Stimulation of Rb+ influx by bradykinin through Na+/K+/Cl- cotransport and Na+/K(+)-ATPase in NIH-3T3 fibroblasts.
Bradykinin receptor stimulation results in G-protein-coupled phospholipase activation, initiating protein kinase C (PKC) stimulation and cytosolic free Ca2+ concentration ([Ca2+]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 [Ca2+]i, not only prevented the bradykinin-induced [Ca2+]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 [Ca2+]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.
Hichami
A
A
Laboratoire de Pharmacologie Moléculaire, UFR des Sciences Pharmaceutiques, Rennes, France.
Anger
J P
JP
Allain
N
N
Vernhet
L
L
Martin
C A
CA
Legrand
A B
AB
D016475:3T3 Cells; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology*; D002352:Carrier Proteins / Q000187:drug effects* / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D005665:Furosemide / Q000494:pharmacology; D017136:Ion Transport; D051379:Mice; D011493:Protein Kinase C / Q000378:metabolism; D012413:Rubidium / Q000494:pharmacology*; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects* / Q000378:metabolism
D002352:Carrier Proteins; D028021:Sodium-Potassium-Chloride Symporters; D005665:Furosemide; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D001920:Bradykinin