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 generati on of inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3) which caused Ca2+ m obilization in human keratinocytes. In this study, BK-induced Ca2+ res ponses were examined in primary cultured human keratinocytes by video imaging fluorescence microscopy using fura-2. Intracellular calcium co ncentration ([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 t o the Ca2+ influx from the external medium, because this disappeared i n the presence of 0.5 mM EGTA. Pretreatment with phorbol-12-myristate- 13-acetate (PMA), a protein kinase C (PKC) activator, significantly re sulted in reduction of the descending shoulder of BK-induced increase in [Ca2+](i). A 20-min pretreatment with PKC inhibitors, H-7 or stauro sporine, reversed the decrease by PMA in the shoulder of BK-induced Ca 2+ response, Furthermore, the BK-induced [Ca-45] 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 di fferences in Ins(1,4,5)P-3 levels at 20 and 60 s among the cells expos ed 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 pr imary cultured human keratinocytes.