N~~~h~~~~ol~g~ Vol. 31, Printed in Great Britain

No. 7,

pp,671-678, 1992

~28-3~~2 $5.00 + 0.00 FkrgamonPressLtd

D, AND D, DOPAMINE RECEPTORS STIMULATE HYPOTHALAM~PITUITARY-ADRENAL ACTIVITY IN RATS BETHBOROWSKY* and CYNTHIAM. KUHN Department of Pharmacology,

DUMC 3813, Duke University Medical Center, Durham, NC 27710, U.S.A (Accepted

20 November

1991)

Summary-A stimulatory role for endogenous dopamine (DA) in the regulation of hypothalamopituit~y-adrenal activity has previously been demonstrated. In the present study, the roles of D, and DZ subtypes of DA receptors in the regulation of activity of the hypo~aiam~it~ta~-ad~nal axis we= investigated. The int~pe~toneal administration of either the D, agonist, SKF 383393 (I-phenyl-2,3,4,5 tetrahydro-(iH)-benzazepine-7,Sdiol HCl, S-20 mg/kg) or the D, agonist quinpiroie (0.05-l m&kg) dosedependently elevated both adrenocorticotropic hormone (ACTH) and corticosterone (CS) in serum. Similarly, administration of either SKF 38393 or quinpirole (l-100 pg) into the third ventricle dose-dependently elevated ACTH in serum. The response of ACTH to intraperitoneal SKF 38393 was blocked by pretreatment with the D, antagonist SCH 23390 (1-chloro-8-hydroxy-3-methyl-I-phenyl-2,3,4,5 tetrahydrolH-3-benzazepine, 0.25 mg/kg, i.p.) but not by the D, antagonist sulpiride (50mg/kg, i.p.). The response of ACTH to intraperitoneal injection of quinpirole was blocked by pretreatment with sulpiride and attenuated slightly by pretreatment with SCH 23390. Further, the co-administration of sub-maximum doses of SKF 38393 and quinpirole caused additive increases in ACTH in serum. These results suggest that both D, and D, subtypes of DA receptors contribute to the do~ner~c regulation of function of the h~oth~am~pitui~~-ad~nal axis and support a role for DA neurons in the hypothalamus in this response. Further, these findings suggest that the D, and D, receptors, mediating the response of the hypothalamopituitary-adrenal axis are not tightly coupled. The involvement of DA neurons in the hypothalamus, as well as the potent effect of quinpirole, suggest that D, receptors may mediate this

response. Key wordsdopamine

receptors, adrenocorticotropic

This laboratory has published several findings supporting a role for endogenous dopamine in the regulation of activity of the hypothalamo-pituitaryadrenal axis (Borowsky and Kuhn, 199la, b, c). Both cocaine and selective inhibitors of the uptake of DA, stimulate secretion of ACTH, an action which is attenuated by DA antagonists. However, the types of DA receptors mediating this response is unclear. The response of the hypothalam~pituit~y-adrenal axis to cocaine was attenuated by either D, or D, DA receptor-selective antagonists, suggesting that both D, and Dz subtypes of DA receptors contribute to DA-mediated activation of the hypothalamopituitary-adrenal axis. A stimulatory effect of DZ agonists on the function of the hypothalamopities-adrenal axis has been demonstrated previously (Fuller and Snoddy, 1981; Fuller, Snoddy, Mason, Clemens and Bemis, 1983; Jezova, Jurcovicova, Vigas, Murgas and Labrie, 1985). However, the response to D, agonists has not been investigated. The dopaminergic regulation of the function of the hypothalamo-pituitary-adrenal axis appears to be mediated by DA neurons in the hypothalamus, that are distinct from the DA neurons in the forebrain, *Present address: Laboratory of Cell Biology, Building 36, Room 3A17, National Institutes of Health, Bethesda, MD 208?‘, !‘.S.A. NP 31,7-E

hormone, HPA axis, corticosterone,

D, , 9.

mediating behavioral activation (Borowsky and Kuhn, 1990,199lc). The purpose of the present study was to determine the relative roles of D, and D2 subtypes of DA receptors in the dopaminergic regulation of function of the hypothalamo-pituitaryadrenal axis. In addition, the potential interaction between D, and DZ receptors, regulating function of the hy~thalam~pituita~-adrenal axis was evaluated to determine if a synergistic interaction existed between these receptors, like that described for recaptors mediating behavioral responses to DA agonists (Breese and Mueller, 1985; Molloy and Waddington, 1984; Mailman, Schultz, Lewis, Staples, Rollema and Dehaven, 1984; Walters, Bergstrom, Carlon, Chase and Braun, 1987). The response of the hypothalamopitui~~-ad~nal axis toeither the D, selective agonist, SKF 38393 or the DZ selective agonist, qu~pirole was determined after both systemic and central administration. Further, the D, selective antagonist, SCH 23390 and the Dz selective antagonist, sulpiride were used to verify the selectivity of these agonists, and to evaluate the independence of D,- and D,-mediated effects. METHODS

The partial D, agonist (+)SKF 38393 NC1 (O’Boyle, Gaitanopoulos, Brenner and Waddington,

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BETH B~ROWSKYand CYNTHIAM. KUHN

1988), theD, antagonist (R( + )SCH 23390 HCI (Iorio, Bamett, Leitz, Houser and Korduba, 1985; Hytell, 1983) and the D2 agonist (-)quinpirole HCl (Tsuruta, Frey, Grewe, Cote, Eskay and Kebabian, 1981) were purchased from Research Biochemical Inc. (Natick, Massachusetts) and dissolved 1 distilled water. Ketamine HCl (Parke-Davis, .. ,rris Plains, New Jersey) and xylazine (Mobay Corporation, Shawnee, Kansas) were obtained from Duke University Medical Center (Durham, North Carolina). Stainless steel guide, internal and dummy cannulae, were purchased from Plastics One (Roanoke, Virginia). Animals

Male Sprague-Dawley rats (Zivic Miller, Allison weighing 200-300 g, were Park, Pennsylvania), housed with free access to food and water. The environment was temperature- and humiditycontrolled and was maintained on a 12-hr light/dark cycle (lights on at 6:00 a.m.). Rats, with indwelling cannulae in the brain were housed singly, while other rats were housed in groups of 3. Cannulation of the brain

Rats were anesthetized with xylazine : ketamine (10: 50 mg/kg) and 26-gauge guide cannulae were implanted with the tips angled 1 mm above the lower III ventricle (1.8 mm posterior to bregma, 8.1 mm below the dura at the midline; Paxinos and Watson, 1982; Matta, Beyer, McAllen and Sharp, 1987) and secured with stainless steel mounting screws and cranioplastic cement. Dummy cannulae were attached to the guides. Administration of dregs

Drugs were administered intraperitoneally in a volume of 1 ml/kg, except for sulpiride, which was injected in a volume of 2 ml/kg. Drugs were administrated

613

D,/D, stimulation of HPA activity Radioimmunoassays

Corticosterone was measured by radioimmune assay (RIA), using an antibody a-d high pressure liquid chromatography (HPLC) purified standards from Radioassay Systems Laboratories (Carson, California) and [H)corticosterone from New England Nuclear (Boston, Massachusetts). Five ~1 samples were assayed in duplicate. Serum proteins were denatured by heating at 98°C for 10 min. Bound and free hormone were separated by dextran-coated charcoal and the samples were counted by liquid scintillation spectrometry, in a scintillation cocktail. The sensitivity of the assay was 1.6 ng/ml and the inter- and intra-assay variances were 9 and 7%, respectively. The level of ACTH was measured by RIA, using an antibody from INCSTAR (Stillwater, Minnesota), directed toward the 14-24 portion of ACTH, which cross-reacts 100% with human ACTH l-74 and porcine ACTH l-39. The [‘2SI]ACTH was ob tined from ICN (Lisle, Illinois). Synthetic ACTH from Peninsula Labs (Belmont, California) was diluted in ACTH-free serum to make the standards. The sensitivity of the assay was 15 pg/ml and the inter- and intra-assay variances were 6 and 6.3%, respectively.

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Statistical analysis One-way analysis of variance (ANOVA), followed by Duncan’s multiple range test, was used to evaluate time-course studies and dose-response relationships. Pretreatment effects were evaluated by two way analysis of variance (ANOVA), followed by post-hoc t-tests. RESULTS

first experiment designed to the response the hypothalamo-pituitary-adrenal axis to systemically administered SKF 38393 and quinpirole, sptective D, and D, agonists, respectively. As shown in Fig. 1 (top), the intraperitoneal injection of SKF 38393 (lOmg/kg) elevated the level of both ACTH and corticosterone, up to 45 min (significant main effect of time: F(4,28) = 4.1, P < 0.01 for ACTH; and F(4,28) = 11.9, P < 0.01 for CS). The response of the hypothalamo-pituitaryadrenal axis to SKF 38393 was dose-dependent, 30min after the injection (significant main effect of dose: F(4,25) =5.4, P < 0.01 for ACTH; and F(4,24) = 12.8, P < 0.01 for CS; Fig. 1, bottom). Figure 2 (top) shows that quinpirole (0.5 mg/kg) also elevated the level of both ACTH and corticosterone, within 15 min after intraperitoneal injection, with baseline values returning by 120 min for ACTH and 360 min for corticosterone (significant main effect of time: F(5,25) = 29.1, P < 0.01 for ACTH; and F(5,29) = 24.7, P < 0.01 for CS; data not shown for 360min time point). Further, the intraperitoneal administration of quinpirole elicited a dose-dependent elevation of both ACTH and corticosterone,

Fig. 1. Time-course (top) and dose-response (bottom) curves for the effects of SKF 38393 (solid symbols) or vehicle (open symbols) on corticosterone and ACTH in serum. Animals received 10 mg/kg of SKF 38393, intraperitoneally for the time-course and were treated 30 min before killing for the dose-response. Each point represents the mean f SEM of 4-9 animals. Significantly different from vehicle control, *P < 0.05.

45 min after the injection (significant main effect of dose: F(5,29) =21.1, P co.01 for ACTH and F(5,29) =27.6, P < 0.01 for CS; Fig. 2, bottom). To determine if the activation of the hypothalamo-pituitary-adrenal axis, after SKF 38393 or quinpirole, was mediated by stimulation of D, and D, DA receptors respectively, the response of ACTH to these agonists was determined in animals, pretreated with selective DA receptor antagonists. In addition, the ability of a D, or D, antagonist to attenuate the response of ACTH to a D, or D, agonist, respectively, was tested to determine if a tonic stimulation at one subtype of receptor was a necessary requirement for a stimulation of the hypothalamo-pituitaryadrenal axis by another subtype of receptor. Despite differences in the magnitude of the response to SKF 38393 on different experimental days, pretreatment with SCH 23390, a selective D, antagonist, but not sulpiride, a selective D, antagonist, significantly blocked the response of ACTH to SKF 38393 (10 mg/kg; interaction effect: F( 1,23) = 9.7, P < 0.01 for SCH 23390; F( 1,26) = 0.2 for sulpiride; Fig. 3). In addition, as shown in Fig. 4, the response of ACTH

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Fig. 2. Time-course (top) and dose-response (bottom) curves for the effectsof quinpirole (solid symbols) or vehicle (open symbols) on corticosterone and ACTH in serum. Animals received 0.5 mg/kg of quinpirole, intraperitoneally for the time-course and were treated 45 min before killing for the dose-response. Each point represents the mean f SEM of 4-8 animals. Significantly different from

vehicle control, *P < 0.05.

quinpirole (0.5 mg/kg) was significantly blocked by sulpiride and slightly but significantly attenuated by SCH 23390 (significant interaction effect: F(1,31) = 42.0, P < 0.01 for sulpiride; F(1,28) = 7.1, P < 0.01 for SCH 23390). to

OControl 600

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The above experiments demonstrated that both D, and Dr receptor agonists stimulated the activity of the hypothalamo-pituitary-adrenal axis and suggest that a tonic stimulation of Dr receptors may be necessary for a full Dr receptor-mediated response. To test further the possibility that D, and D2 receptors act cooperatively to stimulate the activity of the hypothalame-pituitary-adrenal axis, animals were treated with sub-maximum doses of either SKF 38393, quinpirole or both. As shown in Fig. 5, the response of ACTH to the combination of SKF 38393 and quinpirole, was similar to the sum of the responses of ACTH to these compounds, administered separately. It has been demonstrated previously, that DA neurons accessible from the third ventricle, can stimulate the activity of the hypothalamo-pituitaryadrenal axis (Borowsky and Kuhn, 1991c). The next experiment was designed to determine if the response to both Dr and D, selective agonists was mediated by DA receptors, accessible from the third ventricle. Either SKF 38393 or quinpirole (l-1OOpg) was administered to unrestrained animals into the third ventricle through an indwelling cannula. As shown in Fig. 6, both SKF 38393 and quinpirole elicited a dose-dependent elevation in levels of ACTH in serum 10 min after intraventricular administration (significant effect of dose: F(4,22) = 5.3, P < 0.01 for SKF 38393 and F(3,26) = 3.2, P < 0.05 for quinpirole).

DISCUSSION

The major finding of the present study was that both D, and D2 DA receptor agonists stimulated activity of the hypothalamc+pituitary-adrenal axis. While the D, and Dr components of this response do not appear to be cooperative, the results suggest that a certain tone in D, receptors may be required for a full Dz response. Further, both the D, and D, receptors involved in this response were located centrally, at a site accessible from the third ventricle.

ISKF38393

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Fig. 3. Response of ACTH to SKF 38393 (10 mg/kg, i.p., 30 min before killing) after pretreatment with SCH 23390 (left) or sulpiride (right). Each bar represents the mean f SEM of 7-8 animals. Significantly different from control, *P < 0.05, significantly different from vehicle, + P < 0.05.

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Fig. 4. Response of ACTH to quinpirole (0.5 mg/kg, i.p., 45 min before killing) after pretreatment with sulpiride (left) or SCH 23390 (right). Each bar represents the mean f SEM of 7-9 animals. Significantly different from control, *P < 0.05; significantly different from vehicle, +P < 0.05.

A role for endogenous DA in the regulation of function of the hypothalamo-pituitary-adrenal axis was previously established, using indirectly-acting DA agonists (Borowsky and Kuhn, 1991b). In the present study, it has been demonstrated that both D, and D, DA receptors may be involved in this response. The stimulation of the hypothalamr+ pituitary-adrenal axis, after the administration of either D, or Dz selective DA agonists into the third ventricle, suggests that both receptors may be localized in the hypothalamus. Quantitative autoradiographic studies, demonstrating the existence of both D, and D, receptors in the hypothalamus support this conclusion (Bouthenet, Martres, Sales and Schwartz, 1987; Boyson, McGonigle and Molinoff, 1986; Dawson, Barone, Sidhu, Wamsley and Chase, 1988). The present findings confirm and extend earlier pharmacological studies on the response of the hypothalamo-pituitary-adrenal axis to inhibitors of the uptake of DA (Borowsky and Kuhn, 1991b). It was shown previously that the response to cocaine was 0 1000

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significantly attenuated by pretreatment with either SCH 23390 or sulpiride. Similarly, the response to a selective inhibitor of the uptake of DA, was reduced, at least to some extent by either SCH 23390 or sulpiride and was significantly attenuated by the nonselective antagonist, fluphenazine. In addition, the stimulatory effect of D2 agonists, demonstrated in this study, confirms previous studies reporting elevations of corticosterone after D, agonists (Fuller and Snoddy, 1981; Fuller et al., 1983, Jezova et al., 1985). The finding that neither antagonist, used in this study, potently increased secretion of ACTH suggests that previous findings, including the present authors’ (Borowsky and Kuhn, 1991a, b) finding that large doses of haloperidol increased secretion of ACTH, might be best explained by the nondopaminergic actions of large doses of this agent. This study produced conflicting results concerning the interaction between Di and D, receptors involved in stimulation of activity of the hypothalamc+ pituitary-adrenal axis. The finding that SCH 23390

Control Quinpirole

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