Dynamin‐2 is a negative regulator of NOS1β in the collecting duct

Abstract

Nitric oxide synthase 1 (NOS1)/NO pathway in the inner medullary collecting duct (IMCD) promotes natriuresis. Rat IMCD express NOS1α and NOS1β splice variants, while mouse and human IMCD expresses predominantly NOS1β. We reported dynamin 2 (DNM2) activates NO production via direct interaction with NOS1α; however it is undetermined if DNM2 also activates NOS1β. Accordingly, we hypothesized that DNM2 is an activator of NOS1β. To test this hypothesis, we used COS7 cells, which lack endogenous NOS, and mIMCD‐3 immortalized cells that express NOS1β. By immunoprecipitation and western blot, we confirmed that NOS1β interacts with DNM2. Nitrite production (an index of NO production) was assessed by HPLC, and we determined that overexpression of DNM2 in mIMCD‐3 cells resulted in a significant reduction in nitrite production (323 ± 25 pmol/mg pr/h vs. 1734 ± 35, N = 3‐4, P = 0.016). Using a variety of pharmacological inhibitors and control agents targeting different domains of DNM2, we determined that inhibition of DNM2 resulted in a significant increase in mIMCD‐3 nitrite production (ranged from 1410 ± 419 to 1910±105 pmol/mg pr/h) compared to controls (ranged from 665 ± 57 to 839 ± 120 pmol/mg pr/h; n = 4, P < 0.05). Finally, we confirmed our pharmacological experiments by decreasing DNM2 expression with siRNA. DNM2 siRNA resulted in a 95% reduction in mIMCD‐3 DNM2 expression and produced a significant increase in nitrite production (scramble siRNA 112 ± 15 vs DNM2 siRNA 249 ± 21 pmol/ mg pr/ h). In conclusion, these data suggest that DNM2 is a novel negative regulator of NOS1β.