Chronic Hypoxia Increases Inducible NOS-Derived Nitric Oxide in Fetal Guinea Pig Hearts

Abstract

Intrauterine hypoxia impacts fetal growth and organ function. Inducible nitric oxide synthase (iNOS) and neuronal NOS (nNOS) expression was measured to assess the response of fetal hearts to hypoxic (HPX) stress. Pregnant guinea pigs were housed in a hypoxic chamber (10.5% O 2 for 14 d, n ϭ 17) or room air [normoxic (NMX), n ϭ 17͔. Hearts of anesthetized near-term fetuses were removed. mRNA ͓hypoxia-inducible factor, (HIF)-1␣, 1␤, 2␣, 3␣, iNOS, and nNOS͔ and protein levels (HIF-1␣, iNOS, and nNOS) of fetal cardiac left ventricles were quantified by real time polymerase chain reaction (PCR) and Western analysis, respectively. Cardiac nitrite/nitrate levels were measured in the presence/absence of L-N 6 -(1-iminoethyl)-lysine (L-NIL), an iNOS inhibitor, administered to pregnant sows. Hypoxia significantly increased fetal cardiac HIF-1␣ and -2␣ mRNA, HIF-1␣ protein but not HIF-3␣ or -1␤ mRNA levels. Hypoxia increased both iNOS mRNA (by 5ϫ) and protein (by 23%) levels but had no effect on nNOS levels. Nitrite/nitrate levels were increased in HPX hearts by 2.5ϫ and decreased with L-NIL by 67 Ϯ 14%. Thus, up-regulation of iNOS-derived nitric oxide (NO) generation is an important mechanism by which fetal hearts respond to chronic hypoxic stress. T he adaptive response of the fetal heart to intrauterine stress is critical for its survival. Several studies using high altitude (1,2) exposure to acute and chronic hypoxia (2,3), and anemia (4) have demonstrated how the fetal cardiovascular system responds to hypoxic (HPX) stress. Depending on the severity and duration of the HPX conditions, as well as the gestational age of the fetus, cardiac adaptations have been associated with altered coronary blood flow (2,5,6), increased heart size (1), and decreased contractile performance (7). The underlying mechanisms mediating these changes in fetal heart morphology and function are not fully understood.Hypoxia is a potent stimulus for gene activation of several genes (8), including nitric oxide synthase (NOS), the synthetic enzyme that generates nitric oxide (NO) from L-arginine oxidation (9). NO is derived from three isoforms of NOS ͓endothelial NOS (eNOS); neuronal NOS (nNOS); and inducible NOS (iNOS)͔, all of which are expressed in the heart (10,11). Specifically, eNOS is expressed constitutively in both endothelial cells and cardiomyocytes (10), nNOS in both cardiomyocytes and conducting tissue of cardiac ventricles (12), and iNOS in cardiomyocytes (12) and in mature hearts under conditions of hypoxia (12-14), heart failure (15), left ventricular hypertrophy (16) and cardiac cyanosis in children (17). NOS gene expression has been reported to be oxygensensitive, with levels varying among cardiac cells (18,19) and endothelial cells of differing vascular origin (20 -22). For example, hypoxia increases eNOS expression in porcine coronary artery endothelial cells (20), newborn (23-25) and adult rabbit heart ventricles (26), and adult guinea pig ventricles (27), and decreases expression in pulmonary artery endothelial cell...

Metabolic fates and effects of nitrite in brown trout under normoxic and hypoxic conditions: blood and tissue nitrite metabolism and interactions with branchialNOS,Na+/K+-ATPaseandhsp70expression

“…Indeed, we observed an increased expression of NOS mRNA in the gills of hypoxic brown trout (Fig. 4), which may also have occurred in other tissues, and increased NOS mRNA expression is typically translated into increased protein levels (LeCras et al, 1996;McNeill and Perry, 2006;Thompson et al, 2009).…”

“…This bears resemblance to increased nNOS mRNA and protein expression in the brain and posterior cardinal vein of hypoxic rainbow trout (McNeill and Perry, 2006), increased mRNA and protein expression of iNOS and eNOS in hypoxic rat lungs (LeCras et al, 1996) and an increased iNOS and unchanged nNOS expression in hypoxic fetal guinea pig hearts (Thompson et al, 2009). An upregulation of iNOS expression in hypoxia may be mediated by hypoxia inducible factor-1, but other possibilities also exist (Thompson et al, 2009;Robinson et al, 2011). The activity (NO production) of iNOS enzymes is mainly regulated at the transcriptional level (Aktan, 2004).…”

Chronic hypoxia increases peroxynitrite, MMP9 expression, and collagen accumulation in fetal guinea pig hearts

“…To test whether chronic hypoxia induces peroxynitrite formation through an iNOS-mediated pathway, both NMX and HPX animals were treated with LNIL, a selective iNOS inhibitor (1,12,25), administered in the drinking water (1-2 mg/kg/d) for 10 d, 4 d after being placed in the HPX chamber (1). In a separate group, pregnant sows were treated with NAC (500 mg/kg/d) administered in the drinking water using the same protocol for LNIL.…”

“…Protein levels of 3-NT from isolated fetal cardiomyocytes of hearts from NMX and HPX guinea pigs treated with and without LNIL were quantified using western blot analysis (1). Frozen isolated fetal cardiomyocyte preparations were homogenized in 1× lysis buffer (cat.…”

Physiological Neuroprotective Mechanisms in Natural Genetic Systems: Therapeutic Clues for Hypoxia-Induced Brain Injuries

“…The maintenance of normal brain functions require many parameters to work together and contribute to the homeostasis of brain energy demand and supply. Such a combination of parameters that could, in turn, lead to hypoxia protection include regulation of levels of inducible nitric oxide synthase (Thompson and Dong 2005;Thompson et al 2009)and expression of HIF-1a (Trollmann and Gassmann 2009). Other parameters include the activation of extracellular-signal-regulated kinase (Osorio-Fuentealba et al 2009;Wilkerson and Mitchell 2009)and c-Jun N-terminal kinase/stress-activated protein kinases (Comerford et al 2004).…”