Camp. Biochem. Physiol. Vol. 103C, No. I, pp. 87-90, 1992 Printed in Great Britain

0306-4492/92 $5.00 + 0.00 8 1992 Pergamon Press Ltd

THE ANTIFERTILITY AGENT, GOSSYPOL, CHANGES SEVERAL MITOCHONDRIAL FUNCTIONS IN THE

PRESENCE OF Mg2+

FEDERICO MARTiNEZ

Departamento de Bioquimica, Facultad de Medicina, Universidad National Autbnoma de Mtxico, Apdo. Postal 70-159, Coyoacln 04510 Mtxico, D.F., Mexico

(Received 18 March 1992; accepted for publication 18 March 1992)

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

2. Respiration was uncoupled in the presence of gossypol, Mg2+, and K+, whereas in the presence of gossypol and Mg2+ a partial inhibition was observed.

3. Gossypol stimulated ATPase activity in the presence of K+ or Mg*+, but maximal activity was observed when both cations were in the incubation medium.

4. Stimulation of ATPase activity in the presence of Mg2+ was dose related. 5. EDTA reverted the stimulation oroduced bv aossvool on ATPase activitv 6. Gossypol had no effect on the kTPase activity oisubmitochondrial par&es, which suggests an

indirect action of gossypol on the enzyme. 7. Mitochondrial membrane potential showed a higher collapse in the presence of gossypol and

1 mM MgCI, . 8. The observed effects of gossypol could be explained by the collapse of the mitochondrial membrane

potential.

INTRODUCTION

Gossypol, a polyphenolic compound found in cotton seeds (Adams et al., 1960), produces sterility in mammals (Zatuchni and Osborn, 1981; Prasad and Diczfaluzy, 1983). The antifertility effect is time and dose related (Xu, 1981; Abou-Donia et al., 1989). Several proposals have been made to explain its antifertility effects; however, there is no general agree- ment on the mechanism through which gossypol induces infertility in oiuo (Shi et (II., 1987; Quian et al., 1979; Quian et al., 1979; Quian, 1981; Quian and Wang, 1984; Oligati et al., 1984; Hamasaki et al., 1985; Vainio et al., 1985; Nakamura et al., 1988a).

It has been shown, in spermatogenic cells, that the earliest and most conspicuous damage caused by gossypol takes place in mitochondria (Nakamura et al., 1988a; Abou-Donia, 1976; Tso et al., 1982a; Oko and Hrudka, 1982; Tso et al., 1982b; Kim and Waller, 1984; Nakamura et al., 1988b). In rats, the highest incorporation of [4C]gossypol occurs in mito- chondria (Xu, 1981). In addition, gossypol uncouples oxidative phosphorylation in mitochondria from guinea-pig kidney (Tso and Lee, 1981), and rat liver (Abou-Donia and Dieckert, 1974). Also, gossypol stimulates ATPase activity and mitochondrial respir- ation when monovalent cations are added to the incubation media (Martinez et al., 1988). Since the effect of gossypol in different models appears to be related to the presence of cations, we studied the role of Mg2+ and K+ in the action of gossypol in isolated rat liver mitochondria and in submitochondrial particles.

MATERIALS AND METHODS

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,~11 per ml. Control experiments were carried out with equivalent amounts of ethanol. All other reagents and substrates were of the highest purity available.

Liver mitochondria from male Wistar adult rats and submitochondrial particles (SMP) were prepared as de- scribed by Martinez el al. (1988). The mitochondria and SMP were suspended in 0.25 M sucrose, 1 mM EDTA, adjusted to pH 7.3 with Tris-base. Protein was determined by the method of Lowry rr al. (1951), using bovine serum albumin as standard.

Oxygen uptake was measured polarographically using a Clark-type electrode. Mitochondrial protein (3 mg) was added to 3 ml of the reaction mixture at 25C containing: IOmM malate, 1OmM glutamate, 1OmM phosphate pH 7.3, 100 mM KCl, and when Mg2+ was present. 10 mM. In the experiments using potassium-free media, a solution of 0.25 M sucrose. 1 mM EDTA. PH 7.3 was added to main- tain the osmolarity.

ATPase activity was assayed as described by Martinez et al. (1988). After stopping the reaction with 6% trichloro- acetic acid, the released phosphate (Pi) was quantified as reported by Sumner (1944).

Membrane potential in mitochondria was quantified as reported by Akerman and WikstrBm (1976).

RESULTS

Effects of gossypol on oxygen consumption

Figure 1 shows that in the presence of 20 PM gossypol plus K+ and Mg2+, the rate of oxygen

CBP IoPc,I-o 87

88 F. MARTINEZ

Fig. 1. Oxygen consumption in the presence of gossy- pol with or without Mg2+ from rat liver mitochon- dria. The incubation medium contained 1OOmM KCI, 10mM MgCl,, 10 mM phosphate, pH 7.3, 1OmM gluta- mate, 10 mM malate; 2 mg of mitochondrial protein (M) were added to a final volume of 3 ml. Numbers indicate the ngat oxygen consumed mg- min-I. Respiration was stimu- lated with ADP (500 nmol), and 20 nM gossypol (G) was added as depicted. Trace A is the control; in trace C, MgCl,

was omitted.

uptake was increased and the respiration became uncoupled (trace B); in absence of Mg2+ a stimulation followed by an inhibition was observed (trace C), suggesting that the effect of gossypol could be related to the presence of K+ and Mg2+.

ATPase activity in presence of gossypol plus MgZf

ATPase, an enzyme from the inner mitochondrial membrane, is stimulated by gossypol when K+ is present in the incubation medium (Martinez et al., 1988). The following experiment was performed to determine if gossypol could stimulate ATPase in the presence of Mg*+ alone. Table 1 shows that gossypol plus 1OmM MgCl, stimulated ATPase activity around 3-fold. However, when Mg2+ was 1 mM, gossypol stimulated only 2-fold, indicating that the gossypol effect depends on the concentration of MgCI,. Indeed, maximal hydrolysis of ATP was

Table I. Effect of gossypol and Mg*+ on ATPase

activitv

WI (mM)

ATPase activity

nmol Pi released mg-. min. + gossypol (2u~M)

0 17.9 + 3.9 25.9 f 4.6

I 16.3 f 2.0 31.0 * 1.9 IO 36.6 k I I .O 85.5 * 0.5

Experimental conditions were as in Fig. 2. Results

represent the mean value of four individual

determinations.

7 .E 200 E

;

e

B 8 s loo L

ii

5

F = 0

A

0 IO 20 30

MgCl2 tmM)

h I ij .

E 0 I 0 IO 20 30

MgCI;! (mM)

Fig. 2. ATPase activity in rat liver mitochondria in the presence of gossypol and increasing MgCl, concentrations. ATP hydrolysis was assayed in a 1 ml medium containing 10 mM Tris-HCl, pH 7.3, 20 FM gossypol (-_O-), plus 1 mg of mitochondrial protein, and 250mM sucrose (panel A) or 1OOmM KCl (panel B). ATPase activity was started with 2.5 mM ATP after 5 min incubation at 30C. ATPase activity was also assayed in the absence of gossypol

(--).

observed with about 3 mM MgCI, (Fig. 2A). A control experiment was performed without gossypol; Mg2+ alone had no effect on ATPase (Fig. 2A).

Effect of gossypol, h4g2+ and KC, on ATPase activity

Table 2 shows that gossypol plus K+ stimulated ATPase to a level higher than that observed with 10 mM MgCl, alone; however, in the presence of gossypol, K+ and 1 mM MgCl,, ATPase activity was stimulated around 43% with respect to that of gossy- pol and K+. Figure 2B shows that gossypol plus K+ and Mg2 + increased ATPase activity in a dose-related Mg-dependent fashion, reaching maximal ATP hy- drolysis with 1 mM MgCl,. Table 3 shows that EDTA abolishes the simulation effect produced by gossypol plus MgCI, .

Table 2. Effect of gossypol, K+ and Mg+ on

[WI (mM)

ATPase activity

ATPase activity

nmol Pi released mg- min-

0 I

IO

15.3 f 4.4

21.7k2.7

21.4 + 9.9

+ gossypol @PM)

143.1 f 20.5

205.5 + 32.1

89.5 + 4. I

Experimental conditions were as in Fig. 2. Results

represent the mean value of three individual

determinations.

Mitochondrial functions in the presence of Mg*+ 89

Table 3. Effect of EDTA on gossypol-Mg-stimulated ATPase

With K+ Without K+ EDTA Mg2+

(mM) (1 mM) (ZL,

0 141.5 + 17.0 213.3 + 21.0 30.3 _+ 0.1 28.0 + 6.2 I 120.6 + 14.8 150.7 * 13.7 33.7 * 4.1 32.3 + 4.3 2 120.8 k 19.0 131.9 f 30.5 38.8 f 5.7 31.8+0.8 3 120.2 k 20.4 108.5 + 46.3 43.1 f 0.3 35.0 f I .3

Experimental conditions were as in Fig. 2. Results represent the mean value of four individual determinations.

Localization of gossypol effects

To explore whether mitochondrial integrity is re- quired for the effect of gossypol on the ATPase activity, its effect was assayed in SMP. It was observed that despite the presence of Mg2+, with or without K+, ATPase activity was not modified by gossypol (Fig. 3). These findings strongly suggest that gossypol effects on ATPase activity are not directly on the enzyme, but rather are exerted at the membrane level, such as collapse of the membrane potential, which could be responsible for the ATPase activity stimulation. An experiment to determine the membrane potential in the presence of gossypol plus Mg2+, with or without K+, was performed. The results showed that in the presence of K+, gossypol collapsed the membrane potential faster than when only gossypol plus Mg2+ were in the incubation medium (Fig. 4A-C).

DISCUSSION

Previous reports suggest that the effect of gossypol depends on the ionic composition of the reaction medium (Martinez et al., 1988). The results presented in this work show that gossypol increases the uncou- pling of respiration when the combination Mg+, K+, and gossypol were in the respiration medium. With

0 b 0 IO 2d 40 80 loo

[ Gossypol] (FM)

Fig. 3. Effect of different gossypol concentrations on ATPase activity in submitochondrial particles. ATPase ac- tivity was assayed in a 1 ml medium containing 10mM Tris-HCl, pH 7.3, IOmM MgCI,, 1 mg of protein from submitochondrial particles, and 250 mM sucrose (-_O-) or IOOmM KC1 (--). The activity was started with 2.5 mM ATP. Bars indicate the standard deviation of 7

determinations.

respect to the role of Mg2+, an association between divalent cations and gossypol has been described (Ramaswamy and OConnor, 1969), and the addition of iron, in the presence of calcium salts, decrease the concentration of free gossypol in food mixtures (Bressani et al., 1964). Also, the addition of 0.1% Ca(OH), and 0.1% FeSo,-7H,O eliminates the toxicity of gossypol (Jarquin et al., 1966; Braham et al., 1967), suggesting a close relationship between gossypol and cations.

Furthermore, it has been reported that the M$+- gossypol complex exhibits a high antispermatogenic effect in rats, as compared to the effect of free gossypol (Shi et al., 1981). Since gossypol and Mg2+ did not affect ATPase activity, the stimulation of

sue 1 IO mM MgCl

J \ _J

f

J I A= 0 05 50"

Fig. 4. Mitochondrial membrane potential in the presence of gossypol, K+, and M&+. Mitochondria (2 mg) were incubated in a medium containing 1Opg rotenone, 37.5 PM Safranine, and 100mM KC1 or 250 mM sucrose in a final volume of 2 ml. The membrane potential was started by the addition of 5 mM succinate, and followed in an Aminco spectrophotometer (dual mode) using the difference between

5 1 l-533 nm. For each trace, the particular additions are depicted.

90 F. MARTINEZ

ATPase activity in rat liver mitochondria could be explained on the basis that gossypol plus K+ and Mg2+ may form complexes at the membrane level, which produce the increase observed in respiration and ATPase activity. Alternatively, the combination of gossypol and cations increases the mitochondrial permeability to H+, as has been suggested (Martinez et al., 1988).

On the other hand, it has been reported that Mg2+ can regulate the permeability of the inner membrane of mitochondria to monovalent cations, probably by regulating the K+/H+ antiport activity (Garlid, 1988; Brierley et af., 1984). Mg2+ is also involved in the maintenance of mitochondrial membrane structure and integrity (Jung and Brierley, 1986). Our data cannot be explained by an Mg2+ effect, since ATPase activity in the presence of K+ and Mg2+ without gossypol was not stimulated.

Thus, the effect of gossypol could be related to an action on the energy conservation mechanism by mitochondria. Indeed, the rate of the effect of gossy- pol on the potential was faster in the presence of 1 mM MgCl, and KC, than with K+ alone, sucrose, or 10 mM MgCl, plus K +, suggesting a primary effect of gossypol on the membrane potential.

Acknowledgements-The author thanks Dr Armando Gbmez-Puyou for his helpful criticisms of the manuscript, and Federico Martinez, Jr for the illustrations. This work was partially supported by Grant IN2000189 IFCjUNAM from the Direccibn General de Asuntos de1 Personal Aca- dtmico de la Universidad National Autonoma de Mexico.

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