Promiscuous or heterogeneous muscarinic receptors in rat atria? I. Schild analysis with simple competitive antagonists

European Journal of Pharmacology, 191 (1990) 39-48

39

F&vier EIP 51590

Terry P. Kenakin and Cinzia Boselli

*

Division of Pharmacology, Glaxo Research Laboratories, Glaxo Inc.. Fiue Moore Drive, Research Triangie Purk, NC 27709, U.S.A.

Received 13 August 1990, accepted 28 August 1990

Carbachol has been shown to produce a biphasic response in rat left atria. At low concentrations, carbachol depresses basal inotropy, while at high doses a positive inotropic effect is observed. The negative inotropic response can be selectively eliminated by pretreatment of rats with pertussis toxin. The aim of these studies was to determine whether or not evidence could be obtained to show that different muscat-ink receptors produced these different bi~he~~ responses to the agonist carbachol. Schild analysis was used to measure the ~u~b~~ dissociation constant of the antagonist-receptor complex for antagonism of the negative inotropy to carbachol by atropine, scopolamine &liphenylacetoxy-N-methylpiperidine methiodide (CDAMP) and AF-DX 116. The antagonism of the positive inotropic response to carbachol by these antagonists was studied in atria from rats pretreated with pertussis toxin where the negative inotropy was nearly completely abolished. In general, it was found that the antagonists did not produce simple competitive blockade of the positive inotropy but rather a nominal shift to the right of the dose-response curves folIowed by a depression of maximaI responses. However, it was found that when pA, or pK, values could be calculated, they coincided with those determined for the antagonism of the negative inotropy to carbachol. The conclusion drawn from these experiments was that no evidence was obtained to disprove the nuIl hypothesis that a common receptor, interacting with two G-proteins, mediates these two effects of carbachol in rat left atria. The implications of these data for the classification of drug receptors with agonists is discussed.

~usc~c

receptors; Receptor classification; Schild anaIysis; Receptor coupling

1. Introduction Recently it has been shown that muscarinic agonists can elicit a biphasic inotropic response in rat (Imai and Ohta, 1982; Eglen et al., 1989), guinea pig (Eglen et al., 1988) and avian (Brown and Brown, 1984; Brown and Goldstein, 1986) atria1 muscle. These studies suggest that the nega-

’ Present address: Instituto di Pharmacologia, Facolta di Farmacia, Viale Taramelli, 14, 27100 Pavia, Italy. Correspondence to: T.P. Kenakin, Division of Pharmacology, Glaxo Research Laboratories, Glaxo Inc., Five Moore Drive, Research Triangle Park, NC 27709, U.S.A.

tive inotropic effect is due to the activation of the GTP-binding protein (G-protein) modulating potassium channel activation (Gk; Pfaffinger et al., 1985; Breitweiser and Szabo, 1985), while the positive inotropic effect is related to phosphoinositide turnover (Jones et al., 1979; Quist, 1982). This latter response is thought to be related to activation of a putative G-protein (G,). The negative inotropic response can be abolished by pretreatment of prep~ations with pertussis toxin, an endotoxin produced by Burdetela pertussis, which ADP ribosylates the (Y subunit of G in cardiac tissue. In contrast, the positive inotropic effect (activation of Gp) is sensitive to pertussis treatment.

0014-2999#90/$03.50 0 1990 Efsevier Science Publishers B.V. (Biomedicai Division)

aim of this present study was to determine r o: not these two effects were due to w ~~t~~~ati~nof one receptor subtype which interacts with two G-proteins or the activation of two cell surface reseptors which activate the two G-protein respecti~~ely. Schild analysis was used to generate evidence to disprove the nnll hypothesis stating that the same receptor mediated both effects. It boded be expected that if different cell surface eptors mediated these responses, then the difshonld be detected by measurement of ;lihi;rgonist affinity, i.e. it would not be expected that all antagonists wonld have identical affinities for the two receptor types. In contrast, if a single extracellular recognition domain is responsible for drug binding, then a uniform set of antagonists affinities would be expected for the blockade of both responses. The ~ffe~ntiation between these two receptor mechanisms has relevance to the definition of intrinsic efficacy as a drug-related parameter. Z MaterhIs and methods

2.I. Animal pretreaiment Rats (male, Sprague-Dawley, 180-220 g) were depicted of endogenous catecholamines by reserpinization (1 mg/kg i-p., once a day for two days). Some rats were treated with pertussis toxin (40 gg/kg, intracardiac injection) and killed after 48-72 h. These animals were not pretreated with reserpine. Preliminary experiments indicated that reserpine pretreatment 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 (vide infra). 2.2. Tissue preparation Hearts were removed from rats previously killed with carbon dioxide and exanguinated. The hearts were placed in oxygenated (95% 0,-Z% CO,) Krebs-Henseleit solution of composition (mM/i): NaCl 118.4, KC1 4.7, CaCl, 1.8, NaHCO, 25,

MgSO,. 7H,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. The other side of the atrium was tied with 4-O silk to a Grass PI 03 isometric transducer under a resting tension of 0.5 g. The tissue was stimulated electrically (square wave, 1 Hz, 10 ms duration, threshold voltage-t 30%), and the isometric twitch contractions were recorded on a Sensormedics R612 dynograph recorder. 2.3. Experimental protocol Preliminary studies indicated that desensitization to carbachol was evident in left atria from pertussis-treated rats. Therefore, only one cumulative (Van Rossum, 1963) dose-response curve was carried out 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 ruin in the absence of a response. Tissues were ~ui~brat~ with ~tago~sts 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 pmol/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 throughout the experiment. 2.4. Quantification of antagonism Schild analysis was used to quantify the potency of the antago~sts. For both the negative and positive inotropic responses, the ratios (dr) of equieffective concentrations of carbachol in the absence and presence of the antagonist were calculated for at least three different concentrations of antagonist. They then were utilized in a

41

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 following linear equation (~~~shana and Schild, 1959): Log(dr-l)=nLog[B]-LogK,

(1)

where K, is the equilibrium dissociation constant of the antagonist-receptor complex. If the regression was linear and had a slope not significantly different from unity (n = l), it was recalculated with a constrained slope of unity (MacKay, 1978). Under these ~nditions, the intercept was considered to be an estimate of the equilibrium-dissociation constant of the antagonist-receptor complex suitable for receptor classification. If the regression was not linear, the intercept was the PA, (negative logarithm of the molar concentration that produces a dose ratio of 2). This was a measure of ~tago~st potency but could not be considered a chemical term like the K,,. In the instances where one shift of the agonist dose-response curve could be used to quantify antagonist potency, the PA, was calculated with the following equation: pA2= (Log(dr-I)-Log[B]

(2)

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. This program was used to fit the complete set of dose-response curves to logistic functions of common maxima and slope and tested whether or not this was valid statistically. If the dose-response curves were not significantly different with respect to maxima and slope, the data set was fit to the logistic functions and equiactive dose ratios were calculated. Estimates of the error of these dose ratios also were obtained. For the positive inotropic dose-response curves, the dose ratios were calculated from the

linear portion of the concentration responses curves for those curves with maxima that were not significantly different as calculated by a one-way Anova test. These linear portions were recalculated with a mean slope when the slopes of the curves were not significantly different (Auova one-way, RS/l). Analysis of the maxima of the dose-response curves also was analyzed with Anova one way in RS/L 2.6. &ugs

Drugs used in these experiments were carbachol chloride, scopolamine HCi, atropine sulfate and reserpine, all obtained from Sigma Chemical Comply (St. Louis, MO); QDAMP (~phenylacetoxy-N-methylpiperidine me&iodide), AF-DX 116 (11[~2-(diethylam~o)methyl]-l-piperidinyl acetyl)J,1l-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine Gone) were a generous gift from Professor E. Grana, Pharmacology Institute, Pavia, Italy. Pertussis toxin was obtained from List Biological Laboratories Inc. (Campbell, CA).

3. Results

In normal left atria from reserpine-pretreated rats, carbachol produced a biphasic dose-response curve (fig. 1A). At concentrations between 10 and 3000 nmol/l, carbachol produced a dose-dependent depression of basal twitch concentrations. At con~ntrations between 3 and 3000 Fmol/l, a positive inotropic response was observed. In left atria from rats previously treated with pertussis toxin, very little residual negative response was evident, only a dose-dependent positive inotropic effect (fig. 1A). This pattern of response bas been reported previously (Imai and Ohta, 1982). It also was observed that while repeated doseresponse curves to carbachol could be obtained with little differences for the negative portion of the dose-response curve, the same was not true for the positive phase. Figure 1B shows repeated dose-response curves to carbachol in atria from

60

60

40

50

20

40 36

I 2c -2i 1E 91

a

61

-1f

-64

-a

-7

-6

-6

-4

-3

-2

-6

-1

-7

-6

-6

-4

-3

-2

Fig. P. Dose-response curves to carbachot in rat left atria. Abscissas: logarithm of the molar con~n~ation of carbachol. Ordinates: depression of inotmpy expressed as a percentage of the basal twitch contraction. Increases over basal expressed as a percentage of the maximaf positive inotropic response tq forskolin in the same tissue. (A) Responses in atria from normal (e, n “8) and pertussis-pretreated (0. n = 9) rats. (B) Repeat dose-response curves to carbachol in atria from pertussis-treated rats. Fit (0, n = 4) dose-response and repeated curves in the same tissues after 1 (0, n = 4) and 2 h (A, n = 4) of washiug with drug-free medium. Bars represent S.E.M.

pertussis treated rats. A progressive desensitization was obtained which could not be eliminated by re-equilibration with drug-free medium. This necessitated the use of single dose-response curves for each tissue in these analyses.

3-Z. An~Qgon~~of neguriue inorropy fo carb~c~~i The dose-response curves describing the negative inotropy of atria to carbachol in the absence and presence of muscarinic ~tag~s~ were subjected to analysis with the program ALLFIT. In general, atropine, scopolamine, 4DAMP and AFDX 116 produced parallel shifts to the right of the dose-response curves with no change in the maximal response to carbachol. The effects of the antagonists are shown in fig. 2. The Schild regressions for these antagonists all were linear, had slopes not siguificantly different from unity and yielded estimates of respective I& estimates (fig. 3). The data lescribing these Schild regressions are shown i .ble 1.

3.3. Antagonist

of positive inotropic responses

As shown in fig. lA, little negative inotropy to carbachol was obtained in atria from rats pretreated with pertussis toxin. Therefore, the sensi-

TABLE 1 Schild regression parameters for antagonism of the negative inotropic response to carbachol. Antagonist

Slope

PR, =

Atropine

0.8 (0.6-1.0) ’

8.7 b (8.6-8.8) 8.7 (8.6-8.8)

Scopolamine (siCl.1) AF-DX 116 4DAMP

(&.3, 1.2 (0.9-1.4)

(z-7.4) (:::-8.2)

a -Logarithm of the equilibrium dissociation constant of the ~~go~st-r~ptor complex. b Intercepts of regressions of slope constrained to unity. ’ All values in parentheses represent 95% confidence limits as calculated by the linear regression program in RS/l.

43

A

1 -7

-6

-6

t

-3

-4

-2

-6

-7

-6

-5

4

-3

t

-2

100

C t

-6

I

-7

,

-6

,

-5

I

-4

c

-3

a -2

a

i -6

I

-7

I

-6

I

-5

I

4

t

-3

i

-2

Fig. 2. Dose-response curves in rat Ieft atria to carbachol: Depression of inotropy. Ordinates and abscissas as for fig 1. (A) Dose-response curves in the absence (0, n = 4) and presence of atropine, 3 nmol/l (0, n - 4), 10 mnol/l (A, n = 4) and 30 nmolfl (Q n = 4).(B) Dose-response curves in the absence (@, n = 4) and presence of 4-DAMP, 10 nmol/I (0, n = 4), 30 nmol/l (A, n = 4) and 100 nmol/l (Cl, n = 4). (C) Dose-response curves in the absence (#v, n = 4) and presence of scopolamine, 3 nmol/l (0, n = 4). 10 nmol/l (A. n = 4) and 30 nmol/l (Cl, n = 4). (D) Dose-response curves in the absence (e, n = 4) and presence of AF-DX 116.0.3 amol/l (0, n = 4), 1.0 amol/l (A, n = 4) and 3.0 gmol/l (0, n = 4). Bars represent S.E.M. Curves represent the best fit logistic functions with common slope and maxima as calculated with computer program ALLFIT.

tivity of the positive inotropy to muscarinic antagonists could be measured. Figure 4 shows the positive inotropy to carbachol in the absence and presented of various concen~ations of atropine. As can be seen from these curves, atropiue did not produce parallel displacements of the dose-response curves, but rather depressed the maximal responses at ~n~entrations equal to the K,. Due

to the nature of antagonism, Schild analysis could not be used to quantify the potency of atropine. A different pattern was observed with 4-DAMP. While two concentrations (0.03 and 0.1 ~molff) depressed the maximal responses to carbachol in a manner similar to that of atropine, 30 nmol/l produced a shift to the right of the dose-response curve with no si~ific~t depression of the maxi-

50

40 30

20 10

0

-9

-6

-7

-6

-5

Fii. 3. Schild regmssions for antagonism of negative inotropic responses to carbachol in rat atria. Ordinate: logarithms of equiactive dose ratios of carbachol in the absence and presence of antagonist minus one. Abscissa: logarithms of molar concentrations of antagonist. Schild regressions for atropine (0, n = 12). scopolamine (0. n =12). 4-DAMP (A. n =12). and AF-DX 116 (a n =12). AB regmssion fines constrained to a slope or unity. Bars represent S.E.M.

-10

-20 -7

-6

-5

-4

-3

-2

-1

Fig. S. Dose-response curves to carbachol in atria from pertussis-treated rats. Ordinate and abscissa as for fig. 1. Dose-response curves in the absence (0. n = 8) and presence of 4 DAMP, 30 nmol/l (0, n = 4). 100 nmol/L (A, n = 4), and 300 nmol/l (0, n = 4). Bars represent S.E.M.

mal response (fig. 5). A one way analysis of variance indicated that the slopes of these dose-response curves were not statistically different, therefore, the linear portions of the curves were fit to parallel straight lines and the displacement between them (dose ratio) calculated by computer. The resulting pA2 (table 2) of 8.1 was not significantly different from the pK, determined by Schild analysis (table 1) for the negative inotropic phase of the dose-response curve to carbaghol.

10

TABLE 2 Parameters describing response. Antagonist Scopolamine AF-DX 116 4DAMP

Fig. 4. Dose-response curves to carbachol in atria from pertussis-treated rats. Ordinate and scissa as for fig. 1. Dose-response curves in the absence ( n = 8) and presence of atro_ pine, 3 mnoffi (0. n = 4). 10 nmof/f (A, n = 4).and 30 nmoI/l (Cl,n = 4). Bars represent S.E.M.

antago~sm

of the positive inotropic

Slope

PKh a

0.8 (0.6-1-O) =

7.4 (7.2-7.5)

pAzb

8.7

8.1

a -Logarithm of the equilibrium dissociation constant of the antago~st-r~eptor complex. b - ~~thrn of the molar concentration of antagonist that produces a Z-fold shift of the dose-response curve to the agonist. ’ All values in parentheses represent 95% confidence limits as calculated by the linear regression statistical analysis in RS/l.

45

Figure 6 shows the effects of scopolamine on the positive inotropy to carbachol. Two concentrations (3.0 and 10 nmol/l) of scopola~e produced dextral displacement of the dose-response curves with no significant depression of maxima. Higher concentrations (30 and 100 nmol/l) depressed the maxima and reduced the slopes of the dose-response curves. The displacements of the dose-response curves obtained with 3.0 and 10 nmol/l scopolamine were utilized to calculate the pA, for this antagonist. The responses to three doses of carbachol for the control and two doseresponse curves in the presence of scopolamine were fitted to parallelstraight lines and the resulting shifts calculated by computer. These furnished dose ratios which were converted to pA, values. The pA, of 8.7 (table 2) was not si~ic~tly different from the pK, value obtained for the negative inotropic response by Schild analysis. Three concentrations of AF-DX 116 produced dose-related dextral displacements of the dose-response curve to carbachol with no significant depression of the maximal response (fig. 7). Statistical analysis also indicated that the slopes of the

30 -

Fig. 7. Dose-response curves to carbachol in atria from pertussis-treated rats. Ordinate and abscissa as for fig 1. Dose-response curves in the absence (e, n = 8) and presence of AF-DX 116, 0.3 gmoi/l (0, n = 4) 1.0 rmof/l (A, n = 4). and 3.0 nmol/l (Cl,n = 4). Bars represent S.E.M.

linear portions of the dose-responses curves were not different, therefore, parallel straight tines were fitted to these curves and dose ratios calculated by computer. The resulting dose ratios produced a Schild regression which had a slope not significant different from unity and an estimate of the pK, of 7.4 (table 2). This estimate was not significantly different from the pK, for antagonism of the negative inotropic effects of carbachol (table 1).

20 -

4. Discussion

10 -

o-

-10 -

-2oc

-7

-6

-5

4

-3

-2

-1

Fig. 6. Dose-response curves to carbachol in atria from pertussis-treated rats. Ordinate and abscissa as for fig. 1. Dose-ren = 8) and presence of sponse curves in the absence 10 nmolfl (a, n = 4), 30 scopolamine, 3 nmol/l (0, n = nmoifl (0, n = 4), and 100 nmol/l (A, n = 4). Bars represent S.E.M.

There are several aspects of this receptor study which require comment. First, it is clear that while the n~ative~y inotropic responses to carbachol could adequately be quantified and reliably measured, the positive phase of the dose-response curve was problematic. This was critical to the basic premise of this paper, since the conclusions about the pharmacology of these effects must be drawn from the magnitude of the parameters measured by the Schild technique. The major problem with the positive inotropy seen with carbachol in atria from pertussis-treated

rite ~9s thy desensitization to repeated stimulais ~~~u~~ use of a fundamental tool in tion. ~~~~tor clarification studies, namely that Of ma]ytis by null methods. Under the best circ stances, control responses are quantified, an ~ta~o~st ~~~brat~ with the tissue receptors. and the response measured in the presence of the antagonist in the same tissue. This allows for the appellation of tissue effects such as efficiency of receptor coupling and receptor number, which could otherwise modify the translation of agonistreceptor effects to tissue response. Since repeat dose-response curves to carbachol for the positive phase of the dose-response curve could not be achieved, comparison of different tissues was required for the me~urement of receptor effects. This had two consequences, namely a larger within-group error and also the loss of accurate measures of error on the calculated parameters. Only one dose-response curve could be obtained for the positive inotropy, thus for uniformity, the same protocol was utilized for the negative inotropic response as well. This was much less a problem, since the within group error was much smaller, and the program ALLFIT could be used to analyze the dose-response curves as a group and provide estimates of error. ALLFIT is a statistical package which assimilates a collection of dose-response curves and gives statistical estimates of confidence that the curves have a common maximal response and a common slope. When the answer to both of these questions is affirmative, then Schild analysis is valid. ALLFIT then takes the complete data set and fits parallel logistic functions to the data points with a common maximal response. Also, estimates of the dose ratios, with an estimate of the error on each dose ratio, are calculated for use in SchiId analysis. For the negative inotropic responses to carbachol in normalatria, these techniques were adequate, and estimates of K, values with error were obmined. Unfo~~ately, *he positive inotropic responses to carbachol in atria from pertussis-treated rats showed a complex pattern not amenable to analysis by ALLFIT. This was initiated to disprove the null hypothesis that the receptors mediating the negative and positive isotropic responses to carbachol in atria1

tissue were the same. Ideally, it was anticipated that the eq~~b~urn diss~iation constants of a number of muscarinic antagonists could be measured for both responses, and some insight into the properties of the receptors mediating these two effects could be gained. However, except for the antagonism with AF-DX 116, the dose-response curves for the positive inotropy were not shifted in parallel to allow Schild analysis and the estimation of a pK,_ One possible explanation for this is that the inordinately high concentrations of earbachol required for the positive inotropy had other effects which depress the response. This can be seen from the bell-shaped dose-response curve to carbachol. Under these circumstances, it might be expected that no competitive ~tago~st would produce parallel shifts of the dose-response curve with no depression of maxima. Another possibility is that the pertussis toxin pretr~tment did not entirely eliminate G, and that the positive inotropy was modulated by a negative response by residual G, protein. This is suggested in the small but consistent negative inotropic responses to carbachol in atria from pertussis-treated atria. In general, the sensitivity of the carbachol-induced positive inotropy to the muscarinic antagonists is comparable to the sensitivity of the negative inotropy. Thus, atropine depressed the carbachol dose-response curve for pos%ve inotropy at 3.0 nmol/l, a value near the equilibrium dissociation constartt of the atropinsreceptor complex for negative inotropy (2.0 nmoI/l). Similarly, with 4-DAMP, the PA, for blockade of positive inotropy (8.1) was identical to that for blockade of the negative inotropy. The same was true for scopolamine. A partial Schild regression for the antagonism of the positive inotropic response was obtained for AF-DX 116 (7.4), and this was not si~fi~ntIy different from that for antagonism of the negative inotropy (7,3). In this case, estimates of the error of these values also could be calculated. The pharmacologic conclusion from these data, made with the caveat that there were shortcomings in the quantitative analysis, is that no evidence was obtained to disprove the null hypothesis that the receptors mediating the negative and positive inotropic responses to carbachol are the same.

47

This cannot be taken as definitive evidence to prove that the same receptor mediates these two effects, since it is possible that the specific antagonists which would show these receptors to be different were not tested in this study. However, if there were two receptors mediating these responses, these data would suggest that the affinities of atropine, s~pol~e, LGDAMP and AFDX 116 are identical for both. There are conflicting data concerning the identity of the receptors mediating the pleiotropic biochemical responses to muscarinic agonists. In contrast to these present results, Imai and Ohta (1982) found a difference in the potency of atropine in blocking the positive and negative inotropic response to carbachol in rat atria. Gil and Wolfe (1985) found that pirenzepine distinguishes between the muscarinic receptors which mediate phospho~ositide breakdown and i~bition of adenylate cyclase. This antagonist has been analyzed pharmacologically, and it has been found that no difference in potency for that antago~sm of the positive and negative inotropic effects of carbachol could be detected in guinea pig atria (Eglen et al., 1988) and rat left atria (Kenakin and Boselli, 1990). Under present schemes of classification, this would agree with the postulate that the positive inotropic response to muscarinic agonists, as welI as the negative inotropic response, is due to activation of Mz receptors (Eglen et al., 1988). Recent evidence shows that the muscarinic receptors coupled to phosphoinositide turnover and adenylate cyclase are ~distinguishable when studied with the antagonists, pirenzepine, AF-DX 116 and 4-DAMP, but differ considerably in their interaction with agonists (Baumgold and White, 1989). This suggests that the differences may lie in the interaction with G-proteins and not in the recognition domain for antagonists. This is consistent with a pro~cuous muscarinic receptor with a uniform domain for the recognition of drugs but differing capacity to interact with multiple G-proteins. It is known that receptors are capable of interacting with more than one G-protein in recombinant systems (for review see Kenakin, 1988; in press). It still is not resolved whether this occurs under physiological conditions. The results of this

present study are consistent with a single receptor for muscarinic agonists which can interact with varying efficiency in the cardiac cell membrane with G, and Gp to mediate negative and positive inotropy, respectively. However, such data does not negate the possibility of these responses being mediated by two receptor subtypes as well. Further studies with various muscarinic ~~go~sts are needed to evaluate the possibility that heterogeneous receptors mediate these responses and that the definitive antagonists to uncover this heterogeneity have not yet been found. If a single receptor is responsible for the two responses and if various agonists differ in their capacity to activate these G-proteins, then the intrinsic efficacy of agonists will depend not only upon the receptor type present in the membrane, but also upon the type and quantity of the G-proteins present as well. This would negate the concept of intrinsic efficacy as a drug property of value in the process of drug receptor classification (Kenakin, 1988; 1989).

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Imai. X and H. Ohm. 1982, Positive inotropic effects induced by carbachol in rat atria treated with islet-activating protein (IAP) - Association with phosphoinositol breakdown, Br. J. I’barmacoI. 94. 347. Jc~es. L.M.. S. Co&oft and R.H. Mitchell. 1979, Stimulation of pbosphatidyhnositol in various tissues by chohnergic and adrenergic agonists, by histamine and by ;ae_ru!ein. Eiocbem. J. 182.669. Kenakin. T.P.. 1988, Are receptors promiscuous? Intrinsic efficacy G 5 transduction phenomenon, Life ki. 43, 1055 Ken&in. T.P.. 1989, Challenges for receptor theory as a tool for drug and drug receptor classification, Trends PharmacoI. Sci. I@, 18. Kenakin, T.P.. Drugs and receptors: An overview of the current state of knowledge, Drugs (in press). IknaIdn. T-P. and C. Bosehi. 1990, Promiscuous or heteroge-

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muscarinic receptors in rat atria? II. Antagonism of responses to carbachol by pirenzepine. European J. Pharmacol. 191,49. McKay, D.. 1978, How should values of pA, and affinity constants for pharmacological competitive antagonists be estimated? J. Pharm. Phartnacol. 30, 312. Pfaffinger, P.J.. J.M. Martin, D.D. Hunter, N.M. Nathanson and 8. HiIIk 1985. GTP-binding proteins couple cardiac muscatink receptors to a K chart&, Nnturc 317,536. Q&c, E.E.. 1962, Ev%ia;ce for a carbachol stimulated phosphatidyhnositol effect in heart, Biochem. Pharmacol. 31, 3130. Van Rossum, J.M., 1963, Cumulative dose-response curves. II. Technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters, Arch. Int. Pharmacodyn. Ther. 143, 299.