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

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Abstract
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. 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. The inactive isomer 4α‐PMA was less effective and the peak [Ca2+]i increase was significantly smaller than that induced by PMA. In contrast, 4α‐PDD produced a monophasic or biphasic [Ca2+]i increase with a different latency, while 4α‐phorbol had no effect. 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 4α‐PMA, 4α‐PDD or HTS was not significantly affected by BIM. 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. British Journal of Pharmacology (2003) 140, 413–421. doi:10.1038/sj.bjp.0705443