Introduction

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5-Hydroxytryptamine₂ (5-HT_2A and/or 5-HT_2C) receptors play important roles in depression, obsessive compulsive disorder, eating disorders, and schizophrenia (Blier and de Montigny, 1999; Aghajanian and Marek, 2000). Both 5-HT_2A and 5-HT_2C receptors are G protein-linked receptors that couple to phospholipase C as a second messenger and thereby increase diacylglycerol, inositol trisphosphate, and intracellular Ca²⁺ levels (for review, see Barnes and Sharp, 1999). The lack of selective agonists for each 5-HT₂ receptor subtype has hindered a precise differentiation between 5-HT_2A and 5-HT_2C receptor-mediated effects.

The involvement of the 5-HT_2C and/or 5-HT_2A receptors in the regulation of hormone secretion has been examined with a variety of 5-HT₂ receptor agonists, the most common being (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane HCl (DOI). DOI increases the plasma levels of adrenocorticotrophic hormone (ACTH), corticosterone, oxytocin, prolactin, and renin (Rittenhouse et al., 1993, 1994; Van de Kar et al., 2001). DOI was shown to stimulate the secretion of ACTH, corticosterone, oxytocin, prolactin, and renin by specifically activating the 5-HT_2A receptor as the increase in plasma levels of all these hormones was blocked by very low doses of the 5-HT_2A receptor antagonist MDL 100,907 (Van de Kar et al., 2001). Moreover, evidence suggests that 5-HT_2A receptors mediate hormone responses to other 5-HT_2C/2A agonists, such as m-chlorophenylpiperazine (m-CPP), MK-212, and quipazine. The increase in plasma corticosterone levels after injection of MK-212, m-CPP, and quipazine are blocked by MDL 100,907. On the other hand, SB 242084 and SB 200646A, 5-HT_2C receptor antagonists, do not inhibit the m-CPP-, MK-212-, and quipazine-mediated increases in plasma corticosterone levels (Hemrick-Luecke and Fuller, 1996; Hemrick-Luecke and Evans, 2002). Both studies provide evidence for the importance of the 5-HT_2A receptor subtype in the regulation of neuroendocrine responses. However, there is no direct evidence to evaluate the role of the 5-HT_2C receptor subtype in the secretion of ACTH, oxytocin, or prolactin. Thus, we used Ro 60-0175, an agonist with a high affinity for 5-HT_2C receptors, in an attempt to determine the role of 5-HT_2C receptors in hormone secretion.

Ro 60-0175 has a high affinity for human recombinant 5-HT_2C receptors (pK_i = 9.0) and 5-HT_2B receptors (pK_i = 9.3), a lower affinity for 5-HT_2A receptors (pK_i = 7.5), and an even lower affinity for other serotonin receptors (5-HT_1A, pK_i = 5.4; 5-HT_1B, pK_i = 5.3; and 5-HT₃, pK_i = 5.2) (Martin et al., 1998; Cussac et al., 2002). The affinity of Ro 60-0175 for nonserotonergic receptors has not been documented. Ro 60-0175 has been widely used in behavioral studies and shown to be 5-HT_2C receptor selective because the 5-HT_2C receptor antagonist SB 242084 blocked its effects on hypolocomotion and penile grooming, and as a discriminative stimulus (Dekeyne et al., 1999; Higgins et al., 2001). The doses of Ro 60-0175, in the present study, were chosen based on published data showing changes in behavioral responses and in neurotransmitter release (Martin et al., 1998; Millan et al., 1998).

SB 242084 is a selective 5-HT_2C receptor antagonist (pK_i = 9.0), because its affinity for other serotonergic and nonserotonergic receptors is considerably lower (5-HT_2B, pK_i = 7.0; 5-HT_2A, pK_i = 6.8; ₁-adrenergic, pK_i <5.0) (Kennett et al., 1997). The doses of SB 242084 were chosen based on published data showing inhibition of Ro 60-0175-mediated behavior (hypoactivity and penile grooming) and neurotransmitter release (Millan at al., 1998; Higgins et al., 2001).

MDL 100,907 is a selective 5-HT_2A receptor antagonist (pK_i = 9.07). Its affinity for other receptors is separated by at least one log scale (5-HT_2C, pK_i = 7.06; ₁-adrenergic, pK_i = 6.89) (Kehne et al., 1996). The doses of MDL 100,907 used were based on their ability to block DOI-mediated increases in hormone levels and to avoid occupancy of the 5-HT_2C receptor (Smith et al., 1999; Van de Kar et al., 2001).

In the present study, we used the 5-HT_2A receptor antagonist MDL 100,907 and the selective 5-HT_2C antagonist SB 242084 in conjunction with Ro 60-0175 to determine the importance of the 5-HT_2A and/or 5-HT_2C receptor in Ro 60-0175-mediated hormone secretion.

	Materials and Methods

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Animals. Male Sprague-Dawley rats (225-275 g) were purchased from Harlan (Indianapolis, IN). The animals were housed two per cage in an environment controlled for lighting (7:00 AM-7:00 PM), temperature, and humidity. Food and water were available ad libitum. Eight to 10 rats were used per experimental group. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals as approved by the Loyola University Institutional Animal Care and Use Committee.

Drugs. (S)-2-(Chloro-5-fluoro-indol-l-yl)-1-methylethylamine fumarate (Ro 60-0175) was donated by F. Hoffman-La Roche (Basel, Switzerland). (±)-2,3-Dimethoxyphenyl-1-[2-4-(piperidine)-methanol]) (MDL 100,907) was donated by Hoechst Marion Roussel Research Institute (Cincinnati, OH). 6-Chloro-5-methyl-1-[2-(2-methylpyridyl-3-oxy)-pyrid-5-yl carbonyl] indoline (SB 242084) was donated by GlaxoSmithKline (Harlow, UK). The solubilizing agent 2-hydroxypropyl--cyclodextrin was purchased from Sigma-Aldrich (St. Louis, MO).

Experimental Procedures. The rats were handled for 4 days before the treatment/sacrifice day. Rats were randomly assigned to the experimental groups (n = 8-10); cage mates were assigned to the same experimental groups.

Radioimmunoassay of Hormones. Plasma ACTH, corticosterone, oxytocin, and prolactin concentrations were determined by radioimmunoassays as described previously (Li et al., 1993, 1997). Briefly, all assays (except for corticosterone) are double antibody assays (using both primary and secondary antibodies). ACTH antiserum was obtained from IgG Corp. (Nashville, TN) and ¹²⁵I-ACTH tracer was obtained from DiaSorin Inc. (Stillwater, MN). The sensitivity limit is 0.25 pg/tube with intra- and interassay variabilities of 4.2 and 14.6%, respectively. Corticosterone antiserum was obtained from ICN Pharmaceuticals (Costa Mesa, CA) and [³H]Corticosterone tracer was obtained from PerkinElmer Life Sciences (Boston, MA). The sensitivity limit is 0.02 ng/tube and the intra- and interassay variabilities are 4.5 and 11.9%, respectively. Rabbit antioxytocin was donated by Dr. Lanny C. Keil (NASA Ames Research Center, Moffat Field, CA); ¹²⁵I-oxytocin tracer was obtained from PerkinElmer Life Sciences. The intra- and interassay variabilities are 5 and 9%, respectively. Prolactin antiserum was donated by the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases-National Institutes of Health and the tracer was prepared using National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases rPRL-I-5 and ¹²⁵I from PerkinElmer Life Sciences. The sensitivity limit is 30 pg/tube with intra-assay variability of 6.8%. All the assays (except for corticosterone) used a goat anti-rabbit secondary antibody from Calbiochem (San Diego, CA).

Statistical Analyses. All data are presented as the group mean (n = 8-10) ± S.E.M. Hormone data for the Ro 60-0175 time-course and dose-response experiments were analyzed by one-way analyses of variance (ANOVA). A two-way ANOVA was used for the SB 242084 and MDL 100,907 experiments. Newman-Keuls multiple range tests were used to compare group means (Steel and Torrie, 1960). GB-STAT software (Dynamic Microsystems, Inc., Silver Spring, MD) was used for all statistical analyses. Significant differences were defined as p  0.05. ED₅₀ values were calculated using nonlinear regression analysis from the dose-response curves with Prism software (GraphPad Software Inc., San Diego, CA).

	Results

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Ro 60-0175 Time Course (Fig. 1). Injection of Ro 60-0175 (5.0 mg/kg s.c.) produced maximal increases in plasma levels of oxytocin [1593% above basal; F_(6,57) = 42.31, p < 0.0001] and prolactin [641% above basal; F_(6,57) = 34.36, p < 0.0001] 15 min postinjection. A maximal increase in plasma ACTH [1412% above basal; F_(6,57) = 29.89, p < 0.0001] was observed between 15 and 30 min postinjection, and statistical analysis did not reveal a difference between these two time points (p = 0.49) (Fig. 1, A, C, and D). Maximum plasma corticosterone levels (2876% above basal) were observed 60 min post-Ro 60-0175 injection but were significantly elevated above basal (uninjected) levels from 15 min (1346% above basal) onward [F_(6,57) = 99.65, p < 0.0001] (Fig. 1B). No significant difference was observed between hormone levels in the saline-challenged groups and the uninjected group, except for corticosterone levels at 15 min postsaline injection (p < 0.01).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Time course (0-120 min) of the Ro 60-0175-induced increase in plasma levels of ACTH (A), corticosterone (B), oxytocin (C), and prolactin (D). **, significant effect comparing the Ro 60-0175-injected group (filled circles) with the uninjected control (filled triangle), p < 0.01 (one-way ANOVA, Newman-Keuls multiple range test). , significant effect when comparing the saline-injected group (open circle) with uninjected group, p < 0.01 (one-way ANOVA, Newman-Keuls multiple range test).

Ro 60-0175 Dose Response (Fig. 2). Administration of Ro 60-0175 produced a dose-dependent increase in plasma levels of ACTH [F_(5,46) = 31.88, p < 0.0001], corticosterone [F_(5,46) = 20.76, p < 0.0001], oxytocin [F_(5,46) = 47.23, p < 0.0001], and prolactin [F_(5,47) = 7.72, p < 0.0001] (Fig. 2, A-D). Ro 60-0175, at a dose of 5.0 mg/kg, produced a near maximal effect for corticosterone, oxytocin, and prolactin. Values for the dose that causes 50% of the maximal effect (ED₅₀) were calculated for corticosterone (ED₅₀ = 2.43 mg/kg), oxytocin (ED₅₀ = 4.19 mg/kg), and prolactin (ED₅₀ = 4.03 mg/kg). A maximal effect (E_max) for ACTH was not achieved and therefore an ED₅₀ value could not be calculated (Fig. 2A).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effect of various doses of Ro 60-0175 on plasma levels of ACTH (A), corticosterone (B), oxytocin (C), and prolactin (D). **, significant effect of Ro 60-0175-injected group (filled circles) compared with saline-injected group (open circle at 0 mg/kg), p < 0.01 (one-way ANOVA, Newman-Keuls multiple range test).

Pretreatment with the 5-HT_2C Receptor Antagonist SB 242084 (Fig. 3). Administration of SB 242084 alone did not affect the plasma levels of ACTH, corticosterone, oxytocin, or prolactin. Ro 60-0175 (5.0 mg/kg s.c.), as previously shown, increased plasma levels of ACTH, corticosterone, oxytocin, and prolactin. Pretreatment with SB 242084 (0.1, 0.5, 1, and 5 mg/kg i.p.) did not inhibit Ro 60-0175-mediated increases in ACTH [F_(4,73) = 1.37, p > 0.05], corticosterone [F_(4,73) = 0.75, p > 0.05], oxytocin [F_(4,73) = 2.19, p > 0.05], or prolactin levels [F_(4,72) = 0.23, p > 0.05] (Fig. 3, A-D). Whereas SB 242084 seemed to dose dependently decrease the Ro 60-0175-mediated increases in oxytocin levels, the statistical analysis (two-way ANOVA) indicated that this was not a statistically significant interaction [F_(4,73) = 2.19; p > 0.05] (Fig. 3C).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effect of SB 242084 pretreatment (0.1, 0.5, 1.0, and 5.0 mg/kg i.p.) on Ro 60-0175 (5.0 mg/kg s.c.)-induced increases in plasma levels of ACTH (A), corticosterone (B), oxytocin (C), and prolactin (D). **, significant effect comparing the Ro 60-0175-injected group (filled circles) with saline-injected group (open circles), p < 0.01 (two-way ANOVA, Newman-Keuls multiple range test).

Pretreatment with the 5-HT_2A Receptor Antagonist MDL 100,907 (Fig. 4). Administration of MDL 100,907 alone did not alter plasma hormone levels in rats. As was observed in the previous experiments, Ro 60-0175 produced increases in plasma levels of ACTH, corticosterone, oxytocin, and prolactin. Pretreatment with MDL 100,907 (1, 5, and 10 µg/kg s.c.) did not block Ro 60-0175-induced increases in plasma ACTH [F_(3,62) = 1.55, p > 0.05], corticosterone [F_(3,62) = 0.64, p > 0.05], oxytocin [F_(3,62) = 1.31, p > 0.05], or prolactin levels [F_(3,59) = 1.10, p > 0.05] (Fig. 4, A-D).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effect of MDL 100,907 pretreatment (1, 5, and 10 µg/kg s.c.) on Ro 60-0175 (5.0 mg/kg s.c.)-induced increases in plasma levels of ACTH (A), corticosterone (B), oxytocin (C), and prolactin (D). **, significant effect comparing the Ro 60-0175-injected group (filled circles) with their respective saline-injected group (open circles), p < 0.01 (two-way ANOVA, Newman-Keuls multiple range test).

	Discussion

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The present study is the first to demonstrate that Ro 60-0175, a commonly used 5-HT_2C receptor agonist, has an acute effect on the neuroendocrine system. This study clearly indicates that Ro 60-0175 stimulates ACTH, corticosterone, oxytocin, and prolactin secretion by a mechanism independent of the activation of 5-HT_2A or 5-HT_2C receptors. Our current data show that Ro 60-0175 dose dependently increases plasma hormone levels, an effect that is not blocked by the 5-HT_2C receptor antagonist SB 242084 or the 5-HT_2A receptor antagonist MDL 100,907.

The reason for testing Ro 60-0175 was that a debate exists regarding the role of 5-HT_2C receptors in the regulation of hormone release. Therefore, Ro 60-0175 seemed to be a good agonist that would help resolve this question. Previous studies have suggested that another putative 5-HT_2C receptor agonist, m-CPP, stimulates the secretion of several hormones by activating 5-HT_2C receptors (Bagdy et al., 1989; Aulakh et al., 1992). However, m-CPP is not a selective 5-HT_2C receptor agonist (Martin et al., 1998). The effect of m-CPP on ACTH and corticosterone release is blocked by the 5-HT_2A receptor antagonists ketanserin and MDL 100,907, respectively (Jorgensen et al., 1999; Hemrick-Luecke and Evans, 2002). Whereas 5-HT_2A receptors were identified by immunohistochemistry within the paraventricular nucleus of the hypothalamus, no immunohistochemical evidence exists for the presence of 5-HT_2C receptors, although evidence suggests that 5-HT_2C receptor mRNA is located within this area (Wright et al., 1995; Zhang et al., 2002). Considering the fact that both 5-HT_2C and 5-HT_2A receptors are expressed by hypothalamic paraventricular neurons, it was surprising that neither a 5-HT_2C antagonist nor a 5-HT_2A antagonist affected the neuroendocrine responses to Ro 60-0175.

Although we are the first to provide a comprehensive overview of the acute effects of Ro 60-0175 on plasma ACTH, oxytocin, and prolactin, a previous study examined the effect of acute Ro 60-0175 administration on plasma corticosterone levels (Hemrick-Luecke and Evans, 2002). In that study, Ro 60-0175-mediated increases in plasma corticosterone levels was completely inhibited by both MDL 100,907 (ED₅₀ = 0.0126 mg/kg) and SB 242084 (ED₅₀ = 2.0 mg/kg) (Hemrick-Luecke and Evans, 2002). A few differences in the methodological details between this study and our current study may explain the differences in the results. Hemrick-Luecke and Evans (2002) administered both of the antagonists (SB 242084 and MDL 100,907) 15 min before the injection of Ro 60-0175, and sacrificed the rats 1 h after injection of Ro 60-0175. However, as can be seen from Fig. 1, only plasma corticosterone levels would remain elevated at 1 h after Ro 60-0175 administration; all other hormones would have returned to basal levels. Thus, obtaining blood at 15 min post-Ro 60-0175 injection was necessary to examine the response of hormones other than corticosterone. Because corticosterone is regulated by both central and peripheral mechanisms (Alper, 1990), we cannot eliminate the possibility that the inhibition of Ro 60-0175-mediated increases in corticosterone levels by SB 242084 is mediated by a peripheral mechanism. We also tested a dose of 5 mg/kg s.c. of Ro 60-0175, whereas Hemrick-Luecke and Evans (2002) used 3 mg/kg s.c. Thus, the higher dose of Ro 60-0175 may have activated other, as yet unidentified receptors, which might not be blocked by MDL 100,907 and SB 242084. However, the doses of MDL 100,907 (1-10 µg/kg s.c.) we tested completely blocked the effect of DOI (2.5 mg/kg i.p.) on plasma levels of ACTH, corticosterone, oxytocin, prolactin, and renin (Van de Kar et al., 2001). Therefore, we are confident that Ro 60-0175 at the dose used (5 mg/kg s.c.) is exerting its effect by stimulating a mechanism or a receptor other than the 5-HT_2C or 5-HT_2A receptors. In the following paragraphs, we will attempt to discuss other probable mechanisms mediating the Ro 60-0175-induced increases in plasma hormone levels.

Studies in CHO-K1 cells transfected with rat 5-HT_2A, 5-HT_2B, or 5-HT_2C receptors suggest that Ro 60-0175 has the highest potency at 5-HT_2B receptors (pEC₅₀ = 8.6) versus 5-HT_2C (pEC₅₀ = 7.92) and 5-HT_2A receptors (pEC₅₀ = 6.78) (Vickers et al., 2001). The agonist profile of Ro 60-0175 was detected by its ability to increase intracellular calcium levels. A similar rank potency was observed when human 5-HT_2A, 5-HT_2B, or 5-HT_2C receptors were transfected into the same cell line (Porter et al., 1999). There are several reasons to interpret these data with caution. First, although 5-HT_2B receptor mRNA has not been detected in rat hypothalamus, its protein expression has been shown to exist in rat dorsal hypothalamic nucleus (Flanigan et al., 1995; Duxon et al., 1997). No quantitative comparison exists for receptor expression between the 5-HT_2B, 5-HT_2A, and 5-HT_2C receptors in the hypothalamus or any brain region. The transfected CHO cells likely express higher amounts of 5-HT_2B and 5-HT_2C receptors than can be found in the hypothalamus. Also, the importance of 5-HT_2B receptors within the hypothalamus and in neuroendocrine regulation is unknown. At the present time, we have no access to specific 5-HT_2B agonists and antagonists to examine whether the effect of Ro 60-0175 on plasma hormone levels is mediated by 5-HT_2B receptors.

Ro 60-0175 is not likely to increase synaptic concentrations of 5-HT and thereby indiscriminately activate postsynaptic 5-HT receptors mediating hormone secretion. Ro 60-0175 was shown not to increase the extracellular levels of 5-HT in frontal cortex (Millan et al., 1998). The structural similarity of Ro 60-0175 to indolamines allows for the possibility that Ro 60-0175 may act at receptors responsive to indolamines without increasing the synaptic concentration of 5-HT. However, binding studies indicate a low affinity of Ro 60-0175 for 5-HT receptors, other than 5-HT_2C and 5-HT_2B receptors (Martin et al., 1998; Cussac et al., 2002).

It also is possible that catecholamines, such as dopamine and norepinephrine, may play a role in the effects of Ro 60-0175 on plasma hormone levels. Dopamine has an inhibitory effect on prolactin secretion but stimulates oxytocin and ACTH secretion (Meller et al., 1991; Borowsky and Kuhn, 1992; Ben Jonathan and Hnasko, 2001). Cortical dopamine levels decrease after administration of Ro 60-0175 (Millan et al., 1998). By suppressing dopamine levels, Ro 60-0175 may be alleviating the dopamine-mediated inhibition of prolactin secretion. This specific dopamine hypothesis may explain what we observe with prolactin but does not offer an explanation as to how oxytocin and ACTH levels increase. On the other hand, the changes observed in cortical dopamine and norepinephrine levels may not be mirrored within the hypothalamic paraventricular nucleus. For example, an increase in cortical norepinephrine was observed after treatment with SB 242084 (Millan et al., 1998). Norepinephrine stimulates the secretion of ACTH and oxytocin (Feldman and Weidenfeld, 1996). If an increase in norepinephrine was also present within the paraventricular nucleus, we should have observed an increase in plasma ACTH and oxytocin levels after administration of SB 242084. However, we observed no effect of SB 242084 alone on hormone levels. Hence, catecholamines are not likely mediators of the effect of Ro 60-0175 on plasma hormones.

An alternative hypothesis to explain the effects of Ro 60-0175 on plasma hormone levels may be that Ro 60-0175 activates a nonserotonergic receptor, which in turn elevates plasma hormone levels. Although Ro 60-0175 has a high affinity for 5-HT_2C receptors and a lower affinity for 5-HT_2A receptors, its affinities for receptors outside the 5-HT receptor family have not been documented (Martin et al., 1998). Ro 60-0175 may directly activate another receptor, such as an adrenergic or histamine receptor, which mediates the increase in hormone secretion. The ₁-adrenergic receptor antagonist prazosin and -adrenergic receptor antagonist sotalol attenuate stress-induced increases in plasma ACTH and corticosterone levels (Feldman and Weidenfeld, 1996). Furthermore, catecholaminergic neurons mediate stress-induced oxytocin and corticosterone release (Richardson Morton et al., 1990; Knigge et al., 1999). Histamine (H₁) receptors are abundant within the paraventricular nucleus of the hypothalamus and histamine is also known to increase the secretion of ACTH and oxytocin (Kjær et al., 1992). Thus, the possibility that Ro 60-0175 can stimulate receptors and increase hormone secretion cannot be ignored, particularly because no information is presently available regarding the affinity of Ro 60-0175 for these receptors.

In conclusion, our data indicate that Ro 60-0175 dose dependently increases plasma hormone levels by a mechanism independent of direct stimulation of either 5-HT_2C or 5-HT_2A receptors. Thus, the most probable mechanism through which Ro 60-0175 increases hormone release is activation of other receptors that remain to be identified. Hence, consistent with previous reports (Vickers et al., 2001), Ro 60-0175 is not a selective 5-HT_2C receptor agonist.