Sex Differences in [3H]Nitrendipine Binding and Effects of Sex Steroid Hormones in Rat Cardiac and Cerebral Membranes

In this study, we have attempted to charac terize the sex differences of calcium channels, using[ 3H]nitrendipine binding in the cardiac and cerebral membranes, and to examine the effects of sex steroid hormones on calcium channels in gonadectomized rats. Sex Differences in [3H]Nitrendipine Binding and Effects of Sex Steroid Hormones in Rat Cardiac and Cerebral Membranes Kenji ISHII, Takashi KANO and Joichi ANDO Department of Pharmacology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569, Japan Accepted October 16, 1987 Abstract-The sex differences and regulation by sex steroid hormones in calcium channels were studied by using [3H]nitrendipine binding to cardiac and cerebral membranes in 15-week old spontaneously hypertensive rats (SHRs). The maximal number of binding sites (Bmax) in the hippocampus of female SHRs increased by 24.1% over that in male SHRs. In the females, the Bmax values in the cardiac, striatal, thalamic and hippocampal membranes from ovariectomized SHRs de creased by 34.7, 29.9, 29.3 and 26.9%, respectively, compared to normal SHRs. This phenomenon, except for the hippocampus, was inhibited by estradiol but not by testosterone. In the male, the Bmax values in cardiac and cerebral membranes showed almost no changes after orchidectomy or treatment with estradiol or testosterone. After gonadectomy, the Bmax values in the cardiac, striatal and thalamic membranes of females decreased by 30.2, 33.0 and 35.6%, respectively, compared to those in males. The changes in apparent dissociation constant (KD) values were less remarkable than those in the Bmax values. These findings suggest that sex differences exist in the calcium channels of the heart, striatum, thalamus and hippocampus, and they suggest that estradiol, but not testosterone, may play a part in the regulation of the calcium channels in female SHRs. It is well-known that sex differences exist tive inotropic and chronotropic effects or a in structures; e.g., in the size of specific brain decrease in neurotransmitter release. Al regions, in dendritic and axonal branching though calcium channels play an important patterns, and in the distribution of synapses role in cell functions, no reports have been (1), receptors (2-5), catecholamine contents published about sex differences in calcium (6) and neurotransmitter systems (7-9). channels and their regulation by sex steroid It is also well-known that calcium plays an hormones. important role in a variety of functions in both Recently, the binding properties of a neuronal and non-neuronal tissues. In calcium channel antagonist,[ 3H] nitrendi neuronal tissue, calcium influx into pre pine, a derivative of dihydropyridine, have synaptic nerve endings initiates a series of been investigated in homogenates of the events leading to neurotransmitter release, guinea-pig and rat cerebral cortex, heart and whereas in the heart and smooth muscles, ileum (14, 15), and[ 3H]nitrendipine has calcium couples excitation to contraction. been used as a chemical probe for the chemi The entry of calcium into these tissues ap cal (16, 17) and biological (18-20) charac pears to be mediated by a voltage-sensitive terization of calcium channels. calcium channel (10, 11 ). Compounds termed organic calcium channel antagonists" are reported to inhibit voltage-sensitive calcium channels (12, 13). Therefore, some calcium channel antagonists at certain concentrations can cause smooth muscle relaxation, nega 20 min. After the supernatants had been Materials and Methods decanted, the pellets were washed in fresh Animals: Male and female WKYs and SHRs 0.32 M sucrose solution, and then centrifuged aged 4 and 15 weeks were used. Male and at 34,000 x g for 20 min. The pellets (crude female SHRs aged 4 weeks were anaes membrane fractions) were resuspended in thetized with pentobarbitone (30 mg/kg, 50 mM Tris-HCI buffer solution (pH 7.4) to i.p.) and then bilaterally orchidectomized obtain a final protein concentration of 100 and/or ovariectomized through a scrotal and/ ,ug/2 ml and were used for the binding assay. or ventral route, respectively. Then the rats Assay of [3H]nitrendipine binding: A 2 ml were maintained under ordinary environ aliquot of the thus prepared suspension was mental conditions for 15 weeks of age and incubated with 20-800 pM [3H]nitrendi were divided 3 groups of male rats and 3 pine in triplicate or duplicate for 90 min at groups of female rats. The orchidectomy 25'C. The mixture was filtered under vacuum (Orchi) and/or ovariectomy (Ovari) groups through a Whatman GF/C glass fiber filter consisted of orchidectomized and/or ovari and washed with three 3 ml aliquots of 50 ectomized rats without hormonal treatment. mM Tris-HCI buffer solution (pH 7.4). The The remaining 2 groups of male and female trapped radioactivity was measured by liquid rats were alternatively given (s.c.) estradiol scintillation spectrophotometry (Packard, benzoate (estradiol) or testosterone benzoate 300C type). Specific binding was defined as (testosterone) dissolved in sesame oil in a the difference between the total binding and dose of 250 ag/kg or 20 mg/kg, respectively, binding in the presence of 1 flM nifedipine. from 3 to 15 weeks of age. The properties of [3H]nitrendipine binding Tissue preparation for binding assay: The were analyzed by the method of Scatchard heart and brain were quickly excised from (21). untreated (control) male and female WKYs Drugs: [3H] Nitrendipine (77.4 Ci/mmol) and SHRs (n=4, each), from gonadectomized was obtained from the Japan Radioisotope male and female SHRs (n=6, each), and from Association (New England Nuclear, Boston, gonadectomized male and female SHRs U.S.A.). Nifedipine was supplied by Bayer treated with estradiol and/or testosterone Yakuhin Co., Ltd., Osaka, Japan. 173 (n=5, each) aged 15 weeks, respectively. Estradiol 3-benzoate (estradiol), testosterone The hearts were homogenized in a Polytron benzoate (testosterone) and Tris-hydroxy P-10 using three 10 sec bursts in 10 volumes methylaminomethane (Tris) were obtained of 50 mM Tris-HCI buffer solution (pH 7.4) from Sigma Chemical Co., U.S.A. All other in ice water. The resulting homogenates were chemicals were of reagent grade. centrifuged at 1,000 x g for 10 min. The super Other methods: Protein was measured by natants were centrifuged at 105,000 x g for the method of Lowry et al. (22) using bovine 60 min. After the supernatants had been serum albumin as a standard. The results were decanted, the pellets (crude membrane frac expressed as the mean±standard error for tions) resupended in 50 mM Tris-HCI buffer each group. The significance of differences solution (pH 7.4) to obtain a final protein was examined by means of Student's t-test. concentration of 50 ,ag/2 ml, were used for binding assays. The brains were divided into Results cerebral cortex, striatum, hypothalamus, Properties of [3 H]nitrendipine binding: thalamus, hippocampus, cerebellum and The specific [3H]nitrendipine binding to medulla pons. The striatum, thalamus and crude membranes prepared from the heart and hippocampus were homogenized separately various areas of the brain of WKYs and SHRs in a teflon-glass homogenizer (Wheaton, aged 15 weeks was saturated monophasically U.S.A.) using 8 strokes at 3,000 rpm in 10 (Fig. 1A-H). Using 20-800 pM [3H]nitren volumes of 0.32 M sucrose solution in ice dipine yielded apparent dissociation con water. The resulting homogenates were cen stants (KD) of 0.090 to 0.186 nM in the heart, trifuged at 1,000xg for 10 min. The super striatum, thalamus and hippocampus for male natants were centrifuged at 34,000 x g for and female WKYs and SHRs. Scatchard Fig. 1. Composite saturation isotherms of [3H] nitrendipine binding to cardiac and cerebral mem Fig. 2. Scatchard analysis of the [3H]nitrendipine binding to cardiac and cerebral membranes from branes from male and female WKY (40) and SHR (0) WKY (0) and SHR (0) observed in Fig. 1. (A and (15 weeks-old). Heart and various areas of brain B) Heart, (C and D) striatum, (E and F) thalamus were homogenized and incubated with increasing concentrations of [3H]nitrendipine. (A and B) and (G and H) hippocampus. (A, C, E and G) Male Heart, (C and D) striatum, (E and F) thalamus and and (B, D, F and H) female. (G and H) hippocampus. (A, C, E and G) Male and (B, D, F and H) female. female control group showed a significant increase over those in the male control group, analysis revealed a single population with a but the Bma,,values differed very little between Hill coefficient of 0.94-1.06 and showed a the male and female control groups (Fig. 3). maximal number of binding sites (Borax) of In the groups treated with gonadectomy, the 57 to 229 fmol/mg protein in the heart, Borax values in the Ovari group showed a striatum, thalamus and hippocampus for male significant (P<0.001) decrease of 34.7% and female WKYs and SHRs (Fig. 2 A-H). over those in the female control group and a Similar data were obtained from crude decrease of 30.2% over those in the Orchi membrane preparations of gonadectomized group, but the K„ and Borax values differed male and female SHRs and from those of very little between the Orchi group and the gonadectomized male and female SHRs male control group. In the males, the K„ and treated with estradiol and/or testosterone Bma,t values differed very little between the (data not shown). The Borax values of [3H] estradiol and/or testosterone groups and the nitrendipine binding sites in the striatum, control and/or Orchi groups. In the females, thalamus and hippocampus in the male and the KDvalues in the estradiol and testosterone female SHRs and in the heart in the female groups showed significant increases over SHRs significantly increased over those in the respective values in the Ovari group.

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