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.
Xu
Feng
F
Department of Pharmacology, Akita University School of Medicine, 1-1-1 Hondoh, Akita 010-8543, Japan.
Satoh
Eisaku
E
Iijima
Toshihiko
T
D002118:Calcium / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D008301:Maleimides / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010703:Phorbol Esters / Q000737:chemistry / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050916:TRPV Cation Channels; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry / Q000494:pharmacology
D027682:Cation Transport Proteins; D004791:Enzyme Inhibitors; D007038:Hypotonic Solutions; D007211:Indoles; D007473:Ion Channels; D008301:Maleimides; D010703:Phorbol Esters; D050916:TRPV Cation Channels; C416986:TRPV4 protein, human; C008876:phorbol-12,13-didecanoate; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; C088060:bisindolylmaleimide; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium