Nitric oxide production by human proximal tubular cells : modulation by atrial natriuretic factor and angiotensin II
The aims of this study were to: (i) investigate the evidence for the presence of ANF and ANGII receptors on primary cultures of human PT cells (ii) determine whether these cultures could produce NO in response to cytokine stimulation and (iii) whether ANF and ANGII could modulate NO production. ANF significantly increased cGMP production, demonstrating the presence of GC-linked receptors. Scatchard and Hill analysis of data obtained from radioligand binding assays using [125I]ANGII identified two binding sites for ANGII. The largest stimulation of NO production was obtained using a combination of IL-1, TNF- and IFN-. This was inhibited in a concentration-dependent manner by PDGF, TGF- and IL-4. Inhibition of cytokine-stimulated NO production by dexamethasone and L-NMMA suggested that NO was produced via NOS, and RT-PCR produced a 336 b.p. NOS mRNA product from cytokine-stimulated PT cells which upon sequencing, shared high homology with previously published sequences of human iNOS. ANF and C(4-23)ANF significantly increased NO production, suggesting that this action was mediated via the NPR-C; a hypothesis reinforced by the demonstration that 8-bromo-cGMP did not have any effect on NO production, suggesting a mechanism independent of cGMP. BNP and CNP reduced basal NO production at higher concentrations. ANGII and ANGIV stimulated NO production but their actions could not be blocked by losartan and PD123319, suggesting that AT1 and AT2 receptors were not involved. Furthermore, the stimulation of NO production by ANGIV implicated the AT4 receptor. ANF and ANGII-stimulated NO production was abolished by L-NMMA, suggesting that NO was produced via NOS. Overall, this study demonstrates interactions between NO, ANF and ANGII in primary cultures of human PT cells. Furthermore, this cell system provides a simple in vitro model and an excellent tool with which to investigate these interactions in greater detail in order to elucidate the exact intracellular mechanisms involved.