Norepinephrine Regulation of Growth Hormone Release from Goldfish Pituitary Cells. II. Intracellular Sites of Action

Abstract

Previous results suggest that norepinephrine decreases growth hormone (GH) release in goldfish by means of alpha-2 adrenoceptor activation. The intracellular mechanisms by which norepinephrine inhibits GH release were examined in the present study using dispersed goldfish pituitary cells. In 2-h static incubation experiments, norepinephrine and the alpha-2 agonist clonidine decreased basal GH release and the GH responses to stimulation by the dopamine D1 agonist SKF38393 and two native gonadotropin-releasing hormones (GnRH). Norepinephrine also reduced GH responses to the adenylate cyclase activator forskolin, two protein kinase C (PKC) activators (phorbol ester and synthetic diacylglycerol), and two Ca2+ ionophores (ionomycin and A23187). Similarly, norepinephrine applied as a 1-h pulse in cell column perifusion experiments reduced basal GH release and abolished the GH response to a 5-min pulse of arachidonic acid. In goldfish, D1-stimulated GH release is mediated by AC-, arachidonic acid-and Ca2+-dependent pathways, whereas GnRH action is coupled to PKC-and Ca2+-dependent mechanisms. These results suggest that norepinephrine activation of alpha-2 receptors inhibits ligand-induced GH secretion by actions subsequent to activation of these second messenger cascades. To further characterize norepinephrine mechanisms of action on unstimulated hormone release, the ability of norepinephrine and an alpha-2 agonist to affect activation of two second messenger cascades under basal conditions was also investigated. Static incubation with clonidine reduced cAMP production in a time-and dose-dependent manner, suggesting that norepinephrine inhibitory action can also be expressed at the level of cAMP production. Resting intracellular free calcium levels in single, identified goldfish somatotropes was unaffected by norepinephrine. However, the inhibitory effects of norepinephrine on basal GH secretion was not observed in the presence of a voltage-sensitive Ca2+ channel agonist. Whether these channels are targets for norepinephrine action on unstimulated GH release requires further investigation.

Mechanisms for gonadotropin-releasing hormone potentiation of growth hormone rebound following norepinephrine inhibition in goldfish pituitary cells

“…This idea is consistent with our findings that both NE and EP could inhibit GH secretion from goldfish pituitary cells through activation of ␣ 2 -adrenoreceptors (28). These inhibitory actions probably are caused by ␣ 2 -blocking of the GH-releasing effects mediated by cAMP-, PKC-, and Ca 2ϩ -dependent cascades (60). In our previous studies (5), a rebound of GH release was noted following termination of NE treatment, which closely resembles the GH rebound observed after SRIF withdrawal in mammalian models.…”

“…Dopamine, the precursor for NE and EP, can stimulate GH release (56) and body growth (57) in the goldfish by acting through pituitary D 1 receptors (55). In contrast, NE and EP suppress GH secretion via ␣ 2 -adrenoreceptors (28), and ␣ 2 -agonists (e.g., clonidine) can block the GH-releasing effects of sGnRH and cGnRH-II, respectively (60). In our previous studies, a GH rebound was noted after NE withdrawal, and this GH rebound could be markedly enhanced by prior exposure to sGnRH (28) and cGnRH-II (60).…”

“…In goldfish pituitary cells, PKC activators (e.g., TPA and DiC8) can enhance the GH-releasing effect of Bay K8644 (58), whereas blockade of VSCC alleviates the GH responses induced by PKC activation (7), confirming that a cross talk between PKC and VSCC also exists in fish model. Using Ca 2ϩ imaging in goldfish pituitary cells, a transient rise in intracellular Ca 2ϩ was also noted in identified somatotrophs after removal of NE treatment, overlapping with the rebound phase of GH secretion (60). These findings, taken together, have prompted us to speculate that PKC activation induced by GnRH treatment, despite its GH-releasing effect, is inhibited by NE perifusion and can enhance Ca 2ϩ influx via VSCC following NE withdrawal, and subsequent CaMKII activation may contribute to the potentiating effect on GH secretion during the "postinhibition" GH rebound.…”

“…In contrast, NE and EP suppress GH secretion via ␣ 2 -adrenoreceptors (28), and ␣ 2 -agonists (e.g., clonidine) can block the GH-releasing effects of sGnRH and cGnRH-II, respectively (60). In our previous studies, a GH rebound was noted after NE withdrawal, and this GH rebound could be markedly enhanced by prior exposure to sGnRH (28) and cGnRH-II (60). In this study, potentiation of GH rebound following NE inhibition was established in gold- fish pituitary cells by use of a perifusion approach with the GnRH superagonist sGnRHa as a stimulant.…”

“…Besides, PKC inactivation (7) and VSCC blockade (21) attenuate the GHreleasing effects of sGnRH and cGnRH-II, whereas PKA inhibition can reduce basal levels of GH secretion (55). At the pituitary level, NE inhibits GH release with a concurrent drop in cAMP production via activation of ␣ 2 -adrenoreceptors (60). In mammals, AC inactivation and inhibition of VSCC currents are known to be the key elements of the signaling mechanisms for ␣ 2 -adrenoreceptors (48).…”