Nitric oxide synthase regulation in inner medullary collecting duct cells

Abstract

Nitric oxide (NO) is a key regulator of sodium and water excretion in the kidney. It has been shown that ren_al tubules contain abundant nitric oxide synthase (NOS); however, little is known about the regulation of NOS expression and NOS activity in renal tubular cells. In the renal medulla, collecting duct cells produce a high level of endothelin:-1 (ET-1), express caveolin-1 artd protein tyrosine kinases (PTKs), and under certain conditions are exposed to high flows, resulting in an increased shear stress. In the current study, we hypothesize that ET-1 regulates expression of NOS isoforrri.( s) and NOS activity is modulated by caveolin-1, tyrosine phosphorylation and shear stress. Western blot analysis and immunofluorescent staining showed that all three NOS isoforms were shown to be present in inner medullary-collecting duct (IMCD) cells, a mouse IMCD cell line. After the IMCD cells were treated with 50 nM ET-1, NOSl was . significantly and specifically increased, but not NOS 2 and NOS 3 expression. ET .;.1 also increased phosphorylation of p42/p44 MAPK in the IMCD cells. Genistein, a protein tyrosine kinase inhibitor, and PD 98059, a Mekl inhibitor, reduced the effects of ET-1 on phosphorylation of p42/p44 MAPK and up-regulation of NOSl; furthermore, the ETA receptor antagonist, A127722, rather than the ETB receptor antagonist, A1926_21,- · inhibited the ET-1 effects in a concentration-dependent manner. The IMCD cells also express caveolin-1, but none of the NOS isofonns appear to be associated with caveolin-1 by co-immunoprecipitation experiments, suggesting ·that caveolin-1 does not regulate NOS activity in the IMCD cells. NOS 1 is regulated by tyrosine phosphorylation and is shown to be phosphorylated at basal conditions. The non-specific inhibition of protein tyrosine kinases with 100 μM erbstatin A significantly increased nitrite production in the IMCD cell media. The tyrosine phosphorylation of Nos·· 1 was reduced by erbstatin A, and enhanced by vanadate, a protein tyrosine phosphatase inhibitor. When the IMCD cells were exposed to three levels of shear stress, 30, 10, 3.3 dyn/cm2 for 1 hour, a significant increase in nitrite production was detected. L-NAME, a non-specific NOS inhibitor, completely blocked the effect of shear stress on nitrite production in IMCD . cells. Therefore, in IMCD cells, NOS 1 expression is up-regulated by ET-1 through activation of the ET A receptor and p42/p44 MAPK pathway; NO production is stimulated by tyrosine dephosphorylation, and activated by shear stress, but does not appear to be regulated by caveolin-1.