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Benzodiazepine receptors modulate circulating plasma vasopressin concentration
368
Brain Research, 359 (1985) 368-370 Elsevier
BRE 21237
Ben~ne ~rll m o d ~ tire,jilting plasma vNopreuin ~ o e m ~ i o n JAMES H. WIBLE, Jr. 1, ROBERT L. ZERBE2and JOSEPH A. DiMICCO t 1Department of Pharmacology and Toxicology, Indiana University School of Medicine and 2Lilly Laboratories for Clinical Research, Indianapolis, IN 46223 (U.S.A.)
(Accepted August 6th, 1985) Key words: vasopressin- - chlordiazepoxide- - blood pressure - - spinal rat
Chlordiazepoxide pretreatment decreased basal levels of plasma arginine-vasopressin (AVP) and attenuated picrotoxin-induced increases in plasma AVP and blood pressure.compared to saline-pretreated spinal animals. Prior administration of RO 15-1788 blocked the effects of chlordiazepoxide on basal plasma AVP as well as picrotoxin-evoked changes in plasma AVP and blood pressure. Thus, interactions at the benzodiazepine receptor may influence basal and evoked changes in plasma AVP concentration. Pharmacological agents that interact with G A B A ergic systems cause changes in circulating levels of plasma arginine-vasopressin (AVP). Muscimol, a G A B A agonist, or G A B A itself inhibit the stimulated (bilateral carotid occlusion, hypertonic saline or angiotensin II) release of AVp1,5,8, n. Inhibitors of G A B A synthesis decrease endogenous levels of G A B A in the CNS and increase plasma AVP levels. whereas inhibitors of G A B A degradation elevate endogenous G A B A levels and decrease plasma AVp9. Picrotoxin or bicuculline, both G A B A antagonists, increase circulating AVP concentrations when placed on the ventral surface of the feline medulla 4. When systemically administered to spinal rats, these same agents elicit a dose-related increase in plasma AVP which results in an elevation of blood pressure 12. Taken together, these findings suggest the existence of tonic GABAergic synaptic inhibition suppressing the secretion of AVP into the systemic circulation from the neurohypophysis. Benzodiazepines potentiate the action of G A B A at its receptor. These compounds act at a specific set of receptors which are coupled to G A B A receptors in the brain and are thought to create allosteric changes in the G A B A receptor-chloride ionophore
complex which enhance the flux of chloride ions (for review see articles by Costa and Guidotti3 and OIsenl0). Specific actions of the benzodiazepines are blocked by RO 15-1788, a compound which also competes specifically for the binding site thought to represent the benzodiazepine receptor in vitro 7. This agent has served to give a better understanding of the mechanism of action of benzodiazepines. If enhancement of GABAergic tone inhibits the secretion of AVP. then the benzodiazepines should suppress stimulated secretion of AVP. The purpose of this study was to investigate the effect of chlordiazepoxide (CDP), a water soluble benzodiazepine, on the picrotoxin-induced release of AVP in spinal rats and also to investigate the role of the benzodiazepine receptor in mediating this effect by employing the benzodiazepine antagonist RO 15-1788. Male Sprague-Dawley rats ranging m weight from 275 g to 325 g were anesthetized with urethane (1.35 g/kg, i.p.). Cannulation of the left femoral artery allowed for monitoring of blood pressure and sampling of blood, while the cannulated left femoral vein was used for infusion of donor blood and admmistration of drugs. A cardiotachometer triggered by a lead(II) electrocardiogram-monitored heart rate
Correspondence: J.A. DiMicco, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46223, U.S.A.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B .V. (Biomedical Division)
369 which was continuously recorded along with mean blood pressure (MBP) on a Beckman R 511 Dynograph. Rectal temperature was monitored and maintained at 37 + 1 °C by intermittent use of an infrared lamp. Cannulation of the trachea allowed for artificial respiration with room air after transection of the spinal cord between C2 and C3. Animals were allowed to stabilize for one hour before administration of any drug. The experimental protocol of this study incorporated the use of two pretreatments followed by administration of picrotoxin. The first pretreatment consisted of infusion of either RO 15-1788 (10.0 mg/kg, i.v.) or an equivalent volume of an appropriate vehicle (1 part dimethylsulfoxide, 1 part Tween 80 and 18 parts saline). The second pretreatment, administered 20 min after the first, consisted of CDP (5.0 or 20.0 mg/kg, i.v.) or saline. Picrotoxin (6.0 mg/kg, i.v.) was administered 20 min after the second pretreatment. Arterial blood samples (800 /~l) were obtained from each animal 10 min before any drug administration, 10 min after each pretreatment and 6 min after picrotoxin infusion. Each sample was immediately centrifuged, and the plasma was separated and stored at -80 °C until assayed. An equal amount of donor blood was infused after each sample was obtained. Donor blood, collected by decapitation of heparinized rats, was placed on ice until needed and warmed to body temperature before infusion. Plasma AVP concentration was measured by radioimmunoassay as described by Zerbe et a1.13.
Data are presented as the mean + S.E.M. Statistical analysis was performed by A N O V A , Newman-Keuls multiple range test, and paired t-test. Criterion for statistical significance was P < 0.05. The effects of the different pretreatments and treatment on MBP and circulating plasma AVP concentrations in spinal rats are presented in Table I. Baseline plasma AVP levels and MBP in these spinal animals were 51 + 6 pg/ml and 60 + 1 mm Hg, respectively. There were no significant differences in any of the baseline values among the different pretreatment groups. Administration of picrotoxin to control (vehicle + saline) spinal rats evoked a significant increase in plasma AVP levels (+172 + 19 pg/ml, 6 min after picrotoxin) and arterial pressure (+61 + 4 mm Hg, 6 min after picrotoxin). The effects of picrotoxin on plasma AVP suggest that blockade of GABAergic tone may cause the release of AVP into the systemic circulation. Enhancement of GABAergic inhibition by CDP should attenuate the picrotoxin-induced blockade. To test this hypothesis CDP (20.0 mg/kg) was administered prior to picrotoxin infusion. When spinal rats were pretreated with CDP (20.0 mg/kg) picrotoxin infusion caused a significant elevation in circulating plasma AVP concentrations (+102 + 18 pg/ml, 6 min after picrotoxin) and MBP (+17 + 3, 6 min after picrotoxin); however, the increases in both plasma AVP and MBP were significantly attenuated compared to control animals. To demonstrate that the effect of CDP was due to its interaction with the benzodiazepine receptor, the imidazodiazepine RO 15-1788 was
TABLE I The effects of picrotoxin on mean blood pressure (MBP) and plasma vasopressin (p[A VP]) in spinal rats pretreated with vehicle or RO 15-1788 (I0.0 mg/kg) and saline or chlordiazepoxide Pretreatment I
Vehicle RO 15-1788 Vehicle Vehicle RO 15-1788
Pretreatment 2
Saline Saline CDP 5.0mg/kg CDP20.0mg/kg CDP20.0mg/kg
n
Baseline MBP p[AVP] (mm ng) (pg/ml)
3 6 6 6 6
55+3 61+4 59+2 62+1 59+4
43+10 65+18 55+11 48+15 41+7
10 min after pretreatment I
10 rain after pretreatment 2
6 min after picrotoxin
MBP p[AVP1 (mm Hg) (pg/ml)
MBP p[AVPI (ram Hg) (pg/ml)
MBP p[A VP] (ram Hg) (pg/ml)
56+2 60+3 59+2 64+1 57+4
58+2 61+4 57+3 61+1 59+4
119+2a 106+6a 100_3a 78+3a,b 108___4a
36+12 38___9 50+8 56+9 44+11
29+14 43+10 41+18 23+7c 41+6
201+29~ 226+22a 196+31a 124+20~.b 275+30a
a Significantlydifferent from baseline by ANOVA and paired t-test (P < 0.05). b Significantly different from the correspondingvalue of all other pretreatment groups by ANOVA and Newman-Keuls test (P < 0.05). c Significantlydifferent from the basal value 10 min after pretreatment 1 by ANOVA and paired t-test (P < 0.05).
370 employed. Administration of R O 15-1788 prior to pretreatment with C D P (20.0 mg/kg) blocked the suppression of picrotoxin-induced A V P release and blood pressure. Attenuation of the effect of picrotoxin on plasma A V P levels by C D P and blockade of the suppression by R O 15-1788 is consistent with the participation of tonic G A B A e r g i c inhibition in the control of stimulated A V P secretion and functional coupling of the pertinent G A B A receptors to benzodiazepine receptors. Although not significant, the increase in plasma A V P concentrations caused by picrotoxin in animals pretreated with R O 15-1788 and C D P tended to be greater than that seen in vehicleand saline-pretreated animals. An explanation for this trend is not readily apparent but may involve the slight inverse agonistic properties occasionally reported after administration of higher doses of R O 151788 (refs. 2, 6).
Administration of C D P (20.0 mg/kg) also caused a significant decrease in basal circulating plasma A V P concentrations without causing a change in basal MBP. Pretreatment with R O 15-1788 blocked the suppression of basal plasma A V P concentrations by the benzodiazepine. The effect of C D P on plasma A V P levels and its blockade by pretreatment with R O 15-1788 suggest that interactions at the benzodiazepine receptor may influence the basal secretion of AVP. The data from this study support the theory that GABAergic tone inhibits the release of A V P into the systemic circulation. Furthermore, CDP, a commonly used anxiolytic agent, may also suppress the release of A V P from the neurohypophysis by interacting with benzodiazepine receptors to enhance G A B A e r g i c inhibition.
1 Brennan, T.J. and Haywood, J.R., GABA inhibition of central angiotensin II and hypertonic CSF pressor responses, Brain Research, 267 (1983) 261-269. 2 Corda, M.G., Costa, E. and Guidotti, A., Specific proconvulsant action of an imidazobenzodiazepine (RO 15-1788) on isoniazid convulsions, Neuropharmacology, 21 (1982) 91-94. 3 Costa, E. and Guidotti, A., Molecular mechanisms in the receptor action of benzodiazepines, Annu. Rev. Pharmacol. Toxicol., 19 (1979) 531-545. 4 Feldberg, W. and Rocha e Silva, M., Jr., Vasopressin release produced in anaesthetized cats by antagonists of ~,aminobutyric acid and glycine, Br. J. Pharmacol., 62 (1978) 99-106. 5 Feldberg, W. and Rocha e Silva, M., Jr., Inhibition of vasopressin release by y-aminobutyric acid and glycine, Br. J. Pharmacol., 72 (1981) 17-24. 6 File, S.E., Lister, R.G. and Nutt, D.J., The anxiogenic action of benzodiazepine antagonists, Neuropharrnacology, 21 (1982) 1033-1037. 7 Hunkeler, W., M6hler, H., Pieri, L., Polc, P., Bonnetti, E.P., Cumin, R., Schaffner, R. and Haefely, W., Selective antagonists of benzodiazepines, Nature (London), 290 (1981) 514-516.
8 Iovino, M., De Caro, G., Massi, M., Steardo, L. and Poenaru, S., Muscimol inhibits A D H release induced by hypertonic sodium chloride in rats, Pharmacol. Biochem. Behay., 19 (1983) 335-338. 9 Knepel. W.. Nutto. D. and Hertting, G., Evidence for the involvement of a GABA-mediated inhibition in the hypovolaemia-induced vasopressin release. Pflagers Arch., 388 (1980) 177-183. 10 Olsen, R.W., Drug interactions at the GABA receptor-ionophore complex. Annu. Rev. Pharmacol. Toxicol,. 22 (1982) 245-277. 11 Unger, Th., Bles, F.. Ganten, D., Lang, R.E.. Rettig, R. and Schwab. N.. GABAergic stimulation inhibits central action of angiotensin II: pressor responses, drinking and release of vasopressin, Eur. J. Pharmacot., 90 (1983) 1-9. 12 Wible. J.H.. Jr.. Zerbe. R.L. and DiMicco, J.A., Stimulation of vasopressm release by GABA antagonists in spinal cord transected rats. J. PharmacoL Exp. Ther.. 234 (1985) 378-385. 13 Zerbe, R.L., Feuerstein, G., Meyer, D.K. and Kopin, I.J., Cardiovascular, sympathetic and renin-angiotensin system responses to hemorrhage in vasopressin-deficient rats, Endocrinology, 111 (1982) 608-613.
Brain Research, 359 (1985) 368-370 Elsevier
BRE 21237
Ben~ne ~rll m o d ~ tire,jilting plasma vNopreuin ~ o e m ~ i o n JAMES H. WIBLE, Jr. 1, ROBERT L. ZERBE2and JOSEPH A. DiMICCO t 1Department of Pharmacology and Toxicology, Indiana University School of Medicine and 2Lilly Laboratories for Clinical Research, Indianapolis, IN 46223 (U.S.A.)
(Accepted August 6th, 1985) Key words: vasopressin- - chlordiazepoxide- - blood pressure - - spinal rat
Chlordiazepoxide pretreatment decreased basal levels of plasma arginine-vasopressin (AVP) and attenuated picrotoxin-induced increases in plasma AVP and blood pressure.compared to saline-pretreated spinal animals. Prior administration of RO 15-1788 blocked the effects of chlordiazepoxide on basal plasma AVP as well as picrotoxin-evoked changes in plasma AVP and blood pressure. Thus, interactions at the benzodiazepine receptor may influence basal and evoked changes in plasma AVP concentration. Pharmacological agents that interact with G A B A ergic systems cause changes in circulating levels of plasma arginine-vasopressin (AVP). Muscimol, a G A B A agonist, or G A B A itself inhibit the stimulated (bilateral carotid occlusion, hypertonic saline or angiotensin II) release of AVp1,5,8, n. Inhibitors of G A B A synthesis decrease endogenous levels of G A B A in the CNS and increase plasma AVP levels. whereas inhibitors of G A B A degradation elevate endogenous G A B A levels and decrease plasma AVp9. Picrotoxin or bicuculline, both G A B A antagonists, increase circulating AVP concentrations when placed on the ventral surface of the feline medulla 4. When systemically administered to spinal rats, these same agents elicit a dose-related increase in plasma AVP which results in an elevation of blood pressure 12. Taken together, these findings suggest the existence of tonic GABAergic synaptic inhibition suppressing the secretion of AVP into the systemic circulation from the neurohypophysis. Benzodiazepines potentiate the action of G A B A at its receptor. These compounds act at a specific set of receptors which are coupled to G A B A receptors in the brain and are thought to create allosteric changes in the G A B A receptor-chloride ionophore
complex which enhance the flux of chloride ions (for review see articles by Costa and Guidotti3 and OIsenl0). Specific actions of the benzodiazepines are blocked by RO 15-1788, a compound which also competes specifically for the binding site thought to represent the benzodiazepine receptor in vitro 7. This agent has served to give a better understanding of the mechanism of action of benzodiazepines. If enhancement of GABAergic tone inhibits the secretion of AVP. then the benzodiazepines should suppress stimulated secretion of AVP. The purpose of this study was to investigate the effect of chlordiazepoxide (CDP), a water soluble benzodiazepine, on the picrotoxin-induced release of AVP in spinal rats and also to investigate the role of the benzodiazepine receptor in mediating this effect by employing the benzodiazepine antagonist RO 15-1788. Male Sprague-Dawley rats ranging m weight from 275 g to 325 g were anesthetized with urethane (1.35 g/kg, i.p.). Cannulation of the left femoral artery allowed for monitoring of blood pressure and sampling of blood, while the cannulated left femoral vein was used for infusion of donor blood and admmistration of drugs. A cardiotachometer triggered by a lead(II) electrocardiogram-monitored heart rate
Correspondence: J.A. DiMicco, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46223, U.S.A.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B .V. (Biomedical Division)
369 which was continuously recorded along with mean blood pressure (MBP) on a Beckman R 511 Dynograph. Rectal temperature was monitored and maintained at 37 + 1 °C by intermittent use of an infrared lamp. Cannulation of the trachea allowed for artificial respiration with room air after transection of the spinal cord between C2 and C3. Animals were allowed to stabilize for one hour before administration of any drug. The experimental protocol of this study incorporated the use of two pretreatments followed by administration of picrotoxin. The first pretreatment consisted of infusion of either RO 15-1788 (10.0 mg/kg, i.v.) or an equivalent volume of an appropriate vehicle (1 part dimethylsulfoxide, 1 part Tween 80 and 18 parts saline). The second pretreatment, administered 20 min after the first, consisted of CDP (5.0 or 20.0 mg/kg, i.v.) or saline. Picrotoxin (6.0 mg/kg, i.v.) was administered 20 min after the second pretreatment. Arterial blood samples (800 /~l) were obtained from each animal 10 min before any drug administration, 10 min after each pretreatment and 6 min after picrotoxin infusion. Each sample was immediately centrifuged, and the plasma was separated and stored at -80 °C until assayed. An equal amount of donor blood was infused after each sample was obtained. Donor blood, collected by decapitation of heparinized rats, was placed on ice until needed and warmed to body temperature before infusion. Plasma AVP concentration was measured by radioimmunoassay as described by Zerbe et a1.13.
Data are presented as the mean + S.E.M. Statistical analysis was performed by A N O V A , Newman-Keuls multiple range test, and paired t-test. Criterion for statistical significance was P < 0.05. The effects of the different pretreatments and treatment on MBP and circulating plasma AVP concentrations in spinal rats are presented in Table I. Baseline plasma AVP levels and MBP in these spinal animals were 51 + 6 pg/ml and 60 + 1 mm Hg, respectively. There were no significant differences in any of the baseline values among the different pretreatment groups. Administration of picrotoxin to control (vehicle + saline) spinal rats evoked a significant increase in plasma AVP levels (+172 + 19 pg/ml, 6 min after picrotoxin) and arterial pressure (+61 + 4 mm Hg, 6 min after picrotoxin). The effects of picrotoxin on plasma AVP suggest that blockade of GABAergic tone may cause the release of AVP into the systemic circulation. Enhancement of GABAergic inhibition by CDP should attenuate the picrotoxin-induced blockade. To test this hypothesis CDP (20.0 mg/kg) was administered prior to picrotoxin infusion. When spinal rats were pretreated with CDP (20.0 mg/kg) picrotoxin infusion caused a significant elevation in circulating plasma AVP concentrations (+102 + 18 pg/ml, 6 min after picrotoxin) and MBP (+17 + 3, 6 min after picrotoxin); however, the increases in both plasma AVP and MBP were significantly attenuated compared to control animals. To demonstrate that the effect of CDP was due to its interaction with the benzodiazepine receptor, the imidazodiazepine RO 15-1788 was
TABLE I The effects of picrotoxin on mean blood pressure (MBP) and plasma vasopressin (p[A VP]) in spinal rats pretreated with vehicle or RO 15-1788 (I0.0 mg/kg) and saline or chlordiazepoxide Pretreatment I
Vehicle RO 15-1788 Vehicle Vehicle RO 15-1788
Pretreatment 2
Saline Saline CDP 5.0mg/kg CDP20.0mg/kg CDP20.0mg/kg
n
Baseline MBP p[AVP] (mm ng) (pg/ml)
3 6 6 6 6
55+3 61+4 59+2 62+1 59+4
43+10 65+18 55+11 48+15 41+7
10 min after pretreatment I
10 rain after pretreatment 2
6 min after picrotoxin
MBP p[AVP1 (mm Hg) (pg/ml)
MBP p[AVPI (ram Hg) (pg/ml)
MBP p[A VP] (ram Hg) (pg/ml)
56+2 60+3 59+2 64+1 57+4
58+2 61+4 57+3 61+1 59+4
119+2a 106+6a 100_3a 78+3a,b 108___4a
36+12 38___9 50+8 56+9 44+11
29+14 43+10 41+18 23+7c 41+6
201+29~ 226+22a 196+31a 124+20~.b 275+30a
a Significantlydifferent from baseline by ANOVA and paired t-test (P < 0.05). b Significantly different from the correspondingvalue of all other pretreatment groups by ANOVA and Newman-Keuls test (P < 0.05). c Significantlydifferent from the basal value 10 min after pretreatment 1 by ANOVA and paired t-test (P < 0.05).
370 employed. Administration of R O 15-1788 prior to pretreatment with C D P (20.0 mg/kg) blocked the suppression of picrotoxin-induced A V P release and blood pressure. Attenuation of the effect of picrotoxin on plasma A V P levels by C D P and blockade of the suppression by R O 15-1788 is consistent with the participation of tonic G A B A e r g i c inhibition in the control of stimulated A V P secretion and functional coupling of the pertinent G A B A receptors to benzodiazepine receptors. Although not significant, the increase in plasma A V P concentrations caused by picrotoxin in animals pretreated with R O 15-1788 and C D P tended to be greater than that seen in vehicleand saline-pretreated animals. An explanation for this trend is not readily apparent but may involve the slight inverse agonistic properties occasionally reported after administration of higher doses of R O 151788 (refs. 2, 6).
Administration of C D P (20.0 mg/kg) also caused a significant decrease in basal circulating plasma A V P concentrations without causing a change in basal MBP. Pretreatment with R O 15-1788 blocked the suppression of basal plasma A V P concentrations by the benzodiazepine. The effect of C D P on plasma A V P levels and its blockade by pretreatment with R O 15-1788 suggest that interactions at the benzodiazepine receptor may influence the basal secretion of AVP. The data from this study support the theory that GABAergic tone inhibits the release of A V P into the systemic circulation. Furthermore, CDP, a commonly used anxiolytic agent, may also suppress the release of A V P from the neurohypophysis by interacting with benzodiazepine receptors to enhance G A B A e r g i c inhibition.
1 Brennan, T.J. and Haywood, J.R., GABA inhibition of central angiotensin II and hypertonic CSF pressor responses, Brain Research, 267 (1983) 261-269. 2 Corda, M.G., Costa, E. and Guidotti, A., Specific proconvulsant action of an imidazobenzodiazepine (RO 15-1788) on isoniazid convulsions, Neuropharmacology, 21 (1982) 91-94. 3 Costa, E. and Guidotti, A., Molecular mechanisms in the receptor action of benzodiazepines, Annu. Rev. Pharmacol. Toxicol., 19 (1979) 531-545. 4 Feldberg, W. and Rocha e Silva, M., Jr., Vasopressin release produced in anaesthetized cats by antagonists of ~,aminobutyric acid and glycine, Br. J. Pharmacol., 62 (1978) 99-106. 5 Feldberg, W. and Rocha e Silva, M., Jr., Inhibition of vasopressin release by y-aminobutyric acid and glycine, Br. J. Pharmacol., 72 (1981) 17-24. 6 File, S.E., Lister, R.G. and Nutt, D.J., The anxiogenic action of benzodiazepine antagonists, Neuropharrnacology, 21 (1982) 1033-1037. 7 Hunkeler, W., M6hler, H., Pieri, L., Polc, P., Bonnetti, E.P., Cumin, R., Schaffner, R. and Haefely, W., Selective antagonists of benzodiazepines, Nature (London), 290 (1981) 514-516.
8 Iovino, M., De Caro, G., Massi, M., Steardo, L. and Poenaru, S., Muscimol inhibits A D H release induced by hypertonic sodium chloride in rats, Pharmacol. Biochem. Behay., 19 (1983) 335-338. 9 Knepel. W.. Nutto. D. and Hertting, G., Evidence for the involvement of a GABA-mediated inhibition in the hypovolaemia-induced vasopressin release. Pflagers Arch., 388 (1980) 177-183. 10 Olsen, R.W., Drug interactions at the GABA receptor-ionophore complex. Annu. Rev. Pharmacol. Toxicol,. 22 (1982) 245-277. 11 Unger, Th., Bles, F.. Ganten, D., Lang, R.E.. Rettig, R. and Schwab. N.. GABAergic stimulation inhibits central action of angiotensin II: pressor responses, drinking and release of vasopressin, Eur. J. Pharmacot., 90 (1983) 1-9. 12 Wible. J.H.. Jr.. Zerbe. R.L. and DiMicco, J.A., Stimulation of vasopressm release by GABA antagonists in spinal cord transected rats. J. PharmacoL Exp. Ther.. 234 (1985) 378-385. 13 Zerbe, R.L., Feuerstein, G., Meyer, D.K. and Kopin, I.J., Cardiovascular, sympathetic and renin-angiotensin system responses to hemorrhage in vasopressin-deficient rats, Endocrinology, 111 (1982) 608-613.