title: Leptin stimulates catecholamine synthesis in a PKC-dependent manner in cultured porcine adrenal medullary chromaffin cells.
abstract: We have previously shown that murine recombinant leptin directly stimulates catecholamine synthesis through the long form of the leptin receptor (Ob-Rb) expressed in cultured porcine chromaffin cells. Additionally, we found that leptin activates IP3 production after PLC activation. It is well established that activation of PLC elicits IP3 production as well as an increase in diacylglycerol, a compound that stimulates PKC. Therefore, we investigated the involvement of PKC in leptin-induced catecholamine synthesis. Leptin was found to induce significant increases in PKC activity in a dose-dependent manner (1, 10, and 100 nM); chelation of extracellular Ca(2+) by EDTA abolished this PKC stimulatory activity. We also confirmed by Western blot analysis that leptin (at 100 nM) induced significant increases in Ca(2+)-dependent PKC alpha, -beta(I), and -gamma expression. The activity of the rate-limiting enzyme tyrosine hydroxylase (TH) in the biosynthesis of catecholamine is regulated at the transcriptional and posttranscriptional levels. TH enzyme activity and TH mRNA levels induced by 100 nM leptin were significantly inhibited by the PKC inhibitor Ro 32-0432 as well as by EDTA. In addition, increases in TH protein and intracellular catecholamine content stimulated by leptin were completely inhibited by Ro 32-0432. Leptin markedly activated ERKs and, to a lesser extent, JNK; these stimulatory effects on ERKs and JNK were completely inhibited by Ro 32-0432 as well as EDTA. In contrast, leptin did not activate P38 MAPK. Similar to leptin, PMA activated ERK and JNK. Nicardipine and omega-conotoxin GVIA, each at 1 microM, were effective at inhibiting leptin-induced TH enzyme activity, TH mRNA accumulation, PKC activity, and ERK activity. Leptin increased activating protein-1 DNA-binding activity, and this was diminished by Ro 32-0432 as well as EDTA, similar to the reduction of TH mRNA levels. In addition, using supershift analysis, we documented the involvement of c-Fos and, to a lesser extent, c-Jun in leptin-induced activating protein-1 activity. These results indicate that leptin stimulates Ca(2+)-dependent PKC isoform-dependent catecholamine synthesis in porcine chromaffin cells. Previously, we had shown that leptin stimulated cAMP. The present study also showed that H89 (a PKA inhibitor) moderately, but significantly, inhibited leptin-induced ERK and TH mRNA. Consistent with this finding, leptin is shown here to activate novel PKC epsilon, which is assumed to stimulate Raf, upstream of ERKs, via cAMP, supporting the suggestion that Ca(2+)-independent novel PKC may also play some physiological role in regulating catecholamine synthesis.
authors.affiliation: Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, 305-8575, Japan. k-takemd@md.tsukuba.ac.jp
authors.forename: K
authors.initials: K
authors.lastname: Takekoshi
authors.forename: K
authors.initials: K
authors.lastname: Ishii
authors.forename: T
authors.initials: T
authors.lastname: Nanmoku
authors.forename: S
authors.initials: S
authors.lastname: Shibuya
authors.forename: Y
authors.initials: Y
authors.lastname: Kawakami
authors.forename: K
authors.initials: K
authors.lastname: Isobe
authors.forename: T
authors.initials: T
authors.lastname: Nakai
mesh_terms: D000313:Adrenal Medulla / Q000166:cytology*; D000818:Animals; D002395:Catecholamines / Q000096:biosynthesis* / Q000378:metabolism; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D020738:Leptin / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D013552:Swine; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism
chemical_list: D002395:Catecholamines; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D020738:Leptin; D012333:RNA, Messenger; D014446:Tyrosine 3-Monooxygenase; D011493:Protein Kinase C