6
Brain Research, 580 (1992) 6-11 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17699
Repeated injections of cocaine inhibit the serotonergic regulation of prolactin and renin secretion in rats Andrew D. Levy a, Peter A. Rittenhouse a, Qian Li a, Anna M. Bonadonna a, Maria C. Alvarez Sanz a, Janice E. Kerr a, Cynthia L. B e t h e a b and Louis D. van de Kar a ~Department of Pharmacology, Loyola University Chicago, Maywood, 1L 60153 (USA) and bDepartment of Reproductive Biology and Behavior, Oregon Regional Primate Research Center, Beaverton, OR (USA) (Accepted 24 December 1991)
Key words: Cocaine; Chronic; Serotonin; Neuroendocrine; Prolactin; Renin
Alterations in serotonergic function following repeated cocaine injections were examined using neuroendocrine responses to a serotonin (5-HT) releaser and 5-HT agonists. Forty-two hours following administration of cocaine (1-15 mg/kg i.p.) twice daily for 7 or 30 days, male Sprague-Dawley rats were injected with the 5-HT releaser p-chloroamphetamine (PCA; 8 mg/kg i.p.) and blood samples were collected 1 h later for radioimmunoassays of plasma prolactin, plasma renin activity (PRA) and plasma renin concentration (PRC). PCA significantly increased secretion of prolactin and renin. These responses were attenuated in rats pretreated with cocaine for 30 days. In rats receiving cocaine for 7 days, the attenuation of PCA-induced secretion of prolactin and renin was less consistently observed. To determine whether these alterations were due to pre- or postsynaptic effects, rats were injected with cocaine (15 mg/kg i.p.) twice daily for 7 days, and the neuroendocrine responses to the direct 5-HT agonists RU 24969 and m-CPP were examined, 42 h after the last cocaine injection. Pretreatment with cocaine potentiated RU 24969-induced stimulation of plasma prolactin concentration. However, cocaine did not alter the ability of m-CPP to increase plasma prolactin concentrations. The stimulation of renin secretion in response to both 5-HT agonists was not altered by cocaine pretreatment. The data suggest that repeated cocaine impairs the function of serotonergic nerve terminals that regulate these endocrine responses. Furthermore, the 5-HT receptors that mediate prolactin secretion may exhibit supersensitivity. INTRODUCTION Alterations in n e u r o n a l function following repeated cocaine exposure are not well understood. While there has been considerable focus on changes in dopamine (DA) neurons, cocaine is also a highly potent inhibitor of serotonin (5-HT) uptake 6'21'2z'25. Consequently, 5-HT neurotransmission is altered by acute cocaine exposure. The concentration of 5-HT in the synapse is increased 6' ~0, and the firing of 5-HT neurons in the dorsal raphe is inhibited by cocaine 4'12'17'19. Neuroendocrine markers can be utilized to evaluate serotonergic involvement in the actions of acute or chronic cocaine. Serotonergic neurons innervating the hypothalamus exert considerable influence on the secretion of many hormones. Secretion of renin is enhanced by the 5-HT releaser p-chloroamphetamine 34 (PCA), and by various 5-HT agonists 31. Data from these studies suggest that 5-HT2 receptors mediate the serotonergic stimulation of renin secretion. Prolactin secretion is also influenced by 5-HT. In contrast to the inhibitory influence of D A 1'15, 5-HT agonists and releasers increase the se-
cretion of prolactin, probably via activation of 5-HTIB and/or 5-HT 2 receptors 3t. The long-term effects of cocaine on 5-HT neurons are poorly understood. Some studies have observed reduced 5-HT content in brain following chronic cocaine 26'29, while another study did not observe changes in brain 5-HT concentration following repeated cocaine exposure 9, Furthermore, changes in 5-HT receptor densities have not been reported following chronic cocaine exposure. Because of the high affinity of cocaine for 5-HT uptake sites 22, it is likely that chronic cocaine exposure would alter the function of serotonergic neurons. Therefore, the function of 5-HT neurons was investigated following repeated cocaine exposure. The effects of 5-HT agonists, and the 5-HT releasing agent PCA, on prolactin and renin secretion were evaluated in rats pretreated with cocaine twice daily for 7 or 30 days. MATERIALS AND METHODS
Animals Male Sprague-Dawley rats (250-275 g) were obtained from Sasco-King (Oregon, WI). Animals were housed two per cage in a
Correspondence: A.D. Levy, Department of Pharmacology, Loyola University of Chicago, Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153, USA. Fax: (1) (708) 216-6596.
7 temperature, humidity and illumination- (12:12 h light/dark cycle, lights on at 07.00 h) controlled room. Water and food (Wayne Lab Blox, Lab Mills, Inc., Chicago, IL) were available ad libitum. Rats were randomly divided into groups of eight (cage mates were assigned to the same experimental conditions). All procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals as approved by the Loyola University committee on animal care and use. Blood collection. Rats were killed by decapitation in an area outside of the animal room. Trunk blood was collected into chilled centrifuge tubes containing 0.5 ml of 0.3 M EDTA (pH 7.4) for subsequent hormone assays. Blood samples were then centrifuged for 15 min at 1,000 x g. Plasma aliquots of 1.0 ml, 0.05 ml and 0.2 ml were taken for plasma renin activity (PRA), plasma renin concentration (PRC) and prolactin assays, respectively. Plasma samples were frozen at -70°C until hormone assays were initiated. Drugs. All drugs were dissolved in physiological saline, and administered intraperitoneally (i.p.) in volumes of 1 ml/kg. All drug doses are expressed as the salt. Cocaine HCI was obtained from Sigma (St. Louis, MO) or the National Institute of Drug Abuse (NIDA). p-Chloroamphetamine (PCA) was obtained from Sigma (St. Louis, MO). The 5-HT agonist R U 24969 (5-methoxy-3-(1,2, 3,4-tetrahydro-4-pyridinyl)-[1H]-indole) was donated by Roussel Uclaf (Romainville, France), and m-CPP (m-chlorophenylpiperazinc) was purchased from Sigma (St. Louis, MO).
Biochemical determinations Plasma renin activity. PRA was measured by radioimmunoassay for generated angiotensin I. To 1.0 ml of plasma sample were added: 0.5 ml of 0.5 M phosphate buffer pH 6.0, 0.025 ml of 8-hydroxyquinoline (3.4 mM final concentration) and 0.02 ml phenylmethylsulfonylfluoride (PMSF; 25 mM final concentration). Plasma samples were incubated for 3 h at 37°C. The incubation was stopped by addition of 0.5 ml of distilled water and immersion of the samples in boiling water for 3 min. The radioimmunoassay for angiotensin I is described below. Plasma renin concentration (PRC). In the PRC assay, a saturating concentration of renin substrate was added so that the generation of angiotensin I proceeds at VmaX. Renin substrate was obtained from nephrectomized rats which received dexamethasone (0.2 mg/rat) 24 h before sacrifice. To plasma samples (50/A) were added: 0.1 ml of nephrectomized plasma, 0.1 ml of 0.5 M phosphate buffer pH 6.0, and 5/~1 each of PMSF and 8-hydroxyquinoline (to a final concentration of 2.5 and 3.4 mM, respectively). The mixture was incubated at 37°C for 1 h. The reaction was stopped by addition of 0.3 ml of water and immersion of the tubes in a boiling water bath for 3 min. The radioimmunoassay for angiotensin I was performed as previously described 14, with an antiserum against angiotensin I at a dilution of 1:16,000 and a total binding of 30%. The sensitivity limit of the assay is 10 pg angiotensin I per tube, and the intra- and interassay variations were 4.4% and 7.4%, respectively. Plasma prolactin radioimmunoassay. Prolactin radioimmunoassay was performed with reagents provided by the National Institute of Arthritis, Diabetes, Digestive and Kidney disorders (NIADDK). Anti-rat prolactin serum S-8 was used at a dilution of 1:5,000 as described previously 32. Briefly, NIADDK rat prolactin (preparation rPRL-I-5) was used for iodinated tracer and NIADDK rat prolactin (preparation rPRL-RP-3) was used as the reference preparation. The intra-assay variability was 6.8%, and all samples from each experiment were determined together. Procedures In the first two experiments, cocaine was injected twice daily (08.00 h and 16.00 h) for 7 or 30 days, in doses of 0 (saline), 1,5,10 and 15 mg/kg i.p. Forty-two hours after the final cocaine injection, rats received injections of PCA (8 mg/kg i.p.) or saline. Rats were killed 1 h later and blood samples were collected as described above. In subsequent experiments, rats were administered cocaine twice daily (15 mg/kg i.p.) for 7 days. Forty-two hours after the
final cocaine injection, rats were administered the 5-HT agonists RU 24969 (0, 0.2, 1 or 5 mg/kg i.p.) or m-CPP (0, 1, 5 or 20 mg/kg i.p.), and were killed 30 min later for blood collection. Data Analysis. Hormone concentrations from the plasma samples were calculated by a computer program (RIA-AID, Robert Maciel Associates, Arlington, MA). Hormone levels for each group are presented as the mean + standard error of the mean (S.E.M.). Statistical analyses were performed by two-way analysis of variance (ANOVA), and individual group means were compared by Newman-Keuls' tests27 using a computer program (NWA StatPak, Portland, OR).
RESULTS
Pretreatment with cocaine did not alter basal plasma levels of prolactin, or P R A / P R C . In the first experiment,
15
c
,_
0 O
O
~
E&
5
O
n
0 SAL
1
5
10
*
.~ -~ 25
~ ",~ 20 ,_ ~. "~_
15
PCA
15
lo
K" ,.-.,
0 SAL
=
.9
5
10
15
,
50 4o
1
*
30
~.
20
c
"Ez° Q
cn
E ~6") ~ - J
Q n
10 0
SAL
1 5 10 15 Dose of C o c a i n e (mg/kg IP, 2x/day, ..,30 days)
Fig. 1. Inhibition of PCA-induced elevations of prolactin (top), PRA (middle), and PRC (bottom) by 30 days of cocaine exposure. Rats were administered cocaine (1-15 mg/kg i.p., 2x/day) for 30 days. PCA (8 mg/kg i.p.) was administered 42 h after the final cocaine injection and blood samples were collected 60 min later. Mean + S.E.M. for each group of eight rats is presented. Main effect of PCA (two-way ANOVA) on plasma prolactin: F1,63 = 252.2, P < 0.001. PCA increased PRA (F1,69 = 21.09, P < 0.001) and PRC (F1.69 = 24.75, P < 0.001). *Significant from corresponding saline treated (0 dose of PCA) rats (P < 0.05), Newman-Keuls' test).tSignificant from corresponding saline pretreated (0 dose of cocaine) rats (P
Brain Research, 580 (1992) 6-11 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17699
Repeated injections of cocaine inhibit the serotonergic regulation of prolactin and renin secretion in rats Andrew D. Levy a, Peter A. Rittenhouse a, Qian Li a, Anna M. Bonadonna a, Maria C. Alvarez Sanz a, Janice E. Kerr a, Cynthia L. B e t h e a b and Louis D. van de Kar a ~Department of Pharmacology, Loyola University Chicago, Maywood, 1L 60153 (USA) and bDepartment of Reproductive Biology and Behavior, Oregon Regional Primate Research Center, Beaverton, OR (USA) (Accepted 24 December 1991)
Key words: Cocaine; Chronic; Serotonin; Neuroendocrine; Prolactin; Renin
Alterations in serotonergic function following repeated cocaine injections were examined using neuroendocrine responses to a serotonin (5-HT) releaser and 5-HT agonists. Forty-two hours following administration of cocaine (1-15 mg/kg i.p.) twice daily for 7 or 30 days, male Sprague-Dawley rats were injected with the 5-HT releaser p-chloroamphetamine (PCA; 8 mg/kg i.p.) and blood samples were collected 1 h later for radioimmunoassays of plasma prolactin, plasma renin activity (PRA) and plasma renin concentration (PRC). PCA significantly increased secretion of prolactin and renin. These responses were attenuated in rats pretreated with cocaine for 30 days. In rats receiving cocaine for 7 days, the attenuation of PCA-induced secretion of prolactin and renin was less consistently observed. To determine whether these alterations were due to pre- or postsynaptic effects, rats were injected with cocaine (15 mg/kg i.p.) twice daily for 7 days, and the neuroendocrine responses to the direct 5-HT agonists RU 24969 and m-CPP were examined, 42 h after the last cocaine injection. Pretreatment with cocaine potentiated RU 24969-induced stimulation of plasma prolactin concentration. However, cocaine did not alter the ability of m-CPP to increase plasma prolactin concentrations. The stimulation of renin secretion in response to both 5-HT agonists was not altered by cocaine pretreatment. The data suggest that repeated cocaine impairs the function of serotonergic nerve terminals that regulate these endocrine responses. Furthermore, the 5-HT receptors that mediate prolactin secretion may exhibit supersensitivity. INTRODUCTION Alterations in n e u r o n a l function following repeated cocaine exposure are not well understood. While there has been considerable focus on changes in dopamine (DA) neurons, cocaine is also a highly potent inhibitor of serotonin (5-HT) uptake 6'21'2z'25. Consequently, 5-HT neurotransmission is altered by acute cocaine exposure. The concentration of 5-HT in the synapse is increased 6' ~0, and the firing of 5-HT neurons in the dorsal raphe is inhibited by cocaine 4'12'17'19. Neuroendocrine markers can be utilized to evaluate serotonergic involvement in the actions of acute or chronic cocaine. Serotonergic neurons innervating the hypothalamus exert considerable influence on the secretion of many hormones. Secretion of renin is enhanced by the 5-HT releaser p-chloroamphetamine 34 (PCA), and by various 5-HT agonists 31. Data from these studies suggest that 5-HT2 receptors mediate the serotonergic stimulation of renin secretion. Prolactin secretion is also influenced by 5-HT. In contrast to the inhibitory influence of D A 1'15, 5-HT agonists and releasers increase the se-
cretion of prolactin, probably via activation of 5-HTIB and/or 5-HT 2 receptors 3t. The long-term effects of cocaine on 5-HT neurons are poorly understood. Some studies have observed reduced 5-HT content in brain following chronic cocaine 26'29, while another study did not observe changes in brain 5-HT concentration following repeated cocaine exposure 9, Furthermore, changes in 5-HT receptor densities have not been reported following chronic cocaine exposure. Because of the high affinity of cocaine for 5-HT uptake sites 22, it is likely that chronic cocaine exposure would alter the function of serotonergic neurons. Therefore, the function of 5-HT neurons was investigated following repeated cocaine exposure. The effects of 5-HT agonists, and the 5-HT releasing agent PCA, on prolactin and renin secretion were evaluated in rats pretreated with cocaine twice daily for 7 or 30 days. MATERIALS AND METHODS
Animals Male Sprague-Dawley rats (250-275 g) were obtained from Sasco-King (Oregon, WI). Animals were housed two per cage in a
Correspondence: A.D. Levy, Department of Pharmacology, Loyola University of Chicago, Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153, USA. Fax: (1) (708) 216-6596.
7 temperature, humidity and illumination- (12:12 h light/dark cycle, lights on at 07.00 h) controlled room. Water and food (Wayne Lab Blox, Lab Mills, Inc., Chicago, IL) were available ad libitum. Rats were randomly divided into groups of eight (cage mates were assigned to the same experimental conditions). All procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals as approved by the Loyola University committee on animal care and use. Blood collection. Rats were killed by decapitation in an area outside of the animal room. Trunk blood was collected into chilled centrifuge tubes containing 0.5 ml of 0.3 M EDTA (pH 7.4) for subsequent hormone assays. Blood samples were then centrifuged for 15 min at 1,000 x g. Plasma aliquots of 1.0 ml, 0.05 ml and 0.2 ml were taken for plasma renin activity (PRA), plasma renin concentration (PRC) and prolactin assays, respectively. Plasma samples were frozen at -70°C until hormone assays were initiated. Drugs. All drugs were dissolved in physiological saline, and administered intraperitoneally (i.p.) in volumes of 1 ml/kg. All drug doses are expressed as the salt. Cocaine HCI was obtained from Sigma (St. Louis, MO) or the National Institute of Drug Abuse (NIDA). p-Chloroamphetamine (PCA) was obtained from Sigma (St. Louis, MO). The 5-HT agonist R U 24969 (5-methoxy-3-(1,2, 3,4-tetrahydro-4-pyridinyl)-[1H]-indole) was donated by Roussel Uclaf (Romainville, France), and m-CPP (m-chlorophenylpiperazinc) was purchased from Sigma (St. Louis, MO).
Biochemical determinations Plasma renin activity. PRA was measured by radioimmunoassay for generated angiotensin I. To 1.0 ml of plasma sample were added: 0.5 ml of 0.5 M phosphate buffer pH 6.0, 0.025 ml of 8-hydroxyquinoline (3.4 mM final concentration) and 0.02 ml phenylmethylsulfonylfluoride (PMSF; 25 mM final concentration). Plasma samples were incubated for 3 h at 37°C. The incubation was stopped by addition of 0.5 ml of distilled water and immersion of the samples in boiling water for 3 min. The radioimmunoassay for angiotensin I is described below. Plasma renin concentration (PRC). In the PRC assay, a saturating concentration of renin substrate was added so that the generation of angiotensin I proceeds at VmaX. Renin substrate was obtained from nephrectomized rats which received dexamethasone (0.2 mg/rat) 24 h before sacrifice. To plasma samples (50/A) were added: 0.1 ml of nephrectomized plasma, 0.1 ml of 0.5 M phosphate buffer pH 6.0, and 5/~1 each of PMSF and 8-hydroxyquinoline (to a final concentration of 2.5 and 3.4 mM, respectively). The mixture was incubated at 37°C for 1 h. The reaction was stopped by addition of 0.3 ml of water and immersion of the tubes in a boiling water bath for 3 min. The radioimmunoassay for angiotensin I was performed as previously described 14, with an antiserum against angiotensin I at a dilution of 1:16,000 and a total binding of 30%. The sensitivity limit of the assay is 10 pg angiotensin I per tube, and the intra- and interassay variations were 4.4% and 7.4%, respectively. Plasma prolactin radioimmunoassay. Prolactin radioimmunoassay was performed with reagents provided by the National Institute of Arthritis, Diabetes, Digestive and Kidney disorders (NIADDK). Anti-rat prolactin serum S-8 was used at a dilution of 1:5,000 as described previously 32. Briefly, NIADDK rat prolactin (preparation rPRL-I-5) was used for iodinated tracer and NIADDK rat prolactin (preparation rPRL-RP-3) was used as the reference preparation. The intra-assay variability was 6.8%, and all samples from each experiment were determined together. Procedures In the first two experiments, cocaine was injected twice daily (08.00 h and 16.00 h) for 7 or 30 days, in doses of 0 (saline), 1,5,10 and 15 mg/kg i.p. Forty-two hours after the final cocaine injection, rats received injections of PCA (8 mg/kg i.p.) or saline. Rats were killed 1 h later and blood samples were collected as described above. In subsequent experiments, rats were administered cocaine twice daily (15 mg/kg i.p.) for 7 days. Forty-two hours after the
final cocaine injection, rats were administered the 5-HT agonists RU 24969 (0, 0.2, 1 or 5 mg/kg i.p.) or m-CPP (0, 1, 5 or 20 mg/kg i.p.), and were killed 30 min later for blood collection. Data Analysis. Hormone concentrations from the plasma samples were calculated by a computer program (RIA-AID, Robert Maciel Associates, Arlington, MA). Hormone levels for each group are presented as the mean + standard error of the mean (S.E.M.). Statistical analyses were performed by two-way analysis of variance (ANOVA), and individual group means were compared by Newman-Keuls' tests27 using a computer program (NWA StatPak, Portland, OR).
RESULTS
Pretreatment with cocaine did not alter basal plasma levels of prolactin, or P R A / P R C . In the first experiment,
15
c
,_
0 O
O
~
E&
5
O
n
0 SAL
1
5
10
*
.~ -~ 25
~ ",~ 20 ,_ ~. "~_
15
PCA
15
lo
K" ,.-.,
0 SAL
=
.9
5
10
15
,
50 4o
1
*
30
~.
20
c
"Ez° Q
cn
E ~6") ~ - J
Q n
10 0
SAL
1 5 10 15 Dose of C o c a i n e (mg/kg IP, 2x/day, ..,30 days)
Fig. 1. Inhibition of PCA-induced elevations of prolactin (top), PRA (middle), and PRC (bottom) by 30 days of cocaine exposure. Rats were administered cocaine (1-15 mg/kg i.p., 2x/day) for 30 days. PCA (8 mg/kg i.p.) was administered 42 h after the final cocaine injection and blood samples were collected 60 min later. Mean + S.E.M. for each group of eight rats is presented. Main effect of PCA (two-way ANOVA) on plasma prolactin: F1,63 = 252.2, P < 0.001. PCA increased PRA (F1,69 = 21.09, P < 0.001) and PRC (F1.69 = 24.75, P < 0.001). *Significant from corresponding saline treated (0 dose of PCA) rats (P < 0.05), Newman-Keuls' test).tSignificant from corresponding saline pretreated (0 dose of cocaine) rats (P
