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Background and purpose:Agonist apparent affinities (pKI′) in histamine H3-receptor binding assays were higher than expected from apparent affinity values (pKapp) estimated in bioassay. Here, we investigate whether the degree of pKI′ overestimation is related to agonist intrinsic efficacy, by studying the effect of buffer composition on the pKI′ of ligands with varying intrinsic activity.Experimental approach:In the guinea-pig ileum bioassay, intrinsic activity (α) was determined from the maximal inhibition of the contraction produced by increasing agonist concentration. pKapp values were estimated using the method of Furchgott. The pKL of [3H]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 CaCl2, 100 mM NaCl and 100 mM KCl (buffer B(0.07,0.1,0.1)). PKI values were determined in competition studies in both buffers.Key results:[3H]clobenpropit saturation isotherms had n H values of unity in both buffers. In buffer B(0.07,0.1,0.1), agonist pKI′ values were closer to pKapp 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 (pKIL) were comparable to pKapp. Differences between the pKI′ in buffer B(0,0,0) and pKIL values in buffer B(0.07,0.1,0.1) (ΔpK) were correlated with α.Conclusions and implications:H3-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 (pKapp) and intrinsic efficacy. The assay predicts that some ligands previously classified as H3-receptor antagonists may possess residual intrinsic efficacy.British Journal of Pharmacology (2007) 151, 128–143. doi:10.1038/sj.bjp.0707174; published online 12 March 2007 [ABSTRACT FROM AUTHOR]

(1) The alternatively spliced, short and long cholecystokinin receptors (CCK2S and CCK2L) were expressed in NIH3T3 cells, and compared using radioligand-binding assays with identical buffer and incubation conditions. (2) As judged by a saturation analysis, the selective CCK2-receptor antagonist radioligand [3H]-JB93182 did not discriminate between the CCK2S or CCK2L receptors. (3) A global analysis of competition studies, using a range of structurally diverse, CCK-receptor selective ligands, provided further evidence that these receptor subtypes were pharmacologically indistinguishable. However, when analysed individually a number of small, yet significant differences were observed with some of the compounds. (4) These data are consistent with previous study that suggested a possible pharmacological difference between these isoforms, at least in terms of the CCK2-receptor antagonist, L-365,260. However, it would appear that the pharmacological profile of these compounds is not consistent with their affinity at the putative G1/G2 receptors previously described by Harper et al.

1. The pharmacology of the cholecystokinin CCK(1) receptors endogenously expressed in human gallbladder and human ascending colon smooth muscle tissue was compared using radioligand binding assays. 2. Saturation analysis of the interaction between the radiolabelled, selective CCK(1)-receptor antagonist, [(3)H]-L-364,718, and enriched gastrointestinal tissue membranes suggested the presence of multiple binding sites in human colon but not human gallbladder. 3. Competition studies, using a range of structurally diverse, CCK-receptor selective ligands provided further evidence for CCK(1) receptor heterogeneity in human colon tissue (n(H) values significantly less than unity for SR27897=0.77+/-0.07, 2-NAP=0.73+/-0.03, YM220=0.70+/-0.09 and PD-134,308=0.83+/-0.01). Moreover, the competition data for SR27897, 2-NAP and YM220 were consistent with the interaction of these compounds at two binding sites. In contrast, in the human gallbladder assay, a single binding site model provided a good fit of the competition curve data obtained with all the CCK receptor selective compounds. 4. The data obtained are consistent with the presence of a single CCK(1) receptor binding site in the gallbladder but not in the colon. A two-site analysis of the colon data, indicated that one of the two sites was indistinguishable from that characterized in the gallbladder. The molecular basis of the apparent receptor heterogeneity in the colon remains to be established., (British Journal of Pharmacology (2002) 136, 873-882)

Cholecystokinin (CCK) produces contractions of gallbladder and colon in a number of different species. Although the effects of CCK on the human gallbladder are relatively well documented, the CCK receptors in the human colon have not been clearly characterised. Therefore, in this study, the CCK receptors in the human gallbladder and colon were compared using pharmacological techniques. Contraction of specimens of the human tissue was measured using in vitro organ bath bioassay. The effect of selective concentrations of CCK(1) and CCK(2) receptor antagonists (L-364,718 and JB93182, respectively) was determined on agonist concentration-effect (E/[A]) curves obtained by cumulative dosing with sulphated CCK. The CCK(1) antagonist L-364,718 produced a rightward shift of the CCK-8S [E/[A] curve in the human gallbladder (pA(2)=9.15 +/- 0.26) and ascending colon (pA(2)=9.20 +/- .33). In both tissues, the CCK(2) receptor antagonist, JB93182, had no effect on the CCK E/[A] curves. In addition, in the colon, pentagastrin responses were inhibited by L-364,718 but unaffected by JB93182. These data indicate that the CCK-induced contraction of the human colon and gallbladder smooth muscle is mediated solely through the CCK(1) receptor subtype, and the antagonist affinity estimates are consistent with those previously obtained in experiments on animal tissue.

This unit describes three standard in vitro bioassays for studying histamine H₁, H₂ and H₃ receptors in isolated intact tissues removed from the guinea pig. Both the H₁ and H₃ receptor assays are based on preparations of the ileum, whereas the spontaneously beating right atrium assay is used for the H₂-receptor.This unit describes three standard in vitro bioassays for studying histamine H₁, H₂ and H₃ receptors in isolated intact tissue.

In the course of structural explorations around a series of potent CCK2 receptor antagonists, it was noted that simple N-methylation of the indolic N-H in the parent molecule gave rise to behavior in vivo that was consistent with the compound acting as an agonist. Exploration in vitro confirmed this property, and it was shown that the agonist action could be blocked by the reference CCK2 receptor antagonist, L-365,260. Further examples of this type of modification were explored, and a common theme with regard to agonist behavior was uncovered. Some molecular modeling is also presented in an attempt to throw light on the nature of the ligand receptor interactions that may be giving rise to the differing properties of these, apparently, structurally similar molecules.

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.

Novel, potent, and selective non-imidazole histamine H(3) receptor antagonists have been prepared based on the low-affinity ligand dimaprit (pK(I) 7.32 +/- 0.12, pK(B) 5.93 +/- 0.17). Detailed structure-activity studies have revealed that N-(4-chlorobenzyl)-N-(6-pyrrolidin-1-ylhexyl)guanidine (pK(I) 8.38 +/- 0.21, pK(B) 8.39 +/- 0.13), 30, and N-(4-chlorobenzyl)-N-(7-pyrrolidin-1-ylheptyl)guanidine (pK(I) 8.78 +/- 0.12, pK(B) 8.38 +/- 0.10), 31, exhibit high affinity for the histamine H(3) receptor. Antagonists 30 and 31 demonstrate significant selectivity over the other histamine, H(1) and H(2), receptor subtypes and a 100-fold selectivity in the sigma(1) binding assay. Compounds 30and 31 are the most potent, selective non-imidazole histamine H(3) receptor antagonists reported in the literature to date.

Recently, it was demonstrated that 5-HT induces relaxation of human colon circular muscle through activation of 5-HT(4) receptors and 5-HT(7) receptors. The aim of the current study was to develop a new in vitro bioassay of human colon that would facilitate the pharmacological analysis of 5-HT responses mediated solely by 5-HT(4) receptors. Contracting circular muscle strips with KCl (80 mM) yielded a stable contractile tension and, in contrast to muscarinic cholinoceptor agonists and histamine, a profound reduction of spontaneous contractility. This allowed the establishment of reproducible, fully-defined, agonist concentration-response curves by cumulative dosing. Under these conditions, 5-HT induced a concentration-dependent relaxation (pEC(50) 7.31, Hill slope 0.91). Neither methysergide (10 microM) nor granisetron (1 microM) affected the 5-HT-induced relaxation, suggesting that 5-HT(1), 5-HT(2), 5-HT(3), 5-ht(5), 5-HT(6) or 5-HT(7) receptors are not involved. The lack of effect of tetrodotoxin (0.3 microM) indicated a direct effect of 5-HT on the smooth muscle. The selective 5-HT(4) receptor antagonists GR 113808, GR 125487 and RS 39604 competitively antagonized the 5-HT-induced relaxation (pK(B) 9.43, 10.12 and 8.53, respectively). SB 204070 (1 nM) produced a rightward shift (pA(2) 10.34) and depression of the 5-HT curve. These affinity estimates are similar to those previously reported for 5-HT(4) receptors. The selective 5-HT(4) receptor agonists, prucalopride and R076186, induced relaxations (pEC(50) 7.50 and 7.57, respectively), that were blocked by GR 113808 (3 nM), yielding pA(2) estimates of 9.31 and 9.21, respectively. To summarise, in KCl (80 mM)-contracted muscle strips, 5-HT induces relaxation through activation of a homogeneous smooth muscle 5-HT(4) receptor population. This new bioassay allows the focused, pharmacological characterization of human colonic 5-HT(4) receptors in vitro.

1 We have investigated the binding of a novel histamine H3-receptor antagonist radioligand, [3H]- clobenpropit ([3H]-VUF9153), to guinea-pig cerebral cortex membranes. 2 Saturation isotherms for [3H]-clobenpropit appeared biphasic. Scatchard plots were curvilinear and Hill plot slopes were significantly less than unity (0.63+/-0.03; n = 12+/-s.e.mean). The radioligand appeared to label two sites in guinea-pig cerebral cortex membranes with apparent affinities (pKD') of 10.91+/-0.12 (Bmax = 5.34+/-0.85 fmol mg(-1) original wet weight) and 9.17+/-0.16 (Bmax = 23.20+/-6.70 fmol mg(-1)). 3 In the presence of metyrapone (3 mM) or sodium chloride (100 mM), [3H]-clobenpropit appeared to label a homogeneous receptor population (Bmax=3.41+/-0.46 fmol mg-1 and 3.49+/-0.44 fmol mg(-1), pKD' = 10.59+/-0.17 and 10.77+/-0.02, respectively). Scatchard plots were linear and Hill slopes were not significantly different from unity (0.91+/-0.04 and 0.99+/-0.02, respectively). Granisetron (1 microM), rilmenidine (3 microM), idazoxan (0.3 microM), pentazocine (3 microM) and 1,3-di-(2-tolyl)guanidine (0.3 microM) had no effect on the binding of [3H]-clobenpropit. 4 The specific binding of [3H]-clobenpropit appeared to reach equilibrium after 25 min at 21+/-3 degrees C and remained constant for >180 min. The estimated pKD' (10.27+/-0.27; n = 3+/-s.e.mean) was not significantly different from that estimated by saturation analysis in the presence of metyrapone. 5 A series of histamine H3-receptor ligands expressed affinity values for sites labelled with [3H]-clobenpropit which were not significantly different from those estimated when [3H]-R-alpha-MH was used to label histamine H3-receptors in guinea-pig cerebral cortex membranes.

1. The binding of the selective histamine H3-receptor agonist ([3H]-R-alpha-methylhistamine) to sites in guinea-pig cerebral cortex and ileum longitudinal muscle myenteric plexus has been characterized and a comparison made of the apparent affinities of a series of H3-receptor ligands. 2. Saturation analysis suggested that [3H]-R-alpha-methylhistamine labelled a homogeneous population of histamine H3-receptors in guinea-pig cerebral cortex (pKD=9.91+/-0. 07; nH=1.07+/-0.03; n=5) and ileum longitudinal muscle myenteric plexus (pKD=9.75+/-0.21; nH=0.97+/-0.02; n=5). There was no significant difference in the estimated affinity of [3H]-R-alpha-methylhistamine in the two tissues. The cerebral cortex had a significantly higher receptor density (3.91+/-0.37 fmol mg-1 tissue) than the ileum longitudinal muscle myenteric plexus (0. 39+/-0.11 fmol mg-1). 3. Overall, the apparent affinities of compounds, classified as H3-receptor ligands, in cerebral cortex and ileum longitudinal muscle myenteric plexus were well correlated (r=0. 91, P<0.0001) and consistent with the cerebral cortex and ileum longitudinal muscle myenteric plexus expressing histamine H3-receptor population(s) that are pharmacologically indistinguishable by the majority of histamine H3-receptor ligands. However, it was evident that the homologues of histamine within this group of compounds could discriminate between the receptor populations in the two tissues. Thus, the estimated affinity of five imidazole unbranched alkylamines (histamine, homohistamine, VUF4701, VUF4732 and impentamine) were significantly higher in the guinea-pig cerebral cortex than in the ileum longitudinal muscle myenteric plexus assay.

Histamine was converted to a selective histamine H3-receptor antagonist by capping the primary amine with 2-naphthalenesulfonyl chloride. Higher receptor affinity and lower variability in the data from the various bioassays were achieved with the 2-naphthalensulfonamides of histamine homologues.

1. The CCKB/gastrin receptors mediating pentagastrin stimulation of gastric acid secretion by histamine release and by direct stimulation of oxyntic cells have been characterized in the immature rat isolated stomach assay. This was achieved by estimating antagonist affinity values for competitive antagonists from three distinct chemical classes (L-365,260, PD134,308 and JB93190) in the absence and presence of a high concentration of the histamine H2-receptor antagonist, famotidine (30 microM). 2. Pentagastrin produced concentration-dependent stimulation of gastric acid secretion in the absence and presence of famotidine. Famotidine depressed the maximum secretory response to pentagastrin although the degree of depression varied between experimental replicates (25-60%). This variation was attributed to the histamine-release mediated component of acid secretion, as judged by the consistency of the maximum responses obtained in the presence, but not absence, of famotidine. 3. All three CCKB/gastrin receptor antagonists behaved as surmountable antagonists in the absence and presence of famotidine. JB93190 (pKB approximately 9.1, approximately 8.9, in the absence and presence of famotidine, respectively) was approximately 30 fold more potent than either L-365,260 (pKB approximately 7.4, approximately 7.1) or PD134,308 (pKB approximately 7.6, approximately 7.4). 4. It was assumed that the famotidine treatment converted pentagastrin-stimulated acid secretion from a combination of an indirect action due to the release of histamine and a direct action on the oxyntic cell to solely a direct action on the oxyntic cell. A simple mathematical model of this two-receptor system was developed. The direct and indirect components were assumed to sum to produce the total response to pentagastrin obtained in the absence of famotidine. It was found that this model could account quantitatively for the behaviour of the three antagonists without invoking a difference in antagonist affinity for the CCKB/gastrin receptors mediating the direct and indirect actions of pentagastrin. However, a conclusion of receptor homogeneity has to be qualified because the model was also used to generate simulations which indicated that the analysis could only detect antagonist affinity differences of greater than one log-unit between enterochromaffin-like (ECL) and oxyntic cell CCKB/gastrin receptor populations.

1. Since L-365,260 was first described as a selective antagonist at cholecystokinin (CCK)B/gastrin receptors, we have used it periodically as a reference compound in isolated tissue assays of guinea-pig gastric muscle and lumen-perfused stomachs from mouse and immature rat. L-365,260 behaved as a surmountable antagonist and produced parallel rightward shifts of pentagastrin concentration-effect curves' in each of the replicate experiments. The experiments were performed by several different experimenters in the same laboratories over a five year period. 2. In the isolated, lumen-perfused, immature rat stomach assay, L-365,260 behaved as a simple competitive antagonist (Schild plot slope = 1.00 +/- 0.10, pKB = 7.54 +/- 0.03 from a global analysis of the data) acting at a homogeneous population of receptors in five separate, highly-reproducible, experiments. In contrast, the replicate data sets obtained from the interaction in the isolated, lumen-perfused mouse stomach and guinea-pig gastric muscle assays, over the same period, were not consistent with the presence of a single receptor population. The guinea-pig gastric muscle data were relatively reproducible between experiments but some individual Schild plot slopes and the slope estimated from a global analysis of all the data were significantly less than unity (slope = 0.80 +/- 0.07, pA2 = 8.56 +/- 0.05 from the global analysis). The data obtained in the mouse stomach were significantly more variable than that obtained in the same assay, during the same period, from the interaction between histamine and the H2-receptor antagonist, famotidine. The individual Schild plot slopes ranged from being very flat (0.20) to being not significantly different from unity (1.23) and the pA2 values ranged from 7.68 to 8.70. 3. Overall, the data could be accounted for by assuming the variable expression of two receptor subtypes across the assays. The rat stomach appeared to express a single receptor characterized by a low affinity constant for L-365,260 (pKB approximately 7.5). The guinea-pig gastric muscle and mouse stomach data could be explained by the presence of this receptor and a second one characterized by a high affinity constant for L-365,260 (pKB approximately 8.6). The activity of the two proposed receptor subtypes was consistent between experiments in the guinea-pig and the high affinity receptor appeared to be predominant. In contrast, the mouse stomach data could only be simulated by assuming that the proportion and absolute number of each subtype varied significantly between the replicate experiments. 4. The L-365,260 affinity estimates at the inferred receptor subtypes were indistinguishable from those obtained in a corresponding analysis of the behaviour of L-365,260 in CCKB/gastrin receptor radioligand binding experiments in guinea-pig gastric gland and mouse and rat cerebral cortex preparations.

1. For several years, we have used the cholecystokinin (CCK)B/gastrin receptor selective antagonist, L-365,260, as a reference compound in a variety of studies in CCKB/gastrin receptor radioligand binding assays. Here, we have analysed the competition curve data sets obtained between L-365,260 and [125I]-BH-CCK8S in guinea-pig gastric gland and mouse and rat cerebral cortex preparations. 2. Competition curves obtained for L-365,260 in the mouse cortex assay were not different from rectangular hyperbolae (slope = 1.01 +/- 0.02) implying the presence of a single population of binding sites (pKI = 8.41 +/- 0.01; data from 47 experiments, slope constrained to unity). However, in the rat cortex and guinea-pig gastric gland assays, the mean slope of the competition curves was significantly less than one and the mean apparent pKI significantly lower than that obtained in the mouse cortex (slope = 0.85 +/- 0.03, 0.90 +/- 0.03; apparent pKI = 7.98 +/- 0.05, 8.07 +/- 0.05; 48 and 45 experiments, in rat and guinea-pig, respectively). The distribution of the individual pKI and slope estimates of the competition curves in these two assays was consistent with expectations for the variable expression (in terms of absolute number and proportion) of two binding sites. The two sites were characterized by pKI values for L-365,260 of 8.50 +/- 0.04 and 8.48 +/- 0.04 for the high affinity site and 7.32 +/- 0.04 and 7.22 +/- 0.06 for the low affinity site in guinea-pig and rat, respectively. 3. The affinity estimates for L-365,260, although obtained on different tissues, are consistent with data obtained from the analysis of L-365,260 antagonism of pentagastrin-stimulated responses in mouse and rat stomach (acid secretion) and guinea-pig gastric muscle (isotonic contraction) assays. To this extent, these data suggest the existence of two CCKB/gastrin receptor subtypes.

We have studied the effects of idazoxan in rat aorta and small mesenteric artery. In the aorta, idazoxan behaved as a partial agonist (pKA = 6.30). Prazosin produced rightward shift (pA2 = 9.88) and steepening of the idazoxan curve. In contrast, idazoxan had no effect of basal tension in the mesenteric artery, but shifted the noradrenaline curve to the right in a parallel manner (pA2 = 6.12). The selective alpha 1-adrenoceptor agonist, indanidine, also behaved as a partial agonist in the aorta and produced no significant contractions of the small mesenteric artery. Since idazoxan and indanidine have been reported to raise blood pressure in the pithed rat via an action at vascular alpha 1-adrenoceptors, these results call into question the reliability of the small mesenteric artery assay as a predictor for alpha 1-adrenoceptor-mediated pressor activity in vivo.

1. In this study, we examined the interaction between noradrenaline (NA) and phenylephrine (PE) with seven antagonists (prazosin, tamsulosin, phentolamine, WB-4101, 5-methylurapidil, spiperone and HV 723) in an attempt to characterize the alpha 1-adrenoceptor population of the rat isolated small mesenteric artery (SMA) preparation. 2. Six of the seven antagonists investigated produced concentration-dependent, parallel, rightward shift of the NA concentration-effect (E/[A]) curves. The exception was tamsulosin, which produced significant decrease of the upper asymptote. In the case of 5-methylurapidil and HV723, the Schild plot slope parameters were not significantly different from unity over the range of concentrations used. However, the Schild plot slopes obtained for the other antagonists were all significantly greater than unity, inconsistent with expectations for simple competitive antagonism. 3. HV723, prazosin and tamsulosin were also tested using PE as an agonist. All three antagonists produced concentration-dependent, parallel, rightward shifts of the PE curves and Schild analysis yielded slope parameters not significantly different from unity. The pKB estimates obtained for tamsulosin and prazosin were not significantly different from the pA2 values obtained when NA was used as agonist. In the case of HV723, the 95% confidence intervals for the pKB values yielded with NA and PE did not overlap (pKB = 8.80-9.13 and 8.15-8.77 for NA and PE, respectively). 4. In the absence of evidence to indicate that the steep Schild plots were due to failure to satisfy the basic criteria for quantitative analysis in a one-receptor system, we considered the possibility that the complexity was caused by an action of NA at inhibitory D1 receptors. The selective D1 receptor antagonists, SCH-23390 (10 nM), had no significant effect on the NA E/[A] control curve, but the apparent potency of 100 nM prazosin was reduced by approximately 3.5 fold. 5. This study indicates that the steep Schild plots obtained from the interaction between NA and alpha 1-adrenoceptor antagonists were due to the simultaneous activation of inhibitory D1 receptors by NA. Notwithstanding this complexity, our explanatory model of the system (see Appendix) suggests that the antagonist affinity values estimated in the absence of D1 receptor block were not significantly affected by this other action of NA. The low affinity estimate obtained for prazosin suggests that the pharmacologically-defined alpha IL-subtype operates in the SMA.

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.

1. Previously, we reported that noradrenaline (NA), in addition to its alpha 1-adrenoceptor-mediated contractile effect, may relax the rat small mesenteric artery (SMA) in order to account for steep Schild plots obtained with compounds classified as alpha 1-adrenoceptor antagonists. In this study, a relaxant action of NA has been exposed in the rat isolated, endothelium-denuded SMA precontracted by the thromboxane A2-mimetic, U46619. 2. NA, but not the selective alpha 2-adrenoceptor agonist, UK14304, produced concentration-dependent contraction of the SMA (pEC50 = 5.7 +/- 0.1). After precontraction with 0.1 microM U46619, 10 nM-30 microM NA produced a further contraction (pEC50 = 6.1 +/- 0.2), while higher concentrations of NA produced small, but significant, relaxant responses. 3. In the presence of 1 microM prazosin, 0.1-30 microM NA produced concentration dependent relaxation (pIC50 = 5.9 +/- 0.1) after precontraction with 0.1 microM U46619. The NA relaxation concentration-effect curve was completely inhibited by 1 microM of the beta 1/beta 2-adrenoceptor antagonist, timolol. However, when the concentration of prazosin was increased by 10 fold (10 microM), NA once again produced concentration-dependent relaxation (pIC50 = 4.5 +/- 0.2). This relaxation concentration-effect curve was not blocked by a 10 fold higher concentration of timolol (10 microM), nor by the presence of idazoxan (10 microM), cyanopindolol (10 microM), NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), indomethacin (10 microM) or sulpiride (1 microM). However, haloperidol (10 microM) and (+/-)-SCH-23390 (10 nM) produced significant inhibition of the relaxation, suggesting the involvement of dopamine D1 receptors. 4. Dopamine also produced concentration-dependent relaxation following U46619 precontraction (pIC50 = 5.4 +/- 0.1) which was significantly inhibited by haloperidol and (+)-SCH-23390. Pretreatment with 10 microM phenoxybenzamine for 60 min produced a significant inhibition of the dopamine and NA relaxation curves and application of the operational model of agonism yielded estimates of the affinity (pKA = 5.3 +/- 0.2 and 4.4 +/- 0.2) and efficacy (log gamma = 0.06 +/- 0.11 and 0.01 +/- 0.10) for dopamine and NA, respectively, at D1 receptors. 5. HV723 (0.1 and 1 microM), a ligand that yielded a Schild plot slope parameter of unity as an antagonist of NA in the contractile assay, produced concentration-dependent inhibition of the NA-mediated relaxation (pA2 approximately 8). 6. The results of this study indicate that NA can activate D1 receptors mediating relaxation in the rat SMA at concentrations which were encountered in our previous receptor classification experiments using competitive alpha 1-adrenoceptor antagonists.

1. Concentration-effect curves were obtained, in the absence and presence of histamine H2-receptor blockade, to 5-methylfurmethide (5-MeF) and McN-A 343, high efficacy and low efficacy acetylcholine (ACh) M-receptor agonists, respectively, in isolated stomach preparations from the mouse and immature rat and guinea-pig. 2. In the immature guinea-pig assay, the responses to 5-MeF and McN-A 343 were abolished by histamine H2-receptor blockade suggesting that the responses were totally dependent upon gastric mucosal histamine. However, in the mouse and immature rat assays, although the histamine H2-receptor antagonists produced small but significant rightward shifts and, in some cases, depression of the maximum of the agonist concentration-effect curves, a significant secretory response remained, presumed to be due to direct stimulation of oxyntic cells. 3. Previously, by assuming that the histamine H2-receptor blockade alters the mode of agonist-stimulated acid secretion from mainly an indirect action mediated by histamine release to direct stimulation of the oxyntic cell, we applied an operational model of agonism to similar data obtained in the mouse preparation. In that study we were able to account for the behaviour of 5-MeF and McN-A 343 by assuming that the agonists expressed 6 fold higher efficacy, tau in the operational model of agonism, at ACh M-receptors on the histamine-releasing cells than on the oxyntic cells. In this study it was possible to account for the variation in the behaviour of the agonists both between and within assays by simply varying the efficacy expressed by the agonists at each of the cells in the model. The efficacy variation could be due to receptor concentration variation.4. The data and analysis are discussed in terms of contemporary models for the role of histamine in the regulation of gastric acid secretion.

1. In the absence of adenosine uptake inhibition, adenosine produced a concentration-dependent (threshold 30 microM) relaxation of the 5-methylfurmethide pre-contracted guinea-pig taenia caecum. The relaxation was not blocked by 8-phenyltheophylline (8-PT, 3 microM) or 1,3-dipropyl, 8-cyclopentylxanthine (DPCPX, 30 microM). 2. In the presence of the adenosine uptake inhibitor, dipyridamole (Dip, 3 microM), a biphasic adenosine concentration-effect curve was obtained (threshold 0.3 microM). The time course of the responses to adenosine in the absence of Dip was similar to that of the second phase responses in the presence of Dip and occurred over the same adenosine concentration-range. 5'-(N-ethyl) carboxamido-adenosine (NECA) concentration-effect curves (in the absence of Dip) were also biphasic. Only the first phases of the concentration-effect curves obtained with NECA and adenosine (plus Dip) were inhibited by 8-PT. The pA2 values for 8-PT of 6.7 and 7.0 versus adenosine and NECA, respectively, were consistent with actions at P1-purinoceptors. There was a trend towards an increase in the upper asymptote of the first phase of the NECA curve in the presence of increasing concentrations of 8-PT. The A1-purinoceptor selective antagonist, DPCPX, also blocked only the first phase of the NECA concentration-effect curve and produced a significant increase in the upper asymptote. The pA2 value (6.8) obtained was consistent with activation of A2-subtype P1-purinoceptors by the low concentrations of NECA. 3. There was no correlation between A1-purinoceptor affinity and the propensity to cause the increase in the upper asymptote of the first phase of the NECA concentration-effect curves amongst a series of 9-methyl adenine analogues, suggesting that the amplification was not due to inhibition of an underlying A1-purinoceptor-mediated contractile response.4. DPCPX (10 microM) produced a significant increase in the upper asymptote of the NECA concentration effect curve, but had no effect on isoprenaline curves whereas the phosphodiesterase inhibitor Ro20-1724 (30 microM) produced a significant increase in the upper asymptote of both NECA and isoprenaline concentration-effect curves. Therefore the amplification of the first phase responses by DPCPX did not appear to be due to phosphodiesterase inhibition.5. It was not possible to conclude whether second phase responses to adenosine and NECA were mediated by intracellular or extracellular sites of action. However, if intracellular sites of action were involved then adenosine did not apparently gain access by the Dip-sensitive uptake system.

1. The interaction between methotrexate and indomethacin has been examined, at a physiological folate concentration (20 nM), on a human normal lymphoblast-like cell line (RPMI 1788) in vitro. 2. Indomethacin (1 microgram ml-1) increased the reduction of lymphoblast growth caused by methotrexate (10-80 ng ml-1). 3. Indomethacin (0.1 and 1 microgram ml-1) potentiated the cytotoxicity of methotrexate (20 and 40 ng ml-1) after 4 days in culture. 4. Indomethacin (0.4 micrograms ml-1) reduced the accumulation of tritium in lymphoblasts incubated with [3H]-methotrexate after 30 min; therefore initial drug accumulation was not responsible for the potentiation seen after 4 days. 5. If indomethacin increases the killing of human cancer cells by methotrexate in vivo, with a smaller potentiation on lymphoblasts, this combination may be beneficial in treating human malignancy.

1. The results of previous studies have been in conflict with respect to the involvement of specific cholecystokinin (CCKA) and CCKB/gastrin receptors in guinea-pig gastric muscle. Here, in an in vitro, guinea-pig gastric muscle assay, pentagastrin (PG) and tetragastrin (TG) behaved as high potency agonists and produced symmetrical concentration-effect curves. In contrast, cholecystokinin-octapeptide (CCK-8), while also behaving as a high potency agonist, produced flat asymmetrical curves. Unlike recent data reported using this tissue (Boyle et al., 1993), the CCKA receptor-selective antagonist, devazepide (3, 10, 30 nM) produced a rightward shift of the upper region of the CCK-8 curve rendering it biphasic. The lower phase was abolished by the CCKB/gastrin receptor-selective antagonist, L-365260 (300 nM) indicating that the contractile effects of CCK-8 in this tissue are mediated by both receptor types. 2. L-365260 produced a concentration-dependent, parallel rightward displacement of PG concentration-effect curves. However, a flat Schild plot slope parameter (0.77 +/- 0.06) was obtained. Therefore, an empirical pA2 value of 8.64 +/- 0.21 was estimated from the smallest dose ratio. This value is consistent with published values characteristic of an interaction at CCKB/gastrin receptors. 3. TG (1 microM) was used to densensitize selectively the CCKB/gastrin receptors in the gastric muscle assay and thereby expose a population of receptors capable of responding to subsequent stimulation by CCK-8 but not by PG. The selectivity of TG for CCKB/gastrin- over CCKA receptors was demonstrated by its low efficacy compared to CCK-8 in the guinea-pig gallbladder assay, a tissue shown previously to contain a homogeneous population of CCKA receptors. In TG-desensitized gastric muscle, CCK-8 concentration-effect curves were symmetrical and could be displaced in a simple parallel fashion by devazepide at nanomolar concentrations consistent with an interaction at CCKA receptors (pKB approximately 10). 4. These results indicate that the guinea-pig gastric muscle contains both CCKA- and CCKB/gastrin receptors and the effects of CCK-8 are mediated via both of these receptors. Notwithstanding the complexity of the behaviour of L-365260, it was possible to obtain a reasonable description of the system using a simple 2-receptor model in which the effects of individual receptor activation were assumed to be additive. The absence of a simple competitive interaction of PG with L-365260 may indicate, for example, non-homogeneity of CCKB/gastrin receptors or lack of concentration equilibrium between the bath and the receptor biophase.

1. Electrical field stimulation produced a tetrodotoxin-sensitive, frequency-dependent, release of acid from isolated, lumen-perfused, stomach preparations from mouse, immature rat and guinea-pig. 2. In the guinea-pig and mouse preparations, the frequency-dependent response was abolished by hexamethonium, acetylcholine (ACh) muscarinic (M) and histamine H2-receptor blockade, consistent with the hypothesis that the vagal ACh acts indirectly by stimulating the release of endogenous histamine. 3. In contrast, in the rat preparation the frequency-dependent response was partially refractory to all of these inhibitors. However, a combination of H2- and ACh M-receptor blockade did abolish the effect. 4. We conclude that vagal-stimulated acid secretion in the rat, unlike the other two species, behaves as though there is a direct innervation of the oxyntic cells by either cholinergic or noncholinergic neurones.

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.

1. The in vitro pharmacological characterization of the sodium salt of 2-naphthalenesulphonyl 1-aspartyl-(2-phenethyl)amide [2-NAP], a hydrophilic compound derived from the C-terminal aspartate-phenylalanine dipeptide of cholecystokinin (CCK), is described. 2. 2-NAP behaved as a competitive antagonist of sulphated cholecystokinin octapeptide (CCK-8) at CCKA-receptors in both intact tissue bioassays (guinea-pig gall bladder, pancreas and ileum, human and rabbit gall bladder) and a radioligand displacement assay (guinea-pig pancreatic cells). The mean pKB, over assays, was 6.5. 3. Compared to the other assays, the rabbit gall bladder assay gave a significantly higher pKB estimate [7.0] for 2-NAP and a significantly lower estimate [8.9] for devazepide (formerly L-364,718 and MK-329), a well-characterized CCKA-receptor antagonist; these anomalous results suggest that a different class of CCKA-receptors may be involved. 4. 2-NAP, was found to be highly selective, having at least 300 fold greater affinity for CCKA-receptors than for 50 other pharmacological loci, including gastrin/CCKB, as estimated by bioassay or radioligand displacement.

Does gastrin stimulate gastric acid secretion by direct action on oxyntic cells, by releasing histamine, or by being potentiated by histamine? Previous studies in the mouse pointed to gastrin-regulated histamine release. Guinea pig and rat are well known to vary in their sensitivity to histamine. Therefore, the effects of histamine and pentagastrin were compared quantitatively on isolated, lumen-perfused, stomach preparations from these species in the absence and presence of histamine H2-receptor blockade. The loss of potency of histamine in the rat was mirrored by a loss of potency of pentagastrin consistent with the idea that pentagastrin acts by releasing histamine. In the rat, a well-defined pentagastrin curve was obtained in the presence of histamine H2-receptor block as though pentagastrin acts both directly on the oxyntic cell and indirectly by releasing histamine. It was not necessary to invoke a potentiating interaction between histamine and pentagastrin at the oxyntic cell; the two effects appeared simply to add. Potentiation was observed, however, between other combinations of stimuli, for example, between vagal nerve and pentagastrin stimulation. The physiological consequences of these results are discussed.

1. The interactions between angiotensin II (AII), two non-peptide antagonists DuP 753 and IMI, and eight peptide analogues of AII were investigated on the rabbit isolated aorta assay. DuP 753 and IMI behaved as simple competitive antagonists (pKB values 8.4 and 6.8, respectively). To different degrees, all the AII-peptide analogue interactions failed to meet the basic criteria for simple competition. In addition to rightward shift, the most significant feature was a concentration-dependent saturable depression of the upper asymptote of the AII concentration-effect curves. 2. 'Washout' and combined dose-ratio analysis experiments, in which DuP 753 was used as a reference antagonist, indicated that the profile of peptide antagonism was solely due to a reversible and syntopic action at the AII receptor. 3. By use of an operational model of agonism (Black & Leff, 1983) as a starting point, it was possible to account for the data with a new model which describes reversible receptor occupancy and occupied receptor-determined, saturable reduction in the efficacy of AII. Model-fitting gave estimates of pKB values for the peptide analogues and agonist affinity and efficacy parameters for AII. 4. The model was successfully tested by applying it to qualitatively similar results obtained in a cross-tissue analysis on guinea-pig aorta, ileum and stomach. 5. A 'molecular' interpretation of the efficacy changes, based on the concepts of receptor internalisation and expression, is offered.