N.P. Shankley

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

1 Agonist‐antagonist interactions at histamine receptors have been re‐examined using improved techniques, on the mouse isolated, lumen‐perfused, stomach gastric acid assay. 2 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. 3 Oxmetidine significantly depressed basal rate on the atrial assay and the Schild plot slope parameter (0.81) was significantly less than one. 4 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. 5 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 muscarinic receptors coupled to oxyntic cell secretion in the mouse stomach

1 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. 2 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. 3 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. 4 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. 5 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.

Evidence that the apparent complexity of receptor antagonism by angiotensin II analogues is due to a reversible and syntopic action

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.

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

1 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. 2 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. 3 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. 4 The improved assay permitted the full characterization of cumulative agonist concentration‐effect curves in single stomach preparations to histamine, 5‐methylfurmethide, pentagastrin and isobutylmethylxanthine. 5 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.

An improved in vitro bioassay for the study of 5-HT4 receptors in the human isolated large intestinal circular muscle

Recently, it was demonstrated that 5‐HT induces relaxation of human colon circular muscle through activation of 5‐HT4 receptors and 5‐HT7 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‐HT4 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 (pEC50 7.31, Hill slope 0.91). Neither methysergide (10 μM) nor granisetron (1 μM) affected the 5‐HT‐induced relaxation, suggesting that 5‐HT1, 5‐HT2, 5‐HT3, 5‐ht5, 5‐HT6 or 5‐HT7 receptors are not involved. The lack of effect of tetrodotoxin (0.3 μM) indicated a direct effect of 5‐HT on the smooth muscle. The selective 5‐HT4 receptor antagonists GR 113808, GR 125487 and RS 39604 competitively antagonized the 5‐HT‐induced relaxation (pKB 9.43, 10.12 and 8.53, respectively). SB 204070 (1 nM) produced a rightward shift (pA2 10.34) and depression of the 5‐HT curve. These affinity estimates are similar to those previously reported for 5‐HT4 receptors. The selective 5‐HT4 receptor agonists, prucalopride and R076186, induced relaxations (pEC50 7.50 and 7.57, respectively), that were blocked by GR 113808 (3 nM), yielding pA2 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‐HT4 receptor population. This new bioassay allows the focused, pharmacological characterization of human colonic 5‐HT4 receptors in vitro.

Analysis of competitive antagonism when this property occurs as part of a pharmacological resultant

1 In this paper, pharmacological resultant is defined as the net effect of a single compound resulting from the simultaneous expression of two or more specific actions. 2 The principles of concentration‐ratio analysis are extended to develop a method for detecting and quantifying competitive antagonism when this property is a component of a pharmacological resultant. The method is general to the extent that it allows analysis of competitive antagonism in combination with all types of post‐receptor intervention. Essentially it depends on the altered expression of competition by a reference antagonist. It incorporates tests for validating its application and it is independent of agonist concentration‐effect curve shape: in these respects the method is analogous to Schild plot‐analysis of simple competition. 3 The methodology for the practical application of the analysis is exemplified by studying the net effect of a combination of a phosphodiesterase inhibitor (isobutylmethylxanthine) and histamine H2‐ receptor antagonist (metiamide) on histamine‐stimulated tachycardia in guinea‐pig, isolated, right atrium. Cimetidine was used as the reference antagonist. 4 The equation used in this analysis is similar in form to one recently described by Hughes & Mackay (1985) to elucidate the situation when competitive antagonism occurs in combination with functional interactions. The relation between their method and the present analysis is discussed.

Analysis of the activity of α1‐adrenoceptor antagonists in rat aorta

1 In this study, the effects of seven α1‐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 (α = 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, α = 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 α1‐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 α1‐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 α1a/d‐adrenoceptor (now referred to as α1d‐adrenoceptor; Hieble et al., 1995). Therefore, the complexity exposed in this study may be due to the expression of closely‐related forms of the α1d‐adrenoceptor. However, relatively good matches were also found between pKB1 and α1c and between pKB2 and α1b. Therefore, on the basis of these data, it is not possible to rule out the involvement of all three α1‐adrenoceptors. The conflicting reports concerning the characteristics of the α1‐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 α1‐adrenoceptors.

2-Naphthalenesulphonyl L-aspartyl-(2-phenethyl)amide (2-NAP)--a selective cholecystokinin CCKA-receptor antagonist.

1 The in vitro pharmacological characterization of the sodium salt of 2‐naphthalenesulphonyl l‐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.

Correlation between log POCT/H2O and pKB estimates for a series of muscarinic and histamine H2‐receptor antagonists

1 With histamine used as agonist, pKB values were estimated for seventeen histamine H2‐receptor antagonists on assays involving acid secretion by the mouse isolated stomach and contraction frequency of the guinea‐pig right atrium. 2 With the exception of oxmetidine, SK&F 94,826 and SK&F 94,206 on the right atrium assay, the compounds behaved as simple competitive antagonists on both assays. Although the former three compounds produced concentration‐dependent, parallel, displacement of the histamine concentration‐effect curves, subsequent analysis indicated Schild plot slope parameters significantly less than unity. However, the application of a combined dose‐ratio analysis indicated that their antagonistic behaviour did not differ from expectations for simple competition at dose‐ratios of approximately 20, and pKB values were estimated on this basis. 3 In accordance with previously reported data, pKB values were found to be consistently lower on the stomach than atrial assays. The pKB value for tiotidine was underestimated to the same extent on the stomach assay when impromidine was used as agonist. 4 The removal of the serosal muscle from the mouse stomach, achieved by using an isolated, perfused, mucosal sheet preparation, did not significantly affect the underestimation of the pKB value for metiamide. 5 Linear regressional analysis indicated a significant, positive, correlation between lipophilicity (log POCT/H2O) of the antagonists and the degree of antagonist pKB value underestimation on the gastric secretion assay.

Pharmacological analysis of the pentagastrin‐tiotidine interaction in the mouse isolated stomach

1 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. 2 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. 3 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. 4 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. 5 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.