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

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• 作者：Hichami ; Aziz ; Anger ; Jean-Pierre ; Allain ; Nathalie ; Vernhet ; Laurent ; Martin ; Corinne A. E. ; et. al.
• 关键词：protein kinase C ; calcium ; phorbol esters ; K⁺ transport
• 刊名：Life Sciences
• 出版年：1996
• 期刊代码：62_00243205
• 类别：imm
• 出版时间：October 25, 1996
• 卷：59
• 期：22
• 页码：1829-1837
• 文件大小：568 K

摘要

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) (2×10⁻⁷ 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.