Effect of a calcium channel blocker, verapamil, on amino acid uptake stimulated by FSH in rat testes. G.F. Wassermann and E.S. Loss. Centro de Endocrinologia ...
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Med. Sei. Rés., 1989; 17, 779-780
Effect of a calcium channel blocker, verapamil, on amino acid uptake stimulated by FSH in rat testes G.F. Wassermann and E.S. Loss Centro de Endocrinologia Experimental, Instituto de Biociências, UFRGS - Porto Alegre, RS, Brazil
Keywords: Verapamil, follicle-stimuíatmg hormonc, amino acid uptake. calcium channel blocker. Introduction: Follicle-stimulating hormonc (FSH) increase amino acid transport in Sertoli cclls of immature rat testes and this action is cxcrtcd on the Na + dependent amino acid transport system [1]. On the othcr hand, it hás becn reported that glucagon stimulates hcpatic amino acid uptake by mobilízing cellular calcium [2]. It hás also been shown that the increase in amino acid uptake produced by isoprotcrenol in mouse kidney córtex slices is abolishcd by Ca2+ transport inhibitors [3]. The prescnt work was therefore dcsigned to study the influence of verapamil, a spccific Ca2+ slow channel blocker [4], on the stimulatory effect of FSH on amino acid transport. Material and methods: Porcinc FSH and verapamil were purchased from Sigma Chemical Co, St Louis, Mo, USA. a[1-14C] aminoisobutyric acid ([14C] AIB; sp. act. 30 mCi mmoP1) was acquired from Du Pont, NEN Products, Boston, Mass, USA. Fíftcen-day-old Wistar rats wcre killed by cervical dislocation. One gonad, alternatcly left and right, from cach rat was treated with verapamil or/and FSH (experimental) and the contralatcral one was used as the control. Each experimental group consisted of five animais. In ali cases the testes were weighcd, decapsulated and incubated immediately after rcmoval. The wholc gonads were individually prcincubated (60 min) and incubated (60 min) in flasks with l mL of Krcbs Ringer bicarbonate (KRb) buffer in a Dubnoff metabolic incubator. The sampies were maintained at 32°C and pH 7.4 and gassed with O2:CO2 mixturc (95:5; v/v). At the beginning of the incubation pcriod, 0.2 ^Ci of [14C] AIB was added to the sampies while verapamil was includcd both during the pre-incubation and the incubation pcriod. Verapamil was added to the flasks at different concentrations (Figure 1). In anothcr cxperiment, with a verapamil concentration of 2.5 x 10 ~ 4 M, FSH was added at the beginning of the incubation period. Afterwards, these sampies wcrc processed as previously reported [1]. Aliquots (25 jiL) of the tissuc fluid and the incubation médium were taken to measure the [14C] AIB radioactivity. This radioactivity was counted in Aquasol 2 (Du Pont, NEN) in a LKB Rack Beta liquid scintillation spectrometer model 1215 (LKB-Produccr A.B. Bromma, Sweden). The results wcrc cxprcssed as the tissuc/medium (T/M) ratio: cpm mL ' tissue fluid per cpm mL"' incubation médium. Data from the verapamil experiments were analysed by one-way ANO VA (Figure 1). Post hoc tests wcre carried out by Duncan's multiple range test whenever suitable [7]. The rcgression line and the correlation coefficicnt were calculated (Figure l, inscrt). Data for the FSH experimcnt was analysed by two-way ANOVA 2 (control/verapamil) vs 2 (FSH untreated/treated) (Figure 2).
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J Vetapamii
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Figure I: Effect of verapamil on the basal l'4C] AIB uptake in rat testes (means ± SEM, n = 5). A-Statistical analysis (one-way ANOVA) indicated that verapamil produced a significant decrease f*p < 0.02) on amino acid uptake. Post hoc comparisons by Duncaris multiple range tesl indicated that means differ from one another (p < 0.05), except for the fotlowing verapamil doses: 5 x 10 *M vs 7.5 x /(T-'A-/ and 7.5 x W~SM vs 10 x IO"SM. B- The linear relaúonship was analysed with the logarithm of the concentration values and its equation for the regression line was: y = -3.32 -137 x. The value of the correlation coefficient was: r = -0.996.
25xlO~ s M Vsrapamil
Botai
Figure 2: Effect of verapamil on the FSH stimulatory action on amino acid uptake of immature rat testes (means ± SEM, r\=5). Statistical analysis (two-way ANOVA) revealed significant differences (*p < 0.01) between FSH and basal in the control groups; and f**p < 0.001) belween control and verapamil treated groups. No significam difference between basal-verapamil vs FSH-verapamil groups.
Resulte and discussion: Verapamil produccd a dose-dependent decrease of amino acid uptake in immature rat testes (Figure
7SÍI
1). At a dose of 2.5 x 10 4 M, the drug abolished completcly the stimulatory action of FSH on amino acid transport (Figure 2). These results are in agreement with those of Goldstone et a/., who found that verapamíl abolishcd thc action of isoprotcrenol on amino acid transport in mouse kidney córtex sliccs [3], and also with those of Tsang et a/., who rcportcd that the same dose (2.5 x 10~4M) of vcrapamíl inhibited the FSH stimulatory action on progestcrone produetion by granulosa cclls [5], These results indicatc that the stimulatory action of FSH is rclatcd to the voltage-dependent Ca2+ channels.
G.F. Wassermann and li.S. Loss Cruz Curte, A. and Wassermann, G.F. 1985. J. Endocr., 106. 91-94 Kdle>. D.S., Evanson, T. and Poiter. V.R. 1980. Proc. Natl. Acad. Sei, USA, 77,5953-5957 Goldstone, A.D. Koenig, H. Lu, C. Y. et ai. 1983. Biochem. Biophys. Rés. Commun., 114. 913-921 Godfraind, T. Milkr. R. and Wiho, M. 1986. 1'ltarmacol. Rev., 38, 321-416 Tsang. B.K. and Carnegie. J . A . 1983. Endocrinology, 113, 763-769 Tan, K.N. and Tashjian, A.H. 19841. J. Bial. Chem.. 259427-434 Sokal, R.R. and Rohlf, S.K. 1969. In: Biometric. pp. 204-52. 299-342. Freuman, W.H. and Co.. San Francisco. USA Reprinl rcqucsls Io: i)r Guillcrmo Federico Wassermann, Rua Sarmento Leite, 500, 90049 Porto Alegre RS, Brazit.
Paper received; 25th July, 1989.
