178
BIOCHEMICAL SOCIETY TRANSACTIONS
slightly. Tetraenoic diacylglycerols seem to be preferred for phosphatidylcholine synthesis, and the decrease in their labelling after 5min incubation suggests the possible occurrence of transacylation mechanisms. Hexa- and tetra-enoic diacylglycerols on the other hand are the preferred substrates for phosphatidylethanolamine synthesis. The labelling of the hexaenoic species also decreases after a short time, again supporting the idea of the Occurrence of transacylation mechanisms. MacDonald, G., Baker, K.R. & Thompson, W. (1975) J. Neurochem. 24, 655 Porcellati, G. & Binaglia, L. (1976) in Lipids (Paoletti, R., Porecllati, G. & Lacini, G., eds.), vol. 1, Raven Press, New York
The Active Uptake of 5Hydroxytryptaminein Snail (Helix pomatia) Central Nervous System and its Energy Source NEVILLE N. OSBORNE and VOLKER NEUHOFF Max-Planck-Institut fur experimentelle Medizin, Forschungsstelle Neurochemie, 3400 Gottingen, Germany
Large amounts of 5-hydroxytryptamine occur in the snail central nervous system, and evidence exists that the amine functions as a transmitter substance in this situation (Gerschenfeld, 1973; Osborne, 1974; Cottrell & Macon, 1974). Moreover, recent experiments have shown snail ganglia to possess an active uptake mechanism for 5-hydroxytryptamine (Osborne e f al., 1975), which suggests that re-uptake of the amine is the method of inactivatingthe transmitter at the post-synapticmembrane. In order to characterize the nature of the uptake of 5-hydroxytryptamineby snail central ganglia, the role of Na+ has since been investigated (Stahl et al., 1976). The present report summarizes the above and additional data, with the aim of ultimately characterizing the source of energy for the active uptake of 5-hydroxytryptamine. The methods used in this study have been described by Osborne et al. (1975) and Stahl et al. (1976). When snail nervous tissues are incubated at 25°C in media containing low (0.01-0.5p~) amounts of [3H]5-hydroxytryptamine, the tissues rapidly accumulate the amine, resulting in high (30: 1 to 13 :1) tissue/medium ratios. An analysis by Lineweaver-Burk plots of the uptake data from experiments where tissues were incubated with various amounts (0.01-100p~)of 5-hydroxytryptamine showed that the uptake was resolved into at least two components. The high-affinity system had a K, value of 8.5 x 1 0 - 8 ~ , which compares well with the K, values for the high-affnity uptake of 5-hydroxytryptamine in the vertebrates (Shaskan & Snyder, 1970; Snyder et al., 1973). The uptake of 5-hydroxytryptamineis an energy-dependent system, since the rate of uptake shows not only saturation kinetics, but is also temperature-sensitive and Na+dependent. The ions Caz+,Mgz+and K+ had little influenceon the uptake of 5-hydroxytryptamine. Kinetic analysis of data from experiments on the influence of Na+ on the uptake of 5-hydroxytryptaminesuggests that for every molecule of amine taken up, one Na+ ion is necessary. In experiments concerned with the influence of ouabain on the uptake of 5-hydroxytryptamine,it was found that even very high concentrationsof the sub) partially inhibited (30%) 5-hydroxytryptamine uptake. This was stance (1 m ~ only especially interesting, as ouabain is a potent inhibitor of the enzyme Na+ K+-ATPase* (Bonting et al., 1962), and this enzyme in its associationwith the sodium pump is thought to be involved in the energy-dependent uptake mechanisms of a variety of substances (Whittam & Wheeler, 1970; Schwartz et al., 1975). It was therefore expected that the uptake of 5-hydroxytryptamine by snail nervous tissue would somehow involve the Na++ K+-ATPase,but the very mild effect of ouabain shows that this is not the case. Further experiments revealed that the action of ouabain on 5-hydroxytryptamineuptake is complex, as even at O"C, where the sodium pump is inoperable, some inhibition of 5hydroxytryptamine uptake could be demonstrated. However, the Na+ K+-ATPasewas
+
+
Abbreviation: Na++K+-ATPase,(Na++K+)-dependentadenosine triphosphatase. 1977
179
566th MEETING, CAMBRIDGE
Table 1. Effect of various substances on the uptake of [3H]5-hydroxytryptamine by snail suboesophageal ganglia The normal incubation medium consisted of 2ml of snail saline (Meng, 1960),containing ascorbic acid (0.2mg/ml), glucose ( 5 mM) and pargyline (0.01m).Tissues were always preincubated in the normal incubation medium containing the test substance for 15 min and then incubated for 30min. before addition of 1p~-[~H]5-hydroxytryptamine Concn. required to cause Substance General metabolic and glucose-transport inhibitors Ouabain
KCN Ethanol Transport-system inhibitors Probenecid Amantadine Respiratory uncoupling agents Oligomycin Gramicidin D Chloramphenicol Thiol reagents Chlorpromazine N-Ethylmaleimide Electron-transport (redox) enzyme inhibitors 2,4-Dinitrophenol Methylene Blue Actinomycin A
40-50% inhibition of uptake (a)
10 10 10
10 1
10 1 5
0.01 0.05
0.1 0.1 0.05
maximally inhibited by 0.1 m-ouabain, a concentration which does not affect 5hydroxytryptamine uptake by more than 15%. Moreover, the presence of 0.1mMouabain was sufficient to increase the intracellular Na+ concentration of the ganglia by 91 % without affecting 5-hydroxytryptamine uptake, suggesting that the inward-directed Na+ gradient present in cells is not the likely energy source, nor is it related to the mechanism of 5-hydroxytryptamine uptake. In other experiments a variety of substances were tested for their effects on 5-hydroxytryptamine uptake, as summarized in Table 1. The results indicate that the uptake of 5-hydroxytryptamine involves thiol groups and mitochondria1 activity rather than an ionic-pump mechanism, thus supporting the findings from earlier experiments. It is thus suggested that the energy source for 5-hydroxytryptamine uptake might be ATP, but its precise role is unknown. Bonting, S.L., Caravaggio, L. L. & Hawkins, N. M. (1962)Arch.Biochem. Biophys. 98,413419 Cottrell, G. A. & Macon, J. B . (1974) J. Physiol. (London)236,435-464 Gerschenfeld, H. M. (1973) Physiol. Rev. 53, 1-119 Meng, K. (1960) Zool. Juhr. 68, 193-204 Osborne, N. N. (1974) Microchemical Analysis of Nervous Tissue, Pergamon Press,Oxford Osborne, N. N., Hiripi, L. & Neuhoff, V. (1975) Blochem. Pharmucol. 24,2141-2148 Schwartz, A., Lindenmayer, G. E. & Allen, J. C. (1975) Pharmucol. Rev. 27, 3-134 Shaskan, E. & Snyder, S. H. (1970) J. Pharmucol. Exp. Ther. 175,404-418 Snyder, S . H., Shaskan, E. G. & Kuhar, M. T. (1973) in Serotonin andBehavior (Borchas, J. L% Usdin, E., eds.), pp. 97-108, Academic Press, New York Stahl, W. L., Neuhoff, V. & Osborne, N. N. (1976) Comp. Biochem. Physiol. C in the press Whittam, R. & Wheeler, K. P. (1970) Amu. Rev. Physiol. 32, 21-60
VOl. 5
BIOCHEMICAL SOCIETY TRANSACTIONS
slightly. Tetraenoic diacylglycerols seem to be preferred for phosphatidylcholine synthesis, and the decrease in their labelling after 5min incubation suggests the possible occurrence of transacylation mechanisms. Hexa- and tetra-enoic diacylglycerols on the other hand are the preferred substrates for phosphatidylethanolamine synthesis. The labelling of the hexaenoic species also decreases after a short time, again supporting the idea of the Occurrence of transacylation mechanisms. MacDonald, G., Baker, K.R. & Thompson, W. (1975) J. Neurochem. 24, 655 Porcellati, G. & Binaglia, L. (1976) in Lipids (Paoletti, R., Porecllati, G. & Lacini, G., eds.), vol. 1, Raven Press, New York
The Active Uptake of 5Hydroxytryptaminein Snail (Helix pomatia) Central Nervous System and its Energy Source NEVILLE N. OSBORNE and VOLKER NEUHOFF Max-Planck-Institut fur experimentelle Medizin, Forschungsstelle Neurochemie, 3400 Gottingen, Germany
Large amounts of 5-hydroxytryptamine occur in the snail central nervous system, and evidence exists that the amine functions as a transmitter substance in this situation (Gerschenfeld, 1973; Osborne, 1974; Cottrell & Macon, 1974). Moreover, recent experiments have shown snail ganglia to possess an active uptake mechanism for 5-hydroxytryptamine (Osborne e f al., 1975), which suggests that re-uptake of the amine is the method of inactivatingthe transmitter at the post-synapticmembrane. In order to characterize the nature of the uptake of 5-hydroxytryptamineby snail central ganglia, the role of Na+ has since been investigated (Stahl et al., 1976). The present report summarizes the above and additional data, with the aim of ultimately characterizing the source of energy for the active uptake of 5-hydroxytryptamine. The methods used in this study have been described by Osborne et al. (1975) and Stahl et al. (1976). When snail nervous tissues are incubated at 25°C in media containing low (0.01-0.5p~) amounts of [3H]5-hydroxytryptamine, the tissues rapidly accumulate the amine, resulting in high (30: 1 to 13 :1) tissue/medium ratios. An analysis by Lineweaver-Burk plots of the uptake data from experiments where tissues were incubated with various amounts (0.01-100p~)of 5-hydroxytryptamine showed that the uptake was resolved into at least two components. The high-affinity system had a K, value of 8.5 x 1 0 - 8 ~ , which compares well with the K, values for the high-affnity uptake of 5-hydroxytryptamine in the vertebrates (Shaskan & Snyder, 1970; Snyder et al., 1973). The uptake of 5-hydroxytryptamineis an energy-dependent system, since the rate of uptake shows not only saturation kinetics, but is also temperature-sensitive and Na+dependent. The ions Caz+,Mgz+and K+ had little influenceon the uptake of 5-hydroxytryptamine. Kinetic analysis of data from experiments on the influence of Na+ on the uptake of 5-hydroxytryptaminesuggests that for every molecule of amine taken up, one Na+ ion is necessary. In experiments concerned with the influence of ouabain on the uptake of 5-hydroxytryptamine,it was found that even very high concentrationsof the sub) partially inhibited (30%) 5-hydroxytryptamine uptake. This was stance (1 m ~ only especially interesting, as ouabain is a potent inhibitor of the enzyme Na+ K+-ATPase* (Bonting et al., 1962), and this enzyme in its associationwith the sodium pump is thought to be involved in the energy-dependent uptake mechanisms of a variety of substances (Whittam & Wheeler, 1970; Schwartz et al., 1975). It was therefore expected that the uptake of 5-hydroxytryptamine by snail nervous tissue would somehow involve the Na++ K+-ATPase,but the very mild effect of ouabain shows that this is not the case. Further experiments revealed that the action of ouabain on 5-hydroxytryptamineuptake is complex, as even at O"C, where the sodium pump is inoperable, some inhibition of 5hydroxytryptamine uptake could be demonstrated. However, the Na+ K+-ATPasewas
+
+
Abbreviation: Na++K+-ATPase,(Na++K+)-dependentadenosine triphosphatase. 1977
179
566th MEETING, CAMBRIDGE
Table 1. Effect of various substances on the uptake of [3H]5-hydroxytryptamine by snail suboesophageal ganglia The normal incubation medium consisted of 2ml of snail saline (Meng, 1960),containing ascorbic acid (0.2mg/ml), glucose ( 5 mM) and pargyline (0.01m).Tissues were always preincubated in the normal incubation medium containing the test substance for 15 min and then incubated for 30min. before addition of 1p~-[~H]5-hydroxytryptamine Concn. required to cause Substance General metabolic and glucose-transport inhibitors Ouabain
KCN Ethanol Transport-system inhibitors Probenecid Amantadine Respiratory uncoupling agents Oligomycin Gramicidin D Chloramphenicol Thiol reagents Chlorpromazine N-Ethylmaleimide Electron-transport (redox) enzyme inhibitors 2,4-Dinitrophenol Methylene Blue Actinomycin A
40-50% inhibition of uptake (a)
10 10 10
10 1
10 1 5
0.01 0.05
0.1 0.1 0.05
maximally inhibited by 0.1 m-ouabain, a concentration which does not affect 5hydroxytryptamine uptake by more than 15%. Moreover, the presence of 0.1mMouabain was sufficient to increase the intracellular Na+ concentration of the ganglia by 91 % without affecting 5-hydroxytryptamine uptake, suggesting that the inward-directed Na+ gradient present in cells is not the likely energy source, nor is it related to the mechanism of 5-hydroxytryptamine uptake. In other experiments a variety of substances were tested for their effects on 5-hydroxytryptamine uptake, as summarized in Table 1. The results indicate that the uptake of 5-hydroxytryptamine involves thiol groups and mitochondria1 activity rather than an ionic-pump mechanism, thus supporting the findings from earlier experiments. It is thus suggested that the energy source for 5-hydroxytryptamine uptake might be ATP, but its precise role is unknown. Bonting, S.L., Caravaggio, L. L. & Hawkins, N. M. (1962)Arch.Biochem. Biophys. 98,413419 Cottrell, G. A. & Macon, J. B . (1974) J. Physiol. (London)236,435-464 Gerschenfeld, H. M. (1973) Physiol. Rev. 53, 1-119 Meng, K. (1960) Zool. Juhr. 68, 193-204 Osborne, N. N. (1974) Microchemical Analysis of Nervous Tissue, Pergamon Press,Oxford Osborne, N. N., Hiripi, L. & Neuhoff, V. (1975) Blochem. Pharmucol. 24,2141-2148 Schwartz, A., Lindenmayer, G. E. & Allen, J. C. (1975) Pharmucol. Rev. 27, 3-134 Shaskan, E. & Snyder, S. H. (1970) J. Pharmucol. Exp. Ther. 175,404-418 Snyder, S . H., Shaskan, E. G. & Kuhar, M. T. (1973) in Serotonin andBehavior (Borchas, J. L% Usdin, E., eds.), pp. 97-108, Academic Press, New York Stahl, W. L., Neuhoff, V. & Osborne, N. N. (1976) Comp. Biochem. Physiol. C in the press Whittam, R. & Wheeler, K. P. (1970) Amu. Rev. Physiol. 32, 21-60
VOl. 5
