C-terminus of heat shock protein 70-interacting protein-dependent GTP cyclohydrolase I degradation in lambs with increased pulmonary blood flow.

We showed that nitric oxide (NO) signaling is decreased in the pulmonary vasculature before the development of endothelial dysfunction in a lamb model of congenital heart disease and increased pulmonary blood flow (Shunt). The elucidation of the molecular mechanism by which this occurs was the purpose of this study. Here, we demonstrate that concentrations of the endogenous NO synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA), are elevated, whereas the NOS cofactor tetrahydrobiopterin (BH(4)) is decreased in Shunt lambs. Our previous studies demonstrated that ADMA decreases heat shock protein-90 (Hsp90) chaperone activity, whereas other studies suggest that guanosine-5'-triphosphate cyclohydrolase 1 (GCH1), the rate-limiting enzyme in the generation of BH(4), may be a client protein for Hsp90. Thus, we determined whether increases in ADMA could alter GCH1 protein and activity. Our data demonstrate that ADMA decreased GCH1 protein, but not mRNA concentrations, in pulmonary arterial endothelial cells (PAECs) because of the ubiquitination and proteasome-dependent degradation of GCH1. We also found that Hsp90-GCH1 interactions were reduced, whereas the association of GCH1 with Hsp70 and the C-terminus of Hsp70-interacting protein (CHIP) increased in ADMA-exposed PAECs. The overexpression of CHIP potentiated, whereas a CHIP U-box domain mutant attenuated, ADMA-induced GCH1 degradation and reductions in cellular BH(4) concentrations. We also found in vivo that Hsp90/GCH1 interactions are decreased, whereas GCH1-Hsp70 and GCH1-CHIP interactions and GCH1 ubiquitination are increased. Finally, we found that supplementation with l-arginine restored Hsp90-GCH1 interactions and increased both BH(4) and NO(x) concentrations in Shunt lambs. In conclusion, increased concentrations of ADMA can indirectly alter NO signaling through decreased cellular BH(4) concentrations, secondary to the disruption of Hsp90-GCH1 interactions and the CHIP-dependent proteasomal degradation of GCH1.

GTP cyclohydrolase I expression is regulated by nitric oxide: role of cyclic AMP.

Our previous studies have demonstrated that nitric oxide (NO) leads to nitric oxide synthase (NOS) uncoupling and an increase in NOS-derived superoxide. However, the cause of this uncoupling has not been adequately resolved. The pteridine cofactor tetrahydrobiopterin (BH(4)) is a critical determinant of endothelial NOS (eNOS) activity and coupling, and GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme in its generation. Thus the initial purpose of this study was to determine whether decreases in BH(4) could underlie, at least in part, the NO-mediated uncoupling of eNOS we have observed both in vitro and in vivo. Initially we evaluated the effect of inhaled NO levels on GCH1 expression and BH(4) levels in the intact lamb. Contrary to our hypothesis, we found that there was a significant increase in both plasma BH4 levels and peripheral lung GCH1 protein levels. Furthermore, in vitro, we found that exposure to the NO donor spermine NONOate (SPNONO) led to an increase in GCH1 protein and BH(4) levels in both COS-7 and pulmonary arterial endothelial cells. However, SPNONO treatment also caused a significant increase in phospho-cAMP response element binding protein (CREB) levels, as detected by Western blot analysis, and significantly increased cAMP levels, as detected by enzyme immunoassay. Furthermore, utilizing GCH1 promoter fragments fused to a luciferase reporter gene, we found that GCH1 promoter activity was enhanced by SPNONO in a CREB-dependent manner, and electromobility shift assays revealed an NO-dependent increase in the nuclear binding of CREB. These data suggest that NO increases BH(4) levels through a cAMP/CREB-mediated increase in GCH1 transcription and that the eNOS uncoupling associated with exogenous NO does not involved reduced BH(4) levels.

Shear stress paradigm for perinatal fractal arterial network remodeling in lambs with pulmonary hypertension and increased pulmonary blood flow.

Congenital heart disease with increased blood flow commonly leads to the development of increased pulmonary vascular reactivity and pulmonary arterial hypertension by mechanisms that remain unclear. We hypothesized a shear stress paradigm of hemodynamic reactivity and network remodeling via the persistence and/or exacerbation of a fetal diameter bifurcation phenotype [parent diameter d(0) and daughters d(1) >or= d(2) with alpha < 2 in (d(1)/d(0))(alpha) + (d(2)/d(0))(alpha) and area ratio beta < 1 in beta = (d(1)(2)+ d(2)(2))/ d(0)(2)] that mechanically acts as a high resistance magnifier/shear stress amplifier to blood flow. Evidence of a hemodynamic influence on network remodeling was assessed with a lamb model of high-flow-induced secondary pulmonary hypertension in which an aortopulmonary graft was surgically placed in one twin in utero (Shunt twin) but not in the other (Control twin). Eight weeks after birth arterial casts were made of the left pulmonary arterial circulation. Bifurcation diameter measurements down to 0.010 mm in the Shunt and Control twins were then compared with those of an unoperated fetal cast. Network organization, cumulative resistance, and pressure/shear stress distributions were evaluated via a fractal model whose dimension D(0) approximately alpha delineates hemodynamic reactivity. Fetus and Control twin D(0) differed: fetus D(0)=1.72, a high-resistance/shear stress amplifying condition; control twin D(0) = 2.02, an area-preserving transport configuration. The Shunt twin (D(0)=1.72) maintained a fetal design but paradoxically remodeled diameter geometry to decrease cumulative resistance relative to the Control twin. Our results indicate that fetal/neonatal pulmonary hemodynamic reactivity remodels in response to shear stress, but the response to elevated blood flow and pulmonary hypertension involves the persistence and exacerbation of a fetal diameter bifurcation phenotype that facilitates endothelial dysfunction/injury.