Involvement of protein kinase C in bradykinin-induced intracellular calcium increase in primary cultured human keratinocytes

Abstract 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)P 3 ) which caused Ca 2+ mobilization in human keratinocytes. In this study, BK-induced Ca 2+ responses were examined in primary cultured human keratinocytes by video imaging fluorescence microscopy using fura-2. Intracellular calcium concentration ([Ca 2+ 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 [Ca 2+ ] i profile was suggested to be due to the Ca 2+ 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 [Ca 2+ ] i . A 20-min pretreatment with PKC inhibitors, H-7 or staurosporine, reversed the decrease by PMA in the shoulder of BK-induced Ca 2+ response. Furthermore, the BK-induced [ 45 Ca] uptake was inhibited by EGTA and PMA. Ins(1,4,5)P 3 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)P 3 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 Ca 2+ influx, which was shown as shoulder, may be negatively modulated by PKC in primary cultured human keratinocytes.