Stanley S. Greenberg

Nitric oxide inhibition causes intrauterine growth retardation and hind-limb disruptions in rats.

OBJECTIVE Our purpose was to determine the effects of nitric oxide synthase inhibition on maternal and fetal health in the last third of pregnancy. STUDY DESIGN Pregnant rats were treated from gestational day 13 to day 19 or 20 with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester, which was administered in the drinking water ad libitum. Control animals received the inactive enantiomer NG-nitro-D-arginine methyl ester or no treatment. Maternal blood pressure, blood chemistry studies, and placenta and pup size were determined. A separate group of rats received nitroprusside sodium in conjunction with NG-nitro-L-arginine methyl ester. RESULTS NG-nitro-L-arginine methyl ester caused a dose-dependent reduction in placenta and pup size. Amniotic fluid levels of cyclic guanosine monophosphate were significantly reduced at 0.1 mg/ml but not at higher doses. Hemorrhagic necrosis of fetal hind limbs occurred only with treatment with NG-nitro-L-arginine methyl ester and was prevented by coadministration of nitroprusside sodium. Maternal blood pressure and blood and urine chemistry studies were unaffected by NG-nitro-L-arginine methyl ester. CONCLUSION Chronic reductions of nitric oxide production in the last third of pregnancy result in significant intrauterine growth retardation and hemorrhagic disruptions of hind limbs. Maternal complications were minimal and did not mimic preeclampsia.

Nitric oxide production in experimental alcoholic liver disease in the rat : role in protection from injury

BACKGROUND & AIMS Regulation of blood flow and oxygen supply are important pathogenetic factors in alcoholic liver disease. Because nitric oxide may have an important role, its effects on alcoholic liver injury were investigated. METHODS Rats were fed ethanol intragastrically with either saturated fat or corn oil. Spontaneous production of NO by liver nonparenchymal cells was compared in the two dietary groups. Two additional groups of rats fed corn oil and ethanol were treated with either an NO inhibitor (L-NAME) or supplemented with L-arginine. Liver pathology and plasma NO production were evaluated. RESULTS In the corn oil and ethanol group, a progressive decrease in liver nonparenchymal cell NO production and increased plasma NO levels were associated with liver injury. Reduced nicotinamide adenine dinucleotide phosphate diaphorase staining showed increased centrilobular staining of hepatocytes in the corn oil and ethanol group and L-NAME-treated group. Moreover, L-NAME increased the severity, whereas L-arginine supplementation completely prevented liver injury. In the saturated fat and ethanol group, in which there was no liver injury, the levels of NO2- in nonparenchymal supernatant were 5-10-fold higher than in the corn oil and ethanol group. CONCLUSIONS Decreased NO production by nonparenchymal cells may contribute to liver injury in ethanol-fed rats, and the compensatory increase in hepatocyte NO production may contribute to centrilobular liver injury.

Potential Role of Nitric Oxide in a Model of Chronic Colitis in Rhesus Macaques

BACKGROUND/AIMS Excess nitric oxide formation, via the inducible NO synthase isoform, has been implicated in the pathogenesis of experimental and clinical inflammatory bowel disease. The aim of this study was to assess the site, enzyme source, and magnitude of NO production in juvenile rhesus macaques with idiopathic colitis. METHODS NO production was assessed systemically from plasma and urine levels of reactive nitrogen intermediates and locally by the formation of [3H]citrulline from [3H]arginine and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Inducible NO synthase gene expression was assessed by reverse-transcription polymerase chain reaction. RESULTS Plasma and urine levels of reactive nitrogen intermediates were greater in colitic animals than in control monkeys by 13- and 5-fold, respectively. NADPH diaphorase activity in normal animals was confined to the myenteric plexus. In colitis, staining was also apparent in crypt abscesses and superficial epithelial and mucosal bands. Gene expression for inducible NO synthase was only found in colitic specimens. Colonic [3H]citrulline formation was markedly elevated in colitic specimens, and the inducible isoform accounted for 58% of total activity. CONCLUSIONS It is proposed that excess NO, formed via the inducible form of NO synthase, contributes to the mucosal inflammation and symptoms of this idiopathic colitis model.

Ethanol Relaxes Pulmonary Artery by Release of Prostaglandin and Nitric Oxide

Acute-intake of ethanol is associated with vasodilation of vascular smooth muscle (VSM). Relaxation of VSM is dependent, in part, on the actions of nitric oxide (NO) and prostaglandin (PG) produced by endothelial cells (EC) lining the VSM. We examined the effects of endothelium rubbing and inhibition of EC synthesis of NO and PG on ethanol-induced relaxation of bovine pulmonary artery (BPA) and pulmonary vein (BPV) in vitro. Rings of isolated BPA and BPV were mounted in muscle chambers for the isometric recording of force development. Blood vessels were precontracted with an EC50 concentration of the thromboxane receptor mimetic U46619. Ethanol (0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, and 1.28% (w/v) produced concentration-dependent relaxation of BPA and BPV. Ethanol-induced relaxation was attenuated in BPA with rubbed EC and by the NO synthase inhibitors, L-NG monomethylarginine (LNMMA, 50 microM) and L-nitroarginine (NOLA, 10 microM), and the prostaglandin cyclooxygenase inhibitor, ibuprofen (10 microM). In contrast, ethanol-induced relaxation of BPV was not affected by endothelium rubbing or by NOLA or LNMMA, but was partially attenuated by ibuprofen. Nitric oxide was measured with the chemiluminescence technique. Ethanol increased the content of NO released under basal conditions by the BPA but did not effect basal NO release from BPV. However, ethanol enhanced bradykinin-induced release of NO from BPA and BPV and, at low concentrations, augmented bradykinin-induced relaxation of both BPA and BPV.(ABSTRACT TRUNCATED AT 250 WORDS)

Independent suppression of nitric oxide and TNF alpha in the lung of conscious rats by ethanol.

Tumor necrosis factor‐α (TNFα) and nitric oxide (NO) mediate in part the microbicidal response of murine and rodent alveolar macrophages (AM) and recruited neutrophils (PMN) to airborne infections. Ethanol (ETOH) suppresses intrapulmonary TNFα and NO release and impairs pulmonary host defense mechanisms. We tested the concept that ETOH down‐regulates NO by inhibiting production of TNFα. Male rats were given intratracheal (i.t.) saline (PBS), a polyclonal anti‐TNFα antibody (TNFab) or nonimmune IgG (22 mg/kg, i.m.) 2 h before giving i.t. Escherichia coli endotoxin (LPS) to normal rats or rats pretreated with ETOH (5.5 g/kg, i.p.) 30 min before experimentation. AM and PMN were obtained from the bronchoalveolar lavage fluid (BAL) fluid of rats killed 2 and 4 h after administration of LPS. mRNA for inducible NO synthase (iNOS) and TNFα were measured in AM and PMN with competitor equalized RT‐PCR techniques. The BAL fluid, AM, and PMN were assayed for TNFα and NO2–, and NO3– (RNI) with the L929 bioassay and chemiluminescence, respectively. TNFab abolished LPS‐induced increases in TNFα but did not suppress the NO content of the BAL fluid or gene expression for iNOS by AM or PMN. ETOH suppressed LPS‐induced increases in mRNA for iNOS, production of RNI, and BAL fluid TNFα but did not affect LPS‐induced increases in mRNA for TNFα. ETOH‐induced attenuation of LPS‐induced up‐regulation of the iNOS system did not differ in rats pretreated with TNFab or IgG. Thus, ETOH down‐regulates iNOS gene expression and RNI production independent of its effects on TNFα. Acute ETOH administration suppresses iNOS at the level of transcription and TNFα at the level of translation or release of the peptide.—Xie, J., Kolls, J. K., Bagby, G., Greenberg, S. S. Independent suppression of nitric oxide and TNFα in the lung of conscious rats by ethanol. FASEB J. 9, 253–261 (1995)

Ethanol suppresses LPS-induced mRNA for nitric oxide synthase II in alveolar macrophages in vivo and in vitro.

Alcohol abuse increases the incidence and severity of opportunistic lung infections and pneumonias. Inducible nitric oxide (NO) synthase (iNOS II) and NO may be a pivotal system in the intracellular bactericidal activity of macrophages. We tested the hypothesis that acute administration of ethanol (ETOH) suppressed Escherichia coli endotoxin lipopolysaccharide (LPS) mediated upregulation of the iNOS II system in the lung of the rat, in vivo. We also tested the effect of ETOH on alveolar macrophage (AM) production of free NO using microelectrodes. Male Sprague-Dawley rats were given ETOH (5.5 g/kg, IP) 30 min. before giving intratracheal sterile phosphate buffered saline solution (PBS, 0.5 ml) or LPS (1 mg/kg in a total volume of 0.5 ml PBS). The isolated lungs were subjected to bronchoalveolar lavage (BAL) 3.5 hr. later. Aliquots of the BAL fluid were assayed for tumor necrosis factor alpha TNF alpha and reactive nitrogen intermediates (nitrate and nitrite) (RNI) with chemiluminescence. Aliquots of AM were incubated 1 hr ex vivo for spontaneous production of RNI or frozen and assayed for iNOS II mRNA with competitor exchange reverse transcriptase polymerase chain reaction (cERT-PCR). The lung was homogenized and assayed for RNI. LPS increased BAL fluid TNF alpha and RNI, lung RNI, and the spontaneous production of RNI by AM, ex vivo. These effects were inhibited by in vivo administration of inhibitors of iNOS II. LPS increased iNOS mRNA in AM. This was unaffected by iNOS inhibitors. ETOH suppressed LPS-induced BAL fluid TNF, iNOS mRNA and RNI production by AM and the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of PKC and Tyrosine Kinase in Ethanol-Mediated Inhibition of LPS-Inducible Nitric Oxide Synthase

Ethanol increases human and animal susceptibility to opportunistic lung infections in part by suppression of endotoxin (LPS) and bacteria-mediated upregulation of inducible nitric oxide synthase (iNOS) in alveolar macrophages (AM). LPS and cytokine-induced NOS mRNA are dependent on NF-kappaB/Rel (NFkappaB) and Activator Protein-1 (AP-1), which are regulated in turn by protein kinase C and tyrosine kinase-dependent phosphorylation. ETOH does not directly inhibit NFkappaB or AP-1, in vivo, but rather inhibits LPS-induced activation of the MEKK/MAP kinase system and inhibition of inhibitory protein IkappaBalpha required for formation of AP-1 and NFkappaB, respectively. in AM. Both transcription factors are involved iNOS mRNA transcription. LPS-induced upregulation of MEKK/MAP tyrosine kinase upregulates NADPH oxidase activity and oxygen free radical formation required for activation of NFkappaB and AP-1 and phosphorylation of IkappaBalpha. LPS downregulates endogenous calcium-sensitive PKC isozymes (PKCdelta), which repress iNOS mRNA expression. ETOH inhibits LPS-induced upregulation of iNOS mRNA by preventing its ability to decrease PKCdelta and upregulate tyrosine kinase-mediated phosphorylation. This effect of ETOH is prevented by inhibitors of PKC and tyrosine kinase. The data support the hypothesis that ETOH inhibits LPS-induced upregulation of iNOS mRNA by interfering with the phosphorylation processes involved in activation of the nuclear transcription factors NFkappaB and AP-1.

Ethanol metabolism is not required for inhibition of LPS-stimulated transcription of inducible nitric oxide synthase.

We examined the effect of inhibition of ethanol metabolism on ethanol-mediated suppression of Escherichia coli endotoxin (LPS-induced upregulation of transcription and release of inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNFalpha) from rat alveolar macrophages (AM) in vivo. Ethanol (3.45 and 5.5 g/kg/IP) and t-butanol (3.7 g/kg, IP), given 30 min before intratracheal administration of LPS (1.0 mg/kg), inhibited the upregulation of iNOS mRNA and protein, determined by competitor equalized RT-PCR and Western immunoblot, respectively, but not TNFalpha mRNA in AM obtained 2 h after LPS administration by bronchoalveolar lavage (BAL). However, ethanol and t-butanol inhibited LPS-stimulated nitrate and nitrite (RNI) and TNFalpha protein in BAL fluid. Pretreatment of rats with 4-methylpyrazole (100 mg/kg, IP) 2 h before, or disulfiram 30 min before, administration of ethanol (3.45 g/kg, IP) failed to attenuate the inhibitory effect on iNOS mRNA or protein. t-Butyl hydroperoxide (100 mg/kg, IP) given to rats 30 min before administration of LPS enhanced LPS-mediated upregulation of iNOS mRNA and TNFalpha protein in AM and BAL fluid. The inhibitory effect of ethanol on iNOS mRNA was not mediated by an interaction with elevated levels of circulating corticosterone because pretreatment of rats with RU-38486 (100 mg/kg, IM), which inhibited prednisolone (50 mg/kg, IM), induced suppression of LPS-stimulated iNOS mRNA, and failed to attenuate ethanol-mediated inhibition of LPS-stimulated iNOS mRNA in AM. We conclude that metabolism of ethanol to acetaldehyde via alcohol dehydrogenase is not required for ethanol-mediated suppression of LPS-induced iNOS transcription and TNFalpha synthesis/release in AM. Moreover, an interaction of ethanol or acetaldehyde with circulating corticosterone is not involved in ethanol-mediated attenuation of LPS-stimulated iNOS mRNA or protein or TNFalpha protein in the lung. Speculatively, because oxidation of t-butanol to t-butylhydroperoxide results in activation, rather than inhibition, of iNOS and TNF-alpha, the reported ethanol-mediated enhancement of iNOS mRNA may result from the action of the hydroxyethyl radical.

Coronary vasodilator responses to bradykinin in euglycemic and diabetic rats

Diabetes mellitus is associated with endothelial dysfunction that is believed to result in impaired release of vasoconstrictor and vasodilator substances from the endothelium and thereby diminished reactivity of many vascular beds. This study was designed to characterize bradykinin (BK)-induced coronary vasodilation in normal and diabetic rats. Bradykinin-stimulated vasodilation of the rat coronary vasculature is mediated by a cytochrome P450-1A (CYP-1A)- inhibitable metabolite that activates KCa, but not KATP, channels on the coronary vascular smooth muscle. Although BK stimulates the release of nitric oxide from the vascular endothelium, the released nitric oxide and its ability to stimulate guanylate cyclase only modulates the duration of, rather than the magnitude of, BK-induced coronary vasodilation. Twelve weeks of streptozotocin-induced diabetes did not affect the coronary vascular responses to BK or the components that mediate BK-induced vasodilation (ie, K-channel activation, nitric oxide-guanylate cyclase). The data support the conclusions that the coronary vasodilator response of the rat to BK is CYP-1A and KCa-channel mediated, that coreleased nitric oxide only modulates the duration of BK-induced vasodilation, and that these mechanisms are unaffected by moderate diabetes.