Journal of Neurochemrstry Vol. 30, pp. 1627-1628 Pergamon Press Ltd. 1978. Printed in Great Britain 0 International Society lor Neurochemistry Ltd.
0022-3042/78/060 I- I627102.00/0
SHORT COMMUNICATION ~~
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The effect of sodium pentobarbital on some mitochondria1 enzymes (Received 9 September 1977. Accepted 19 September 1977)
THEBARBITURATE interference with the carbohydrate metabolism and energy-rich metabolites has been described previously (GATFIELD et al., 1966). Moreover, the barbiturates were reported not only to interfere with cellular respiration but to disturb the oxidative phosphorylation of mitochonet al., dria (BRODY& BAIN, 1954; COHEN,1973; PERSKY 1950). In this communication we will describe a transient pentobarbital inhibition of monoamine oxidase (MAO) and cytochrome oxidase (Cy 0)activities which were observed during the investigation of ischemic effect on some mitochondrial enzymes. Mongolian gerbils in groups of 12 animals were anesthetized with intraperitoneal injection of Na pentobarbital (20 mg/kg body weight) and kept alive for %270 min as well as for 24 h. At the end of each experimental period the gerbils were killed by decapitation. The brain was removed quickly and homogenized immediately in 0.32 M-sucrose at pH 7.0. The mitochondrial fraction was prepared according to the method of WHI-ITAKER& BARKER (1972). The M A 0 activity was assayed by microfluorometric techniques using Baird spectrophotofluorometer which measures the conversion of kynuramine to 4-hydroxyquinoline (KRAJL, 1965). The Cy 0 activity was determined by ultra microspectrophotometric method of HES & POPE(1953) based on oxidation of reduced cytochrome c. Additional 12 animals without barbiturate treatment were processed the same way and served as controls. The proteins were determined by LOWY'Stechnique (1951). Progressively decreased activities of both enzymes were observed up to 150min postbarbiturate treatment as illustrated in the table. M A 0 was affected more significantly than Cy 0 activity at 90min when each of the enzymes was compared with its respective controls. However, M A 0 recovery occurred earlier than that of Cy 0 activity. This pentobarbital depression of M A 0 and Cy 0 activities although transient is noteworthy especially since we have not observed any significant additional decrease in the activity of both brain enzymes until 5 h of continuous deprivation of cerebral blood supply or 20 h after 1 h of transient ischemia (unpublished observations). Besides the ischemic reduction of glycolytic activity in the brain of pentobarital treated animals described by GATFIELD et al. (1966) can be excluded in these animals since the recovery from such effect was seen within 2 h in the nonischemic
TABLE1. MIT~CHONDRIAL ENZYME Postinjection time Control 90 min 150 min 210 min 24 h
MA0 (1.4.3.4.H 22.2 f 0.18 18.3 If: 0.18* 17.1 f 0.58* 21.8 f 0.50 22.3 f 0.79
ACTIVITY
CY 0 (1.9.3.1)tJ 22.1 f 0.75 19.1 f 0.66** 17.3 f 0.23" 19.5 f 0.76** 21.6 f 1.02
* P < 0.01 **P < 0.05 t Each number represents M.E.M. f S.E. of nmol 4HOQ/mg protein/h obtained from 12 determinations. J Each number represents M.E.M. i S.E.of pg Cy C/y protein/h obtained from 12 determinations.
cerebral hemispiere of gerbils injected with higher dose of pentobarbital [(35 mg/kg body weight) MRSULJAet al., 19751 than the one used here. Nevertheless, the function of both enzymes has been reported to be affected by anoxia (SPECTOR,1963). On the other hand the barbiturates were described to depress the oxygen uptake in cell culture (FINKet al., 1969) but were also observed to stimulate the M A 0 activity in in uitro studies (BELLIN& SORRENTINO,1974). Such a discrepancy could be the result of different modes of investigation in each case. The findings described here are in support of the former observation suggestive of barbiturate interference with oxygen uptake, especially since the Cy 0 has been affected the same way as the MAO. Moreover, other anesthetics such as chloroform and carbon dioxide inhibit the activity of M A 0 too (FELDMAN & ROCHE,1976). In view of these observations, pentobarbital and other anesthetics may conceivably affect the level of neurotransmitters in the brain. Section on Neurocytobiology, Laboratory of Neuropathology and Neuroanatomical Sciences, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, M D 20014, U.S.A.
D. MICIC I. KLATZO M. SPATZ'
REFERENCES
BELLINJ. S. & SORRENTINO G. M. (1974) Res. Commun. chem. Path. Pharmac. 9, 673-680. BRODYT. M. & BAINJ. A. (1954) J . Pharmac. exp. Ther. I To whom correspondence should be sent. 110, 148-156. Abbreviations used: MAO, monoamine oxidase; Cy 0, COHENP. J. (1973) Anesthesiology 39, 153-164. cytochrome oxidase. 1627
1628
Short communication
FELi)MAN J. M. & ROCHE J. M. (1976) Pharmac. Biochem. Behav. 4, 447-453. FINKB. R., KENNYG . E. & SIMPSON W. E. (1969) Anesthesiology 30, 15@l55. GATFIELDP. D., LQWRY 0. H., SCHULZ D. W. & PASSONNCAU J. V. (1966) J. Neurochem. 13, 18519.5. HESSH. H. & POPEA. (1953) J. hiol. Chrm. 204, 295306. KRAJLM. (1965) Biochrm. Pharmac. 14, 1683-1685.
LOWRY 0. H., ROSEBROUGH N. J., FARRA. L. & RANDALL J. R. (1951) J . hiol. Chem. 193, 265-275. MRSULJAB. B., MRSULJAB. J., ITO U., WALKER, J. T., JR., SPATZM. & KLATZOI. (1975) Aciu nruropath. 33, 91-103. PERSKV H., GOLDSTEIN M .S. & LEVINER. (1950) J. Pharmac. exp. Ther. 100, 273-283 SPECTORR. G . (1963) Bri. J. exp. Path. 44, 251-254. WHITTAKER V. P. & BARKER I. A. (1972) Methods of Neurochemistry 2, 2-52.
0022-3042/78/060 I- I627102.00/0
SHORT COMMUNICATION ~~
~
~
~~
The effect of sodium pentobarbital on some mitochondria1 enzymes (Received 9 September 1977. Accepted 19 September 1977)
THEBARBITURATE interference with the carbohydrate metabolism and energy-rich metabolites has been described previously (GATFIELD et al., 1966). Moreover, the barbiturates were reported not only to interfere with cellular respiration but to disturb the oxidative phosphorylation of mitochonet al., dria (BRODY& BAIN, 1954; COHEN,1973; PERSKY 1950). In this communication we will describe a transient pentobarbital inhibition of monoamine oxidase (MAO) and cytochrome oxidase (Cy 0)activities which were observed during the investigation of ischemic effect on some mitochondrial enzymes. Mongolian gerbils in groups of 12 animals were anesthetized with intraperitoneal injection of Na pentobarbital (20 mg/kg body weight) and kept alive for %270 min as well as for 24 h. At the end of each experimental period the gerbils were killed by decapitation. The brain was removed quickly and homogenized immediately in 0.32 M-sucrose at pH 7.0. The mitochondrial fraction was prepared according to the method of WHI-ITAKER& BARKER (1972). The M A 0 activity was assayed by microfluorometric techniques using Baird spectrophotofluorometer which measures the conversion of kynuramine to 4-hydroxyquinoline (KRAJL, 1965). The Cy 0 activity was determined by ultra microspectrophotometric method of HES & POPE(1953) based on oxidation of reduced cytochrome c. Additional 12 animals without barbiturate treatment were processed the same way and served as controls. The proteins were determined by LOWY'Stechnique (1951). Progressively decreased activities of both enzymes were observed up to 150min postbarbiturate treatment as illustrated in the table. M A 0 was affected more significantly than Cy 0 activity at 90min when each of the enzymes was compared with its respective controls. However, M A 0 recovery occurred earlier than that of Cy 0 activity. This pentobarbital depression of M A 0 and Cy 0 activities although transient is noteworthy especially since we have not observed any significant additional decrease in the activity of both brain enzymes until 5 h of continuous deprivation of cerebral blood supply or 20 h after 1 h of transient ischemia (unpublished observations). Besides the ischemic reduction of glycolytic activity in the brain of pentobarital treated animals described by GATFIELD et al. (1966) can be excluded in these animals since the recovery from such effect was seen within 2 h in the nonischemic
TABLE1. MIT~CHONDRIAL ENZYME Postinjection time Control 90 min 150 min 210 min 24 h
MA0 (1.4.3.4.H 22.2 f 0.18 18.3 If: 0.18* 17.1 f 0.58* 21.8 f 0.50 22.3 f 0.79
ACTIVITY
CY 0 (1.9.3.1)tJ 22.1 f 0.75 19.1 f 0.66** 17.3 f 0.23" 19.5 f 0.76** 21.6 f 1.02
* P < 0.01 **P < 0.05 t Each number represents M.E.M. f S.E. of nmol 4HOQ/mg protein/h obtained from 12 determinations. J Each number represents M.E.M. i S.E.of pg Cy C/y protein/h obtained from 12 determinations.
cerebral hemispiere of gerbils injected with higher dose of pentobarbital [(35 mg/kg body weight) MRSULJAet al., 19751 than the one used here. Nevertheless, the function of both enzymes has been reported to be affected by anoxia (SPECTOR,1963). On the other hand the barbiturates were described to depress the oxygen uptake in cell culture (FINKet al., 1969) but were also observed to stimulate the M A 0 activity in in uitro studies (BELLIN& SORRENTINO,1974). Such a discrepancy could be the result of different modes of investigation in each case. The findings described here are in support of the former observation suggestive of barbiturate interference with oxygen uptake, especially since the Cy 0 has been affected the same way as the MAO. Moreover, other anesthetics such as chloroform and carbon dioxide inhibit the activity of M A 0 too (FELDMAN & ROCHE,1976). In view of these observations, pentobarbital and other anesthetics may conceivably affect the level of neurotransmitters in the brain. Section on Neurocytobiology, Laboratory of Neuropathology and Neuroanatomical Sciences, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, M D 20014, U.S.A.
D. MICIC I. KLATZO M. SPATZ'
REFERENCES
BELLINJ. S. & SORRENTINO G. M. (1974) Res. Commun. chem. Path. Pharmac. 9, 673-680. BRODYT. M. & BAINJ. A. (1954) J . Pharmac. exp. Ther. I To whom correspondence should be sent. 110, 148-156. Abbreviations used: MAO, monoamine oxidase; Cy 0, COHENP. J. (1973) Anesthesiology 39, 153-164. cytochrome oxidase. 1627
1628
Short communication
FELi)MAN J. M. & ROCHE J. M. (1976) Pharmac. Biochem. Behav. 4, 447-453. FINKB. R., KENNYG . E. & SIMPSON W. E. (1969) Anesthesiology 30, 15@l55. GATFIELDP. D., LQWRY 0. H., SCHULZ D. W. & PASSONNCAU J. V. (1966) J. Neurochem. 13, 18519.5. HESSH. H. & POPEA. (1953) J. hiol. Chrm. 204, 295306. KRAJLM. (1965) Biochrm. Pharmac. 14, 1683-1685.
LOWRY 0. H., ROSEBROUGH N. J., FARRA. L. & RANDALL J. R. (1951) J . hiol. Chem. 193, 265-275. MRSULJAB. B., MRSULJAB. J., ITO U., WALKER, J. T., JR., SPATZM. & KLATZOI. (1975) Aciu nruropath. 33, 91-103. PERSKV H., GOLDSTEIN M .S. & LEVINER. (1950) J. Pharmac. exp. Ther. 100, 273-283 SPECTORR. G . (1963) Bri. J. exp. Path. 44, 251-254. WHITTAKER V. P. & BARKER I. A. (1972) Methods of Neurochemistry 2, 2-52.
