Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates catecholamine secretion from cultured porcine adrenal medullary chromaffin cells in a dose-dependent manner with the half-maximal and maximal doses of 30 nM and 1 microM, respectively. Either removal of extracellular Ca2+ or addition of Gd3+, an inorganic Ca2+ channel blocker, very potently inhibits PACAP-induced catecholamine secretion. Both nicardipine (1 microM) and methoxyverapamil (1 microM), blockers of voltage-dependent Ca2+ channels, are also effective in inhibiting PACAP-induced catecholamine secretion. When the intracellular free Ca2+ concentration ([Ca2+]i) is measured in a fura 2-loaded single chromaffin cell, PACAP is found to cause a sustained increase in [Ca2+]i by mobilizing Ca2+ from both extra- and intracellular pools. It is also found that PACAP stimulates the production of inositol phosphates in a dose-dependent manner, which is not abolished by removal of extracellular Ca2+ unlike the case of nicotine. PACAP increases cAMP content in chromaffin cells in a dose-dependent manner. Removal of extracellular Ca2+ enhances PACAP-induced cAMP production but strongly inhibits PACAP-induced catecholamine secretion. Pretreatment of cells with adenosine-3':5'-monophosphothioate, cyclic, Rp-isomer, a cAMP antagonist, does not block PACAP-induced catecholamine secretion. The addition of forskolin or 3-isobutyl-1-methylxanthine does not enhance the PACAP-induced catecholamine secretion. These results indicate that PACAP activates voltage-dependent Ca2+ channels and phospholipase C as well as adenylate cyclase in cultured porcine adrenal medullary cells and strongly suggest that PACAP-induced catecholamine secretion is mainly mediated by activation of voltage-dependent Ca2+ channels.

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Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors (VEGFRs). Very recently, sunitinib has been shown to be an active agent for the treatment of malignant pheochromocytomas. However, it is unclear whether sunitinib acts only through an antiangiogenic mechanism or whether it may also directly target tumor cells. Sunitinib markedly induced apoptosis of PC12 cells in a dose-dependent and time-dependent manner. Furthermore, in support of these findings, we found that sunitinib induced a reduction in the expression of the antiapoptotic molecule Bcl-2 as well as dephosphorylation of the proapoptotic molecule BAD, which results in the activation of BAD in these cells. Consistent with these apoptotic effects, our results showed that sunitinib inhibited phosphorylation of Akt and mTOR and was followed by a reduction of S6K1, which is a well-known target of mTOR. Knockdown of VEGFR-2 attenuated the sunitinib-induced effects, including apoptosis and inhibition of signaling pathways such as the phosphorylation of Akt as well as mTOR, and Bcl-2, which confirmed that these effects could be mediated by VEGFR-2. In addition, silencing of S6K1 induced apoptosis accompanied by a decrease in the phosphorylation of BAD and Bcl-2, similar to that observed with sunitinib treatment. Thus, these results together suggest that sunitinib initially exerts its apoptotic effect through the inhibition of VEGFR-2, which, when followed by reduction of its downstream effectors, including Akt/mTOR/S6K1, may lead to inhibition of the antiapoptotic molecule Bcl-2 and activation of the proapoptotic molecule BAD in PC12 cells. However, PC12 cells do not precisely reflect the pathogenesis of malignant cells. Therefore, we confirmed the key findings by replicating these experiments in human neuroblastoma SK-N-SH cells.

Recently, the insulin-sensitizing adipokine adiponectin and the insulin resistance-inducing adipokine tumor necrosis factor- (TNF-) were reported to inhibit each other's production in adipocytes. We investigated the effects of two  3-adrenoceptor agonists, 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL-316,243) and (±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid (BRL37344), on the gene expression of adiponectin, two adiponectin receptors, and TNF-in adipose tissues of C57BL/6J mice. CL-316,243 and BRL37344 downregulated adiponectin, but upregulated adiponectin receptor 2 (not receptor 1) in epididymal or/and subcutaneous white adipose tissues and in brown adipose tissue. TNF-expression was upregulated only in epididymal adipose tissue. To further explore these effects, we treated differentiated 3T3-L1 adipocytes with the non-selective -adrenoceptor agonist isoproterenol. As a result, adiponectin receptor 2 (but not receptor 1) gene expression and TNF- protein expression increased, but gene expression and secretion of adiponectin decreased. The upregulation of adiponectin receptor 2 by isoproterenol is most likely via  2 , 3-adrenoceptors, adenylyl cyclases, and protein kinase A (PKA). However, the accompanying activation of AMP-activated protein kinase (AMPK) may inhibit this upregulation. Our results suggest that upregulation of TNF- and downregulation of adiponectin by -adrenoceptor activation may contribute to the pathogenesis of catecholamine-induced insulin resistance, and that upregulation of adiponectin receptor 2 may be a feedback result of reduced adiponectin.