Proc. NatL Acad. Sci. USA Vol. 78, No. 9, pp. 5554-5558, September 1981 Biochemistry

Molecular regulation of receptors: Interaction of (3- and with the muscarinic system (cholinergic //receptor communication) MORDECHAI SOKOLOVSKY*, YAACOV EGOZI, AND SOFIA AvIssAR Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, 69978 Tel-Aviv, Israel Communicated by Alton Meister, June 18, 1981 ABSTRACT The effects of various substrates on the binding MATERIALS AND METHODS of agonists to muscarinic receptors were studied in the rat hypo- thalamus and adenohypophysis by competition experiments using Materials. 3H-Labeled N-methyl-4-piperidyl benzilate the highly specific tritiated muscarinic antagonist N-methyl-4-pi- ([3H]4NMPB) (68.7 Ci/mmol; 1 Ci = 3.7 x 10's becquerels) peridyl benzilate. It was found that agonist binding properties was prepared by catalytic tritium exchange as described (3). Its were affected only by the steroid sex hormones (1-estradiol and purity was >97%. Other compounds used were oxotremorine progesterone), both of which resulted in a decrease in the pro- from Aldrich; atropine sulfate, 17p-estradiol, progesterone, portion of high-affinity binding sites and a decrease in the disso- acid, and cyclic ciation constant. This suggests a link between the muscarinic sys- cholesterol, corticosterone, y-amino-n-butyric tem and the mechanism by which these steroids exert their AMP and cyclic GMP from Sigma; D-Ala2-Met5-enkephalin gonadotropin-releasing effect on the adenohypophysis. We pro- from UCN, Belgium; and gonadotropin-releasinghormone gen- pose a model to depict the putative relationship between the mus- erously donated by Aliza Eshkol. All compounds were of the carinic system and other receptor systems, including that which best grade available. controls the steroid sex hormones. Animals. Adult male and female rats of the CD strain were supplied by Levinstein's Farm (Yokneam) and maintained in an In a recent study, we demonstrated the presence ofmuscarinic air-conditioned room at 24 + 20C for 14 hr under fluorescent receptors in the rat adenohypophysis and described their bio- illumination (0500-1900 hr) and in darkness for 10 hr daily. chemical characteristics (1). Our results showed that (i) in con- Food from Assia Maabarot Ltd. and water were supplied ad lib. trast to other brain regions, antagonist binding was heteroge- After an adjustment period of at least 4 weeks, daily vaginal neous in this area, with the existence ofat least two subclasses smears were taken of all female rats, and only those having a ofsites; (ii) agonist binding is characterized by a two-site model regular 4-day estrous cycle were used. The rats were then 3 to specifying a high and a low affinity state; and (iii) the female rat 4 months old and weighed 190-250 g. They were decapitated is characterized during the proestrous stage by a lower degree and theirbrains were rapidly removed, and the brain areas were ofagonist high-affinity binding and by an increase (almost dou- dissected out in a cold room, after identification with the aid ble) in the proportion ofhigh-affinity sites in comparison with of ref. 4. female rats at other stages of the cycle and with male rats. Pi- Binding Assays. Full details concerning homogenate prep- tuitary responsiveness to muscarinic binding therefore fluc- aration and antagonist binding assay techniques using the fil- tuates during the estrous cycle. It is significant that we detected tration method for the brain areas investigated and the cen- (2) similar features in the characteristics of agonist binding to trifligation method for the adenohypophysis, are described muscarinic receptors in the preoptic area (which houses the elsewhere (1, 2, 5). The fact that the ligand-muscarinic receptor biological clock regulating hormone release)-i.e., the popu- complex dissociates much more rapidly in the adenohypophysis lation of agonist high-affinity sites at the proestrous stage was than in other brain regions creates difficulties with the filtration much higher than at other stages of the estrous cycle (66% vs. technique; therefore, the centrifugation method was used for 38%). In vivo endocrinic manipulations of the estrous cycle at the adenohypophysis. Binding of [3H]4NMPB that was inhib- the estrogenic level, such as ovariectomy ofadult cyclic females ited by 5 ,uM of unlabeled atropine was considered to be or androgenization of newborn females, were reflected by al- specific. terations in the muscarinic system at the adenohypophysis (un- Binding ofagonists in the absence or presence ofthe various published data). These results suggest that the muscarinic re- substrates tested was inferred by their ability to compete with ceptors play apart in the positive or negative (orboth) regulation specific binding of 2.0 nM [3H]4NMPB (1, 2, 6). of on sex hormone secretion. Data Analysis. The data obtained from direct binding assays To investigate this possibility, we have studied the mecha- with the antagonist were analyzed by a nonlinear least-squares nism of fluctuation of pituitary muscarinic responsiveness by curve-fitting procedure using a generalized model for complex of a number of noncholi- ligand-receptor systems as described (1, 2). Computer analysis examining the effects endogenous indicated that binding ofantagonists was best explained by two nergic substrates, including steroids, on in vitro binding mech- total anisms of both agonist and antagonist to these muscarinic re- affinity sites of the receptor (1); 29% of the receptor pop- The effects ofthe substrates were in male and ulation in the proestrous stage is ofhigh affinity (63 + 5 fmol/ ceptors. analyzed mg ofprotein) with a Kd of 1.1 ± 0.03 nM and 74% is oflower proestrous female rats, as the sex dimorphism ofthe muscarinic 11.3 ± 0.1 is then affinity (155 + 10 fmol/mg ofprotein) with a Kd of characteristics apparent (1, 2). nM. The inhibition curves were obtained at 2 nM [3H]4NMPB using an average value ofthe two dissociation constants for the The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: [3H14NMPB, 3H-labeled N-methyl4-piperidyl benzilate. 5554 Downloaded by guest on October 2, 2021 Biochemistry: Sokolovsky et at Proc. NatL Acad. Sci. USA 78 (1981) 5555 analysis as discussed in ref. 1. Theoretical. competition curv'es a ,8estradiol concentration of 5 ng/ml, an increase in affinity were fitted to the experimental data points by using the non- was apparent at a concentration of0.5 ng/ml. linear least-squares regression computer program BMDPAR Similar effects on agonist binding in the female adenohy.- (November 1978 revision) as described (7, 8). (The program was pophysis could be induced by progesterone and cortisol (Table developed at the Health Science Computing. Facility of the 1). It is worth noting that these effects were already seen at University of California, Los Angeles; the facility is sponsored 25-50 ng/ml, which was within the physiological range ofthese by National Institutes of Health Special Research Resources steroids (10). Unlike P-estradiol, however, these two substrates Grant RR-3). Agonist. binding parameters were evaluated sta- had no detectable effect on male adenohypophysis (Table 1). tistically by using Student's t test. No effects of,estradiol; progesterone, or cortisol were ob- served in other brain regions investigated, such as the cortex, RESULTS medulla-pons, median, and posterior hypothalamus (not shown). A large number ofendogenous substrates were tested for pos- Interestingly, in the preoptic area offemales but not of males, sible effects on the cholinergic muscarinic system in the ade- although neither (3estradiol. nor cortisol had any effect, pro- nohypophysis. Competition experiments were carried out in gesterone influenced oxotermorine binding in a manner similar the presence and absence ofthe following: 0.1 mM cyclic AMP, to its effect on the pituitary (Table 2). Here again, a significant 0.1 mM cyclic GMP, luteinizing hormone-releasing hormone decrease in the population. ofhigh-affinity sites (a = 23 ± 3% at 10 ng/ml, thyrotropin-releasing hormone at 10 ng/ml, 10 AM vs. a = 67 + 7% in control proestrous females) was accom- D-Ala2-Met5-enkephalin, 0.1 mM a-aminobutyric acid, choles- panied by an increase in the high-affinity dissociation constant terol at 50 ng/ml, cortisol at 50 ng/ml, f-estradiol at 0.1-50 ng/ (KH = 4.5 + 0.6 nM vs. KH = 27.2 + 0.4 nM in control.proes- ml, and progesterone at 25 ng/ml. The binding characteristics trous females). of the highly specific muscarinic antagonist [ H]4NMPB re- Cholesterol had no observable effect on muscarinic agonist mained unchanged in the presence and absence ofeach ofthese parameters in either the pituitary or the preoptic area. agents (data not shown). Agonist binding characteristics, on the other hand, did show changes, but only in the presence of,the DISCUSSION steroid substrates. Fig. 1A shows Scatchard plots ofcompetition The effect of steroids on agonist binding, as shown in. compe- experiments between 2 nM [3H]4NMPB and various concen- tition binding studies using oxotremorine and carbamoylcholine trations ofthe agonist oxotremorine in the presence and absence (data not shown) as muscarinic agonists, indicates tissue spec- of 70 nM (50 ng/ml) P-estradiol; a marked effect on the-binding ificity-i.e., the effect is seen only in the pituitary and preoptic parameters in the presence of f-estradiol can be seen for both areas and not in the median or posterior hypothalamus, cortex, female and male adenohypophyses (Table 1). In Fig. 1B, on.the or medulla-pons. Furthermore, the effect, which is character- other hand, in which antagonist binding is shown, no such effect ized by a marked decrease in the proportion of agonist high- is observed. Assuming the existence of two binding states for affinity sites accompanied by an increase in their affinity, is in- agonist binding, as described (1, 9), analysis ofthe curves in Fig. duced by substrates having major physiological roles in these 1A indicates that (i) the proportion ofhigh-affinity binding sites regions. One can therefore justifiably assume that there is an decreased from 85% in the control to 20% in the presence of interaction between the muscarinic system and the ovarian hor- ,3-estradiol; (ii) the high dissociation constant (KH) decreased to mones P-estradiol and progesterone, -which are implicated in 20 nM in the presence of (3-estradiol, as compared with the atleast two levels ofthe gonadotropin secretion feedback system control value of 1.3 AM; and (iii) in low-affinity sites, KL, no- (10-12)-namely, the hypothalamus and the anterior pituitary significant differences were found in the absence (control) or (12, 13). This supports the hypothesis that muscarinic receptors presence of 6-estradiol. In other words, the presence of P-es- are physiologically involved in the hypothalamus-pituitary-ovary tradiol resulted in a significant decrease in the proportion of axis, most probably as modulators.. Further support comes from high-affinity sites (a) and, at the same time, in a two order of the observation that cholesterol, which-is a structural precursor magnitude decrease in. the dissociation constant of these sites. ofprogesterone, , and estrogens, but cannot be con- The effect of 3-estradiol on the proportion ofmuscarinic ag- verted into these derivatives in either the pituitary or the hy- onist binding.sites was dose-dependent, as shown in Fig. 2 A pothalamus, indeed has no effect on the binding ofeither mus- and B. Although the change in proportion of high- and low-af- carinic agonists or antagonists. finity binding site populations reached its maximum effect at It has been suggested that the stimulation of the synthesis

9.

m

M: 0

50 100 50 100 150 [3HJ4NMPB displaced, % [3H]4NMPB bound, fmol/mg of protein FIG. 1. Scatchard analysis of displacement of [5H]4NMPa3P by various concentrations of oxotremorine (A) and binding of [3H]4NMPB (B) in adenohypophyseal homogenates of proestrous female rats in the absence (o) and presence (0) of 70 nM P-estradiol. Downloaded by guest on October 2, 2021 5556 Biochemistry: Sokolovsky et aL Proc. Nad Acad. Sci. USA 78 (1981)

Table 1. Characteristics of oxotremorine binding in the adenohypophysis in the presence of steroids Control Progesterone at 25 ng/ml Cortisol at 50 ng/ml O-Estradiol at 50 ng/ml Male Proestrous Male Proestrous Male Proestrous Male Proestrous a, % 42 ± 4 85 ± 8 45 ± 3 17 ± 2* 42 ± 2 17 ± 1* 19 ± lt 22 ± 2* KH,nM 115 ± 20 1370 ± 30 130 ± 15 39 ± 2* 127 ±11 60 ± 5* 16 ± 3t 21 ± 1* KL,ILM 7.0± 0.8 8.3± 0.1 5.7± 0.8 9.5 ± 1.1 7.5 0.5 11.6±2 6±0.7 6.2± 1 Results are average ± SD for at least three experiments and were calculated by the nonlinear least-squares regression procedure for a two-site model. a, Proportion of high-affinity binding sites; KH and KL, Kds for binding of oxotremorine to high- and low-affinity sites, respectively. * P > 0.001 vs. control females at proestrous. tP > 0.005 vs. control males. and secretion ofpituitary hormones might be mediated through induced interconversion of agonist binding states (17-19), as cyclic nucleotides (14). In investigating this possibility, how- discussed below, prompts us to propose the putative model ever, we were unable to detect any effect on the binding prop- outlined in Fig. 3. We make the assumption that the muscarinic erties ofthe muscarinic receptors in the presence ofcyclic AMP receptor (M) and a membranal component (Y) are modulated or cyclic GMP. via a common unit (X) that regulates both M and Y. This ar- These data also indicate substrate specificity. The role ofpro- rangement-i.e., M ... X ... Y-is typical of certain tissues, gesterones in the pituitary and hypothalamus is not entirely while other tissues may be characterized by the presence of M clear. It has been assumed to exert its effect at the hypothalamic coupled to X (M-X), free uncoupled M, or free uncoupled Y. level; our finding that only progesterone has an effect in the We further assume that the regulatory unit X is essential for the preoptic area seems to suggest that it might indeed operate at activation of the M system and, although it is not essential for this level through muscarinic interaction. In addition, our ob- the specific functioning ofthe Y system, coupling ofY to X (Y-X) servation that progesterone causes an alteration in the musca- will cause perturbation of this system. For example, if one of rinic binding parameters in the adenohypophysis is in agree- the specific actions ofY concerns an internalization process (as ment with recent work showing a direct action ofprogesterone indicated by the arrow at the upper left of Fig. 3), this will be on the rat anterior pituitary involving the control offollicle-stim- inhibited by coupling of Y to X. Thus, the model does not spec- ulating hormone release (15, 16). We suggest, on the basis of ify whether the membrane components are freely mobile or results of endocrinic manipulations that, at the level of the pi- organized in domains. tuitary gland, estrogens exert at least one oftheir feedback ac- The identity ofthe components is inferred from biochemical tions (negative or positive or both) on the secretion of sex hor- observations. In the muscarinic system, it is assumed that bind- mones through the muscarinic system (unpublished results). ing ofantagonists and ofagonists differ in their response to the The results described here in regard to the effect of 3estradiol M-X coupling. Thus, antagonists will bind similarly to both M support this suggestion. and M-X, while agonist binding will be characterized as low- As steroids are by definition noncholinergic agents and as affinity state for the former and high-affinity state for the latter. they do not interfere with antagonist binding, it follows that A candidate for X is, for example, the GTP regulatory unit, their effect was not exerted through the muscarinic recognition which need notbe the same as that coupled to adenylate cyclase, site. In a functional sense, receptors are to be regarded as com- while candidates for Y are other receptor systems, such as the posed ofat least two components: a recognition site and an am- receptors, or an ionic channel. It is noteworthy that plification site. These two functions may be incorporated in the recent reports have described the existence ofmembranal high- same macromolecule or be carried out by quite separate mac- affinity binding sites for sex steroids in several preparations romolecules; in either case, linkage must exist between the two (20-22) not to be confused with the cytosol . functions. The results reported here regarding both substrate Evidence for the proposed model is so far circumstantial but specificity and target specificity lend support to the second pos- can be exemplified and summarized for the case of interaction sibility-i.e., that a second macromolecule is involved. This, between the muscarinic and the steroid sex hormone systems taken together with (i) our recent findings on the mode ofaction as follows. of the drug clomiphene on the muscarinic recep- (i) In the presence of muscarinic agonist, the coupling is ba- tors (unpublished data) and (i) recent observations in regard to sically between M and X. Under such conditions, the Y mole-

100 A B 1-6

50-

10-8 [I]t j FIG. 2. Quantitative representation of binding parameters of oxotermorine to muscarinic sites in the female rat adeno- 10-9 hypophysis at the proestrous stage in the Control 0.5 2.5 5 50 Control 0.5 2.5 5.0 50 presence of various concentrations of P- estradiol. a, Proportion of high-afinity sites; KH, high-affinity dissociation P-Estradiol, ng/ml constant. Downloaded by guest on October 2, 2021 Biochemistry: Sokolovsky et aL Proc. Natd Acad. Sci. USA 78 (1981) 5557

Table 2. Characteristics of oxotremorine binding in the preoptic area in the presence of steroids Control Progesterone at 25 ng/ml -Estradiol at 50 ng/ml Male Proestrous Male Proestrous Male Proestrous a, 96 38 ± 1 66 ± 7 37 ± 4 23 ± 3* 30 ± 2 59 ± 4 KH, nM 3.7 ± 2 27.2 ± 0.4 4.05 ± 0.15 4.5 ± 0.6* 3.9 ± 0.2 21.1 ± 1 KL, AM 1.2 ± 0.07 1.7 ± 0.04 1.8 ± 0.2 0.42 ± 0.3 1.7 ± 0.1 1.2 ± 0.02 Results are average ± SD. a, KH, and KL are as described in the legend to Table 1. *P > 0.005 vs. control females at proestrous.

cule becomes uncoupled and, in the presence ofits endogenous (iv) Binding of muscarinic antagonist to its receptor is in- substrate (for example, 1estradiol), the binding might be fol- dependent of X or Y, as has been observed, with GTP, (3-es- lowed by internalization and release of the estrogen into the tradiol, progesterone, etc. cytosol. It follows that, in the presence of muscarinic agonist, (v) Now, let us assume the existence ofa ligand that can bind (3-estradiol activity should increase, and experiments carried to X and that this unit can become coupled to either M or Y or out in the presence of oxotremorine do indeed indicate an in- to both. Thus, in atissue containing these elements, this M-X-Y crease in luteinizing hormone release (23, 24). The presence of coupling will modify some of the properties of both M and Y; muscarinic antagonist should therefore result in an opposite in a tissue in which only M or Y exists, the binding properties effect; reports in the literature indicate that antimuscarinic ofligands to their respective receptor (M or Y)will be modified. drugs such as atropine, acting at -the hypothalamic-pituitary Acandidate foran X-coupling ligand mightbe the antiestrogenic level, inhibit ovulation (24, 25). nonsteroidal drug clomiphene, which affects the muscarinic (ii) According to the scheme, progesterone or 3-estradiol in- system (unpublished data); this effect was exerted by more than teraction with Y will cause coupling between Y and X, thereby one clomiphene molecule and in a cooperative manner. The causing dissociation of M from the three-component complex. most likely explanation is that the first clomiphene molecule In such a case-i.e., uncoupling ofan M-X complex we would binds to X, leading to conformational changes such that, when expect that M would exhibit the low-affinity agonist state while the second clomiphene molecule enters the 4NMPB zone of retaining its inherent antagonistic character. Indeed, in tissue binding (Fig. 3), the complex formed has a much higher dis- or cells in which Y is the estrogen receptor (e.g., in the pitui- sociation constant, koff, and dissociation of [3H]4NMPB is facil- tary), the presence of E2 results in interconversion ofthe mus- itated (unpublished data). Furthermore, unlike the steroid sex carinic agonist binding into low affinity state. The fact that there hormones, which exert their effects via the Y component, clom- is no such effect in the preoptic area or the medulla-pons in- iphene is unlikely to discriminate between the brain regions and dicates that Y in these brain regions either does not exist or is will affect all muscarinic systems containing M-X, as was ob- specific for another substrate (e.g., progesterone); the effect served. Similarly, binding of 17(3-estradiol or progesterone to caused by this ligand on the muscarinic system in the preoptic the membranal component Y should beaffected by the presence area and pituitary is indicative ofthe presence of"Y-progester- of clomiphene. Indeed, an acceleration occurs in dissociation one" in these regions, while in the medulla-pons Y probably of the receptor-ligand complex (25). does not exist, at least for any ofthe steroids investigated so far. This model, which offers an explanation for the effects ofag- (iii) In those cases where X is sensitive to, e.g., guanyl nu- onists or antagonists specific for one system on those of other cleotides or temperature their effect is on the M-X (or Y-X) systems, proposes a general modus operandi for physiological arrangement, which is characterized as stated above by agonist dialogue among different systems. Thus, it could account for high-affinity state. Uncoupling of this arrangement, for exam- the observed interaction between, for example, the sex hor- ple, by GTP will result in interconversion to a low-affinity state mones and the muscarinic receptors, as well as the interplay as recently reported by us (17-19). Consequently, the effect of between cholinergic and other neurotransmitter systems. This transition metal ions is coupling of M and X (19). hypothesis, taken together with the observations ofmuscarinic antagonist-receptor complex isomerization, interconversion between high and low agonist binding states, and supersensi- tivity and subsensitivity reviewed recently (26), should help to 20 GM M, X-Y-0 V, . -X design further experiments aimed at elucidating the mode of action of the muscarinic complex in the nervous system. 0 Enlightening discussions with Drs. Nava Zisapel, Aliza Eshkol, and Daniel Michaelson are gratefully acknowledged. Mrs. Ronit Galron provided excellent technical assistance. This workwas supported in part WM - x V-Y - X VY - -M----X VY by the Recanati Foundation for Medical Research (Israel).

I , .. 1 Et o 1. Avissar, S., Egozi, Y. & Sokolovsky, M. (1981) Neuroendocrinol- EM-X -Y ,M - X-Y gs M- ogy 32, 303-309. 2. Avissar, S., Egozi, Y. & Sokolovsky, M. (1981) Neuroendocrinol- C C C ogy 32, 295-302. 3. Kalir, A., Maayani, S., Rehavi, M., Elkavetz, R., Pri-Bar, I., FIG. 3. Model depicting the putative relationship between the Buchman, 0. & Sokolovsky, M. (1978) Eur. J. Med. Chem. 13, muscarinic system and other receptor systems. o and o, Y agonist and 17-24. antagonist, respectively; * and *, muscarinic agonist and antagonist, 4. DeGroot, J. (1972) The Rat Forebrain in Stereotaxic Coordinates respectively; M, muscarinic receptor; Y, membranal component (e.g., (North Holland, Amsterdam). receptor for sex hormones); X, protein molecule that regulates both M 5. Kloog, Y., Egozi, Y. & Sokolovsky, M. (1979) Molt PharmacoL 15, and Y; C, ligand that binds to X and simultaneously or thereafter to 545-558. M or Y (e.g., clomiphene citrate). 6. Kloog, Y. & Sokolovsky, M. (1977) Brain Res. 134, 167-172. Downloaded by guest on October 2, 2021 5558 Biochemistry: Sokolovsky et aL Proc. Natd Acad. Sci. USA 78 (1981)

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