Activation of protease calpain by oxidized and glycated LDL increases the degradation of endothelial nitric oxide synthase

Oxidation and glycation of low-density lipoprotein (LDL) promote vascular injury in diabetes; however, the mechanisms underlying this effect remain poorly defined. The present study was conducted to determine the effects of 'heavily oxidized' glycated LDL (HOG-LDL) on endothelial nitric oxide synthase (eNOS) function. Exposure of bovine aortic endothelial cells with HOG-LDL reduced eNOS protein levels in a concentration- and time-dependent manner, without altering eNOS mRNA levels. Reduced eNOS protein levels were accompanied by an increase in intracellular Ca(2+), augmented production of reactive oxygen species (ROS) and induction of Ca(2+)-dependent calpain activity. Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL. Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA). Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect. HOG-LDL may impair endothelial function by inducing calpain-mediated eNOS degradation in a ROS- and Ca(2+)-dependent manner.

Hyperoxia Depletes (6R)-5,6,7,8-Tetrahydrobiopterin Levels in the Neonatal Retina: Implications for Nitric Oxide Synthase Function in Retinopathy

Retinopathy of prematurity is a sight-threatening complication of premature birth caused by nitrooxidativeinsult to the developing retinal vasculature during therapeutic hyperoxia exposure and laterischemia-induced neovascularization on supplemental oxygen withdrawal. In the vasodegenerativephase, during hyperoxia, defective endothelial nitric oxide synthase (NOS) produces reactive oxygenand nitrogen free radicals rather than vasoprotective nitric oxide for unclear reasons. More important,NOS critically depends on the availability of the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4).Because BH4 synthesis is controlled enzymatically by GTP cyclohydrolase (GTPCH), we used GTPCHdepletedmice [hyperphenylalanaemia strain Q4 (hph1)] to investigate the impact of hyperoxia on BH4bioavailability and retinal vascular pathology in the neonate. Hyperoxia decreased BH4 in retinas,lungs, and aortas in all experimental groups, resulting in a dose-dependent decrease in NOS activityand, in the wild-type group, elevated NOS-derived superoxide. Retinal dopamine levels were similarlydiminished, consistent with the dependence of tyrosine hydroxylase on BH4. Despite greater depletionof BH4, the hphþ/ and hph1/ groups did not show exacerbated hyperoxia-induced vessel closure,but exhibited greater vascular protection and reduced progression to neovascular disease. This vasoprotectiveeffect was independent of enhanced circulating vascular endothelial growth factor (VEGF),which was reduced by hyperoxia, but Q5 to local ganglion cell layerederived VEGF. A constitutively higherlevel of VEGF expression associated with retinal development protects GTPCH-deficient neonates fromoxygen-induced vascular damage.

Specific role of neuronal nitric-oxide synthase when tethered to the plasma membrane calcium pump in regulating the beta-adrenergic signal in the myocardium

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates beta-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser(16) phospholamban (PLB) phosphorylation as well as Ser(22) and Ser(23) cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the beta-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.

Upregulation of adrenomedullin and its receptor components during cardiomyocyte hypertrophy induced by chronic inhibition of nitric oxide synthesis in rats

Adrenomedullin may provide a compensatory mechanism to attenuate left ventricular hypertrophy (LVH). Nitric oxide synthase inhibition, induced by chronic administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) to rats, induces cardiac hypertrophy in some, but not all cases; there are few reports of direct assessment of cardiomyocyte parameters. The objective was to characterize hypertrophic parameters in left (LV) and right ventricular (RV) cardiomyocytes after administration of L-NAME to rats for 8 wk and to determine whether adrenomedullin and its receptor components were upregulated. After treatment with L-NAME (20 and 50 mg x kg(-1) x day(-1)), compared with nontreated animals, 1) systolic blood pressure increased (by 34.2 and 104.9 mmHg), 2) heart weight-to-body wt ratio increased 24.1% at the higher dose (P