Bombesin-induced HPA and sympathetic activation requires CRH receptors

Peptides 22 (2001) 57– 65

Bombesin-induced HPA and sympathetic activation requires CRH receptors Pam Kenta, Tania Be´darda, Samir Khana, Hymie Anismanc, Zul Meralia,b,* a

School of Psychology, University of Ottawa, 11 Marie Curie, Ottawa, Ontario, Canada Department of Cellular & Molecular Medicine, University of Ottawa, 11 Marie Curie, Ottawa, Ontario, Canada c Institute of Neuroscience, Carleton University, Ottawa, Ontario, Canada

b

Received 6 July 2000; accepted 16 October 2000

Abstract Central administration of bombesin (BN) (into the ventricular system) increased circulating levels of ACTH, corticosterone, epinephrine, norepinephrine and glucose, indicating that this peptide activates the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system. We then assessed the potential contribution of corticotropin-releasing hormone (CRH) system, in the mediation of these BN effects. Blockade of CRH receptors with ␣h-CRF (10 ␮g) attenuated or blocked the BN-induced rise in plasma ACTH, epinephrine, norepinephrine, glucose and corticosterone levels. These findings support the notion that BN-induced HPA axis and sympathetic activation are mediated, at least in part, via activation of CRH neurons. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Gastrin-releasing peptide; ACTH; Corticosterone; Norepinephrine; Epinephrine; Glucose

1. Introduction Originally extracted from the skin of the European frog bombina bombina, bombesin (BN)-like peptides have been found to be widely distributed within the mammalian central nervous system and gastrointestinal tract [26 –28,31]. The administration of BN-related peptides to experimental animals produces a wide spectrum of biologic effects including thermoregulation, satiety, smooth muscle contraction and chemotaxis [10,12,21,22,37]. More recent evidence suggests that BN-like peptides may also influence the activity of the hypothalamic-pituitary-adrenal (HPA) axis as well as the sympathetic division of the autonomic nervous system. In this context, it has been reported that exogenous administration of BN, or its mammalian counterparts, gastrin-releasing peptide (GRP) and neuromedin B (NMB), can potently stimulate the release of ACTH from the anterior pituitary and/or corticosterone from the adrenal medulla [13,14,20,30]. Moreover, BN-related peptides appear to activate the autonomic nervous system, as i.c.v. injection of * Corresponding author. School of Psychology, University of Ottawa, 11 Marie Curie, Room 214 (Vanier Bldg.), Ottawa, Ontario, Canada, K1N 6N5. Tel.: ⫹1-613-562-5800 ext. 4848; fax: ⫹1-613-562-5356. E-mail address: [email protected] (Z. Merali).

BN dose-dependently increases plasma epinephrine, norepinephrine and glucose levels [2,3,29,33]. While the neuronal mechanisms underlying these BNelicited effects remain unclear, it appears that BN-like peptides may mediate some of these effects by recruiting ACTH secretagogue(s), in particular corticotropin-releasing hormone (CRH). Indeed, BN-like peptides share many similarities with CRH in terms of anatomic distribution, endocrine, autonomic and behavioral effects [17]. Moreover, our laboratory has recently shown that pretreatment with the CRH antagonist, ␣h-CRF, blocked BN-induced behavioral effects (suppression of food intake and enhanced grooming behavior) [34]. There is limited data suggesting that the endocrine and/or autonomic responses to BN may also require the participation of CRH neurons. Indeed, BN and GRP potentiate CRH-induced ACTH release [1,11,30]. In addition, i.v. infusion of CRH antiserum or central administration of ␣h-CRF attenuates GRP-stimulated plasma ACTH and corticosterone release [13,30]. The aim of the present study was to characterize the role of CRH in the mediation of BN-induced activation of both the HPA axis and sympathetic nervous system. Thus, in an initial study, we assessed whether 1) central administration of BN would affect plasma ACTH, corticosterone, norepinephrine, epinephrine and glucose levels, and 2) whether

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blockade of CRH receptors (with ␣h-CRF) could attenuate these BN-induced effects. While pretreatment with ␣h-CRF blocked or attenuated the BN-induced increases in plasma ACTH, norepinephrine, epinephrine and blood glucose levels, it failed to alter the rise in plasma corticosterone levels at the 30 min interval. In light of the observation that ACTH (the primary corticosterone secretagogue) release was attenuated by CRH receptor blockade but not corticosterone release, we decided to further explore this interaction. It was thought that the inability of the CRH antagonist to influence corticosterone may be attributable to 1) direct effects of exogenous BN administration on the adrenal cortex to evoke corticosterone release, 2) the dose of BN (or CRH antagonist) selected, or 3) the single time point analysis i.e. the effects may have preceded or followed the 30 min time point selected. In order to address some of these possibilities, a second more dynamic experiment was performed where a lower dose of BN was used and blood samples were collected at different time points to assess the effects of BN on blood corticosterone and glucose levels in the absence and presence of ␣h-CRF.

2. Materials and methods 2.1. Animals Male Sprague-Dawley rats (weighing between 325 and 400 g), obtained from Charles River (St. Constant, Quebec) were used in both experiments. All animals were individually housed and maintained on a 12-h light/dark cycle (with lights on at 6:00 a.m.), in a temperature (23°C) and humidity (60%) controlled room. Animals had free access to food and water. 2.2. Surgical procedure All rats were anesthetized with pentobarbital (60 mg/kg; i.p.) and stereotaxically implanted with permanent guide cannulae (22 gauge) aimed at the 3rd ventricle. The placement coordinates [32] were 4.4 mm posterior to bregma, 0 mm lateral, and 4.4 mm ventral to the skull surface. The cannulae were anchored to the skull surface using 4 stainless steel screws and acrylic dental cement and plugged with removable stainless steel stylettes. During the recovery period, animals were acclimated to handling as well as mock central injections. 2.3. Experimental protocol 2.3.1. Experiment 1a. Effects of central BN administration on plasma ACTH, corticosterone, norepinephrine, epinephrine and glucose levels All experiments were performed between 9:00 and 12:00 A.M. On test day, rats were randomly divided into 3 groups

(n ⫽ 7–10/group) and were microinjected with either vehicle (saline), or one of two doses of BN (0.25 ␮g or 0.5 ␮g; i.c.v.). Bombesin (Bachem, California) was freshly dissolved in 0.9% saline prior to administration. The microinjections were delivered in a 3 ␮l volume infused over 60 s via an injection cannula (0.5 mm longer than the guide cannula) connected by polyethylene tubing to an infusion pump (Harvard Apparatus, MA). Rats were sacrificed 30 min post injection and trunk blood was collected in tubes containing EDTA, centrifuged and the plasma was frozen at ⫺70°C. Blood glucose levels were determined (from the whole trunk blood) using a portable glucometer Elite (Ames, Miles Canada) as previously described by Messier and Kent [25]. Plasma ACTH and corticosterone levels were measured using commercial radioimmunoassay (RIA) kits (ICN Pharmaceuticals, CA). Plasma epinephrine and norepinephrine levels were determined by HPLC using a modification of the method of Seegal et al. [38]. 2.3.2. Experiment 1b. Effects of CRH receptor blockade on BN-induced increases in plasma ACTH, corticosterone, norepinephrine, epinephrine and glucose levels The design of this experiment was identical to that of experiment 1a except that rats were randomly divided into 4 groups (n ⫽ 7–10/group) and were microinjected with either vehicle (saline), ␣h-CRF (10 ␮g/kg), saline followed 20 min later with BN (0.25 ␮g; i.c.v.) (saline ⫹ BN), or ␣h-CRF (10 ␮g/kg) followed 20 min later with BN (0.25 ␮g/kg) (␣h-CRF ⫹ BN). 2.3.3. Experiment 2. Effects of CRH receptor blockade on BN-induced increases in corticosterone and glucose as a function of time The design of this experiment was identical to that of experiment 1 except that the dose of BN used was 0.2 ␮g (i.c.v.) and blood samples were collected at 0, 15, 30, 60 and 120 min (following drug or saline injection). Blood samples were collected by lancing the rat’s tail close to the tip with a razor blade and then wiping the first droplet of blood away. Blood droplets (totaling 20 ␮l) were then blotted onto preprinted circles on S&S filter paper (Schleicher & Schuell, Mandel Scientific), allowed to dry at room temperature and stored at ⫺20°C. Blood was eluted from the filter paper by placing one 2.5 mm punch (per time interval) of filter paper in a 12 ⫻ 75 culture tube containing 100 ␮l of Dulbecco’s phosphate-buffered saline (PBS) (Sigma, USA) and then shaking the tubes on an automatic shaker (50 rpm) for 1 h at room temperature. Tubes were stored overnight in a refrigerator (at 4°C). On the following day, tubes were again agitated on an automatic shaker for 1 h at room temperature and corticosterone levels in the eluted blood samples were determined using a commercial RIA kit (ICN Pharmaceuticals, CA).

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Fig. 1. Plasma levels of ACTH, corticosterone, norepinephrine, epinephrine and glucose (mean ⫾ S.E.M.) following central BN administration. The open columns represent control animals injected with vehicle (saline, 3 ␮l; i.c.v.) whereas the hatched and solid columns represent animals injects with BN at 0.25 ␮g and 0.5 ␮g (i.c.v.), respectively. * significantly different from saline values at P ⬍ 0.05. † significantly different from low dose BN (0.25 ␮g) values at P ⬍ 0.05.

2.4. Statistical analysis All results are expressed as means ⫾ S.E.M. In experiments 1a and 1b, blood glucose, plasma ACTH, corticosterone, epinephrine and norepinephrine values were analyzed separately by one factor (Treatment) analyses of variance (ANOVA) followed by Newman-Keuls multiple comparisons. In experiment 2, blood corticosterone and glucose levels were analyzed separately using two factor repeated measures ANOVAs. The between-groups factor was Treatment condition (saline, ␣h-CRH, saline ⫹ BN and ␣h-CRH ⫹ BN) and the within-subjects factor was Time (0, 15, 30, 60 and 120 min). Post hoc comparisons were conducted using Newman-Keuls multiple comparisons. A value of P ⬍ 0.05 was considered statistically significant. 3. Results 3.1. Experiment 1a. Effects of central BN administration on plasma ACTH, corticosterone, norepinephrine, epinephrine and glucose levels Fig. 1 shows plasma ACTH, corticosterone, norepineph-

rine, epinephrine and glucose levels in rats centrally injected with saline, or one of two doses of BN (0.25 or 0.5 ␮g). ANOVA revealed significant effects of BN treatment on plasma levels of ACTH (F(2, 21) ⫽ 20.30; P ⬍ 0.0001), corticosterone (F(2, 22) ⫽ 27.29; P ⬍ 0.0001), norepinephrine (F(2, 19) ⫽ 6.23; P ⬍ 0.01), epinephrine (F(2, 19) ⫽ 7.66; P ⬍ 0.005) and glucose (F(2, 17) ⫽ 17.44; P ⬍ 0.0002). Newman-Keuls multiple comparisons of the simple effects revealed significant elevations in plasma concentrations of ACTH following administration of the higher dose of BN (0.5 ␮g) and significant increases in plasma corticosterone, norepinephrine, epinephrine and blood glucose levels following administration of both doses of BN (0.25 and 0.5 ␮g). 3.2. Experiment 1b. Effects of CRH receptor blockade on BN-induced increases in plasma ACTH, corticosterone, norepinephrine, epinephrine and glucose levels Plasma ACTH, corticosterone, norepinephrine, epinephrine and glucose levels in rats centrally injected with either vehicle (saline), ␣h-CRF (10 ␮g/kg), saline ⫹ BN (0.25 ␮g;

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Fig. 2. Plasma levels of ACTH, corticosterone, norepinephrine, epinephrine and glucose (mean ⫾ S.E.M.) in rats that received central injections of either saline, ␣h-CRF (10 ␮g/kg), saline ⫹ BN (0.25 ␮g; i.c.v.) or ␣h-CRF (10 ␮g/kg) ⫹ BN (0.25 ␮g; i.c.v.). * significantly different from saline values at P ⬍ 0.05. † significantly different from saline ⫹ BN values at P ⬍ 0.05.

i.c.v.) or ␣h-CRF (10 ␮g/kg) ⫹ BN (0.25 ␮g; i.c.v.) are depicted in Fig. 2. Individual ANOVAs revealed significant Treatment effects on plasma levels of ACTH (F(3, 28) ⫽ 3.66; P ⬍ 0.025), corticosterone (F(3, 27) ⫽ 6.067; P ⬍ 0.003), norepinephrine (F(3, 23) ⫽ 4.65; P ⬍ 0.012), epinephrine (F(3, 25) ⫽ 14.50; P ⬍ 0.0001) and blood glucose levels (F(3, 23) ⫽ 17.07; P ⬍ 0.0001). Newman-Keuls comparisons of the simple effects revealed that saline ⫹ BN administration significantly increased plasma levels of ACTH, corticosterone, norepinephrine, epinephrine and blood glucose levels. Whereas, ␣h-CRF was without effect on its own, its administration prior to BN injection blocked the BN-induced rise in plasma epinephrine and ACTH levels, and attenuated the BN-induced increases in circulating norepinephrine and blood glucose levels. Pretreatment with ␣h-CRF had no effect on BN-elicited changes in plasma corticosterone levels.

3.3. Experiment 2. Effects of CRH receptor blockade on BN-induced increases in corticosterone and glucose as a function of time Figs. 3 and 4 illustrate blood corticosterone and glucose levels respectively, at 0, 15, 30, 60 and 120 min following drug administration. Rats received the following central treatments: 1) injections of saline (control), 2) ␣h-CRF (10 ␮g/kg), 3) saline ⫹ BN (0.2 ␮g; i.c.v.) or ␣h-CRF (10 ␮g/kg) ⫹ BN (0.2 ␮g; i.c.v.). The overall two factor repeated measures ANOVA revealed that blood corticosterone levels varied as a function of Treatment ⫻ Time interaction (F(12, 124) ⫽ 9.66; P ⬍ 0.0001). NewmanKeuls multiple comparisons of the simple effects for this interaction revealed that blood corticosterone levels were significantly elevated above baseline following all treatment conditions, however the rise in corticosterone levels was

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Fig. 3. Blood concentrations of corticosterone (mean ⫾ S.E.M.) at the 0, 15, 30, 60 and 120 min time intervals (following drug administration) in rats that received central injections of either vehicle (saline), ␣h-CRF (10 ␮g/kg), saline ⫹ BN (0.2 ␮g; i.c.v.) or ␣h-CRF (10 ␮g/kg) ⫹ BN (0.2 ␮g; i.c.v.). Closed symbols represent points that are significantly different from (within-treatment condition) baseline values at P ⬍ 0.05. * significantly different from (between-treatment condition) time-point matched saline values at P ⬍ 0.05. † significantly different from (between-treatment condition) time-point matched saline ⫹ BN values at P ⬍ 0.05.

more pronounced and protracted following administration of saline ⫹ BN or ␣h-CRF ⫹ BN as compared to the rise observed following administration of either saline or ␣hCRF alone. For the saline ⫹ BN and ␣h-CRF ⫹ BN treatment conditions, levels of corticosterone were significantly elevated above baseline values by 15 min post drug injection and remained elevated throughout the 120 min testing period. In contrast, administration of saline or ␣h-CRF was associated with a rise in corticosterone levels above baseline values by 15 min post drug administration which only remained significantly elevated until 30 min post injection for ␣h-CRF and 60 min post injection for saline. NewmanKeuls comparisons further revealed that in the absence of drug administration, levels of corticosterone were comparable for all treatment conditions. At the 15 min interval, levels of corticosterone were significantly elevated above the saline condition following saline ⫹ BN administration, and ␣h-CRF attenuated the BN-induced rise in blood corticosterone levels as indicated by the significant difference in blood corticosterone concentrations between the saline ⫹

BN and ␣h-CRF ⫹ BN conditions. At the 30, 60 and 120 min intervals, plasma corticosterone levels were significantly elevated above the saline condition following both saline ⫹ BN and ␣h-CRF ⫹ BN administration, and ␣hCRF attenuated the BN-induced rise in blood corticosterone levels as indicated by the significant difference in blood corticosterone concentrations between the saline ⫹ BN and ␣h-CRF ⫹ BN conditions. The overall two factor repeated measures ANOVA revealed that blood glucose levels also varied as a function of the Treatment ⫻ Sample interaction (F(12, 124) ⫽ 5.31; P ⬍ 0.0001). Newman-Keuls multiple comparisons of the simple effects for this interaction indicated that under all drug treatment conditions, blood glucose levels were significantly elevated above baseline levels and peaked at the 30 min time interval. In the absence of drug administration, glucose levels were comparable for all drug treatment conditions. As compared to the control condition, BN administration increased blood glucose levels at 15, 30, 60 and 120 min time intervals. Pretreatment with ␣h-CRF attenu-

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Fig. 4. Blood glucose levels (mean ⫾ S.E.M.) at the 0, 15, 30, 60 and 120 min time intervals in rats that received central injections of either vehicle (saline), ␣h-CRF (10 ␮g/kg), saline ⫹ BN (0.2 ␮g; i.c.v.) or ␣h-CRF (10 ␮g/kg) ⫹ BN (0.2 ␮g; i.c.v.). Closed symbols represent points that are significantly different from (within-treatment condition) baseline values at P ⬍ 0.05. * significantly different from (between-treatment condition) time-point matched saline values at P ⬍ 0.05. † significantly different from (between-treatment condition) time-point matched saline ⫹ BN values at P ⬍ 0.05.

ated the BN-induced rise in blood glucose levels (at the 15, 30 60 and 120 min time intervals).

4. Discussion CRH is well established as the primary endogenous regulator of ACTH secretion from the anterior pituitary [9,36, 40]. Evidence also suggests that CRH may be involved in autonomic activation [4,18]. It is known that many different stimuli elicit CRH release including systemic- (infections, altered blood pressure) and processive-type (psychological) stressors, however, the neurochemical mechanism(s) mediating the release of this peptide remain largely unknown [24,35,42]. Given the considerable functional and anatomic overlap between BN and CRH systems, the present study aimed to elucidate whether BN-induced pituitary-adrenocortical and autonomic activation are mediated via CRH neurons. In support of this contention, results from the

present study demonstrated that 1) central BN administration dose-dependently increased plasma levels of ACTH, corticosterone, norepinephrine, epinephrine and blood glucose levels and 2) that pretreatment with ␣h-CRF blocked or attenuated these BN-induced effects on HPA and autonomic systems. Since in the initial experiment ␣h-CRF attenuated a variety of effects with the exception of the BN-induced elevation of plasma corticosterone levels (measured at a single 30 min time point), we decided to explore the possibility that this may have been due to the doses used or the time of sampling. Specifically, it remained possible that the effects of ␣h-CRF may have preceded or followed the 30 min time point selected. Furthermore, it was possible that at the dose of BN used, clear blockade was not possible. Thus, in the second experiment, a slightly lower dose of BN was used and blood samples were collected at different time points to assess the time-dependent changes in blood corticosterone and glucose levels. Results revealed that follow-

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ing all drug injections (saline, ␣h-CRF, saline ⫹ BN and ␣h-CRF ⫹ BN), levels of corticosterone and glucose were significantly elevated. It is likely that the rise in blood corticosterone and glucose levels observed following the control injections (saline or ␣h-CRF) were attributable to the stress associated with the tail lance procedure. It should be noted however, that the elevations in corticosterone levels observed following both saline ⫹ BN and ␣h-CRF ⫹ BN administrations were significantly higher than those observed following the control injections. Similarly, blood glucose levels were significantly greater following saline ⫹ BN condition than following all other treatment injections. These findings suggest that the stress associated with the tail lance did not mask the drug-induced effects. Finally, in contrast to the result in our initial single time point study, repeated sampling at various time points revealed that ␣hCRF significantly attenuated the stimulatory effect of BN (0.2 ␮g) on corticosterone secretion. Similarly, ␣h-CRH attenuated the BN-elicited rise in blood glucose levels. This observation is consistent with results from our initial experiment demonstrating that pretreatment with ␣h-CRF significantly attenuated the BN-induced rise in blood glucose levels at 30 min post BN. Garrido et al. (1998) [13] recently reported that central ␣h-CRF administration completely blocked the stimulatory effect of GRP on plasma ACTH and corticosterone levels (at 30 min post injection). Although we found a similar effect with ACTH, the ability of ␣h-CRF to attenuate the BN-induced rise in plasma corticosterone was only demonstrable in the second experiment where a slightly lower dose of BN (0.2 ␮g instead or 0.25 ␮g) was used. A likely explanation for this discrepancy is that Garrido and colleagues used a much lower dose of GRP (0.01 ␮g) as compared to the doses of BN (0.2 or 0.25 ␮g) that we used in our experiments. Although BN and GRP share an almost identical active C-terminal decapeptide region (differing in only 1 amino acid), GRP is in fact less potent than BN [39]. In terms of other differences, BN stimulates both GRP (BB2) and neuromedin B (BB1) receptors (whereas GRP has a higher affinity for BB2 receptors), and BN is also thought to be more resistant to enzymatic degradation as compared to GRP [19,23,41]. Not only was ␣h-CRF effective at attenuating the stimulatory effect of BN on the HPA axis, but it significantly inhibited the autonomic activation induced by BN as well. To our knowledge, there is only one other study which has investigated the effects of ␣h-CRF on the BN-induced rise in circulating levels of norepinephrine, epinephrine and glucose [5]. Contrary to our results, they found that pretreatment with ␣h-CRF had no effect on the BN-induced elevations in plasma concentrations of norepinephrine, epinephrine and glucose. It should be noted however that the doses of BN and ␣h-CRH used in that experiment were substantially higher, 1 ␮g and 50 ␮g respectively, than those in the current study. In keeping with this possibility, the BN-induced rise in plasma norepinephrine, epinephrine

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and glucose levels in that study were more than double to those observed in the present investigation. While our results suggest that BN mediates its effects, at least in part, via activation of central CRH neurons, they do not reveal the site(s) of action for these peptidergic interactions. It is well established that CRH, produced in the neurons emanating from the paravocellular division of the PVN, is released at the median eminence terminals (ME) to regulate ACTH secretion from the anterior pituitary [15,16]. It is possible that BN activates the HPA axis by interacting with receptors located on CRH-expressing neurons at the PVN. This nucleus contains high levels of both immunoreactivity as well as binding sites for both BN-like peptides and CRH [7,8,26,31]. Moreover, microinjection of BN into the PVN (but not other hypothalamic sites) elicits a rise of plasma corticosterone [14]. In addition, we have recently shown using in vivo push-pull perfusion, that central BN administration elicits the release of CRH from the ME (unpublished findings). In terms of autonomic activation, it has previously been observed that direct microinjection of BN into the NTS (but not other brain structures) elicits a dramatic increase in plasma catecholamine levels [3,6]. It is possible that BN mediates these effects by interacting with receptors located on CRH neurons at the NTS or other brain stem structures. Indeed, high levels of both BN-like peptide and CRH immunoreactivity as well as high densities of binding sites for both of these peptides have been observed at the NTS [7,8,26,31]. Moreover, we have recently shown that central BN administration elicits a significant reduction in levels of immunoreactive CRH at the NTS (unpublished findings). In addition to our previous findings demonstrating that pretreatment with a CRH receptor antagonist (␣h-CRF) blocks BN-induced satiety and enhanced grooming, results from the present investigation show that pretreatment with ␣h-CRF attenuates BN-induced HPA axis and sympathetic activation as well. These findings provide further support for the contention that BN-like peptides mediate their effects, at least in part, through activation of CRH neurons. Interestingly, we have recently demonstrated the release of both BN-like peptides and CRH occurs not only upon exposure to a stressor, but also in response to an appetitive event (ingestion of a meal) [24]. There is evidence to suggest that these peptides (particularly BN-related peptides) may serve a physiological role in the regulation of food intake, by signaling meal termination (satiety peptide(s)). Although at first blush it may seem counterintuitive to have the same peptidergic systems mediating the stress-response on the one hand, and participating in potentially pleasurable events, one can hypothesize that under situations requiring a ‘fight or flight’ response, it would be advantageous to concurrently activate brain circuits that bring about cessation of food intake. Thus, it would appear that these two peptidergic systems may act in concert to prepare organisms to deal with biologically significant events.

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Acknowledgments This work was supported by a grant from the Medical Research Council of Canada and an Ontario Graduate Student Scholarship.

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