Abstract

In this study, the role of tyrosine phosphorylation in agonist-stimulated cAMP accumulation and GH release in rat anterior pituitary cells was investigated. It was found that genistein, a tyrosine kinase inhibitor, while having no effect on its own, potentiated GHRH-stimulated cAMP accumulation in a concentration-dependent manner. In comparison, daidzein, an inactive analogue of genistein, was ineffective and vanadate, a phosphotyrosine phosphatase inhibitor, reduced GHRH-stimulated cAMP accumulation. Additional structurally unrelated tyrosine kinase inhibitors, erbstatin and tyrphostins, also potentiated GHRH-stimulated cAMP accumulation. To determine the site of action of the tyrosine kinase inhibitors, pituitary adenylate cyclase-activating polypeptide (PACAP), cholera toxin and forskolin were used to increase cAMP accumulation. Genistein enhanced the PACAP-, cholera toxin- or forskolin-stimulated cAMP accumulation, suggesting that the site of action is at the post-receptor level. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine, genistein did not potentiate and vanadate did not inhibit GHRH-stimulated cAMP accumulation, indicating that phosphodiesterase is a probable site of action for the inhibitor. Genistein and erbstatin also enhanced GHRH-stimulated GH release and the effect of vanadate was inhibitory. These results indicate that tyrosine kinase inhibitors enhance cAMP accumulation through their action on phosphodiesterase activity in rat anterior pituitary cells and the tyrosine kinase pathway appears to be involved in the control of GH release.

Journal of Endocrinology (1997) 152, 193–199

ABSTRACT

In this study, we examined the effect of changes in intracellular pH (pH_i) on basal and GH-releasing factor (GRF)-stimulated cyclic AMP (cAMP), intracellular Ca²⁺ and GH release using a static monolayer culture prepared from dispersed rat anterior pituitary cells. To modulate pH_i, two approaches were used: variation of extracellular pH (pH_o) and addition of sodium propionate and ammonium chloride which alter pH_i directly. Direct pH_i measurement with 2′7′-bis(carboxyethyl)-5(6)-carboxyfluorescein showed that for pH_o values between 6·9 and 7·6, a change in pH_o of 0·1 units resulted in a change in pH_i of 0·045 units. Sodium propionate (30 mmol/l) reduced pH_i by 0·06 units whereas ammonium chloride (30 mmol/l) increased pH_i by 01 units. Increasing pH_o from 6·6 to 7·8 enhanced the maximal GRF-stimulated cAMP and GH responses by 80% and 300% respectively, indicating that the GRF-stimulated cAMP and GH release were both pH-dependent. Acute elevation of pH_o from 6·6 to 7·8 also increased basal GH release by sixfold. Reduction of pH_i by sodium propionate, however, had no significant effect on GRF-stimulated cAMP levels while the corresponding GRF-stimulated GH release was reduced by up to 40%. In comparison, elevation of pH_i by ammonium chloride enhanced the GRF-stimulated cAMP release by up to 75% and the corresponding increase in GH was less than 20%. When the relationship between pH_i and intracellular Ca²⁺ was determined with the fluorescent Ca²⁺ indicator, Fura-2, it was found that increasing pH_o and treatment with ammonium chloride increased intracellular Ca²⁺, while sodium propionate and reducing pH_i had no effect on intracellular Ca²⁺. These results indicate that activation of adenylate cyclase and mobilization of intracellular Ca²⁺, two intracellular signalling pathways of importance to GH secretion, are both sensitive to changes in pH_i.

Journal of Endocrinology (1992) 135, 343–352

Abstract

Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) share 68% homology and function as neurotransmitters or neuroendocrine factors. Although VIP immunoreactivity has been detected in bone cells, the presence of PACAP or PACAP receptors in bone has not been determined. In this study, we investigated the role of PACAP and VIP in regulating cAMP accumulation in the UMR 106 osteoblast-like tumor cell line.

PACAP 27 (10⁻⁹ to 3 × 10⁻⁷ m), PACAP 38 (10⁻⁹ to 3 × 10⁻⁷ m) and VIP (10⁻⁸ to 10⁻⁶ m) stimulated cAMP accumulation up to eightfold. PACAP 27 was slightly more potent than PACAP 38, and both were tenfold more potent than VIP. Both PACAP- and VIP-stimulated cAMP accumulation were potentiated by 4β-phorbol 12-myristate 13-acetate, an activator of protein kinase C. Two PACAP antagonists, PACAP 6–27 (3 × 10⁻⁶ m) and PACAP 6–38 (3 × 10⁻⁶ m), blocked PACAP- and VIP-stimulated cAMP accumulation. Two VIP antagonists ([Lys¹,Pro^2,5,Arg^3,4,Tyr⁶]-VIP, and 4 Cl-d-Phe⁶,Leu¹⁷]-VIP) did not reduce the PACAP-or VIP-stimulated cAMP accumulation. Pretreatment with PACAP 27, PACAP 38 or VIP equally blocked PACAP- and VIP-stimulated cAMP accumulation.

These results suggest that PACAP is a more potent stimulator of cAMP accumulation than VIP in UMR 106 cells. PACAP and VIP may share a role in the paracrine or neuroendocrine regulation of bone metabolism.

Journal of Endocrinology (1996) 149, 287–295