0013-7227/78/1033-0912$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 3 Printed in U.S.A.
Effect of Intraventricular Administration of Antisomatostatin y-Globulin on the Lethal Dose-50 of Strychnine and Pentobarbital in Rats* KAZUO CHIHARA, AKIRA ARIMURA, MIEKO CHIHARA, AND ANDREW V. SCHALLY Department of Medicine, Tulane University School of Medicine, and Endocrine and Polypeptide Laboratories, Veterans Administration Hospital, New Orleans, Louisiana 70146 ABSTRACT. Effects of intraventricular injection of sheep anti-somatostatin y-globulin (anti-SSG) on strychnine-induced seizures, strychnine LD5o, and pentobarbital LDso were examined in male rats under light ether anesthesia. Ten microliters of anti-SSG given 2 h earlier significantly decreased the duration of strychnine-induced seizures as compared with that in the control rats pretreated with normal sheep y-globulin (NSG). This effect of anti-SSG seemed to be specific, as there was no difference in seizure duration between sheep anti-LHRH y-globulin (anti-LHRHG)- and NSG-
S
OMATOSTATIN, a tetradecapeptide isolated from ovine (1) and porcine (2) hypothalami, is found throughout the central nervous system (CNS) in a significant concentration (3) and in the cerebrospinal fluids (CSF) (4). There is accumulating evidence that this peptide exerts various neurotropic actions on the CNS. The intracerebroventricular injection of somatostatin in rats results in a decrease in spontaneous motor activity (5), sedation (6), potentiation of the L-dopa pargyline test (7), and reduction of both slow wave sleep and rapid eye movement (REM) sleep (8). Somatostatin also increases the LD50 of strychnine and decreases the LD50 of pentobarbital in rats when administered iv in a large dose (9). Although these findings suggest the possible role of somatostatin as a neurotropic substance, the physiological role of en-
pretreated rats. Survival rates in anti-SSG-pretreated rats after injection of strychnine and pentobarbital were significantly larger (P < 0.01 and P < 0.05, respectively) than those in the control rats receiving NSG. The administration of anti-SSG resulted in 26.7% and 22.9% increases in the LDso of strychnine and pentobarbital,
respectively. These results indicate that endogenous somatostatin in the cerebrospinal fluids and/or the periventricular tissue modulates the response of the central nervous system to strychnine and pentobarbital in rats. (Endocrinology 103: 912, 1978)
dogenous somatostatin in the CNS and CSF is unclear. In the present study, we examined the effect of passive immunization with anti-somatostatin y-globulin on LD50 of strychnine and pentobarbital in order to clarify the physiological role of the endogenous somatostatin in the CSF and the periventricular tissue. Materials and Methods
Male rats of Charles River CD strain, weighing 180-220 g, were maintained in an air-conditioned room (24 ± 2 C) under a controlled artificial illumination (light on from 0500-1900 h). They were given access to Purina laboratory chow and tap water ad libitum. The antiserum to somatostatin was generated in sheep, as described previously (10). The antiserum (no. 774) used in the present study bound 85% of [125I-Tyr']somatostatin (SA, 330 mCi/mg) at 1:70 dilution. At 1:70,000 dilution of the antiserum, the Received November 28, 1977. Address all correspondence and requests for reprints tracer-antibody binding was inhibited in a doseto: Dr. Akira Arimura, Department of Medicine, Tulane related manner by unlabeled synthetic somatoUniversity School of Medicine, 1430 Tulane Avenue, New statin ranging from 16-2082 pg/tube, while the Orleans, Louisiana 70112. binding was not inhibited by the presence of either * This study was supported in part by USPHS Research Grants AM-09094 and AM-07467, by the Veterans TRH, LHRH, rat GH, PRL, TSH, LH, or FSH. Administration, and by the Naito Research Grant for The affinity constant (Ka) of this antiserum was 5.7 1976. X 109 liters/mol, and each microliter would be 912 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 July 2015. at 02:05 For personal use only. No other uses without permission. . All rights reserved.
SOMATOSTATIN ON STRYCHNINE AND PB LD50 capable of binding 4.8 ng somatostatin by calculation on the Scatchard plot (11). The iv injection of 1 ml this antiserum resulted in the blunting of stress-induced decrease in serum rat GH (10) and caused an increase in the basal as well as TRHstimulated TSH secretion in rats (12, 13). The antiserum to LHRH (no. 772) was also obtained by immunization of sheep, as described previously (14). The administration of this antiserum in cycling hamsters suppressed follicular maturation as well as the surge of gonadotropins in the afternoon of proestrus and completely blocked ovulation (15). Sheep anti-somatostatin y-globulin (anti-SSG), sheep anti-LHRH y-globulin (anti-LHRHG), and normal sheep y-globulin (NSG) were purified from respective antiserum by two-step precipitation with ammonium sulfate, as described elsewhere (16). Immediately before use, the lyophilized y-globulin was dissolved in saline to reconstruct the original concentration in the serum. Ten microliters of either anti-SSG, antiLHRHG, or NSG were injected into the right lateral ventricle of the ether-anesthetized rats by means of Krieg's stereotaxic instrument, as described previously (17). Two hours after the administration of y-globulin, strychnine sulfate or sodium pentobarbital dissolved in saline was injected in a volume of 0.1 ml/100 g BW via the jugular vein of the lightly ether-anesthetized rat. All experiments were carried out in a randomized block design. Ten animals were used to determine the effect of one dose of the drug on seizure duration and/or survival. The seizure duration after strychnine injection was expressed as the mean ± SE in minutes. Differences between treatments were analyzed by Duncan's new multiple range test (18). Survival rates after the administration of either strychnine or pentobarbital were converted to binominal data which were subjected to analysis of variance (19, 20) and then to Duncan's new multiple range test (18).
913
Results Effect of anti-SSG on seizure duration and survival after strychnine injection Within a few minutes after iv injection of strychnine sulfate at a dose larger than 50 jug/100 g BW, general motor seizures were observed in all rats examined. When anti-SSG was administered into the lateral ventricle 2 h before the strychnine injection, the seizure duration was significantly shortened as compared with those in rats pretreated with NSG (Table 1). There was no significant difference in the duration of seizures between NSG- and anti-LHRHG-pretreated rats (Table 1). Severe convulsions observed after the injection of higher doses of strychnine caused subsequent death. The survival rates of anti-SSGpretreated rats after strychnine injection in doses of 79 and 126 jug/100 g BW were significantly greater than those of NSG-pretreated rats (P < 0.01 and P < 0.01, respectively). Analysis of variance indicated that the effect of anti-SSG administration on survival rates was highly significant (P < 0.005). The LD50 dose for strychnine was 86 jug/100 g BW in NSG-pretreated rats when determined graphically, as shown in Fig. 1. The LD50 in animals that received anti-SSG was 109 jug/100 g BW, indicating a 26.7% rise above the dose in NSGtreated rats. Effect of anti-SSG on survival after pentobarbital injection The iv injection of 4 mg pentobarbital/100 g BW resulted in the death of 3 out of 10 rats receiving NSG, while it did not cause death in
TABLE 1. Effects of anti-SSG, anti-LHRHG, and NSG on the duration of strychnine-induced seizure Dose of strychnine Duration of seizure activity (min) sulfate Anti-SSG Anti-LHRHG NSG (jug/100 g iv) 1.53 ± 0.46 (0) 1.94 ± 0.44 (0) 50 3.88 ± 0.92 (0) 2.19 ± 0.45 (0) 4.13 ± 0.66 (0) 63 14.32 ± 2.08 (3) 7.21 ± 1.10 (1)" 16.41 ± 2.53 (4) 79 9.21 ± 3.01 (7)" 16.95 ± 1.49 (10) 126 14.17 ± 1.86 (10) 17.03 ± 3.35 (10) 159 y-Globulins were administered into the lateral ventricle 2 h before strychnine injection. Each value is the mean ± SE in 10 animals. The number of rats that died after the strychnine injection is shown in parentheses. " Significant differences (vs. NSG; P < 0.01).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 July 2015. at 02:05 For personal use only. No other uses without permission. . All rights reserved.
Endo Vol 103
CHIHARA ET AL.
914
1978 No 3
50 Strychnine Sulfate ( pg/100g BW) FIG. 1. Effects of intraventricular administration of antiSSG and NSG on survival after strychnine injection. Each point was calculated from 10 animals. *, Significant differences (vs. NSG; P < 0.01).
any rats that were pretreated with anti-SSG 2 h earlier. When the dose of pentobarbital was increased to 6.3 mg/100 g BW, the subsequent death was observed in 9 out of 10 rats receiving NSG and 6 out of 10 rats receiving anti-SSG. Survival rates in anti-SSG-pretreated rats after the injection of pentobarbital in doses of 4.0 and 6.4 mg/100 g BW were significantly greater than those of NSG-pretreated rats (P< 0.05, respectively). All of the rats pretreated with either NSG or anti-SSG succumbed with a dose of 10.0 mg pentobarbital/100 g BW. As shown in Fig. 2, the administration of anti-SSG resulted in a 22.9% increase in the LD50 dose of pentobarbital. Analysis of variance indicated that the effect of anti-SSG pretreatment on the survival rate in pentobarbital-treated rats was significant (P < 0.025). Discussion The present study demonstrated that the intraventricular injection of anti-SSG decreased the duration of strychnine-induced seizures and increased the LD50 of strychnine. The treatment also increased the LD50 of pentobarbital. This suggests that endogenous somatostatin in the CSF and/or the periventricular tissue affects the CNS responses to these drugs. According to calculation on the Scat-
4.0
6.3
10.0
Sodium Pentobarbital ( mg/100gBW ) FIG. 2. Effects of intraventricular administration of antiSSG and NSG on survival after pentobarbital injection. Each point was calculated from 10 animals. *, Significant differences (vs. NSG; P < 0.05).
chard plot (11), 10 jtil anti-SSG used in this experiment would be capable of binding 48 ng somatostatin. Although the concentration of somatostatin in the CSF is unknown in the rat, those in humans have been reported to be in the range of 15-55 pg/ml (4). The total volume of the CSF is considered to be approximately 500 ]Ltl in the rat (21). Assuming that somatostatin concentrations in the rat CSF are nearly the same as those in the human, 10 /xl anti-SSG would be more than sufficient to neutralize endogenous somatostatin in the rat CSF. The fact that anti-LHRHG was ineffective in causing any significant change in duration of strychnine-induced seizures suggests a specific effect of the anti-SSG. Brown and Vale (9) have observed that iv injection of somatostatin (1 mg/kg BW) resulted in a decrease of the pentobarbital LD50 and a decrease of strychnine-induced seizure duration with an increase of the LD50 for strychnine, suggesting that somatostatin depresses CNS functions. The present study supports in part their observation, as the imraunological neutralization by anti-SSG of the action of somatostatin in the CSF and/or the periventricular tissue caused an increase in the LD50 of pentobarbital. In our study, how-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 July 2015. at 02:05 For personal use only. No other uses without permission. . All rights reserved.
SOMATOSTATIN ON STRYCHNINE AND PB LD« ever, the intraventricular injection of antiSSG shortened the duration of seizures induced by strychnine and increased the LD50 of strychnine, indicating that endogenous somatostatin in the CSF may play a facilitatory role in the mechanisms of a strychnine-induced seizure. Rezek et al. (22) have recently observed that the administration of small doses of somatostatin (0.01 and 0.1 fig) into the neostriatal complex of rats results in behavioral excitation, whereas the greater doses (1.0 and 10.0 fig) caused drowsiness and inhibition of normal behavior, suggesting that the effect of somatostatin on behavior varies depending on the dose of peptide administered. If this is true, the discrepancy between the findings of Brown and Vale (9) and ours might be explained by the differential effects of large and small doses of somatostatin on a strychnine-induced seizure. Behavioral inhibition after the injection of large doses of somatostatin may coincide with the suppression of a strychnine-induced seizure observed when this peptide was injected in a dose of 1 mg/kg BW into the jugular vein (9). Reversely, the small doses of somatostatin may lead to behavioral excitation as well as the stimulation of a strychnine-induced seizure. As the content of somatostatin in the whole hypothalamus of the rat is approximately 60 ng, the excitatory effect of small doses of somatostatin seems likely to represent the physiological action of the peptide. The mechanism of a strychnine-induced seizure is complex and unknown, but the possibility that the ventricular or periventricular somatostatin is involved in the induction of the seizure by strychnine cannot be ruled out. If so, the immunological neutralization by antiserum of the action of endogenous somatostatin could diminish the seizure response. Alternatively, we cannot rule out the possibility that somatostatin administered exogenously may exert its CNS action indirectly through an activation of other neurotropic substances in the brain. In any case, the finding in the present study indicates that endogenous somatostatin in the CSF and/or periventricular tissue indeed modulates CNS response to strychnine and pentobarbital.
915
References 1. Brazeau, P., W. Vale, R. Burgus, N. Ling, M. Butcher, J. River, and R. Guillemin, Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone, Science 179: 77, 1973. 2. Schally, A. V., A. Dupont, A. Arimura, T. W. Redding, N. Nishi, G. L. Linthicum, and D. H. Schlesinger, Isolation and structure of somatostatin from porcine hypothalami, Biochemistry 15: 509, 1976. 3. Brownstein, M., A. Arimura, H. Sato, A. V. Schally, and J. S. Kizer, The regional distribution of somatostatin in the rat brian, Endocrinology 96: 1456, 1975. 4. Patel, Y. C, K. Rao, and S. Reichlin, Somatostatin in human cerebrospinal fluid, N Engl J Med 296: 529, 1977. 5. Segal, D. S., and A. J. Mandell, Differential behavior effects of hypothalamic polypeptides, In Prange, A. J., Jr. (ed.), The Thyroid Axis, Drugs, and Behavior, Raven Press, New York, 1974, p. 129. 6. Cohn, M. L., Cyclic AMP, thyrotropin releasing factor and somatostatin: key factors in the regulation of the duration of narcosis, In Fink, B. R. (ed.), Molecular Mechanism of Anesthesia, Raven Press, New York, 1975, p. 485. 7. Plotnikoff, N. P., A. J. Kastin, and A. V. Schally, Growth hormone release inhibiting hormone: neuropharmacological studies, Pharmacol Biochem Behav 2: 693, 1974. 8. Rezek, M., V. Havlicek, K. R. Hughes, and H. Friesen, Central site of action of somatostatin (SRIF): role of hippocampus, Neuropharmacology 15: 499, 1976. 9. Brown, M., and W. Vale, Central nervous system effects of hypothalamic peptides, Endocrinology 96: 1333, 1975. 10. Arimura, A., W. D. Smith, and A. V. Schally, Blockade of the stress-induced decrease on blood GH by antisomatostatin serum in rats, Endocrinology 98: 540, 1976. 11. Scatchard, G., The attractions of proteins for small molecules and ions, Ann NY Acad Sci 51: 660, 1949. 12. Arimura, A., and A. V. Schally, Increase in basal and thyrotropin-releasing hormone (TRH)-stimulated secretion of thyrotropin (TSH) by passive immunization with antiserum to somatostatin in rat, Endocrinology 98: 1069, 1976. 13. Gordin, A., A. Arimura, and A. V. Schally, Effect of thyroid hormone excess and deficiency on serum thyrotropin in rats immunized passively with antiserum to somatostatin, Proc Soc Exp Biol Med 153: 319, 1976. 14. Arimura, A., M. Shino, K. G. de la Cruz, E. G. Rennels, and A. V. Schally, Effect of active and passive immunization with luteinizing hormone-releasing hormone on serum luteinizing hormone and follicle-stimulating hormone levels and the ultrastructure of the pituitary gonadotrophs in castrated male rats, Endocrinology 99: 291, 1976. 15. de la Cruz, A., A. Arimura, K. G. de la Cruz, and A. V. Schally, Effect of administration of anti-serum to
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 July 2015. at 02:05 For personal use only. No other uses without permission. . All rights reserved.
916
16. 17.
18.
19.
CHIHARA ET AL.
luteinizing hormone-releasing hormone on gonadal function during the estrous cycle in the hamster, Endocrinology 98: 490, 1976. Weir, D. M., Handbook of Experimental Biology, F. A. Davis, Co., Philadelphia, 1967, p. 4. Chihara, K., Y. Kato, S. Ohgo, Y. Iwasaki, H. Abe, K. Maeda, and H. Imura, Stimulating and inhibiting effects of thyrotropin-releasing hormone on growth hormone release in rats, Endocrinology 98: 1047, 1976. Steel, R. D. G., and J. H. Torrie, Principles and Procedures of Statistics, McGraw-Hill, New York, 1966, p. 107. Hsu, T., and L. S. Feldt, The effect of limitation on
Endo Vol 103
1978 No 3
the number of criterion score values on the significance level of the F-test, Am Ed Res J 6: 515, 1969. 20. Seeger, P., and A. Gabrielson, Application of the cochran 0 test and the F-test for statistical analysis of dichotomous data for dependent samples, Psychol Bull 69: 269, 1968. 21. Davson, H., Physiology of the Cerebrospinal Fluid, Churchill, London, 1967, p. 120. 22. Rezek, M., V. Havlicek, L. Leybin, C. Pinsky, E. A. Kroeger, K. R. Hughes, and H. Friesen, Neostriatal administration of somatostatin: differential effect of small and large doses on behavior and motor control.
CanJPhysiol Pharmacol 55: 234, 1977.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 July 2015. at 02:05 For personal use only. No other uses without permission. . All rights reserved.
Vol. 103, No. 3 Printed in U.S.A.
Effect of Intraventricular Administration of Antisomatostatin y-Globulin on the Lethal Dose-50 of Strychnine and Pentobarbital in Rats* KAZUO CHIHARA, AKIRA ARIMURA, MIEKO CHIHARA, AND ANDREW V. SCHALLY Department of Medicine, Tulane University School of Medicine, and Endocrine and Polypeptide Laboratories, Veterans Administration Hospital, New Orleans, Louisiana 70146 ABSTRACT. Effects of intraventricular injection of sheep anti-somatostatin y-globulin (anti-SSG) on strychnine-induced seizures, strychnine LD5o, and pentobarbital LDso were examined in male rats under light ether anesthesia. Ten microliters of anti-SSG given 2 h earlier significantly decreased the duration of strychnine-induced seizures as compared with that in the control rats pretreated with normal sheep y-globulin (NSG). This effect of anti-SSG seemed to be specific, as there was no difference in seizure duration between sheep anti-LHRH y-globulin (anti-LHRHG)- and NSG-
S
OMATOSTATIN, a tetradecapeptide isolated from ovine (1) and porcine (2) hypothalami, is found throughout the central nervous system (CNS) in a significant concentration (3) and in the cerebrospinal fluids (CSF) (4). There is accumulating evidence that this peptide exerts various neurotropic actions on the CNS. The intracerebroventricular injection of somatostatin in rats results in a decrease in spontaneous motor activity (5), sedation (6), potentiation of the L-dopa pargyline test (7), and reduction of both slow wave sleep and rapid eye movement (REM) sleep (8). Somatostatin also increases the LD50 of strychnine and decreases the LD50 of pentobarbital in rats when administered iv in a large dose (9). Although these findings suggest the possible role of somatostatin as a neurotropic substance, the physiological role of en-
pretreated rats. Survival rates in anti-SSG-pretreated rats after injection of strychnine and pentobarbital were significantly larger (P < 0.01 and P < 0.05, respectively) than those in the control rats receiving NSG. The administration of anti-SSG resulted in 26.7% and 22.9% increases in the LDso of strychnine and pentobarbital,
respectively. These results indicate that endogenous somatostatin in the cerebrospinal fluids and/or the periventricular tissue modulates the response of the central nervous system to strychnine and pentobarbital in rats. (Endocrinology 103: 912, 1978)
dogenous somatostatin in the CNS and CSF is unclear. In the present study, we examined the effect of passive immunization with anti-somatostatin y-globulin on LD50 of strychnine and pentobarbital in order to clarify the physiological role of the endogenous somatostatin in the CSF and the periventricular tissue. Materials and Methods
Male rats of Charles River CD strain, weighing 180-220 g, were maintained in an air-conditioned room (24 ± 2 C) under a controlled artificial illumination (light on from 0500-1900 h). They were given access to Purina laboratory chow and tap water ad libitum. The antiserum to somatostatin was generated in sheep, as described previously (10). The antiserum (no. 774) used in the present study bound 85% of [125I-Tyr']somatostatin (SA, 330 mCi/mg) at 1:70 dilution. At 1:70,000 dilution of the antiserum, the Received November 28, 1977. Address all correspondence and requests for reprints tracer-antibody binding was inhibited in a doseto: Dr. Akira Arimura, Department of Medicine, Tulane related manner by unlabeled synthetic somatoUniversity School of Medicine, 1430 Tulane Avenue, New statin ranging from 16-2082 pg/tube, while the Orleans, Louisiana 70112. binding was not inhibited by the presence of either * This study was supported in part by USPHS Research Grants AM-09094 and AM-07467, by the Veterans TRH, LHRH, rat GH, PRL, TSH, LH, or FSH. Administration, and by the Naito Research Grant for The affinity constant (Ka) of this antiserum was 5.7 1976. X 109 liters/mol, and each microliter would be 912 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 July 2015. at 02:05 For personal use only. No other uses without permission. . All rights reserved.
SOMATOSTATIN ON STRYCHNINE AND PB LD50 capable of binding 4.8 ng somatostatin by calculation on the Scatchard plot (11). The iv injection of 1 ml this antiserum resulted in the blunting of stress-induced decrease in serum rat GH (10) and caused an increase in the basal as well as TRHstimulated TSH secretion in rats (12, 13). The antiserum to LHRH (no. 772) was also obtained by immunization of sheep, as described previously (14). The administration of this antiserum in cycling hamsters suppressed follicular maturation as well as the surge of gonadotropins in the afternoon of proestrus and completely blocked ovulation (15). Sheep anti-somatostatin y-globulin (anti-SSG), sheep anti-LHRH y-globulin (anti-LHRHG), and normal sheep y-globulin (NSG) were purified from respective antiserum by two-step precipitation with ammonium sulfate, as described elsewhere (16). Immediately before use, the lyophilized y-globulin was dissolved in saline to reconstruct the original concentration in the serum. Ten microliters of either anti-SSG, antiLHRHG, or NSG were injected into the right lateral ventricle of the ether-anesthetized rats by means of Krieg's stereotaxic instrument, as described previously (17). Two hours after the administration of y-globulin, strychnine sulfate or sodium pentobarbital dissolved in saline was injected in a volume of 0.1 ml/100 g BW via the jugular vein of the lightly ether-anesthetized rat. All experiments were carried out in a randomized block design. Ten animals were used to determine the effect of one dose of the drug on seizure duration and/or survival. The seizure duration after strychnine injection was expressed as the mean ± SE in minutes. Differences between treatments were analyzed by Duncan's new multiple range test (18). Survival rates after the administration of either strychnine or pentobarbital were converted to binominal data which were subjected to analysis of variance (19, 20) and then to Duncan's new multiple range test (18).
913
Results Effect of anti-SSG on seizure duration and survival after strychnine injection Within a few minutes after iv injection of strychnine sulfate at a dose larger than 50 jug/100 g BW, general motor seizures were observed in all rats examined. When anti-SSG was administered into the lateral ventricle 2 h before the strychnine injection, the seizure duration was significantly shortened as compared with those in rats pretreated with NSG (Table 1). There was no significant difference in the duration of seizures between NSG- and anti-LHRHG-pretreated rats (Table 1). Severe convulsions observed after the injection of higher doses of strychnine caused subsequent death. The survival rates of anti-SSGpretreated rats after strychnine injection in doses of 79 and 126 jug/100 g BW were significantly greater than those of NSG-pretreated rats (P < 0.01 and P < 0.01, respectively). Analysis of variance indicated that the effect of anti-SSG administration on survival rates was highly significant (P < 0.005). The LD50 dose for strychnine was 86 jug/100 g BW in NSG-pretreated rats when determined graphically, as shown in Fig. 1. The LD50 in animals that received anti-SSG was 109 jug/100 g BW, indicating a 26.7% rise above the dose in NSGtreated rats. Effect of anti-SSG on survival after pentobarbital injection The iv injection of 4 mg pentobarbital/100 g BW resulted in the death of 3 out of 10 rats receiving NSG, while it did not cause death in
TABLE 1. Effects of anti-SSG, anti-LHRHG, and NSG on the duration of strychnine-induced seizure Dose of strychnine Duration of seizure activity (min) sulfate Anti-SSG Anti-LHRHG NSG (jug/100 g iv) 1.53 ± 0.46 (0) 1.94 ± 0.44 (0) 50 3.88 ± 0.92 (0) 2.19 ± 0.45 (0) 4.13 ± 0.66 (0) 63 14.32 ± 2.08 (3) 7.21 ± 1.10 (1)" 16.41 ± 2.53 (4) 79 9.21 ± 3.01 (7)" 16.95 ± 1.49 (10) 126 14.17 ± 1.86 (10) 17.03 ± 3.35 (10) 159 y-Globulins were administered into the lateral ventricle 2 h before strychnine injection. Each value is the mean ± SE in 10 animals. The number of rats that died after the strychnine injection is shown in parentheses. " Significant differences (vs. NSG; P < 0.01).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 July 2015. at 02:05 For personal use only. No other uses without permission. . All rights reserved.
Endo Vol 103
CHIHARA ET AL.
914
1978 No 3
50 Strychnine Sulfate ( pg/100g BW) FIG. 1. Effects of intraventricular administration of antiSSG and NSG on survival after strychnine injection. Each point was calculated from 10 animals. *, Significant differences (vs. NSG; P < 0.01).
any rats that were pretreated with anti-SSG 2 h earlier. When the dose of pentobarbital was increased to 6.3 mg/100 g BW, the subsequent death was observed in 9 out of 10 rats receiving NSG and 6 out of 10 rats receiving anti-SSG. Survival rates in anti-SSG-pretreated rats after the injection of pentobarbital in doses of 4.0 and 6.4 mg/100 g BW were significantly greater than those of NSG-pretreated rats (P< 0.05, respectively). All of the rats pretreated with either NSG or anti-SSG succumbed with a dose of 10.0 mg pentobarbital/100 g BW. As shown in Fig. 2, the administration of anti-SSG resulted in a 22.9% increase in the LD50 dose of pentobarbital. Analysis of variance indicated that the effect of anti-SSG pretreatment on the survival rate in pentobarbital-treated rats was significant (P < 0.025). Discussion The present study demonstrated that the intraventricular injection of anti-SSG decreased the duration of strychnine-induced seizures and increased the LD50 of strychnine. The treatment also increased the LD50 of pentobarbital. This suggests that endogenous somatostatin in the CSF and/or the periventricular tissue affects the CNS responses to these drugs. According to calculation on the Scat-
4.0
6.3
10.0
Sodium Pentobarbital ( mg/100gBW ) FIG. 2. Effects of intraventricular administration of antiSSG and NSG on survival after pentobarbital injection. Each point was calculated from 10 animals. *, Significant differences (vs. NSG; P < 0.05).
chard plot (11), 10 jtil anti-SSG used in this experiment would be capable of binding 48 ng somatostatin. Although the concentration of somatostatin in the CSF is unknown in the rat, those in humans have been reported to be in the range of 15-55 pg/ml (4). The total volume of the CSF is considered to be approximately 500 ]Ltl in the rat (21). Assuming that somatostatin concentrations in the rat CSF are nearly the same as those in the human, 10 /xl anti-SSG would be more than sufficient to neutralize endogenous somatostatin in the rat CSF. The fact that anti-LHRHG was ineffective in causing any significant change in duration of strychnine-induced seizures suggests a specific effect of the anti-SSG. Brown and Vale (9) have observed that iv injection of somatostatin (1 mg/kg BW) resulted in a decrease of the pentobarbital LD50 and a decrease of strychnine-induced seizure duration with an increase of the LD50 for strychnine, suggesting that somatostatin depresses CNS functions. The present study supports in part their observation, as the imraunological neutralization by anti-SSG of the action of somatostatin in the CSF and/or the periventricular tissue caused an increase in the LD50 of pentobarbital. In our study, how-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 July 2015. at 02:05 For personal use only. No other uses without permission. . All rights reserved.
SOMATOSTATIN ON STRYCHNINE AND PB LD« ever, the intraventricular injection of antiSSG shortened the duration of seizures induced by strychnine and increased the LD50 of strychnine, indicating that endogenous somatostatin in the CSF may play a facilitatory role in the mechanisms of a strychnine-induced seizure. Rezek et al. (22) have recently observed that the administration of small doses of somatostatin (0.01 and 0.1 fig) into the neostriatal complex of rats results in behavioral excitation, whereas the greater doses (1.0 and 10.0 fig) caused drowsiness and inhibition of normal behavior, suggesting that the effect of somatostatin on behavior varies depending on the dose of peptide administered. If this is true, the discrepancy between the findings of Brown and Vale (9) and ours might be explained by the differential effects of large and small doses of somatostatin on a strychnine-induced seizure. Behavioral inhibition after the injection of large doses of somatostatin may coincide with the suppression of a strychnine-induced seizure observed when this peptide was injected in a dose of 1 mg/kg BW into the jugular vein (9). Reversely, the small doses of somatostatin may lead to behavioral excitation as well as the stimulation of a strychnine-induced seizure. As the content of somatostatin in the whole hypothalamus of the rat is approximately 60 ng, the excitatory effect of small doses of somatostatin seems likely to represent the physiological action of the peptide. The mechanism of a strychnine-induced seizure is complex and unknown, but the possibility that the ventricular or periventricular somatostatin is involved in the induction of the seizure by strychnine cannot be ruled out. If so, the immunological neutralization by antiserum of the action of endogenous somatostatin could diminish the seizure response. Alternatively, we cannot rule out the possibility that somatostatin administered exogenously may exert its CNS action indirectly through an activation of other neurotropic substances in the brain. In any case, the finding in the present study indicates that endogenous somatostatin in the CSF and/or periventricular tissue indeed modulates CNS response to strychnine and pentobarbital.
915
References 1. Brazeau, P., W. Vale, R. Burgus, N. Ling, M. Butcher, J. River, and R. Guillemin, Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone, Science 179: 77, 1973. 2. Schally, A. V., A. Dupont, A. Arimura, T. W. Redding, N. Nishi, G. L. Linthicum, and D. H. Schlesinger, Isolation and structure of somatostatin from porcine hypothalami, Biochemistry 15: 509, 1976. 3. Brownstein, M., A. Arimura, H. Sato, A. V. Schally, and J. S. Kizer, The regional distribution of somatostatin in the rat brian, Endocrinology 96: 1456, 1975. 4. Patel, Y. C, K. Rao, and S. Reichlin, Somatostatin in human cerebrospinal fluid, N Engl J Med 296: 529, 1977. 5. Segal, D. S., and A. J. Mandell, Differential behavior effects of hypothalamic polypeptides, In Prange, A. J., Jr. (ed.), The Thyroid Axis, Drugs, and Behavior, Raven Press, New York, 1974, p. 129. 6. Cohn, M. L., Cyclic AMP, thyrotropin releasing factor and somatostatin: key factors in the regulation of the duration of narcosis, In Fink, B. R. (ed.), Molecular Mechanism of Anesthesia, Raven Press, New York, 1975, p. 485. 7. Plotnikoff, N. P., A. J. Kastin, and A. V. Schally, Growth hormone release inhibiting hormone: neuropharmacological studies, Pharmacol Biochem Behav 2: 693, 1974. 8. Rezek, M., V. Havlicek, K. R. Hughes, and H. Friesen, Central site of action of somatostatin (SRIF): role of hippocampus, Neuropharmacology 15: 499, 1976. 9. Brown, M., and W. Vale, Central nervous system effects of hypothalamic peptides, Endocrinology 96: 1333, 1975. 10. Arimura, A., W. D. Smith, and A. V. Schally, Blockade of the stress-induced decrease on blood GH by antisomatostatin serum in rats, Endocrinology 98: 540, 1976. 11. Scatchard, G., The attractions of proteins for small molecules and ions, Ann NY Acad Sci 51: 660, 1949. 12. Arimura, A., and A. V. Schally, Increase in basal and thyrotropin-releasing hormone (TRH)-stimulated secretion of thyrotropin (TSH) by passive immunization with antiserum to somatostatin in rat, Endocrinology 98: 1069, 1976. 13. Gordin, A., A. Arimura, and A. V. Schally, Effect of thyroid hormone excess and deficiency on serum thyrotropin in rats immunized passively with antiserum to somatostatin, Proc Soc Exp Biol Med 153: 319, 1976. 14. Arimura, A., M. Shino, K. G. de la Cruz, E. G. Rennels, and A. V. Schally, Effect of active and passive immunization with luteinizing hormone-releasing hormone on serum luteinizing hormone and follicle-stimulating hormone levels and the ultrastructure of the pituitary gonadotrophs in castrated male rats, Endocrinology 99: 291, 1976. 15. de la Cruz, A., A. Arimura, K. G. de la Cruz, and A. V. Schally, Effect of administration of anti-serum to
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 July 2015. at 02:05 For personal use only. No other uses without permission. . All rights reserved.
916
16. 17.
18.
19.
CHIHARA ET AL.
luteinizing hormone-releasing hormone on gonadal function during the estrous cycle in the hamster, Endocrinology 98: 490, 1976. Weir, D. M., Handbook of Experimental Biology, F. A. Davis, Co., Philadelphia, 1967, p. 4. Chihara, K., Y. Kato, S. Ohgo, Y. Iwasaki, H. Abe, K. Maeda, and H. Imura, Stimulating and inhibiting effects of thyrotropin-releasing hormone on growth hormone release in rats, Endocrinology 98: 1047, 1976. Steel, R. D. G., and J. H. Torrie, Principles and Procedures of Statistics, McGraw-Hill, New York, 1966, p. 107. Hsu, T., and L. S. Feldt, The effect of limitation on
Endo Vol 103
1978 No 3
the number of criterion score values on the significance level of the F-test, Am Ed Res J 6: 515, 1969. 20. Seeger, P., and A. Gabrielson, Application of the cochran 0 test and the F-test for statistical analysis of dichotomous data for dependent samples, Psychol Bull 69: 269, 1968. 21. Davson, H., Physiology of the Cerebrospinal Fluid, Churchill, London, 1967, p. 120. 22. Rezek, M., V. Havlicek, L. Leybin, C. Pinsky, E. A. Kroeger, K. R. Hughes, and H. Friesen, Neostriatal administration of somatostatin: differential effect of small and large doses on behavior and motor control.
CanJPhysiol Pharmacol 55: 234, 1977.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 July 2015. at 02:05 For personal use only. No other uses without permission. . All rights reserved.
