Stimulation of Rb+ influx by bradykinin through Na+/K+/Cl− cotransport and Na+/K+-atpase in NIH-3T3 fibroblasts

Abstract Bradykinin receptor stimulation results in G-protein-coupled phospholipase activation, initiating protein kinase C (PKC) stimulation and cytosolic free Ca 2+ concentration ([Ca 2+ ]i) rises as signalling pathways. Using Rb + as a tracer for K + , we have studied the mechanisms involved in bradykinin-stimulated Rb + influx in NIH-3T3 fibroblasts. The furosemide-sensitive Na + /K + /Cl − cotransport and the ouabain-sensitive Na + /K + -ATPase were both involved in Rb + influx under resting conditions with a ratio Na + /K + /Cl − cotransport / Na + /K + -ATPase (r) = 0.73. Bradykinin stimulated Rb + influx (+82.6%) through both systems without changing their ratio (r = 0.72). PKC stimulation by a 15-min-treatment with phorbol 12-myristate 13-acetate (PMA) (2x10 −7 M) increased Rb + influx in resting cells by 75.7% without affecting r (0.75). PKC inhibition by H-7, and PKC down-regulation by 24-h PMA (10 −6 M) treatment decreased the bradykinin-induced stimulation of Rb + influx (+31% and +14.9% above control, respectively). Both down-regulation and inhibition of PKC dramatically reduced the furosemide-sensitive Na + /K + /Cl − cotransport, as r fell to 0.239 and 0.032 in bradykinin-stimulated cells after H-7 and 24-h PMA treatments, respectively. BAPTA/AM pretreatment (10 −4 M, 60 min), which complexed with [Ca 2+ ]i, not only prevented the bradykinin-induced [Ca 2+ ]i raise, but also partially inhibited bradykinin-induced Rb + influx stimulation (+39% above control), without modifying r (0.76). We conclude that stimulation of PKC is a major pathway involved in bradykinin stimulation of Rb + influx in NIH-3T3 fibroblasts, and that rises in [Ca 2+ ]i participate in bradykinin signalling, possibly through PKC activation. Our data also suggest that active PKC is required for basal and bradykinin-stimulated Na + /K + /Cl − cotransport activity in these cells.