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Stimulatory effect of somatostatin on norepineprhine release from rat brain cortex slices
Life Sciences, Vol. 28, pp. 903-910 Printed in the U.S.A.
Pergamon Press
STIMULATORY EFFECT OF SOMATOSTATIN ON NOREPINEPRBINE RELEASE FROM RAT BRAIN CORTEX SLICES Akira Tsujimoto and Shokichl Tanaka Department of Pharmacology, Hiroshima University School of Dentistry Hiroshima, Japan (Received in final form December ii, 1980) S,-,-~ry The effect of somatostatln (SRIF) on norepinephrlne (NE) release from the brain tissue was determined on the superfused rat cerebral cortex slices preloaded with 3H-NE. SRIF (0.38 uM- 1.53 uM) was found to stimulate dose-dependently tritium (38) overflow evoked electrically by 30 % - 116 % although SRIF did not affect on the spontaneous 3H overflow. SRIF at the concentrations which exhibited the stimulatory effect inhibited scarecely the uptake of 3H-}~ by cortex slices, while the reference drug, cocaine (50 uM, i0 uM) markedly depressed the uptake. The stimulatory effect of SRIF was not reduced by phentolamine (3.14 ~M), s-adrenoceptor blocker, which increased the evoked 3H overflow from the slices itself. These resul£s suggest that SRIF does not produce its stlmulatory effect by inhibiting the NE reuptake mechanisms or by interacting with the presynaptic s-adrenoceptors. Elevating of Ca 2+ concentrations from 0.75 mM to 2.25 mM in the superfuslon fluid reduced the stimulatory effect of SRIF. It is possible that SRIF stimulates NE release by facilitating the availability of Ca 2+ for the release mechanisms. The tetradecapeptide somatostatin first isolated from hypothalamus and characterized as a potent inhibitor ~pituitary growth hormone secretion (i) has been shown to cause a profound inhibitory effect on a wide variety of hormone secretion including insulin, glucagon and gastrln (2). Furthermore, somatostatin (SRIF) was found to distribute widely throughout brain (3), to localize to the nerve terminals (4), to modify animal behavlour (5), and neuronal excitability (5, 6). In addition, an increase in DOPA, 5-hydroxytryptophan and 5-hydroxyindoleacetlc acid brain levels in rats administered SRIF intracerebroventriculary suggesting stlmulatlon of the monoamlne turnover has been shown (7). These findings imply that SRIF may function as neurotransmitter or neuromodulator at specific synapses in the CNS. The present paper reports that SRIF stimulates the evoked 3H-norepinephrine release in the' rat cerebral cortex and the possible mechanisms of its stimulatory effect. Materials and Methods 3H-norepinephrine release Male Wistar rats weighing 120- 150 g were decapitated, and their brains were dissected out and cooled in the fluid mentioned below. Slices of 0.3 to 0.4 mm thickness of the cerebral cortex were prepared by a razor blade on a moist filter paper. The slices were transferred into 1.0 ml of the fluid 0024-3205/81/080903-O8502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
904
Somatostatin and Norepinephrine
Release
Vol. 28, No. 8, 1981
of the following composition (8) in ~ : NaCI, 123; NaHCO3, 26, NaH2PO~, 1.2; MgSO~, 1.8; KCI, 3.6; CaCI2, 0.75 and glucose, I0: saturating with 95% 0 2 + 5 % CO2, pH 7.4. As described by Farnebo and Hamberger (9), brain tissues were loaded with 3H-norepinephrine (3H-NE) 50 nM (SA = 5 Ci/mmol) for 30 min at 37°C under 95 % O 2 + 5 % CO 2 atmosphere. After loading, the tissues were washed twice with 5.0 ml of fresh medium and then placed into the superfusion chamber. The make of chamber and the superfusion were carried out according to the method described by Carmichael and Israel (8). The chamber with a capacity of 0.5 ml of polypropylene make was mounted in water bath at 37°C. In the chamber~ two platinum wire (diameter 0.5 mm) electrodes are put at 4 mm intervals. The slices (20 - 26 mg) held on the mesh discs of nylon between the two electrodes were superfused at pressure of 50 cm water with the superfusion fluid mentioned above at 37°C continuously bubbled with 95 % 02 + 5 % CO 2 at a rate of 0.3 ml per minute for 20 minutes to allow stabilization of tritium (3H), after which successive 3-minute samples (0.9 ml) were collected into test tubes. Four samples were collected to determine spontaneous release after which the tissues were electrically stimulated for 3 minutes. The frequency of stimulation was 25 Hz, with pulses of 0.5 msec duration and 25 V amplitude. The application of drugs was done 6 minutes before the electrical stimulation until the end of experiments. 3H-Radioactivity in perfusates was determined in a Liquid Scintillation Spectrometer (Aloka, LSC-903) after addition of I0 ml of Bray's scintillation solution (I0) to 0.5 ml of the sample. The radioactivity remaining in the slices at the end of the perfusion was determined as above after slices were dissolved in 0.2 ml of N-NaOH and neutralized with 0.2 ml of N-HCI. The labeled material that is overflowed per 3-mlnute sample is expressed as a percenrage of the total radioactivity in the slices at the start of the collection period. This total radioactivity is the sum of the radioactivity in all collected samples and the radioactlvity in slices at the end of the perfus%on. Unchanged 3 H - ~ in the material released spontaneously or by the electrical stimulation with or without SRIF accounted for 81 - 84 % of the total radioactivity. The presence of pargyline IO-SM, a monoamine oxidase inhibitor, in the superfusion fluid did not affect the spontaneous or the stimulated overflow of 3H, and did not raise the percentage of unchanged NE in the released material. Therefore, the monoamine oxidase inhibitor was not used in d ~ present experiments. Identification
of noreplnephrine
Identification of unchanged 3H-NE was performed using thin layer chromatography (ii). After scraping slica gel layers corresponding to NE and the remaining layers divided into each 5 ~ , individual samples were counted as above. 3H-Norepinephrine
uptake
The cerebral cortex slices were preincubated for 5 minutes at 37°C in the above mentioned medium in the presence or absence of drugs under 95 % 02 + 5 % CO 2 atmosphere. After the addition of 3H-NE (final concentrations: 50 nM), the incubation was continued for an additional 5 min and the slices were washed 3 times with ice-cold medium. Finally 3H-radioactivity in slices was determined as above. Druss and chemicals The following drugs were used: cyclic somatostatin (Ayerst Research Lab., Montreal and Peptide Institute Protein Reseach Foundation, Osaka), 3H-norepinephrine (the Radiochemical Center, Amersham), cocaine hydrochloride (Sankyo Co., Ltd.), norepinephrine (Fluka AG), pargyline hydrochloride (Abbott Lat.),phentolamine hydrochloride (Ciba-Geigy). The final molar concentration of each drug is expressed in terms of the base.
Vol. 28, No. 8, 1981
Somatostatin and Norepinephrine Release
905
Results and Discussion Effect on 3H-norepinephrine releas e The spontaneous overflow of 3H from the cerebral cortex slices which had previously been incubated with 3H-NE during the superfusion process was observed. This basal 3H overflow was enhanced about three times by the electrical stimulation (FIG. 1 left panel). When slices were perfused with Ca 2+ omitted fluid from 20 minutes before the electrical stimulation., the spontaneous 3H overflow was not significantly altered but the electrical stimulation enhanced scarcely the 3H overflow indicating that the evoked 3H overflow is dependent on extracellular Ca 2~ (data not shown).
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FIG. i Left panel: Effect of somatostatin (SRIF) on the tritium (3H) overflow from superfused brain cortex slices. Abscissa, time period of efflux in minutes, with 3-minute electrical stimulation indicated. 3H overflow during each 3 minute collection period is expressed as a percentage of the total radioactivity in the tissue at the start of the collection period. SRIF (1.53 ~M) was present in the superfusion fluid from 6 minutes before the electrical stimulation until the end of the experiments. See Methods on details of experiments. O---O, control; O - - Q , SRIF; ~ , electrical stimulation. Each point represents the mean ± S. E. M. of twelve experiments. * p<0. O01; •* p<0.005, *** p<0.01. Right panel: Concentration-response curve for the stimulatory effect of SRIF on 3H overflow evoked by the electrical stimulation. Values represent a percent increase to the control response during 3-minute electrical stimulation in experiments of the type shown in FIG. i left panel. Each point represents the mean ± S. E. M. of seven to twelve experiments.
906
Somatostatin and Norepinephrine
Release
Vol. 28, No. 8, 1981
SRIF was found to stimulate 3H overflow from the cerebral cortex slices evoked by the electrical stimulation. SRIF, 1.53 uM had no effect on the spontaneous release, but produced a significantly greater increase in the stimulation-induced overflow of 3H (FIG. 1 left panel). The concentration-effect relationship for the stimulatory effect of SRIF (0.38 ~ M - 1 . 5 3 ~M) on 3H overflow is shown in FIG. 1 (right panel). "It is evident that the SRIF stimulation is dose dependent. SRIF has been reported to stimulate acetylcholine (ACh) release from rat hippocampal synaptosomes only at high concentration, 100 ~g/ml, with or without extrac e llular Ca 2+ and SRIF-induced ACh release was accompanied by an efflux of lactate dehydrogenase, cytoplasmic enzyme marker suggesting that the stimulatory effect is non-speclfic and is due to membrane damage (12). In the present experiments, the concentrations [0.38 uM (0.62 ~g/ml) - 1.53 uM (2.5 ~g/ml)] of SRIF which exhibited the stimulatory effect were much lower than the concentration shown to release ACh and were the same order as the concentration known to inhibit glucose-lnduced insulin release (13). Therefore, it is conceivable the stimulatory effect of SRIF on NE release is not due to a non-specific effect, llke membrane damage, which only becomes evident at high concentration. The present results provide the first direct evidence that SRIF stimulates the release of norepinephrine, a putative neurotransmitter in the central nervous system. Garcia-Sevilla et al. (7) reported changes of the brain monoamine and its precursor levels in rats received SRIF suggesting an increase of adrenergic monoamine turnover. The stimulation of the evoked NE release by SRIF may account for the increase of adrenergic monoamine turnover. Effect on 3H-norepinephrine
uptake
To determine whether the increase in 3H overflow by SRIF is due to an inhibition of neurotransmitter reuptake mechanisms or not, an effect of SRIF on 3H-NE uptake by cerebral cortex slices was examined. Cocaine which specifically inhibits NE uptake, thereby apparently increases NE release (14) was used as a reference drug. SRIF at concentrations which exhibited the stimulatory effect on 3H overflow did not inhibit 3H-NE uptake by cortex slices with the exception of 1.53 uM showing a slight inhibition. Cocaine at concentrations 50 ~M and i0 ~M markedly depressed the uptake of 3H-NE (FIG. 2). Thus, these data indicate that the stimulatory effect of SRIF on NE release is not caused by a reuptake inhibition of NE released. Effect of ~-adrenoceptor
blocker
It has been suggested thata negative feedback system operates in the central nervous system on the basis of observation that a-adrenoceptor agonists decrease while e-adrenoceptor blocking agents increase the release of 3H-NE induced by electrical field stimulation or by potassiLm~ in brain slices (15). Therefore, to determine whether the stimulation of 3H overflow induced by SRIF results from a blockade of presynaptic a-adrenoceptors or not, an effect of SRIF on the sitmulated 3H overflow from the brain slices during exposure to phentola~Line, an a-blocker, was examined. The concentration of phentolamlne which is strong enough to block presynaptic a-adrenoceptors in the cerebral cortex slices was used (16). The electrically stimulated 3H overflow from superfused slices was significantly increased by 3.14 uM phentolamine. The stlmulatory effect of SRIF was not reduced by the same concentration of phentolamine (FIG. 3). Thus, these results suggest that the SRIF does not cause its stimulatory effect by acting on presynaptic e-adrenoceptors. Effect of alteration of Ca 2+ concentration SRIF-induced inhibition on insulin secretion could be counteracted by elevating the extracellular Ca 2+ concentrations (17). Therefore, it is con-
Vol. 28, No. 8, 1981
Somatostatin and Norepinephrine Release
907
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FIG. 2 Effect of somatostatln and cocaine on 3H-noreplnephrine uptake by brain cortex slices. Slices prelncubated for 5minuteswlthor without drugs were incubated for an addltlonal 5mlnutes after the addition of 3H-noreplnephrlne (50 nM). The uptake is expressed as a percentage of total radloactlvltles in control slices. Vertical bars represent the mean ± S. E. M. of 5 determina~iorB. * p<0.001; ** p<0.05.
sidered that the stimulatory effect of SRIF might be affected by a change in the level of Ca 2+ in the superfuslon fluid. As shown in FIG. 4, increasing Ca 2+ concentrations~ from 0.75 mM to 1.25 mM, 1.8 mM and 2.25 mM, produced the overflow of the greater amount of 3H and reduced the stimulatory effect of SRIF in a dose related manner. On the contrary, decreasing Ca 2÷ concentrations from 0.75 mM to 0.25 mM caused a slight reduction of 3H overflow and a slight augment of the stlmulatory effect of SRIF. The depolarizing influence of invading action potentials couple Ca 2+ influx to the secretory event and it may be assumed that the Ca 2+ influx into the terminal nerve fibers increases with increasing the extracellular Ca 2+ concentrations (18, 19). Thus modulation of Ca 2+ availability for stimulus-release coupling appears to be more effective, when t~e transmembrane inward current of Ca 2+ is smaller. It is possible that SR~F stimulates NE release by facilitating the availability of Ca 2+ for the release mechanisms.
908
Somatostatin and Noreplnephrine
Release
Vol. 28, No. 8, 1981
The stimulatory effect of SRIF was reduced when Ca 2+ concentration in the superfusion fluid was raised close to total concentration of the mmmmalian serum. It has been, however, considered that the activity of extracellular Ca is much lower than total concentration ( 1 8 ) . The ionized Ca in serum of some mammalians including human is approximately half of total concentration, 2.38 - 2.60 mM ( 20, 21, 22, 23 ). Although the values of Ca concentration in the extracellular fluid in the central nervous system are not available, it has been shown that the cerebrospinal fluid Ca concentration is 1.6 mM (23)
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FIG. 3 Effect of phentolamlne on the somatostatin (1.53 uM) stimulation of the electrically evoked 3H overflow from brain cortex slices. Experiments were carried out as described in the legend FIG. i left panel except the application of phentolamine. Phentolamine (3.14 uM) was present in the superfuslon fluid from 12 minutes before the electrical stimulation until the end of the experiment. Values represent a percent increase to the control response during 3-minute electrical stimulation. Vertical bars represent the m e a n ± S. E. M. of four experiments except somatostatln alone (twelve experiments). PHENT: phentolamine. with bound form of at least i0 percent (24). Thus, the meaning of the Ca 2+ concentration in the experimental fluid not containing substances bound to Ca such as protein should be estimated in due consideration of these knowledge. The studies on the relations of SRIF to Ca 2+ including the effect of SRIF on Ca 2+ flux are in progress.
Vol. 28, No. 8, 1981
Somatostatln and Norepinephrine Release
909
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FIG. 4 Effect of alteration of Ca 2÷ concentrations on the somatostatln (SRIF) stlmulatlon of the electrlcally evoked 3H overflow from brain cortex slices. Experiments were carried out as described in the legend FIG. 1 left panel except for the variation in the concentration of Ca 2+ in the superfusion fluid. O---O, 3H overflow in the absence of SRIF during 3-mlnute electrical stimulation expressed as described in the legend FIG. 1 left" panel; e----e, an increase in 3H overflow by SRIF (1.53 ~M) during 3-mlnute electrlcal stimulation as a percent increase to the control response. Each point represents the mean ± S. E. M. of four to twelve experiments. * p
Acknowledgements i!
The authors wlsh to thank Dr. M. Goz, Ayerst Research Laboratories, ClbaGelg~ and Drs. G. M. Everett and A. O. Gelszler, Abbott Laboratories for generously providing cyclic somatostatin, phentolamlne and pargyllne respectively. References i.
P. BRAZEAU, W. VALE, R. BURGUS, N. LING, M. BUTCHER, J. RIVER and R. GUILLEMIN, Science 179 77-79 (1973).
910
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Sonmtostatin and Norepinephrine Release
Vol. 28, No. 8, 1981
R. LUFr, S. EFENDIC and T. HOKFELT, Diabetologia 14 1-13 (1978). Y. C. PATEL and S. REICHLIN, Endocrinology 102 523-530 (1978). J. EPELBAUM, P. BRAZEAU, D. TSANG, J. BRANER and J. B. MARTIN, Brain Res. 126 309-323 (1977). M. REZEK, V. HAVLICEK, K. R. HUGHES and H. FRIESEN, Neuropharmacology i_~6 157-162 (1977). L. P. RENAUD, J. B. MARTIN and P. BRAZEAU, Nature 255 233-235 (1975). J. A. GARCIA-SEVILLA, T. MUGNASSON and A. DARLSSON, Brain Res. 155 159-164 (1978). F. J. CARMICHAEL and Y, ISRAEL, J. Pharmacol. Exp. Therap. 193 824-834 (1975). L. O. FARNEBOand B. HA~ERGER, Acta Physiol. Scand. Suppl. 371 35-44 (1971) G. A. BRAY, Analyt. Biochem. i 279-285 (1960). R. M. FLEMINGand W. G. CLARK, J. Chromatog. 52 305-312 (1970). E. F. NEMETHand J. R. COOPER, Brain Res. 165 I.66-170 (1979). K. G. M. M. ALBERTI, S. E. CRRISTENSEN, J. IVERSEN, K. SEYER-HANSEN, N. J. CHRISTENSEN, A. PRANGE-HANSEN, K. LUNDBAEK and H. ~RSKOV, Lancet 2 12991301 (1973). H. THOENEN, A. HUERLIMANN., W. RAEFELY, J. Pharmacol. Exp. Therap. 143 5763 (1964). S.Z. LANGER, Brit. J. Pharmacol. 60 481-497 (1977). M. G~THERT, I. M. POHL and E. W E ~ N G , Naynyn-Schmiedberg's Arch. Pharmacol. 307 21-27 (1979). D. L. CURRY, L. L. BENNETT, Biochem. Biophys. Res. Commun. 60 1015-1019 (1974). C. W. COTMAN, J. W. HAYCOCK and W. F. WHITE, J. Physiol. 254 475-505 (1976). M. G~THERT, Naunyn-Schmiedeberg's Arch. Pharmak. 307 29-37 (1979). R. W. STRAUB, Trends in Pharmacol. Sei. -1 106-109 (1979). J. TOFFALETTI, H. J. GITELMAN and J. SAVORY, Clin. Chem. 22 1968-1972 (1976). D.M. GREENBERG, Adv. Protein Chem. 1 121-151 (1944). R.N. LOLLEY, J. Neurochem. i0 665-6~6 (1963). J. PAUPE, Path. et Biol. (Semaine Hosp., Paris) 7 1665-1674 (1959).
Pergamon Press
STIMULATORY EFFECT OF SOMATOSTATIN ON NOREPINEPRBINE RELEASE FROM RAT BRAIN CORTEX SLICES Akira Tsujimoto and Shokichl Tanaka Department of Pharmacology, Hiroshima University School of Dentistry Hiroshima, Japan (Received in final form December ii, 1980) S,-,-~ry The effect of somatostatln (SRIF) on norepinephrlne (NE) release from the brain tissue was determined on the superfused rat cerebral cortex slices preloaded with 3H-NE. SRIF (0.38 uM- 1.53 uM) was found to stimulate dose-dependently tritium (38) overflow evoked electrically by 30 % - 116 % although SRIF did not affect on the spontaneous 3H overflow. SRIF at the concentrations which exhibited the stimulatory effect inhibited scarecely the uptake of 3H-}~ by cortex slices, while the reference drug, cocaine (50 uM, i0 uM) markedly depressed the uptake. The stimulatory effect of SRIF was not reduced by phentolamine (3.14 ~M), s-adrenoceptor blocker, which increased the evoked 3H overflow from the slices itself. These resul£s suggest that SRIF does not produce its stlmulatory effect by inhibiting the NE reuptake mechanisms or by interacting with the presynaptic s-adrenoceptors. Elevating of Ca 2+ concentrations from 0.75 mM to 2.25 mM in the superfuslon fluid reduced the stimulatory effect of SRIF. It is possible that SRIF stimulates NE release by facilitating the availability of Ca 2+ for the release mechanisms. The tetradecapeptide somatostatin first isolated from hypothalamus and characterized as a potent inhibitor ~pituitary growth hormone secretion (i) has been shown to cause a profound inhibitory effect on a wide variety of hormone secretion including insulin, glucagon and gastrln (2). Furthermore, somatostatin (SRIF) was found to distribute widely throughout brain (3), to localize to the nerve terminals (4), to modify animal behavlour (5), and neuronal excitability (5, 6). In addition, an increase in DOPA, 5-hydroxytryptophan and 5-hydroxyindoleacetlc acid brain levels in rats administered SRIF intracerebroventriculary suggesting stlmulatlon of the monoamlne turnover has been shown (7). These findings imply that SRIF may function as neurotransmitter or neuromodulator at specific synapses in the CNS. The present paper reports that SRIF stimulates the evoked 3H-norepinephrine release in the' rat cerebral cortex and the possible mechanisms of its stimulatory effect. Materials and Methods 3H-norepinephrine release Male Wistar rats weighing 120- 150 g were decapitated, and their brains were dissected out and cooled in the fluid mentioned below. Slices of 0.3 to 0.4 mm thickness of the cerebral cortex were prepared by a razor blade on a moist filter paper. The slices were transferred into 1.0 ml of the fluid 0024-3205/81/080903-O8502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
904
Somatostatin and Norepinephrine
Release
Vol. 28, No. 8, 1981
of the following composition (8) in ~ : NaCI, 123; NaHCO3, 26, NaH2PO~, 1.2; MgSO~, 1.8; KCI, 3.6; CaCI2, 0.75 and glucose, I0: saturating with 95% 0 2 + 5 % CO2, pH 7.4. As described by Farnebo and Hamberger (9), brain tissues were loaded with 3H-norepinephrine (3H-NE) 50 nM (SA = 5 Ci/mmol) for 30 min at 37°C under 95 % O 2 + 5 % CO 2 atmosphere. After loading, the tissues were washed twice with 5.0 ml of fresh medium and then placed into the superfusion chamber. The make of chamber and the superfusion were carried out according to the method described by Carmichael and Israel (8). The chamber with a capacity of 0.5 ml of polypropylene make was mounted in water bath at 37°C. In the chamber~ two platinum wire (diameter 0.5 mm) electrodes are put at 4 mm intervals. The slices (20 - 26 mg) held on the mesh discs of nylon between the two electrodes were superfused at pressure of 50 cm water with the superfusion fluid mentioned above at 37°C continuously bubbled with 95 % 02 + 5 % CO 2 at a rate of 0.3 ml per minute for 20 minutes to allow stabilization of tritium (3H), after which successive 3-minute samples (0.9 ml) were collected into test tubes. Four samples were collected to determine spontaneous release after which the tissues were electrically stimulated for 3 minutes. The frequency of stimulation was 25 Hz, with pulses of 0.5 msec duration and 25 V amplitude. The application of drugs was done 6 minutes before the electrical stimulation until the end of experiments. 3H-Radioactivity in perfusates was determined in a Liquid Scintillation Spectrometer (Aloka, LSC-903) after addition of I0 ml of Bray's scintillation solution (I0) to 0.5 ml of the sample. The radioactivity remaining in the slices at the end of the perfusion was determined as above after slices were dissolved in 0.2 ml of N-NaOH and neutralized with 0.2 ml of N-HCI. The labeled material that is overflowed per 3-mlnute sample is expressed as a percenrage of the total radioactivity in the slices at the start of the collection period. This total radioactivity is the sum of the radioactivity in all collected samples and the radioactlvity in slices at the end of the perfus%on. Unchanged 3 H - ~ in the material released spontaneously or by the electrical stimulation with or without SRIF accounted for 81 - 84 % of the total radioactivity. The presence of pargyline IO-SM, a monoamine oxidase inhibitor, in the superfusion fluid did not affect the spontaneous or the stimulated overflow of 3H, and did not raise the percentage of unchanged NE in the released material. Therefore, the monoamine oxidase inhibitor was not used in d ~ present experiments. Identification
of noreplnephrine
Identification of unchanged 3H-NE was performed using thin layer chromatography (ii). After scraping slica gel layers corresponding to NE and the remaining layers divided into each 5 ~ , individual samples were counted as above. 3H-Norepinephrine
uptake
The cerebral cortex slices were preincubated for 5 minutes at 37°C in the above mentioned medium in the presence or absence of drugs under 95 % 02 + 5 % CO 2 atmosphere. After the addition of 3H-NE (final concentrations: 50 nM), the incubation was continued for an additional 5 min and the slices were washed 3 times with ice-cold medium. Finally 3H-radioactivity in slices was determined as above. Druss and chemicals The following drugs were used: cyclic somatostatin (Ayerst Research Lab., Montreal and Peptide Institute Protein Reseach Foundation, Osaka), 3H-norepinephrine (the Radiochemical Center, Amersham), cocaine hydrochloride (Sankyo Co., Ltd.), norepinephrine (Fluka AG), pargyline hydrochloride (Abbott Lat.),phentolamine hydrochloride (Ciba-Geigy). The final molar concentration of each drug is expressed in terms of the base.
Vol. 28, No. 8, 1981
Somatostatin and Norepinephrine Release
905
Results and Discussion Effect on 3H-norepinephrine releas e The spontaneous overflow of 3H from the cerebral cortex slices which had previously been incubated with 3H-NE during the superfusion process was observed. This basal 3H overflow was enhanced about three times by the electrical stimulation (FIG. 1 left panel). When slices were perfused with Ca 2+ omitted fluid from 20 minutes before the electrical stimulation., the spontaneous 3H overflow was not significantly altered but the electrical stimulation enhanced scarcely the 3H overflow indicating that the evoked 3H overflow is dependent on extracellular Ca 2~ (data not shown).
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FIG. i Left panel: Effect of somatostatin (SRIF) on the tritium (3H) overflow from superfused brain cortex slices. Abscissa, time period of efflux in minutes, with 3-minute electrical stimulation indicated. 3H overflow during each 3 minute collection period is expressed as a percentage of the total radioactivity in the tissue at the start of the collection period. SRIF (1.53 ~M) was present in the superfusion fluid from 6 minutes before the electrical stimulation until the end of the experiments. See Methods on details of experiments. O---O, control; O - - Q , SRIF; ~ , electrical stimulation. Each point represents the mean ± S. E. M. of twelve experiments. * p<0. O01; •* p<0.005, *** p<0.01. Right panel: Concentration-response curve for the stimulatory effect of SRIF on 3H overflow evoked by the electrical stimulation. Values represent a percent increase to the control response during 3-minute electrical stimulation in experiments of the type shown in FIG. i left panel. Each point represents the mean ± S. E. M. of seven to twelve experiments.
906
Somatostatin and Norepinephrine
Release
Vol. 28, No. 8, 1981
SRIF was found to stimulate 3H overflow from the cerebral cortex slices evoked by the electrical stimulation. SRIF, 1.53 uM had no effect on the spontaneous release, but produced a significantly greater increase in the stimulation-induced overflow of 3H (FIG. 1 left panel). The concentration-effect relationship for the stimulatory effect of SRIF (0.38 ~ M - 1 . 5 3 ~M) on 3H overflow is shown in FIG. 1 (right panel). "It is evident that the SRIF stimulation is dose dependent. SRIF has been reported to stimulate acetylcholine (ACh) release from rat hippocampal synaptosomes only at high concentration, 100 ~g/ml, with or without extrac e llular Ca 2+ and SRIF-induced ACh release was accompanied by an efflux of lactate dehydrogenase, cytoplasmic enzyme marker suggesting that the stimulatory effect is non-speclfic and is due to membrane damage (12). In the present experiments, the concentrations [0.38 uM (0.62 ~g/ml) - 1.53 uM (2.5 ~g/ml)] of SRIF which exhibited the stimulatory effect were much lower than the concentration shown to release ACh and were the same order as the concentration known to inhibit glucose-lnduced insulin release (13). Therefore, it is conceivable the stimulatory effect of SRIF on NE release is not due to a non-specific effect, llke membrane damage, which only becomes evident at high concentration. The present results provide the first direct evidence that SRIF stimulates the release of norepinephrine, a putative neurotransmitter in the central nervous system. Garcia-Sevilla et al. (7) reported changes of the brain monoamine and its precursor levels in rats received SRIF suggesting an increase of adrenergic monoamine turnover. The stimulation of the evoked NE release by SRIF may account for the increase of adrenergic monoamine turnover. Effect on 3H-norepinephrine
uptake
To determine whether the increase in 3H overflow by SRIF is due to an inhibition of neurotransmitter reuptake mechanisms or not, an effect of SRIF on 3H-NE uptake by cerebral cortex slices was examined. Cocaine which specifically inhibits NE uptake, thereby apparently increases NE release (14) was used as a reference drug. SRIF at concentrations which exhibited the stimulatory effect on 3H overflow did not inhibit 3H-NE uptake by cortex slices with the exception of 1.53 uM showing a slight inhibition. Cocaine at concentrations 50 ~M and i0 ~M markedly depressed the uptake of 3H-NE (FIG. 2). Thus, these data indicate that the stimulatory effect of SRIF on NE release is not caused by a reuptake inhibition of NE released. Effect of ~-adrenoceptor
blocker
It has been suggested thata negative feedback system operates in the central nervous system on the basis of observation that a-adrenoceptor agonists decrease while e-adrenoceptor blocking agents increase the release of 3H-NE induced by electrical field stimulation or by potassiLm~ in brain slices (15). Therefore, to determine whether the stimulation of 3H overflow induced by SRIF results from a blockade of presynaptic a-adrenoceptors or not, an effect of SRIF on the sitmulated 3H overflow from the brain slices during exposure to phentola~Line, an a-blocker, was examined. The concentration of phentolamlne which is strong enough to block presynaptic a-adrenoceptors in the cerebral cortex slices was used (16). The electrically stimulated 3H overflow from superfused slices was significantly increased by 3.14 uM phentolamine. The stlmulatory effect of SRIF was not reduced by the same concentration of phentolamine (FIG. 3). Thus, these results suggest that the SRIF does not cause its stimulatory effect by acting on presynaptic e-adrenoceptors. Effect of alteration of Ca 2+ concentration SRIF-induced inhibition on insulin secretion could be counteracted by elevating the extracellular Ca 2+ concentrations (17). Therefore, it is con-
Vol. 28, No. 8, 1981
Somatostatin and Norepinephrine Release
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sidered that the stimulatory effect of SRIF might be affected by a change in the level of Ca 2+ in the superfuslon fluid. As shown in FIG. 4, increasing Ca 2+ concentrations~ from 0.75 mM to 1.25 mM, 1.8 mM and 2.25 mM, produced the overflow of the greater amount of 3H and reduced the stimulatory effect of SRIF in a dose related manner. On the contrary, decreasing Ca 2÷ concentrations from 0.75 mM to 0.25 mM caused a slight reduction of 3H overflow and a slight augment of the stlmulatory effect of SRIF. The depolarizing influence of invading action potentials couple Ca 2+ influx to the secretory event and it may be assumed that the Ca 2+ influx into the terminal nerve fibers increases with increasing the extracellular Ca 2+ concentrations (18, 19). Thus modulation of Ca 2+ availability for stimulus-release coupling appears to be more effective, when t~e transmembrane inward current of Ca 2+ is smaller. It is possible that SR~F stimulates NE release by facilitating the availability of Ca 2+ for the release mechanisms.
908
Somatostatin and Noreplnephrine
Release
Vol. 28, No. 8, 1981
The stimulatory effect of SRIF was reduced when Ca 2+ concentration in the superfusion fluid was raised close to total concentration of the mmmmalian serum. It has been, however, considered that the activity of extracellular Ca is much lower than total concentration ( 1 8 ) . The ionized Ca in serum of some mammalians including human is approximately half of total concentration, 2.38 - 2.60 mM ( 20, 21, 22, 23 ). Although the values of Ca concentration in the extracellular fluid in the central nervous system are not available, it has been shown that the cerebrospinal fluid Ca concentration is 1.6 mM (23)
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FIG. 3 Effect of phentolamlne on the somatostatin (1.53 uM) stimulation of the electrically evoked 3H overflow from brain cortex slices. Experiments were carried out as described in the legend FIG. i left panel except the application of phentolamine. Phentolamine (3.14 uM) was present in the superfuslon fluid from 12 minutes before the electrical stimulation until the end of the experiment. Values represent a percent increase to the control response during 3-minute electrical stimulation. Vertical bars represent the m e a n ± S. E. M. of four experiments except somatostatln alone (twelve experiments). PHENT: phentolamine. with bound form of at least i0 percent (24). Thus, the meaning of the Ca 2+ concentration in the experimental fluid not containing substances bound to Ca such as protein should be estimated in due consideration of these knowledge. The studies on the relations of SRIF to Ca 2+ including the effect of SRIF on Ca 2+ flux are in progress.
Vol. 28, No. 8, 1981
Somatostatln and Norepinephrine Release
909
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Acknowledgements i!
The authors wlsh to thank Dr. M. Goz, Ayerst Research Laboratories, ClbaGelg~ and Drs. G. M. Everett and A. O. Gelszler, Abbott Laboratories for generously providing cyclic somatostatin, phentolamlne and pargyllne respectively. References i.
P. BRAZEAU, W. VALE, R. BURGUS, N. LING, M. BUTCHER, J. RIVER and R. GUILLEMIN, Science 179 77-79 (1973).
910
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Sonmtostatin and Norepinephrine Release
Vol. 28, No. 8, 1981
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