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Both prenalterol and pirbuterol are partial agonists (when compared to isoproterenol) in rat left atria. Schild analysis with the beta-1 adrenoceptor selective antagonist atenolol indicated that all three agonists stimulate beta-1 adrenoceptors in this preparation. Atria from rats implanted with mini-osmotic pumps which delivered isoproterenol (400 micrograms kg-1 hr-1) s.c. for 4 days were 9 times less sensitive than control, to isoproterenol, and produced no responses to either prenalterol or pirbuterol. Schild analysis with atenolol on these desensitized atria indicated a homogeneous population of beta-1 adrenoceptors. In the desensitized atria, both prenalterol and pirbuterol functioned as competitive antagonists of responses to isoproterenol and Schild analysis yielded estimates of the equilibrium dissociation constants (Kp) of prenalterol (0.09 micron) and pirbuterol (2 micron) in these tissues. The estimates of Kp were utilized to calculate the relative efficacy of pirbuterol and prenalterol; pirbuterol was shown to have 2.3 times the efficacy of prenalterol on beta-1 adrenoceptors. The implications of this method as a means of calculating the Kp for partial agonists are discussed. These data also serve as a caveat to the use of pirbuterol in the determination of functional cardiac beta adrenoceptor subtypes in experimental animals and in man.

Ambenonium is known to be an inhibitor of acetylcholinesterase, and recent data have shown this drug to antagonize muscarinic receptors as well. This latter property was confirmed by Schild analyses of ambenonium-induced blockade of responses to bethanechol in guinea-pig ileal longitudinal smooth muscle, taenia caeci, trachea and rat anococcygeus muscle. Statistical analysis showed ambenonium to be a simple competitive antagonist of responses to bethanechol in these tissues with pKB values in each tissue not significantly different from each other (mean pKB = 6.0). However, considerable variability in pKB estimates was encountered when ambenonium was utilized to block responses to acetylcholine. Ambenonium was a less potent antagonist of tissue responses to acetylcholine, and the underestimation in the pKB (as compared to that obtained with bethanechol) could be eliminated by prior treatment of tissues with the acetylcholinesterase inhibitor neostigmine. These data suggested that ambenonium had a dual effect on tissue responses to acetylcholine-producing potentiation by blockade of acetylcholinesterase and concomitant antagonism by blockade of muscarinic receptors. The Schild regressions obtained for ambenonium antagonism of acetylcholine responses formally satisfied criteria for simple competitive antagonism of a homogenous population of receptors (linear regression, slope equal to unity). The fact that these regressions yielded erroneous apparent pKB values suggests how two properties of a drug in one molecule could provide misleading information about drug receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

A mathematical model is presented that simulates the steady state kinetics of agonists interacting with a promiscuous receptor. The model system consists of a single receptor that forms a ternary complex with either of two transducer proteins (G proteins). At a given agonist concentration, the concentrations of the two ternary complexes are determined by the relative quantities of the two G proteins and the ratio of the dissociation constants for the two ternary complexes. Accordingly, the potency of an agonist is dependent upon the relative quantities of the G proteins. If receptors are truly promiscuous and if the distribution of G proteins varies with tissue type, then the agonist potency ratio would be tissue dependent as well as receptor dependent. Experimental data from literature studies are reviewed in the context of the promiscuous receptor model, and implications of the model regarding pharmacologic classification of receptors are discussed.

The antagonism of muscarinic receptor-mediated contraction of rat trachea by a range of muscarinic antagonists was quantified by Schild and resultant analysis. Dose-response curves to carbachol, muscarine and oxotremorine were shifted to the right by gallamine and pirenzepine in a parallel manner with no change in maximal response ostensibly indicating simple competitive inhibition. However, Schild analysis indicated differences in the blockade and estimated pKb values with each agonist for both gallamine and pirenzepine. This suggested either that the responses to these three agonists were mediated by a heterogeneous receptor population in this tissue or that the blockade produced by gallamine and by pirenzepine was not competitive. Further Schild analysis with the muscarinic antagonists scopolamine, atropine, 4-diphenylacetoxy-N-methyl piperidine methiodide and (11 [(2-[(diethylamino)methyl]-1-piperidinyl]acetyl)-5,11- dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine 6-one) with carbachol, muscarine and oxotremorine indicated simple competitive antagonism of a homogeneous population of muscarinic receptors. Therefore, the competitivity of binding of atropine, gallamine and pirenzepine with the scopolamine binding site was measured with the recently reported technique of resultant analysis. With this method the effect of various concentrations of the test antagonist on the antagonism produced by specified concentrations of the reference antagonist scopolamine was measured and the equilibrium dissociation constant of the test antagonist-receptor complex estimated. These data indicated that atropine and scopolamine bind to a common binding site on the muscarinic receptor, but that scopolamine and both gallamine and pirenzepine bind to mutually exclusive sites. This result is mine and pirenzepine are allosteric modulators of muscarinic receptors which bind at sites other than that utilized by agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

G protein-coupled receptors initiate signaling cascades after associating with heterotrimeric G proteins. This is typically initiated by agonist binding, but can also occur spontaneously, particularly in receptors bearing distinct missense mutations. Two such mutations in the parathyroid hormone receptor are associated with constitutive activity, manifesting clinically as Jansen's metaphyseal chondroplasia. We introduce analogous mutations separately and together into the secretin receptor to explore their impact on another family member. Constructs were expressed transiently in COS cells, and had binding and signaling (cAMP generation) studied. Each construct was processed appropriately to lead to cell surface expression and signaling. Secretin bound to the wild-type receptor with two affinity states recognized, 1% of sites in the high affinity state (Ki = 0.5 +/- 0.1 nM) and 99% in the low affinity state (Ki = 23 +/- 3 nM). Mutant receptor binding best fit a single affinity state, having values for Ki of 5 +/- 1 nM (H156R), 8 +/- 1 nM (T322P) and 6 +/- 1 nM (H156R/T322P), with each of these demonstrating a shift to higher affinity than the predominent low affinity state of the wild-type receptor. Each mutant receptor expressed small to moderate constitutive activity, with basal levels of cAMP activity greater than control (P < .01): H156R, 1.4-fold; T322P, 4.5-fold and H156R/T322P, 6.8-fold. The level of basal activity of even the most active construct was only 15% of the maximal response of wild-type receptor. Although each of the single site mutants responded to secretin by increasing their cAMP levels in a concentration-dependent manner, the dual mutant decreased its cAMP in response to hormone (EC50 = 13 nM). Thus, a natural agonist had become an inverse agonist at this unique construct. Because this could reflect reduced normal coupling with Gs or increased aberrant coupling with Gi, the mechanism was further explored using pertussis toxin and a stable analogue of GTP. Although ligand-binding determinants were retained in the dual receptor mutant, the conformation of this receptor upon secretin binding effected a reduction in its basal coupling with Gs, thereby resulting in inverse agonism.

The relative effects of drugs which elevate cytosolic cyclic AMP on inotropy and diastolic relaxation (lusitropy) of guinea pig atria were quantified in vitro. There was a temporal difference between these responses in that inotropy reached peak response considerably faster than lusitropy. Also, although the relaxation response was sustained to an elevated steady state, the inotropic responses to beta adrenoceptor agonists were transient and returned to base line over 90 min. However, the inotropic responses to forskolin and dibutyryl cyclic AMP (cAMP) were sustained. For all of the drugs tested, the lusitropic response was at least 4 times more sensitive than the inotropic response (i.e., the concentration response curve for relaxation was shifted to the left of the curve for inotropy). In the case of beta adrenoceptor agonists, these differences were greater, presumably because of the fading inotropic response over 90 min. It was found that although high efficacy beta adrenoceptor agonists such as isoproterenol (and the direct activator of adenylate cyclase forskolin) produced both inotropy and lusitropy, lower efficacy agonists produced predominant lusitropy. The low efficacy agonist prenalterol produced insignificant inotropy but 60% maximal lusitropy. These data were modeled mathematically by a "differential coupling model" which assumed that a uniform cytosolic level of elevated cAMP activated two biochemical processes of differing sensitivity. Thus, the lusitropic response (phosphorylation of phospholamban) was coupled more efficiently to the cAMP response than the inotropic response (phosphorylation of calcium channels). A second model ("differential messenger concentration model") which calculated the effects of a compartmentalization of cAMP concentration within the cardiac cell by restricted diffusion and/or selective degradation by phosphodiesterases also was used.(ABSTRACT TRUNCATED AT 250 WORDS)

Schild regressions in isolated tissues are powerful tools for receptor classification only if they yield equillibrium dissociation constants of antagonists which can, in turn, occur only when measurements are made under equillbrium conditions. Regressions with slopes different from unity of nonlinear regressions indicate noncompliance to equillbrium. This paper describes a condition whereby Schild regressions apparently comply with criteria which signify equillbrium conditions but in fact are regressions obtained at nonequilibrium steady states yielding significant underestimations of the potency of the antagonist. Specifically, if agonist uptake processes in isolated tissues are inhibited by uptake inhibitors with receptor blocking properties, the experimental data and a theoretical model suggest that the resulting Schild regression will be linear, have a slope of unity and be shifted to the right of the true Schild regression, thereby significantly underestimating antagonist potency. Experiments with the effects of neuronal uptake inhibition (with desmethylimipramine and amitriptylene) on the phentolamine-inhibition of norepinephrine responses in rat anococcygeus muscle and the effects of metanephrine on the propranolol-inhibition of isoproterenol relaxation of guinea-pig trachea provided data to compare to a theoretical model. The data and the model help define the selectivity of the uptake inhibitor fo uptake over receptors needed to achieve steady states approaching true equilibrium, a condition required for satisfactory estimation of antagonist potency.

Both tolazoline and clonidine produce agonist effects sensitive to histamine H2-receptor antagonism in whole animals and a variety of isolated tissues. In guinea-pig right atria, tolazoline concentration-response curves were shifted to the right and progressively depressed by cimetidine. Clonidine produced a submaximal histamine-like tachycardia which was converted by cimetidine to a depression of sinus rate. Neither tolazoline nor clonidine stimulated histamine H2-receptors in rabbit right atria. Instead, these agents were very weak inhibitors of histamine with a pA2 for tolazoline of 3.3 and for clonidine of 3.7. No appreciable histamine H1-receptor activity was evident for either drug in experiments with guinea-pig ileum. The weak histamine H2-receptor affinity is inconsistent, from a quantitative point of view, with the hypothesis that the agonist activity results from direct stimulation of histamine receptors, but all data re compatible with previous evidence which suggests that these drugs release endogenous histamine.

A model is described that predicts the behavior of competitive antagonists in tissues with more than one receptor mediating response. The receptor stimuli for two receptor types are summed and processed, via a cellular stimulus-response mechanism, into tissue response. The primary receptor is described by a standard Langmurian isotherm, and a secondary receptor input with a variable maximal strength, for which the agonist has variable sensitivity, is added. The prediction of drug effects in this system does not depend on the way in which the two stimuli are combined or on the absolute magnitudes of the parameters used to make the calculations. The model is maximally flexible, in that no pharmacological significance is put on the magnitudes of the inputs from the secondary receptor system (i.e., they can vary with either agonist intrinsic efficacy, receptor number, or efficiency of stimulus-response coupling). The theoretical Schild regressions for selective antagonists in two-receptor systems are calculated for various secondary receptor inputs. These regressions generally are curvilinear whenever the secondary receptor significantly contributes to agonist response. These calculated data also indicate that minor variations in biological input from secondary receptor systems would obscure curvature in the Schild regression and result in a seemingly linear regression with a slope of less than unity. However, further calculations indicate three possible ways to use Schild analysis to detect receptor heterogeneity in tissues. One indicator of receptor heterogeneity is a change in the slope of the dose-response curve for the agonist in the presence of a selective antagonist. A second indicator would be a marked heteroscedasticity of errors in the Schild regression, i.e., the magnitude of the standard errors in the ordinate values would depend upon the concentration of the antagonist. A third, and most experimentally accessible, aspect of heterogeneous receptor systems predicts that changes in the overall sensitivity of organ response mechanisms will differentially alter the relative strength of two receptor inputs. This would be observed as a change in the potency of an antagonist. Under these circumstances, differences in the stimulus-response characteristics of a tissue would result in a change in the Schild regression for a selective antagonist. These concepts are discussed in terms of the use of Schild analysis in functional systems for the detection of physiologically relevant mixtures of receptors and the possible advantages over biochemical binding data.

The striking resistance of norepinephrine contractions of rat splenic strips to antagonism by the selective alpha-1 adrenoceptor antagonist prazosin was examined by Schild analysis. Prazosin was a simple competitive antagonist of contractions to phenylephrine indicating that this tissue possesses alpha-1 adrenoceptors. In contrast, the Schild regression for prazosin, with norepinephrine as the agonist, was nonlinear and had an overall slope of 0.24. These data indicated that norepinephrine activated a prazosin-resistant adrenoceptor in this tissue. As a working hypothesis, it was assumed that the prazosin-resistant receptor was an alpha-2 adrenoceptor; the concomitant addition of yohimbine, in concentrations below those required to block alpha-1 adrenoceptors, converted the atypical Schild regression for prazosin (norepinephrine as agonist) to a linear regression identical with that found for antagonism of phenylephrine responses. Selective alkylation of alpha-1 adrenoceptors with phenoxybenzamine (POB) eliminated responses to phenylephrine but not those to norepinephrine. After POB-alkylation and in the presence of a concentration of prazosin that was sufficient to produce a profound blockade of alpha-1 adrenoceptors, a response to norepinephrine remained. It was determined that the POB/prazosin-resistant response most likely was mediated by a homogeneous population of receptors by the finding that the Schild regressions for both yohimbine and idazoxan were identical with respect to slope and elevation when either norepinephrine or cobefrin were utilized as agonists, i.e., a difference in the regressions for these antagonists would be expected if the two agonists activated a heterogeneous receptor population.(ABSTRACT TRUNCATED AT 250 WORDS)

It was observed experimentally that both theophylline and isobutylmethylxanthine (IBMX) produced surmountable antagonism of the agonist effects of 2-chloroadenosine on purine receptors in rat vas deferens and guinea pig atria. In the case of IBMX, there was a statistically significant difference between the Schild regressions in the two tissues, ostensibly indicating a possible purine receptor heterogeneity with respect to the binding of this antagonist. However, the analysis was complicated by the fact that both theophylline and IBMX produced positive inotropic responses in guinea pig atria (presumably by inhibition of cardiac phosphodiesterase), making the calculation of dose ratios subjective and ambiguous. To determine whether the phosphodiesterase blocking property of theophylline and IBMX was interfering with the observation of purine receptor antagonism in guinea pig atria, a new technique described as pharmacologic resultant analysis was utilized to measure the purine receptor blocking properties of theophylline and IBMX in guinea pig atria in the presence of phosphodiesterase blockade. Based on the principle of additive dose ratios for two antagonists competing for one receptor, pharmacologic resultant analysis measures the effects of a test antagonist (in this case theophylline or IBMX) on the competitive blockade of a reference antagonist; for these studies, the reference antagonist was 8-sulfophenyltheophylline. Under these circumstances, the direct effects of the test antagonist (positive inotropy) are expressed throughout the experiment and cancel with the normal null methods used for measurement of competitive antagonism. Using this technique, it was found that the potencies of both theophylline and IBMX on the purine receptors in rat vas deferens and guinea pig atria were identical. The use of this method in the delineation of multiple drug activities is discussed.

In rat anococcygeus muscle, dobutamine produced concentration-related submaximal contractions which were antagonized competitively by phentolamine (pKB = 8.3) and dobutamine antagonized norepinephrine-induced contractions in a competitive manner with an equilibrium dissociation constant for the alpha adrenoceptor of 20 nM (pKB = 7.7). Therefore, dobutamine satisfied criteria for a partial agonist of alpha adrenoceptors having an affinity for alpha adrenoceptors 25 times that of norepinephrine (pKA = 6.3) in this tissue. An estimate of the relative efficacy of dobutamine showed one-fortieth the the efficacy of norepinephrine at the alpha adrenoceptors. Dobutamine contracted rabbit aorta and produced concentration-related relaxations at 1000 times greater concentrations after alkylation of alpha adrenoceptors by phenoxybenzamine. In noncontracted canine saphenous vein, dobutamine had no visible agonist activity but did produce contractions after propranolol. In partially contracted saphenous vein, dobutamine produced a small contraction which was converted to a propranolol-sensitive relaxation of tone after phentolamine. Dobutamine was a full beta adrenoceptor agonist in guinea-pig trachea under spontaneous tone but a partial agonist after strong contraction by bethanechol. This allowed measurement of the pKB of dobutamine at beta adrenoceptors (pKB = 5.35) and estimation of efficacy at beta adrenoceptors relative to isoproterenol (eDob/eIso = 1/20). No evidence for beta adrenoceptor selectivity was found in studies of potency ratios and relative efficacy using isoproterenol for comparison. Dobutamine showed a slight (2-fold) selectivity for inotropy in vitro when compared to isoproterenol in guinea-pig right and left atria. This selectivity was removed by phentolamine suggesting a cardiac alpha-like adrenoceptor effect; this finding was confirmed in propranolol-treated guinea-pig left atria. These results are discussed in terms of the in vivo effects of dobutamine and its use as a tool for classification of beta adrenoceptors, particularly the putative presynaptic beta adrenoceptor.

The histamine-induced relaxation of bethanechol-contracted rabbit trachea was analyzed pharmacologically in experiments designed to determine whether this tissue contains relaxant histamine H2-receptors or an atypical histamine receptor. Relaxation to histamine was potentiated by inhibition of histamine H1-receptors with chlorpheniramine. A cimetidine-resistant relaxant effect of high concentrations of histamine was observed which correlated with concomitant decreases in organ bath pH upon addition of histamine. Neutralization of the histamine solution eliminated the cimetidine-resistant effects of histamine. Cimetidine, metiamide and burimamide were competitive antagonists of histamine-responses with pKB values of 6.6, 6.1 and 5.4, respectively. These estimates are not significantly different from literature values for antagonism of H2-receptors by these drugs in guinea-pig right atria. The potency ratio of histamine and 4-methylhistamine (a selective H2-receptor agonist) was not significantly different from that obtained in rabbit right atria, an H2-receptor-containing tissue. These data from studies with antagonists and agonists indicated that histamine produced relaxation of rabbit trachea by activation of histamine H2-receptors.

The concentration-related sensitization of guinea-pig left atria to adenosine produced by six benzodiazepines and dipyridamole was analyzed with a quantitative procedure which theoretically yields the pKI (-log equilibrium dissociation constant) of an uptake inhibitor for the site of uptake. Four benzodiazepines produced sensitization of atria to adenosine and no alteration of responses to 2-chloroadenosine (a purine agonist which is not a substrate for adenosine uptake or degradation), the rank order of potency being diazepam (pKI = 5.6) much greater than oxazepam (pKI = 4.6) greater than clonazepam (pKI = 4.3) = lorazepam (pKI = 4.2). Chlordiazepoxide and prazepam produced little sensitization to adenosine, but a concentration-related antagonism of responses to 2-chloroadenosine. Prior inhibition of adenosine uptake with dipyridamole unmasked antagonism of responses to adenosine by these two benzodiazepines as well, indicating a dual self-cancelling effect (for inhibition of adenosine uptake; chlordiazepoxide, pKI = 5.0; prazepam, pKI = 4.8). Although benzodiazepines have been shown to inhibit uptake of adenosine in the central nervous system, the potency of these drugs and dipyridamole is more in accord with inhibition of a myocardial uptake process distinct from the synaptosomal transport of adenosine.

Agonist effects of the reputedly beta-1 selective adrenoceptor agonists prenalterol, dobutamine and tazolol were compared, in rat uterus, with those of the reportedly beta-2 selective agonist, terbutaline. Butoxamine was a simple competitive antagonist of responses to all agonists with a 10 to 16 times greater potency in rat uterus than in guinea-pig left atria. Similarly, atenolol was 50 to 80 times less potent as an antagonist of all agonists in rat uterus than guinea-pig left atria. The Schild regressions for both antagonists, when subjected to analyses of covariance of regression lines, yielded no evidence to suggest that the reputedly beta-1 selective agonists activated receptors different from those activated by terbutaline in rat uterus. These data indicated that the responses produced by these agonists in rat uterus were due to stimulation of beta-2 adrenoceptors under current classifications. An analysis of the relative intrinsic efficacy of prenalterol on beta-2 as opposed to beta-1 adrenoceptors indicated a nonselective efficacy at the receptor level. The implications of these data in terms of tissue-related efficacy (i.e., intrinsic activity) and receptor-related intrinsic efficacy (as defined by Furchgott) are discussed as a caveat to ascribing tissue selectivity to receptor selectivity without the appropriate data. Specifically, estimates of selective agonist efficacy at receptors should be made on the basis of selective intrinsic efficacy and not on measurements of agonists potency or maximal responses.