Endothelin-1 stimulates NO production and inhibits cAMP accumulation in rat inner medullary collecting duct through independent pathways

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Abstract
Endothelin-1 (ET-1) inhibition of vasopressin (AVP)-stimulated cAMP accumulation in the collecting duct has been hypothesized to be mediated, at least in part, by nitric oxide (NO). To examine this, the effect of ET-1 on NO production by acutely isolated rat inner medullary collecting duct (IMCD) cell suspensions and the role of NO in mediating ET-1 effects on AVP-stimulated cAMP accumulation were studied. ET-1 dose dependently (first evident at 100 pM ET-1) increased IMCD NO production as determined by DAF-FM fluorescence. ETBreceptor (BQ-788), but not ETAreceptor (BQ-123), antagonism blocked this effect. Nonspecific NO synthase (NOS) inhibitors [ NG-nitro-l-arginine methyl ester (l-NAME) or NG-monomethyl-l-arginine] or NOS-1 inhibitors (SMTC or VNIO) inhibited the ET-1 response, whereas NOS-2 or NOS-3 inhibitors (l-NAA or 1400W) were ineffective. ET-1 also increased cGMP accumulation. ET-1 caused a 35% reduction in AVP-stimulated cAMP levels; however, this response was not affected by l-NAME or SMTC. The addition of l-arginine, NADPH, tetrahydrobiopterin, or tempol (to reduce superoxide-dependent conversion of NO to peroxynitrate) did not affect the response. NO donors (SNAP or spermine NONOate), at concentrations that stimulated DAF-FM fluorescence and increased cGMP levels, did not alter AVP-stimulated cAMP accumulation in the IMCD cell suspensions. In conclusion, ET-1 stimulates IMCD NO production through activation of the ETBreceptor and NOS-1. However, neither ET-1-mediated NO production nor NO donors inhibit AVP-stimulated cAMP accumulation, indicating that NO does not mediate ET-1 inhibition of cAMP production by the IMCD.