CONTRACEPTION

VERAPAMIL PLASMA

STIMULATES MEMBRANE

R. Junejal,

Ca++-UPTAKE

VESICLES

OF

AND

GUINEA

I. Gupta', A. Wali' R.N. S. Majumdak2

Ca++-ATPase PIG

IN

SPERMATOZOA

Chakravarti2

and

'Dep3rtment of Obstetrics and Gynecology and Department of Experimental Medicine Postgraduate Institute of Medical Education and Research Chandigarh 160 012, India

Abstract Verapamil, a potent calcium channel blocker, was administered orally at three different doses to guinea pigs for both short(4 weeks) and long-term (12 weeks) eff;zza. treatment stimulated Ca++-transport The drug and activated ATPase in isolated p$iasma membrane vesicles of guinea spermatozoa. Ca -uptake studies exhibited pig partial to complete restoration of stimulated Ca++-transport during recovery period, whereas the CA++-activated ATPase system remained stimulated even after 4 and 6 weeks of withdrawal of the drug treatment. The lack of inhibitory effect of verapamil on Ca++ -uptake ruled out the involvement of calcium channels in spermatozoa1 calcium uptake in guinea The stimulatory effect of the drug on CA++-uptake, on pigs. the other hand, might indicate the possible capability of this lipophilic compound to induce favourable changes in the lipid microenvironment of the membrane, wherein the integral membrane proteins operate. Submitted for publication Accepted for publication

APRIL

1990 VOL. 41 NO. 4

November 21, 1989 December 14, 1989

419

CONTRACEPTION

Introduction It is well documented that intracellular calcium within a narrow range of concentration has a regulatory role in sperm motility acrosome and reaction (l-6)., Indirect evidence suggests that increased spermatozoa1 membrane to ca++ is a YIZEi%ilZpoG?&ttE~ capacitation that allows subsequent the occurrence of acrosome reaction and motility activation (7,8). Although there is no doubt that the entry of extracellular Ca++ is essential to induce the acrosome reaction of mammalian spermatozoa (g-11), the mechanism of Ca++ entry is not fully understood. Further, research to understand the mechanism of calcium uptake by mammalian spermatozoa suggests that intracellular Ca++ concentration is regulat.$d by 1) ATPdependent Ca++ pump (12-14) and by 2) Na /Ca antiporter of the plasma membrane (15,16) and of the mitochondria (17). D-600, a structural analogue of verapamil, results in the induction of accelerated acrosome reaction and Ca++ build up in guinea pig spermatozoa in vitro (18 Verapamil and D600 are able to block in vitro a Na+/Ca -!i antiporter present in ram sperm fla ella (15), thereby leading to a rise in intracellular Ca+' concentration. The adverse effect of an excessively high intracellular Ca++ concentration on the sperm motility has been well recognised (19,20). The paucity+ of information on effects of verapamil on spermatozoa1 Ca -transport and Ca++ -ATPase system prompted us to elucidate the effect of this drug on the Ca++transport system present in sperm plasma membrane of guinea pigs. Methods Adult male guinea pigs of the Institute's colony with body weight x 500 g were maintained in the laboratory for one month on pellet chow (Lipton India, Ltd., Bombay) for acclimatization. Water was given & libitum and ascorbic acid was supplemented to all the animals. Ten animals were (groups Iput in the normal control group, whereas 5 groups each were subjected to drug treatment. V) of 30 animals Each of these groups were divided into 3 sub-groups of 10 animals each. Verapamil suspended in saline (verapamil hydrochloride, Lyka Lab., Bombay, India) was administered orally for 12 weeks in 0.5 ml volume at doses of 1.0, 5.0 and 10.0 b.w./day (groups III-V), respectively. mg/kg Whereas, to group I and II, the drug was given at doses of 1 respectively, for 4 weeks only. and 5 mg/kg b.w./day, Control animals received an equal volume of saline. Ten animals from each experimental group along with the controls were then sacrificed. The remaining animals were allowed to drug recover after withdrawal of the treatment and sacrificed at intervals of 4 and 6 weeks thereafter.

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CONTRACEPTION

The membrane Prenaration of soerm wlasma membrane vesicles: preparations were based on the method of Rufo et al. (16) The animals were sacrificed under ether with modifications. anaesthesia and the sperm were collected by flushing the vas buffer containing 0.25 M epididymis with deferens and sulfonic sucrose, 0.2 mM MgC12 and 10 mM morpholinopropane The sperm cells were washed two times acid (MOPS), pH 7.4. with the same buffer by centrifuging at 1,500 G for 10 min. resuspended in the same buffer washed cells were The and disrupted thoroughly by an containing 1 mM EDTA ultrasonifier (B Braun Instruments) with a microtip probe at ice-water environment. The sonicated speed in an high suspensions were centrifuged at 600 G for 10 min and the of the pellet was washed two times with an equal volume above mentioned buffer minus MgCl The combined 600 G supernatant fractions were centrl Y2' uged at 6,000 G for 10 was then layered on a min. The 6,000 G supernatant discontinuous sucrose gradient composed of 40% (d They were cen$~r:u~~~ and 15% (dzo--1.05) sucrose solution. in a swing out rotor at 35,000 G for 40 min. The membrane fraction located at the interface of d20=1.17/1.05 layer was removed and diluted four times with 0.25 M sucrose and 10 mM sperm plasma membrane was pelleted out by MOPS. The centrifuging at 35,000 G for 40 min and suspended in the same buffer. The membrane preparation showed a 7-g-fold enrichment in comparison to the whole cell homogenates of the plasma membrane-specific marker enzyme NafK+-ATPase and 5'-nucleotidase, confirming purity and ensuring enzyme activity and integrity of the isolated plasma membrane vesicles. The protein concentration of the membrane was estimated by the sodium dodecyl sulphate-Lowry procedure of Lees and Paxman using crystalline bovine serum albumin as the standard (2 1). All operations were carried out at 4OC. Measurement of Ca++-uwtake: Ca++-uptake by spermatozoa1 plasma membrane vesicles was measured as described by us earlier for rat intestinal brush border membrane vesicles a rapid filtration technique with Millipore (22), adopting filters of 0.45 pm size (23). Assav of ATPase activitv: The Ca++-ATPase activity was measured as described by us earlier for human sperm plasma membrane vesicles (24). Inorganic phosphorus liberated from the reaction was determined by the method of Chen et al. The Na+ -K+-ATPase activity was measured as the (25). ouabain-sensitive portion of the ATPase and was calculated as the difference in Pi release in the presence and absence of 0.1 mM ouabain. Estimation of calcium content: Total cellular calcium was estimated in infusate by calcium analyser which sperm incorporates a fluorometric titration (CALCETTE Model 4008 Systems, MA). A value of 3% of total and 4009, Precision

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1990 VOL. 41 NO. 4

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CONTRACEPTION

calcium content was considered to represent (20) which had been confirmed at the beginning

ionic calcium of the study.

5'-Nucleotidase activity was measured by the method of Heppel and Hilmoe (26). About 6-8 sperm suspensions obtained from different animals of the same experimental group were pooled together for plasma membrane preparativ$s in a sinsle experiment. The results on Ca++-UPtake. CA ATPase and other enzymes have been based on two-independent membrane preparations, analyzed in triplicate. Results Ca++-uDtake: The sperm plasma membrane repared from verapamil-treated sequestered animals at a faster rate and to a greater extent than those prepared from the control groups of animals (Fig. l-3). The uptake followed saturation-type kinetics, and maximal uptake for both types of membrane preparations occurred within 60 min at 37OC. After long-term (12 weeks) treatment at a dose of verapamil exhibited a marked stimulation Afm&@+b.w./day, -uptake by sperm plasma membrane vesicles (Fig. 1), and this effect of the drug was found to be less pronounced at 5 and 10 mg/kg b.w./day doses of verapamil (Fig. 1 and Short-term (4 weeks) treatment resulted in lesser 2) stimulation of the uptake system (Fig. 3). Partial to complete restoration of stimulated calcium uptake was achieved after withdrawal of the drug therapy which was earlier administered for a different duration of time. Ca++-ATPase activitv: A dose-dependent enhancement in the Ca++-ATPase activity was observed after 12 weeks of treatment with different doses of verapamil (Table I). Recovery studies after the long-term treatment of animals at doses of 1 and 5 mg/kg b.w./day exhibited a further increase activity. long-term treatment with in enzyme However, verapamil at a dose of 10 mg/kg b.w./day resulted in marked stimulation of Ca++-ATPase activity, even immediately at the end of the drug treatment. Verapamil treatment at a dose of 1 mg/kg b.w./day could not alter the enzyme levels, whereas a daily dose of 5 mg/kg b.w. resulted in stimulation of Ca++-ATPase activity. The recovery studies showed a negligible restoration of stimulatory levels of Ca++-ATPase activity. Cellular calcium content: Short-term (4 weeks) treatment using different doses of verapamil resulted in an increase in cellular calcium content which, however, was not found to be statistically significant. In addition, a significant decline in calcium content as compared to the control values was noted during the recovery study. Twelve weeks of treatment with verapamil at a dose of 1 mg/kg b.w./day exhibited a marked stimulation of calcium content without any noticeable recovery even after 6 weeks of withdrawal of

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CONTRACEPTION

Fig.

APRIL

1.

guinea 45Ca++-uptake by Kinetics of pig vesicles. membrane spermatozoan plasma (a) : Plasma membrane vesicles prepared from animals of (1.0 mg/kg b.w./day 0 Control, 0 verapamil-treated A 4 weeks recovery, and U 6 weeks for 4 weeks), recovery experimental groups were incubated in a medium containing 110 mM NaCl, 5 mM KCl, 10 mM 37oc. Following a 5-min MOPS, pH 7.4, at (45CaC12, Sp.Ac=1.83 preincubation, calcium Amersham) was added at a concentration mCi/mmol, At the designated time points, of 2 pCi/assay. duplicate aliguots of vesicle suspension (25 I.rg) as described in the were withdrawn and counted, text. Data are expressed as means + S.D. of at determinations on two individual least three preparations of plasma membrane. (b): the assay above except for was performed as verapamil administered at a dose of 5 mg/kg b.w./day for 12 weeks.

1990 VOL. 41 NO. 4

423

CONTRACEPTION

10

20 TlME

Fig.

Fig.

424

30

40

so

60

(MINUTES)

2.

Effect of verapamil administration (10 mg/kg b.w./day for 12 weeks) on the kinetics of Ca++uptake by guinea pig spermatozoa1 plasma membrane vesicles. Experimental details are as described in Fig. 1.

3.

Effect of short-term (4 weeks) verapamil treatment on the kinetics of Ca++-uptake by guinea pig spermatozoa1 plasma membrane vesicles. The drug was administered at a dose of: (a) 1.0 mg/kg b.w./day and (b) 5.0 mg/kg b.w./day for 4 weeks. Experimental details are as described in Fig. 1.

APRIL 1990 VOL. 41 NO. 4

in

guinea

: Results of

v’erapami 1 pig-spermatozoa administration

on

different

Groups

9.86*0.49** 4.88*0.21*5.82*0.29**

Group -___ III (1.0 mg/kg ____ 12 Weeks Treatment 4 Weeks Recovery 6 Weeks Recovery

Group __V (10 mg/kg b.w./day) ---12 Weeks Treatment 4.53?0.26** 4 Weeks Recovery 4.25kO.93 6 Weeks Recovery 4.55+1.18** 3~“____‘__*4__-_-______-_-___-______-_--_______~__-----_~-_-__~_~~~-_~~~~~-~_~~~~---~__ p co.05 p < 0.01 Values are Mean*S.D. of 7-8 observations.

b.w.lday)

b.w./day)

3.7OiO.46 2.91*0.38** 3.24+0.07**

3.58kO.48 3.8OkO.23 3.74kO.12

Control (Saline) ___--__ 12 Weeks Treatment 4 Weeks Recovery 6 Weeks Recovery

(5.0 mglkg Treatment Recovery Recovery

4.61*0.32* 3.86fO.26 3.45fO.19

GrouP -_-II (5.0 mglkg ---4 Weeks Treatment 4 Weeks Recovery 6 Weeks Recovery

Group __-IV ---12 Weeks 4 Weeks 6 Weeks

4.42tO.45 3.41kO.75 2.58kO.39*

Group __I (1.0 mg/kg --__ 4 Weeks Treatment 4 Weeks Recovery 6 Week Recovery b.w./day)

3.99kO.22 3.83eO.21 3.71*0.75

Control (Saline) -___-__--_______ 4 Weeks Treatment 4 Weeks Recovery 6 Weeks Recovery

b.w./day)

Calcium

Cat+ -ATPase

biochemical

parameters

136.36*7.23** 70.73+2.19** 36.82*1.38**

31.20+3.28** 34.22+1.76** 69.65*3.76*’

31.20*3.28** 34.22+1.76*! 69.65+3.76**

13.78kO.89 16.84*1.28 16.56kO.78

38.54*2.34* 44.36+1.98** 48.43iO.93**

18.87t1.46 82.72+4.38** 72.35*2.08**

13.82k1.39 15.76*0.75 15.82kO.79

(n r-m1es Img_ p_r_o~_ei_nl__

Verapamil stimulates Ca(++)-uptake and Ca(++)-ATPase in plasma membrane vesicles of guinea pig spermatozoa.

Verapamil, a potent calcium channel blocker, was administered orally at three different doses to guinea pigs for both short- (4 weeks) and long-term (...
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