Inhibition of bradykinin-induced increases of cytosolic Ca⁺⁺ by a novel amiloride analog

The present study was undertaken to determine whether activation of the Na⁺/H⁺ antiport regulates the bradykinin (BK)-induced Ca⁺⁺ transient in Madin-Darby canine kidney cells. An amiloride analog, 5-N-N-ethyl[2-methoxy 5-nitrobenzyl]amiloride (L651,548), led to a dose-dependent inhibition of the BK-induced Ca⁺⁺ transient (K(i) = 9 μM), and this effect was eliminated in high external Na⁺. Zero external Na⁺ failed to inhibit the BK-induced Ca⁺⁺ release, suggesting L651,548 is not acting through inhibition of the Na⁺/H⁺ antiport. Use of monovalent ionophores to adjust the intracellular pH demonstrated changes in peak Ca⁺⁺ release by BK only over wide pH ranges. Furthermore, studies revealed L651,548 was much more potent at inhibiting pH recovery (K(i) = 55 nM) than BK-induced Ca⁺⁺ release. Studies in saponin-permeabilized cells demonstrated no effect of L651,548 on inositol trisphosphate-induced Ca⁺⁺ release. Whole cell [³H] BK binding studies indicated a dose-dependent inhibition by L651,548. We conclude that the Na⁺/H⁺ antiport does not play a critical role in control of BK-induced Ca⁺⁺ release in Madin-Darby canine kidney cells. Also, the amiloride analog L651,548 reduces BK Ca⁺⁺ release by inhibition of BK receptor binding in Madin-Darby canine kidney cells.