Camp. Biochem. Physid. Vol. 104C. No. 1, pp. 16St69, 1993 Printed in Great Britain

0306~4492/93 $6.00 + 0.00 0 1993 Pergamoo Press Ltd

THE ANTIFERTILITY AGENT, GOSSYPOL, RELEASES CALCIUM FROM RAT LIVER MITOCHONDRIA

FEDERICO MARTiNEZ,* REBECA MILAN, MA. TERFISA ESPINOSA-GARCiA and JUAN PABLO PARDO

Departamento de Bioquimica, Facultad de Medicina, Universidad National Autbnoma de Mkxico, Apdo. Postal 70-159 Coyoak, MCxico, D.F.

(Received 10 July 1992; accepted for publication 21 August 1992)

Abstract-l. The effect of gossypol in the presence of K+ or Caz+, or both, was studied on respiration, 2+ ATPase activity, and Ca transport of rat liver mitochondria.

2. The uncoupled respiration induced by gossypol was inhibited by CaZ+. 3. This inhibition was lowered by the presence of Mg 2+. ATPase activity was stimulated when gossypol

and Ca2+ were in the incubation medium, in a Ca’+-dose related fashion. 4. Mitochondrial CaZ+ was released by gossypol, an effect associated with the membrane fluidity. 5. The results suggest that gossypol modifies the mitochondrial concentration of Ca2+.

INTRODUCTION

The transport of Ca*+ in the spermatozoa cells is an essential requirement for the fertilization process, since Ca*+ is involved in several steps, such as the acrosomal reaction and the control of the spermato- zoal motility (Yanagimachi, 1981). The acrosomal reaction is a Ca*+-dependent process and allows the fusion between the plasma and acrosomal mem- branes (Russel et al., 1979). The net Ca*+ uptake during the capacitation event has been clearly estab- lished (Singh et al., 1978); however, there is not enough information about the Ca*+-transport kinetics, although several transport mechanisms have been involved in both plasma and acrosomal mem- branes from spermatozoa cells, for the regulation of Ca2+ concentration, such as the Ca2+ pump or the Mg’+/Ca*+ATPase from ram flagellar membrane, and a Ca’+ATPase described in the acrosomal mem- brane from rodents, boar, and bull (Paterson et al., 1983).

Gossypol is a potential non-steroid antifertility agent for male (Chang et al., 1980). It has been demonstrated that gossypol inhibits the Mg*+/ Ca*+ATPase in spermatic cells (Kalla and Vasudev, 1981) and the lactate dehydrogenase C4 from testis (Montamat et al., 1982; Eliasson and Virji, 1983). For several enzymatic activities, such as the enzyme in- volved in the metabolism of nucleotides, gossypol is a competitive inhibitor (Olgiati et al., 1984), although, in the kidney, Na+/K’ATPase gossypol showed a non-competitive inhibition (Xiopeng et al., 1981). In addition, it has been reported that gossypol inhibits the Ca*+ transport and Ca’+ATPase of plasma mem- brane from human spermatozoa (Kanwar et al.,

1989).

*Author to whom correspondence should be sent.

On the other hand, the mechanism by which gossypol produces its infertility effect is unknown. It has been demonstrated that mito~hondria is the subcellular organelle that shows the highest incorpor- ation of gossypol, as well as the highest structural and functional damage (Xue, 1980). Gossypol un- couples mitochondrial respiration (Abou-Donia and Dieckert, 1974), and its effects are associated with the presence of monovalent cations (Martinez et al., 1988). This paper reports the effect of gossypol on respiration, Ca*+ transport, and ATPase activity in rat liver mitochondria (a well studied and known biological system) to get insight on the bioenergetic mechanism of action of gossypol.

MATERIALS AND METHODS

All chemical reagents were purchased from Sigma Chemical, Co. (St Louis, MO, U.S.A.). Gossypol acetic acid, 99% pure, was a kind gift from Mr Soulat Sufi, Principal Biochemist, Chelsea Hospital for Women (London). Gossypol was dissolved in ethanol and used in aliquots not higher than 40~1 per ml. Control experiments were performed with equivalent amounts of ethanol.

Rat liver mitochondria were isolated by differential centrifugation, as previously reported (Martinez et af., 1988). Protein dete~ination was quantified using the method described by Lowry et al. (1951), using bovine serum albumin as standard.

Mitochondriai respiration was determined in a medium containing 100 mM KCl, 10 mM Pi, 10 mM malate, 10mM glutamate, 20pM gossypol, and 10mM MgCl,, when present, in a final volume of 3 ml at 25°C; adjusted to pH 7.3 with Tris-base, containing 1 mg of mitochondrial protein/ml. Respir- ation was stimulated with 500nmol ADP.

165

166 F. MARTINEZ et al.

[Co++] /rM

Fig. 1. Effect of gossypol on mitochondrial respiration in the presence of increasing Ca*+ concentrations. Mitochondria were incubated in the respiration medium as described under Material and Methods in the presence of the Car+ concentrations observed in the figure (0). After approxi- mately 1 min, 20 PM gossypol was added and the oxygen consumption was recorded. The uncoupling effect produced by gossypol alone was considered 100%. The percentage of uncoupled respiration in the presence of Mg*+ is presented

with close symbols (a).

Mitochondrial Ca*+ uptake was determined in 100 mM KCl, 10 mM succinate, 10 mM Tris-HCl, pH 7.3, 10 mM Tris-acetate, 10 pg rotenone, 10 PM of radioactive Ca*+ (specific activity 100,000 cpm/ nmol), and a protein concentration of 1 mg/ml. At different times of incubation, an aliquot of 50 ~1 was removed and deposited on a Millipore membrane (0.22 pm). Mitochondria were immediately washed with 10ml of cold 10mM CaCl, to eliminate the unspecific Ca2+ bound to the outer surface. After 3 min incubation, 20 PM gossypol was added and samples were taken each min. The dry Millipore filters were counted in a scintillation vial in a Packard spectrometer, model 3255, with an efficiency of 60%.

Mitochondrial Ca2+ movements were also followed in an Aminco DW-2a spectrophotometer in 10mM succinate, 1Opg rotenone, 10mM HEPES, 50pM Tris-acetate, 10 PM ADP, 50 PM Arsenazo III, and 1 mg/ml of mitochondrial protein. The reaction was started by the addition of 50pM Ca2+.

An artificial system of two phases was used to determine the Ca2+ movement. The aqueous phase contained 10mM Tris-HCl, pH 7.3, 50pM radio- active Ca*+ , and different KC1 concentrations. The organic phase contained chloroform/methanol (60/40, v/v). Gossypol (100 PM) was added and the system vortexed vigorously for 1 min at 25°C. The mixture was spun out at 2,500g for 10 min in a refrigerated Sorvall RT600 centrifuge to separate the two phases. Aliquots from each phase were taken and the radioactivity in the organic phase was measured.

ATPase activity was performed in 1 ml of a medium containing 100 mM KC1 or 250 mM sucrose, 20pM gossypol, 10mM Tris-HCl, pH 7.3 at 30°C. After 5 min of incubation, 1 mg of mitochondrial protein was added, and incubated 2 more min, followed by the addition of 2.5 mM ATP. ATP

hydrolysis was stopped after 4 min of incubation by the addition of 6% trichloroacetic acid. The mixture was centrifuged at 2,500g for 10 min, and the super- natant collected to determine the phosphate released as described by Sumner (1944).

RESULTS

Figure 1 shows that 20 pM gossypol uncouples (100% in Y-axis) the mitochondrial respiration in the absence of Ca2+. When mitochondria were incubated with different concentrations of Ca2+ and gossypol (20 PM) a partial inhibition of the uncoupling effect induced by gossypol was observed, reaching a 50% inhibition at 50pM Ca2+. Figure 1 also shows that in the presence of Mg2+ the inhibitory effect of Ca2+ was diminished, suggesting that the effect of Ca2+ on respiration could be associated with its transport.

Mitochondrial respiration was assayed in the ab- sence of Ca2+. It was observed that the uncoupled respiration induced by 20 PM of gossypol was inhib- ited when Ca2+ was added (Fig. 2). It is worthwhile mentioning that the effect of Ca2+ required a time to inhibit the gossypol-stimulated respiration, and fur- ther ADP additions did not increase oxygen con- sumption (Fig. 2).

As reported, gossypol increases mitochondrial ATPase activity when monovalent cations are present (Martinez et al., 1988). The experiment of Fig. 3

’

2 min

Fig. 2. Ca*+ and gossypol effect upon the oxygen consump- tion from rat liver mitochondria. Experimental conditions are similar to Fig. I, except that Ca*+ was added where indicated. Trace A is the control. ADP concentration in each addition was 500 nmol, except in trace B, where the last ADP addition was 1,000 nmol. Numbers indicate ngatoms

of oxygen/mg/min. G = 20 nM gossypol.

Gossypol releases Ca2+ from mitochondria 167

’ 0!2 Ob 0!4 O!Cr O!S

[Co**] mM Fig. 3. Mit~hond~al ATPase activity in the presence of gossypol and increasing Caz+ concentrations. The activity of ATPase was assayed as described under Material and Methods determining the phosphate (Pi) released from ATP in the presence (0) or absence (a) of KCI. Bars indicate the

standard deviation of four experiments.

shows the effect of gossypol on ATPase activity at different Ca*+ concentrations in the presence or absence of KCI; 20pM gossypol plus Ca2+ did not modify ATP hydrolysis. However, when ATPase activity was assayed with 100 mM KCl, the presence of Ca2+ (below 200 ~1 M) increased ATPase activity in a bell-shape fashion.

It is well known that mitochondria have a specific Ca2 + transport system. Figure 4 shows that the addition of gossypol releases Ca*+ from the mitochondrial matrix, only in the presence of KC1 (Fig. 5). In contrast to ionophore A23187,

A- I

Time (mid Fig. 4. Intramitochondrial Caz+ release induced by gossypol. Radioactive Caa+ uptake into mit~hondria was followed in time by the Miili~~ membrane filtration method. G indicates the addition of 20 yM gossypol (O), and the close symbols are the control (0). This is a

representative experiment.

the Ca*+ efIlux induced by gossypol was a tem~~ture-de~ndent mechanism, since at 4”C, gossypol did not release Ca*+ from mitochondria (Fig. 5).

Gossypol was able to translocate Ca2+ from the aqueous to the organic phase in an artificial mem- brane system, reaching equilibrium values of 36 to 37 prnol of Ca2*jml in the organic phase, whereas in the absence of gossypol, Ca*+ concentration in the organic phase was only 2.1 to 2.3 pmol. Under these conditions, the presence of KC1 increased the amount of Ca2+ transferred to the organic phase from 38 to 52.4 ~mol/ml.

Y

I 0.02

3187

Fig. 5. Ca2* uptake in rat liver mitochondria. The uptake of Ca*+ was followed after the addition of mitochondria (M) by the changes of absorbance with Arsenaxo III. Where indicated, 66 mM KC1 was added. In trace B, lOpM gossypol (G) was added each time, except in the other traces where the addition

was 20 )IM. The ionophore A23187 (IO#g) was added as indicated.

168 F. MART&Z ei a(.

DISCUSSION

We studied the effect of gossypol on several mito- chondrial functions in the presence of Ca’+, and observed that Ca2+ inhibited the uncoupling effect induced by gossypol. It has been reported that gossy- pol binds to metallic cations, such as Mg2+ (Shi et al.,

1981), Mn2+ (Vishwanath and White, 1987), Fe”- (Muzaffaraddin and Saxena, 19661, and Cu*+ (Ra- maswamy and O’Connor, 1969). The results pre- sented in this work could be explained by the formation of gossypol-Ca*+ complexes, which, in turn, diminish free gossypol, inhibiting the stimulated respiration. On the other hand, Ca*+ might modulate the dehydrogenase activities of the tricarboxylic cycle, as reported by Hansford (1985) inhibiting the respiration. However, the fact that Mg’+ was able to modify the Ca 2+ effect on respiration suggests that gossypol is involved in the transport of cations.

The stimulation of ATPase activity could be ex- plained by the uncoupling effect of gossypol, although the swelling produced during the Ca*+ transport, increasing membrane permeability and disrupting the selectivity mechanism of the mitochon- drial membrane, could also explain the increase in the ATPase activity. The inhibition of ATPase activity at high Ca2+ concentrations could be due to the for- mation of the Ca2+-ATP complex instead of the Mg2+-ATP substrate.

In this work, we evidence that gossypol induces the release of Ca2+ from rat liver mitochondria; similar data have been reported in plasma membrane vesicles isolated from ram and bull spermatozoa cells (Breitbart et al., 1984). The mechanism of Ca”’ release could be associated with the formation of complexes with gossypol; since, as observed with the artificial membrane system, gossypol transfers CaZf to the organic phase. This is an important event, since gossypol modifies several Ca2+-dependent enzymatic activities in the spermatozoa, such as, adenylate cyclase (Vishwanath and White, 1986; Olgiati er a& 1984), protein kinase (Kimura et at., 19851, Ca’+ATPase (Kanwar et al., 1989), as well as other spermatic systems, as the metabolism of adenine nucleotides (Tso et al., 1982; Reyes et al.,

1988; Reyes and Benos, 1988) and the acrosomal reaction (Tso and Lee, 1982).

As mentioned before, it has been established that Ca2+ has very important roles in several spermatic processes (Yanagimachi, 1981; Vijayasarathy et al., 1980) and, recently, it has been reported that regu- lation for Ca2+ accumulation in spermatic mitochon- dria depends on the redox state of pyridine nucleotides (Vijayaraghavan et al., 1989) and lactate dehydrogenase X. Our results suggest that the modifi- cation of some mitochondrial functions by gossypol is related to membrane fluidity, since gossypol was unable to release the intramitochondrial Ca*+ at 4°C. In addition, these results support the assumption that Ca2+ and gossypol form complexes that lower the

concentration of free Ca2+ and affect the spermatic metabolism.

Ackn#wledgeme~ts-The authors thank Federico Martinez Jr for the illustrations. This work was partially supported by Grant IN200189 IFCjUNAM from the Direction General de Asuntos de1 Personal Academic0 de la Universidad National Autbnoma de Mexico.

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