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.

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).

Considerable evidence has recently been presented that suggests that nitric oxide (NO) is a nonadrenergic noncholinergic (NANC) neurotransmitter in gastrointestinal tissues. One of the criteria that must be satisfied before this hypothesis can be accepted is that enteric neurons must be shown to contain the enzymatic apparatus necessary to synthesize NO. Specific antibodies have been developed for NO synthase (NOS) isolated from rat cerebellum, and studies have shown that NOS copurifies and colocalizes with NADPH diaphorase activity, a commonly used neural marker. We used antibodies raised against the cerebellar NOS to determine the distribution of NOS-like immunoreactivity (NOS-LI) in enteric neurons of the canine proximal colon. We also tested whether NADPH diaphorase staining would label the population of neurons containing NOS-LI in this species. A subpopulation of neurons in myenteric and submucosal ganglia displayed NOS-LI and were colabeled with NADPH diaphorase. Labeled neurons had morphological characteristics similar to the Dogiel type I morphology. Cryostat sections showed NOS-positive nerve trunks throughout the circular and longitudinal muscle layers, but a high density of NOS-LI was observed within the submucosal pacemaker region, as predicted from physiological studies. These studies provide the first morphological support for the hypothesis that NO serves as a NANC neurotransmitter in the canine colon. The study also shows that the NADPH diaphorase reaction provides a useful method to label cells with NOS-LI.

No abstract

No abstract

To better understand the role of nitric oxide (NO) in fetal lung development, specifically in the transition of the fetal circulation at birth, we studied the timing of cell-specific expression of NO synthase (NOS) isoforms from formation of lung buds (13th day of gestation) to 7 days postnatal. Expression of NOS was studied using immunohistochemical labeling with antibodies against the three known NOS isoforms and the NADPH diaphorase technique (NADPH-d). Endothelial NOS (eNOS) immunoreactivity was found in the cells of the 14-day fetal lung. As gestation proceeded, the quantity of these immunopositive cells increased, and they coalesced to form an inner (endothelial) layer of pulmonary vessels. This process of angiogenesis marked by eNOS-positive cells was seen from 15 days of gestation to at least 7 days postnatal. A majority of the eNOS immunoreactivity appeared densely in one focal spot in the cytoplasm, indicating that during development the eNOS may be primarily located in a cytoplasmic organelle. Epithelial cells of the rat airway from the same developmental period were positively stained with both brain NOS antibody (bNOS) and NADPH-d at the beginning of 13 days of gestation. Then the intensity of stainings began to decrease and reached the lowest level in the 16-day fetal lung. However, the NOS stainings of the epithelium, especially in small canalicular structures of the airways, began to increase at 18 days of gestation and was dramatically elevated at 20 days of gestation (term is 22 days). Postnatally, NOS in epithelium was decreased in distal airways in conjunction with the formation of alveolar structure. Inducible NOS (iNOS) immunoreactivity was also found in the epithelium of rat lung airways after 16 days of gestation. Unlike the bNOS staining, iNOS immunoreactivity exhibited a pattern of a small dot-like staining within epithelial cytoplasm during gestation and the first day postnatal, then changed to a pattern of diffuse cytoplasmic staining by the 7th postnatal day. This study concludes that 1) expression of three isoforms of NOS is present and regulated during lung development; 2) markedly increased NOS in epithelium near term supports a role for NO in mediating the pulmonary transition from fetal to neonatal life; and 3) eNOS immunohistochemistry serves as an effective marker to follow the process of pulmonary angiogenesis and suggests the concept of in situ formation of endothelial vesicles in developing mesenchyme.

Recently, we reported the presence of a local renal aldosterone production. In the present study, we tested the hypothesis that local aldosterone production in the kidney contributes to renal inflammation, matrix formation and albuminuria associated with diabetes. We evaluated changes in renal aldosterone content (RAC), aldosterone synthase expression, nuclear factor κB (NFκB), tumour necrosis factor α (TNFα), interleukin-6 (IL-6), transforming growth factor β (TGFβ), glomerular fibronectin, collagen type IV and urinary albumin extraction (UAE) in response to the aldosterone synthase inhibitor FAD286. Studies were conducted in adrenalectomized, normoglycaemic (control) or diabetic rats for 14 weeks. The FAD286 was administered during the last 10 weeks of the study. Plasma aldosterone levels were not detectable in any of the study groups. Compared with control rats, diabetic rats had higher levels of RAC by 488% (P < 0.01), NFκB by 293% (P < 0.01), TNFα by 356% (P < 0.01), IL-6 by 378% (P < 0.01), TGFβ by 337% (P < 0.01) and UAE by 1122% (P < 0.01), and increased glomerular fibronectin and collagen type IV immunostaining. In diabetic rats, FAD286 reduced RAC (P < 0.01), UAE (P < 0.05), NFκB mRNA, TNFα mRNA, IL-6 mRNA and TGFβ mRNA by 51, 41, 41 and 52% and also their proteins and decreased glomerular fibronectin and collagen type IV immunostaining. In conclusion, diabetes increases local aldosterone production in the kidney, which contributes to development of renal inflammation, matrix formation and albuminuria. Inhibition of aldosterone production in the kidney could be helpful in management of diabetic nephropathy.

Abstract-Renin is regulated by angiotensin subtype 1 (AT 1 ) receptor, but it is unknown whether angiotensin subtype 2 (AT 2 ) receptor contributes to this regulation. We hypothesized that AT 2 receptors inhibit angiotensin II (Ang II) through inhibition of renin biosynthesis. We monitored changes in renal Ang II, renin mRNA and protein expression, and plasma renin concentration (PRC) in response to renal cortical administration of the AT 1 receptor blocker valsartan or the AT 2 receptor blocker PD 123319 (PD) in conscious rats administered low sodium intake (LS (Ang II) is the major effector hormone of the renin-angiotensin system (RAS). Most of the known physiological functions and pathologic effects associated with Ang II are mediated by the angiotensin subtype 1 (AT 1 ) receptor, including renin inhibition. 1 In contrast, the functions of the angiotensin subtype 2 (AT 2 ) receptor are still being elucidated. The AT 2 receptor is expressed abundantly during fetal development and declines after birth. [1][2][3] In mammalian adults, the AT 2 receptor had been reported in multiple organs, including the kidney. 4 -6 Currently, the AT 2 receptor has several described functions related to inhibition of cell growth, promotion of cell differentiation, and stimulation of apoptosis. [7][8][9][10][11][12] Previously, we demonstrated that during sodium depletion, the AT 2 receptor mediates renal production of bradykinin, nitric oxide (NO), and cGMP. [13][14][15][16][17] Unlike the well-known effect of AT 1 receptor on renin production, 18 -19 the influence of the AT 2 receptor on renal renin biosynthesis and Ang II production is not known.In this study, we tested the hypothesis that the AT 2 receptor inhibits renal renin synthesis and Ang II production. We used multiple techniques including renal interstitial microdialysis, [13][14][15][16][17] real-time quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and enzymelinked immunoassays for monitoring changes in renal Ang II, renin mRNA, renin protein expression, and plasma renin concentration to investigate whether intrarenal AT 2 receptors regulate renin production and Ang II formation. We monitored renal interstitial levels of Ang II in conscious rats during sodium restriction, a condition known to increase AT 2 receptor expression, and during AT 1 and AT 2 receptor blockade. 5 A distinct advantage of the microdialysis technique 13 is the ability to monitor renal Ang II levels in conscious rats without undesirable hemodynamic changes. In this study, we demonstrate for the first time to our knowledge that AT 2 receptors regulate the activity of the RAS through inhibition of renin biosynthesis in young rats. Methods Renal Microdialysis TechniqueFor the determination of renal interstitial fluid (RIF) Ang II, we constructed a microdialysis probe as previously described. [13][14][15][16][17] In vitro, best recovery for Ang II was observed with a perfusion rate of 3 L/min, and was Ϸ53%. Animal PreparationThe experiments were approved by the U...