligands for either CCK(1) or CCK(2) receptors. Manipulation of the hydrophobic residues at positions 31 and 33 gave a series of CCK(1) tripeptide antagonists, typified by N-t-BOC-Trp-2-Nal-Asp-2-(phenyl)ethylamide (pK(B) 6.8 +/- 0.3). Molecular modeling was used to identify the bioactive conformation of these CCK(1)-selective compounds and prompted the design of new peptoid structures. We aimed to maintain the conformation of the parent series by exploiting patterns of hydrogen-bonding and pi-stacking interactions present in the original molecule, rather than introducing additional covalent bonds. The prototype, N-(succinyl-D-Asp-2-phenylethylamido)-L-Trp-2-(2-naphthyl)ethylami de, was a potent and selective CCK(1) antagonist (pK(B) 7.2 +/- 0.3). Furthermore, the new series showed patterns of biological activity that mirrored those of the parent tripeptides. These compounds contain elements of both peptide primary and secondary structure and represent a novel approach to designing peptidomimetics. Interesting results were obtained from comparing models of a representative tripeptide CCK(1) antagonist with a conformation of CCK(30)(-)(33) that others have proposed to be responsible for its activity at the CCK(2) receptor. The results suggest that CCK(1) and CCK(2) receptors recognize enatiomeric dispositions of the Trp(30) indole, Asp(32) carboxylic acid, and C-terminal phenyl groups arrayed about a common backbone configuration. This "functional chirality" may underpin the mechanism by which these closely related receptor systems bind CCK(30)(-)(33) and explain patterns of selectivity observed with optical isomers of a number of peptoid and nonpeptide ligands.

Pharmacological analysis of the muscarinic receptors involved when McN-A 343 stimulates acid secretion in the mouse isolated stomach.

In view of the recent M1 and M2 subclassification of muscarinic receptors and the suggestion of separate populations of muscarinic receptors on oxyntic and histamine cells in the gastric mucosa, we have analysed the effects of McN-A 343, classified as an M1-selective agonist, on gastric acid secretion by the mouse, isolated, lumen-perfused stomach assay. Acid secretion stimulated by McN-A 343 was not inhibited by tetrodotoxin pretreatment, although it was competitively antagonized by atropine (pKB 7.90), suggesting a muscarinic site of action between postganglionic neurones and the final secretory event. Acid secretion stimulated by McN-A 343 was more sensitive than 5-methylfurmethide-stimulated secretion to H2-receptor blockade: the profile of inhibition was consistent with expectations for a model of indirect agonism, suggesting that McN-A 343 preferentially stimulated the release of endogenous histamine from mucosal histamine cells. In view of this selective action the McN-A 343-pirenzepine interaction was studied, the latter being classified as an M1-selective antagonist. Results were consistent with expectations for a competitive interaction but the pKB (6.69) was not significantly different from the value obtained at the oxyntic cell, using 5-methylfurmethide as agonist in the presence of H2-receptor blockade, in a previous study. We suggest that there is no need to postulate differences in oxyntic and histamine cell muscarinic receptors to account for the selective stimulant activity of McN-A 343 observed in this study and the relatively selective inhibition of gastric acid secretion by pirenzepine in vivo. McN-A 343 selectivity may be accounted for by a higher muscarinic receptor density on the histamine cell and pirenzepine selectivity by a smaller degree of loss into the gastric secretion compared to atropine.

The isolated stomach preparation of the mouse: a physiological unit for pharmacological analysis.

Although oxyntic cell secretion can be studied at many organisation levels between isolated cell suspensions and non-invasive techniques in animals, the isolated, lumen-perfused, stomach preparation of the mouse represents a hierarchical level which eliminates extrinsic regulatory influences but retains all the cellular architecture known to be necessary for physiological responses and so can be defined as the physiological unit of acid secretion. The feeding pattern before and the distending pressure during an experiment have been identified as the main determinants of basal secretion: the combination of an intragastric pressure of 12 cmH2O and the fasted state generated a stable basal secretion over 2 h providing a satisfactory basis for bioassays. Basal acid secretion was lowered by treatment with omeprazole and sodium thiocyanate but not with tetrodotoxin, N-methylatropine or tiotidine, suggesting that basal secretion does not involve nervous stimulation or the local release of histamine under these experimental conditions. The improved assay permitted the full characterization of cumulative agonist concentration-effect curves in single stomach preparations to histamine, 5-methylfurmethide, pentagastrin and isobutyl-methylxanthine. Interestingly, pentagastrin produced sustained stimulation of gastric acid secretion under conditions when there was no pharmacological evidence that histamine secretion was taking place. This finding is discussed in relation to the role of histamine in the control of gastric acid secretion.

Pharmacological analysis of muscarinic receptors coupled to oxyntic cell secretion in the mouse stomach.

In the light of recent attempts to subclassify muscarinic receptors, agonist-antagonist interactions at muscarinic receptors have been re-examined using improved techniques, on the mouse, isolated, lumen-perfused stomach gastric acid assay. Using 5-methylfurmethide as the muscarinic agonist, the pKB estimated for atropine was significantly lower on the stomach assay (7.78) than on the guinea-pig trachea (8.93). However pKB values for N-methylatropine, the quaternary ammonium derivative of atropine, at concentrations producing dose-ratios above 20 on the stomach assay (pKB = 9.67), and over the full concentration range studied on the trachea (pKB = 9.69) were not significantly different. The deviation from simple competitive behaviour at low dose-ratios with N-methylatropine in the stomach assay is consistent with the effects of a saturable uptake mechanism for quaternary ammonium compounds. The pKB values for pirenzepine on the stomach (6.67) and the trachea (6.87) were not significantly different suggesting that pirenzepine behaves more like N-methylatropine in terms of expressed affinity. We conclude that the oxyntic cell muscarinic receptors are homogeneous with those in the guinea-pig trachea. An initial exploration suggests that there is a relationship between the lipophilicity (log P) of the antagonists and the degree of apparent underestimation of antagonist affinity in the stomach assay. This supports the hypothesis that the underestimation of antagonist affinity is due to the loss of antagonist into the gastric secretion from the receptor compartment. Apparently, relatively selective inhibition of acid secretion, compared to atropine, could be explained without the need to postulate heterogeneity of muscarinic receptor populations.

Analysis of anomalous pKb values for metiamide and atropine in the isolated stomach of the mouse.

1 In the isolated, lumen-perfused, stomach preparation of the mouse, metiamide was found by kinetic analysis to behave like a simple competitive antagonist of histamine-stimulated acid secretion. However, the pK(B) estimate of 5.08 was significantly lower than that found in guinea-pig atrium (6.0) or rat uterus (6.1) suggesting that H(2)-receptors might not be homogeneous.2 A similar analysis showed that atropine also behaved like a simple competitive antagonist of bethanechol-stimulated acid secretion and the estimated pK(B) (7.65) was significantly lower than the standard estimate of this parameter in guinea-pig ileum (9.0). Either the muscarinic cholinoceptors in mouse stomach were also anomalous or the preparation was introducing a systematic error. Lumen perfusion might distort this type of kinetic analysis by allowing steady-state conditions but not true equilibrium to develop at the receptor compartment due to loss of antagonist into the gastric secretion. Drug interactions at receptors in the muscle layers of the stomach would be expected to be much less sensitive to this error.3 When the atropine-bethanechol interaction was measured on the contraction of the isolated, lumen-perfused, stomach of the mouse the necessary conditions for simple competition were not met even though the sensitivity to atropine was obviously increased. The criteria for the expected simple competition were being obscured by events at low antagonist concentrations. Alterations in agonist or antagonist concentrations could be more or less eliminated so that physiological antagonism, perhaps by release of 5-hydroxytryptamine, was considered. This was supported, to some extent, by finding that, when stomachs from animals pretreated with reserpine were used, the kinetic analysis was normalized and gave a pK(B) of 8.99. Apparently, the muscarinic receptors in mouse stomach are homogeneous with those in other tissues.4 Therefore, we conclude that our results no more point to heterogeneity among histamine receptors than they point to differences in muscarinic cholinoceptors because this type of kinetic analysis can be readily distorted by special features of the measuring system.

Pharmacological analysis of the inhibition by pirenzepine and atropine of vagal-stimulated acid secretion in the isolated stomach of the mouse.

The muscarinic receptors involved in the vagal stimulation of gastric acid secretion in the mouse isolated stomach assay have been examined by analysing the effects of pirenzepine and atropine on fully-defined frequency-effect curves. Both atropine and pirenzepine produced concentration-dependent inhibition of vagal-stimulated acid secretion in a manner consistent with a model describing competitive antagonism of endogenous acetylcholine, which was assumed to be released by vagal stimulation. The results obtained are quite compatible with the hypothesis that vagal stimulation involves muscarinic receptors which are homogeneous with those previously found on histamine and oxyntic cells in the mouse stomach assay. These results find no evidence for muscarinic receptor heterogeneity and reinforce the hypothesis that the selectivity of pirenzepine in vivo relative to atropine is due to the loss of atropine into the gastric secretion.

Further analysis of anomalous pKB values for histamine H2-receptor antagonists on the mouse isolated stomach assay.

Agonist-antagonist interactions at histamine receptors have been re-examined using improved techniques, on the mouse isolated, lumen-perfused, stomach gastric acid assay. Using histamine as agonist, pKB values have been estimated for burimamide, metiamide, cimetidine, ranitidine, oxmetidine and famotidine on both the gastric and guinea-pig isolated right atrium assays. With the exception of oxmetidine on the atrial assay, these compounds behaved as competitive antagonists on both assays. Oxmetidine significantly depressed basal rate on the atrial assay and the Schild plot slope parameter (0.81) was significantly less than one. The pKB values estimated on the gastric assay were lower than those on the atrial assay. However, the difference between the values on the gastric and atrial assays was not constant. The difference between the two assays for famotidine was not significant. We conclude that the apparent varying selectivity of the antagonists for gastric and atrial histamine H2-receptors may be explained by the differential loss of antagonists into the gastric secretion from the receptor compartment and that there is no need to postulate heterogeneity of histamine H2-receptors.

Pharmacological analysis of the pentagastrin-tiotidine interaction in the mouse isolated stomach.

The pentagastrin-tiotidine interaction has been analysed, using improved techniques, in the mouse isolated, lumen-perfused, stomach assay. For comparison and quantification of the H2-receptor blocking activity of tiotidine, histamine-tiotidine interactions have also been analysed in the mouse stomach and guinea-pig isolated right atrial preparation. Tiotidine behaved as a competitive antagonist of histamine both in the guinea-pig right atrium (pKB 7.57) and mouse stomach (pKB 6.96). The difference in pKB was attributed to the loss of tiotidine into the gastric secretion. On the stomach assay, pentagastrin concentration-effect curves were significantly flatter with lower maximal responses than those obtained to histamine. In addition the profile of inhibition observed with tiotidine was different in that the pentagastrin curve maxima were depressed with only a small concomitant dextrad shift. A mathematical model has been developed which accounts for the differences in agonist concentration-effect curves and describes in a quantitative manner the expectations for the competitive antagonism of endogenous histamine assumed to be released by pentagastrin. Fitting of the pentagastrin-tiotidine data to this model provided a reasonable goodness-of-fit. The results are discussed in terms of the role of endogenous histamine in gastrin-stimulated acid secretion. We conclude that the results are consistent with the hypothesis that pentagastrin stimulates acid secretion via the release of endogenous histamine under the present experimental conditions.

Estimation of pKB values for histamine H2-receptor antagonists using an in vitro acid secretion assay.

1 Histamine H2-receptor antagonism by burimamide, metiamide and cimetidine was analysed under apparent equilibrium conditions in the lumen-perfused isolated stomach preparation of the mouse. 2 The behaviour of these compounds was not incompatible with simple competitive antagonism but the estimated pKB values (-log KB) were all significantly lower, by about 1 log unit, than reference values reported for guinea-pig atrium or rat uterus. 3 There seems no need to propose that the parietal cell receptors are somehow different from the others; metiamide continually appears in the gastric juice so that a steady-state but not equilibrium can presumably be reached with respect to the bath concentration and this could keep the antagonist concentration artificially low in the region of the receptors.

Pharmacological analysis of beta-adrenoceptor-mediated agonism in the guinea-pig, isolated, right atrium.

The recently developed, operational model of pharmacological agonism defines the efficacy of agonists by tau = [Ro]/KE, where [Ro] is the total functional concentration of receptors and KE is the concentration of agonist-occupied receptors for half-maximal effect. Theoretically, variations in [Ro] and KE affect tau and in turn, E/[A] curve profiles similarly. Using the beta-adrenoceptor mediated chronotropic responses of the guinea-pig isolated right atrial preparation we have investigated the consequences of experimental [Ro] and KE variation. Bromoacetylalprenolol menthane (M-75) produced displacements of isoprenaline and dichloroisoprenaline E/[A] curves consistent with [Ro] reduction. Cholera toxin produced displacements consistent with decreases in KE. The operational model provides a simple conceptual framework for the prediction and interpretation of changes in E/[A] curve profile resulting from experimental interventions at the post-receptor (KE) level as well as at the receptor ([Ro]) level.

An operational model of pharmacological agonism: the effect of E/[A] curve shape on agonist dissociation constant estimation.

An operational model of pharmacological agonism has been analysed to predict the behaviour of rectangular hyperbolic and non-hyperbolic agonist-concentration effect, E/[A], curves with variation in receptor concentration, [Ro]. Irreversible antagonism is predicted to cause E/[A] curve gradient changes in non-hyperbolic cases but not in hyperbolic cases; in both cases estimation of agonist dissociation constants (KAS) is theoretically valid. 5-Hydroxytryptamine (5-HT) produced "steep' E/[A] curves in contracting the rabbit isolated aorta preparation. Irreversible antagonism by phenoxybenzamine (Pbz) produced a flattened E/[A] curve, consistent with theoretical predictions. Fitting 5-HT E/[A] curves in the presence and absence of Pbz to the model provided an estimate of KA for 5-HT which was not significantly different from the estimate obtained using Furchgott's null method. The operational model of agonism appears to account qualitatively and quantitatively for the effects of [Ro] changes on hyperbolic and non-hyperbolic E/[A] curves. Under conditions where irreversible antagonism may be used to estimate KAS, fitting the operational model directly to E/[A] data represents a valid, economical and analytically simple alternative to the conventional null method.

Cardiac-specific overexpression of human beta2 adrenoceptors in mice exposes coupling to both Gs and Gi proteins.

1. Left atrial strips from transgenic (TG4) mice with cardiac-specific overexpression ( approximately 200-fold) of the beta(2) adrenoceptor (beta(2)AR) were isolated, and their isometric force of contraction (F(c)) in response to electrical stimulation was measured. 2. The betaAR agonist isoprenaline elicited negative inotropic responses in all left atrial strips; in 6/11 preparations, it also had a small positive inotropic effect. This 'up-phase' was observed from 0.1 to 10 nM, with the 'down-phase' occurring at higher concentrations. Both phases were mediated by beta(2)AR, as shown by their sensitivity to the beta(2)AR antagonist ICI-118,551 (100 nM; pA(2) 8.60+/-0.07, 8.45+/-0.19, for 'up-phase' and 'down-phase,' respectively), but not the beta(1)AR antagonist CGP-20712A (100 nM). Conversely, nontransgenic littermate preparations responded to isoprenaline treatment solely by an increase in F(c), which was beta(1)AR-mediated. 3. Pretreatment of left atrial strips with either 10 nM isoprenaline or 1 mM 8-bromo-cAMP significantly attenuated the TG4 'up-phase', while having no effect on either the TG4 'down-phase' or the littermate controls' responses. B. pertussis toxin treatment of the animals prevented isoprenaline's negative inotropic effects in TG4 preparations, but had no effect in littermate controls. 4. The findings imply that the responses of TG4 left atrium to isoprenaline are because of beta(2)AR coupling to G(s) and G(i) proteins, consistent with the model of Daaka et al., in which protein kinase A phosphorylation of the beta(2)AR causes a switch from G(s) to G(i) protein coupling.

Comparative study of the control of basal acid output from rodent isolated stomachs.

1. Isolated, lumen-perfused, whole stomach preparations from mouse and immature rat produced a stable basal acid output which, although not blocked by histamine H2-, acetylcholine M- or CCKB/gastrin receptor antagonists, was almostly completely blocked by the H+/K(+)-ATPase inhibitor, omeprazole, and the metabolic inhibitor, sodium thiocyanate (NaSCN). 2. Fully-defined concentration-effect curves could be obtained on both assays with the phosphodiesterase inhibitor, isobutyl methylxanthine (IBMX) and with dibutyryl cyclic AMP. 3. On the rat stomach assay, histamine H2-receptor blockade had no effect on the IBMX curve. In contrast, the IBMX response in the mouse was abolished by histamine H2-receptor blockade. On both assays responses to dibutyryl cyclic AMP were resistant to H2-receptor blockade. 4. In the absence of suprathreshold endogenous histamine, it is argued that H+/K(+)-ATPase mediated basal acid secretion from the mouse stomach assay is regulated by something other than cyclic AMP.

Analysis of the activity of alpha 1-adrenoceptor antagonists in rat aorta.

1. In this study, the effect of seven alpha 1-adrenoceptor antagonists (tamsulosin, phentolamine, prazosin, WB-4101, 5-methylurapidil, spiperone and HV723) have been examined on the contractile response to noradrenaline (NA) and phenylephrine (PE) in rat isolated aorta. 2. NA and PE, when administered using a cumulative dosing schedule, both produced concentration-dependent contraction of aortic rings. It was possible to fit the individual concentration-effect (E/[A]) curve data to the Hill equation to provide estimates of the curve midpoint location (p[A]50 = 7.74 +/- 0.10 and 7.14 +/- 0.18), midpoint slope (nH = 0.82 +/- 0.03 and 0.99 +/- 0.10) and upper asymptote (alpha = 3.2 +/- 0.3 and 3.1 +/- 0.2 g) parameters for NA and PE, respectively. However, the Hill equation provided a better fit to the E/[A] curve data obtained with another contractile agent, 5-hydroxytryptamine (5-HT) (p[A50] = 6.09 +/- 0.08, nH = 1.49 +/- 0.09, alpha = 2.6 +/- 0.3 g), as judged by calculation of the mean sum of squares of the differences between the observed and predicted values. 3. All of the antagonists investigated produced concentration-dependent inhibition of the contractile responses of the aorta to NA and PE. Although no significant effects on the upper asymptotes of the E/[A] curves of any of the antagonists tested were detected, only tamsulosin and 5-methylurapidil did not have a significant effect on the slope (nH) of the NA and PE E/[A] curves. The other antagonists produced significant steepening of the curves obtained with NA and/or PE. 4. Notwithstanding the fact that one of the basic criteria for simple competitive antagonism at a single receptor class was not always satisfied, the individual log [A]50 values estimated in the absence and presence of antagonist within each experiment were fitted to the competitive model. The Schild plot slope parameters for the antagonism of NA and PE by phentolamine and HV723 were found to be significantly less than unity. The Schild plot slope parameters for the other antagonists were not significantly different from unity. 5. In the absence of evidence to suggest that the deviations from simple competitive antagonism were due to failure to satisfy basic experimental conditions for quantitative analysis, an attempt was made to see whether the data could be accounted for by an existing two-receptor model (Furchgott, 1981). The goodness-of-fit obtained with the two-receptor model was significantly better than that obtained with the one-receptor model. Furthermore, with the exception of the data obtained with phentolamine, the pKB estimates for the two receptors were independent of whether NA or PE was used as agonist. 6. To determine which alpha 1-adrenoceptor subtypes may be associated with those defined by the two receptor model, the mean pKB estimates obtained from the two-receptor model fit were compared with affinities measured by Laz et al. (1994) for rat cloned alpha 1-adrenoceptor subtypes expressed in COS-7 cells. The sum of squared differences of the data points from the line of identity was smallest for both pKB1 and pKB2 in the case of the alpha 1a/d-adrenoceptor (now referred to as alpha 1d-adrenoceptor; Hieble et al., 1995). Therefore, the complexity exposed in this study may be due to the expression of closely-related forms of the alpha 1d-adrenoceptor. However, relatively good matches were also found between pKB1 and alpha 1c and between pKB2 and alpha 1b. Therefore, on the basis of these data, it is not possible to rule out the involvement of all three alpha 1-adrenoceptors. The conflicting reports concerning the characteristics of the alpha 1-adrenoceptor population mediating contraction of the rat aorta may, at least in part, be due to the lack of highly selective ligands and to between-assay variation in the expression of multiple alpha 1-adrenoceptors.