INCREASE IN ACCUMULATION OF L-DOPA (3,4-DIHYDROXY PHENYLALANINE) IN BRAIN SLICES BY ALCOHOL

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1 INCREASE IN ACCUMULATION OF L-DOPA (3,4-DIHYDROXY PHENYLALANINE) IN BRAIN SLICES BY ALCOHOL KENICHI KANIIKE* AND HIROSHI YOSHIDA Department of Pharmacology, Faculty of Medicine, Osaka University, Osaka Received for publication June 10, 1963 In our previous paper it was described that an amino acid, L-dopa (3,4-dihydroxy phenylalanine) was accumulated in brain slices against concentration gradient and an energy dependent active transport process was assumed to be participated in this pheno menon (1, 4). The carrier system for this active transport of L-dopa was assumed to divide at least into two parts : a specific site to combine with L-dopa and the Na+, K+ activated ATP-ase which seems to play a common important role for many active trans - port processes. Moreover it was discussed on a role of intracellular K+ concentration in this active transport process (1-4). In this paper, effect of alcohol on accumulation of dopa in brain slices is examined, for alcohol is a typical neurotropic and lipotropic agent and it is assumed that alcohol may exert a certain influence on the phenomenon and it may be possible to get a clue to clarify a mode of action of alcohol on nervous system. 1. Incubation of brain slices with dopy METHODS Brain cortex slices of adult guinea pigs were incubated at 37 C for 30 min with 0.1 mm L-dopa in glucose Ringer solution at ph 7.0, and then amount of dopa accumulated in the slices was estimated according to the methods described in our previous paper (1). In this experiments incubation tubes were closed with stoppers to prevent evapora tion of alcohol during incubation. 2. Procedures to examine intracellular distribution of dopa After incubation with dopa for 1 hr, the slices were washed twice with ice cold Ringer solution and transferred into glass homogenizer. Then, the slices were homo genized with cold 0.25 M sucrose and fractionated as follows : the homogenate was centrifuged for 10 min at 600 x g in a refrigerated centrifuge to remove debris and the supernatant was centrifuged for 10 min at 8,500 x g and the sediment was taken as mito chondria) fraction. Microsomal and soluble fractions were divided by further centrifuga tion of the supernatant for 1 hr at 100,000 x g. Amount of dopa in each fraction was measured by the method described in our previous paper (1) after extraction with 0.4 N perchloric acid. * Present address : Department of Pharmacology, Nara Medical College, Kashiwara.

2 3. Experiment on influx of 14C-dopa Effect of alcohol on influx of 14C-dopa into brain slices under conditions where there was no net transport of dopa was examined as follows: Brain slices were pre incubated for 60 min with 0.1 mm nonradioactive L-dopa in presence or absence of alcohol, and then transferred into media containing the same concentration of 14C-dopa instead of nonradioactive dopa with or without alcohol. Reincubation was carried for 10 min. Absolute amount and radioactivity of dopa present in the incubated slices were estimated with fluorometric method and with gas-flow-counter (Nuclear-Chicago) after washing the slices twice with ice cold Ringer. TABLE 1. Stimulatory effect of alcohol on dopa accumulation in brain slices. * standard deviation ** number of experiments FIG. 1. Time course of stimulatory effect of ethanol on dopa accumulation in brain slices. RESULTS 1. Stimulatory effect of alcohol on dopa accumulation in brain cortex slices As shown in Table 1 and Fig. 1, ethanol showed a remarkable stimu latory effect on the accumulation of L-dopa in brain slices at concentra tions of %. n-butanol also caused a stimulation at 0.1%. 2. Intracellular distribution of dopa in brain slices It is considered that amount of substance inside the cell are regulated by processes which include influx into the cell, efflux from the cell and bind ing with the cellular constituents, even if the substance is metabolically inert. Dopa is metabolized in brain slices, but the amount of dopa in the slices under our experimental condition is not so strongly depend

3 on its metabolic destruction as described in our previous paper (1). Therefore, intra cellular distribution of dopa in brain slices was examined to know whether binding with particles concerned or not with the increase in amount of dopa accumulated in brain slices caused by ethanol. As shown in Table 2, the major part of dopa was found in soluble fraction in both the control and the ethanol treated slices. Therefore, it seemed unlikely to consider that binding of dopa with the cellular constituents might play an important role in accumulation of dopa in brain slices and in an increase of its ac cumulation by ethanol. TABLE 2. Intracellular distribution of dopa accumulated in brain slices. * mean value of 3 experiments ± standard deviation 3. Influence of ethanol on influx of 14C-dopa into brain cortex slices The accumulation of dopa in brain slices was increased by addition of ethanol as shown in Fig. 1 and Table 1, but such a result can be obtained by an increase in influx and also by a decrease in efflux of dopa from the cell. To distinguish the above two possibilities, influx of 14C-dopa was measured for 10 min after 60 min of preincubation. Results showed that the influx of dopa into brain slices was rather decreased by ethanol (Table 3). TABLE 3. Effect of ethanol on influx of 14C-dopa into brain slices. * number of experiments ** mean value ::L standard deviation 4. Inhibition on leak out of dopa by ethanol Above described expriments led us to consider that the increase in dopa accumula tion by ethanol seemed to be due to an inhibition of efflux. Thus effect of ethanol on efflux of dopa from the brain slices was examined according to the method described in our previous paper (2). As shown in Table 4, presence of ethanol resulted in an inhibition of efflux of dopa from the brain slices apparently.

4 TABLE 4. Effect of ethanol on leak out of dopa from the accumulated brain cortex slices. * mean value,-.l standard deviation (number of experiments) DISCUSSION An accumulated amount of amino acid inside the cell is regulated by active influx, passive efflux, binding with cellular components, metabolism of amino acid and others. In the results presented in this paper it is clearly demonstrated that amount of dopa accumulated in brain slices is increased by addition of ethanol. The site of ethanol action to cause an increase in dopa accumulation should be present in above described points. Conversion of dopa to dopamine and noradrenaline in brain slices and autoxida tion of dopa in the medium do not seem to have an important influence on amount of dopa accumulated in brain slices under our experimental conditions as pointed in our previous paper (1). In this paper it is shown that the major part of dopa in the slices is present in free form and binding of dopa with the intracellular particulate components does not seem to play an important role even in presence of ethanol. Another problem to be taken into considerations is swelling of the slices. Wet weight of brain slices is increased by incubation and this swelling is promoted by ethanol. But the increase in swelling by ethanol was within 5% and this is not enough to explain the remarkable increase in accumulation of dopa in brain slices by ethanol. Therefore, it is thought that active influx and passive efflux may be important to cause the increase in accumulation of dopa. However our data presented in Table 3 show that influx of 14C-dopa was rather decreased by addition of ethanol. Though exchange reaction of dopa might be observed under our experimental conditions, it is generally considered that exchange reaction may have a similar mechanism with process of active net uptake (5, 6). Therefore, the result can not support an assumption that ethanol would increase active uptake of dopa by the slices. Accordingly, it seemes more reasonable to consider that ethanol caused an increase in accumulation of dopa by an inhibition of passive efflux. The finding that the increase in accumulation of dopa by ethanol become manifest with time as shown in Fig. 1 may support above consideration and the data presented in Table 4 show directly that ethanol decreases efflux of dopa from the slices. Effect of alcohol on permeability of cell membrane has been studied by a number of investigators. For example, Jacobs (7) suggested that butanol increased permeability

5 of erythrocyte to cations and decreased to anions, and also to non-electrolyte when an active process was involved in the movement. On transport of amino acid, Johnstone and Quastel (9) reported that lipotropic agents such as tribromethanol, chloretone, pentothal, indole and skatol, inhibited transport of glycine into Ehrlich's ascites cells and also inhibited the efflux from the cells. These results are agreed with our data presented in this paper, but in their case the amount of glycine accumulated in the cell was decreased by the lipotropic agents. In our preliminary experiments it was shown that chloretone and amytal also had a tendency to decrease the amount of dopa accu mulated in brain slices. Therefore, it is considered that alcohol and other lipotropic agents may have a common property to inhibit both transmembrane transports of amino acid into the cells and from the cells, and whether they cause an increase or decrease in content of the amino acid inside the cells may be determined by difference of in hibitory degrees on these two processes. It seems reasonable to consider that active transport mechanism and passive per meability of the cell membrane are closely associated with properties of lipoprotein in the membrane and that lipotropic agents alter transport and permeability by interact ing with the lipoprotein components. SUMMARY 1. Ethanol caused a remarkable increase in accumulation of L-dopa in brain cortex slices at concentrations of 0.5-4%. Butanol also stimulated the accumulation at 0.1%. 2. The major part of dopa accumulated in the slices was found to be present in cytoplasmic fraction. The intracellular distribution of dopa was not changed by ad dition of ethanol. 3. Influx of dopa into brain cortex slices was not stimulated, rather decreased by ethanol. 4. Efflux of dopa from the slices was observed to be decreased by ethanol. From these results, it was considered that the increase in accumulation of dopa in brain cortex slices caused by ethanol was mainly due to an inhibition on efflux of dopa from the slices. Acknowledgement : The authors wish to express their cordial gratitude to Prof. R. Imaizumi for his kind advice and encouragement. REFERENCES 1) YOSHIDA, H., NAMBA, J., KANIIKE, K. AND IMAIZUMI, R. : THIS JOURNAL 13, 1 (1963) 2) YOSHIDA, H., KANIIKE, K. AND NAMBA, J.: Ibid. 13, 10 (1963) 3) YOSHIDA, H. AND KANIIKE, K. : Ibid. 13, 122 (1963) 4) YOSHIDA, H., KANIIKE, K. AND NAMBA, J. : Nature 198, 191 (1963) 5) HEINZ, E. AND WALSH, P.O. : J. biol. Chem. 233, 1488 (1958) 6) JOHNSTONE, R.M. AND QIASTEL, J.H. : Biochim. biophys. acta 46, 527 (1961) 7) JACOBS, M.H.: Biol. Bull., Woods Hole 101, 210 (1951) 8) HUNTER, F.R.: J. cell. comp. Physiol. 58, 203 (1961) 9) JOHNSTONE, R.M. AND QUASTAL, J.H.: Biochem. biophys. acta 46, 514 (1961)