Dynamin interacts with NOS1 in the renal collecting duct and activates NO production

Abstract

Within the kidney, the inner medullary collecting duct (IMCD) has the highest total NOS activity. We hypothesized that NOS isoforms may be regulated by dynamin (DNM) in the IMCD. Thus, the aims of this study were to determine which DNM isoforms (DNM1, DNM2, DNM3) are expressed in IMCDs, whether DNM interacts with NOS in the renal inner medulla (IM) and IMCD, and finally whether DNM activates NO production from NOS1. DNM2 and DNM3 are highly expressed in the rat IMCD, while DNM1 is localized outside of the IMCD. We performed immunoprecipitation experiments to probe for DNM/NOS interactions in the IM and IMCD of male Sprague Dawley rats. We found that DNM1 interacts with NOS1α, NOS1β, and NOS3 in the IM. DNM2 interacts with NOS1α in the IMCD, while DNM3 interacts with both NOS1α and NOS1β in the IMCD. DNM2 and DNM3 do not interact with NOS3 in the rat IM. Furthermore, we transfected mIMCD3 and COS7 cells with NOS1α and DNM2-GFP constructs and determined that NOS1α interacts with DNM2. Finally, nitrite production in COS7 cells (N=6) expressing NOS1α (193 ± 20 pmol nitrite/mg protein/hr) or NOS1α/DNM2-GFP (390 ± 65 pmol/mg protein/hr) was significantly higher compared to DNM2-GFP only transfected cells (not detectable; p <0.001). Upon stimulation with ionomycin, nitrite production further increased in the NOS1α/DNM2-GFP transfected cells (2122 ± 592 pmol nitrite/mg protein/hr; N=4) compared to NOS1α only (574 ± 34 pmol nitrite/mg protein/hr; N=5). In conclusion, these data demonstrate that DNM interacts with NOS1 to activate NO production in the IMCD.