• Calcium entry;
  • phorbol esters;
  • protein kinase C;
  • TRPV channels
  • 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