Protein kinase C-mediated Ca2+ entry in HEK 293 cells transiently expressing human TRPV4

1 We investigated whether protein kinase C (PKC) activation stimulates Ca2+ entry in HEK 293 cells transfected with human TRPV4 cDNA and loaded with fura-2. 2 Phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, increased the intracellular Ca2+ concentration ([Ca2+](i)) in a dose-dependent manner, with an EC50 value of 11.7 nM. Exposure to a hypotonic solution (HTS) after PMA further increased [Ca2+](i). Two other PKC-activating phorbol esters, phorbol 12,13-didecanoate (PDD) and phorbol 12,13-dibutyrate, also caused [Ca2+](i) to increase. 3 The inactive isomer 4alpha-PMA was less effective and the peak [Ca2+](i) increase was significantly smaller than that induced by PMA. In contrast, 4alpha-PDD produced a monophasic or biphasic [Ca2+](i) increase with a different latency, while 4alpha-phorbol had no effect. 4 The PMA-induced [Ca2+](i) increase was abolished by prior exposure to bisindolylmaleimide (BIM), a PKC-specific inhibitor, and suppressed by the nonspecific PKC inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine. The [Ca2+](i) increase induced by 4alpha-PMA, 4alpha-PDD or HTS was not significantly affected by BIM. 5 These results suggest that both PKC-dependent and -independent mechanisms are involved in the phorbol ester-induced activation of TRPV4, and the PKC-independent pathway is predominant in HTS-induced Ca2+ entry.