Proc. NatL Acad. Sci. USA Vol. 78, No. 9, pp. 5554-5558, September 1981 Biochemistry Molecular regulation of receptors: Interaction of (3-estradiol and progesterone with the muscarinic system (cholinergic receptor/sex hormone/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 estrogens 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.