Dynamin-2 is a novel NOS1β interacting protein and negative regulator in the collecting duct - PubMed (original) (raw)

Comparative Study

Dynamin-2 is a novel NOS1β interacting protein and negative regulator in the collecting duct

Kelly A Hyndman et al. Am J Physiol Regul Integr Comp Physiol. 2016.

Abstract

Nitric oxide synthase 1 (NOS1)-derived nitric oxide (NO) production in collecting ducts is critical for maintaining fluid-electrolyte balance. Rat collecting ducts express both the full-length NOS1α and its truncated variant NOS1β, while NOS1β predominates in mouse collecting ducts. We reported that dynamin-2 (DNM2), a protein involved in excising vesicles from the plasma membrane, and NOS1α form a protein-protein interaction that promotes NO production in rat collecting ducts. NOS1β was found to be highly expressed in human renal cortical/medullary samples; hence, we tested the hypothesis that DNM2 is a positive regulator of NOS1β-derived NO production. COS7 and mouse inner medullary collecting duct-3 (mIMCD3) cells were transfected with NOS1β and/or DNM2. Coimmunoprecipitation experiments show that NOS1β and DNM2 formed a protein-protein interaction. DNM2 overexpression decreased nitrite production (index of NO) in both COS7 and mIMCD-3 cells by 50-75%. mIMCD-3 cells treated with a panel of dynamin inhibitors or DNM2 siRNA displayed increased nitrite production. To elucidate the physiological significance of IMCD DNM2/NOS1β regulation in vivo, flox control and CDNOS1 knockout mice were placed on a high-salt diet, and freshly isolated IMCDs were treated acutely with a dynamin inhibitor. Dynamin inhibition increased nitrite production by IMCDs from flox mice. This response was blunted (but not abolished) in collecting duct-specific NOS1 knockout mice, suggesting that DNM2 also negatively regulates NOS3 in the mouse IMCD. We conclude that DNM2 is a novel negative regulator of NO production in mouse collecting ducts. We propose that DNM2 acts as a "break" to prevent excess or potentially toxic NO levels under high-salt conditions.

Keywords: collecting duct; dynamin-2; human; nitric oxide; nitric oxide synthase 1 splice variant.

Copyright © 2016 the American Physiological Society.

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Figures

Fig. 1.

Fig. 1.

Protein expression of NOS1 splice variants in human cortical and medullary homogenates by Western blot analysis. A: five different human samples (see

methods

for demographic information) express both NOS1α (155 kDa) and NOS1β (130 kDa). B: COS7 cells transfected with either NOS1α or NOS1β as a positive control for the COOH-terminal, NOS1-specific antibody.

Fig. 2.

Fig. 2.

Dynamin-2 (DNM2) and NOS1β interactions in COS7 and mouse inner medullary collecting duct-3 (mIMCD-3) cells. A: COS7 cells were transfected with either NOS1β ± DNM2-GFP. Coimmunoprecipitation experiments were performed by immunoprecipitation (IP) with anti-GFP or mouse IgGs and immunoblot (IB) with anti-NOS1. An interaction is observed in the NOS1β+DNM2 cells only. B: confirmation of the anti-GFP IP with IB with anti-GFP. GFP only expressed in the DNM2-GFP transfected cells. C: mIMCD-3 cells transfected with empty vector or DNM2-GFP. Coimmunoprecipitation experiments were performed by IP with anti-NOS1 and IB with anti-DNM2. NOS1β interacts with both the endogenous DNM2 (100 kDa) and transfected DNM2-GFP (130 kDa), as shown in the bottom panel. D: confirmation of the anti-NOS1 IP with IB with anti-NOS1 (n = 3). L, molecular ladder; V, empty vector transfected cells.

Fig. 3.

Fig. 3.

Nitrite production in COS7 and mIMCD-3 cells. A: COS7 cells were transfected with NOS1β ± DNM2 and DNM2 significantly reduced nitrite production (n = 3, *P = 0.007). B: mIMCD-3 cells overexpressing DNM2 produced significantly less nitrite compared with vector control cells (n = 3; *P = 0.04). C: acute stimulation of transfected COS7 cells resulted in a significant increase in nitrite in the NOS1β transfected cells, but no significant effect in the NOS1β+DNM2 cells. *P < 0.05 compared with vehicle. †P < 0.05 compared with corresponding NOS1β vehicle or ionomycin treated cells.

Fig. 4.

Fig. 4.

The domains of DNM2 and pharmacological inhibition of DNM2. A: illustration of the different domains in mouse DNM2. PH, plekstrin homology; GED, GTPase effector domain; PRD, proline-rich domain; B: acute pharmacological blockade of the GTPase domain of DNM2 with a panel of inhibitors, resulted in a significant increase in mIMCD-3 nitrite production, compared with their respective controls. C: acute pharmacological inhibition of the PH domain of DNM2 resulted in a significant increase in mIMCD-3 nitrite production, compared with control (P = 0.01). Two-factor ANOVA with Sidak's multiple (6) comparisons were used. *Significant difference compared with control.

Fig. 5.

Fig. 5.

Knockdown of DNM2 by siRNA in mIMCD-3 cells. A: representative image of DNM2 knockdown with 0, 10, or 60 pmol of DNM2 siRNA. B: DNM2 siRNA knockdown results in a significant increase in mIMCD-3 nitrite production compared with scramble siRNA. *P < 0.05 compared with scramble siRNA.

Fig. 6.

Fig. 6.

NOS1β and DNM2 form a protein-protein interaction in vivo. Freshly isolated IMCD from flox and collecting duct-specific NOS1 knockout (CDNOS1KO) mice on different salt diets for 7 days. A: coimmunoprecipitation (IP) experiments were performed with anti-DNM2 or rabbit IgGs followed by immunoblot (IB) with anti-NOS1 or anti-DNM2 with IMCD from flox control mice on a HS diet. An interaction is observed between NOS1β and DNM2. B: nitrite production from freshly isolated IMCDs from flox and CDNOS1KO mice on a 7-day high-salt diet (n = 4–10; see

methods

for detailed explanation of sample). Acute 30-min inhibition of DNM2 resulted in a significant increase in nitrite production by flox and tended to increase nitrite production in CDNOS1KO mice (CDNOS1KO vehicle compared with dynasore P > 0.05); however, it was blunted in the CDNOS1KO IMCD. Two-factor ANOVA with Sidak's multiple (6) comparisons were used. *P < 0.05 compared with flox vehicle. †P < 0.05 compared with flox dynasore. C: flox control mice fed a HS diet for 7 days had significantly less inner medullary DNM2 than flox mice on NS diet or CDNOS1KO mice on HS diet (n = 4 mice per group). Two-factor ANOVA with Sidak's multiple (6) comparisons were used. *P < 0.05 compared with flox NS. ‡P < 005 compared with flox HS.

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