Intracellular calcium changes induced by the endozepine triakontatetraneuropeptide in human polymorphonuclear leukocytes: role of protein kinase C and effect of calcium channel blockers

The PKC activator 12-O-tetradecanoylphorbol-13-acetate (PMA) concentration-dependently inhibited TTN-induced [Ca++]i rise, and this effect was reverted by the PKC inhibitors rottlerin (partially) and Ro 32-0432 (completely). PMA also inhibited TTN-induced IL-8 mRNA expression. In the absence of PMA, however, rottlerin (but not Ro 32-0432) per se partially inhibited TTN-induced [Ca++]i rise. The response of [Ca++]i to TTN was also sensitive to mibefradil and flunarizine (T-type Ca++-channel blockers), but not to nifedipine, verapamil (L-type) or ω-conotoxin GVIA (N-type). In agreement with this observation, PCR analysis showed the expression in human PMNs of the mRNA for all the α1 subunits of T-type Ca++ channels (namely, α1G, α1H, and α1I).In human PMNs TTN activates PKC-modulated pathways leading to Ca++ entry possibly through T-type Ca++ channels.Triakontatetraneuropeptide [diazepam-binding inhibitor (DBI) 17–50, TTN] is one of the major endogenous peptides generated through the cleavege of DBI, a neuropeptide also known as acyl-CoA-binding protein [1]. DBI and DBI-derived peptides are also called endozepines, after the ability of at least some of them to recognize the diazepam binding site on the GABAA receptor, and are widely distributed in the CNS and in peripheral organs [2,3]. In particular, immune tissues express DBI [4] and evidence exists that TTN and its related peptides octadecaneuropeptide (DBI 33–50, ODN) and eiksoneuropeptide (DBI 51–70, ENP) can affect the immune response. In particular, TTN and ODN stimulate the production of tumor necrosis factor (TNF) alpha, interleukin (IL)-1 beta, IL-8, granulocyte/macrophage colony-stimulating factor, IL-6 and IL-8 in human monocytes [5,6], and ODN enhances the LPS-induced secretion of IL-6 in human peripheral blood mononuclear cells [7].In human polymorphonuclear leukocytes (PMNs) we previously showed that TTN rises intracellular calcium ([Ca++]i) and stimulates chemotaxis, O2- generation, phagocytosis and IL