Effect of scorpion venom, tityustoxin, on the uptake of calcium by isolated nerve ending particles from brain

182

Brain Research, 93 (1975) 182 -I 87 t ) Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in The N e t h e r l a n d s

Effect of scorpion venom, tityustoxin, on the uptake of calcium by isolated nerve ending particles from brain

M. C E L E S T E H E N R I Q U E S

AND M A R C U S V. G O M E Z

Department of Biochemistry and lmmunology, Institute of Biological Sciences, UFMG, Belo Horizonte, Minas:Gerais (Brazil)

(Accel~tedApril 10th, 1975)

Synaptosomes, isolated nerve ending particles from the brain, accumulate calcium by a process dependent on ATP2,14. The calcium concentration gradient may play an important role in neuronal function1. Calcium not only affects the excitability of the nerve cell membrane 17, but also is an important constituent of the membrane itself ~8. Calcium entry at the presynaptic nerve terminals may be an early step in the process of transmitter release l°,H. Tityustoxin (TsTX), a toxin purified from the venom of the scorpion Tityus serrulatus, increases the release of cellular acetylcholine (ACh) from cortical slices of rat brain 5. This toxin effect is dependent on calcium and sodium in the incubation medium7. The mechanism of ACh release is calcium-coupled and the entry of this cation into the nerve ending is necessary for the release of ACh 9. This suggests that the site of action of TsTX might be the calcium-transport system of the nerve ending. Thus, it would be interesting to study the effect of tityustoxin on the calcium-transport system of the nerve ending. Tityustoxin was purified from Tityus serrulatus venom by a combination of extraction and chromatographic techniques using Sephadex G-25 and carboxymethylcellulose; the procedure allowed the isolation of a highly purified toxin which is homogeneous by polyacrylamide gel electrophoresis6. Tityus venom was a gift of Dr. Carlos R. Diniz, Ribeir~o Preto, SP, Brazil. Isolated presynaptic nerve terminals were prepared from rat brain cortex according to the procedure described by Gray and Whittaker8, using discontinuous sucrose density gradient centrifugation. Synaptosomes were incubated in the presence of 0.32 M sucrose and an incubation medium containing 0.1 mM CaC12, 3 mM MgC12, 3 mM ATP, 50 mM Tris-HC1, pH 7.5, in a final volume of 1.0 ml. Usually, after a 10 min period of pre-incubation in the presence of TsTX, 0.1-O.2/zCi of 45CaCIz was added (Amersham-Searle, Arlington Heights, I11.), specific activity 1 Ci/mmole. Unless otherwise indicated, the synaptosomes were incubated for 6 min at 37 °C in a Dubnoff shaker bath (60 oscillations/min). The reaction was terminated by the addition of 6 ml of ice-cold stopping solution (0.32 M sucrose plus 0.1 mM CaCI2). "[he synaptosomes were separated rapidly from the medium by suction filtration through

183 I

I

I

I

I

I

20O d

Z

8 150 b_ 0

~ I00

--. 50

I

I

I

I

I

I

5

I0

15

20

25

30

TIME (min)

Fig. 1. The tityustoxin (TsTX) effect on the uptake of calcium by synaptosomes plotted as a function of the incubation time. O, without tityustoxin (control); O, with tityustoxin 2.8/,M. Synaptosomes containing 0.12 mg of protein/ml of medium were incubated at 37 °C. The results are means for 3 experiments, and they are expressed as percent of the calcium uptake by the control synaptosomes, incubated for 6 min without TsTX. For other details see the text.

a Millipore filter (0.45/~m). The filters were then rinsed twice with 6 ml and 3 ml of ice-cold stopping solution, dried and immersed in 10 ml of scintillation solution 16. Radioactivity was counted in a Beckman liquid scintillator, Model LS-150. The counting efficiency was corrected for each sample by the external standard method. The amount of 45Ca was calculated from the radioactivity found per mg of synaptosomal protein divided by the specific radioactivity of the isotope present in the incubation medium. Protein was assayed by the method of Lowry et al. la. Lactate dehydrogenase I

I

IOO n~ hZ

8 75 ~5 - ' 50

~ 25 o, I

o

5

I

I0 TIME (rnin)

15

Fig. 2. The effect of TsTX on the uptake of calcium by synaptosomes plotted as a function of preincubation time. O, without tityustoxin (control); O, with tityustoxin 2.8/~M. Synaptosomal protein concentration was 0.13 mg/ml of medium. The results are means for 3 experiments and they are expressed as percent of the calcium uptake, by the control synaptosomes, incubated for 6 rain without TsTX.

184 I©o'

i

i

r

~

I

I

]

--

i

g z5

,,, 50 Nf B_

25

0 0

I

r

ID 20 20 40 TITYUSTOXlN (u,M)

50

Fig. 3. Effect of several concentrations of TsTX on calcium uptake by rat brain synaptosomes. ©, without tityustoxin (control); 0 , with TsTX. 0.12 mg of synaptosomal protein was used per ml of medium. The results are means for 3 experiments, and they are expressed as percent of the calcium uptake by the control synaptosomes, incubated for 6 min without TsTX. For other details see the text.

(L-lactate; NAD oxidoreductase; EC 1.1.1.27) was determined spectrophotometrically by the procedure of Kubowitz and Ott:L The synaptosome accumulation of 45Ca averaged 10.74 4- 1.06 #moles/g protein/6 min (n -= 14). In the presence of TsTX the value was 6.10 4- 0.71/~moles/g protein/6 rain (n -= 14). Thus, for brain synaptosomes, the average reduction of Ca 2 ~ uptake by TsTX was 43 ~. Since the ability of different synaptosomal preparations to accumulate Ca z+ varied, the results are expressed as percent of control. The time course of the effect of TsTX on calcium uptake by synaptosomes is shown in Fig. 1. TsTX causes a reduction in calcium accumulation by nerve ending I

I

I

I

i

I

I

I

5.0

Ld .¢, 0~; 1.0 _D

I

0.1

0.2 05 04 PROTEIN, mg

I

0.5

Fig. 4. The effect of TsTX on calcium uptake as a function of synaptosomal protein concentration. ©, without tityustoxin (control); 0 , with TsTX 2.8 #M. At protein concemrations of 0.10, 0.19 and 0.28 mg/ml of incubation medium the differences were statistically significant, P ~< 0:001. For the other points the differences were not significant. The results are means i $.E.M. for 3 experiments. For other details see the text.

185 TABLE I RELEASE OF LACTATE DEHYDROGENASE FROM SYNAPTOSOMES

Synaptosomes were incubated for 16 min at 37 °C in the medium for measuring 45Ca uptake, in the presence of 2.8/~M tityustoxin, 0.1% Triton X-100, after freezing-thawing (3 ×, 0 °C), or none of these treatments (control). All samples were filtered through a Millipore filter (0.45 /,m) and the filtrate was assayed for lactate dehydrogenase. For each condition duplicate samples were incubated and assayed. Lactate dehydrogenase is expressed as/~moles of substrate utilized per min per mg of protein. Synaptosomal protein concentration was 0.130mg/mi of medium. The values are the means S.D. for duplicate samples. Treatment

Lactate dehydrogenase activity released

% Synaptosomes disrupted

Triton X-100 Freezing and thawing Tityustoxin Control

0.524 -4- 0.11 0.517-4- 0.09 0.065 4- 0.04 0.068 -4-0.03

100 98.6 12.4 12.9

particles. The effect of TsTX varied with the incubation time with a maximum at 30 min. After incubations for 2, 4, 6, 10, 15 and 30 min, TsTX reduced 45Ca uptake by 21, 20, 37, 27, 35 and 56 ~o, respectively. The effect of the pre-incubation time of the tityustoxin on the uptake of 45Ca is shown in Fig. 2. After pre-incubations for 2, 6, 10 and 15 rain, followed by the addition of 45Ca and incubation for additional 6 min, TsTX reduced calcium uptake by 28, 47 54 and 55 ~o, respectively. The reduction of the uptake of calcium caused by TsTX was a function of the tityustoxin concentration (Fig. 3). There was no measurable effect of TsTX at a dose of 1.0 #g/ml of the incubation medium, which corresponds to a molar concentration of 0.14 #M. At a concentration of 0.70/~M, TsTX reduced calcium uptake by 20 ~ . The maximum effect, 41.2 ~ of inhibition could be observed at a concentration of 2.8 #M. The rate of 45Ca uptake and the effect of 2.8/~M TsTX on this accumulation was proportional to the synaptosomal protein concentration (Fig. 4) up to 300 #g/ml. At high synaptosomal protein concentrations 2.8 # M TsTX had little or no effect on the uptake of 45Ca. However, in separate experiments (unpublished observations) it could be demonstrated that the inhibitory effect of TsTX is dependent on the existing concentration rates of TsTX to synaptosomal protein. In an attempt to determine whether TsTX reduces 45Ca accumulation by disruption of brain synaptosomes during the incubation, we measured the release of lactate dehydrogenase, a marker of synaptosomal cytoplasm 4. For these experiments, the activity of lactate dehydrogenase, in the presence of Triton X-100, was assumed to represent 100~o of the synaptosome disruption s. Table I shows that TsTX did not cause extensive synaptosome disruption since the values obtained for the release of lactate dehydrogenase from synaptosomes incubated in the presence or absence of TsTX are identical.

186 Tityustoxin increases A C h release and this effect is dependent on the presence of Ca z~ and N a t ions in the incubation medium 5. TsTX also increases the uptake of 4~Ca and Z4Na by cortical rat brain slices 7. Since calcium entry into the nerve endings is necessary for the release of A C h to take place 9 -11, it was surprising to observe the inhibition by tityustoxin o f the calcium uptake by synaptosomes. However, in view of the recent observations that TsTX releases A C h from incubated synaptosomes (unpublished data), the present results may be taken as supporting the notion that TsTX causes A C h release by a mechanism involving the transport o f calcium across the nerve ending membrane. An intact m e m b r a n e seems to be required for the observed inhibition of calcium influx by TsTX. Thus, we observed that in a synaptosomal preparation that was frozen and thawed, the uptake o f 4~Ca was reduced to 15 ~ o f the control value and the inhibitory effect by TsTX was no longer demonstrable. Since the synaptosomal fraction used in the present experiments is contaminated with fragmented membranes and free mitochondria 1~, and in view of the fact that brain mitochondria actively accumulate calcium 19, one might question whether the inhibition o f 45Ca uptake by TsTX is into the synaptic terminals or into the intrasynaptic mitochondria or both. Experiments are in progress to answer this question. Alternatively, the inhibitory effect o f TsTX on 45Ca uptake by the synaptosomes might be related to and possibly underlie an effect o f the toxin on rates o f calcium efflux from nerve endings and for mitochondria and in more general terms, on calcium movement across physiologically active membranes. This paper was supported by grants from the Conselho Nacional de Pesquisas, Conselho de Pesquisas da U F M G and Bunco Nacional do Desenvolvimento Econ6mico (FUNTEC/199). The authors are grateful to Drs. Otto Z. Sellinger, David Lee Nelson and EniD Cardillo Vieira for their criticism o f the manuscript. We are grateful to Mr. A n t o n i o Soares Usual dos Santos for the expert technical assistance.

1 BRINK, F. JR., The role of calcium ions in neural processes, Pharmacol. Rev., 6 (1954) 243-298. 2 DIAMOND,I., AND GOLDBERG,A. L., Uptake and release of 45Ca by brain microsomes, synaptosomes and synaptic vesicles, J. Neurochem., 18 (1971) 1419-1431. 3 FISZER,S., ANDDE ROBERTlS,E., Action of Triton X-100 on ultrastructure and membrane-bound enzymes of isolated nerve endings from rat brain, Brain Research, 5 (1967) 31--44. 4 FONNUM, F., The distribution of glutamate decarboxylase and aspartate transaminase in subcellular fractions of rat and guinea-pig brain, Biochem. J., 106 (1968) 401-412. 5 GOMEZ,M. V., DAI, M. E. M., AND DINIZ, C. R., Effect of scorpion venom, tityustoxin, on the release of acetylcholine from incubated slices of rat brain, J. Neurochem., 20 (1973) 1051-106I. 6 GOMEZ,M. V., ANDDINIZ, C. R., Separation of toxic components from the Brazilian scorpion Tityus serrulatus - venom, Mere. Inst. Butantan, 33 (1968) 899-902. 7 GOMEZ,M. V., DINIZ,C. R., ANDBARBOSA,J. S., A comparison of the effects of scorpion venom, tityustoxin, and ouabain on the release of acetylcholine from incubated slices of rat brain, J. Neurochem., 24 (1975) 331-336. 8 GRAY,E. G., AND WHITTAKER,V. P., The isolation of nerve endings from brain. An electron microscopic study of cell fragments derived by homogenization and centrifugation, J. Anat. (Lond.), 96 (1962) 79-88. 9 HODGKIN,A. L., AND KEYNES, R. D., Movements of labeled calcium in squid giant axons, J. Physiol. (Lond.), 138 (1957) 253-281.

187 10 KATZ, B., AND MILEDI, R., The effect of calcium on acetylcholine release from motor nerve terminal, Proc. roy. Soc. B, 161 (1965) 496-503. 11 KATZ, B., AND MILEDI, R., The release of acetylcholine from nerve endings by graded electric pulses, Proc. roy. Soc. B, 167 (1967) 23-38. 12 KUBOWlTZ, F., UND OTT, P., Isolierung und Krystallisation eines G~irungferments aus Tumoren, Biochem. Z., 314 (1943) 94-117. 13 LOWRY,O. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL, R. J., Protein measurement with phenol reagent, J. biol. Chem., 193 (1951) 265-275. 14 LUST, W. D., AND ROBINSON,L. D., Calcium accumulation by isolated nerve ending particles from brain. I. The site of energy dependent accumulation, J. Neurobiol., 1 (1970) 303-316. 15 MZCHAELSON,J. A., AND WHITTAKER, V. P., The subcellular localization of 5-hydroxytryptamine in guinea-pig brain, Biochem. Pharmacol., 12 (1963) 203-211. 16 PATTERSON, M. S., AND GREENE, R. C., Measurement of low energy beta-emitters in aqueous solution by liquid scintillation counting emulsion, Anal. Chem., 37 (1965) 854-857. 17 SHANES,A. M., Electrochemical aspects of physiological and pharmacological actions in excitable cells. I. The resting cell and its alterations by extrinsic factors, Pharmacol. Rev., 10 (1958) 59-164. 18 ToalAS, J. M., Experimentally altered structure related to function in the lobster axon: extrapolation to molecular mechanisms in excitation, J. cell comp. Physiol., 52 (1958) 89-100. 19 TOWER, D. B., Ouabain and the distribution of calcium and magnesium in cerebral tissues in vitro, Exp. Brain Res., 6 (1968) 273-283.