Involvement of protein kinase C in bradykinin-induced intracellular calcium increase in primary cultured human keratinocytes.
Bradykinin (BK) is one of the key mediators of inflammation and a weak mitogen. We have previously demonstrated that BK induced the generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) which caused Ca2+ mobilization in human keratinocytes. In this study, BK-induced Ca2+ responses were examined in primary cultured human keratinocytes by video imaging fluorescence microscopy using fura-2. Intracellular calcium concentration ([Ca2+]i) level increased to a peak within 30 s after BK addition and decreased gradually to the basal level. The existence of the broad shoulder in the [Ca2+]i profile was suggested to be due to the Ca2+ influx from the external medium, because this disappeared in the presence of 0.5 mM EGTA. Pretreatment with phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, significantly resulted in reduction of the descending shoulder of BK-induced increase in [Ca2+]i. A 20-min pretreatment with PKC inhibitors, H-7 or staurosporine, reversed the decrease by PMA in the shoulder of BK-induced Ca2+ response. Furthermore, the BK-induced [45Ca] uptake was inhibited by EGTA and PMA. Ins(1,4,5)P3 generation induced by BK peaked at 20 s and returned to the basal level at 60 s. There were no significant differences in Ins(1,4,5)P3 levels at 20 and 60 s among the cells exposed to BK alone, BK with PMA pretreatment (20 min) and BK with PMA+H-7 pretreatment. These results suggest that the BK-induced Ca2+ influx, which was shown as shoulder, may be negatively modulated by PKC in primary cultured human keratinocytes.
Aoyama
Y
Y
Department of Dermatology, Gifu University School of Medicine, Japan.
Seishima
M
M
Mori
S
S
Kitajima
Y
Y
Okano
Y
Y
Nozawa
Y
Y
D000328:Adult; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009994:Osmolar Concentration; D011493:Protein Kinase C / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D002118:Calcium