166

Brain Research, 165 (I 979) 166-170 ~¢3Elsevier/North-Holland Biomedical Press

Effect of somatostatin on acetylcholine release from rat hippocampal synaptosomes

E. F. NEMETH* and J. R. COOPER Department of Pharmacology, Yale University School of Medicine, New Haven, Conn. 06510 (U.S.A.)

(Accepted November 16th, 1978)

The hypothalamic peptide hormones, in addition to their established role in regulating secretions of the anterior pituitary, have actions throughout the central nervous system where they may function as neurotransmitters or neuromodulators 1. Somatostatin (somatotropin release-inhibiting factor, SRIF) is particularly interesting in this respect for it is widely distributed throughout nervous and non-nervous tissues 10 and produces a profound inhibitory effect on a wide variety of secretory cells (for a recent review see: Luft et al.8). The fact that SRIF influences secretion in such a diverse group of secretory cells suggests that it may interact with some step common to the secretory mechanism of all cells. In the present study we have followed this line of reasoning by examining the effect of SRIF on the release of acetylcholine (ACh) from synaptosomes. We have chosen the hippocampus for this investigation as it receives a dense cholinergic innervation 7 and contains a relatively high concentration of SRIF which has been shown to reside in terminals impinging upon the pyramidal cells in close proximity to the cholinergic endingslL Furthermore, in the peripheral nervous system SRIF inhibits electrically-induced release of ACh 5. Male, Sprague-Dawley rats were decapitated, hippocampi removed and a 'P2' fraction prepared as previously described 13. After washing twice, the synaptosomes were resuspended in Krebs-Ringer-bicarbonate buffer containing (mM): NaC1, 124; KCI, 5.6; NaHCO3, 25; MgC12, 1 ; NaH2PO4, 1 ; CaC12, 2; pH 7.4. Synaptosomes were loaded with [3H]ACh by incubating with [methyl-3H]choline 13. Aliquots (0.25 ml) of the synaptosomal suspension (containing 20 #M eserine) were preincubated in plastic tubes (0.5-0.7 mg/ml protein, final volume 0.4 ml) at 37 °C under 95 ~ 02-5 ~ CO2 with or without test substances. ACh release was induced by the addition of KCI (to final 55.6 or 15.6 mM) and incubation continued for an additional 15 min. 'Basal' release was determined in samples where Tris-base was substituted for KC 1. The reaction was stopped by cooling on ice for 5 min followed by centrifugation at 3000 g for 15 min. * To whom reprint requests should be addressed.

167 ACh in the supernatants was separated from choline either by high-voltage paper electrophoresis14 or an enzymatic liquid-cation exchange method 4 modified as follows: a 0.04 ml aliquot of the supernatant was incubated in a final volume of 0.12 ml containing 80 mM glycylglycine buffer, pH 8.5, 12.3 mM MgCI2, 5.1 mM ATP and 0.3 U/ml choline kinase (Sigma) for 30 min at 37 °C. The reaction was stopped by dilution to 0.52 ml with ice-cold water. 1.0 ml chilled tetraphenylboron (Sigma) (10 mg/ml 3-heptanone) was added and the tubes thoroughly mixed. Aqueous and organic phases were separated by centrifugation and [3H]ACh in an aliquot of the organic phase was determined by liquid scintillation counting. Under these conditions, there was a good correlation between results obtained by electrophoresis and liquid-cation exchange methods. Neither Trasylol (Sigma) nor SRIF interfered with the separation methods. Lactate dehydrogenase (LDH) release 6 was expressed as percent LDH activity in supernatants from samples treated with 0.1 ~ Triton X-100. Cyclic somatostatin was dissolved in 0.9 ~o NaC1 containing 0.5 mg/ml bovine serum albumin and stored at --20 °C; solutions were used within one week of preparation. [Methyl-SH]choline, specific activity 69.5 Ci/mmole, was obtained from New England Nuclear. When synaptosomes were preincubated for 10 min in the presence of 0.01 to 0.5 /~g/ml SRIF and then depolarized with 55.6 mM K + for 15 min, no effect was found on either basal or K+-evoked [3H]ACh release (data not shown). Since a lack of effect could be due to rapid proteolytic breakdown of SRIF 9, we added Trasylol (500 KIU/ml) to all samples; yet again low concentrations of SRIF were without effect. At higher concentrations SRIF has been reported to alter calcium fluxes in synaptosomeslz and, given the critical role of calcium in neurotransmitter release, we considered that at these levels an effect of SRIF may become apparent. Preincubation of synaptosomes with 100 #g/ml SRIF for 10 min again failed to significantly alter [3H]ACh release in response to either maximal or submaximal K+-depolarization. However, SRIF augmented both basal and K+-evoked release; this effect was small (never exceeding 15 ~ over controls) but reproducible and varied quantitatively from one lot of peptide to another. In certain secretory cells, SRIF-induced inhibition of secretion could be overcome by raising the extracellular calcium concentrationL By analogy, we thought that lowering the external calcium concentration might further increase the stimulatory effect of SRIF. This was indeed the case; when the calcium concentration was lowered to 0.2 mM, and preincubation extended to 25 rain, SRIF-treated samples showed enhanced release when compared to similarly treated samples in 2 mM calcium. This enhancement was due to the fact that control [SH]ACh release was reduced by 30 ~ whereas [3H]ACh of the corresponding SRIF-treated samples was only slightly reduced (about 4 ~). In the presence of 0.2 mM extracellular calcium, the magnitude of this stimulatory effect did not vary with SRIF concentrations of 1-10 /~g/ml but increased dramatically at 100/~g/ml (Fig. 1). When calcium was omitted from the medium and replaced with 4 mM EGTA, the stimulatory effect of SRIF was still present though not further augmented (data not shown).

168

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Fig. 1. Dose-effect relationship of the stimulatory effect of somatostatin on [aH]ACh release. Synaptosomes were preincubated for 30 rain in buffer containing 0.2 mM Ca 2+ and 500 KIU/ml Trasylol. Per cent release is expressed as the per cent increase over controls, in which basal release was 15.16 ~ 1.05 CPM/mg protein'10 -4 and that of K+-stimulated controls was 21.89 & 1.03. Each point is the mean ± S.E.M. of duplicate determinations from 2 to 4 separate experiments. Data obtained with Bachem, lot R1207-A.

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Fig. 2. Effect of somatostatin on lactate dehydrogenase and [aH]ACh release. Synaptosomes were treated as in Fig. 1 (SRIF = 100/~g/mi). Per cent release is expressed as the per cent increase over controis in which the basal L D H activity was 0.60 i 0.11 AAa40 ~ / m i n / m g protein and basal [SH]ACh release was 18.07 ~ 0.27 CPM/mg protein. 10 -4. The bars represent the mean of duplicate determinations from three separate experiments. The standard error in all cases was < 2 % of the mean. Data obtained with Bachem, lot R1207-B and Wyeth, lot C11873.

169 The lack of a dependence on extracellular calcium of SRIF-induced [aH]ACh release suggested to us that either SRIF mobilized an intracellular pool of calcium not depleted by EGTA, or that the stimulatory effect was a reflection of a non-specific release process. To test the latter, we assayed all samples for the release of a cytoplasmic enzyme marker, lactate dehydrogenase. In 3 experiments, we found that control synaptosomes released 8.7 ~o of their total L D H and this rose only slightly when challenged with either 15.6 or 55.6 mM K + (to 8.8 and 9.1 ~, respectively), even though [aH]ACh release increased by 30 and 90 ~o. In contrast, incubation with SRIF alone markedly increased LDH release and this increase was proportional to that of released [aH]ACh (Fig. 2). The fact that SRIF-induced [3H]ACh release was accompanied by an efflux of L DH suggests that the effect is non-specific and suggestive of membrane damage. The lack of calcium dependence further supports this notion. Sawano et al. 12 have reported that SRIF, at the high concentrations used in the present study, 'paradoxically increased' the basal release of growth hormone and thyroid stimulating hormone from perfused pituitary glands, whereas low concentrations clearly inhibited secretion. It is conceivable that this stimulatory effect of SRIF, like that obtained by us on [aH]ACh release from hippocampal synaptosomes, may result from a non-selective change in membrane permeability. Similarly, the reported effect of SRIF on calcium fluxes in synaptosomes lz, which was obtained at 100 #g/ml, may be due to the non-specific effect of this peptide which only becomes evident at high concentrations. Our data showing a lack of response of ACh release to physiological concentrations of SRIF lend support to recent results showing that low concentrations of iontophoretically applied SRIF depolarized hippocampal pyramidal cells, implicating a postsynaptic effect of the peptide 3. The present study suggests that presumptive postsynaptic effects of SRIF, at least in the hippocampus, are not mediated via presynaptic release of ACh. The present study also provides a cautionary note to investigators who employ a high concentration of this peptide. At the present time we cannot offer an explanation for this curious effect of SRIF in inducing leakage from synaptosomal preparations. We thank the following companies for generously supplying the somatostatin used in this investigation: Ayerst Research Laboratories (lot AY-24,910); Wyeth Laboratories (lot C11873); and Bachem Fine Chemicals (lot 9427 and R1207). Supported by USPHS grant NS-09836 and by the Dysautonomia Foundation.

1 Barker, J. L. and Smith, T. G., Peptides as neurohorrnones. In W. M. Cowan and J. A. Furendelli (Eds.), Approaches to the Cell Biology o f Neurons, Society for Neuroscience Symposia, VoL 11, Society for Neuroscience,Bethesda, 1977, pp. 340--373. 2 Curry, D. L. and Bennett, L. L., Reversalofsomatostatin inhibition of insulin secretion by calcium, Biochem. biophys. Res. Commun., 60 (1974) 1015-1019. 3 Dodd, J. and Kelly, J. S., Is somatostatin an excitatory transmitter in the hippocampus? Nature (Lond.), 273 (1978) 674--675. 4 Goldberg, A. M. and McCaman, R. E., The determination of picomole amounts of acetylcholine in mammalian brain, J. Neurochem., 20 (1973) 1-8.

170 5 Guillemin, R., Somatostatin inhibits the release ofacetylcholine induced electrically in the myenteric plexus, Endocrinology, 99 (1976) 1653-1654. 6 Kornberg, A., Lactic dehydrogenase of muscle. In S. P. Colowick and N. O. Kaplan (Eds.), Methods in Enzymology, Fol. I, Academic Press, New York, 1955, pp. 441-443. 7 Lewis, P. R. and Shute, C. C. D., The cholinergic limbic system: Projections to hippocampal tbrmation, medial cortex, nuclei of the ascending cholinergic reticular system, and the subfornical organ and supraoptic crest, Brain, 90 (1967) 521-540. 8 Luft, R., Efendi6, S. and H6kfelt, T., Somatostatin - - Both hormone and neurotransmitter? Diabetologia, 14 (1978) 1-13. 9 Marks, N. and Stern, F., Inactivation of somatostatin (GH-RIH) and its analogs by crude and partially purified rat brain extracts, FEBS Lett., 55 (1975) 220-224. 10 Patel, Y. C. and Reichlin, S., Somatostatin in hypothalamus, extrahypothalamic brain, and peripheral tissues of the rat, Endocrinology, 102 (1978) 523-530. 11 Petrusc, P., Sar, M., Grossman, G. H. and Kizer, J. S., Synaptic terminals with somatostatin-like immunoreactivity in the rat brain, Brain Research, 137 (1977) 181-187. 12 Sawano, S., Kokubu, T. and Ohashi, S., Variety of GH and TSH responses to somatostatin in perfused rat pituitaries in vitro, Endocr. Japonica, 23 (1976) 541-545. 13 Sen, I., Grantham, P. A. and Cooper, J. R., Mechanism of action of ~-bungarotoxin on synaptosomal preparations, Proc. nat. Acad. Sci., U.S.A., 173 (1976) 2664-2668. 14 Sgaragli, B. P. and Cooper, J. R., Effect of coUagenase pretreatment on choline and acetylcholine release from slices of bovine superior cervical sympathetic ganglia, Biochem. Pharrnacol., 23 (1974) 911-916. 15 Tan, A. T., Tsang, D., Renaud, L. P. and Martin, J. B., Effect of somatostatin on calcium transport in guinea pig cortex synaptosomes, Brain Research, 123 (1977) 193-196.