Department of Biochemistry, Imperial College, Prince Consort Road,. London ... Bradford, 1972a) and noradrenaline (Blaustein et al., 1972) from cerebral cortex, glycine ... [L-tyrosine and DL-dopa (3,4-dihydroxyphenylalanine)] was measured.
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This work was partially supported by an M.R.C. grant to S. P. R. R. Rose, S.P. R. (1967) Biochem. J. 102,3343 Rose, S. P. R. & Sinha, A. K. (1969) J. Neurochem. 16,1319-1328 Sellinger, 0. Z., Azcurra, J. M., Johnson, D. E., Ohlsson, W. C. & Lodin, Z. (1971) Nurure (London)New Biol. 230,293-296
The Release of Endogenous 3,6Dihydroxyphenethylamine from Synaptosomes Isolated from Corpus Striatum J. S. DE BELLEROCHE and H. F. BRADFORD Department of Biochemistry, Imperial College, Prince Consort Road, London S. W.7, U.K. Synaptosomes are now known to possess a high degree. of metabolic independence during incubation in physiological medium, containing glucose as a substrate (see review by de Belleroche & Bradford, 1973). Under these conditions, depolarizing agents induce the calcium-dependent release of physiologically active compounds, including acetylcholine (de Belleroche &Bradford, 19726),aspartate, glutamate, y-aminobutyrate (de Belleroche & Bradford, 1972a) and noradrenaline (Blaustein et al., 1972) from cerebral cortex, glycine, aspartate and y-aminobutyrate from spinal cord (Osborne et al., 1973), and releasingfractors from hypothalamus (Edwardson et al., 1972). In the present study, this experimental system was used to study the metabolism and release of dopamine (3,4dihydroxyphenethylamine) from synaptosomes prepared from corpus striatum where it is likely to act as an inhibitory transmitter as judged by histochemical, electrophysiological and biochemical evidence (see review by Hornykiewicz, 1973). Both electrical pulses and amphetamine induce dopamine release in vivo from corpus striatum (McLennan, 1965; Portig &Vogt, 1969; Riddell & Szerb, 1971; Beeson etal., 1971) and in uitro either from striatal slices (Baldessarini & Kopin, 1966; Farnebo et af., 1971) or from striatal synaptosomes pre-loaded with radioactively labelled dopamine (Ferris et al., 1972). To characterize nerve-terminal compartments of dopamine (Javoy & Glowinski, 1971) involved in specific release processes, the release of both endogenous dopamine and dopamine synthesized in situ from radioactively labelled precursors [L-tyrosine and DL-dopa (3,4-dihydroxyphenylalanine)] was measured. Synaptosomes were isolated from sheep corpus striatum by conventional methods (Gray & Whittaker, 1962) and incubatedin the formof a'synaptosomebed'aspreviously described (de Belleroche & Bradford, 1972a). Oxygen consumption by the striatal synaptosomes was linear over at least 90min of incubation at a rate of 61.0k4.0 (6)pmol of oxygen consumed/h per lOOmg of protein, which was comparable with the rates for synaptosomes isolated from other brain regions. Endogenous dopamine was measured fluorimetically after separation with alumina by the methods of Anton & Sayre (1962) and Laverty & Taylor (1968). Radioactively labelled dopamine was isolated together with non-radioactive carrier by using a cation-exchange resin (Zeocarb 225) before liquid-scintillation counting. Electrical pulses, elevated potassium ( 5 6 m ~ and ) D- and L-amphetamine (0.11 9 m ~ ) induced the release of endogenous dopamine from synaptosome beds substantially above the control amounts (Table 1). By using radioactively labelled DL-dopa, radioactive dopamine was readily formed. Labelled dopamine was released during incubation and the amount released was increased by amphetamine but not by depolarizing agents during incubation periods of up to 30min. The D-isomer was most potent in this respect. However, with incubation times of greater than 30min potassium stimulation in the presence of D-amphetamine caused an increase in the release of radioactive dopamine. This may be taken as evidence of serial labelling of different dopamine compartments within the nerve endings. Amphetamine appears to induce release from a compartment which becomes very rapidly labelled and the depolarizing agents release dopamine from a compartment characterized by a lower rate of labelling.
vol. 3
Y
Expt. A
(nmol of dopamine/ lOOmg of protein) 40 Condition Time of incubation (min) ... Control 1.22 f 0.21 (5) Potassium stimulation 2.06 f 0.23 (6) 2.76 k 0.22 (3) Electrical stimulation D-Amphetamine (0.1 19mM) 5.26f 0.45 (4) Potassium stimulation with 5.94 f 1.23 (4) D-amphetamine (0.1 19mM)
I
401.6 2 35.0 (6) 466.0 f 23.7 (5)
40 -
336.02 41.2 (3) 242.0 f 19.4 (4)
-
Y
-
DL-Dopa
DL-Dopa 20 70.9 f 15.8 (5) 69.8 f 12.0 (6)
I
Expt. B (nCi of dopamine/lWmg of protein)
Release of dopamine \
Synaptosome beds (approx. lOmg of protein) were incubated in all experimentsin Krebs bicarbonate medium (Umbreit et al., 1964)containing glucose (10mM) and L-ascorbate ( 0 . 5 m ~ at ) 37°C. Potassium stimulation was by raising the medium potassium concentration to 5 6 m with ~ KCl and electrical stimulation was with alternating pulses of lOV, 0.4ms duration and 1OO/s. Both types of stimulation were carried out during the final lOmin of incubation. In Expt. A, synaptosome beds were incubated for 4Omin and the dopamine in the medium was measured fluorimetrically after alumina purification. The recovery from this procedure was 72 % (range 68-76 %) but a correction for recovery was not made. In Expt. B, synaptosome beds were preincubated for 40min in medium (5ml) containing 1pCi of DL-dopa (~~-3,4-dihydroxy[2-'~C]phenylalanine, 51 mCi/mmol). The beds were then transferred to fresh medium and the incubation continued for a further 20 or 40min. Radioactively labelled dopamine in the incubation medium was purified on a cation-exchangeresin, the recovery for this procedure being greater than 95 %. The values are means+s.a.M. for the number of experiments in parentheses.
Table 1. Dopamine release from synaptosome beds to the incubation medium
5 z m
c
% 9
j;3
-
c(
2cl
5iz 8 F
!c,5
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This work was supported by an M.R.C. programme grant. Anton, A. H. & Sayre, D. F. (1962) J. Pharmacol. Exp. Ther. 138,360-375 Baldessarini, R. J. & Kopin, I. J. (1966) Science 152,1630-1631 Beeson, M., Cheramy, A., Feltz, P. & Glowinski, J. (1971) Brain Res. 32,407-424 Blaustein, M. P., Johnson, E. M., Jr. &Needleman, P. (1972) Proc. Nut. Acad. Sci. U.S.69, 2237-2240 de Belleroche, J. S. &Bradford, H. F. (1972~)J. Neurochem. 19,505-602 de Belleroche, J. S. &Bradford, H. F. (19726) J. Neurochem. 19,1817-1819 de Belleroche, J. S. &Bradford, H. F. (1973)Progress in Neurobiology (Kerkut, G. A. & Phillis, J. W., eds.), vol. 1, pp. 275-298, Pergamon Press, Oxford Edwardson, J., Bennett, G. &Bradford, H. F. (1972) Nature (London) 240,554-556 Farnebo, L. O., Hamberger, B. & Jonsson, G. (1971)J.Neurochem. 18,2491-2500 Ferris, R. M., Tang, F. & Maxwell, R. (1972)J.Pharmacol. Exp. Ther. 181,407416 Gray, E. G. & Whittaker, V. P. (1962) J. Anat. 96,79-87 Hornykiewicz, 0. (1973) Brit. Med. Bull. 29,172-178 Javoy, F. & Glowinski, J. (1971) J. Neurochem. 18,1305-1311 Laverty, R. &Taylor, K. M. (1968) Anal. Biochem. 22,269-279 McLennan, H . (1965) Experientia 21,725-726 Osborne, R. H., Bradford, H. F. & Jones, D. G. (1973) J. Neurochem. 21,407419 Portig, P. J. & Vogt, M. (1969) J. Physiol. (London) 204,687-715 Riddell, D. & Szerb, J. C. (1971) J. Neurochem. 18,989-1006 Umbreit, W. W., Burris, R. H. & Stauffer, J. F. (1964) in Manometric Techniques and Tissue Metabolism, 4th edn., p. 132, Burgess Publishing Co., Minneapolis.
The Role of Potassium Ion Loss in the Anoxic Impairment of Respiration of Rat Cerebral-Cortex Slices NOSHIRWAN J. PATEL* and LESLIE M. FIXTER Department of Biochemistry, University of Glasgow, Glasgow GI2 8QQ, U.K. It has been shown that slices of rat cerebral cortex have a decreased rate of respiration, with glucose as substrate, after a period of anaerobic preincubation (Dickens & Greville, 1933; Elliot & Rosenfeld, 1958). This anoxic impairment of respiration in calcium-free phosphate-buffered salines is caused by the loss of glutamine and aspartate from the slices during the anaerobic preincubation and the addition of these compounds to the preincubation saline protects the slices against this effect of anoxia (Phizackerley & Fixter, 1973). However, it has been found that glutamine and aspartate will not protect brain slices against this anoxic damage in salines containing CaZ+and the present work was undertaken to see whether the loss of any slice component played a role in the anoxic impairment of respiration in Caz+-containing salines. Rat cerebral-cortex slices, 0.3 mm thick, were prepared and preincubated as previously described (Phizackerley & Fixter, 1973). The salines used were based on the phosphatebuffered saline of Krebs (1950) except that Tris-HC1 buffer, pH7.4, was substituted for the phosphate buffer and CaClz wasadded to a final concentration of 1mM. After a 60min preincubation the rate of respiration of the brain slices in saline containing 1 1 . 5 m ~ glucose was determined manometrically at 37°C. K+ content of slices was determined by the method of Varon & Mcllwain (1961). High-speed supernatants were prepared from brain homogenates (50%, w/v) in lOmM-Tris-HC1, pH7.4, by centrifugation at 1OOOOOg,. for 2h. After 60min preincubation aerobically in glucose saline, the rate of respiration was 10.2~1 of Oz/h per mg dry wt., whereas after a similar preincubation in basal saline the rate of respiration was 1 . 8 ~ of 1 0,jh per mg dry wt. This decrease in respiration could be prevented by changes in the anaerobic preincubation condition. The preincubation of slices at 0°C in basal saline, or the preincubation of slices in brain homogenate supernatants at 37"C, did not cause anoxic impairment of respiration. Similarly the preincubation * Present address: Department of Pharmacology, Biocentre of the University of Basle, Basle, Switzerland.
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