Ca(2+) mobilization, tyrosine hydroxylase activity, and signaling mechanisms in cultured porcine adrenal medullary chromaffin cells: effects of leptin.

Leptin acts as a satiety factor, but there is also evidence that it affects energy expenditure. Leptin's effects are mediated by its receptors, which function as activators of a Janus family of tyrosine kinases-signal transducer and activator of transcription (JAK-STAT) pathway. We have previously shown that murine recombinant leptin markedly induces both the release of catecholamine and tyrosine hydroxylase (TH) (rate-limiting enzyme in the biosynthesis of catecholamine)-messenger RNA (mRNA) levels, probably through Ob-Rb expressed in cultured porcine chromaffin cells. In the present study, we examined the effect of leptin on Ca(2+) mobilization, TH enzyme activity, and signaling. Ca(2+) channel blockers, nicardipine and omega-Conotoxin GVIA, each at 1 microM, were effective in inhibiting leptin-induced catecholamine secretion. When intracellular Ca(2+) ([Ca(2+)](i)) was measured in fura 2-loaded chromaffin cells, leptin was found to cause a sustained increase of Ca(2+) by mobilizing Ca(2+) from both extra- and intracellular pools. Additionally, leptin significantly stimulated inositol 1.4.5-triphosphate IP(3) production in a dose-dependent manner. TH-activity is regulated by both TH enzyme activity and increased TH-mRNA levels accompanied by increased TH protein synthesis. Leptin (>/=1 nM) significantly stimulated TH enzyme activity and increased the TH protein level, indicating that it stimulates catecholamine biosynthesis. In addition, removal of external Ca(2+) completely inhibited leptin (100 nM)-induced TH enzyme activity. Leptin (>/=1 nM) caused an increase in the activity of mitogen-activated protein kinases (MAPKs) that was accompanied by increased phosphorylation of STAT-3 and -5, but not STAT-1. Moreover, MAPK activity evoked by leptin(100 nM) and TH-mRNA caused by leptin (10 nM) were inhibited by 50 and 30 microM of PD-98059 (the MAP kinase kinase-1 inhibitor), respectively. These findings indicate that leptin activates voltage-dependent Ca(2+) channels (VDCC), presumably L-type and N-type Ca(2+) channels, as well as phospholipase C, and suggest that leptin-induced catecholamine secretion is mainly mediated by activation of VDCC. In addition, leptin stimulates the JAK-STAT pathway as well as increasing the levels of TH-mRNA levels through the MAPK pathway in porcine chromaffin cells.