Aleksandra Glavaski-Joksimovic

Stimulatory Effect of Ghrelin on Isolated Porcine Somatotropes

Research on the mechanism for growth hormone secretagogue (GHS) induction of growth hormone secretion led to the discovery of the GHS receptor (GHS-R) and later to ghrelin, an endogenous ligand for GHS-R. The ability of ghrelin to induce an increase in the intracellular Ca2+ concentration – [Ca2+]i – in somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by application of human growth hormone releasing hormone. Ghrelin increased the [Ca2+]i in a dose-dependent manner in 98% of the cells that responded to human growth hormone releasing hormone. In the presence of (D-Lys3)-GHRP-6, a specific receptor antagonist of GHS-R, the increase in [Ca2+]i evoked by ghrelin was decreased. Pretreatment of cultures with somatostatin or neuropeptide Y reduced the ghrelin-induced increase of [Ca2+]i. The stimulatory effect of ghrelin on somatotropes was greatly attentuated in low-calcium saline and blocked by nifedipine, an L-type calcium channel blocker, suggesting involvement of calcium channels. In a zero Na+ solution, the stimulatory effect of ghrelin on somatotropes was decreased, suggesting that besides calcium channels, sodium channels are also involved in ghrelin-induced calcium transients. Either SQ-22536, an adenylyl cyclase inhibitor, or U73122, a phospholipase C inhibitor, decreased the stimulatory effects of ghrelin on [Ca2+]i transiently, indicating the involvement of adenylyl cyclase-cyclic adenosine monophosphate and phospholipase C inositol 1,4,5-trisphosphate pathways. The nonpeptidyl GHS, L-692,585 (L-585), induced changes in [Ca2+]i similar to those observed with ghrelin. Application of L-585 after ghrelin did not have additive effects on [Ca2+]i. Preapplication of L-585 blocked the stimulatory effect of ghrelin on somatotropes. Simultaneous application of ghrelin and L-585 did not cause an additive increase in [Ca2+]i. Our results suggest that the actions of ghrelin and synthetic GHS closely parallel each other, in a manner that is consistent with an increase of hormone secretion.

Effects of Leptin on Intracellular Calcium Concentrations in Isolated Porcine Somatotropes

Leptin, the product of the obese gene, is a protein that is secreted primarily from adipocytes. Leptin can influence the function of the pituitary gland through its action on the hypothalamus, but it can also directly act at the level of the pituitary gland. The ability of leptin to induce an increase in intracellular Ca2+ concentration ([Ca2+]i) in somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by the application of human growth hormone releasing hormone. Leptin increased [Ca2+]i in porcine somatotropes in a dose-dependent manner. The application of 100 nM leptin for 3 min did not have a significant effect on [Ca2+]i, while a 3-min application of 1 µM leptin increased [Ca2+]i in about 50% of the somatotropes (p < 0.01). The application of a second leptin challenge (1 µM) evoked a response in only 18% of the observed somatotropes. The stimulatory effect of leptin was abolished in low calcium saline and blocked by nifedipine, an L-calcium channel blocker, suggesting an involvement of calcium channels. Pretreatment of the cultures with AG 490, a specific Janus kinase inhibitor, and with SB 203580, a mitogen-activated protein kinase (MAP kinase) inhibitor, abolished the increase in [Ca2+]i evoked by leptin. In the presence of Nω-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, the magnitude of the increase in [Ca2+]i evoked by 1 µM leptin was not significantly changed. However, in the presence of L-NAME only 24% of the somatotropes responded to leptin, while in parallel control cultures 70% of the somatotropes responded to leptin. These results imply an involvement of Janus kinase/signal transducer and activator or transcription, MAP kinase and NOS-signaling pathways in the stimulatory effect of leptin on porcine somatotropes.

Subpopulations of Somatotropes with Differing Intracellular Calcium Concentration Responses to Secretagogues

Multiple secretagogues stimulate the release of growth hormone (GH). The present studies examined the ability of chicken somatotropes to respond to GH secretagogues with increased intracellular calcium concentrations ([Ca2+]i). It was hypothesized that there are subsets of the somatotrope population with different responsiveness to the various secretagogues. Somatotropes were identified and distinguished from other adenohypophyseal cells, by their unique ability to respond to GH-releasing hormone with increased [Ca2+]i with immunocytochemistry used as a post-hoc confirmatory test. Large increases in [Ca2+]i (222 ± 16 nM) were evoked by thyrotropin-releasing hormone in only 73% of the somatotropes. Similarly, [Ca2+]i was increased by perifusion with pituitary adenylate cyclase-activating peptide in 85% and by leptin but only in 51% of somatotropes. Ghrelin acutely increased [Ca2+]i in only 21% of somatotropes. Perfusion with gonadotropin-releasing hormone elevated [Ca2+]i, but in only 40% of somatotropes. The kinetics of calcium transients and the magnitude of the response differed from those observed in the presumptive gonadotropes. It is concluded that there are subsets of the somatotrope population in the anterior pituitary gland with differences in their ability to respond to various secretagogues.