Novel aspects of autocrine/paracrine regulation of growth hormone secretion and synthesis in grass carp pituitary cells

Abstract

(Uncorrected OCR) Abstract of thesis entitled

NOVEL ASPECTS OF AUTOCRINEIP ARACRINE REGULATION OF GROWTH HORMONE SECRETION AND SYNTHESIS IN GRASS CARP PITUITARY CELLS

Submitted by

ZHOUHONG

for the degree of Doctor of Philosophy at The University of Hong Kong

in March 2003

In this study, autocrine/paracrine regulation of growth hormone (GH) synthesis and secretion by local interactions of gonadotrophs and somatotrophs was examined in vitro in pituitary cells prepared from Chinese grass carp (Ctenopharyngodon idellus). Treatment with exogenous OH and gonadotropin (OTH) resulted in a dose-dependent increase in basal GH release, GH production, and GH mRNA levels. However, the opposite effects were observed by removing endogenous OR and OTH using immunoneutralization. Furthermore, GR and OTH immunoneutralizations at the pituitary level were effective in blocking the stimulatory influence on GH mRNA expression induced by GH-releasing factors in fish, including GnRH, dopamine, and PACAP38�Apparently" GH-induced GH gene expression was mediated by increasing the T1/2 ofGH mRNA in the cytoplasm and enhancing the production of GH primary transcripts in the nucleus. Since GH-induced OR mRNA gene expression could be blocked by inhibiting JAK2, P42144MAPK, P38MAPK, and PI3K, it is likely that the JAK/MAPK and JAK/PI3K pathways are involved in the GH receptor signaling. Similarly, exogenous GTH increased the production ofGH primary transcripts. However, it did not improve OR mRNA stability but rather enhanced the turnover of GH transcripts. GTR also increased cAMP production in carp pituitary cells. GTH-induced GH mRNA expression Was mimicked by activating cAMP synthesis and blocked by inhibiting adenylate cyclase (AC) and PKA.. GTH-induced OR mRNA expression was also sensitive to inhibition of JAKz, P42/44MAPK, P3SM.AP1C and PI3K. Similar inhibitions, except for PI3K, were all effective in blocking OR mRNA expression

induced by activation of cAMP synthesis. These results indicate that GTH may induce GR gene expression through the AC/ cAMP/PKA pathway secondary coupled to JAK.2 andlor MAPK. Apparently, a cAMP-independent PI3K component is also involved in the post-receptor signaling. Using a colunm perifusion approach, the dynamic interactions between somaotrophs and gonadotrophs were examined. In this case, exogenous OTR induced a rapid rise in basal GH secretion, whereas exogenous GR was found to inhibit basal GTR release. In parallel studies, GTHinduced OR mRNA expression was abolished by OR immunoneutralization. Similarly, GTR immunoneutralization blocked GR-induced OR mRNA expression in carp pituitary cells. These results, as a whole, indicate that endogenously secreted OH and GTR, besides their functions as endocrine hormones, serve as novel autocrine/paracrine factors at the pituitary level to modulate GH secretion, OH production, OH gene expression, and somatotroph sensitivity to stimulation by hypothalamic regulators. These stimulatory influences of GH and GTR on OR gene expression axe exerted at the level of GR rnRNA stability and OH gene transcription, presumably via a direct coupling to the JAK/MAPK and JAKiPI3K cascades or an indirect coupling via the AC/cAMP/PKA pathway. Apparently, a local il1trapituitary feedback loop is present. In this case, GTH released from gonadotrophs stimulates GH secretion in neighboring somatotrophs. GR release from somatotrophs is essential to maintain basal GH synthesis and secretion and also exerts a negative feedback on basal GTB release. This intrapituitary feedback loop formed by local interactions between gonadotrophs and somatotrophs may represent a novel mechanism to control OR gene expression in lower vertebrates.