European Journal of Pharmacology, 191 (1990)49-57 Elsevier
49
E?JP51591
miscuous or heterogeneous muscariuic receptors iu rat II. A~~oni§m of ~~s~n~~ to e~~aeho~ by aired Cinzia Boselli ’ and Terry P. Kenakin DivSon of Pharmacokgy, GIaxo Research Laboratories, Gluxo Inc., Five Maore Drive, Research Trim&e Park,NC 27709. &Ql, Received 13 August 1990, accepted 28 August 1990
Carbachol produces both negative and positive inotropy in rat left atria. It is not clear whether these two effects are mediated by two separate cell surface auscarinic receptors or a siugle receptor interacting with two coupling proteins in the cell membrane. Pirenzepine, known to selectively block some biochemical muscarinic responses, was used in this study to block the biphasic response to carbachol in rat left atria. The negative inotropy to carbachol was blocked by pirenzepine, and Schild analysis indicated a -log dissociation constant (pK,) for the pirenzepine-receptor complex of 6.2. However, the Schild analysis may have been complicated by positive inotropy observed with pirenzepiue. This positive inotropic effect was sensitive to blockade by other muscarinic antagonists. In atria from rats pretreated with pertussis toxin, carbachol produced a positive inotropic effect. SchiId analysis with pirenzepine for ~~go~~ of this response indicated a -log equilibrium dissociation constant for the pirenzepine-receptor complex of 6.7, significantly different from that for antagonism of negative inotropy. This ostensibly suggested a difference in the receptors mediating these responses. In view of the possible complicating effects of the positive inotropic effects of pirenzepine in this assay, an alternative method for the measurement of pirenxepine affinity was utilized. Resultant analysis was used to measure the pK, for pirenzepine antagonism of negative inotropy to carbachoi. This method had the advantage of cancelling the positive inotropy to pirenzepine. Under these circumstances, pirenzepine had a pKt, of 6.9, a value not significantly different from for antagonism of the positive inotropy to carbachol. The relevance of these findings is discussed in terms of a single pro~~uous musearinic receptor or heterogeneous receptors in this tissue. These data do not support the hypothesis that two separate receptors mediate these two effects.
Muscarinic receptors; Receptor classification; Schild analysis; Pirenzepine; (Resultant analysis) 1. IntrodWian
Previc~us studies have indicated that the biphasic responses of rat left atria (negative and positive inotropy) are equally sensitive to blockade by ~mpetitive music ~ta~o~sts, atropine, scopolamine, ~diph~lylacetoxy-~-methylpipe~-
’ Present address: Instituto di Pharmacologia, Facolta di Farmacia, Viale Taramelli, 14,27100 Pavia, Italy. Correspondence to: T.P. Kenakin, Division of Pharmacology, G&o Research Laboratories, GtaXo Inc., Five Moore Drive, Research Triangle Park, NC 27709, U.S.A. ~1~2999/90/$03.5~
dine m&iodide (4-DAMP) and AF-DX 116 (Kenakin and &s&i, 1990). This is consistent with a ph~a~lo~c scheme whereby a single muscariuic receptor interacts with two G-proteins (Gk and Gr) to mediate both responses. This has broad imp~cation for the use of intrinsic efficacy of agonists for drug and receptor classification. A caveat to such a conclusion is the possibility that the appropriate muscarinic antagonists have not yet been tested to demonstrate a receptor difference. There is evidence to suggest that pirenzepine selectively discriminates between the muscarinic receptors mediating i~bit~on of adenylate cyclase
Q 1990 Elsevier Science Publishers B.V, (Biomedical Division)
asing ~bospboi~ositide turnover 119385). In contrast, Eglen et al. that the psitiw and negative ints of c~bachol in guinea pig atria y sensitive to antagonism by pirenzeview of these data. the effect of pirenzethe bi~~as~c response of rat atria to was studied q~~titatively. These studaimed at disproving the null hypothesis same muscarinic receptor mediates both sitise and negative inotropic responses to c~bacboI in this tissue.
E
Rats (male, Sprague-Dawley. 180-220 g) were treated with reserpine (1 mg/kg i.p., once a day for two days) to deplete endogenous catecholamines. Some rats were treated with pertussis toxin yg/kg. intracardiac injection) and killed after 48-72 h. Previously, it had been determined that rese~i~ation did not modify the response in pertussis-treated rats but did contribute significantly to animal mortality. To cancel possible effects of endogenous catecholamines, these atria were studied in the presence of propranolol and phentolamine. 2.2. Tissue pre~uration Hearts were removed from rats previously killed with carbon dioxide and exanguinated. The hearts were placed in oxygenated (95% 0,-S% CO,) Krebs-Henseleit solution of composition (mM/l): NaCl 118.4, KC1 4.7, CaCl* 1.8, NaHCO, 25, gS0, *? H,O 1.19, KH,PO, 1.18 and d-glucose 11.1. The left atria were dissected, tied to Perspex holders across two platinum electrodes and placed in heated (34°C) 20 ml organ baths. Silk suture (4-O) was used to tie the atrium to a Grass FT 03 isometric transducer under a resting tension of 0.5 g. The tissues were stimulated electricahy (square wave. 1 Hz, 10 ms duration, threshold voltage +30%) and the isometric twitch contractions re-
corded on a Sensormedics corder.
R612 dynograph
re-
There was a rapid desensitization to repeated dose-response curves to carbachol in atria from pertussis-treated rats (Kenakin and Boselli, 1990}, therefore, only one cumulative (Van Rossum, 1963) dose-response curve was obtained in each tissue. Agonist concentrations were added to the organ bath (in volumes equal to or less than 200 ~1) in 0.5 log unit multiple increments. Increasing doses were added after attainment of a steady state response to the previous dose or after 5 tnin in the absence of a response. Tissues were equilibrated with antagonists for at least 120 min before measurement of responses to carbachol. Negative inotropic responses were expressed as percentages of the basal twitch contraction and positive effects as percentages of the maximal positive inotropic response to forskolin (10 ~mol/l) in the same tissue. All tissues were allowed to equilibrate for at least 60 min (with repeated changes in bath fluid), and the resting tension was readjusted to 0.5 g t~ou~out the entire experiment. In atria from non-reserpinized rats, propranolol (0.3 pmol/l) and phentolamine (0.3 pmol/l) were added to the bathing medium. 2.4. Quantification of antagonism Schild analysis was used to quantify the potency of the antagonists. For both the negative and positive inotropic responses, the ratios of equieffective concentrations of carbachol in the absence and presence of the antagonist were calculated. These dose ratios (dr) then were utilized in a regression of Log (dr - 1) upon the molar concentration of antago~st ([B]), which produced the given dose ratio. This regression was compared to the Schild equation (Arunlakshana and Schild, 1959): Log(dr-l)=nLog[B]-LogK,
(1)
where K, is the equilibrium dissociation constant of the ~tago~st-receptor complex. If the regression was linear and had a slope not significantly
51
different from unity (n = l), it was recalculated with a constrained slope of unity (MacKay, 1978). Under these conditions, the intercept was considered to be an estimate of the equilibrium-dissociation constant of the antagonist-receptor complex suitable for receptor classification. The computer program (RS/l, BBN Software Systems), ALLFIT, was used to calculate dose ratios for these dose-response curves. This program analyzed the complete data set and determined whether the maxima and slopes of the curves were significantly different from each other when fit to a general logistic function. If these parameters were not different, the program fit the complete data to a set of parallel logistic functions with a common maxima. ALLFIT then calculated the dose ratios from the independent curves with an estimate of the error of the dose ratio. These values then were utilized in a Schild regression to estimate the pK, (-log Kb).
2.5. Resultant ana&sis The interaction between pirenzepine and scopolamine as muscarinic antagonists was studied with the technique of resultant analysis (Black et al., 1986). With this method, the additive blockade produced by various concentrations of pirenzepine on the calculated antagonism by scopolamine was measured. Thus, atria were equilibrated with a given concentration of pirenzepine and pirenzepine plus scopolamine. The effects of pirenzepine on the Schild regression to scopolamine then were used to calculate the equilibrium dissociation constant of the pirenzepine-receptor complex. Schild regressions were obtained for scopolamine alone and scopolamine in the presence of pirenzepine (1.0, 3.0 and 10 pmol/l). All of these regression had a slope not significantly different from unity, therefore, they all were recalculated with a slope constrained to unity (MacKay, 1978). The pirenzepine-induced dextral displacements of the scopolamine Schild regressions were used in a regression according to the following equation (Black et al., 1986): Lo&y-l)
=Log[C]-LogK,
(2)
where y refers to the ratio of equiactive dose ratios produced by scopolamine in the absence and presence of pirenzepine, [C] refers to the molar concentration of pirenzepine, and K, to the equilibrium dissociation constant of the pirenzepinereceptor complex. If the regression was linear and had a slope not significantly different from unity, then the intercept was considered to be a valid estimate of Log K,. 2.6. Statistical analysis Linear regression slopes were calculated and analyzed using RS/l, a data analysis package from BBN Software Product Corporation, Cambridge, MA 02238. Ratios of equieffective concentrations of carbachol mediating negative inotropic responses in the absence and presence of antagonists were calculated using ALLFIT, a data software package also in RS/l. For the positive inotropic dose-response curves, the dose ratios were calculated from the linear portion of the concentration-response curves for those curves with maxima that were not significantly different. These linear portions were recalculated with a mean slope when the slopes of the curves were significantly different (Anova one-way, RS/l). Analysis of the maxima of the dose-response curves also was analyzed with Anova one-way in RS/l. 2.7. Drugs Drugs used in these experiments were carbachol chloride, scopolamine HCI and reserpine, all obtained from Sigma Chemical Company (St. Louis, MO); and pirenzepine diHC1 (Boeluinger Ingelheim Pharmaceuticals, Ridgefield, CT). AF-DX 116 (11[(2-(diethylamino)methyl]-1-piperidinyl acetyl)-5,l I-dihydro-6H-pyrido[ 2,3-b][1,4]benzodiazepine 6-one) and 4-DAMP (Cdiphenylacetoxy-N-methylpiperidine methiodide) were generous gifts from Professor E. Grana, Pharmacology Institute, Pavia, Italy. Pertussis toxin was obtained from List Biological Laboratories Inc. (Campbell, CA).
xepine 0)) Ieft atria ly. pirenzepine produced a positive e in left atria from reserpine-prepositive inotropy was not blocked 0.3 ~rnol~~~, and phentola~ne l/l). began immediately after pirenzepine ed to the organ bath and achieved a steady state within 10-15 min. The positive in~tr~pic effect was s~st~ned for 20-30 min and then waned back to control basal levels by 60 min. s positive inotropic response to pirenzepine was not observed in atria from rats pretreated toxin. Figure I shows the steady responses to 1.0 and 3.0 Pmol/l pirenzepine in atria from normal (reset-pinked) and ~~~~is-tr~ted rats. The positive inotropy to pirenzepine was blocked by low concentrations of scarinic antagonists atropine (1.0 nmol/l), (1.0 nmol/l), &DAMP (10 nmol,‘l) and AF-DX 116 (0.3 pmol/l) {see fig. 2).
Fig. 1. Positive inotropy to pirenzepine. Positive inotropy expressed as a percentage of the maximal positive inotropy to forskolin in the same tissues. Negative inotropy expressed as a percentage of basal twitch contraction. Responses to 1.0 and 3.0 pmol/l pirenzepine in atria from normal reserpinized (open bars) and pertussis-pretreated (shaded bars) rats. Bars represent S.E.M. (n = 4 for each group). Ordinate: % max. Abscissa: pirenzepine.
-10
Fig. 2. Dose-response curves to pirenzepine. Ordinate: positive inotropy to pirenzepine expressed as percentage of the maximal response to forskolin. Abscissa: logarithm of the molar concentration of pirecuepine. Curves in the absence ( and presence of atropine 1.0 nmol/l (0, n = 4), scopolamine 1.0 nmol/l (A, n = 4), AF-DX 116 0.3 pmol/l (0, n = 4) and QDAMP 10 mnol/l {A, n = 4). Bars reprwent SEM.
3.2. Antagonism of negative inotropy to carbuchol As shown in the previous paper (Kenakin and Boselli, 1990) and by others, carbachol produces a biphasic dose-response curve producing negative inotropy at concentrations between 10 and 3000 nmol/l and positive inotropy at concentrations between 3 and 3000 pmol/l. Dose-response curves for the negative response were obtained for carbachol in the absence and presence of pirenzepine (fig. 3) and the resulting dose ratios used for a Schild regression. The mean data points are shown as well as logistic functions with a ~~on slope and maxima as calculated by ALLFIT. Tissue were equilibrated with pirenzepme for > 60 min, therefore, the positive inotropic effects of this antagonist were not an evident complication in the analysis. The Schild regression was linear and had a slope not significantly different from unity. The regression was recalculated with a constrained slope of unity and yielded an estimate of the equilibrium dissociation constant for the pirenzepine-receptor complex of 0.6 pmol/l (pKt, = 6.2; table 1).
53
04 8
, -I
L
I
-6
-5
I
I
-4
-3
fig. 3. Dose-responsecures in rat left atria to carbacbok depression of inotropy. Ordinate: inotropy expressed as percentage of tbe
basal. Abscissa: logarithm or molar concentrations of carbacbol. Dose-response curves in the absence n = 4) and presence of pkuepiie, 1.0 rrnolfi (0, n = 4),3 firnol/l (v, n = 4) and 10 rmol/l (Cl,n = 4). Bars represent S&M. Curves represent the beat fit logistic functions with common slope and maxima as calculated with computer program ALLFIT.
3.3. Antagonism of posit&e inatropic responses Little negative inotropy to carbachol was obtained in atria from rats pretreated with pertussis toxin, therefore, the sensitivity of the positive inotropy to pirenzepine could be measured. Figure 4 shows the positive inotropy to carbachol in the absence and presence of various ~n~ntrations of pirenzepine. No statistically significant depressions of maximal responses to carbachol were TABLE 1 Parameters for ~tago~sm of the responses to carbachol by scopolamine and pirenzepine. Antaeonist
Normal rats
Scopolamine
Schiid PK, 8.7 a (M-8.8)
Pirenzepine
6.2 (6.0-6.4)
Resultant PK,
Pertussis-treated Schild pKb
8.7 a
b (Z-7.3)
6.7 (656.9)
a Kenakin and Boselfi (1990). b AlI values in parentheses represent 95% confidence limits as calculated by tbe BS/l linear regression program (B3N Software Inc.).
-20 L -7
-6
-!j
-4
-3
-2
-1
Fig. 4. Dose-response curves to carbachol in atria from pertuss&-treated rats. Ordinate as for fig. 1. Abscissa: Logarithms of molar concentrations of carbachol. Dose-response curves in the absence 5n = 4) and presence of pirenzepine 1.0 rmol/l (0, n = 4), 3.0 pmol/i (A, n = 4), and 10 rrnog/l (Cl, n = 4). Bars represent S.E.M.
blockade of the negative inotropic responses to carbachol by scopolamine was measured in the presence of pirenzepine 1.0, 3.0 and 10 Fmol/l. The resulting Schild regression all were linear and had slopes not si~fi~ntly different from unity, therefore, all were recalculated with constrained slopes of unity (fig. 6A) and used to furnish estimates of y for the resultant plot shown in fig. 6B.
-0.5
-7 bild regressions for antagonism of negative inotropic Fig and positive ~rtu~s-F~~t~) responses to (no carbachol by pirenzepine in rat atria. Ordinate: logarithms of eqmactive dose ratios of carbachol in the absence and presence of pirenzepine minus one. Abscissa: iogarithms of molar con. Sshild regressions for antagonism wntations of pirenz n = 12) and negative inotropy (0. of positive ino~p~ n = 12). Regression tines constrained to a slope of unity. Bars represent S.E.M.
produced by ~~~ep~e
(Anova, one-way analysis), therefore, the linear portions of the dose-response curves were fitted to straight lines of common slope. From these lines, dose ratios were calculated by computer and utilized in a Schild regression shown in fig. 5. The Child regression was linear and had a slope not significantly different from unity. Therefore, the regression was recalculated with a slope constrained to unity and yielded an estimate of the plc, of 6.95. This value was significantly larger than that found for the ~tago~sm of the negative carbachol responses by pirenzepiue (fig. 5).
-0.5
L -9
-8
-7
-6
3.4. Resultant analysis of pirenzepine antagonism A major concern about the Schild analysis described above was the possible complicating effect of the positive inotropy to pirenzepine or the effects of the desensitization to that inotropy seen over 60 mm. Therefore, the equilibrium dissociation constant of pirenzepine was obtained by resultant analysis. This had an advantage in that the reference antagonist, scopolamine, completely blocked the positive inotropy to pirenzepine (fig. 2). This neutralized the property of pirenzepine that could obfuscate the receptor antago~sm. The
Fig. 6. Resultant analysis of pirenzepine antagonism of negative inotropy to carbachol. (A) Schild regressions for scopolamine. Ordinate: logarithms of equiactive dose ratios of carbachol in the absence and presence of either scopolamine or scopo~~e plus pirenzepine minus one. Abscissa: lofty of molar concentrations of scopolamine. Schild regression for scopolamine alone ( n = 12) and scopolamine in the presence of pirenzepine 1.0 irmol/l (0, n -12). 3.0 pmol/l (A, n =12) and 10 pmol/l (0, n = 12). Bars represent S.E.M. (B) Resultant plot of data from {A). Ordinate: equiactive dose ratios produced by scopolamine in the absence and presence of pirenzepine minus 1 (according to equation 2). Abscissa: logarithms of molar concentrations of pirenzepine. Slope = 1.07.
55
The resultant plot had a slope not sig~ficantly different from unity and yielded a pK, for pirenzepine ~tago~sm of negative ~otropy to carbachol of 6.9 (table 1). This value was not sig~fic~tly different from that rn~ur~ for the antagonism of positive inotropy to carbachol in atria from pertussis-treated rats. However, it was different from that found by simple Schild analysis with pirenzepine antagonism of negative inotropy to carbachol (table 1).
4. Discmion The aim of these experiments and those described in the pr~ous paper (Kenakin and Boselli, 1990) was to disprove the null hypothesis that the negative and positive inotropy to carbachol in rat left atria was mediated by the same muscarinic receptor. Studies with atropine, scopolamine, 4DAMP and AF-DX 116 did not furnish data to refute this hypothesis (Kenakin and Bose& 1990).. However, pirenzepine, in these present studies, appeared to differentiate the receptors which mediated negative and positive inotropy to carbachol, respectively. Before the possible significance of these findings is discussed, it is worth considering the limitations of the data from a qu~titative point of view. As with the previous study, the within-group error of the dos~respon~ curves for positive inotropy was relatively high. This was due to the fact that only one dose-response curve to carbachol could be obtained in a tissue. In the case of the negative inotropy, the computer program ALLFIT was used to advantage. This program tested whether the antagonists changed the maximal response and whether the curves were shifted in a parallel manner. For both the Schild data with pirenzepine and the resultant data for scopolamine plus pirenzepine, the analysis indicated that both conditions, required for Schild analysis, were met. Accordingly, the data were fit to a set of curves of common slope and rn~rn~ response and the dose ratios calculated by computer.
Another advantage of ALLFIT was the estimate of between-group error on the dose ratios. The data describing the positive inotropy to carbachol in atria from pertussis-treated rats were more complex and not amenable to analysis by ALLFIT. In this case, the maxima of the dose-response curves in the absence and presence of pirenzepine were compared by one-way Anova to determine whether responses were depressed. The linear portions of the dose-response curves then were analyzed and found to be parallel. Linear regressions of common slope then were fitted to the data and dose ratios calculated for use in a Schild regression. It should be noted that the resulting dose ratios differed very little from those calculated diiectly from the dose-response curves but that the analysis described removed the bias produced by level of the response. With the obvious cautions to the quantitative analysis in mind, the data indicated that pirenzepine was three times more potent at blocking the positive inotropy to carbachol than the negative inotropy. This suggested that different cell surface receptors were mediating these two responses. However, one possible complicating factor in this analysis was the antagonist-sensitive positive inotropy of left atria to pirenzepine. There were two striking features of this effect. The first was the equation of the response by pretreatment of rats with pertussis toxin. This indicated a possible role of the G-protein mediating ~t~sium entry into cardiac cells (Gk, Pfaffiuger et al., 1985; Br~tweiser and Szabo, 1985) or the G-protein mediating negative control of adenylate cyclase fGi, Oliana et al., 1983). Both of these G-proteins are sensitive to ADP ribosylation by pertussis toxin. The second interesting aspect of this effect was the sensitivity to low concentrations of the muscarinic antagonists, atropine, scopolamine 4DAMP and AF-DX 116. This might suggest that pirenzepine is a weak partial agonist for the muscarinic receptor pathway leading to phosphoinositol breakdown (positive inotropy). However, the complete elimination of the effect by concentrations below the pK,, for muscarinic receptor ~~go~srn by these ~tago~sts does not support such a conclusion. For the present, the
of this positive inotropy to pirenzepine f cwncem was the possible complicating effect at the positive inotropy to pirenzepinecould OIP the Schild analysis of pirenzepine f negative carbachol responses. method of resultant analysis Was used to eliminate this effect of pirenzepine. Resultant analysis is a powerful ph~acolo~c tool which can be used to measure the affinity of drugs with more than one property for receptors (Black et al., 1986; Leff and Morse, 1987; Kenakin and Beck, 1987) or the detection of mutuahy exclusive binding sites for antagonists and/or allosteric effectors (Kenakin and Boselh, 1989; Kenakin, in press a). The particular advantage of this method in these experiments was that the reference antagonist, scopolamine, completely eiimiuated the complicating positive inotropy seen with pirenzepine. With this technique, the ~~~b~~ dissociation constant of the pirenzepine-receptor complex was found to be different from that found by simple Schild analysis. This would suggest that the property of pirenzepine which produced the positive inotropy also affected the measurement of pireuzepine affinity for the muscarinic receptor as measured by Scbild analysis. If it is assumed that resultant analysis yielded a less ambiguous estimate of the pK, for pirenzepine antagonism of the negative inotropy to carbachol than simple Schild analysis, then pirenzepiue also did not furnish data to disprove the null hypothesis that a single muscarinic receptor mediates both the negative and positive inotropic responses to carbachol in this tissue. If this is correct, then it would suggest that this receptor is pro~~uous with respect to which G-protein with which it interacts in the cell membrane. This, in turn, would seriously question the concept of intrinsic efficacy as a drug-related parameter useful for drug receptor classification (Kenakin, 1988; in press b), since the agonist profile for a receptor in a given tissue would depend not only upon the particular agonist and receptor, but also upon the type and relative quannty of the G-protein present in the membrane with which the activated receptor could bind. Since the relative quantities of receptor and G-protein
are known to vary between tissues and even in pathological conditions, the effects of agonists woutd vary as well. This would make agonist potency ratios not only receptor-dependent but also tissue-de~ndent.
References ArunIakshana. 0. and H-0. Schild, 1959. Some quantitative: usesof drug antagonists. Br. J. Pharmacol. 14,48. Black, J.W., V.P. Gerskowitch, P. Leff and N.P. ShankIey, 1986, Analysis of competitive antagonism when this property occurs as part of a pharmacological resultant, Br. J. Pharmacol. 89, 547. Breitweiser, G.E. and G. Srabo, 1985, Un~up~g of cardiac muscarinic and #I-adrenergic receptors from ion channels by a guanine nucieotide anaIogue+Nature 317,538. EgIen, R.M., W.W. Montgomery and R.L. Whiting, 1988, Negative and positive inotropic responses to muscarinic agonists in guinea pig and rat atria in vitro, J. Pharmacol. Exp. Tber. 247.911. GiII. D.W. and B.B. Wolfe, 1985. Pimnzepine distinguishes between muscarinic receptor mediated pbosphoinositide breakdown and inhibition of adenylate cyclase, J. Pharmacol. Bxp. Ther. 232,608. Kenakin, T-P., 1988. Are receptors promiscuous? Intrinsic efficacy as a transduction phenomenon, Life Sci. 43,109s. Kenakin, T-P., 1989, Challenges for receptor theory as a tool for drug and drug receptor classification, Trends Pharmacol. Sci. 10, 18. Kenakin, T-P., Drugs and receptors: An overview of the current state of knowledge, Drugs (in press a). Kenakin, T-P.. AIIosteric effects in isolated tissues, Trends Pharmacol. Sci. (in press b). Kenakin, T.P. and D. Beck, 1987, Measurement of antagonist affinity for purine receptors of drugs producing concomitant phosphodiesterase blockade: The use of pharmacologic resultant analysis, J. PharmacoI. Bxp. Ther. 243,482. Kenakin, T.P. and C. Bose& 1989, Pharmacologic discrimination between receptor heterogeneity and aUosteric interaction: Resultant analysis of gaBamine and pirenzepine antagonism of muscarinic responses in rat trachea, J. Pharmacol. Exp. Ther. 250,944. Kenakin, T.P. and C. Bosehi, 1990, Promiscuous or heterogeneous muscarinic receptors in rat atria? I. SchiId analysis with simple ~m~titive antagonists, European J. Pharmacol. 191. 39. Leff, P. and J.M. Morse, 1987, Resultant pharmacoIogicaI actions of verapam& Quantification of competitive 5-hydroxytryptamine antagonism in combination with calcium ~tago~sm, J. Pharmacol. Bxp. Ther. 24O. 284. MacKay, D., 1978, How should values of PA, and affinity constants for pharmacological competitive antagonists be estimated?, J. Pharm. Pharmacol 3O, 312.
57
Oliana, MC., P. Onali, N.H. Neff and E. Costa, 1983, Adenylate cyclase activity of synaptic membranes from rat striaturn. Inhibition by muscarinic agonists, Mol. Pharmacol. 23, 393. Pfaffinger, P.J., J.M. Martin, D.D. Hunter, N.M. Nathanson and B. Hillie, 1985, GTP-binding proteins couple cardiac muscarinic receptors to a K channel, Nature 317, 536.
Van Rossum,J-M., 1963, Cumulative dose-response curves. II. Technique for making of dose-response curves in isolated organs and the evaluation of drug parameters, Arch. Int_ Pharmacodyn. Ther. 143,299.
49
E?JP51591
miscuous or heterogeneous muscariuic receptors iu rat II. A~~oni§m of ~~s~n~~ to e~~aeho~ by aired Cinzia Boselli ’ and Terry P. Kenakin DivSon of Pharmacokgy, GIaxo Research Laboratories, Gluxo Inc., Five Maore Drive, Research Trim&e Park,NC 27709. &Ql, Received 13 August 1990, accepted 28 August 1990
Carbachol produces both negative and positive inotropy in rat left atria. It is not clear whether these two effects are mediated by two separate cell surface auscarinic receptors or a siugle receptor interacting with two coupling proteins in the cell membrane. Pirenzepine, known to selectively block some biochemical muscarinic responses, was used in this study to block the biphasic response to carbachol in rat left atria. The negative inotropy to carbachol was blocked by pirenzepine, and Schild analysis indicated a -log dissociation constant (pK,) for the pirenzepine-receptor complex of 6.2. However, the Schild analysis may have been complicated by positive inotropy observed with pirenzepiue. This positive inotropic effect was sensitive to blockade by other muscarinic antagonists. In atria from rats pretreated with pertussis toxin, carbachol produced a positive inotropic effect. SchiId analysis with pirenzepine for ~~go~~ of this response indicated a -log equilibrium dissociation constant for the pirenzepine-receptor complex of 6.7, significantly different from that for antagonism of negative inotropy. This ostensibly suggested a difference in the receptors mediating these responses. In view of the possible complicating effects of the positive inotropic effects of pirenzepine in this assay, an alternative method for the measurement of pirenxepine affinity was utilized. Resultant analysis was used to measure the pK, for pirenzepine antagonism of negative inotropy to carbachoi. This method had the advantage of cancelling the positive inotropy to pirenzepine. Under these circumstances, pirenzepine had a pKt, of 6.9, a value not significantly different from for antagonism of the positive inotropy to carbachol. The relevance of these findings is discussed in terms of a single pro~~uous musearinic receptor or heterogeneous receptors in this tissue. These data do not support the hypothesis that two separate receptors mediate these two effects.
Muscarinic receptors; Receptor classification; Schild analysis; Pirenzepine; (Resultant analysis) 1. IntrodWian
Previc~us studies have indicated that the biphasic responses of rat left atria (negative and positive inotropy) are equally sensitive to blockade by ~mpetitive music ~ta~o~sts, atropine, scopolamine, ~diph~lylacetoxy-~-methylpipe~-
’ Present address: Instituto di Pharmacologia, Facolta di Farmacia, Viale Taramelli, 14,27100 Pavia, Italy. Correspondence to: T.P. Kenakin, Division of Pharmacology, G&o Research Laboratories, GtaXo Inc., Five Moore Drive, Research Triangle Park, NC 27709, U.S.A. ~1~2999/90/$03.5~
dine m&iodide (4-DAMP) and AF-DX 116 (Kenakin and &s&i, 1990). This is consistent with a ph~a~lo~c scheme whereby a single muscariuic receptor interacts with two G-proteins (Gk and Gr) to mediate both responses. This has broad imp~cation for the use of intrinsic efficacy of agonists for drug and receptor classification. A caveat to such a conclusion is the possibility that the appropriate muscarinic antagonists have not yet been tested to demonstrate a receptor difference. There is evidence to suggest that pirenzepine selectively discriminates between the muscarinic receptors mediating i~bit~on of adenylate cyclase
Q 1990 Elsevier Science Publishers B.V, (Biomedical Division)
asing ~bospboi~ositide turnover 119385). In contrast, Eglen et al. that the psitiw and negative ints of c~bachol in guinea pig atria y sensitive to antagonism by pirenzeview of these data. the effect of pirenzethe bi~~as~c response of rat atria to was studied q~~titatively. These studaimed at disproving the null hypothesis same muscarinic receptor mediates both sitise and negative inotropic responses to c~bacboI in this tissue.
E
Rats (male, Sprague-Dawley. 180-220 g) were treated with reserpine (1 mg/kg i.p., once a day for two days) to deplete endogenous catecholamines. Some rats were treated with pertussis toxin yg/kg. intracardiac injection) and killed after 48-72 h. Previously, it had been determined that rese~i~ation did not modify the response in pertussis-treated rats but did contribute significantly to animal mortality. To cancel possible effects of endogenous catecholamines, these atria were studied in the presence of propranolol and phentolamine. 2.2. Tissue pre~uration Hearts were removed from rats previously killed with carbon dioxide and exanguinated. The hearts were placed in oxygenated (95% 0,-S% CO,) Krebs-Henseleit solution of composition (mM/l): NaCl 118.4, KC1 4.7, CaCl* 1.8, NaHCO, 25, gS0, *? H,O 1.19, KH,PO, 1.18 and d-glucose 11.1. The left atria were dissected, tied to Perspex holders across two platinum electrodes and placed in heated (34°C) 20 ml organ baths. Silk suture (4-O) was used to tie the atrium to a Grass FT 03 isometric transducer under a resting tension of 0.5 g. The tissues were stimulated electricahy (square wave. 1 Hz, 10 ms duration, threshold voltage +30%) and the isometric twitch contractions re-
corded on a Sensormedics corder.
R612 dynograph
re-
There was a rapid desensitization to repeated dose-response curves to carbachol in atria from pertussis-treated rats (Kenakin and Boselli, 1990}, therefore, only one cumulative (Van Rossum, 1963) dose-response curve was obtained in each tissue. Agonist concentrations were added to the organ bath (in volumes equal to or less than 200 ~1) in 0.5 log unit multiple increments. Increasing doses were added after attainment of a steady state response to the previous dose or after 5 tnin in the absence of a response. Tissues were equilibrated with antagonists for at least 120 min before measurement of responses to carbachol. Negative inotropic responses were expressed as percentages of the basal twitch contraction and positive effects as percentages of the maximal positive inotropic response to forskolin (10 ~mol/l) in the same tissue. All tissues were allowed to equilibrate for at least 60 min (with repeated changes in bath fluid), and the resting tension was readjusted to 0.5 g t~ou~out the entire experiment. In atria from non-reserpinized rats, propranolol (0.3 pmol/l) and phentolamine (0.3 pmol/l) were added to the bathing medium. 2.4. Quantification of antagonism Schild analysis was used to quantify the potency of the antagonists. For both the negative and positive inotropic responses, the ratios of equieffective concentrations of carbachol in the absence and presence of the antagonist were calculated. These dose ratios (dr) then were utilized in a regression of Log (dr - 1) upon the molar concentration of antago~st ([B]), which produced the given dose ratio. This regression was compared to the Schild equation (Arunlakshana and Schild, 1959): Log(dr-l)=nLog[B]-LogK,
(1)
where K, is the equilibrium dissociation constant of the ~tago~st-receptor complex. If the regression was linear and had a slope not significantly
51
different from unity (n = l), it was recalculated with a constrained slope of unity (MacKay, 1978). Under these conditions, the intercept was considered to be an estimate of the equilibrium-dissociation constant of the antagonist-receptor complex suitable for receptor classification. The computer program (RS/l, BBN Software Systems), ALLFIT, was used to calculate dose ratios for these dose-response curves. This program analyzed the complete data set and determined whether the maxima and slopes of the curves were significantly different from each other when fit to a general logistic function. If these parameters were not different, the program fit the complete data to a set of parallel logistic functions with a common maxima. ALLFIT then calculated the dose ratios from the independent curves with an estimate of the error of the dose ratio. These values then were utilized in a Schild regression to estimate the pK, (-log Kb).
2.5. Resultant ana&sis The interaction between pirenzepine and scopolamine as muscarinic antagonists was studied with the technique of resultant analysis (Black et al., 1986). With this method, the additive blockade produced by various concentrations of pirenzepine on the calculated antagonism by scopolamine was measured. Thus, atria were equilibrated with a given concentration of pirenzepine and pirenzepine plus scopolamine. The effects of pirenzepine on the Schild regression to scopolamine then were used to calculate the equilibrium dissociation constant of the pirenzepine-receptor complex. Schild regressions were obtained for scopolamine alone and scopolamine in the presence of pirenzepine (1.0, 3.0 and 10 pmol/l). All of these regression had a slope not significantly different from unity, therefore, they all were recalculated with a slope constrained to unity (MacKay, 1978). The pirenzepine-induced dextral displacements of the scopolamine Schild regressions were used in a regression according to the following equation (Black et al., 1986): Lo&y-l)
=Log[C]-LogK,
(2)
where y refers to the ratio of equiactive dose ratios produced by scopolamine in the absence and presence of pirenzepine, [C] refers to the molar concentration of pirenzepine, and K, to the equilibrium dissociation constant of the pirenzepinereceptor complex. If the regression was linear and had a slope not significantly different from unity, then the intercept was considered to be a valid estimate of Log K,. 2.6. Statistical analysis Linear regression slopes were calculated and analyzed using RS/l, a data analysis package from BBN Software Product Corporation, Cambridge, MA 02238. Ratios of equieffective concentrations of carbachol mediating negative inotropic responses in the absence and presence of antagonists were calculated using ALLFIT, a data software package also in RS/l. For the positive inotropic dose-response curves, the dose ratios were calculated from the linear portion of the concentration-response curves for those curves with maxima that were not significantly different. These linear portions were recalculated with a mean slope when the slopes of the curves were significantly different (Anova one-way, RS/l). Analysis of the maxima of the dose-response curves also was analyzed with Anova one-way in RS/l. 2.7. Drugs Drugs used in these experiments were carbachol chloride, scopolamine HCI and reserpine, all obtained from Sigma Chemical Company (St. Louis, MO); and pirenzepine diHC1 (Boeluinger Ingelheim Pharmaceuticals, Ridgefield, CT). AF-DX 116 (11[(2-(diethylamino)methyl]-1-piperidinyl acetyl)-5,l I-dihydro-6H-pyrido[ 2,3-b][1,4]benzodiazepine 6-one) and 4-DAMP (Cdiphenylacetoxy-N-methylpiperidine methiodide) were generous gifts from Professor E. Grana, Pharmacology Institute, Pavia, Italy. Pertussis toxin was obtained from List Biological Laboratories Inc. (Campbell, CA).
xepine 0)) Ieft atria ly. pirenzepine produced a positive e in left atria from reserpine-prepositive inotropy was not blocked 0.3 ~rnol~~~, and phentola~ne l/l). began immediately after pirenzepine ed to the organ bath and achieved a steady state within 10-15 min. The positive in~tr~pic effect was s~st~ned for 20-30 min and then waned back to control basal levels by 60 min. s positive inotropic response to pirenzepine was not observed in atria from rats pretreated toxin. Figure I shows the steady responses to 1.0 and 3.0 Pmol/l pirenzepine in atria from normal (reset-pinked) and ~~~~is-tr~ted rats. The positive inotropy to pirenzepine was blocked by low concentrations of scarinic antagonists atropine (1.0 nmol/l), (1.0 nmol/l), &DAMP (10 nmol,‘l) and AF-DX 116 (0.3 pmol/l) {see fig. 2).
Fig. 1. Positive inotropy to pirenzepine. Positive inotropy expressed as a percentage of the maximal positive inotropy to forskolin in the same tissues. Negative inotropy expressed as a percentage of basal twitch contraction. Responses to 1.0 and 3.0 pmol/l pirenzepine in atria from normal reserpinized (open bars) and pertussis-pretreated (shaded bars) rats. Bars represent S.E.M. (n = 4 for each group). Ordinate: % max. Abscissa: pirenzepine.
-10
Fig. 2. Dose-response curves to pirenzepine. Ordinate: positive inotropy to pirenzepine expressed as percentage of the maximal response to forskolin. Abscissa: logarithm of the molar concentration of pirecuepine. Curves in the absence ( and presence of atropine 1.0 nmol/l (0, n = 4), scopolamine 1.0 nmol/l (A, n = 4), AF-DX 116 0.3 pmol/l (0, n = 4) and QDAMP 10 mnol/l {A, n = 4). Bars reprwent SEM.
3.2. Antagonism of negative inotropy to carbuchol As shown in the previous paper (Kenakin and Boselli, 1990) and by others, carbachol produces a biphasic dose-response curve producing negative inotropy at concentrations between 10 and 3000 nmol/l and positive inotropy at concentrations between 3 and 3000 pmol/l. Dose-response curves for the negative response were obtained for carbachol in the absence and presence of pirenzepine (fig. 3) and the resulting dose ratios used for a Schild regression. The mean data points are shown as well as logistic functions with a ~~on slope and maxima as calculated by ALLFIT. Tissue were equilibrated with pirenzepme for > 60 min, therefore, the positive inotropic effects of this antagonist were not an evident complication in the analysis. The Schild regression was linear and had a slope not significantly different from unity. The regression was recalculated with a constrained slope of unity and yielded an estimate of the equilibrium dissociation constant for the pirenzepine-receptor complex of 0.6 pmol/l (pKt, = 6.2; table 1).
53
04 8
, -I
L
I
-6
-5
I
I
-4
-3
fig. 3. Dose-responsecures in rat left atria to carbacbok depression of inotropy. Ordinate: inotropy expressed as percentage of tbe
basal. Abscissa: logarithm or molar concentrations of carbacbol. Dose-response curves in the absence n = 4) and presence of pkuepiie, 1.0 rrnolfi (0, n = 4),3 firnol/l (v, n = 4) and 10 rmol/l (Cl,n = 4). Bars represent S&M. Curves represent the beat fit logistic functions with common slope and maxima as calculated with computer program ALLFIT.
3.3. Antagonism of posit&e inatropic responses Little negative inotropy to carbachol was obtained in atria from rats pretreated with pertussis toxin, therefore, the sensitivity of the positive inotropy to pirenzepine could be measured. Figure 4 shows the positive inotropy to carbachol in the absence and presence of various ~n~ntrations of pirenzepine. No statistically significant depressions of maximal responses to carbachol were TABLE 1 Parameters for ~tago~sm of the responses to carbachol by scopolamine and pirenzepine. Antaeonist
Normal rats
Scopolamine
Schiid PK, 8.7 a (M-8.8)
Pirenzepine
6.2 (6.0-6.4)
Resultant PK,
Pertussis-treated Schild pKb
8.7 a
b (Z-7.3)
6.7 (656.9)
a Kenakin and Boselfi (1990). b AlI values in parentheses represent 95% confidence limits as calculated by tbe BS/l linear regression program (B3N Software Inc.).
-20 L -7
-6
-!j
-4
-3
-2
-1
Fig. 4. Dose-response curves to carbachol in atria from pertuss&-treated rats. Ordinate as for fig. 1. Abscissa: Logarithms of molar concentrations of carbachol. Dose-response curves in the absence 5n = 4) and presence of pirenzepine 1.0 rmol/l (0, n = 4), 3.0 pmol/i (A, n = 4), and 10 rrnog/l (Cl, n = 4). Bars represent S.E.M.
blockade of the negative inotropic responses to carbachol by scopolamine was measured in the presence of pirenzepine 1.0, 3.0 and 10 Fmol/l. The resulting Schild regression all were linear and had slopes not si~fi~ntly different from unity, therefore, all were recalculated with constrained slopes of unity (fig. 6A) and used to furnish estimates of y for the resultant plot shown in fig. 6B.
-0.5
-7 bild regressions for antagonism of negative inotropic Fig and positive ~rtu~s-F~~t~) responses to (no carbachol by pirenzepine in rat atria. Ordinate: logarithms of eqmactive dose ratios of carbachol in the absence and presence of pirenzepine minus one. Abscissa: iogarithms of molar con. Sshild regressions for antagonism wntations of pirenz n = 12) and negative inotropy (0. of positive ino~p~ n = 12). Regression tines constrained to a slope of unity. Bars represent S.E.M.
produced by ~~~ep~e
(Anova, one-way analysis), therefore, the linear portions of the dose-response curves were fitted to straight lines of common slope. From these lines, dose ratios were calculated by computer and utilized in a Schild regression shown in fig. 5. The Child regression was linear and had a slope not significantly different from unity. Therefore, the regression was recalculated with a slope constrained to unity and yielded an estimate of the plc, of 6.95. This value was significantly larger than that found for the ~tago~sm of the negative carbachol responses by pirenzepiue (fig. 5).
-0.5
L -9
-8
-7
-6
3.4. Resultant analysis of pirenzepine antagonism A major concern about the Schild analysis described above was the possible complicating effect of the positive inotropy to pirenzepine or the effects of the desensitization to that inotropy seen over 60 mm. Therefore, the equilibrium dissociation constant of pirenzepine was obtained by resultant analysis. This had an advantage in that the reference antagonist, scopolamine, completely blocked the positive inotropy to pirenzepine (fig. 2). This neutralized the property of pirenzepine that could obfuscate the receptor antago~sm. The
Fig. 6. Resultant analysis of pirenzepine antagonism of negative inotropy to carbachol. (A) Schild regressions for scopolamine. Ordinate: logarithms of equiactive dose ratios of carbachol in the absence and presence of either scopolamine or scopo~~e plus pirenzepine minus one. Abscissa: lofty of molar concentrations of scopolamine. Schild regression for scopolamine alone ( n = 12) and scopolamine in the presence of pirenzepine 1.0 irmol/l (0, n -12). 3.0 pmol/l (A, n =12) and 10 pmol/l (0, n = 12). Bars represent S.E.M. (B) Resultant plot of data from {A). Ordinate: equiactive dose ratios produced by scopolamine in the absence and presence of pirenzepine minus 1 (according to equation 2). Abscissa: logarithms of molar concentrations of pirenzepine. Slope = 1.07.
55
The resultant plot had a slope not sig~ficantly different from unity and yielded a pK, for pirenzepine ~tago~sm of negative ~otropy to carbachol of 6.9 (table 1). This value was not sig~fic~tly different from that rn~ur~ for the antagonism of positive inotropy to carbachol in atria from pertussis-treated rats. However, it was different from that found by simple Schild analysis with pirenzepine antagonism of negative inotropy to carbachol (table 1).
4. Discmion The aim of these experiments and those described in the pr~ous paper (Kenakin and Boselli, 1990) was to disprove the null hypothesis that the negative and positive inotropy to carbachol in rat left atria was mediated by the same muscarinic receptor. Studies with atropine, scopolamine, 4DAMP and AF-DX 116 did not furnish data to refute this hypothesis (Kenakin and Bose& 1990).. However, pirenzepine, in these present studies, appeared to differentiate the receptors which mediated negative and positive inotropy to carbachol, respectively. Before the possible significance of these findings is discussed, it is worth considering the limitations of the data from a qu~titative point of view. As with the previous study, the within-group error of the dos~respon~ curves for positive inotropy was relatively high. This was due to the fact that only one dose-response curve to carbachol could be obtained in a tissue. In the case of the negative inotropy, the computer program ALLFIT was used to advantage. This program tested whether the antagonists changed the maximal response and whether the curves were shifted in a parallel manner. For both the Schild data with pirenzepine and the resultant data for scopolamine plus pirenzepine, the analysis indicated that both conditions, required for Schild analysis, were met. Accordingly, the data were fit to a set of curves of common slope and rn~rn~ response and the dose ratios calculated by computer.
Another advantage of ALLFIT was the estimate of between-group error on the dose ratios. The data describing the positive inotropy to carbachol in atria from pertussis-treated rats were more complex and not amenable to analysis by ALLFIT. In this case, the maxima of the dose-response curves in the absence and presence of pirenzepine were compared by one-way Anova to determine whether responses were depressed. The linear portions of the dose-response curves then were analyzed and found to be parallel. Linear regressions of common slope then were fitted to the data and dose ratios calculated for use in a Schild regression. It should be noted that the resulting dose ratios differed very little from those calculated diiectly from the dose-response curves but that the analysis described removed the bias produced by level of the response. With the obvious cautions to the quantitative analysis in mind, the data indicated that pirenzepine was three times more potent at blocking the positive inotropy to carbachol than the negative inotropy. This suggested that different cell surface receptors were mediating these two responses. However, one possible complicating factor in this analysis was the antagonist-sensitive positive inotropy of left atria to pirenzepine. There were two striking features of this effect. The first was the equation of the response by pretreatment of rats with pertussis toxin. This indicated a possible role of the G-protein mediating ~t~sium entry into cardiac cells (Gk, Pfaffiuger et al., 1985; Br~tweiser and Szabo, 1985) or the G-protein mediating negative control of adenylate cyclase fGi, Oliana et al., 1983). Both of these G-proteins are sensitive to ADP ribosylation by pertussis toxin. The second interesting aspect of this effect was the sensitivity to low concentrations of the muscarinic antagonists, atropine, scopolamine 4DAMP and AF-DX 116. This might suggest that pirenzepine is a weak partial agonist for the muscarinic receptor pathway leading to phosphoinositol breakdown (positive inotropy). However, the complete elimination of the effect by concentrations below the pK,, for muscarinic receptor ~~go~srn by these ~tago~sts does not support such a conclusion. For the present, the
of this positive inotropy to pirenzepine f cwncem was the possible complicating effect at the positive inotropy to pirenzepinecould OIP the Schild analysis of pirenzepine f negative carbachol responses. method of resultant analysis Was used to eliminate this effect of pirenzepine. Resultant analysis is a powerful ph~acolo~c tool which can be used to measure the affinity of drugs with more than one property for receptors (Black et al., 1986; Leff and Morse, 1987; Kenakin and Beck, 1987) or the detection of mutuahy exclusive binding sites for antagonists and/or allosteric effectors (Kenakin and Boselh, 1989; Kenakin, in press a). The particular advantage of this method in these experiments was that the reference antagonist, scopolamine, completely eiimiuated the complicating positive inotropy seen with pirenzepine. With this technique, the ~~~b~~ dissociation constant of the pirenzepine-receptor complex was found to be different from that found by simple Schild analysis. This would suggest that the property of pirenzepine which produced the positive inotropy also affected the measurement of pireuzepine affinity for the muscarinic receptor as measured by Scbild analysis. If it is assumed that resultant analysis yielded a less ambiguous estimate of the pK, for pirenzepine antagonism of the negative inotropy to carbachol than simple Schild analysis, then pirenzepiue also did not furnish data to disprove the null hypothesis that a single muscarinic receptor mediates both the negative and positive inotropic responses to carbachol in this tissue. If this is correct, then it would suggest that this receptor is pro~~uous with respect to which G-protein with which it interacts in the cell membrane. This, in turn, would seriously question the concept of intrinsic efficacy as a drug-related parameter useful for drug receptor classification (Kenakin, 1988; in press b), since the agonist profile for a receptor in a given tissue would depend not only upon the particular agonist and receptor, but also upon the type and relative quannty of the G-protein present in the membrane with which the activated receptor could bind. Since the relative quantities of receptor and G-protein
are known to vary between tissues and even in pathological conditions, the effects of agonists woutd vary as well. This would make agonist potency ratios not only receptor-dependent but also tissue-de~ndent.
References ArunIakshana. 0. and H-0. Schild, 1959. Some quantitative: usesof drug antagonists. Br. J. Pharmacol. 14,48. Black, J.W., V.P. Gerskowitch, P. Leff and N.P. ShankIey, 1986, Analysis of competitive antagonism when this property occurs as part of a pharmacological resultant, Br. J. Pharmacol. 89, 547. Breitweiser, G.E. and G. Srabo, 1985, Un~up~g of cardiac muscarinic and #I-adrenergic receptors from ion channels by a guanine nucieotide anaIogue+Nature 317,538. EgIen, R.M., W.W. Montgomery and R.L. Whiting, 1988, Negative and positive inotropic responses to muscarinic agonists in guinea pig and rat atria in vitro, J. Pharmacol. Exp. Tber. 247.911. GiII. D.W. and B.B. Wolfe, 1985. Pimnzepine distinguishes between muscarinic receptor mediated pbosphoinositide breakdown and inhibition of adenylate cyclase, J. Pharmacol. Bxp. Ther. 232,608. Kenakin, T-P., 1988. Are receptors promiscuous? Intrinsic efficacy as a transduction phenomenon, Life Sci. 43,109s. Kenakin, T-P., 1989, Challenges for receptor theory as a tool for drug and drug receptor classification, Trends Pharmacol. Sci. 10, 18. Kenakin, T-P., Drugs and receptors: An overview of the current state of knowledge, Drugs (in press a). Kenakin, T-P.. AIIosteric effects in isolated tissues, Trends Pharmacol. Sci. (in press b). Kenakin, T.P. and D. Beck, 1987, Measurement of antagonist affinity for purine receptors of drugs producing concomitant phosphodiesterase blockade: The use of pharmacologic resultant analysis, J. PharmacoI. Bxp. Ther. 243,482. Kenakin, T.P. and C. Bose& 1989, Pharmacologic discrimination between receptor heterogeneity and aUosteric interaction: Resultant analysis of gaBamine and pirenzepine antagonism of muscarinic responses in rat trachea, J. Pharmacol. Exp. Ther. 250,944. Kenakin, T.P. and C. Bosehi, 1990, Promiscuous or heterogeneous muscarinic receptors in rat atria? I. SchiId analysis with simple ~m~titive antagonists, European J. Pharmacol. 191. 39. Leff, P. and J.M. Morse, 1987, Resultant pharmacoIogicaI actions of verapam& Quantification of competitive 5-hydroxytryptamine antagonism in combination with calcium ~tago~sm, J. Pharmacol. Bxp. Ther. 24O. 284. MacKay, D., 1978, How should values of PA, and affinity constants for pharmacological competitive antagonists be estimated?, J. Pharm. Pharmacol 3O, 312.
57
Oliana, MC., P. Onali, N.H. Neff and E. Costa, 1983, Adenylate cyclase activity of synaptic membranes from rat striaturn. Inhibition by muscarinic agonists, Mol. Pharmacol. 23, 393. Pfaffinger, P.J., J.M. Martin, D.D. Hunter, N.M. Nathanson and B. Hillie, 1985, GTP-binding proteins couple cardiac muscarinic receptors to a K channel, Nature 317, 536.
Van Rossum,J-M., 1963, Cumulative dose-response curves. II. Technique for making of dose-response curves in isolated organs and the evaluation of drug parameters, Arch. Int_ Pharmacodyn. Ther. 143,299.
