Mechanism of action of the growth hormone secretagogue, L-692,585, on isolated porcine somatotropes

Abstract

The effects of a GH secretagogue, L-692,585 (L-585), and human GH-releasing hormone (hGHRH) on calcium transient and GH release were investigated in isolated porcine pituitary cells using calcium imaging and the reverse hemolytic plaque assay (RHPA). Somatotropes were functionally identified by the application of hGHRH. All cells that responded to hGHRH responded to L-585 application. Perfusion application of 10 µM hGHRH and L-585 for 2 min resulted in an increase in intracellular calcium concentrations ([Ca 2+ ] i ) of 53 1 nM (mean S.E.M.) (P<0·01) and 68 2 nM (P<0·01) respectively. The L-585 response was characterized by an initial increase in [Ca )-GRF (1-29) amide) decreased the stimulatory effect of hGHRH. These failed to block the stimulatory effect of L-585, suggesting a different receptor for L-585 from the GHRH receptor. The hGHRHinduced calcium transients and initial peak increase induced by L-585 were significantly decreased by removal of calcium from the bathing medium or the addition of nifedipine, an L-calcium channel blocker. The plateau component of L-585-induced calcium change was abolished by removal of calcium and nifedipine. These results suggest an involvement of calcium channels in GH release. Either SQ-22536, an adenylate cyclase inhibitor, or U73122, a phospholipase C (PLC) inhibitor, blocked the stimulatory effects of hGHRH and L-585 on [Ca 2+ ] i transient, indicating the involvement of adenylate cyclasecAMP and PLC-inositol triphosphate pathways. These results further suggested that calcium mobilization from internal stores during the first phase of the L-585 response induced an increase in [Ca 2+ ] i whereas calcium influx during the second phase is a consequence of somatotrope depolarization.

Effects of Leptin on Intracellular Calcium Concentrations in Isolated Porcine Somatotropes

“…The increase in [Ca 2+ ] i evoked by leptin in porcine somatotropes is likely to be accompanied by an elevated GH release. In previous experiments on isolated porcine somatotropes with simultaneous measurements of GH release and calcium transients, we have shown that L-692,585, a nonpeptidyl GHS, evoked an increase in [Ca 2+ ] i that coincided with GH release [40]. In addition, it has been shown that leptin stimulates the basal GH release from cultured porcine somatotropes [30, 36].…”

“…The tissue was transferred to sterile cold (4°C) DMEM-0.1% BSA medium. Primary cell cultures from anterior pituitary glands were established using a method previously employed in our laboratory [40]. Tissue from 4 animals was incubated for 50 min at 37°C in 2 ml of EBSS papain solution (1.54 mg/ml).…”

“…We have previously reported the effects of L-692,585, a nonpeptidyl GH secretagogue (GHS), and ghrelin on intracellular calcium [40, 41]and GH secretion [40, 42]in somatotropes of newborn and 3-month-old pigs. In these studies, we determined that qualitatively results did not differ in isolated pituitary cells from animals of different ages; however, the pituitary cells from newborn animals were much easier to maintain in culture.…”

Subpopulations of Somatotropes with Differing Intracellular Calcium Concentration Responses to Secretagogues

“…The tissue was transferred to a sterile cold (4°C) DMEM-0.1% BSA medium. Primary cell cultures from anterior pituitary glands were established using a method previously employed in our laboratory with neonatal pig anterior pituitary tissue [11]. Tissue from 3 chickens was incubated for 50 min at 37°C in 2 ml of EBSS-papain solution (1.54 mg/ml).…”

“…The present studies examine the response of individual somatotropes to secretagogues. The technique of loading pituitary cells with fura-2/AM followed by radiometric imaging [10, 11] allows determination of secretagogue-induced changes in intracellular calcium concentrations ([Ca 2+ ] i ) in individual cells . …”