Post-translational inactivation of endothelial nitric oxide synthase in the transgenic sickle cell mouse penis

Abstract

Introduction: Sickle cell disease (SCD)-associated priapism is characterized by endothelial nitric oxide synthase (eNOS) dysfunction in the penis. However, the mechanism of decreased eNOS function/activation in the penis in association with SCD is not known.

Aims: Our hypothesis in the present study was that eNOS is functionally inactivated in the SCD penis in association with impairments in eNOS post-translational phosphorylation and the enzyme's interactions with its regulatory proteins.

Methods: Sickle cell transgenic (sickle) mice were used as an animal model of SCD. Wild-type (WT) mice served as controls. Penes were excised at baseline for molecular studies. eNOS phosphorylation on Ser-1177 (positive regulatory site) and Thr-495 (negative regulatory site), total eNOS, and phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177) expressions, and eNOS interactions with heat-shock protein 90 (HSP90) and caveolin-1 were measured by Western blot. Constitutive NOS catalytic activity was measured by conversion of L-[14C]arginine-to-L-[14C]citrulline in the presence of calcium.

Main outcome measures: Molecular mechanisms of eNOS dysfunction in the sickle mouse penis.

Results: eNOS phosphorylated on Ser-1177, an active portion of eNOS, was decreased in the sickle mouse penis compared with WT penis. eNOS interaction with its positive protein regulator HSP90, but not with its negative protein regulator caveolin-1, and phosphorylated AKT expression, as well as constitutive NOS activity, were also decreased in the sickle mouse penis compared with WT penis. eNOS phosphorylated on Thr-495, total eNOS, HSP90, and caveolin-1 protein expressions in the penis were not affected by SCD.

Conclusions: These findings provide a molecular basis for chronically reduced eNOS function in the penis by SCD, which involves decreased eNOS phosphorylation on Ser-1177 and decreased eNOS-HSP90 interaction.

Figure 1

Effect of SCD on P-eNOS (Ser-1177) (A), P-eNOS (Thr-495) (B) and total eNOS (C) expressions in the penis of WT and sickle mice at baseline. Upper panels are representative Western immunoblots. Lower panels represent quantitative analyses of P-eNOS (Ser-1177), P-eNOS (Thr-495), and total eNOS in penes of WT and sickle mice. Each bar represents the mean ± SEM. *, P < 0.05. n= 6–8

Figure 2

Effect of SCD on P-AKT in the penis of WT and sickle mice at baseline. Upper panel is a representative Western immunoblot, and lower panel represents a quantitative analysis of P-AKT in penes of WT and sickle mice. Each bar represents the mean ± SEM. *, P < 0.05. n=8

Figure 3

Effect of SCD on HSP90 binding to eNOS (A) and total HSP90 protein expression (B) in the penis of WT and sickle mice at baseline. Upper panels are representative Western blots, and lower panels represent quantitative analyses of HSP90/eNOS (indicative of HSP90 binding to eNOS) and HSP90 protein expression in penes of WT and sickle mice. Each bar represents the mean ± SEM. *, P < 0.05. n=5

Figure 4

Effect of SCD on caveolin-1 binding to eNOS (A) and total caveolin-1 protein expression (B) in the penis of WT and sickle mice at baseline. Upper panels are representative Western blots, and lower panels represent quantitative analyses of caveolin-1/eNOS (indicative of caveolin-1 binding to eNOS) and caveolin-1 protein expression in penes of WT and sickle mice. Each bar represents the mean ± SEM. n=5–8

Figure 5

Model for altered eNOS regulatory mechanisms in the SCD mouse penis. eNOS phosphorylation on Ser-1177, an active portion of eNOS, is decreased in the SCD mouse penis. This effect is due to decreased eNOS interaction with its positive protein regulator HSP90 (but not increased interaction with caveolin-1), which decreases AKT activation and AKT-mediated eNOS phosphorylation on Ser-1177, and reduces constitutive NOS activity. Decreased eNOS interaction with HSP90 in the sickle mouse penis does not affect dephosphorylation of eNOS on Thr-495.