The effects of ovarian hormones on beta-adrenergic and muscarinic receptors in rat heart

L i f e S c i e n c e s , Vol. 42, pp. 2307-2314 P r i n t e d i n t h e U.S.A.

Pergamon P r e s s

THE EFFECTSOF OVARIAN HORMONESON BETA-ADRENERGIC AND MUSCARINIC RECEPTORS IN RAT HEART Borpit Klangkalya and Albert Chan Department of Pharmacologyand Toxicology University of Mississippi Medical Center Jackson, Mississippi 39216 (Received in final form April 4, 1988)

Summar~ The in vitro and in vioo effects of estrogen and progesterone on muscarinic and B-adrenergic receptors of cardiac tissue were studied in ovariectomized (OVX) rats. The binding assay for muscarinic receptors was performed under a nonequilibrium condition; whereas the binding assay for B-adrenergic receptors, under an equilibrium condition. Estrogeniccompounds and progesterone were found to have no effect on the binding of the radioligand, [SH]-dihydroalprenolol, to B-adrenergic receptors in vitro. However, progestins but not estrogenic compounds inhibited the binding of the radioligand, [SH]-quinuclidinyl benzilate, to muscarinic receptors in v i t r o , with progesterone as the most potent inhibitor (ICso = 37 pM, apparent Ki = 13 pM). Progesterone was found to decrease the apparent a f f i n i t y of muscarinic receptors for [SH](-)QNB in vitro. Daily treatment of OVX rats with estradiol benzoate (4 ~g) or progesterone (2.5 mg) for 4 days had no effect on the muscarinic or B-adrenergic receptors with respect to the binding a f f i n i t y and receptor density. However, administrations of these hormonestogether for 4 days caused an increase in the receptor density of muscarinic receptors without a significant effect on their apparent binding a f f i nity; also these hormones induced a decrease in the binding a f f i nity and an increase in the receptor density of B-adrenergic receptors. The results of this study demonstrate that progestins are capable of interacting with the cardiac muscarinic receptors in vitro, and indicate that estrogen and progesterone have a synergistic effect to increase the receptor densities of muscarinic and B-adrenergic receptors as well as to cause a decrease in the binding a f f i n i t y of B-adrenergic receptors in vivo. Many reports have been accumulated which suggest that ovarian hormones have effects in altering muscarinic and B-adrenergic receptors in a variety of tissues. Administration of ovarian hormones has been shownto modify B-adrenergic receptors in the brain ( I - 3 ) , uterus (4,5), bladder (6,7), and lung (8) as well as muscarinic receptors in the brain (9) and bladder (7). On the basis of these previous findings and the fact that B-adrenergic and muscarinic receptors are also present in the heart tissue for mediating the response to autonomic a c t i v i t y , the possibility exists that ovarian hormones might have similar effects on muscarinic and B-adrenergic receptors there. There are two lines of evidence to support this idea. First, estrogen and 0024-3205/88 $3.00 + .00

Copyright (c) 1988 Pergamon Press plc

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progesterone have been shown to be capable of blocking the extraneuronal uptake of catecholamines in the isolated preparation of rat heart with estradiol being nineteen times more potent than progesterone (10). Such an attenuation in the release of catecholamines might have a bearing on the characteristics of 8-adrenergic receptors. Second,myocardium has also been shown to concentrate estradiol (11) and progesterone (12), both of which are well known to have an effect on protein synthesis via a genomic action (13). Thus, the present study was undertaken to study the in vitro and in uivo effects of estrogen and progestins on B-adrenergic and muscarinic receptors of the cardiac tissue. Methods L-[Benzilic-4,4'-3H]-Quinuclidinyl benzilate ([3H](-)QNB, 30.1Ci/mmol) and Levo-[ring, propyl-3H(N)]-Dihydroalprenolol hydrochloride ([3H]DHA, 96.1 Ci/mmol) were obtained from New England Nuclear (Boston, MA). Oxotremorine, atropine, d,Z-propranolol, and all steroids used were purchased from Sigma (St. Louis, MO). Female Sprague-Dawley rats (Charles River Lab, Wilmington, MA) weighing 300-350 g were housed in a temperature-controlled room with a light/dark cycle of 12 hr light and 12 hr dark (dark period starting at 11:00 AM). All animals were fed Purina Chowand water ad libitum. They were ovariectomized (OVX) at least three weeks before sacrifice or receiving hormonal treatment. They were then divided into four groups to receive each of the following daily treatments for 4 days: (1) 4 ~g estradiol benzoate, (2) 2.5 mg progesterone, (3) a mixture of 4 vg estradiol benzoate and 2.5 mg progesterone, and (4) corn oil. All hormones were dissolved in corn oil for subcutaneous injections. The animals were sacrificed 24 hr after the last injection by decapitation and the hearts were removed quickly for the preparation of cardiac membranes. Cardiac membranes were prepared according to the procedure of Baker and Potter (14). The final membrane pellets were resuspended either in 60 mM sodium potassium phosphate buffer (pH 7.4) for the binding assay for muscarinic receptors, or in 50 mM HEPESbuffer (pH 8.0) for the binding assay for B-adrenergic receptors. We have previously found from in vitro studies that progesterone caused a 50% decrease in the association rate constant of [3H](-)QNB with muscarinic receptors of hypothalamic membranes without any effect on the dissociation rate constant (15). Similar findings were also obtained in our preliminary studies with cardiac membranes. Therefore, the [3H](-)QNB binding assay was performed under a nonequilibrium condition for the experimental convenience of a shorter incubation time and a greater sensitivity for detecting the effect of progesterone (16). However, under this condition for a radioligand such as [3H](-)QNB with a negligible dissociation rate at 25 °C (17), a change in the apparent dissociation constant (Kd, app) only reflects a change in the association rate constant (18). The binding assay was carried out according to the procedure of Yamamura and Snyder (17) with minor modifications. Cardiac membranes (about 0.1 mg protein) were incubated in quadruplicate with 0.01 to 2 nM of [~H](-)QNB (7-9 different concentrations) in 60 mM sodium potassium phosphate buffer in a final volume of 0.25 ml. For determining the nonspecific binding, 100 vM oxotremorine was included in the incubation mixture. The incubation was carried out at 25 °C for 60 min and terminated by adding 4 ml of ice-cold 60 mM sodium potassium phosphate buffer (pH 7.4), filtering through a Whatman GF/B glass-fiber f i l t e r , and washing the f i l t e r twice with 4 ml of the same buffer. The radioactivity of the f i l t e r s

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was determined by s c i n t i l l a t i o n spectrometry. The binding assay for B-adrenergic receptors was performed under an equilibrium condition and according to the procedures of Cervoni and co-workers (19) with minor modifications. Cardiac membranes (about 0.2 mg protein) were incubated in quadruplicate with 1 to 100 nM of [3H]DHA (7-9 different concentrations) in 50 mM HEPESbuffer (pH 8.0) containing 4 mMMgCl2 in a final volume of 0.25 ml for 20 min at 25 °C. For determining the nonspecific binding, 20 pM of d,Z-propranolol was added to the incubation mixture. The termination of the incubation was performed in the sameway as that used for the [3H](-)QNB binding assay with the exception that 25 mM HEPES buffer (pH 8.0) was used instead of sodium potassium phosphate buffer. For in v i t r o testing, the steroids were dissolved in 50% propanediol and then added to the incubation medium. The final concentration of propanediol in the incubation medium was adjusted to 4%. Such a concentration was found to cause less than 10% inhibition on [3H](-)QNB binding or [3H]DHA binding. Therefore, propanediol was always added to the incubation medium as a control for the steroid compounds to be tested. Protein content in each membrane preparation was assayed by the method of Lowry et al. (20) using bovine serum albumin as a standard. All data analyses were performed with a microcomputer. The binding constants, the dissociation constant (Kd) or apparent dissociation constant (Kd, app), and the receptor density (Bmx), were obtained by analyzing the binding data with Scatchard plots which were constructed with linear regression analysis. In the event that the correlation coefficient was not equal or less than 0.95, the data were discarded. The Lineweaver-Burke plots for the kinetic analysis of binding data were constructed with a graphical method. For all statistical tests as indicated, the values for Kd, Kd, app and the apparent Michaelis-Menten constant (Km,app) were assumedto have a log-normal distribution and those for B~x were assumed to have a normal distribution. Results In Y~tro Effects

For all the in v i t r o studies, the cardiac membraneswere prepared from untreated OVX rats. As shown in table I, all progestins at a concentration of 100 pM significantly inhibited [3H](-)QNB binding. However, estrogenic compounds such as S-estradiol, 17~-estradiol, ethynylestradiol, d i e t h y l s t i l bestrol, estradiol-17B-acetate, S-estradiol diacetate, S-estradiol-17B-hemisuccinate, and estrone were found to be devoid of this effect (data not shown). As shown in figure IA, the inhibitory effect of the two most active progestins, progesterone and 16a-methylprogesterone, was found to be concentration-dependent. An estimation of the values of their apparent Ki (Kl,app) with the equation of Cheng and Prusoff (21) yielded 13 pM (ICso = 37 pM) and 40 pM (ICso = 113 pM), respectively. Atropine, a knownmuscarinic antagon i s t , was found to decrease [aH](-)QNB binding with a Ki, app of 0.82 nM (ICso = 23. riM). I n regard to the B-adrenergic receptors (fig. IB), (-)-isoproterenol decreased [~H]DHA binding with an ICso value of 0.14 pM and a Ki value of 54 nM; whereas estrogen and progesterone were found to have no effect (fig. IB). Similar findings on S-adrenergic receptors were also observed with those progestins tested in table I and other estrogenic compounds mentioned above (data not shown). In both studies as shown in figures 1A and IB, no attempt was made to test the steroids at a concentration greater than 200 pM, since they become p a r t i a l l y insoluble at this higher concentration. The inhibition of [3H](-)QNB binding did not require preincubation as

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TABLE I The In Vitro Effects of Progestins on Specific Binding of [3H](-)QNB to Cardiac Membranes Prepared from Ovariectomized Rats Addition

Specific binding (% of control) ontro] (4% propanedio]) I00.0 + 2.8 Progesterone 18.0 -+ 0.7* 16a-Methylprogesterone 51.7 ± 3.5* Norethindrone 62.3 ± 6.4* Norethynodrel 73.3 ± 1.5" D(-)norgestrel 80.0 ± 2.6* The data are presented as mean ± S.E. of six determinations combined from two separate experiments. For muscarinic receptor binding assay, the concentration of [3H](-)QNB used in the incubation medium was 0.2 nM and the specific [3H](-)QNB binding for the control group was 140 ± 4 fmol/mg protein. * Indicates P < 0.05 when compared to the control group as determined by Student's t test.

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FIG. 1. (A) Displacement of specific [3H](-)QNB binding by atropine ( 0 ), progesterone ( ~ ), and 16a-methylprogesterone ( o ). The concentration of [SH](-)QNB used in the incubation medium was 0.2 nM and the specific [3H](-)QNB binding for the control group was 122 ± 6 fmol/mg protein. (B) Displacement of specific [3H]DHA binding by (-)-isoproterenol, estrogen, and progesterone. The concentration of [3H](-)DHA used in the incubation medium was 15 nM and the specific [3H](-)DHA binding for the control group was 104 ± 8 fmol/mg protein. In both (A) and (B), the data are presented with standard errors of respective means of 4 separate experiments. demonstrated in the following experiment. When cardiac membranes were preincubated with 40 ~M progesterone for 15 min and [3H](-)QNB binding was subsequently initiated by the addition of [3H](-)QNB, the extent of inhibition by progesterone was found to be the same as that observed when i t

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Ovarian Hormones and Cardiac Receptors

(B)

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FIG. 2. (A) A typical Scatchard plot of specific [SH](-)QNB binding to cardiac membranes in the presence of 40 pM progesterone ( o ) or 4% propanediol as a control ( • ). Eachdata point represents the meanof 4 determinations. (B) A typical Lineweaver-Burke plot on the inhibition of specific [aH](-)QNB binding by 40 pM progesterone. The membranes were incubated in the presence of 4% propanediol as a control ( • ) or 40 pM progesterone ( o ). The data are presented with standard errors of 4 determinations of corresponding means. For both (A) or (B), similar results were obtained with three other separate experiments. A summary of the data is presented in table I I . TABLE I I The In V ¢ ~ Effect of Progesterone on the Binding and Kinetic Constants of [3H](-)QNB Binding to Cardiac Membranes from Rats Addition Control

Scatchard Plot Kd, app Bmx (pM) (fmol/mgprotein) IZ5 ± 6 135 ± 7

Lineweaver-BUrke Plot Ks, app Bmx 6±(PM)7 (fmol/mg protein) 11 137 + 3

40 ~M Progesterone 230 ± 8* 129 ± 6 244 ± g* 136 ± 3 presentedwith standard errors of the corresponding means of 4 separate experiments. * Indicates P < 0.05 when compared to the corresponding control group in the same column as determined by a two-tailed ¢ test for matched pairs.

The data are

was tested without the preincubation (data not shown). Furthermore, the inhibition was found to be complete]~ reversible in the following experiment. After cardiac membranes were preincubatedwith 40 pM progesterone in I0 mM sodium potassium phosphate buffer (pH 7.4) containing 4% propanediol for 30 min and then washed (polytronized for 10 sec and centrifuged at 48,000 g for 10 min) twice w i t h the same buffer, they exhibited the sameextent of bindin9 as those membranes preincubated without progesterone in the subsequent [3H](-)QNB binding assay (data not shown).

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TABLE I l l The Effects of Treatment of Ovariectomized Rats with Ovarian Hormones on Cardiac [3H](-)QNB and [3H]DHA Binding Sites Treatment [~H](-)QNB binding OVX OVX P OVX E OVX EP

N 9 6 9 6

63 64 74 156

± ± ± ±

11 13 18 48

Kd (nM)

210 168 146 371

± 12 ± 6 ± 15 ± 60*

Bmax (fmol/m 9 protein)

[3H]DHA binding OVX 7 10 ± 2 121 ± 14 OVX P 7 10 ± I 103 ± 7 OVX E 7 12 ± 3 142 ± 18 OVX EP 6 58 ± 7* 260 ± 20* OVX = ovariectomized, P = progesterone, E = estradio] benzoate, and N = number of separate experiments, each of which consists of a single animal. The data are presented with standard errors of respective means. * Indicates significant difference from the other three groups with B < 0.05 as determined by the analysis of variance followed by the Newman-Keuls multiple comparisons test.

Saturation studies on [3H](-)QNB binding were performed on cardiac membranes prepared from OVX rats. The membranes were incubated with varying concentrations of [3H](-)QNB in the presence of 40 ~M progesterone and 4% propanediol, or 4% propanediol alone. Scatchard Analysis of the binding data ( f i g . 2A, table I I ) indicated that progesterone caused a significant increase in the apparent dissociation constant without a significant effect on the receptor density. Further analysis of the data with Lineweaver-Burke plots revealed that the inhibition of [3H](-)QNB binding by progesterone was competitive in nature, since progesterone caused a significant increase in the apparent Michaelis-Menten constant without a change in the maximal binding capacity (fig 2B, table I I ) . I n Vi~o Effects

Table I I I summarizes the effects of ¢n oivo treatment of OVX rats with estrogen and progesterone. Hormonal treatment of OVX animals for 4 days with estrogen or progesterone, or both had no significant effect on the apparent dissociation constant of cardiac muscarinic receptors for [3H](-)QNB. Even though concomitant administrations of estrogen and progesterone appeared to have a slight effect in decreasing the binding a f f i n i t y of muscarinic receptors, the decrease was not s t a t i s t i c a l l y significant when the data were s t a t i s t i c a l l y tested with the assumption that the values of Kd, app had a log-normal distribution. In regard to the receptor density of muscarinic receptors, simultaneous administrations of estrogen and progesterone caused a significant increase; whereas treatment with either hormone alone had no effect. In the case of [3H]DHA binding sites, treatment with estrogen and progesterone together caused a significant decrease in the binding a f f i n i t y of these binding sites as well as a significant increase in the receptor density. However, treatment with either hormone alone had no effect on both of these binding constants.

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Discussion As shown in fig. IB, estrogen or progesterone when tested ~n v~tmo had no effect on the binding of ESH]DHA to B-adrenergic receptors; whereas ( - ) - i s o proterenol, a known B-adrenergic agonist, competed with [aH]DHA for the B-receptors with a Kt value of 54 nM, which is comparable in term of order of magnitude to the previously reported value obtained from brain tissue (22). The lack of ~n y e S , effect of these steroid hormones on [3H]DHA binding in the heart tissue as observed in t h i s study is similar to the previous results obtained from the brain and p i t u i t a r y tissues (23). In regard to the finding that progesterone was capable of d i r e c t l y i n h i b i t i n g the [3H](-)QNB binding, i t should be noted that progesterone possesses no functional group resembling the quarternary ammonium group in the acetylcholine molecule nor the t e r t i a r y amine group in the quinuclidinyl ring of QNB molecule, yet progesterone was able to i n h i b i t the E3H](-)QNB binding in a rapid and reversible manner. When the values of apparent Kt of progesterone and 16a-methylprogesterone are compared to that of atropine, atropine is 16 times more potent than progesterone and 49 times more potent than 16a-methylprogesterone in displacing [3H](-)QNB from i t s binding sites. The ICso value (2.3 nM) of atropine as estimated from figure 1A is comparable to the previously reported value obtained from membranes of rat brain with a similar assay condition (17). As revealed by Lineweaver-Burke plot (fig. 2B, table I I ) , the inhibition by progesterone on E3H]QNB binding appears to be competitive. However, the possibility that progesterone might have an allosteric effect in altering the conformation of the receptor cannot be excluded, since a complete displacement of E3H]QNB from i t s binding sites cannot be experimentally demonstrated due to the partial insolubilit~ of the steroid at concentrations greater than 200 pM (fig. IA). Sincethe [3H](-)QNB binding experiments in this study were performed under a nonequilibrium condition, the increase in the apparent dissociation constant ( f i g . 2A) was simply a reflection of a decrease in the association rate constant (18). This observation is in agreement with our preliminary results that progesterone caused a 50% decrease in the association rate constant but had no effect on the dissociation rate constant (data not shown). Even though progesterone had an ~n pitro effect of inhibiting [3H](-)QNB binding as noted above, i t had no (n v~vo effect on the binding constants of ESH](-)QNB or [3H]DHA binding sites when i t was administered alone to OVX animals for four days. A similar lack of Ca v~vo effect was also observed with estrogen. However,administrations of estrogen and progesterone together to OVX rats resulted in a different profile of effects on muscarinic and B-adrenergic receptors. For the [3H](-)QNB binding sites, the receptor density was increased; whereas, for the [3H]DHA binding sites, both the dissociation constant and the receptor density were increased. The values of Kd for B-adrenergic receptors determined from untreated, estrogen-treated, and progesterone-treated OVX rats are similar to the previously reported values obtained from rat hearts (19, 24). The precise mechanism responsible for the synergistic effect of estrogen and progesterone on these two different receptors is not known and is probably a result of multiple effects. I t is possible that, under the influence of estrogen ~n ~vo, the direct effect of progesterone on cardiac muscarinic receptors might be expressed. Alternat i v e l y , estrogen and progesterone might have a synergistic action on the synthesis of muscarinic and B-adrenergic receptors since these steroids are known to have an effect on protein synthesis (13). In view of the finding that estrogen or progesterone was found to have no Cn vC~ro effect on cardiac B.adrenergic receptors, the alteration of these receptors by these hormones administered together might be due to their indirect effect in altering adrenergic a c t i v i t y via a complex cardiovascular regulation. Such an alteration might result from the possible synergistic effect of estrogen and progeste-

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rone on cardiac muscarinic receptors as noted above. Further studies are required to delineate the precise mechanism involved in the synergistic effects of these hormones on the cardiac tissue. In summary, our present findings demonstrate that progestins but not estrogen-related compounds are able to d i r e c t l y interact with cardiac muscar i n i c receptors ~n v l t r o , and indicate that concomitant administrations of estrogen and progesterone to ovariectomized rats for 4 days result in an increase in the receptor density of cardiac muscarinic receptors as well as an increase in the dissociation constant and the receptor density of cardiac B-adrenergic receptors. The pharmacologic significance of these steroidal effects with respect to cardiac function remains yet to be elucidated. Acknowledgements The authors wish to thank S. Winstead and C.L. Harrell for t hei r technical assistance and Jeri D. Martin for her help in the preparation of the manuscript. This work was supported by a grant (MS84G4) awarded to A. Chart from American Heart Association, Mississippi A f f i l i a t e . References 1. M. WILKINSON, H. HERDON,M. PEARCEand C. WISON, Brain Res. 168 652-655 (1979). 2. A. BIEGON, A. RECHES, L. SNYDERand B.S. MCEWEN, Life Sci. 32 2015-2021 (1983). 3. A. MAGGI, I. ZUCCHI and J. PEREZ, Pharmacol. Res. Commun. 17 283-291

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(1985). J.M. ROBERTS, P.A. INSEL and A GOLDFIEN, Mol. Pharmacol. 20 52-58 (1981). T. KANO, Jap. J. Pharmacol. 32 535-549 (1982). E. SHAPIRO, J. Urol. 135 108~1087 (1986). R.M. LEVIN, F.S. SHOFEIT-andA.J. WEIN, J. Pharmacol. Exp. Ther. 215 614-618 (1980). A.H. MOAWAD, L.P. RIVER and S.J. KILPATRICK, Am J. Obstet. Gynecol. 144 608-613 (1982). T.C. RAINBOW, V. DEGROFF, V.N. LUINE and B.S. MCEWEN, Brain Res. 198 239-243 (1980). L.L. IVERSEN and P.J. SALT, Br. J. Pharmacol. 40 528-530 (1970) W.E. STUMPF, M. SARRand G. AUMULLER, ScienceT~6 320-321 (1977) R.j. FALK and W.C. BARDIN, Endocrinology8.6 10~'~JT-1063 (1970). B.W. O'MALLEY, W.T. SCHRADERand M.-J. Tsal, Adv. Exp. Med. Biol. 196 I-I0 (1986) S.P. BAKER and L.T. POTTER, Br. J. Pharmaco]. 68 65-70 (1980). B. KLANGKALYAand A. CHAN, Neuroendocrinology,~n press. H.J. MOTULSKYand L.C. MAHAN, Mol. Pharmacol. 25 1-9 (1984). H.I. YAMAMURAand S.H. SNYDER, Proc. Natl. Aca~7. Sci. USA. 71 1725-1728 (1974). P. ARANYI, Biochim. Biophys. Acta 584 529-537 (1979) P. CERVONI, H. HERZLINGER, F.M. LA~[-'~nd T. TANIKELLA, Br. J. Pharmaco]. 74 517-523 (1981). ~7.H. LOWRY, N.J. ROSEBROUGH,A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). ~CHENG and W.H. PRUSOFF, Biochem. Pharmacol. 22 3099-3108 (1973). D.B. Bylund, Brain Res. 152 391-395 (1978). C.M. PADEN, B.S. MCEWEN,~FISHMAN, L. SNYDERand V. DEGROFF, J. Neurochem. 39 512-520 (1982). L.T. WILLIA]~F~and R.J. LEFKOWITZ, J. Biol. Chem. 252 2787-2789 (1977).