N omega-nitro-L-arginine Methyl Ester Research Articles

Urotensin‐II Induces Ear Flushing in Rats

While investigating the effects of systemic urotensin II (U-II), a potent vasoactive peptide acting at the UT receptor, we observed ear pinna flushing after systemic administration to conscious rats. In the present study, U-II-induced ear flushing was quantified in terms of ear pinna temperature change and potential mechanisms were explored. U-II-induced ear flushing was quantified by measuring lateral ear pinna temperature changes and compared to that of calcitonin gene-related peptide (CGRP), a known cutaneous vasodilator. Further, the effects of a variety of pharmacological agents on U-II-induced ear flushing were explored. Subcutaneous injection of U-II (9 microg kg(-1))produced localized ear pinna flushing with an onset of approximately 15 min, a duration of approximately 30 min and a maximal temperature change of 9 degrees C. In contrast, CGRP caused cutaneous flushing within multiple cutaneous beds including the ear pinna with a shorter onset and greater duration than U-II. A potent UT receptor antagonist, urantide, blocked U-II-induced ear flushing but did not affect CGRP-induced ear flushing. Pretreatment with indomethacin or L-Nomega-nitroarginine methylester (L-NAME) abolished U-II-induced ear flushing. Mecamylamine or propranolol did not affect this response to U-II. Direct intracerebroventricular injection studies suggested that the ear flushing response to U-II was not mediated directly by the CNS. Our results suggest that U-II-induced ear flushing and temperature increase is mediated by peripheral activation of the UT receptor and involves prostaglandin- and nitric oxide-mediated vasodilation of small capillary beds in the rat ear pinna.

Overexpression of regucalcin suppresses apoptotic cell death in cloned normal rat kidney proximal tubular epithelial NRK52E cells: Change in apoptosis‐related gene expression

The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death and apoptosis was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 24-72 h in a medium without BS containing either vehicle, tumor necrosis factor-alpha (TNF-alpha; 0.1 or 1.0 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml), Bay K 8644 (10(-9)-10(-7) M), or thapsigargin (10(-9)-10(-7) M). The number of wild-type cells was significantly decreased by culture for 42-72 h in the presence of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-5) M), or thapsigargin (10(-8) or 10(-7) M). The effect of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-6) M), or thapsigargin (10(-7) M) in decreasing the number of wild-type cells cultured for 24-72 h was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with LPS (1.0 microg/ml), Bay K 8644 (10(-7) M), or thapsigargin (10(-8) M) for 24 h, and this DNA fragmentation was significantly suppressed in transfectants. DNA fragmentation in adherent cells was not seen by culture with TNF-alpha (1.0 ng/ml). TNF-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M), while LPS- or Bay K 8644-induced decrease in cell number was significantly prevented by caspase-3 inhibitor or N omega-nitro-L-arginine methylester (NAME) (10(-5) M), an inhibitor of nitric oxide (NO) synthase. Thapsigargin-induced decrease in cell number was not prevented in the presence of two inhibitors. Bcl-2 and Akt-1 mRNA levels were significantly increased in transfectants cultured for 24 h as compared with those of wild-type cells, while Apaf-1, caspase-3, or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions were not significantly changed in transfectants. Culture with TNF-alpha (1.0 ng/ml), LPS (1.0 microg/ml), Bay K 8644 (l0(-7) M), or thapsigargin (10(-8) M) caused a significant increase in caspase-3 mRNA levels in wild-type cells. LPS (1.0 microg/ml) significantly decreased Bcl-2 mRNA expression in the cells. Their effects on the gene expression of apoptosis-related proteins were not significantly changed in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death and apoptosis induced by various factors which their action are mediated through many intracellular signaling pathways, and that it modulates the gene expression of apoptosis-related proteins.

Overexpression of regucalcin suppresses cell death and apoptosis in cloned rat hepatoma H4‐II‐E cells induced by insulin or insulin‐like growth factor‐I

The role of regucalcin, a regulatory protein in intracellular signaling pathway, in cell death was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin/pCXN2 transfectants were cultured for 72 h in a medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 24-72 h in a medium containing either vehicle, insulin (10(-8) or 10(-7) M) or insulin-like growth factor-I (IGF-I; 10(-9) or 10(-8) M) in the absence of FBS. The number of wild-type cells was significantly decreased by culture for 24, 48, or 72 h in the presence of insulin (10(-8) or 10(-7) M) or IGF-I (10(-9) or 10(-8) M). Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with insulin or IGF-I. The effect of insulin or IGF-I in stimulating cell death and DNA fragmentation in hepatoma cells (wild-type) was significantly prevented in transfectants overexpressing regucalcin. Meanwhile, epinephrine (10(-6) or 10(-5) M) or transforming growth factor-beta1 (10(-13) or 10(-12) M) did not cause cell death of hepatoma cells. Insulin-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M), although the effect of IGF-I was not inhibited. The effect of insulin or IGF-I in inducing the death of hepatoma cells (wild-type) was significantly prevented in the presence of N omega-nitro-L-arginine methylester (NAME), an inhibitor of nitric oxide synthase. Genistein (10(-6) M), an inhibitor of protein tyrosine kinase, or vanadate (10(-5) M), an inhibitor of protein tyrosine phosphatase, caused a significant decrease in the number of hepatoma cells (wild-type). The effect of insulin in inducing the death of wild-type cells was not seen in the presence of genistein or vanadate. The effect of IGF-I on the death of wild-type cells was observed in the presence of genistein or vanadate. The effect of genistein on cell death was significantly prevented in transfectants. Such effect was not seen with vanadate. This study demonstrates that insulin or IGF-I stimulates cell death and apoptosis in the hepatoma cells, and that overexpression of regucalcin has a suppressive effect on cell death induced by insulin or IGF-I that is mediated through different signaling pathway.

Relaxation effects of adrenomedullin in isolated rabbit corpus cavernosum smooth muscle.

To clarify the pharmacological effects of adrenomedullin, a potent vasodilator and hypotensive peptide isolated from human phaeochromocytoma cells, on corpus cavernosal smooth muscle in vitro, as the intracavernosal injection of adrenomedullin induces penile erection in the anaesthetized cat. The effects of adrenomedullin were investigated in isolated muscle strips from New Zealand rabbit corpus cavernosum smooth muscle pre-contracted with phenylephrine alone, in the presence of indomethacin (cyclooxygenase inhibitor), Nomega-nitro l-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), and K+-channel blockers. Adrenomedullin caused relaxation of isolated pre-contracted rabbit corpus cavernosum strips in a concentration-dependent manner. The response of corpus cavernosum was unaffected L-NAME, indomethacin and K+-channel blockers. The relaxation exerted by adrenomedullin in rabbit corporal tissue may arise from the effect of the drug on its specific receptors and/or calcitonin gene-related peptide-1 receptors. The relaxant effect of adrenomedullin might lead to novel clinical applications for erectile dysfunction.

Activation of Nitric Oxide—Cyclic Guanosine Monophosphate Signaling in Kidney by Extracorporeal Shock Wave Therapy

We defined whether extracorporeal shock wave therapy (ESWT) to the kidney activates the nitric oxide (NO)-cyclic 3',5'-guanosine monophosphate (cGMP) pathway. A total of 90 male rabbits were randomly divided into group 1--pretreated with normal saline, group 2--pretreated intravenously (i.v.) with Nomega nitro-L-arginine-methyl ester (NAME) (100 mg/kg), group 3--pretreated i.v. with NAME and L-arginine (300 mg/kg) with ESWT to 1 kidney, group 4--pretreated IV with ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) (20 microg/kg) and group 5--pretreated with normal saline with ESWT to the bladder. Plasma nitrite, NO metabolite and cGMP were analyzed in peripheral blood samples before, immediately after, and 30 and 60 minutes after ESWT. ESWT to the kidney but not to the bladder caused an increase before, immediately after, and 30 and 60 minutes after ESWT in plasma nitrite in group 1 (186.1 +/- 20.6, 217.5 +/- 21.6, 241.9 +/- 28.4 and 230.5 +/- 25.3 nM) and group 5 (149.0 +/- 14.7, 155.6 +/- 18.4, 131.8 +/- 13.6 and 140.0 +/- 15.7 nM), and in cGMP in group 1 (24.2 +/- 1.9, 33.8 +/- 3.2, 32.9 +/- 2.2 and 29.4 +/- 1.9 pmol/ml) and group 5 (25.5 +/- 2.1, 27.5 +/- 2.5, 28.7 +/- 3.1 and 25.5 +/- 2.6 pmol/ml, respectively). In group 2 NAME significantly inhibited the production of nitrite (113.4 +/- 18.6, 118.2 +/- 19.9, 114.6 +/- 18.3 and 112.5 +/- 17.6 nM) and cGMP (19.4 +/- 2.6, 20.6 +/- 2.8, 19.3 +/- 2.7 and 18.6 +/- 2.6 pmol, respectively). In group 3 inhibited nitrite and cGMP production caused by NAME was recovered with L-arginine. In group 4 ODQ significantly inhibited cGMP production. The results show that ESWT increases the level of NO and cGMP released by the kidney in an animal model.

Nitric oxide scavenging, alone or with nitric oxide synthesis inhibition, modulates vascular hyporeactivity in rats with intraperitoneal sepsis.

Elevated levels of nitric oxide (NO) may be a primary cause of the vascular hyporeactivity (vasoplesia) and refractory hypotension in sepsis. This study was initiated to determine the efficacy of NO scavenging with acellular hemoglobin (Hb) solution in modulating sepsis-mediated vasoplesia. Male Sprague Dawley rats were subjected to sepsis by cecal ligation and puncture (CLP). Twenty-four hours post-CLP, the animals were randomly assigned to one of four groups (n = 5-6 each) and given an intravenous injection of 0.5 mL bovine serum albumin (BSA; 5 g/dL), 0.5 mL human Hb (7g/dL), 50 microL Nomega-nitro L-arginine methyl ester (NAME; 1 M), or both Hb and NAME. Blood pressure (BP), cardiac output, systemic vascular resistance, and vascular reactivity (VR) to norepinephrine (NE; 40 ng/Kg) were assessed before and after an experimental treatment. In some animals, inducible NO synthase (iNOS) mRNA expression was assessed in selected tissue samples harvested at the conclusion of experiment using a reverse transcription-polymerase chain reaction (RT-PCR) method. Treatment with Hb, NAME, or NAME + Hb elicited a significant improvement in mean BP and VR compared with the control (BSA) group (P < 0.05, analysis of variance and Neuman-Keuls tests). Tissue samples from 24-h CLP rats clearly exhibited iNOS gene expression; higher iNOS gene expression in the intestine compared with aorta suggests that the intestine may be a major source of the elevated NO level in this model. In conclusion, NO scavenging with Hb, alone, or in combination with NO synthesis inhibition, appears to be effective in modulating sepsis-mediated vascular hyporeactivity and may reduce complications associated with global NO synthesis inhibition.

Role of endogenous nitric oxide in liver-specific functions and survival of cultured rat hepatocytes

1. The role of endogenous nitric oxide in rat hepatocyte functionality and survival in cell culture was examined. Towards this aim, cytochrome P450 activities (CYP1A1/2, 2B1, 2A1, 2C11, 2D1, 2E1 and 3A1), liver-specific metabolic functions and cell survival were comparatively evaluated in hepatocytes isolated from the male Sprague-Dawley rat and/or cultured in control conditions or in the presence of N-omega-nitro-l-arginine methyl ester (NAME), an inhibitor of nitric oxide synthesis. 2. Suppression of nitric oxide production by NAME paralleled a substantial preservation of hepatocyte phenotype in culture. The presence of NAME was particularly important during isolation and/or the 6-24 h culture. By 24 h, beneficial effects were evident in parameters particularly unstable in culture (glycogen content, P450), whereas no changes were produced in well-preserved functions (glucose, urea and albumin synthesis, glutathione, drug-conjugating enzymes). 3. Long-term treatment of hepatocytes with NAME also produced a reduction in caspase 3 activation and in the percentage of spontaneous apoptotic cells, and an increase in cell survival and transcriptional activity as shown by attached cellular protein content and the protein-DNA ratio respectively. 4. In conclusion, inhibition of early endogenous nitric oxide formation is an efficient procedure for obtaining hepatocyte cultures with stable expression of differentiated functions, high cell survival and few signs of cell senescence.

Guinea pig airway hyperresponsiveness induced by blockade of the angiotensin II type 1 receptor. Role for endogenous nitric oxide.

Losartan is the first angiotensin II type 1 (AT1) receptor antagonist to become available for the treatment of hypertension. However, recent reports have revealed several cases of losartan-induced bronchoconstriction. We investigated to determine the mechanism of losartan-induced bronchoconstriction, considering in particular the involvement of endogenous nitric oxide (NO). In this study, we examined the effects of losartan on airway obstruction and endogenous NO production using anesthetized guinea pigs and cultured airway epithelial cells. Five minutes after administration of angiotensin II (Ang II), the bronchoconstriction induced by acetylcholine was not changed. In contrast, Ang II in the presence of losartan caused a significant increase in the acetylcholine responsiveness. Pretreatment with L-N omega-nitroarginine-methylester (L-NAME) potentiated acetylcholine-induced bronchoconstriction 5 min after administration of Ang II, and L-arginine reversed this action of L-NAME on the acetylcholine responsiveness. Moreover, Ang II administration increased NO concentration in expired air (12.5 +/- 1.5 ppb for saline, 40 +/- 5 ppb for Ang II, p < 0.01), and losartan significantly inhibited Ang II-stimulated NO release (20 +/- 3.5 ppb) from guinea pig airway. In cultured airway epithelial cells, Ang II also increased NO release (160 +/- 25 nM), and the effect of this Ang II-induced NO release was significantly inhibited by pretreatment with losartan (25 +/- 8 nM, p < 0.01). These findings suggest that losartan-induced bronchoconstriction may result from inhibition of endogenous NO release in the airway.

Prostaglandins maintain renal vasodilation and hyperfiltration during chronic nitric oxide synthase blockade in conscious pregnant rats.

Rats demonstrate renal vasodilation and hyperfiltration in pregnancy. Because both NO and cGMP biosynthesis are increased in gravid rats and because acute administration of NO synthase inhibitors abrogates renal vasodilation and hyperfiltration, NO most likely mediates the renal circulatory changes of gestation. In the present study, we tested the effect of chronic inhibition of NO synthase on effective renal plasma flow (ERPF) and glomerular filtration rate (GFR) in chronically instrumented, conscious, gravid rats. Because gestation is a relatively long-term condition, we postulated that chronic withdrawal of NO would result in sustained inhibition of renal vasodilation and hyperfiltration. Contrary to our hypothesis, the renal circulatory changes of pregnancy were maintained during chronic blockade of NO synthase. That is, subcutaneous administration of 10 micrograms/min N omega-nitro-L-arginine methyl ester (NAME) for 48 hours did not significantly reduce GFR in either virgin or pregnant rats; thus, hyperfiltration persisted in the latter despite chronic NO synthase blockade. In contrast, ERPF was reduced and effective renal vascular resistance (ERVR) increased in both groups of rats during NAME administration but in a parallel fashion, such that renal vasodilation persisted in the gravid animals despite chronic inhibition of NO synthase. However, with superimposition of acute prostaglandin synthesis inhibition (meclofenamate, 10 mg/kg IV), renal vasodilation and hyperfiltration were abolished; ie, the combined treatments of chronic NO synthase blockade and acute prostaglandin synthesis inhibition led to the equalization of GFR, ERPF, and ERVR in conscious virgin and pregnant rats. Inhibition of prostaglandin synthesis alone had little affect on the renal circulation, as previously reported. In summary, prostaglandins are recruited to maintain renal vasodilation and hyperfiltration during chronic NO synthase blockade in conscious pregnant rats.

TNF-alpha-induced inhibition of PC synthesis by human type II pneumocytes is sequentially mediated by PGE2 and NO.

Tumor necrosis factor-alpha (TNF-alpha)-induced inhibition of surfactant synthesis participates in the pathogenesis of the acute respiratory distress syndrome. We examined the ability of human type II pneumocytes to produce nitric oxide (NO) in the presence of TNF-alpha as well as the role of NO and prostaglandin (PG) E2 in the transduction of the cytokine signal. Multiple organ donors were used as a source of lung tissue. After 24-h preculture, type II pneumocytes were cultured for 18 h in the presence or absence of additives. The D-[U-14C] glucose incorporation into phosphatidylcholine (PC) was selectively inhibited by TNF-alpha, PGE2, sodium nitroprusside (SNP), or 8-bromoguanosine 3',5'-cyclic monophosphate. The effect of TNF-alpha was attenuated by indomethacin, N omega-nitro-L-arginine methyl ester (NAME), or methylene blue (MB). The effect of PGE2 was attenuated by NAME, while that of SNP was reversed by MB but not by indomethacin. TNF-alpha induced an increase in PGE2 and guanosine 3',5'-cyclic monophosphate cell content and in the NO release to the medium. NAME did not affect PGE2 production, while indomethacin blunted NO generation. Our results suggest that NO generation, secondary to PGE2 production, is responsible for the TNF-alpha-induced inhibition of PC synthesis by human type II pneumocytes.

L-arginine minimizes reperfusion injury in a low-flow, reflow model of liver perfusion.

A low-flow, reflow model of liver perfusion was used in the rat to investigate the effects of L-arginine on reperfusion injury in the absence of blood elements. In contrast to in vivo studies, L-arginine cannot minimize hypoxia by improving the microcirculation under these special conditions, but rather can only increase oxygen delivery upon reflow. During reflow, lactate dehydrogenase (LDH) release reached a new steady-state value of 35 ± 3 U/g/h in livers perfused in the absence of L-arginine. L-Arginine (0.1 mmol/L) significantly reduced LDH release during reflow to 14 ± 1 U/g/h; higher concentrations (1.0-3.0 mmol/L) were less effective and the arginine analogue Nomega-nitro L-arginine methyl ester (L-NAME, 0.3 mmol/L) reversed the protective effect completely. Infusion of the biologically inactive isomer D- arginine (0.1 mmol/L had no effect on the observed reperfusion injury. Malondialdehyde (MDA) release during reflow averaged 92 ± 10 nmol/g/h and was decreased significantly to 47 ± 13 nmol/g/h with L-arginine (0.1 mmol/L). Oxygen uptake during reflow was not significantly altered by L-arginine, although the time required to reach steady state values upon reflow was reduced significantly (about twofold), indicating improved microcirculation. Trypan blue distribution time, which is used to index the hepatic microcirculation, was decreased significantly from 330 ± 17 to 227 ± 31 seconds by L-arginine, an effect also blocked by L-NAME. Additionally, L-arginine, significantly increased both the rate of entry and exit of fluorescein-dextran, a dye confined to the vascular space, by approximately 50%, also reflecting improved microcirculation. Collectively , these findings indicate that L-arginine protects against hypoxia/reoxygenation injury in a blood-free perfusion model specifically during the reoxygenation period. It is likely that L-arginine rapidly removes substrates for free radical generation by improving the microcirculation. (Hepatology 1996 Jul;24(1):163-8)

L-arginine minimizes reperfusion injury in a low-flow, reflow model of liver perfusion.

A low-flow, reflow model of liver perfusion was used in the rat to investigate the effects of L-arginine on reperfusion injury in the absence of blood elements. In contrast to in vivo studies, L-arginine cannot minimize hypoxia by improving the microcirculation under these special conditions, but rather can only increase oxygen delivery upon reflow. During reflow, lactate dehydrogenase (LDH) release reached a new steady-state value of 35 +/- 3 U/g/h in livers perfused in the absence of L-arginine. L-Arginine (0.1 mmol/L) significantly reduced LDH release during reflow to 14 +/- 1 U/g/h; higher concentrations (1.0-3.0 mmol/L) were less effective and the arginine analogue Nomega-nitro L-arginine methyl ester (L-NAME, 0.3 mmol/L) reversed the protective effect completely. Infusion of the biologically inactive isomer D-arginine (0.1 mmol/L had no effect on the observed reperfusion injury. Malondialdehyde (MDA) release during reflow averaged 92 +/- 10 nmol/g/h and was decreased significantly to 47 +/- 13 nmol/g/h with L-arginine (0.1 mmol/L). Oxygen uptake during reflow was not significantly altered by L-arginine, although the time required to reach steady state values upon reflow was reduced significantly (about twofold), indicating improved microcirculation. Trypan blue distribution time, which is used to index the hepatic microcirculation, was decreased significantly from 330 +/- 17 to 227 +/- 31 seconds by L-arginine, an effect also blocked by L-NAME. Additionally, L-arginine, significantly increased both the rate of entry and exit of fluorescein-dextran, a dye confined to the vascular space, by approximately 50%, also reflecting improved microcirculation. Collectively , these findings indicate that L-arginine protects against hypoxia/reoxygenation injury in a blood-free perfusion model specifically during the reoxygenation period. It is likely that L-arginine rapidly removes substrates for free radical generation by improving the microcirculation.

Both prostaglandin E2 and nitric oxide sequentially mediate the tumor necrosis factor alpha-induced inhibition of surfactant synthesis by human type II pneumocytes.

Tumor necrosis factor alpha (TNF-alpha)-induced inhibition of surfactant synthesis seems to participate in the pathogenesis of the adult respiratory distress syndrome. To examine the ability of human type II pneumocytes to produce nitric oxide (NO) in the presence of TNF-alpha and, in addition, to explore the role of this radical in the transduction of the cytokine signal. Multiple organ donors were the source of lung tissue specimens. Type II pneumocytes were isolated by enzymatic digestion, adherence separation of macrophages, and gradient purification. After 24-hour preculture, cells were cultured for 24 hours in the presence or absence of TNF-alpha (100 ng/mL), sodium nitroprusside (100 mumol/L), N omega-nitro-L-arginine methyl ester (NAME) (1 mmol/L), methylene blue (10 mumol/L),8-bromo-3',5'-cyclic guanosine monophosphate (8-Br-cGMP) (1 mmol/L), prostaglandin E2 (PGE2) (0.1 mumol/L), indomethacin (30 mumol/L), and combinations. The NO release to the medium and cGMP and PGE2 contents of the cells were measured. The incorporation of 14C-labeled glucose (D-[U-14C]glucose) into phosphatidylcholine and phosphatidylglycerol was selectively inhibited either by 8-Br-cGMP or in the presence of TNF-alpha, PGE2, or nitroprusside, all of which caused an increase in the intracellular levels of cGMP. The inhibitory effect of TNF-alpha was partially reverted by indomethacin, NAME, N-monomethyl arginine, or methylene blue. The inhibitory effect of PGE2 was partially reverted by NAME, while that of nitroprusside was reverted by methylene blue, but not by indomethacin. Tumor necrosis factor alpha induced an increase in PGE2 (4.31 +/- 0.27 vs 1.65 +/- 0.17-pg/microgram protein, n = 10, P < .01) and cGMP (0.238 +/- 0.012 vs 0.109 +/- 0.014-pmol/microgram protein, n = 10, P < .01) cell content and in the NO release to the medium (3.10 +/- 0.14 vs 1.19 +/- 0.11-nmol/microgram protein, n = 10, P < .01). The basal NO release to the medium was also increased in the presence of PGE2. The NAME, which blocked NO generation and cGMP increase, did not affect PGE2 production in response to TNF-alpha. However, indomethacin, which blocked PGE2 production, also blunted NO generation and cGMP increase. The NO generation, secondary to PGE2 production, seems responsible for the TNF-alpha-induced inhibition of phosphatidylcholine synthesis by human type II pneumocytes. Nitric oxide seems to exert this effect through activation of guanylyl cyclase.

Effects of Pulmonary Hypertension on Vasoconstrictor Responses to Endothelin-1 and Sarafotoxin S6C and on Inherent Tone in Rat Pulmonary Arteries

Vasoconstrictor responses to endothelin-1 (ET-1) and the ETB receptor agonist sarafotoxin S6c (SXS6c) were investigated in the main pulmonary artery and pulmonary artery branch removed from rats previously exposed to 10% O2 [chronic hypoxic (CH) rats] or room air (control rats) for 2 weeks. The effects of nitric oxide synthase (NOS) inhibition with L-Nomega-nitroarginine methyl ester (L-NAME) (100 microM) on ET receptor-induced responses in these arteries were also investigated. In control rats, in rings of main pulmonary arteries and pulmonary artery branches. ET-1 induced vasoconstrictor responses. These responses were mediated by the ETA receptor as they were antagonized by the ETA receptor antagonist FR 139317 whereas SXS6c did not vasoconstrict. Chronic hypoxia had no effect on the sensitivity of the main pulmonary arteries to ET-1, whereas small vasoconstrictor responses to SCS6c were evident. ET-1 was more potent in the CH rat pulmonary artery branches than in controls. SXS6c also caused vasoconstriction with a maximum response 30% of that to ET-1 in both endothelium-intact and endothelium-denuded vessels. L-NAME increased the sensitivity to ET-1 in the CH rat main pulmonary arteries and increased the responses to low concentrations of ET-1 in the control rat main pulmonary arteries but did not affect any ET-1 responses in any other vessels. It did disclose responses to SXS6c in control rat main pulmonary arteries. L-NAME itself increased vascular tone to a greater extent in CH rat pulmonary arteries than in controls. In preconstricted pulmonary arteries, however, relaxations to acetylcholine (ACh) were diminished in the CH rats as compared with their controls. All pulmonary artery branches, denuded of their vascular endothelium, relaxed to sodium nitroprusside (SNP) and therefore exhibited endogenous vascular tone. This effect was greatest in the pulmonary artery branches from the CH rats. The results suggest that rat large pulmonary artery responses to ET-1 are normally mediated by ETA receptors. Pulmonary hypertension can potentiate ETA receptor-mediated vasoconstriction and facilitate ETB receptor-mediated vasoconstriction. Endogenous NO may normally suppress ETA receptor-mediated responses in rat main pulmonary arteries. Rat pulmonary arteries exhibit endogenous tone, which is increased by exposure to chronic hypoxia.

Electron transfer in the nitric-oxide synthases. Characterization of L-arginine analogs that block heme iron reduction.

Heme iron reduction in the nitric-oxide synthases (NOSs) requires calmodulin binding and is associated with increased NO synthesis and NADPH oxidation (Abu-Soud, H. M., and Stuehr, D. J. (1993) Proc. Natl. Acad. Sci., U. S. A. 90, 10769-10772). Here, we examined how L-arginine and the analogs N omega-methyl-L-arginine (NMA), N omega-nitro-L-arginine methyl ester (NAME), and d-(thioureido)-L-norvaline (thiocitrulline) affect electron flux through neuronal and macrophage NOS. L-Arginine and NMA increased or decreased NOS NADPH consumption depending on the isoform, while thiocitrulline and NAME decreased NADPH oxidation in both NOS by 73-86% relative to their ligand-free rates. Kinetic studies showed that thiocitrulline and NAME inhibited NOS NADPH consumption through binding within the substrate binding site. Thiocitrulline and NAME did not affect the NADPH-dependent reduction of NOS flavins nor NOS cytochrome c reduction, indicating that they blocked electron flux at a point beyond the flavins in the electron transfer sequence. Thiocitrulline and NAME inhibited both NADPH-dependent and dithionite-mediated heme iron reduction in the NOS isoforms relative to the substrate-free NOS, whereas L-arginine and NMA did not. Thus, L-arginine and NMA increase or decrease electron flux through the NOS by coupling NADPH oxidation to NO synthesis (L-arginine), or by occupying the substrate binding site with minimal catalytic coupling (NMA). In contrast, thiocitrulline and NAME decrease electron flux through both NOS isoforms by decreasing the reduction potential of the heme iron. Inhibition of heme iron reduction by substrate analogs is unusual and represents a new means to modulate electron flow through the NOS.

Endothelin ETA- and ETB-receptor-mediated vasoconstriction in rat pulmonary arteries and arterioles.

We investigated the endothelin (ET) receptors involved in the vasoconstrictor responses to ET-1 in rat pulmonary arteries and arterioles and the effect of endothelium removal, nitric oxide (NO) synthase inhibition, and hypoxia on ET-1-induced responses in the arteries. In isolated rat pulmonary artery rings (2-3 mm ID) prepared from the pulmonary artery branch before its entry into the lung, ET-1-induced vasoconstrictor responses. These responses were mediated by the ETA receptor as they were competitively antagonized by the ETA receptor antagonist FR 139317, and the ETB-receptor agonist sarafotoxin S6c (SXS6c) was a very weak vasoconstrictor in these vessels, inducing maximum contractions only 9% of those of ET-1. In contrast, in rat intrapulmonary resistance arteries (100-150 microns ID), SXS6c induced FR 139317-resistant contractions, and these vessels were more sensitive to SXS6c than to ET-1. SXS6c produced maximum contractions 92% those of ET-1, suggesting that ET-1-induced contractions were mediated by the ETB receptor in these resistance vessels. In the larger pulmonary arteries, the NO synthase inhibitor L-N omega nitroarginine methyl ester (L-NAME) (100 microM) potentiated responses to ET-1, an effect that was reversed by FR 139317. Endothelium removal also potentiated response to ET-1, and L-NAME had no effect on ET-1 responses in endothelium-denuded vessels, suggesting that in these vessels the ETA receptor-mediated responses to ET-1 are normally suppressed by endothelium-derived NO.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelium-derived relaxing factor regulates renin release in vivo.

Endothelium-derived relaxing factor (EDRF), through its inhibitory second messenger guanosine 3',5'-cyclic monophosphate (cGMP), inhibits renin release in vitro. To determine whether EDRF affects renin in vivo, we tested whether EDRF synthesis inhibition could stimulate renin secretion in intact rats. Because EDRF synthesis inhibition increases blood pressure and consequently withdraws sympathetic activity (both renin inhibitory signals), we also studied the effect of L-N omega-nitroarginine methyl ester (L-NAME) when renal perfusion pressure was controlled and during beta-adrenergic blockade. Mean blood pressure (BP), heart rate (HR), and plasma renin activity (PRA) were measured in anesthetized rats before and after EDRF synthesis inhibition by a 10 mg/kg body wt bolus of L-NAME. L-NAME decreased PRA by 67% [from 11.0 +/- 2.7 to 3.7 +/- 0.8 ng angiotensin I (ANG I).ml-1.h-1, n = 12; P less than 0.001], increased BP by 20 +/- 2 mmHg (P less than 0.001), and decreased HR from 332 +/- 8 to 312 +/- 9 beats/min (P less than 0.005). We repeated our experiment in rats instrumented with an intra-aortic balloon catheter to control renal perfusion pressure and pretreated with propranolol to eliminate the beta-adrenergic effect. Under these conditions, L-NAME now increased PRA by 55% (from 6.9 +/- 1.9 to 10.8 +/- 2.6 ng ANG I.ml-1.h-1, n = 12; P less than 0.02), whereas renal perfusion pressure was unchanged (91 +/- 4 vs. 90 +/- 4 mmHg). HR increased slightly from 308 +/- 5 to 315 +/- 3 beats/min (P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)