Serotonin interferes with Ca²⁺ and PKC signaling to reduce gonadotropin-releasing hormone-stimulated GH secretion in goldfish pituitary cells

Abstract

In goldfish, two endogenous gonadotropin-releasing hormones (GnRH), salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), are thought to stimulate growth hormone (GH) release via protein kinase C (PKC) and subsequent increases in intracellular Ca²⁺ levels ([Ca²⁺]_i). In contrast, the signaling mechanism for serotonin (5-HT) inhibition of GH secretion is still unknown. In this study, whether 5-HT inhibits GH release by actions at sites along the PKC and Ca²⁺ signal transduction pathways leading to hormone release were examined in primary cultures of goldfish pituitary cells. Under static incubation and column perifusion conditions, 5-HT reduced basal, as well as sGnRH- and cGnRH-II-stimulated, GH secretion. 5-HT also suppressed GH responses to two PKC activators but had no effect on the GH-releasing action of the Ca²⁺ ionophore ionomycin. Ca²⁺-imaging studies with identified somatotropes revealed that 5-HT did not alter basal [Ca²⁺]_i but attenuated the magnitude of the [Ca²⁺]_i responses to the two GnRHs. Prior treatment with 5-HT and cGnRH-II reduced the magnitude of the [Ca²⁺]_i responses induced by depolarizing levels of K⁺. Similar inhibition, however, was not observed with prior treatment of 5-HT and sGnRH. These results suggest that 5-HT, by direct actions at the somatotrope level, interferes with PKC and Ca²⁺ signaling pathways to reduce the GH-releasing effect of GnRH. 5-HT action may occur at the level of PKC activation or its downstream signaling events prior to the subsequent rise in [Ca²⁺]_i.. The differential Ca²⁺ responses by depolarizing doses of K⁺ is consistent with our previous findings that sGnRH and cGnRH-II are coupled to overlapping and yet distinct Ca²⁺-dependent mechanisms.

Introduction

Since its identification as a neurotransmitter, serotonin (5-HT) has been implicated as a regulator of hypothalamic functions in mammals, including stress, satiation, mood, sleep and body temperature (Struder and Weicker, 2001). Among the actions of 5-HT on the hypothalamo-hypophysial axis, the dynamic interplay between 5-HT and the hypothalamus-pituitary-adrenal axis has been extensively studied because of the importance of 5-HT in regulating stress (Lowry, 2002). In addition, 5-HT has also been shown to regulate growth hormone (GH) release although its effects are controversial. Early studies with in vivo treatment of 5-HT or 5-HT receptor agonists and antagonists suggested a stimulatory effect of 5-HT on GH release in mammals (Arnold and Fernstrom, 1978, Arnold and Fernstrom, 1981, Collu et al., 1979); however, Spencer et al. (1991) and Muller et al. (1976) reported that 5-HT inhibits GH release in sheep and dog, respectively. In birds, evidence indicates that 5-HT inhibits GH secretion by reducing hypothalamic GH-releasing activity (Hall, 1982, Carew et al., 1983). The lack of direct 5-HT effects at the level of the pituitary in most mammalian studies supports the view that 5-HT indirectly affects GH release via actions on GH-releasing hormone (GHRH), pituitary adenylate cyclase-activating polypeptide (PACAP) and somatostatin neurons in the hypothalamus (Willoughby et al., 1987, Conway et al., 1990, Radcliff et al., 2003). However, recent findings with rat pituitary cell aggregates suggest that direct action at the level of the pituitary is also possible (Papageorgiou and Denef, 2007).

In teleosts such as goldfish, rainbow trout and Atlantic croaker, 5-HT immunoreactive fibers have been detected in the pars distalis of the pituitary gland (Kah and Chambolle, 1983, Frankenhuis-van den Heuvel and Nieuwenhuys, 1984, Khan and Thomas, 1993), suggesting that 5-HT can directly act at the level of the pituitary in bony fishes. In support of the hypothesis of direct action at the level of the pituitary, 5-HT reduced GH release from goldfish pituitary fragments and cultured pituitary cells in vitro (Somoza and Peter, 1991, Wong et al., 1998). This ability of 5-HT to inhibit GH release was not confined to basal secretion but was also observed with stimulated GH secretion in goldfish (Wong et al., 1998). The exact physiological condition(s) under which 5-HT influence on GH release is important has not been elucidated. However, sex steroids such as estradiol are known to elevate serum GH levels in several fish species, including the goldfish and rainbow trout (Canosa et al., 2007). In rainbow, estradiol induced an increase in pituitary 5-HT level and decreased 5HIAA/5-HT ratio, suggesting that 5-HT turnover is suppressed (Hernandez-Rauda and Aldegunde, 2002). Whether estradiol feedback stimulation of pituitary GH release involves the removal of a 5-HT inhibitory influence is a possibility that is worth investigating.

Extensive information is available on how 5-HT acts in mammals. To date, at least seven distinct serotonergic receptor subtypes (5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7) have been identified or cloned in mammals. In addition, the 5-HT1, 5-HT2 and 5-HT5 have been further subclassified (Bockaert et al., 2006). In mammals, the stimulation role of 5-HT on the GH regulation has been attributed to 5-HT1B/D and 5-HT2 receptors (Mota et al., 1995, Katz et al., 1996, Valverde et al., 2000, Papageorgiou and Denef, 2007). Using pharmacological manipulation of 5-HT receptors, it was reported that 5-HT2-like receptors may be involved in inhibition of 5-HT on basal GH release in goldfish (Wong et al., 1998). However, very little is known with regard to the molecular mechanisms responsible for this inhibitory action of 5-HT on GH release in goldfish.

In teleosts, hypothalamic neurons directly innervate the pars distalis. In goldfish, GH release is stimulated by many hypothalamic neuroendocrine factors, including gonadotropin-releasing hormone (GnRH), GHRH, dopamine and PACAP (reviewed in Canosa et al., 2007). Two GnRHs are found in, and released from, the pituitaries of goldfish, these being GnRH forms initially identified in salmon and chicken, namely, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). Among these neuroendocrine stimulators, the mechanisms of action for the two GnRHs, are the best characterized. Both sGnRH and cGnRH-II stimulate GH release via activation of protein kinase C (PKC), increases in intracellular free Ca²⁺ levels ([Ca²⁺]_i), and activation of voltage-sensitive Ca²⁺ channels (VSCC); however, the involvement of intracellular Ca²⁺ stores in their GH-releasing actions are not identical. Although a caffeine-sensitive Ca²⁺ store participates in both sGnRH and cGnRH-II stimulation of GH release, only sGnRH uses an IP3-sensitive mechanism, whereas cGnRH-II utilizes a ryanodine-sensitive Ca²⁺ signaling pathway. In addition, only sGnRH-induced GH release is attenuated by mitochondrial Ca²⁺ buffering. These and other results not only indicate that multiple pharmacologically distinct intracellular Ca²⁺ stores are present in goldfish somatotropes but that the two GnRHs mobilizes Ca²⁺ from dissimilar intracellular stores (Canosa et al., 2007). In this study, we examined the signal transduction mechanisms mediating 5-HT inhibition of GH release from primary cultures of goldfish pituitary cells under basal condition and GnRH stimulation. The possible involvement of PKC was examined by studying 5-HT action on the GH-releasing effects by PKC activators including the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; Liu and Heckman, 1998) and the synthetic diacylglycerol dioctanoylglycerol (DiC8; Nishizuka, 1986). Involvement of [Ca²⁺]_i was also examined in Ca²⁺-imaging experiments with morphologically identified goldfish somatotropes (Johnson et al., 2002) and in hormone release experiments with the Ca²⁺ ionophore ionomycin (Kwong and Chang, 1997). Our results suggest that 5-HT can modulate GnRH-stimulated increase in [Ca²⁺]_i and inhibit GH release responses to PKC activation in goldfish somatotropes.