Free Intracellular Ca²⁺Concentration ([Ca²⁺]_i) and Growth Hormone Release from Purified Rat Somatotrophs. I. GH-Releasing Factor-Induced Ca²⁺Influx Raises [Ca²⁺]_i*

Abstract

This study was carried out to investigate the role of free intracellular Ca2+ ([Ca2+]i) in the action of GH-releasing factor (GRF) by determining whether GRF causes and increase in [Ca2+]i and whether this increase results from changes in Ca2+ influx/efflux and/or mobilization of intracellular Ca2+ stores. We used a purified preparation of normal rat somatotrophs and examined the changes in 45Ca uptake, [Ca2+]i measured with indo-1, intracellular cAMP, and GH release induced by GRF. GRF stimulated a concentration-related biphasic increase in [Ca2+]i. Both the GRF-dependent increase in [Ca2+]i and GH release were blocked by incubation in low Ca2+ medium and by the organic Ca2+ antagonists nifedipine and diltiazem. The measurement of 45Ca uptake, in both steady state and nonsteady state conditions, demonstrated directly that GRF stimulates Ca2+ influx into somatotrophs. These data demonstrate that the GRF-stimulated increase in [Ca2+]i is dependent on Ca2+ influx. Redistribution of intracellularly stored Ca2+ could not be detected, even though intracellular Ca2+ stores were present. Therefore, the increase is due to Ca2+ influx, and the biphasic nature of the increase in [Ca2+]i induced by GRF is due to a difference in the rate of activation of Ca2+ influx and Ca2+ removal from the cytosol.

Expression of Ca2+-Mobilizing EndothelinAReceptors and Their Role in the Control of Ca2+Influx and Growth Hormone Secretion in Pituitary Somatotrophs

“…Also, the stimulation of adenylyl cyclase by GHRH and several other G s -coupled receptors in somatotrophs elevates cAMP production, leading to the activation of the depolarizing pacemaker current and the generation of APs (Cuttler et al, 1992;Bluet-Pajot et al, 1998). This, in turn, stimulates voltagegated Ca 2ϩ influx and GH secretion (Lussier et al, 1991b). Finally, somatostatin inhibits both adenylyl cyclase and electrical activity in somatotrophs (Sims et al, 1991).…”

“…In mixed populations of anterior pituitary cells, ϳ50% of cells are somatotrophs, and their identification in our experiments was based on the actions of somatostatin (typical for somatotrophs and some lactotrophs), TRH (typical for lactotrophs and thyrotrophs), and GnRH (typical for gonadotrophs). In purified cultures, ϳ94% of cells were somatotrophs (Lussier et al, 1991b). The procedure used for the purification of cells separates a subfraction of high-density cells, which previously have been shown to respond to GHRH by the facilitation of Ca 2ϩ influx in an adenylyl cyclase-dependent manner (Lussier et al, 1991a,b;Ramirez et al, 1998).…”

Characterization of a Plasma Membrane Calcium Oscillator in Rat Pituitary Somatotrophs

“…The [Ca 2ϩ ] i responses to GnRH, TRH, and GHRH, applied at the end of experiments, were used for identification of cells from a mixed population. Cell purification was done by a Percoll discontinuous density gradient centrifugation (27).…”

“…GHRH-controlled [Ca 2ϩ ] i Signals-In further experiments, cells were stimulated with GHRH, a well established agonist for somatotrophs (27,35,36). Addition of 1 nM GHRH initiated Ca 2ϩ spiking in quiescent cells (Fig.…”

International Union of Pharmacology. XXXV. The Glucagon Receptor Family

“…In somatotroph cells, GHRH also stimulates an influx of calcium (Holl et al, 1988;Lussier et al, 1991) most likely through voltagesensitive Ca 2ϩ channels (Chen et al, 1994). Although GHRH is reported to stimulate the phospholipase Cinositol phosphate-calcium mobilization pathway in pituitary cells in some studies (Canonico et al, 1983;Ohlsson and Lindstrom, 1990), other studies report no activation of this pathway (Escobar et al, 1986;French et al, 1990), and no coupling of the cloned GHRH receptor to this signaling pathway has yet been detected in transfected cells (Miller et al, 1999).…”