Activation of Na+/Ca2+ Exchanger in Kinin B1 Receptor-Stimulated Human Fibroblast Is Associated with Collagen Production
The arterial wall in hypertension is characterized by thickening of the media, in part due to increased deposition of connective tissue. Autocrine and paracrine factors may participate in this process; including products of the kallikrein-kinin system. We evaluated early signal transduction events and effects on collagen formation in B1-stimulated human myofibroblast cells (IMR-90). We measured cytosolic calcium (Cacyt) levels in cells loaded with FURA-2AM. Gene expression of connective tissue growth factor (CTGF) and α1(I) collagen was determined by estimating mRNA levels using Northern analysis of B1 stimulated cells. Activation of the B1 receptor with des-arg10-kallidin stimulated a three-fold increase in CTGF mRNA by increasing its stability. Furthermore, B1 receptor activation caused an increase in α1(I) collagen mRNA and a four-fold increase in type I collagen synthesis in these cells; events not observed in B2 receptor-stimulated cells. Activation of the B1 receptor stimulated a dose dependent rise in Cacyt (EC50=1.9nM) which was completely inhibited by des-arg10-[leu9]-kallidin (100nM), a B1 receptor antagonist. Isosmotic replacement of extracellular Na+ with N-methyl,D-glucamine blocked > 90% of the B1 stimulated rise in Cacyt. A similar effect was observed when Ca2+ was removed from the extracellular media, suggesting a role for the plasma membrane Na+/Ca2+ exchanger (NCX). To further define a role for the NCX on CTGF formation we used dichlorobenzamil (DCB) and KB-R7943, two specific NCX inhibitors. DCB completely blocked the activation of B1 receptor induced increase in CTGF mRNA stability while not affecting basal CTGF mRNA levels. In contrast, preincubation with EIPA, an amiloride analog, did not affect basal or stimulated CTGF mRNA levels. Furthermore, 60μM KB-R7943 blocked the B1 stimulated rise in Cacyt. NCX isoform 1 was identified in these cells using RT-PCR and immuno-detection. Thus, B1 receptor stimulation increases fibrogenesis through a mechanism that involves modulation of cation metabolism via reverse-mode activation of NCX.