Abstract
BackgroundThe endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca++]i) changes followed by activation in human polymorphonuclear leukocytes (PMNs). The present study was undertaken to investigate the role of protein kinase (PK) C in the modulation of the response to TTN by human PMNs, and to examine the pharmacology of TTN-induced Ca++ entry through the plasma membrane of these cells.ResultsThe 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).ConclusionsIn human PMNs TTN activates PKC-modulated pathways leading to Ca++ entry possibly through T-type Ca++ channels.
Highlights
The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca++]i) changes followed by activation in human polymorphonuclear leukocytes (PMNs)
In human polymorphonuclear leukocytes (PMNs) we previously showed that TTN rises intracellular calcium ([Ca++]i) and stimulates chemotaxis, O2- generation, phagocytosis and IL-8 production [8,9]
The results show that the stimulatory effect of TTN is profoundly affected by PKC activation, and suggest that different PKC isoforms may play distinct roles
Summary
Effect of PKC ligands In agreement with previous studies [8,9], in human PMNs TTN 100 μM induced a rapid and transient rise of [Ca++]i and increased the expression of IL-8 mRNA. The PKC activator PMA (but not its inactive analogue α-PMA) reduced the effect of TTN 100 μM on [Ca++]i rise in a concentration-dependent fashion (Fig. 1). Panel B: Concentration-response relationship for the effect of PMA (empty circles) and lack of effect of α-PMA (empty square) on TTN 100 μM-induced [Ca++]i rise. In the absence of PMA, rottlerin (but not Ro 32-0432) per se was able to inhibit TTN 100 μM-induced [Ca++]i rise in a concentration-dependent fashion (Fig. 2). PMA, α-PMA, Ro 32-0432, and rottlerin at the concentrations used had per se no significant effect on the parameters under study (data not shown). FCl0ine4irodg3iunu2cr(e(recdniget2[hrnCattrapiao+al+np]-eiarrlne,iseseplm,oepnmtsyepctryiercslaqletuisoa)nraesnhsd)ipolafnockrTtTohfNeefe1ffe0fce0tcμtoMof fR-root3t2-Concentration-response relationship for the effect of rottlerin (right panel, empty circles) and lack of effect of Ro 320432 (central panel, empty squares) on TTN 100 μM-. Data are expressed as percentage of the effect of TTN alone (left panel, filled circle). Northern blot analysis provided evidence for the expression of the mRNA for all the α1 subunits of T-type Ca++ channels (namely, α1G, α1H, and α1I) in human PMNs (Fig. 4)
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