Non-selective NOS Inhibitor L-NAME Research Articles

The Muscarinic Acetylcholine M2 Receptor-Induced Nitration of p190A by eNOS Increases RhoA Activity in Cardiac Myocytes.

p190RhoGAP, which exists in two paralogs, p190RhoGAP-A (p190A) and p190RhoGAP-B (p190B), is a GTPase activating protein (GAP) contributing to the regulation of the cellular activity of RhoGTPases. Recent data showed that M2 muscarinic acetylcholine receptor (M2R) stimulation in neonatal rat cardiac myocytes (NRCM) induces the binding of p190RhoGAP to the long isoform of the regulator of G protein signaling 3 (RGS3L). This complex formation alters the substrate preference of p190RhoGAP from RhoA to Rac1. By analyzing carbachol-stimulated GAP activity, we show herein that p190A, but not p190B, alters its substrate preference in NRCM. Based on data that the RhoGAP activity of p190A in endothelial cells is diminished upon nitration by endothelial nitric oxide synthase (eNOS)-derived peroxynitrite, we studied whether carbachol-induced NO/peroxynitrite formation contributes to the carbachol-induced RhoA activation in NRCM. Interestingly, the carbachol-induced RhoA activation in NRCM was suppressed by the eNOS-preferring inhibitor L-NIO as well as the non-selective NOS inhibitor L-NAME. Using L-NIO, we firstly verified the carbachol-induced NO production concurrent with eNOS activation and, secondly, the carbachol-induced nitration of p190A in NRCM. By co-immunoprecipitation, the carbachol-induced complex formation of eNOS, p190A, RGS3L and caveolin-3 was detected. We thus conclude that the NO production by M2R-induced eNOS activation in caveolae in NRCM is required for the nitration of p190A, leading to the binding to RGS3L and the change in substrate preference from RhoA to Rac1. In line with this interpretation, the disruption of caveolae in NRCM by methyl-β-cyclodextrin suppressed carbachol-induced RhoA activation in NRCM to a similar extent as the inhibition of NO production.

Induction of Secretagogue Independent Gastric Acid Secretion via a Novel Aspirin-Activated Pathway.

Aspirin has been widely recommended for acute and chronic conditions for over 2,000 years. Either single or repetitive doses are commonly used for analgesic and antipyretic reasons and to prevent heart attacks, stroke, and blood clot formation. Recent studies show that it can also be used chronically to dramatically reduce the risk of a variety of cancers. However, prolonged usage of aspirin can cause severe damage to the mucosal barrier, increasing the risk of ulcer formation and GI-bleeding events. In the present study, we show the effects of acute low-dose aspirin exposure as an active secretagogue-inducing gastric acid secretion. Studies were carried out with isolated gastric glands using the pH-sensitive dye BCECF-AM to assess acid secretion. The non-selective NOS inhibitor L-NAME (30 μM), or the specific inhibitor ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) was applied while monitoring intracellular pH. The effects of basolateral exposure to aspirin (acetylsalicylic acid, ASA) caused activation of gastric acid secretion via the H+, K+-ATPase. Our data suggest that aspirin increases nitric oxide (NO) production, which in turn activates acid secretion. Exposure of gastric glands to either the non-selective NOS inhibitor L-NAME, and the highly selective, soluble guanylyl cyclase inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) effectively inhibited aspirin-dependent gastric acid secretion. Aspirin can be considered as a novel secretagogue, in the way that it activates the H+, K+-ATPase. With increased daily aspirin consumption, our findings have important implications for all individuals consuming aspirin even in low doses and the potential risks for increased acid secretion.

Role of K+ channels and NOS on human eccrine sweat gland function

The non‐selective NOS inhibitor L‐NAME has been shown to reduce cholinergic‐mediated sweating in humans. We tested the hypothesis that the effect of NOS inhibition on sweat gland out is mediated through the ability of NO to activate KCa1.1 channels. We generated stimulus response curves using: lactated‐Ringers, 50 mM TEA, 20 mM L‐NAME and a cocktail of 50 mM TEA plus 20 mM L‐NAME (n=10). Local sweat rate (SR) was measured by passing dry gas through a small sweat capsule mounted on the skin. The skin was stimulated at a constant current intensity of 2.5 mA for 30 s at frequencies of 0.2, 1, 2, 4, 6, 8, 12, 16, 32, and 64 Hz using two small stainless‐steel stimulating electrodes. We plotted the area under the SR‐time curve versus stimulus frequency. All data were expressed as the area under the local SR‐time curve (AUC) and normalize to the peak AUC response during Saline control trials (% AUC peak). During Ringers perfusion, peak local sweat output averaged 99.6 ± 5.7 AUC % peak which was significantly (p<0.0001) reduced by application of 50 mM TEA to 61.7 ± 22.7 AUC %peak. Addition of 20 mM L‐NAME to the 50 mM TEA solution produced an additional 20% reduction in peak SR to 42.5 ± 21.1 %AUC peak (p <0.05). These data support the hypothesis that sudomotor control of sweat gland activity is modulated by the presence of TEA sensitive K+ channels and the activity of an intrinsic NOS enzyme in the epithelial cells of the human sweat glands. Further, the ability of L‐NAME to produce an additional reduction in peak SR following 50 mM TEA indicates that the influence of NOS on local SR is not likely mediated via a modulation of TEA sensitive K+ channels.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P248: Overexpression of an Intracellular Angiotensin II Fusion Protein Selectively in the Mitochondria of Mouse Proximal Tubule Cells Induces Mitochondrial Stress and Glycolytic Responses: Roles of Nitric Oxide and Superoxide

Endocrine and paracrine angiotensin II ( ANG II) activates cell surface AT 1 receptors to induce mitochondrial superoxide (O 2 - ) production and uncoupling of eNOS, leading to ANG II-induced renal and hypertensive injury. Little is known, however, whether ANG II is internalized to the mitochondria to activate AT 1 or AT 2 receptors. We tested the hypothesis that ANG II in the mitochondria exerts important dual roles on mitochondrial stress and glycolytic responses via the AT 1a /NADPH oxidase/O 2 .- and AT 2 /eNOS/NO signaling pathways. To test the hypothesis, a mitochondria-targeting ANG II protein (mito-ECFP/ANG II) was expressed selectively in the mitochondria of mouse proximal tubule cells, and treated with or without AT 1 receptor blocker losartan (10 μM), AT 2 receptor blocker PD123319 (10 μM), a non-selective NOS inhibitor L-NAME (10 mM), or a scavenger of mitochondrial superoxide, mito-TEMPO (10 μM) for 48 h, respectively. The mitochondrial stress and glycolytic responses were then determined using Seahorse XF e 24 Extracellular Flux Analyzer. Expression of mito-ECFP/ANG II alone significantly increased oxygen consumption rate (OCR) (Control: 259.8 ± 52.2 vs. mito-ECFP/ANG II: 512.5 ± 75.6 pmol/min; p <0.01, n=8) and extracellular acidification rate (ECAR) (Control: 13.5 ± 2.2 vs. mito-ECFP/ANG II: 19.3 ± 2.4 mpH/min; p <0.01, n=8), respectively. These mito-stress responses were blocked by losartan ( p <0.01, n=8), but not by PD123139 ( n.s. ). Furthermore, the mito-stress response of mito-ECFP/ANG II was significantly attenuated by mito-TEMPO (288.1 ± 10.3 pmol/min; p <0.01, n=8), and augmented by L-NAME (623.5 ± 8.7 pmol/min, p <0.05, n=8). Finally, the mito-stress response to mito-ECFP/ANG II expression was associated with significant increases in mitochondrial redox carries, Complex I (NADH coenzyme Q reductase), Complex II (succinate dehydrogenase), Complex III (cytochrome bc 1 complex) and Complex IV proteins (cytochrome c oxidase) ( p <0.01). We concluded that ANG II in the mitochondrial induces important mitochondrial stress and glycolytic responses primarily via the AT 1a /NADPH oxidase/O 2 .- signaling pathways in mouse proximal tubule cells.

Laminar shear stress suppresses vascular smooth muscle cell proliferation through nitric oxide-AMPK pathway

In healthy condition, vascular smooth muscle cells (VSMCs) are not directly exposed to shear stresses, because they are shielded by endothelial cell (EC) layer that lines blood vessels. After injury to EC layer caused by rupture of atherosclerotic lesions or invasive techniques such as angioplasty, VSMCs are directly exposed to blood flow which modulate molecular signaling and function. In endothelium, exposure to fluid shear stress has been reported to induce AMP-activated protein kinase (AMPK) phosphorylation and nitric oxide (NO) production. However, the influence of laminar shear stress on exposed VSMC is not defined. In this study, we investigated whether laminar shear stress regulates AMPK phosphorylation in VSMC and tried to identify underlying signaling pathway. NO production was increased by shear stress. The expression of NOS isoforms was increased 1 h after exposure to shear stress, and AMPK phosphorylation started to increase after 2 h. AMPK and LKB1, the upstream kinases of AMPK, phosphorylation were decreased by the non-selective NOS inhibitor l-NAME and the selective iNOS inhibitor aminoguanidine despite exposure to shear stress. On the other hand, compound C, a specific AMPK inhibitor, did not affect the expression of NOS isoforms. In addition, PDGF-induced VSMC proliferation was decreased by shear stress and restored by l-NAME. These findings suggest that shear stress upregulated AMPK phosphorylation in VSMC via NOS expression may be a beneficial route to prevent pathogenesis in the vascular system.

Abstract 111: Xanthine Oxidoreductase Plays a Key Role in Nitrate, Nitrite and Nitric Oxide Homeostasis When the Endothelial Nitric Oxide Synthase is Compromised

Xanthine oxidoreductase (XOR) is generally known as a source of superoxide production, but this enzyme has also been suggested to mediate NO production via reduction of inorganic nitrate (NO 3 - ) and nitrite(NO 2 - ). This pathway for NO generation is of particular importance during certain pathologies, whereas endothelial NO synthase (eNOS) is the primary source of vascular NO generation under normal physiological conditions. The exact interplay between the NOS and XOR-derived NO is not yet fully elucidated. The aim of the present study was to investigate if eNOS deficiency is partly compensated by XOR upregulation and sensitization of the NO 3 - - NO 2 - - NO pathway. NO 3 - and NO 2 - were similar between naïve eNOS KO and wildtype (wt) mice, but reduced upon chronic treatment with the non-selective NOS inhibitor L-NAME (wt: 25.0±5.2, eNOS KO: 39.2±6.4, L-NAME: 8.2±1.6 μ NO 3 - -, wt: 0.38±0.07, eNOS KO: 0.42±0.04, L-NAME: 0.12±0.02 μ NO 2 - ). XOR function was upregulated in eNOS KO compared with wt mice [(mRNA: wt 1±0.07, eNOS KO 1.38±0.17), (activity: wt 825±54, eNOS KO 1327±280 CLU/mg/min), (uric acid: wt 32.87±1.53, eNOS KO 43.23±3.54 μ)]. None of these markers of XOR activity was increased in nNOS KO and iNOS KO mice. Following acute dose of NO 3 - (10 mg/kg bw, i.p.), the increase of plasma NO 2 - was more pronounced in eNOS KO (+0.51±0.13 μ) compared with wt (+0.22±0.09 μ), and this augmented response in the eNOS KO was abolished by treatment with the highly selective XOR inhibitor febuxostat (FEB). Liver from eNOS KO had higher reducing capacity of NO 2 - to NO compared with wt, and this effect was attenuated by FEB (Δppb of NO: wt +8.7±4.2, eNOS KO +44.2±15.0, wt+FEB +22.2±9.6, eNOS KO+FEB +26.8±10.2). Treatment with FEB increased blood pressure in eNOS KO (ΔMAP:+10.2±5.6 mmHg), but had no effect in wt (ΔMAP:-0.6±3.3 mmHg). Supplementation with NO 3 - (10 mM, drinking water) reduced blood pressure in eNOS KO (ΔMAP: -6.3±2.2 mmHg), and this effect was abolished by FEB (ΔMAP: +1.1±1.9 mmHg). In conclusion, upregulated and altered XOR function in conditions with eNOS deficiency can facilitate the NO 3 - - NO 2 - - NO pathway and hence play a significant role in vascular NO homeostasis.

TRPA1 activation leads to neurogenic vasodilatation: involvement of reactive oxygen nitrogen species in addition to CGRP and NO.

Background and PurposeTransient receptor potential ankyrin‐1 (TRPA1) activation is known to mediate neurogenic vasodilatation. We investigated the mechanisms involved in TRPA1‐mediated peripheral vasodilatation in vivo using the TRPA1 agonist cinnamaldehyde.Experimental ApproachChanges in vascular ear blood flow were measured in anaesthetized mice using laser Doppler flowmetry.Key ResultsTopical application of cinnamaldehyde to the mouse ear caused a significant increase in blood flow in the skin of anaesthetized wild‐type (WT) mice but not in TRPA1 knockout (KO) mice. Cinnamaldehyde‐induced vasodilatation was inhibited by the pharmacological blockade of the potent microvascular vasodilator neuropeptide CGRP and neuronal NOS‐derived NO pathways. Cinnamaldehyde‐mediated vasodilatation was significantly reduced by treatment with reactive oxygen nitrogen species (RONS) scavenger such as catalase and the SOD mimetic TEMPOL, supporting a role of RONS in the downstream vasodilator TRPA1‐mediated response. Co‐treatment with a non‐selective NOS inhibitor L‐NAME and antioxidant apocynin further inhibited the TRPA1‐mediated vasodilatation. Cinnamaldehyde treatment induced the generation of peroxynitrite that was blocked by the peroxynitrite scavenger FeTPPS and shown to be dependent on TRPA1, as reflected by an increase in protein tyrosine nitration in the skin of WT, but not in TRPA1 KO mice.Conclusion and ImplicationsThis study provides in vivo evidence that TRPA1‐induced vasodilatation mediated by cinnamaldehyde requires neuronal NOS‐derived NO, in addition to the traditional neuropeptide component. A novel role of peroxynitrite is revealed, which is generated downstream of TRPA1 activation by cinnamaldehyde. This mechanistic pathway underlying TRPA1‐mediated vasodilatation may be important in understanding the role of TRPA1 in pathophysiological situations.

Meningeal transient receptor potential channel M8 activation causes cutaneous facial and hindpaw allodynia in a preclinical rodent model of headache.

Migraine headache is a neurological disorder affecting millions worldwide. However, little is known about the mechanisms contributing to migraine. Recent genome-wide association studies have found single nucleotide polymorphisms in the gene encoding transient receptor potential channel M8. Transient receptor potential channel M8 is generally known as a cold receptor but it has been implicated in pain signaling and may play a role in migraine pain. In order to investigate whether transient receptor potential channel M8 may contribute to the pain of migraine, the transient receptor potential channel M8 activator icilin was applied to the dura mater using a rat behavioral model of headache. Cutaneous allodynia was measured for 5 hours using Von Frey filaments. Dural application of icilin produced cutaneous facial and hind paw allodynia that was attenuated by systemic pretreatment with the transient receptor potential channel M8-selective antagonist AMG1161 (10 mg/kg p.o.). Further, the anti-migraine agent sumatriptan (0.6 mg/kg s.c.) or the non-selective NOS inhibitor L-NAME (20 mg/kg i.p.) also attenuated allodynia when given as a pretreatment. These data indicate that transient receptor potential channel M8 activation in the meninges produces behaviors in rats that are consistent with migraine and that are sensitive to pharmacological mechanisms known to have efficacy for migraine in humans. The findings suggest that activation of meningeal transient receptor potential channel M8 may contribute to the pain of migraine.

PSD95 Coupling of NMDA Receptor Activation and Nitric Oxide Activity in the Hypothalamic PVN

PVN regulation of sympathetic nerve activity (SNA) and mean arterial pressure (MAP) strongly depends on the balance of glutamatergic excitation by NMDAR and GABAergic inhibition facilitated by nitric oxide (NO). The adaptor protein PSD95 regulates NO production by tethering nNOS to Ca2+‐permeable GluN2B‐containing NMDA receptors. To determine the importance of PSD95 in NMDA‐NO auto‐inhibitory feedback in PVN, lentivirus encoding PSD95 shRNA or scrambled shRNA (control) was injected into PVN (400 nL/side) of male rats. Rats were anesthetized 3‐4 weeks later and NMDA was injected into PVN. Consistent with diminished NMDA‐mediated stimulation of NO in LV‐PSD95‐shRNA rats, PVN NMDA evoked greater increases of renal SNA (PSD95 shRNA: 80±15% vs control: 28±5%) and MAP (PSD95 shRNA: 13±3 mmHg vs control: 10±1 mmHg) compared to controls. Whereas the non‐selective NOS inhibitor L‐NAME in PVN had no effect on baseline renal SNA or MAP, it reduced PVN NO as measured in controls by amperometry. During L‐NAME suppression of NO in controls, PVN NMDA caused greater increases of renal SNA (NMDA: 28±5% vs NMDA+L‐NAME: 70±12%) and MAP (NMDA: 10±1 mmHg vs NMDA+L‐NAME: 21±1 mmHg). Consistent with reduced capacity for NMDA stimulation of NO in LV‐PSD95‐shRNA rats, PVN L‐NAME did not enhance renal SNA (NMDA: 62±16% vs NMDA+L‐NAME: 59±13%) or MAP (NMDA: 13±1 mmHg vs NMDA+L‐NAME: 13±1 mmHg) responses to NMDA. Results indicate that PVN PSD95 couples NMDAR activation to NO production, which restrains NMDAR driven responses. In the context of neurogenic cardiovascular diseases, decreased PVN PSD95 expression/function could contribute to loss of NO inhibition and to exaggerated glutamatergic/NMDAR driven SNA. HL 088052 & 102310 (GMT), T32 07446 (MEB)

Enhanced Nitric Oxide Synthase‐Nitric Oxide (NOS‐NO) signaling underlies sildenafil amelioration of cyclosporine‐induced nephrotoxicity in rats

Studies have demonstrated that the potent immunosuppressive effect of cyclosporine A (CsA) is associated with severe nephrotoxicity. However, the mechanism(s) of CsA‐mediated nephrotoxicity are understudied. This study aimed to investigate the possible protective effect of sildenafil on CsA‐induced nephrotoxicity and whether this effect is mediated via enhanced NOS‐NO signaling. Wistar rats were administered sildenafil (5 mg/kg, p.o) for 21 days alone or in combination with CsA 20 mg/kg, s.c. or with the non‐selective NOS inhibitor L‐NAME; 10 mg/kg, i.p. or both. Control groups were treated with either vehicle, CsA or L‐NAME. Renal functions (serum urea and creatinine, creatinine clearance and albumin‐creatinine ratio), indices of antioxidant activities (catalase, reduced glutathione and malondialdehyde), total nitrate/nitrite (NOx) were measured. Apoptotic and inflammatory markers (caspase‐3 and TNF‐alpha) were measured by. CsA administration for 21 days resulted in significant renal oxidative stress, deterioration of renal functions, reduction of NOx levels, marked apoptotic effect and elevation of nephritic TNF‐alpha expression. Concomitant treatment with sildenafil significantly improved CsA‐renal dysfunction, elevated NOx levels and reduced nephritic oxidative stress. Furthermore, sildenafil attenuated CsA‐evoked apoptosis and TNF‐alpha expression. Interestingly, L‐NAME reversed sildenafil protective effects against CsA‐mediated nephrotoxicity. Taken together, the current study highlights the clinically significant sildenafil protective effects against CsA‐induced nephrotoxicity that might be mediated via enhanced NOS‐NO signaling.

ROLE OF NITRIC OXIDE PRODUCED BY CONSTITUTIVE AND INDUCIBLE NITRIC OXIDE SYNTHASES IN THE MOUSE GASTRIC FUNDUS

In the present study, the role of nitric oxide (NO) produced by constitutive and inducible nitric oxide synthases (cNOS and iNOS, resepctively) on the contraction and relaxation of fundus in normal and lipopolysaccharide (LPS)-treated mice was examined. A whole fundic ring isolated from mice pretreated with reserpine was mounted in an organ bath containing Krebs' solution with 0.001 mmol/L atropine. Rings were contracted initially by 5-hydroxytryptamine (5-HT; 0.03 mmol/L) before relaxation was induced using ATP (0.03 mmol/L), ADP (0.03 mmol/L), pentoxifylline (0.002 mmol/L), electrical field stimulation (EFS; 50 V, 1 msec, 50 Hz, 3 min) and L-arginine (0.05 mmol/L). All drugs and EFS induced significant relaxation of isolated rings. The relaxations induced were significantly inhibited by N(G)-nitro-L-arginine methyl ester (L-NAME; 1.0 mmol/L). However, the iNOS inhibitors L-N(6)-(1-iminoethyl) lysine hydrochloride (L-NIL; 1.0 mmol/L) and amino guanidine (AMG; 1.0 mmol/L) had no significant effect on tissue relaxation. Then, the relaxant effects of 0.03 mmol/L ATP were tested on precontracted isolated fundic rings taken from 10 mg/kg LPS-treated animals. The non-selective NOS inhibitor L-NAME (10 mg/kg), the iNOS inhibitors L-NIL (3 mg/kg) and AMG (20 mg/kg) and betamethasone (0.1 mg/kg) were used to examine the role of NO produced by iNOS in the relaxation responses. It was found that the level of contraction induced by 0.03 mmol/L 5-HT in rings isolated from LPS-treated animals was significantly (P < 0.5) less than that in rings from untreated mice. However, precontracted tissues from LPS-treated mice were significantly relaxed by ATP and the relaxation response to ATP was significantly inhibited by L-NIL, ANG and betamethasone, but not by L-NAME. We suggest that, in LPS-treated mice, the production of NO from iNOS produces a reduction in the contractile response, as well as a decrease in NO formation by cNOS, resulting in changes to smooth muscle cell function.

Role of the nitric oxide pathway and the endocannabinoid system in neurogenic relaxation of corpus cavernosum from biliary cirrhotic rats

Relaxation of corpus cavernosum, which is mediated by nitric oxide (NO) released from non-adrenergic non-cholinergic (NANC) neurotransmission, is critical for inducing penile erection and can be affected by many pathophysiological conditions. However, the peripheral effect of liver cirrhosis on erectile function is as yet unknown. The aim of the present study was to investigate the effect of biliary cirrhosis on NANC-mediated relaxation of rat corpus cavernosum and the possible roles of endocannabinoid and nitric oxide systems in this model. Cirrhosis was induced by bile duct ligation. Controls underwent sham operation. Four weeks later, strips of corpus cavernosum were mounted in a standard organ bath and NANC-mediated relaxations were obtained by applying electrical field stimulation. The NANC-mediated relaxation was enhanced in corporal strips from cirrhotic animals. Anandamide potentiated the relaxations in both groups. Either AM251 (CB(1) antagonist) or capsazepine (vanilloid VR(1) antagonist), but not AM630 (CB(2) antagonist), prevented the enhanced relaxations of cirrhotic strips. Either the non-selective NOS inhibitor L-NAME or the selective neuronal NOS inhibitor L-NPA inhibited relaxations in both groups, but cirrhotic groups were more resistant to the inhibitory effects of these agents. Relaxations to sodium nitroprusside (NO donor) were similar in tissues from the two groups. Cirrhosis potentiates the neurogenic relaxation of rat corpus cavernosum probably via the NO pathway and involving cannabinoid CB(1) and vanilloid VR(1) receptors.

Inducible nitric oxide synthase inhibition improves intestinal microcirculatory oxygenation and CO2 balance during endotoxemia in pigs

We examined whether selective inhibition of inducible nitric oxide synthase (iNOS) promotes intestinal microvascular oxygenation (microPO2) and CO2 off-load after endotoxic shock. Prospective, controlled experimental study in a university animal research laboratory. 13 domestic pigs. After baseline measurements shock was induced by 1 microg kg-1 h-1 endotoxin until mean arterial pressure fell below 60 mmHg. After 30 min in shock the animals were resuscitated with either fluid alone (control, n=6) or fluid and the iNOS inhibitor N-[3-(aminomethyl)benzyl]acetamidine hydrochloride (1400W, n=7). As final experimental intervention all animals received the nonselective NOS inhibitor L-NAME. Systemic and regional hemodynamic and oxygenation parameters were measured at baseline, during endotoxemia and shock, hourly for 3 h of 1400W therapy, and 30 min after the final L-NAME administration. microPO2 was assessed by the Pd-porphyrin phosphorescence technique, and the arterial to intestinal PCO2 gap was determined by air tonometry. Endotoxemia and shock resulted in a decrease in ileal mucosal and serosal microPO2 and a rise in PCO2 gap. The combination of 1400W and fluid resuscitation, but not fluid alone, normalized both the serosal microPO2 and the intestinal PCO2 gap. Administration of L-NAME decreased cardiac output and oxygen delivery and intestinal microPO2 and blood flow in both groups. Partial blockade of NO production by 1400W increased serosal microvascular oxygenation and decreased the intestinal CO2 gap. This findings are consistent with the idea that 1400W corrects pathological flow distribution and regional dysoxia within the intestinal wall following endotoxic shock.

Role of nitric oxide synthase against MPTP neurotoxicity in mice

1-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic pathway injury similar to that observed in Parkinson's disease. Many hypotheses have been proposed to explain the mechanisms underlying MPTP neurotoxicity. Previous work showed that the inhibitor of neuronal nitric oxide synthase (nNOS) might produce protection against MPTP-induced dopaminergic toxicity. To exactly test the role of NO in MPTP neurotoxicity, we examined the effect of nNOS inhibitor 7-nitroindazole, in comparison with that of nonselective NOS inhibitor (L-NAME), immunosuppressant (FK-506), monoamine oxidase (MAO) inhibitors (clorgyline and pargyline), N-methyl-D-aspartate receptor antagonist (MK-801) and Ca2+ antagonist (amlodipine). Among seven compounds, 7-nitroindazole produced dose-dependent protection against MPTP-induced depletion of striatal dopamine and its metabolite 3,4-dihydroxyphenyl acetic acid (DOPAC) in mice. Clorgyline and pargyline also showed a significant effect on MPTP-induced dopamine depletion in the mouse striatum. However, both compounds did not protect against MPTP-induced depletion of striatal DOPAC. Our immunohistological study with tyrosine hydroxylase (TH) and microtuble-associated protein 2 (MAP 2) showed that 7-nitroindazole or pargyline can protect against MPTP-induced depletion of TH and MAP 2 immunostained neurons in the substantia nigra. Furthermore, these compounds reduced a marked increase in GFAP-positive astrocytes of the mouse striatum after MPTP treatments. The present study demonstrates that nNOS inhibitor 7-nitroindazole as well as MAO inhibitors clorgyline and pargyline can produce dose-dependent neuroprotection against the dopaminergic neurotoxicity of MPTP. However, nonselective NOS inhibitor L-NAME, immunosuppressant FK-506, NMDA receptor antagonist MK-801 and Ca2+ antagonist amlodipine did not show a beneficial effect on MPTP neurotoxicity.

Roles of endogenous prostaglandins and nitric oxide in gastroduodenal ulcerogenic responses induced in rats by hypothermic stress

We examined the roles of endogenous prostaglandins (PGs) and nitric oxide (NO) in the gastroduodenal ulcerogenic responses to hypothermic stress (28∼30 °C) in anesthetized rats. Lowering body temperature provoked damage in the gastroduodenal mucosa, with an increase of gastric acid secretion and motility. These responses were completely abolished by bilateral vagotomy or atropine, while 16,16-dimethyl PGE 2 decreased the mucosal ulcerogenic response with no effect on acid secretion. The non-selective COX inhibitors, indomethacin or aspirin, worsened these lesions with enhancement of gastric motility and no effect on acid secretion, while the selective COX-2 inhibitor NS-398 did not affect any of these responses. On the other hand, the non-selective NOS inhibitor L-NAME but not aminoguanidine (a relatively selective inhibitor of iNOS), significantly potentiated the acid secretory and mucosal ulcerogenic responses in the stomach but reduced the duodenal damage in response to hypothermia, the effects being antagonized by co-administration of L-arginine. Hypothermia itself decreased duodenal HCO 3 – secretion under both basal and mucosal acidification-stimulated conditions. Both indomethacin and aspirin further decreased the HCO 3 – response to the mucosal acidification, while L-NAME significantly increased the HCO 3 – secretion even under hypothermic conditions, similar to 16,16-dimethyl PGE 2. These results suggest that 1) hypothermic stress caused an increase of acid secretion and motility as well as a decrease of duodenal HCO 3 – secretion, resulting in damage in both the stomach and duodenum, 2) the COX-1 but not COX-2 inhibition worsened these lesions by enhancing gastric motility and further decreasing duodenal HCO 3 – response, 3) the cNOS but not iNOS inhibition worsened gastric lesions by increasing acid secretion but decreased duodenal damage by increasing HCO 3 – secretion. Thus, it is assumed that the gastroduodenal ulcerogenic and functional responses to hypothermic stress are modified by cNOS/NO as well as COX-1/PGs.