Leptin stimulates catecholamine synthesis in a PKC-dependent manner in cultured porcine adrenal medullary chromaffin cells

We have previously shown that murine recombinant leptin directly stimulates catecholamine synthesis through the long form of the leptin receptor (Ob-R b) expressed in cultured porcine chromaffin cells. Additionally, we found t hat leptin activates IP3 production after PLC activation. It is well establ ished that activation of PLC elicits IP3 production as well as an increase in diacylglycerol, a compound that stimulates PKC. Therefore, we investigat ed the involvement of PKC in leptin-induced catecholamine synthesis. Leptin was found to induce significant increases in PKC activity in a dose-depend ent manner (1, 10, and 100 wi); chelation of extracellular Ca2+ by EDTA abo lished this PKC stimulatory activity. We also confirmed by Western blot ana lysis that leptin (at 100 nM) induced significant increases in Ca2+-depende nt PKC alpha,beta (1), and -gamma expression. The activity of the rate-limi ting enzyme tyrosine hydroxylase (TH) in the biosynthesis of catecholamine is regulated at the transcriptional and posttranscriptional levels. TH enzy me activity and TH mRNA levels induced by 100 mi 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 activate d 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, l eptin did not activate P38 MAPK. Similar to leptin, PMA activated ERK and J NK. Nicardipine and omega -conotoxin GVIA, each at 1 muM, were effective at inhibiting leptin-induced TH enzyme activity, TH mRNA accumulation, PKC ac tivity, and ERK activity. Leptin increased activating protein-1 DNA-binding activity, and this was diminished by Ro 32-0432 as well as EDTA, similar t o 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 le ptin-induced activating protein-1 activity. These results indicate that lep tin stimulates Ca2+-dependent PKC isoform-dependent catecholamine synthesis in porcine chromaffin cells. Previously, we had shown that leptin stimulat ed cAMP. The present study also showed that H89 (a PKA inhibitor) moderatel y, but significantly, inhibited leptin-induced ERK and TH mRNA. Consistent with this finding, leptin is shown here to activate novel PKC is an element of, which is assumed to stimulate Raf, upstream of ERKs, via cAMP, support ing the suggestion that Ca2+-independent novel PKC may also play some physi ological role in regulating catecholamine synthesis.