Sodium channels are required during in vivo sodium chloride hyperosmolarity to stimulate increase in intestinal endothelial nitric oxide production

NaCl hyperosmolarity increases intestinal blood flow during food absorption due in large part to increased NO production. We hypothesized that in vivo, sodium ions enter endothelial cells during NaCl hyperosmolarity as the first step to stimulate an increase in intestinal endothelial NO production. Perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature at rest and under hyperosmotic conditions, 330 and 380 mosM, respectively, before and after application of bumetanide (Na(+)-K(+)-2Cl(-) cotransporter inhibitor) or amiloride (Na(+)/H(+) exchange channel inhibitor). Suppressing amiloride-sensitive Na(+)/H(+) exchange channels diminished hypertonicity-linked increases in vascular [NO], whereas blockade of Na(+)-K(+)-2Cl(-) channels greatly suppressed increases in vascular [NO] and intestinal blood flow. In additional experiments we examined the effect of sodium ion entry into endothelial cells. We proposed that the Na(+)/Ca(2+) exchanger extrudes Na(+) in exchange for Ca(2+), thereby leading to the calcium-dependent activation of endothelial nitric oxide synthase (eNOS). We blocked the activity of the Na(+)/Ca(2+) exchanger during 360 mosM NaCl hyperosmolarity with KB-R7943; complete blockade of increased vascular [NO] and intestinal blood flow to hyperosmolarity occurred. These results indicate that during NaCl hyperosmolarity, sodium ions enter endothelial cells predominantly through Na(+)-K(+)-2Cl(-) channels. The Na(+)/Ca(2+) exchanger then extrudes Na(+) and increases endothelial Ca(2+). The increase in endothelial Ca(2+) causes an increase in eNOS activity, and the resultant increase in NO increases intestinal arteriolar diameter and blood flow during NaCl hyperosmolarity. This appears to be the major mechanism by which intestinal nutrient absorption is coupled to increased blood flow.     

Hippocampal functional hyperemia mediated by NMDA receptor/NO signaling in rats during mild exercise

Current studies have demonstrated that exercise increases regional cerebral blood flow (rCBF), an index of neuronal activity. However, neuronal regulation of the increased rCBF in the brain parenchyma is poorly understood. We developed a running model with rats for monitoring hippocampal cerebral blood flow (Hip-CBF) and found that mild treadmill running increases Hip-CBF in a tetrodotoxin-dependent manner, suggesting that functional hyperemia, an increase in rCBF in response to neuronal activation, occurs in the running rat's hippocampus (Nishijima T and Soya H. Neurosci Res 54: 186-191, 2006). To further support our hypothesis, it was important to discover the neurogenic pathways behind the increase in Hip-CBF that occurred during running. Here, we examine the possible role of N-methyl-d-aspartate (NMDA) receptor/nitric oxide (NO) signaling and group I metabotropic glutamate receptors in mediating the Hip-CBF increase. Hip-CBF during running was measured by laser-Doppler flowmetry. Intrahippocampal drug administration was performed by microdialysis. Mild treadmill running (10 m/min) increased Hip-CBF, which was remarkably attenuated by either NMDA receptor antagonists (1 mM MK-801) or NO synthase inhibitors (2 mM N(G)-nitro-l-arginine methyl ester). However, group I metabotropic glutamate receptor antagonists {1 mM 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester + 1 mM 2-methyl-6-(phenylethynyl)pyridine hydrochloride} augmented the running-induced Hip-CBF increase. We also found that rCBF in the olfactory bulb was unchanged with running. These results strongly suggest that Hip-CBF during mild exercise is regulated locally under hippocampal neuronal activity, mediated mainly through NMDA receptor/NO signaling. Collectively, these results, together with our previous findings, support our hypothesis that mild exercise elicits neuronal activation, which then triggers functional hyperemia in the rat hippocampus.     

Chemistry of the diazeniumdiolates. I. Structural and spectral characteristics of the [N(O)NO]- functional group.

Ions of structure X[N(O)NO]-, examples of which have seen increasing use as probes for studying the biology of nitric oxide (NO) over the past decade, have a varied chemical history spanning nearly two centuries. Nevertheless, they have not been widely appreciated for their physicochemical similarities. Here we begin a series of systematic inquiries into the fundamental chemistry of such compounds aimed at identifying both the characteristics that justify considering them as a group and the factors that contribute to observed differences in their physicochemical properties. In the present paper, X-ray structures in which X is SO3- (1), O- (2), Ph (3), and Et2N (5), as well as that of the gem-disubstituted carbon derivative CH2[N(O)NO]2-(2) (4), are compared. All their O-N-N-O systems are essentially planar, with cis oxygens and an N-N linkage exhibiting considerable double-bond character. The ultraviolet spectrum of the isolated chromophore consists of a relatively intense ( approximately 6-10 mM(-1) x cm(-1) per [N(O)NO]- group) absorption at 248-250 nm (for 2 and 5) that is red shifted by through-space Stark interactions (e.g., by approximately 10 nm in 1 and 4) as well as by conjugative interaction with X (lambda(max) = 284 nm for 3). Infrared and Raman spectra for the widely used pharmacological probe 5 were determined, with analysis of vibrational modes being aided by comparison with the spectra of the [15N(O)15NO]- isotopomer and density functional theory calculations at the B3LYP/6-311++G** level. To address confusion that has arisen in the literature resulting from rather widespread use of differing trivial designations for this class of compounds, a unifying nomenclature system is recommended in which compounds containing the [N(O)NO]- moiety are named as diazeniumdiolates. It is hoped that these and other efforts to understand and predict the physicochemical similarities and differences among different members of the diazeniumdiolate class will aid in reaping their full potential in the area of rational drug design.     

Lactate dilates cochlear capillaries via type V fibrocyte-vessel coupling signaled by nNOS

Transduction of sound in the inner ear demands tight control over delivery of oxygen and glucose. However, the mechanisms underlying the control of regional blood flow are not yet fully understood. In this study, we report a novel local control mechanism that regulates cochlear blood flow to the stria vascularis, a high energy-consuming region of the inner ear. We found that extracellular lactate had a vasodilatory effect on the capillaries of the spiral ligament under both in vitro and in vivo conditions. The lactate, acting through monocarboxylate transporter 1 (MCT1), initiated neuronal nitric oxide (NO) synthase (nNOS) and catalyzed production of NO for the vasodilation. Blocking MCT1 with the MCT blocker, α-cyano-4-hydroxycinnamate (CHC), or a suppressing NO production with either the nonspecific inhibitor of NO synthase, N(G)-nitro-L-arginine methyl ester (L-NAME), or either of two selective nNOS inhibitors, 3-bromo-7-nitroindazole or (4S)-N-(4-amino-5[aminoethyl]aminopentyl)-N'-nitroguanidine (TFA), totally abolished the lactate-induced vasodilation. Pretreatment with the selective endothelial NO synthase inhibitor, L-N(5)-(1-iminoethyl)ornithine (L-NIO), eliminated the inhibition of lactate-induced vessel dilation. With immunohistochemical labeling, we found the expression of MCT1 and nNOS in capillary-coupled type V fibrocytes. The data suggest that type V fibrocytes are the source of the lactate-induced NO. Cochlear microvessel tone, regulated by lactate, is mediated by an NO-signaled coupling of fibrocytes and capillaries.     

Nitric oxide regulates actin reorganization through cGMP and Ca(2+)/calmodulin in RAW 264.7 cells

Nitric oxide (NO) has been reported to be involved in the regulation of pseudopodia formation, phagocytosis and adhesion in macrophages through the reorganization of actin. In the present study, we directly separated the globular (G) and filamentous (F) actin from quiescent or NO-stimulated macrophage-like cell line RAW 264.7 cells in order to investigate the dynamic redistribution of actin pools. We also focused on the regulatory mechanisms of actin assembly, induced by NO and its possible subsequent signaling pathway. We showed that predominant G-actin coexisted with Triton X-100-insoluble filamentous (TIF) and Triton X-100-soluble filamentous actin in resting RAW 264.7 cells. The exogenous NO produced by (+/-)-(E)-2-[(E)-hydroxyimino]-6-methoxy-4-methyl-5-nitro-3-hexenamide (NOR1), the endogenous NO induced by lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma), and dibutyryl-cGMP increased the contents of TIF-actin in dose- and time-dependent manners and altered its morphology. The increase in the TIF-actin contents induced by NOR1 or LPS plus IFNgamma was efficiently blocked by the radical scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or the arginine analogue N(G)-monomethyl-L-arginine acetate, respectively. Preincubation with the calmodulin antagonist W-7 almost completely blocked the NO-induced TIF-actin increase and morphological change. On the other hand, preincubation with C3 transferase, an inhibitor of Rho protein, efficiently prevented the change in cell morphology, but had no effect on the TIF-actin increase. We postulate that cGMP and subsequent Ca(2+)/calmodulin may be key regulators of actin reorganization in NO-stimulated RAW 264.7 cells.     

Time course of nitric oxide production after endotoxin challenge in mice

Nitric oxide (NO) regulates numerous processes during endotoxemia and inflammation. However, the sequential changes in whole body (Wb) nitric oxide (NO) production during endotoxemia in vivo remain to be clarified. Male Swiss mice were injected intraperitoneally with saline (control group) or lipopolysaccharide (LPS group). After 0, 2, 4, 6, 9, 12, and 24 h, animals received a primed constant infusion of L-[guanidino-(15)N(2)-(2)H(2)]arginine, L-[ureido-(15)N]citrulline, L-[5-(15)N]glutamine, and L-[ring-(2)H(5)]phenylalanine in the jugular vein. Arterial blood was collected for plasma arginine (Arg), citrulline (Cit), glutamine (Gln), and phenylalanine (Phe) concentrations and tracer-to-tracee ratios. NO production was calculated as plasma Arg-to-Cit flux, Wb de novo Arg synthesis as plasma Cit-to-Arg flux, and Wb protein breakdown as plasma Phe flux. LPS reduced plasma Arg and Cit and increased Gln and Phe concentrations. Two peaks of NO production were observed at 4 and 12 h after LPS. Although LPS did not affect total Arg production, de novo Arg production decreased after 12 h. The second peak of NO production coincided with increased Wb Cit, Gln, and Phe production. In conclusion, the curve of NO production in both early and late phases of endotoxemia is not related to plasma Arg kinetics. However, because Wb Cit, Gln, and Phe fluxes increased concomitantly with the second peak of NO production, NO production is probably related to the catabolic phase of endotoxemia.     

Nitric oxide synthase inhibition and glutamate binding in quinolinate-lesioned rat hippocampus

The effect of lesions induced by bilateral intracerebroventricular (i.c.v.) injection of quinolinate (250 nmol of QUIN/ventricle), a selective N-methyl-D-aspartate (NMDA) receptor agonist, on [3H]glutamate ([3H]Glu) binding to the main types of both ionotropic and metabotropic glutamate receptors (iGluR and mGluR) was investigated in synaptic membrane preparations from the hippocampi of 50-day-old rats. The membranes from QUIN injured brains revealed significantly lowered binding in iGluR (by 31%) as well as in mGluR (by 22%) as compared to the controls. Using selected glutamate receptor agonists as displacers of [3H]Glu binding we found that both the NMDA-subtype of iGluR and group I of mGluR are involved in this decrease of binding. Suppression of nitric oxide (NO) production by N(G)-nitro-L-arginine (50 nmol of NARG/ventricle) or the increase of NO generation by 3-morpholinylsydnoneimine (5 nmol of SIN-1/ventricle) failed to alter [3H]Glu or [3H]CPP (3-((D)-2-carboxypiperazin-4-yl)-[1,2-(3)H]-propyl-1-phosphonic acid; NMDA-antagonist) binding declines caused by QUIN-lesions. Thus, our findings indicate that both the NMDA-subtype of iGluR and group I of mGluR are susceptible to the QUIN-induced neurodegeneration in the rat hippocampus. However, the inhibition of NO synthesis did not reveal any protective action in the QUIN-evoked, NMDA-receptor mediated decrease of [3H]Glu binding. Therefore, the additional mechanisms of QUIN action, different from direct NMDA receptor activation/NO production (e.g. lipid peroxidation induced by QUIN-Fe-complexes) cannot be excluded.     

Renal cortical and medullary blood flow responses to L-NAME and ANG II in wild-type, nNOS null mutant, and eNOS null mutant mice

Experiments in wild-type (WT; C57BL/6J) mice, endothelial nitric oxide synthase null mutant [eNOS(-/-)] mice, and neuronal NOS null mutant [nNOS(-/-)] mice were performed to determine which NOS isoform regulates renal cortical and medullary blood flow under basal conditions and during the infusion of ANG II. Inhibition of NOS with N(omega)-nitro-l-arginine methyl ester (l-NAME; 50 mg/kg iv) in Inactin-anesthetized WT and nNOS(-/-) mice increased arterial blood pressure by 28-31 mmHg and significantly decreased blood flow in the renal cortex (18-24%) and the renal medulla (13-18%). In contrast, blood pressure and renal cortical and medullary blood flow were unaltered after l-NAME administration to eNOS(-/-) mice, indicating that NO derived from eNOS regulates baseline vascular resistance in mice. In subsequent experiments, intravenous ANG II (20 ng x kg(-1) x min(-1)) significantly decreased renal cortical blood flow (by 15-25%) in WT, eNOS(-/-), nNOS(-/-), and WT mice treated with l-NAME. The infusion of ANG II, however, led to a significant increase in medullary blood flow (12-15%) in WT and eNOS(-/-) mice. The increase in medullary blood flow following ANG II infusion was not observed in nNOS(-/-) mice, in WT or eNOS(-/-) mice pretreated with l-NAME, or in WT mice administered the nNOS inhibitor 5-(1-imino-3-butenyl)-l-ornithine (1 mg x kg(-1) x h(-1)). These data demonstrate that NO from eNOS regulates baseline blood flow in the mouse renal cortex and medulla, while NO produced by nNOS mediates an increase in medullary blood flow in response to ANG II.     

NOS3 is involved in the increased protein and arginine metabolic response in muscle during early endotoxemia in mice

Sepsis is a severe catabolic condition. The loss of skeletal muscle protein mass is characterized by enhanced release of the amino acids glutamine and arginine, which (in)directly affects interorgan arginine and the related nitric oxide (NO) synthesis. To establish whether changes in muscle amino acid and protein kinetics are regulated by NO synthesized by nitric oxide synthase-2 or -3 (NOS2 or NOS3), we studied C57BL6/J wild-type (WT), NOS2-deficient (NOS2-/-), and NOS3-deficient (NOS3-/-) mice under control (unstimulated) and lipopolysaccharide (LPS)-treated conditions. Muscle amino acid metabolism was studied across the hindquarter by infusing the stable isotopes L-[ring-2H5]phenylalanine, L-[ring-2H2]tyrosine, L-[guanidino-15N2]arginine, and L-[ureido-13C,2H2]citrulline. Muscle blood flow was measured using radioactive p-aminohippuric acid dilution. Under baseline conditions, muscle blood flow was halved in NOS2-/- mice (P < 0.1), with simultaneous reductions in muscle glutamine, glycine, alanine, arginine release and glutamic acid, citrulline, valine, and leucine uptake (P < 0.1). After LPS treatment, (net) muscle protein synthesis increased in WT and NOS2-/- mice [LPS vs. control: 13 +/- 3 vs. 8 +/- 1 (SE) nmol.10 g(-1).min(-1) (WT), 18 +/- 5 vs. 7 +/- 2 nmol.10 g(-1).min(-1) (NOS2-/-); P < 0.05 for LPS vs. control]. This response was absent in NOS3-/- mice (LPS vs. control: 11 +/- 4 vs. 10 +/- 2 nmol.10 g(-1).min(-1)). In agreement, the increase in muscle arginine turnover after LPS was also absent in NOS3-/- mice. In conclusion, disruption of the NOS2 gene compromises muscle glutamine release and muscle blood flow in control mice, but had only minor effects after LPS. NOS3 activity is crucial for the increase in muscle arginine and protein turnover during early endotoxemia.     

Nitric oxide-cGMP-mediated vasoconstriction and effects of acetylcholine in the branchial circulation of the eel

Information about the presence and effects of nitric oxide (NO) in fish vasculature is scant and contradictory. We have studied the NO/cGMP system in the branchial circulation of the teleost Anguilla anguilla using a branchial basket preparation under basal conditions and cholinergic stimulation. The effects of endogenous and exogenous NO were tested with L-arginine, the nitric oxide synthase (NOS) substrate, and the NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), respectively. L-arginine (from 10(-11) to 10(-6) M) and the NO donors (starting from 10(-14) M) caused dose-dependent vasoconstriction. Conversely, in the ACh-pre-contracted preparations both donors elicited vasodilation. SIN-1-induced vasoconstriction was due to NO generation: it was increased by superoxide dismutase (SOD) and blocked by NO scavenger hemoglobin. Pre-treatment with sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) inhibited the effects of SIN-1 and SNP. The stable cGMP analogue 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br cGMP) induced dose-dependent vasoconstriction. Unexpectedly, three NOS inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-monomethyl-L-arginine (L-NMMA), L-N(5)-(1-iminoethyl) ornithine (L-NIO), caused mild vasoconstriction. ACh caused vasoconstriction, but at pico- and nanomolar concentrations it caused mild but significant vasodilation in 40% of the preparations. Both responses, blocked by atropine and pirenzepine, required an intact endothelium. The ACh-induced vasoconstriction was substantially independent of a NO-cGMP mechanism.     

Major role for neuronal NO synthase in curtailing choroidal blood flow autoregulation in newborn pig.

We examined whether nitric oxide (NO) generated from neuronal NO synthase (nNOS) contributes to the reduced ability of the newborn to autoregulate retinal blood flow (RBF) and choroidal blood flow (ChBF) during acute rises in perfusion pressure. In newborn pigs (1-2 days old), RBF (measured by microsphere) is autoregulated over a narrow range of perfusion pressure, whereas ChBF is not autoregulated. N(G)-nitro-L-arginine methyl ester (L-NAME) or specific nNOS inhibitors 7-nitroindazole, 3-bromo-7-nitroindazole, and 1-(2-trifluoromethyl-phenyl)imidazole as well as ganglionic blocker hexamethonium, unveiled a ChBF autoregulation as observed in juvenile (4- to 6-wk old) animals, whereas autoregulation of RBF in the newborn was only enhanced by L-NAME. All NOS inhibitors and hexamethonium prevented the hypertension-induced increase in NO mediator cGMP in the choroid. nNOS mRNA expression and activity were three- to fourfold higher in the choroid of newborn pigs than in tissues of juvenile pigs. It is concluded that increased production of NO from nNOS curtails ChBF autoregulation in the newborn and suggests a role for the autonomic nervous system in this important hemodynamic function, whereas, for RBF autoregulation, endothelial NOS seems to exert a more important contribution in limiting autoregulation.     

Isotope effects and intermediates in the reduction of NO by P450(NOR)

The mechanism of the heme-thiolate-dependent NADH-NO reductase (P450(NOR)) from Fusarium oxysporum was investigated by kinetic isotope effects including protio, [4S-2H]-, [4R-2H]-, [4,4(2)H(2)]-NADH and stopped-flow measurements. The respective kinetic isotope effects were measured at high NO concentrations and were found to be 1.7, 2.3 and 3.8 indicating a rate-limitation at the reduction step and a moderate stereoselectivity in binding of the cofactor NADH. In a different approach the kinetic isotope effects were determined directly for the reaction of the Fe(III)-NO complex with [4R-2H]- and [4S-2H]-NADH by stopped-flow spectroscopy. The resulting isotope effects were 2.7+/-0.4 for the R-form and 1.1+/-0.1 for the S-form. In addition the 444 nm intermediate could be chemically generated by addition of an ethanolic borohydride solution to the ferric-NO complex at -10 degrees C. In pulse radiolysis experiments a similar absorbing species could be observed when hydroxylamine radicals were generated in the presence of Fe (III) P450(NOR). Based on these results we postulate hydride transfer from NADH to the ferric P450-NO complex resulting in a ferric hydroxylamine-radical or ferryl hydroxylamine-complex and this step, as indicated by the kinetic isotope effects, to be rate-limiting at high concentrations of NO. However, at low concentrations of NO the decay of the 444 nm species becomes the rate-limiting step as envisaged by stopped-flow and optical kinetic measurements in a system in which NO was continuously generated. The last step in the catalytic cycle may proceed by a direct addition of the NO radical to the Fe-hydroxylamine complex or by electron transfer from the NO radical to the ferric-thiyl moiety in analogy to the postulated mechanisms of prostacyclin and thromboxane biosynthesis by the corresponding P450 enzymes. The latter process of electron transfer could then constitute a common step in all heme-thiolate catalyzed reactions.     

Hemodynamic and renal effects of acute and progressive nitric oxide synthesis inhibition in anesthetized dogs

This study evaluated the effects of progressive nitric oxide (NO) inhibition in the regulation of systemic and regional hemodynamics and renal function in anesthetized dogs. The N(G)-nitro-L-arginine methyl ester group (n = 9) received progressive doses of 0.1, 1, 10, and 50 microg. kg(-1). min(-1). Renal (RBF), mesenteric (MBF), iliac (IBF) blood flows, mean arterial pressure (MAP), pulmonary pressures, cardiac output (CO), and systemic and pulmonary vascular resistances were measured. During N(G)-nitro-L-arginine methyl ester infusion, MAP and systemic vascular resistances increased in a dose-dependent manner. Mean pulmonary pressure and pulmonary vascular resistances increased in both the N(G)-nitro-L-arginine methyl ester and the control group, but the increase was more marked in the N(G)-nitro-L-arginine methyl ester group during the last two infusion periods. CO decreased progressively, before any significant change in blood pressure was noticeable in the N(G)-nitro-L-arginine methyl ester group. IBF decreased significantly from the first N(G)-nitro-L-arginine methyl ester dose, whereas RBF and MBF only decreased significantly during the highest N(G)-nitro-L-arginine methyl ester dose. Urinary volume and sodium excretion only increased significantly in the time control group during the two last time periods. The pulmonary vasculature was more sensitive than the systemic vasculature, whereas skeletal muscle and renal vasculatures showed a greater sensitivity to the inhibition of NO production than the mesenteric vasculature. NO synthesis inhibition induces a progressive antidiuretic and antinatriuretic effect, which is partially offset by the increase in blood pressure.     

Denitrosation of carcinostatic nitrosoureas by purified NADPH cytochrome P-450 reductase and rat liver microsomes to yield nitric oxide under anaerobic conditions

The nitrosoureas, CCNU (1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea) and BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) are representatives of a class of N-nitroso compounds which undergo denitrosation in the presence of NAD(P)H and deoxygenated hepatic microsomes from rats to yield nitric oxide (NO) and the denitrosated parent compound. Formation of NO during microsomal denitrosation of CCNU and BCNU was determined by three methods. With one procedure, NO was measured and concentration shown to increase over time in the head gas above microsomal incubations with BCNU. Two additional methods utilized NO binding to either ferrous cytochrome P-450 or hemoglobin to form distinct Soret maxima at 444 and 415 nm, respectively. Incubation of either BCNU or CCNU in the presence of NAD(P)H and deoxygenated microsomes resulted in the formation of identical cytochrome P-450 ferrous · NO optical difference spectra. Determination of the P-450 ferrous · NO extinction coefficient by the change in absorbance at 444 minus 500 nm allowed measurement of rates of denitrosation by monitoring the increase in absorbance at 444 nm. The rates of BCNU and CCNU denitrosation were determined to be 4.8 and 2.0 nmol NO/min/mg protein, respectively, for phenobarbital (PB) induced microsomes. For the purpose of comparison, the rate of [¹⁴C]CCNU (1-(2-[¹⁴C]chloroethyl)-3-(cyclohexyl)-1-nitrosourea turnover was examined by the isolation of [¹⁴C]CCU (1-(2-[¹⁴C] chloroethyl)-3-(cyclohexyl)-1-urea) from incubations that contained NADPH and deoxygenated PB-induced microsomes. These analyses showed stoichiometric amounts of NO and [¹⁴C]CCU being formed at a rate of 2.0 nmol/min/mg protein. Denitrosation catalysis by microsomes was enhanced by phenobarbital pretreatment and partially decreased by cytochrome P-450 inhibitors, SKF-525A, α-naphthoflavone (ANF), metyrapone, and CO, suggesting a cytochrome P-450-dependent denitrosation. However, in the presence of NADPH and purified NADPH cytochrome P-450 reductase reconstituted in dilauroylphosphatidylcholine, [¹⁴C]CCNU was shown to undergo denitrosation to [¹⁴C]CCU. Thus, NADPH cytochrome P-450 reductase could support denitrosation in the absence of cytochrome P-450.     

Contribution of adenosine to compensatory dilation in hypoperfused contracting human muscles is independent of nitric oxide

We previously demonstrated that nitric oxide (NO) contributes to compensatory vasodilation in the contracting human forearm subjected to acute hypoperfusion. We examined the potential role of an adenosine-NO interaction to this response in 17 male subjects (25 ± 2 yr). In separate protocols subjects performed rhythmic forearm exercise (20% of maximum) while hypoperfusion was evoked by balloon inflation in the brachial artery above the elbow. Each trial included exercise before inflation, exercise with inflation, and exercise after deflation (3 min each). Forearm blood flow (FBF; ultrasound) and local [brachial artery catheter pressure (BAP)] and systemic [mean arterial pressure (MAP); Finometer] arterial pressure were measured. In protocol 1 (n = 10), exercise was repeated during nitric oxide synthase inhibition [N(G)-monomethyl-L-arginine (L-NMMA)] alone and during L-NMMA-aminophylline (adenosine receptor blockade) administration. In protocol 2, exercise was repeated during aminophylline alone and during aminophylline-L-NMMA. Forearm vascular conductance (FVC; ml·min(-1)·100 mmHg(-1)) was calculated from blood flow (ml/min) and BAP (mmHg). Percent recovery in FVC during inflation was calculated as (steady-state inflation + exercise value - nadir)/[steady-state exercise (control) value - nadir]. In protocol 1, percent recovery in FVC was 108 ± 8% during the control (no drug) trial. Percent recovery in FVC was attenuated with inhibition of NO formation alone (78 ± 9%; P < 0.01 vs. control) and was attenuated further with combined inhibition of NO and adenosine (58 ± 9%; P < 0.01 vs. L-NMMA). In protocol 2, percent recovery was reduced with adenosine receptor blockade (74 ± 11% vs. 113 ± 6%, P < 0.01) compared with control drug trials. Percent recovery in FVC was attenuated further with combined inhibition of adenosine and NO (48 ± 11%; P < 0.05 vs. aminophylline). Our data indicate that adenosine contributes to compensatory vasodilation in an NO-independent manner during exercise with acute hypoperfusion.     

Hypotensive mechanism of [Leu13]motilin in dogs in vivo and in vitro.

The effects of [Leu13]motilin were examined in vivo after its intravenous administration into anesthetized dogs and in vitro with isolated preparations of canine mesenteric artery. [Leu13]Motilin (0.1-10 nmol x kg(-1), i.v.) induced both strong and clustered phasic contractions in the gastric antrum and duodenum. At doses of over 1 nmol x kg(-1), [Leu13]motilin also produced transient decreases in arterial blood pressure, left ventricular pressure, maximum rate of rise of left ventricular pressure, and total peripheral resistance, and an increase in aortic blood flow and heart rate. A selective motilin antagonist, GM-109 (Phe-cyclo[Lys-Tyr(3-tBu)-betaAla] trifluoroacetate), completely abolished the gastric antrum and duodenal motor responses induced by [Leu13]motilin. In contrast, hypotension induced by [Leu13]motilin (1 nmol x kg(-1)) was unchanged in the presence of GM-109. In isolated mesenteric artery preparations precontracted with U-46619 (10(-7) M), [Leu13]motilin (10(-8)-10(-5) M) induced an endothelium-dependent relaxation, and this was inhibited by a pretreatment with N(omega)-nitro-L-arginine, a competitive inhibitor of NO synthase (10(-4) M). A high dose (10(-4) M) of GM-109 slightly decreased [Leu13]motilin-induced relaxation, and shifted the concentration-response curve of [Leu13]motilin to the right. However, the pA2 value (4.09) of GM-109 for [Leu13]motilin in the present study was conspicuously lower than that previously demonstrated in the rabbit duodenum (7.37). These results suggest that [Leu13]motilin induces hypotension via the endothelial NO-dependent relaxation mechanism and not through the receptor type that causes upper gastrointestinal contractions.     

Nitrite is an alternative source of NO in vivo

In this study, we investigated whether orally administered nitrite is changed to NO and whether nitrite attenuates hypertension in a dose-dependent manner. We utilized a stable isotope of [15N]nitrite (15NO2-) as a source of nitrite to distinguish between endogenous nitrite and that exogenously administered and measured hemoglobin (Hb)-NO as an index of circulating NO in whole blood using electron paramagnetic resonance (EPR) spectroscopy. When 1 mg/kg Na15NO2 was orally administered to rats, an apparent EPR signal derived from Hb15NO (A(Z) = 23.4 gauss) appeared in the blood. The peak blood HbNO concentration occurred at the first measurement after intake (5 min) for treatment with 1 and 3 mg/kg (HbNO: 4.93 +/- 0.52 and 10.58 +/- 0.40 microM, respectively) and at 15 min with 10 mg/kg (HbNO: 38.27 +/- 9.23 microM). In addition, coadministration of nitrite (100 mg/l drinking water) with N(omega)-nitro-L-arginine methyl ester (L-NAME; 1 g/l) for 3 wk significantly attenuated the L-NAME-induced hypertension (149 +/- 10 mmHg) compared with L-NAME alone (170 +/- 13 mmHg). Furthermore, this phenomenon was associated with an increase in circulating HbNO. Our findings clearly indicate that orally ingested nitrite can be an alternative to L-arginine as a source of NO in vivo and may explain, at least in part, the mechanism of the nitrite/nitrate-rich Dietary Approaches to Stop Hypertension diet-induced hypotensive effects.     

Identification of manganese superoxide dismutase as a NO-regulated gene in rat glomerular mesangial cells by 2D gel electrophoresis.

The course of inflammatory glomerular diseases is accompanied by changes in the expression of matrix-associated proteins, growth factors, and mediators in renal mesangial cells. Furthermore, the production of nitric oxide (NO) by the inducible isoform of nitric oxide synthase (iNOS) is enhanced after stimulation with pro-inflammatory cytokines. NO has been demonstrated to be a potent modulator of gene expression. To identify NO-regulated genes, we compared the expression patterns of mesangial cells treated for 24h with 500 microM (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) with those of un-stimulated controls by applying a proteomics approach. One protein found to be NO-modulated by 2D gel electrophoresis is the manganese superoxide dismutase (Mn-SOD). Immunoblot and Northern blot analysis demonstrated a dose- and time-dependent induction of Mn-SOD expression by S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and DETA-NO on both the protein and the mRNA levels. An upregulation of Mn-SOD expression by NO was accompanied by an increased Mn-SOD activity. Immunoblots of extracts of IL-1beta-treated cells cultivated with or without the iNOS inhibitor N(G)-monomethyl-L-arginine and the inhibitor of soluble guanylyl cyclase (sGC) 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) demonstrated that the upregulation of the Mn-SOD by NO is due to a NO-dependent activation of sGC. The upregulation of Mn-SOD mRNA expression by NO was confirmed in vivo by Northern blot analysis in kidneys from rats treated with lipopolysaccharide (LPS) either in presence or absence of the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (l-NIL).     

NO production by cNOS and iNOS reflects blood pressure changes in LPS-challenged mice

Increased nitric oxide (NO) production is the cause of hypotension and shock during sepsis. In the present experiments, we have measured the contribution of endothelial (e) and inducible (i) nitric oxide synthase (NOS) to systemic NO production in mice under baseline conditions and upon LPS treatment (100 microg/10 g ip LPS). NO synthesis was measured by the rate of conversion of l-[guanidino-15N2]arginine to l-[ureido-15N]citrulline, and the contribution of the specific NOS isoforms was evaluated by comparing NO production in eNOS-deficient [(-/-)] and iNOS(-/-) mice with that in wild-type (WT) mice. Under baseline conditions, NO production was similar in WT and iNOS(-/-) mice but lower in eNOS(-/-) mice [WT: 1.2 +/- 0.2; iNOS(-/-): 1.2 +/- 0.2; eNOS(-/-): 0.6 +/- 0.3 nmol. 10 g body wt-1. min-1]. In response to the challenge with LPS (5 h), systemic NO production increased in WT and eNOS(-/-) mice but fell in iNOS(-/-) mice [WT: 2.7 +/- 0.3; eNOS(-/-): 2.2 +/- 0.6; iNOS(-/-): 0.7 +/- 0.1 nmol. 10 g body wt-1. min-1]. After 5 h of LPS treatment, blood pressure had dropped 14 mmHg in WT but not in iNOS(-/-) mice. The present findings provide firm evidence that, upon treatment with bacterial LPS, the increase of NO production is solely dependent on iNOS, whereas that mediated by cNOS is reduced. Furthermore, the data show that the LPS-induced blood pressure response is dependent on iNOS.     

Isotopic determination of amino acid-urea interactions in exercise in humans

We recently reported that in light exercise (30% VO2max) the oxidation of [1-13C]leucine was significantly increased but the rate of urea production was unchanged (J. Appl. Physiol: Respirat. Environ. Exercise Physiol. 52: 458-466, 1982). We have therefore tested three possible explanations for this apparent incongruity. 1) We infused NaH13CO3 throughout rest and exercise and found that, although altered bicarbonate kinetics in exercise resulted in greater recovery of 13CO2, the difference between rest and recovery was small compared with the increase in the rate of 13CO2 excretion during exercise when [1-13C]leucine was infused. 2) We infused [15N]leucine and isolated plasma urea N to determine directly the rate of incorporation of the 15N. During exercise there was no increase in the rate of 15N incorporation. Simultaneously, we infused [2,3-13C]alanine and quantified the rate of incorporation of 15N in alanine. We found that [15N]alanine production from [15N] leucine more than doubled in exercise, and by deduction, alanine production from other amino acids also doubled. 3) We tested our previous assumption that [1-13C]leucine metabolism in exercise was representative of the metabolism of other essential amino acids by infusing [1-13C] and [alpha-15N]lysine throughout rest and exercise. We found that the rate of breakdown of lysine during exercise was not increased in a manner comparable to that of leucine. Thus, these data confirm our original findings that leucine decarboxylation is enhanced in light exercise but urea production is unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)