The effect of chronic hypoxia-induced pulmonary hypertension on nitric oxide synthase (NOS) in the lung is controversial. To clarify the regulation of endothelial and inducible NOS (eNOS and iNOS) expression in the chronically hypoxic lung, Northern and Western blot analyses were performed on mRNA and total protein from lungs of rats exposed to 3 wk of hypoxia (10% O2, normobaric) or normoxia. Expression of the mRNA and protein for eNOS was significantly increased (1.6-fold and 2.1-fold, respectively) by hypoxia. Immunohistochemistry with an isoform-specific antibody demonstrated de novo expression of eNOS in the endothelium of resistance vessels in the pulmonary vasculature of the hypoxic rats. eNOS was detected in the endothelium of large vessels in both normoxic and hypoxic rat lungs. The level of mRNA and protein for iNOS was also found to be significantly increased (1.9-fold and 1.4-fold, respectively). In addition to the 4.4-kilobase (kb) iNOS mRNA species, a novel 4.0-kb species was also induced by hypoxia. We conclude that expression of eNOS and iNOS was increased in the lungs of rats subjected to chronic hypoxia, and that there was de novo expression of eNOS protein in the microvascular endothelium.

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Alterations in nitric oxide signaling have been hypothesized to have an etiologic role in the development of hypoxic pulmonary hypertension. However, changes in the expression of nitric oxide synthase (NOS) in hypoxic lungs remains controversial. In this study, we used (1) Northern and Western analyses to measure NOS mRNA and protein expressions, (2) lung histology together with measurements of lung and heart weights to monitor pulmonary vascular remodeling, and (3) immunohistochemistry to localize NOS proteins. The data demonstrated that endothelial NOS mRNA and protein were upregulated over 1 to 7 days of hypoxia that temporally correlated with and preceded the vascular remodeling that occurred in the course of the development of hypoxic pulmonary hypertension. Hypoxia also induced brain NOS in bronchial epithelium and inducible NOS in vascular smooth muscle but did not affect inducible NOS expression in macrophages or basal guanylyl cyclase activity in the lung. These findings showed that upregulation of endothelial NOS was tightly correlated with the vascular remodeling induced by hypoxia, suggesting a role for nitric oxide in the development of pulmonary hypertension.

Expression and localization of nitric oxide synthase (NOS) in the lungs of chronically hypoxic and normoxic rats were studied using both immunohistochemistry and NADPH diaphorase (NADPH-d) staining techniques. In the normoxic and in the hypoxic rat, NOS was detected by both methods in the endothelium of large pulmonary vessels and in the epithelium of bronchi and bronchioli. NOS expression was not detected in the endothelium of normoxic pulmonary resistance vessels but was prominently expressed in the endothelium of these vessels after 2-4 wk of chronic hypoxia. In contrast to small pulmonary vessels, the endothelium of small bronchial vessels exhibited NOS immunostaining in both normoxic and hypoxic lungs. Hypoxia was also found to induce de novo NOS expression in the smooth muscle of large and small pulmonary vessels and in bronchial smooth muscle. NOS enzyme activity in lung homogenates was assessed by [3H]arginine to [3H]citrulline conversion. The activity of soluble NOS, but not particulate NOS, was increased in the hypoxic lungs. These results demonstrate chronic hypoxia-induced upregulation of NOS protein expression and activity in the rat lung, suggesting a potentially important role of nitric oxide in adaptation of the pulmonary circulation to chronic hypoxia. The lack of immunostaining in small pulmonary resistance vessels is also consistent with physiological studies suggesting that NO may not be involved in the mechanism for maintaining the normally low pulmonary vascular resistance.

Abstract-CYP11B2 is the enzyme responsible for aldosterone synthesis mainly in the adrenal gland. In this study, we hypothesized that CYP11B2 gene, protein, and aldosterone are produced locally in kidney and regulated by low salt intake, angiotensin II type 1 (AT 1 ) receptor and insulin-deficient diabetes hyperglycemia. We used real-time RT-PCR, immunohistochemistry staining, and microdialysis techniques to monitor changes in renal CYP11B2 mRNA and protein and aldosterone production in normal, adrenalectomized, or streptozotocin-induced insulin-deficient diabetic hyperglycemic rats. In normal kidney, CYP11B2 mRNA and protein were localized mainly in the renal cortex and upregulated by angiotensin II and low salt intake. The key enzyme for aldosterone synthesis is CYP11B2, a member of cytochrome P450 family with molecular weight 48.5 kDa. The CYP11B2 gene is Ϸ45 kb, contains 9 exons and is located on chromosome 8. 5 Aldosterone stimulates cellular hypertrophy, matrix formation, 6,7 and cell death. 8 Although aldosterone can be produced in tissues other than the adrenal gland, 9 its production in the kidney has never been reported. The demonstrated benefits of aldosterone receptor blockers in diabetic nephropathy, 6,10 -12 despite reported normal [13][14][15] or low 16 -19 levels of plasma aldosterone, suggest that this hormone may be produced locally within the kidney. Thus, we hypothesized that there is a local renal aldosterone system. Identification of different components of an aldosterone system in the kidney would boost this hypothesis. In this study, we confirmed that CYP11B2 gene, protein, and aldosterone production are locally present in kidney and regulated by low salt intake, Ang II type 1 (AT 1 ) receptor, and insulin-deficient diabetes hyperglycemia. Methods Animal PreparationStudy protocols were approved by the University of Virginia animal research committee. Sprague-Dawley rats (Harlan Teklad; Madison, Wis) weighing 245 to 255 g were used in this study. Rats were housed in a well-ventilated room (21Ϯ1°C; 12-hour light/dark cycle). Animals were weighed at the end of each study week. Adrenalectomy and Microdialysis ProceduresBilateral adrenalectomy was performed according to previously published methods. 20 A single microdialysis probe was inserted into left renal cortex as described previously. 21 Each animal in the control group was instrumented by microdialysis probe but did not have adrenalectomy (sham). After adrenalectomy, rats received dexamethasone (Sigma; 250 mg/kg per day SC). On the fifth day after surgery, renal interstitial fluid (RIF) samples 21 were collected from normal (nϭ6) and adrenalectomized (nϭ6) rats. Salt Intake and Renal Aldosterone Synthase ExpressionThe effects of different levels of salt intake on the renal expression of aldosterone synthase were studied by placing animals for 1 week on low-salt (0.05% NaCl), normal-salt (0.5% NaCl), or high-salt (4% NaCl) diet (nϭ8 for each group).

A highly conductive, ultralight, neat and versatile nitrogen-doped GNRs aerogel has been fabricated by a new hydrothermal method for the first time. The newly developed aerogel shows a very promising performance when used as a novel ORR catalyst in both alkaline and acidic solutions.

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The KAT5 (Tip60/Esa1) histone acetyltransferase is part of NuA4, a large multifunctional complex highly conserved from yeast to mammals that targets lysines on H4 and H2A (X/Z) tails for acetylation. It is essential for cell viability, being a key regulator of gene expression, cell proliferation, and stem cell renewal and an important factor for genome stability. The NuA4 complex is directly recruited near DNA double-strand breaks (DSBs) to facilitate repair, in part through local chromatin modification and interplay with 53BP1 during the DNA damage response. While NuA4 is detected early after appearance of the lesion, its precise mechanism of recruitment remains to be defined. Here, we report a stepwise recruitment of yeast NuA4 to DSBs first by a DNA damage-induced phosphorylation-dependent interaction with the Xrs2 subunit of the Mre11-Rad50-Xrs2 (MRX) complex bound to DNA ends. This is followed by a DNA resection-dependent spreading of NuA4 on each side of the break along with the ssDNA-binding replication protein A (RPA). Finally, we show that NuA4 can acetylate RPA and regulate the dynamics of its binding to DNA, hence targeting locally both histone and nonhistone proteins for lysine acetylation to coordinate repair.