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Hypoxic modulation of nitric oxide (NO) production pathways in the cutaneous microvasculature and its interaction with cold-induced reflex vasoconstriction, independent of local cooling, have yet to be identified. This study assessed the contribution of NO to nonglabrous microvasculature perfusion during hypoxia and whole body cooling with concomitant inhibition of NO synthase [NOS; via N G-nitro-L-arginine methyl ester (L-NAME)] and the nitrite reductase, xanthine oxidase (via allopurinol), two primary sources of NO production. Thirteen volunteers were exposed to independent and combined cooling via water-perfused suit (5°C) and normobaric hypoxia (FI O2, 0.109 + 0.002). Cutaneous vascular conductance (CVC) was assessed across four sites with intradermal microdialysis perfusion of 1) lactated Ringers solution (control), 2) 20 mmol L-NAME, 3) 10 mol allopurinol, or 4) combined L-NAME/allopurinol. Effects and interactions were assessed via four-way repeated measures ANOVA. Independently, L-NAME reduced CVC (43%, P < 0.001), whereas allopurinol did not alter CVC (P = 0.5). Cooling decreased CVC (P = 0.001), and the reduction in CVC was consistent across perfusates (~30%, P = 0.9). Hypoxia increased CVC (16%, P = 0.01), with this effect abolished by L-NAME infusion (P = 0.04). Cold-induced vasoconstriction was blunted by hypoxia, but importantly, hypoxia increased CVC to a similar extent (39% at the Ringer site) irrespective of environmental temperature; thus, no interaction was observed between cold and hypoxia (P = 0.1). L-NAME restored vasoconstriction during combined cold-hypoxia (P = 0.01). This investigation suggests that reflex cold-induced cutaneous vasoconstriction acts independently of NO suppression, whereas hypoxia-induced cutaneous vasodilatation is dependent on NOS-derived NO production.

Background Myocardial perfusion during adenosine-induced hyperemia is used both in clinical diagnosis of coronary heart disease and for scientific investigations of the myocardial microcirculation. The objective of this study was to clarify whether adenosine-induced hyperemia is dependent on endothelial NO production or is influenced by adrenergic mechanisms. Methods and Results In 12 healthy men, myocardial perfusion was measured with PET in 2 protocols performed in random order, each including 3 perfusion measurements. First, perfusion was measured at rest. Second, either saline or the NO synthase inhibitor N G -nitro- l -arginine methyl ester (L-NAME, 4 mg/kg) was infused, and perfusion during adenosine-induced hyperemia was determined. Last, in both protocols, the α-receptor blocker phentolamine was infused, and perfusion during adenosine-induced hyperemia was determined again. Resting perfusion was similar in the 2 protocols (0.69±0.14 and 0.66±0.18 mL · min −1 · g −1 ). L-NAME increased mean arterial blood pressure by 12±7 mm Hg ( P P −1 · g −1 ) was attenuated by L-NAME (1.50±0.55 mL · min −1 · g −1 , P −1 · g −1 , P =NS). In the presence of L-NAME, however, when the adenosine response was attenuated, phentolamine was able to increase hyperemic perfusion (2.05±0.44 mL · min −1 · g −1 , P Conclusions Inhibition of endogenous NO synthesis attenuates myocardial perfusion during adenosine-induced hyperemia, indicating that coronary vasodilation by adenosine is partly endothelium dependent. α-Adrenergic blockade has no effect on adenosine-induced hyperemia unless NO synthesis is inhibited.

Aim: To investigate the role of two separate enzymatic pathways [soluble (sGC) vs. particulate (pGC) guanylyl cyclase] in the synthesis of cyclic GMP (cGMP) in cultured human retinal pigment epithelial (RPE) cells. Methods: cGMP accumulation was evaluated by quantitative analysis of cGMP immunoreactivity. RPE cells were also stained for inducible nitric oxide synthase (iNOS), ANP and β1- and α2-subunits of sGC. Results: We showed nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and iNOS immunoreactivity in RPE cells. Incubation of the cells in the presence of 1 mM IBMX to inhibit phosphodiesterase activity, and the simultaneous inhibition of NOS activity with L-NAME suggested the involvement of sGC in maintaining a low level of cGMP in the RPE cells. The involvement of sGC was further supported by detection of the β1- and α2-subunits of sGC. Incubation of the cells in the presence of atrial natriuretic peptide (ANP) to stimulate pGC strongly increased cGMP immunoreactivity. We also demonstrated the presence of ANP in all RPE cells. Conclusion: Cultured human RPE cells are capable of producing cGMP after stimulation of sGC or pGC. The presence of iNOS and ANP in all cells suggests two different autocrine pathways of stimulating cGMP production in these cells. The possible role of cGMP in the regulation iNOS gene expression and in the regulation of ANP is discussed.

Type 2 diabetic patients are characterized by a reduced adenosine-induced hyperemic myocardial perfusion, which may contribute to their increased cardiovascular morbidity. We hypothesized that the reduced hyperemia can be explained by functional changes in endothelial or autonomic nervous regulation. In 12 type 2 diabetic patients without signs of ischemic heart disease and 14 age-matched control subjects, myocardial perfusion was measured at rest, during adenosine, and during adenosine and α-receptor blockade (phentolamine) using positron emission tomography on two separate days: 1) with, and 2) without nitric oxide (NO) inhibition with NG-nitro-L-arginine methyl ester. Myocardial perfusion during adenosine was lower in type 2 diabetic patients compared with control subjects (P = 0.05). No significant effect of NO inhibition on myocardial perfusion during adenosine was found in any of the groups. In control subjects, α-receptor blockade increased hyperemic myocardial vascular resistance during NO inhibition, whereas no effect was observed in type 2 diabetic patients. At rest, a significant correlation was observed between rate-pressure product and myocardial perfusion in control subjects. NO inhibition and type 2 diabetes abolished this correlation. Endothelial and cardiac autonomic nerve function seems to play only a minimal role in the reduced hyperemic myocardial perfusion in type 2 diabetic patients. However, the linear correlation between resting perfusion and cardiac work appears to be abolished in type 2 diabetes and during NO synthase inhibition.

Previously, we showed that neonatal induced chronic partial unilateral ureteral obstruction (PUUO) of the multipapillary pig kidney decreased glomerular filtration rate (GFR) of the obstructed kidney. We hypothesized that ANG II and nitric oxide (NO) are important for the changes in renal function and in the present study we examined the effects of chronic AT1 receptor blockade using CV-11974 (0.12 mg/h candesartan from age 23 to 30 days) on kidney function development after PUUO was induced in 2-day-old piglets. Moreover, the effect of superimposed acute NO inhibition using NG-nitro-l-arginine methyl ester (l-NAME; 15 mg/kg) was examined to identify if this has diagnostic potential. PUUO significantly increased GFR in the nonobstructed contralateral kidney independent of candesartan. In candesartan-treated piglets, the l-NAME-induced GFR reduction seen in normal and nonobstructed kidneys was absent in the partial obstructed kidneys. Urine output and fractional excretion of water were increased from the partial obstructed kidneys. Consistent with this immunohistochemical analyses showed a reduced aquaporin-2 labeling in the collecting duct principal cells. Moreover, renal sodium handling was compromised by PUUO evidenced by an increased fractional excretion of sodium which was enhanced by candesartan treatment. In conclusion, our findings suggest that the counterbalance between AT1 receptor-mediated vasoconstriction and NO-mediated vasodilatation which maintain GFR in normal young porcine kidneys is changed by neonatal induced chronic PUUO. This may have diagnostic potential in children with suspected congenital obstruction. Our results also demonstrate compromised tubular functions in response to chronic PUUO despite preservation of glomerular function.

Whereas previous studies have shown that opening of the mitochondrial ATP-sensitive K+ (mitoKATP) channel protects the adult heart against ischemia-reperfusion injury, it remains to be established whether this mechanism also operates in the developing heart. Isolated spontaneously beating hearts from 4-day-old chick embryos were subjected to 30 min of anoxia followed by 60 min of reoxygenation. The chrono-, dromo-, and inotropic disturbances, as well as alterations of the electromechanical delay (EMD), reflecting excitation-contraction (E-C) coupling, were investigated. Production of reactive oxygen species (ROS) in the ventricle was determined using the intracellular fluorescent probe 2′,7′-dichlorofluorescin (DCFH). Effects of the specific mitoKATP channel opener diazoxide (Diazo, 50 μM) or the blocker 5-hydroxydecanoate (5-HD, 500 μM), the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME, 50 μM), the antioxidant N-(2-mercaptopropionyl)glycine (MPG, 1 mM), and the PKC inhibitor chelerythrine (Chel, 5 μM) on oxidative stress and postanoxic functional recovery were determined. Under normoxia, the baseline parameters were not altered by any of these pharmacological agents, alone or in combination. During the first 20 min of postanoxic reoxygenation, Diazo doubled the peak of ROS production and, interestingly, accelerated recovery of ventricular EMD and the PR interval. Diazo-induced ROS production was suppressed by 5-HD, MPG, or l-NAME, but not by Chel. Protection of ventricular EMD by Diazo was abolished by 5-HD, MPG, l-NAME, or Chel, whereas protection of the PR interval was abolished by l-NAME exclusively. Thus pharmacological opening of the mitoKATP channel selectively improves postanoxic recovery of cell-to-cell communication and ventricular E-C coupling. Although the NO-, ROS-, and PKC-dependent pathways also seem to be involved in this cardioprotection, their interrelation in the developing heart can differ markedly from that in the adult myocardium.

Fluid shear stress can be increased either by increasing the flow rate or perfusing increasing doses of norepinephrine (NE) at a constant flow rate. Concomitantly, increased fluid shear stress at the surface of endothelial cells releases nitric oxide (NO). To better understand the role of NO released by shear stress in regulating intrahepatic vascular resistances, we increased fluid shear stress either by changing the flow rate or by perfusing increasing doses of NE at a constant flow rate in perfused livers isolated from normal rats. When concentration-response curves to NE were studied at low, mild, and high flow rates, portal pressure increased during NE perfusion. The higher the flow rate, the lower the response to NE. NO synthase inhibition similarly increased the response to NE at each flow rate. Thus, NO was released by NE-induced increased shear stress, but other vasodilators are likely to be responsible for the flow-induced increased shear stress. In additional experiments, when flow rate was decreased while infusing increasing doses of NE to maintain the portal pressure constant, shear stress remained steady and NO was not released. Hepatic NO production in the different conditions of shear stress could not be detected. Our data are consistent with the fact that in the liver, NO released by shear stress decreases the vasoconstriction to NE and regulates the intrahepatic vascular resistances.

Transforming growth factor (TGF)- b 1 is a growth factor involved in the mechanisms of lung repair and fibrosis that follow inflammatory processes. We sought to examine the link between the generation of reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI) by inflammatory cells and the expression of TGF- b 1 by alveolar epithelial cells. Exposure of the A549 lung epithelial cell line to either an ROI generating system (xanthine and xanthine oxidase) or an RNI donor (S-nitroso- N -acetyl-penicillamine [SNAP]) promoted a time- and dose-dependent increase in TGF- b 1 release, as measured by a specific enzyme-linked immunosorbent assay. At the peak, the levels of TGF- b 1 were twice the control values. The induction of TGF- b 1 release by ROI was blunted by catalase and unaffected by superoxide dismutase, indicating the involvement of hydrogen peroxide. The response was also blunted by 5,6dichloro-1- b - D -ribofuranosyl benzimidazole (DRB), a specific RNA polymerase II inhibitor, and accompanied by a corresponding increase in TGF- b 1 messenger RNA, as measured by quantitative/competitive reverse transcription polymerase chain reaction, suggesting the involvement of transcriptional mechanisms and possibly other downstream mechanisms. In contrast, RNI-induced TGF- b 1 release was unaffected by DRB and blunted by the protein synthesis inhibitor cycloheximide, suggesting the involvement of translational and post-translational mechanisms. This response required cyclic guanosine monophosphate (cGMP)‐ mediated processes because ( 1 ) immunoreactive cGMP accumulated in the culture medium of SNAPtreated cells; ( 2 ) SNAP-induced TGF- b 1 release was blunted by KT 5823, an inhibitor of cGMP-dependent protein kinase; and ( 3 ) similar increase in TGF- b 1 release was obtained by cell exposure to membrane-permeable dibutyryl-cGMP or to atrial natriuretic factor, a known agonist of particulate guanylate cyclase. These data suggest that in vitro exposure of human alveolar epithelial cells to ROI and RNI enhances TGF- b 1 release through different mechanisms. In vivo , this control may constitute a molecular link between inflammatory and fibrotic processes. Bellocq, A., E. Azoulay, S. Marullo, A. Flahault, B. Fouqueray, C. Philippe, J. Cadranel, and L. Baud. 1999. Reactive oxygen and nitrogen intermediates increase transforming growth factor‐ b 1 release from human epithelial alveolar cells through two different mechanisms. Am. J. Respir. Cell Mol. Biol. 21:128‐136.

Peritoneal adhesions continue to be a significant cause of postoperative complications. The purpose of the present study was to investigate the effect of nitric oxide in preventing postoperative adhesion formation in rats. Three randomized groups of Sprague-Dawley rats were subjected to a standardized lesion by cecal abrasion and parietal peritoneal defect. 0.9% NaCl (control, group 1), L-arginine (300 mg/kg, group 2) and Nω-nitro arginine methyl ester (L-NAME; 25 mg/kg, group 3) were administered intraperitoneally before abdominal closure and during 3 consecutive days after surgery. Two weeks after surgery, a relaparotomy was performed and the extent of adhesion formation was determined. In groups 1 and 3 heavy adhesions were detected. In the L-arginine group, adhesion formation was significantly less than in the other groups (p < 0.05). This study showed that L-arginine reduced adhesion formation.