Pulmonary hypertension (PH) is an unremitting disease defined by a progressive increase in pulmonary vascular resistance leading to right-sided heart failure. Using mice with genetic deletions of caveolin 1 (Cav1) and eNOS (Nos3), we demonstrate here that chronic eNOS activation secondary to loss of caveolin-1 can lead to PH. Consistent with a role for eNOS in the pathogenesis of PH, the pulmonary vascular remodeling and PH phenotype of Cav1 -/-mice were absent in Cav1 -/-Nos3 -/-mice. Further, treatment of Cav1 -/-mice with either MnTMPyP (a superoxide scavenger) or l-NAME (a NOS inhibitor) reversed their pulmonary vascular pathology and PH phenotype. Activation of eNOS in Cav1 -/-lungs led to the impairment of PKG activity through tyrosine nitration. Moreover, the PH phenotype in Cav1 -/-lungs could be rescued by overexpression of PKG-1. The clinical relevance of the data was indicated by the observation that lung tissue from patients with idiopathic pulmonary arterial hypertension demonstrated increased eNOS activation and PKG nitration and reduced caveolin-1 expression. Together, these data show that loss of caveolin-1 leads to hyperactive eNOS and subsequent tyrosine nitration-dependent impairment of PKG activity, which results in PH. Thus, targeting of PKG nitration represents a potential novel therapeutic strategy for the treatment of PH.
These findings confirm the inhibitory role of n-LDL and ox-LDL on NO generation and suggest that lipoproteins may induce a decreased uptake of L-arginine. The local depletion of the L-arginine substrate may derange the NO synthase, leading to overproduction of O(2)(-) from oxygen, the other substrate of NO synthase.
Abstract-Caveolin-1, the caveolae scaffolding protein, binds to and negatively regulates eNOS activity. As caveolin-1 also regulates caveolae-mediated endocytosis after activation of the 60-kDa albumin-binding glycoprotein gp60 in endothelial cells, we addressed the possibility that endothelial NO synthase (eNOS)-dependent NO production was functionally coupled to caveolae internalization. We observed that gp60-induced activation of endocytosis increased NO production within 2 minutes and up to 20 minutes. NOS inhibitor N G -nitro-L-arginine (L-NNA) prevented the NO production. To determine the role of caveolae internalization in the mechanism of NO production, we expressed dominant-negative dynamin-2 mutant (K44A) or treated cells with methyl--cyclodextrin. Both interventions inhibited caveolae-mediated endocytosis and NO generation induced by gp60. We determined the role of signaling via Src kinase in the observed coupling of endocytosis to eNOS activation. Src activation induced the phosphorylation of caveolin-1, Akt and eNOS, and promoted dissociation of eNOS from caveolin-1. Inhibitors of Src kinase and Akt also prevented NO production. In isolated perfused mouse lungs, gp60 activation induced NO-dependent vasodilation, whereas the response was attenuated in eNOS Ϫ/Ϫ or caveolin-1 Ϫ/Ϫ lungs. Together, these results demonstrate a critical role of caveolae-mediated endocytosis in regulating eNOS activation in endothelial cells and thereby the NO-dependent vasomotor tone. Key Words: caveolin-1 Ⅲ endocytosis Ⅲ vasomotor tone Ⅲ albumin Ⅲ transcytosis E ndothelial NO synthase (eNOS) is modified by N-myristoylation and palmitoylation, which targets the enzyme to caveolae, 1 the plasma membrane cholesterol-rich microdomains. 2,3 Multiple mechanisms are involved in regulating NO production following eNOS activation. eNOS activity is regulated by Ca 2ϩ -calmodulin, phosphorylation activated by kinases such as Src and interactions with caveolin-1, dynamin, and heat shock protein 90 (HSP90). 4 The effects of phosphorylation are complex. Phosphorylation of Ser116 and Thr497 negatively regulates eNOS activity, whereas phosphorylation at Ser635 and Ser1179 has the opposite effect. 5-7 Src kinase activated eNOS by inducing phosphorylation at Tyr83. 8 Phosphorylation at Ser617 functions by affecting the phosphorylation of the above residues. 9 Insulin, estrogen, and shear stress were shown to induce phosphorylationdependent activation of eNOS at Ser1179 independent of increased intracellular [Ca 2ϩ ] 10 -12 eNOS in caveolae is held inactive by its association with caveolin-1, 13 but eNOS activity can be increased by Ca 2ϩ /calmodulin 3 and binding to HSP90 and dynamin-2. 14,15 HSP90 facilitates the phosphorylation of eNOS by forming a ternary complex with eNOS and Akt. 16 Dynamin-2 regulates eNOS activity through the binding of its proline-rich domain to the FAD domain of eNOS, promoting electron transfer between the bound flavins of the reductase domain and increasing NO production. 15 We have shown that activation of t...
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