nitroarginine and Ischemia

We have shown recently that repetition of ischemic preconditioning stimulus augments endothelium-dependent vasodilation in forearm circulation of healthy subjects through increases in NO production and the number of circulating progenitor cells under a local condition. The purpose of this study was to evaluate the "late" effect of ischemic preconditioning on endothelial function in smokers. Ischemic preconditioning was induced by upper-limb ischemia 6 times a day for 1 month. We evaluated forearm blood flow responses to acetylcholine and sodium nitroprusside before and after ischemic preconditioning stimulus in 15 male smokers (27+/-7 years) and 15 male nonsmokers (26+/-5 years). Forearm blood flow was measured by using a strain-gauge plethysmography. The ischemic preconditioning stimulus resulted in significant increases in the circulating level of circulating progenitor cells from 1029+/-261 to 1232+/-341 mL (P=0.02), cell migration response to vascular endothelial growth factor from 38+/-16 to 52+/-17 per high-power field (P=0.02), and forearm blood flow response to acetylcholine from 25.1+/-5.2 to 32.4+/-6.6 mL/min per 100 mL of tissue (P=0.002) in nonsmokers, but these did not change in the smoker group. The forearm blood flow responses to sodium nitroprusside before and after the ischemic preconditioning stimulus were similar. Intra-arterial infusion of N(G)-monomethyl-l-arginine, an NO synthase inhibitor, completely eliminated the ischemic preconditioning stimulus-induced augmentation of forearm blood flow responses to acetylcholine in nonsmokers. These findings suggest that repetition of ischemic preconditioning stimulus may be a simple, safe, and feasible therapeutic technique for endothelial protection of peripheral vessels. However, smoking abolishes ischemic preconditioning stimulus-induced augmentation of endothelium-dependent vasodilation.

Topics: Acetylcholine; Adult; Endothelium, Vascular; Enzyme Inhibitors; Forearm; Hematopoietic Stem Cells; Humans; Infusions, Intra-Arterial; Ischemia; Ischemic Preconditioning; Male; Nitroarginine; Nitroprusside; Plethysmography; Smoking; Vascular Endothelial Growth Factor A; Vasodilation; Vasodilator Agents; Young Adult

The contribution of endothelial function to tissue oxygenation is not well understood. Muscle blood oxygen level-dependent MRI (BOLD MRI) provides data largely dependent on hemoglobin (Hb) oxygenation. We used BOLD MRI to assess endothelium-dependent signal intensity (SI) changes.. We investigated mean BOLD SI changes in the forearm musculature using a gradient-echo technique at 1.5 T in 9 healthy subjects who underwent a protocol of repeated acetylcholine infusions at 2 different doses (16 and 64 microg/min) and N(G)-monomethyl-L-arginine (L-NMMA; 5 mg/min) into the brachial artery. Sodium nitroprusside was used as a control substance. For additional correlation with standard methods, the same protocol was repeated, and forearm blood flow was measured by strain gauge plethysmography. We obtained a significant increase in BOLD SI during acetylcholine infusion (64 microg/min) and a significant decrease for L-NMMA infusion (P<0.005 for both). BOLD SI showed a different kinetic signal than did blood flow, particularly after intermittent ischemia and at high flow rates.. In standard endothelial function tests, BOLD MRI detects a dissociation of tissue Hb oxygenation from blood flow. BOLD MRI may be a useful adjunct in assessing endothelial function.

Topics: Acetylcholine; Adult; Endothelium, Vascular; Enzyme Inhibitors; Humans; Infusions, Intra-Arterial; Ischemia; Magnetic Resonance Imaging; Male; Models, Cardiovascular; Muscle, Skeletal; Nitroarginine; Nitroprusside; Oxygen; Plethysmography; Regional Blood Flow; Vasodilation; Vasodilator Agents

The purpose of this study was to determine whether autologous mesenchymal stem cells (MSCs) implantation improves endothelial dysfunction in a rabbit ischemic limb model.. We evaluated the effect of MSC implantation on limb blood flow (LBF) responses to acetylcholine (ACh), an endothelium-dependent vasodilator, and sodium nitroprusside (SNP), an endothelium-independent vasodilator, in rabbits with limb ischemia in which cultured MSCs were implanted (n = 20) or saline was injected as a control group (n = 20). LBF was measured using an electromagnetic flowmeter. A total of 10(6) MSCs were implanted into each ischemic limb.. Histological sections of ischemic muscle showed that capillary index (capillary/muscle fiber) was greater in the MSC implantation group than in the control group. Laser Doppler blood perfusion index was significantly increased in the MSC implantation group compared with that in the control group. LBF response to ACh was greater in the MSC group than in the control group. After administration of N(G)-nitro-L-arginine, a nitric oxide synthase inhibitor, LBF response to ACh was similar in the MSC implantation group and control group. Vasodilatory effects of SNP in the two groups were similar.. These findings suggest that MSC implantation induces angiogenesis and augments endothelium-dependent vasodilation in a rabbit ischemic model through an increase in nitric oxide production.

Topics: Acetylcholine; Animals; Bone Marrow Cells; Hemodynamics; Hindlimb; Ischemia; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Neovascularization, Physiologic; Nitroarginine; Nitroprusside; Rabbits; Recovery of Function; Transplantation, Autologous; Vasodilation; Vasodilator Agents

The influence of carotid artery occlusion (10, 30 and 60 min) on regulatory mechanisms implicated in the vasorelaxant responses of isolated glandular branch of rabbit facial artery to acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) was examined.. In organ bath studies with arterial rings precontracted with phenylephrine (1 microM), before and after carotid artery occlusion, changes in isometric tension were recorded.. Endothelium-dependent vasorelaxation by ACh and endothelium-independent vasorelaxation by VIP were significantly reduced, started from 30 and 10 min of carotid occlusion, respectively. Inhibitory effect of indomethacin on ACh vasorelaxation was enhanced whilst effect of N(G)-nitro-L-arginine reduced, started from 30 min of carotid occlusion. Sodium nitroprusside-induced vasorelaxation was not changed after carotid occlusion. Inhibition of VIP vasorelaxation by L-N(omega)-nitroarginine-2,4-L-diaminobutyric-amide, was reduced, started from 30 min of carotid occlusion. Forskolin enhanced VIP-induced vasorelaxation in control rings but this effect was reduced started from 30 min of occlusion. In the presence of VIP, vasorelaxant effect of ACh was increased; the increase was reduced, started from 10 min of carotid occlusion.. The present investigation provides evidence for the decreased responsiveness to both, ACh-endothelium-dependent and VIP-endothelium-independent vasorelaxation in rabbit facial artery after carotid occlusion. In addition, the data suggest that ischaemia alters contribution of endothelial nitric oxide (eNO) and prostaglandin to ACh, and vascular smooth muscle's cAMP and neuronal NO to VIP vasorelaxant effects.

Topics: Acetylcholine; Animals; Arteries; Bradykinin; Carotid Artery Diseases; Carotid Artery, Common; Colforsin; Cyclic AMP; Endothelium, Vascular; Epoprostenol; Female; Indomethacin; Ischemia; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitroarginine; Nitroprusside; Phenylephrine; Rabbits; Sialadenitis; Signal Transduction; Submandibular Gland; Vasoactive Intestinal Peptide; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

The involvement of matrix metalloproteinases (MMPs) in ischemic tissue damage and remodeling has been reported by many investigators. Our study was designed to investigate the involvement of MMPs and of tissue inhibitors of metalloproteinases (TIMPs) in rat retinal ischemic injury, the effect of nitric oxide synthase (NOS) inhibitors on MMPs' activity in this model and whether minocycline (an MMP inhibitor) is protective in retinal ischemia.. Ninety-four rats were used in the study. Ischemia was induced by 90 min elevation of intraocular pressure. MMPs' activities and the effect of NOS inhibitors [aminoguanidine (AG) or N-nitro-L-arginine (NNA)] and minocycline on MMPs' activities were assessed by zymography and TIMPs expression by Western analysis. Morphological damage was quantified by morphometry of hematoxylin and eosin-stained retinal sections.. Retinal extracts exhibited activities of proMMP-9 and proMMP-2. The activity of proMMP-9 increased immediately post ischemia (PI) and peaked to 4.6 times that of normal untreated controls in ischemic retinas and to 2.6 times that of controls in retinas of fellow sham-treated eyes at 24 h PI. The relative amount of TIMP-1 increased to 1.9-fold following ischemia and 2.5-fold in fellow sham-treated eyes at 24 h PI. ProMMP-2 activity increased more than two-fold immediately, at 24 h and at 48 h PI in ischemic retinas, and insignificantly in fellow sham-treated eyes. Treatment with 25 mg/kg AG or NNA caused a non-significant increase in proMMP-9 activity at 24 h PI (3.7- and 2.9-fold, respectively, p>0.6). There was no effect of AG or NNA on the activity of proMMP-2. Minocycline significantly attenuated the retinal ischemic damage, primarily by partially preserving ganglion cells and the inner plexiform layer. Minocyline (0.5 mg/ml or 5 mg/ml) inhibited MMPs' activities in ischemic retinal extracts in vitro.. MMPs participated in morphological ischemic damage to rat retina. Treatment with minocycline dramatically attenuated damage to the retina.

Topics: Animals; Blotting, Western; Disease Models, Animal; Enzyme Inhibitors; Guanidines; Ischemia; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Minocycline; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Sprague-Dawley; Retinal Diseases; Retinal Vessels; Tissue Inhibitor of Metalloproteinase-1

Brief ischemia was reported to protect retinal cells against injury induced by subsequent ischemia-reperfusion with de novo protein synthesis, and this phenomenon is known as late ischemic preconditioning. The aims of the present study were to determine whether nitric oxide synthase (NOS) was involved in the mechanism of late ischemic preconditioning in rat retina using pharmacological tools. Under anesthesia with pentobarbital sodium, male Sprague-Dawley rats were subjected to 60 min of retinal ischemia by raising intraocular pressure to 130 mm Hg. Ischemic preconditioning was achieved by applying 5 min of ischemia 24 hrs before 60 min of ischemia. Retinal sections sliced into 5 microm thick were examined 7 days after ischemia. Additional groups of rats received NG-nitro-L-arginine and NG-monomethyl-L-arginin, non-selective NO synthase inhibitors, 7-nitroindazole, a neuronal NOS inhibitor, and aminoguanidine and L-N6-(1-iminoethyl) lysine, inducible NO synthase (iNOS) inhibitors before preconditioning, and were subjected to 60 min of ischemia. In the non-preconditioned group, cell loss in the ganglion cell layer and thinning of the inner plexiform and inner nuclear layer were observed 7 days after 60 min of ischemia. Ischemic preconditioning for 5 min completely protected against the histological damage induced by 60 min of ischemia applied 24 hrs thereafter. Treatment of rats with aminoguanidine and L-N6-(1-iminoethyl) lysine, but not NG-nitro-L-arginine, NG-monomethyl-L-arginine or 7-nitroindazole, wiped off the protective effect of ischemic preconditioning. The inhibitory effect of aminoguanidine was abolished by L-arginine, but not D-arginine. The results in the present study suggest that NO synthesized by iNOS is involved in the histological protection by late ischemic preconditioning in rat retina.

Topics: Animals; Enzyme Inhibitors; Guanidines; Indazoles; Ischemia; Ischemic Preconditioning; Lysine; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitroarginine; Rats; Rats, Sprague-Dawley; Retina; Retinal Vessels

1. We compared the effects of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine (L-NOARG) and tetraethylammonium (TEA), a blocker of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, on vasodilator responses to endothelium-dependent (acetylcholine; ACh) and -independent (sodium nitroprusside; SNP) vasodilators. The mechanism of the vasodilator responses was determined in rat hindquarters under normal conditions (sham ischaemia) and after 2 h ischaemia followed by reperfusion with physiological saline. 2. In sham ischaemia, the responses to ACh were significantly reduced by L-NOARG (1 mmol/L) and TEA (1 mmol/L) and there was a further reduction in response the presence of both agents. Dilator responses to SNP were significantly enhanced by L-NOARG, whereas TEA did not alter the SNP-induced vasodilatation when given either alone or in the presence of L-NOARG. 3. After ischaemia, L-NOARG caused a similar inhibition of ACh-induced dilatation to that observed in sham ischaemia. However, TEA alone or combined with L-NOARG caused a significantly greater inhibition of the ACh-induced vasodilatation after ischaemia than observed in the sham ischaemia group. Tetraethylammonium alone did not affect the responses to SNP, but it did attenuate the enhanced dilatation observed in the presence of L-NOARG. 4. In the rat hindquarters vasculature, both nitric oxide and the opening of TEA-sensitive K(+) channels contribute to ACh-induced endothelium-dependent dilatation. In addition, a TEA-sensitive mechanism was not involved in the SNP-induced dilatation under normal conditions but, after ischaemia, if there is a further inhibition of endogenous nitric oxide by L-NOARG, exogenous nitric oxide causes dilatation that is sensitive, in part, to TEA. Thus, the contribution of the opening of BK(Ca) channels to endothelium-dependent vasodilatation assumes greater importance after ischaemia and reperfusion. This may reflect an increased ability of nitric oxide or cGMP to open BK(Ca) channels after ischaemia.

Topics: Animals; Calcium Channel Blockers; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; Hindlimb; Ischemia; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Nitric Oxide; Nitroarginine; Potassium Channels; Potassium Channels, Calcium-Activated; Rats; Rats, Sprague-Dawley; Vasodilation

Pre-conditioned rats underwent one cycle of intestinal ischemia/reperfusion with subsequent assessment of the heart remote ischemic pre-conditioning (IPC). A local IPC exerted an obvious protective action whereas a remote IPC was ineffective. The local IPC depended partially on the nitric oxide (NO) synthesis. L-arginine administration reduced the intestinal injury. The findings suggest that the ischemic pre-conditioning of intestine depends partially on the NO synthesis and that the remote IPC in intestine does not exist.

Topics: Adaptation, Physiological; Animals; Enzyme Inhibitors; Hindlimb; Intestine, Small; Ischemia; Ischemic Preconditioning; Male; Microcirculation; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Rats; Reperfusion Injury

Chronic nitric oxide (NO) synthase inhibition in rats causes hypertension, renal vascular injury, and proteinuria. NO deficiency increases superoxide (O(2)(-)) activity, but the effects of antioxidant treatment on renal injury have not been studied in this model. Exposure of rats to N omega-nitro-L-arginine (L-NNA) for 4 d markedly decreased NO-dependent relaxation in aortic rings and increased glomerular and renal interstitial monocyte influx, but renal O(2)(-) activity was not increased. After 7 d, BP and proteinuria were significantly increased. After 21 d of L-NNA treatment, rats displayed severe hypertension, decreased GFR, marked proteinuria, glomerular ischemia, renal vascular and tubulointerstitial injury, and complete loss of NO-dependent relaxation. Renal O(2)(-) activity was markedly increased [lucigenin-enhanced chemiluminescence (LEC), 279 +/- 71 versus 50 +/- 7 counts/10 mg, P < 0.01; electron paramagnetic resonance spectroscopy, 0.57 +/- 0.05 versus 0.34 +/- 0.04 U/10 mg, P < 0.05]. Apocynin, a specific inhibitor of NADPH oxidase, and diphenyleneiodonium, an inhibitor of flavin-containing enzymes, completely inhibited LEC signals in vitro, whereas allopurinol had no effect, indicating that NAD(P)H oxidase plays a major role in superoxide production in the kidney. Endothelial function remained impaired during cotreatment with alpha-tocopherol and there was no effect on hypertension or tubulointerstitial injury, but glomerular ischemia, decreases in GFR, and renal vascular injury were prevented and proteinuria was ameliorated. Renal LEC signals were intermediate between control and L-NNA-alone values (181 +/- 84 counts/10 mg). Chronic NO synthase inhibition in rats results in marked increases in renal cortical O(2)(-) activity, mediated by flavin-dependent oxidases. The absence of early increases in renal O(2)(-) activity, in the presence of endothelial dysfunction and macrophage influx, indicates that increased renal O(2)(-) activity is neither attributable to NO deficiency per se nor solely related to macrophage influx. The improvement of glomerular function and amelioration of renal vasculitis and proteinuria with vitamin E cotreatment indicate that oxidants are involved in the pathogenesis of renal injury in this model. However, markedly impaired endothelial function and unabated hypertension persist with vitamin E treatment and seem to be directly attributable to NO deficiency.

Topics: Animals; Antioxidants; Aorta; Blood Pressure; Enzyme Inhibitors; Hypertension; Ischemia; Kidney; Kidney Cortex; Kidney Glomerulus; Macrophages; Male; Monocytes; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Proteinuria; Rats; Rats, Sprague-Dawley; Superoxides; Systole; Time Factors; Vasodilation; Vitamin E

The onset of reperfusion and the recovery of the ERG b-wave following retinal ischemia was examined among three groups of rats: group 1 (n = 12) and group 2 (n = 6) received pretreatment with NG-nitro-L-arginine (20 mg/kg, i.p., 2 h before ischemia) followed by intravenous injection of saline (group 1) or of 200 mg/kg L-arginine (group 2) 5 min before the end of ischemia; group 3 (n = 7) received saline pretreatment followed by intravenous injection of saline as a control. Group 1 showed delayed onset of reperfusion compared to the other two groups and a reduction in the rate of the b-wave recovery compared to the control on the 1st day after reperfusion (group 1 vs. group 3; p = 0.0357). The L-arginine posttreatment significantly increased the b-wave recovery (group 2 vs. group 1; p = 0.0005 on day 1 and p < 0.0006 on day 3). The rate of the b-wave recovery in group 1 was inversely proportional to the time to establish complete reperfusion. Inhibition of nitric oxide synthase during the initial phase of reperfusion may worsen the recovery of the b-wave following retinal ischemia, at least in part, by inhibiting establishment of reperfusion.

Topics: Animals; Disease Models, Animal; Electroretinography; Enzyme Inhibitors; Ischemia; Male; Microcirculation; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Sprague-Dawley; Reperfusion; Retinal Diseases; Retinal Vessels; Treatment Outcome

Recent studies have reported that nitric oxide (NO) acts as a cytoprotective mediator in ischemia-reperfusion (IR) lung injury. We hypothesized that the addition of L-arginine to the perfusate would attenuate the increases in microvascular permeability and pulmonary vascular resistance.. Isolated rabbit lungs were reperfused for 60 min after 120 min warm ischemia. Lung injury was assessed using the fluid filtration coefficient (Kf), pulmonary vasucular resistance (PVR) before ischemia and after reperfusion, and a wet-to-dry lung weight ratio (W/D).. The Kf of the control group (without L-arginine) was significantly increased after reperfusion. Lungs perfused with L-arginine showed attenuation of the IR-induced increases in Kf and PVR. Addition of Nomega-nitro-L-arginine (L-NA), a NO synthase inhibitor, to the perfusate reduced the beneficial effects of L-arginine. The lungs perfused with dibutyryl-cyclic GMP (dbcGMP) showed attenuation of IR-induced increases in Kf and PVR. There were no significant differences in the W/D ratio between these groups.. These results demonstrate that L-arginine has beneficial effects on IR lung injury, perhaps due to enhancement of endothelial cGMP levels.

Topics: Animals; Arginine; Body Fluids; Capillary Permeability; Cyclic GMP; Enzyme Inhibitors; Hemodynamics; In Vitro Techniques; Ischemia; Lung; Nitroarginine; Pressure; Pulmonary Circulation; Rabbits; Reperfusion Injury; Trachea; Vascular Resistance

To assess the roles of endothelium-derived vasodilators and K(+) channels on metabolic ischemia-induced vasodilation from diameter changes in choroidal arterioles of the guinea-pig.. The choroid was isolated from the guinea-pig eye-ball, pinned flat on a silicone rubber plate, and superfused with oxygenated warmed (35 degrees C) Krebs solution. Diameters of choroidal arterioles were measured using video microscopy and a computer program for analysis. Vasodilatory effects were examined after the choroid was exposed to glucose-free/NaCN solutions for 10 minutes. The effects of Nomega-nitro-L-arginine (nitroarginine), indomethacin, and K(+) channel inhibitors (glibenclamide [Glib] and charybdotoxin [ChTX]) on ischemic vasodilation were assessed.. Reversible vasodilation was observed when the choroid was exposed to glucose-free/NaCN (10(-3) M) solutions. Nitroarginine (10( -4) M), Glib (2x10(-5) M) and ChTX (10(-7) M) significantly inhibited glucose-free/NaCN (10(-3) M)-induced vasodilation by 47%, 62%, and 24%, respectively. No significant inhibitory effect was observed with indomethacin (10(-5) M). Simultaneous application of Glib and ChTX reduced vasodilation by 77%. When Glib and ChTX were added together to nitroarginine, dilation was reduced by 86%. With high K(+) ([K]o = 47.2 mM) Krebs solution, ischemia caused a slight vasodilation (11%), which was significantly inhibited by nitroarginine.. In guinea-pig choroidal arterioles, glucose-free/ NaCN-induced ischemic vasodilation was mainly mediated by NO and K(ATP) channels. A part of NO-mediated vasodilation was induced independent of the opening of K(+) channels.

Topics: Animals; Arterioles; Charybdotoxin; Choroid; Endothelium, Vascular; Enzyme Inhibitors; Glucose; Glyburide; Guinea Pigs; Ischemia; Male; Nitroarginine; Potassium Channel Blockers; Potassium Channels; Sodium Cyanide; Vasodilation; Vasodilator Agents

Nitric oxide (NO) modulation of ischemia-reperfusion injury was investigated by measuring lipid peroxide and neutrophil accumulation in rat stomachs treated with NG-nitro-L-arginine (L-NNA), a specific NO synthase inhibitor. Ischemia-reperfusion injury was induced in the rat stomach. Treatment with L-NNA for 3 days at a dose of 3 mg/kg/day significantly enhanced this injury. This enhancement was reversed by the simultaneous administration of L-arginine at a dose of 30 mg/kg/day. Both thiobarbituric acid (TBA)-reactive substances, an index of lipid peroxidation, and myeloperoxidase (MPO) activity, an index of tissue-associated neutrophil accumulation, were increased in the gastric mucosa after ischemia-reperfusion. L-NNA treatment enhanced these increases in TBA-reactive substances and MPO activity. The increase in the area of gastric erosions correlated closely with accumulation of TBA-reactive substances as well as the increase in MPO activity. Enhancement of ischemia-reperfusion injury by L-NNA treatment was inhibited by injection with anti-neutrophil antibody, anti-platelet activating factor (PAF) antagonist, and anti-leukotriene B4 (LTB4) receptor antagonist. In addition, the increase in TBA-reactive substances and MPO activity was decreased by these antibodies or antagonists. Enhancement of reperfusion-induced gastric mucosal injury associated with inhibition of NO synthesis may involve neutrophil infiltration and lipid peroxide accumulation in the gastric mucosa, mediated by PAF and LTB4.

Topics: Animals; Enzyme Inhibitors; Gastric Mucosa; Immune Sera; Ischemia; Leukotriene B4; Lipid Peroxidation; Male; Neutrophils; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Platelet Aggregation Inhibitors; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Stomach; Thiobarbituric Acid Reactive Substances

A free radical gas, nitric oxide, has many useful functions when produced under physiological conditions by neurons and endothelial cells. However, excess nitric oxide has been reported to exert cytotoxic effects by direct toxicity or by reaction with superoxide. The effect of nitric oxide on the microcirculation in the periphery of a flap remains unclear, and its effect on flap survival is also unknown because nitric oxide has a dual action. Thus, we attempted to clarify the effect of nitric oxide on survival of rat random pattern skin flaps by the use of an endothelial constitutive nitric oxide synthase inhibitor (i.p. administration of 50 mg/kg N(G)-nitro-L-arginine) and the substrate of nitric oxide synthase (i.p. administration of 1 g/kg L-arginine). Three kinds of experiments were done using a total number of 120 animals: (1) time course measurement of blood flow in the flap periphery was performed using a laser Doppler flowmeter (30 rats), (2) the length of the surviving area of flaps was measured 1 week after raising the flap (60 rats), and (3) Western blot analysis was used to determine the time course of changes in the amount of endothelial constitutive nitric oxide synthase and the formation of 3-nitro-L-tyrosine, which is a marker of peroxynitrite-mediated (i.e., nitric oxide-dependent) tissue damage (30 rats). Inhibition of endothelial constitutive nitric oxide synthase by N(G)-nitro-L-arginine significantly decreased the length of the surviving area of skin flap (p < 0.01 compared with the control), which was associated with a decrease in the blood flow of the proximal portion of the flap. On the other hand, exogenous L-arginine increased the survival length of skin flap significantly (p < 0.01 compared with the control), which was associated with an increase in blood flow of the distal portion of the flap even though there was nitric oxide-mediated oxidative tissue damage. These results suggest that nitric oxide produced by endothelial constitutive nitric oxide synthase plays a role in maintaining circulation in the skin flap periphery and that L-arginine administration contributes to reduction of ischemic necrosis in the skin flap.

Topics: Animals; Arginine; Blotting, Western; Endothelium, Vascular; Enzyme Inhibitors; Free Radicals; Graft Survival; Injections, Intraperitoneal; Ischemia; Laser-Doppler Flowmetry; Male; Microcirculation; Necrosis; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxidation-Reduction; Rats; Rats, Wistar; Regional Blood Flow; Skin; Skin Transplantation; Tyrosine

To clarify the role of nitric oxide (NO) in the pathogenesis of renovascular hypertension, we examined the effects of long-term oral administration of either the precursor substrate L-arginine or the NO synthesis inhibitor Nomega-nitro-L-arginine (L-NA) on systemic and renal hemodynamics in dogs with chronic two-kidney, one-clip (2K-1C) renovascular hypertension. Furthermore, the importance of NO in maintaining kidney function in chronic renal ischemia was evaluated. Chronic inhibition of NO production aggravated the rise in blood pressure (L-NA 117.7+/-6.8 vs. control 107.2 3.3 mmHg, p < 0.05 on day 1) and stimulated marked bradycardia (L-NA 84.9+/-3.2 vs. control 94.6+/-2.6 beats/min, p < 0.05 on day 1). These changes were associated with significant reductions in renal plasma flow (RPF, L-NA 0.03+/-0.02 vs. control 0.85+/-0.20 ml/min/kg, p < 0.01) and glomerular filtration rate (GFR, L-NA 0.02+/-0.01 vs. 0.22+/-0.05 ml/min/kg, p < 0.01) in the ischemic kidney. In contrast, in the contralateral non-clipped kidney, chronic inhibition of NO production induced a significant reduction in RPF with no significant change in GFR. Oral administration of L-arginine had no effect on the magnitude of hypertension. L-arginine significantly improved RPF (2.76+/-0.49 ml/min/kg) and GFR (0.61+/-0.08 ml/min/kg) in the ischemic kidney, whereas the elevation of RPF and GFR in the non-clipped kidney was not significant. Unilateral renal artery occlusion in these hypertensive dogs resulted in diffuse atrophic tubulointerstitial changes in the ischemic kidney. These changes were markedly aggravated by NO synthesis inhibition. On the other hand, L-arginine treatment significantly protected against the morphological changes of renal ischemia. These data show that NO plays a key role in the maintenance of renal function during the evolution of hypertension induced by chronic renal ischemia. In addition, these data demonstrate that renovascular hypertension is associated with a compensatory increase in the vasodilator function of the vascular endothelium.

Topics: Animals; Arginine; Blood Pressure; Dogs; Enzyme Inhibitors; Glomerular Filtration Rate; Heart Rate; Hormones; Hypertension, Renovascular; Ischemia; Kidney; Male; Nitric Oxide; Nitroarginine; Renal Circulation

The contribution of nitric oxide (NO) to ischemic acute renal failure is unclear. Because polymorphonuclear neutrophils (PMN) accentuate injury in kidneys subjected to ischemia-reperfusion and because NO has potent vascular and PMN effects, we examined the contribution of NO to PMN-mediated injury in isolated perfused rat kidneys. Nonischemic and ischemic kidneys were perfused by the isolated kidney technique in the presence or absence of PMN and NO agonists [sodium nitroprusside (SNP), L-arginine (L-Arg)] or a NO synthase inhibitor [N omega-nitro-L-arginine (L-NNA)]. In nonischemic kidneys, the NOS antagonist decreased perfusion flow rate by 25% without affecting glomerular filtration rate (GFR) or tubular sodium reabsorption (TNa), whereas NOS agonist treatment had no effects. After 20 min of ischemia/60 min reperfusion in the absence of PMN NO agonist treatment potentiated ischemia-reperfusion-induced loss of GFR and TNa, whereas adding the NO antagonist lessened glomerular and tubular injury. Reperfusion of ischemic kidneys with PMN resulted in PMN retention and potentiated ischemic injury. However, increases in PMN retention as well as decreases in GFR and TNa caused by PMN were prevented by SNP and worsened by L-NNA. Moreover, in nonischemic kidneys, activated PMN caused renal injury and PMN retention, which were prevented by SNP and worsened by L-NNA. In conclusion, 1) NO worsens ischemic injury in the absence of PMN, and 2) NO prevents the PMN component of ischemic renal injury by blocking PMN retention and the deleterious effects of activated PMN on glomerular and tubular function.

Topics: Absorption; Acute Kidney Injury; Animals; Arginine; Enzyme Inhibitors; Glomerular Filtration Rate; Ischemia; Kidney; Neutrophils; Nitric Oxide; Nitroarginine; Nitroprusside; Rats; Reference Values; Renal Circulation; Reperfusion Injury; Sodium

During the postischemic flow response (PFR), vasodilator mediators such as nitric oxide (NO) and histamine are liberated, influencing the blood flow rate at the onset of reperfusion. The possible roles of these two mediators, and the relationship between their release, were examined during segmental intestinal ischemia of different durations and subsequent reperfusion in two series of anesthetized dogs. In series I (untreated ischemia), 15, 30, 60, and 120 min ischemia and 2 h reperfusion were studied. In series II, the same experimental protocol was repeated after pretreatment with the NO synthase inhibitor N-nitro-L-arginine (NNA, 10 mumol/kg, i.e., 2.19 mg/kg). Intramucosal pH (pHi), segmental blood flow and effluent histamine levels were measured, and segmental vascular resistance (SVR) and PFR volumes were calculated. The ischemic periods caused a considerable fall in pHi. Reperfusion resulted in an early return to normal pHi levels following a 15 or 30 min ischemia, but this process took longer after longer occlusions. In the later phase of reperfusion, SVR was elevated. The PFR volume increased in proportion to the duration of occlusion, except after the 120 min ischemia. At the onset of reperfusion, peak histamine levels rose in parallel with the duration of ischemia. During reperfusion, a prolonged decrease in pHi, an increase in SVR, and a reduction in PFR volume, with no significant histamine level elevation, were observed in the NNA-treated groups. This study indicates that both NO and histamine take part in the PFR in the canine small intestine. Inhibition of NO synthesis prevents the postischemic release of histamine.

Topics: Animals; Blood Flow Velocity; Dogs; Enzyme Inhibitors; Female; Histamine Release; Intestine, Small; Ischemia; Male; Mesentery; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Time Factors

The dose effects of two nitric oxide synthase (NOS) inhibitors: NG-nitro-L-arginine (L-NNA) and N omega-monomethyl-L-arginine (L-NMMA), were evaluated in an established rat model of retinal ischemia using morphometry of the inner retina: inner retinal thickness (IRT) measurements and retinal ganglion cell counts (RGCCs) of the posterior and peripheral retina. By IRT and RGCCs of the posterior retina, there were dose dependent beneficial effects of both inhibitors. However, by RGCCs of the peripheral retina, there was no significant beneficial effect by either inhibitor. In addition, L-NMMA at 0.3 mg/kg aggravated the loss of RGC in both the posterior and peripheral retina. An important role and a possible differential site of action of NOS in the pathophysiology of retinal ischemia are implicated.

Topics: Animals; Cell Count; Enzyme Inhibitors; Intraocular Pressure; Ischemia; Male; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; omega-N-Methylarginine; Rats; Rats, Inbred Lew; Reperfusion Injury; Retina; Retinal Ganglion Cells

Changes in nitric oxide (NO) levels were determined in ischaemic acute renal failure in streptozotocin-induced diabetes mellitus rats. Two weeks after streptozotocin administration and immediately after right nephrectomy, the left renal artery was occluded for 60 min. Similar procedures were carried out in non-diabetic rats. The nitrite (NO2) + nitrate (NO3) levels were measured in plasma and urine. The effects of chronic oral supplementation with L-arginine and an NO synthase inhibitor (N-omega-nitro-L-arginine) were also studied in both diabetic and non-diabetic rats before and after renal artery clamping. The rats with diabetic acute renal failure had a much lower creatinine clearance (90 +/- 22 microliters.min-1. 100g body weight-1, p < 0.005), and higher fractional excretion of sodium (FENa)% (10.90 +/- 4.2, p < 0.001) and protein excretion (2078 +/- 69 micrograms/ml creatinine clearance, p < 0.001) compared with the respective values in the non-diabetic groups (163 +/- 30; 1.46 +/- 86; 453.3 +/- 31). The plasma and urine NO2 + NO3 levels were significantly higher in the untreated diabetic rats compared with the untreated normal rats before ischaemia (p < 0.001). The ischaemic acute renal failure in non-diabetic rats increased the plasma and urinary NO2 + NO3 excretion after ischaemia. The urinary excretion of these metabolites decreased significantly and their plasma levels remained unchanged in the ischaemic diabetic rats. The L-arginine administration resulted in a small but significantly higher creatinine clearance after clamping in the non-diabetic rats. The NO synthase inhibitor caused deterioration in renal function in all ischaemic and non-ischaemic groups. In summary, the greater vulnerability to ischaemia of the diabetic kidney seems to be associated with both impaired response to and impaired production of NO.

Topics: Acute Kidney Injury; Analysis of Variance; Animals; Arginine; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Ischemia; Nephrectomy; Nitrates; Nitric Oxide; Nitrites; Nitroarginine; Rats; Rats, Wistar; Reference Values; Renal Artery; Renal Circulation; Sodium

We tested the hypothesis that an endogenous nitric oxide synthase (NOS) inhibitor released from ischemic hindlimbs increases the activity of calcium channels in vascular smooth muscle, thus contributing to the increased contractile response to calcium agonists. Hindlimb ischemia was generated in rats by infrarenal aortic cross clamping for 5 h, after which plasma was obtained from femoral vein blood. Incubating naive aortic rings (endothelium intact) for 2 h in plasma collected from ischemic rats significantly reduced relaxation to acetylcholine in precontracted rings and increased contraction to the calcium channel agonist, BAY K 8644. However, in isolated smooth muscle cells (without endothelium) loaded with fura-2, no difference was noted in BAY K 8644-stimulated intracellular calcium concentration. The contractile responses to sodium fluoride, serotonin, and calcium ionophore A23187 were not different in either ischemic or control plasma-incubated rings. The augmentation of the contractile response to BAY K 8644 was significantly inhibited by nitroglycerin (10-8 M) and by exposure to calcium-free solution. N omega-nitro-L-arginine (without plasma incubation)-pretreated rings also demonstrated hyperresponsiveness to BAY K 8644. The increase in responsiveness to BAY K 8644 exhibited a negative correlation with the maximal relaxation to acetylcholine (r = -0.99), suggesting that the apparent increase in activity of calcium channels is mediated through inhibition of nitric oxide by an endogenous NOS inhibitor on endothelium.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Acetylcholine; Animals; Blood Pressure; Calcium; Calcium Channels; Heart Rate; Hindlimb; Ischemia; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Sprague-Dawley; Serotonin; Vasoconstrictor Agents; Vasodilator Agents

Elevation of the ocular pressure in the anterior chamber of the rat eye caused major ischemic damage, manifested as changes in retinal morphology. The two most affected structures were the inner plexiform layer, which decreased in thickness by 90%, and the number of ganglion cells, which decreased by 80%. Pretreatment of the animals with N omega-nitro-L-arginine, a nitric oxide (NOS) inhibitor, almost completely abolished the ischemic damage. Administration of aminoguanidine, a NOS inhibitor selective for the inducible enzyme, partially abolished the ischemic damage. Moreover, administration of the NOS inhibitors 1 h after ischemia, also protected the retina from damage, suggesting that similarly acting drugs could be used clinically to limit ischemic injury in humans. We conclude that NOS, and therefore NO, may be involved in the mechanism of ischemic injury to the retina.

Topics: Animals; Arginine; Cell Count; Enzyme Inhibitors; Guanidines; Intraocular Pressure; Ischemia; Male; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Sprague-Dawley; Retinal Diseases; Retinal Ganglion Cells; Retinal Vessels

We tested the hypotheses that maintaining the activity of nitric oxide by L-arginine infusion would counteract the release of an endogenous nitric oxide synthase inhibitor, improve survival, and decrease intraoperative hypertension after infrarenal aortic cross-clamp surgery. Hindlimb ischemia was generated by infrarenal aortic cross-clamping and tying of the left femoral artery for 5 hours in rats with bilateral femoral and sciatic nerves cut. Mean blood pressure significantly increased during the 5-hour ischemic period in ischemic rats (no drug treatment). Baroreceptor function was inhibited in ischemic rats assessed by intravenous dose response to phenylephrine and nitroprusside after 5 hours of ischemia, suggesting baroreceptor resetting. In ischemic rats infused with L-arginine the intraoperative hypertension was prevented during the 5-hour period, suggesting that this hypertension may be mediated by nitric oxide inhibition. The rates of survival and arrhythmias 2 hours after declamping were 50% in ischemic rats and 100% in ischemic rats treated with N omega-nitro-L-arginine (a nitric oxide synthase inhibitor) 10 minutes before declamping. In ischemic rats infused with L-arginine the survival rate was significantly increased to 100% and the arrhythmic rate was inhibited. We conclude that L-arginine prevents hypertension during cross-clamping and decreases the mortality rate and arrhythmias after declamping by maintaining nitric oxide synthesis. These results suggest that humoral factors released from the ischemic hindlimb may inhibit endogenous nitric oxide production, thus contributing to intraoperative hypertension, arrhythmias, and high mortality rate after aortic cross-clamp surgery.

Topics: Animals; Arginine; Hindlimb; Hypertension; Ischemia; Male; Nitric Oxide; Nitroarginine; Pressoreceptors; Rats; Rats, Sprague-Dawley

There is clinical and experimental evidence to indicate that cigarette smoking may increase the risk of skin ischemic necrosis in flap surgery but the pathogenic mechanism remains unclear. The objectives of this project were to investigate the potential deleterious effects and mechanism of action of nicotine, a major by-product of cigarette smoking, in skin flap surgery in the pig. It was observed that 4-5 weeks of intramuscular nicotine injections (4 mg/kg; twice daily) significantly (p < 0.05) decreased the skin flap capillary blood flow and the length and area of skin flap viability in the pig. This nicotine treatment also induced a 1.6-fold increase in skin flap tissue content of norepinephrine compared with the saline-treated control. The estimated mean wet skin tissue content of norepinephrine (5 x 10(-7) M) was much higher than the circulating level of norepinephrine (1.8 x 10(-9) M) in nicotine-treated pigs. This level of norepinephrine (5 x 10(-7) M) was seen to induce a significant vasoconstrictor effect (75% increase over basal perfusion pressure) in isolated perfused pig skin flaps. It was also observed that the vasoconstrictor effect of norepinephrine was significantly (p < 0.05) enhanced in the presence of 10(-4) M N omega-monomethyl-L-arginine or NG-nitro-L-arginine, an endothelium-derived relaxing factor--nitric oxide (EDRF/NO) synthesis inhibitor. This vasoconstrictor effect was further enhanced in the presence of NG-nitro-L-arginine and 10(-5) M indomethacin, a cyclooxygenase inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arginine; Blood Pressure; Body Temperature; Cardiac Output; Catecholamines; Dermatologic Surgical Procedures; Ischemia; Microcirculation; Necrosis; Nicotine; Nitric Oxide; Nitroarginine; Norepinephrine; omega-N-Methylarginine; Regional Blood Flow; Skin; Surgical Flaps; Swine

Acute renal failure in rats was induced by transient occlusion of bilateral renal arteries and veins to investigate whether insulin-like growth factor-I (IGF-I) has an effect on the damaged renal function or not. Administration of IGF-I at 0.01, 0.1 and 1 mg/kg by s.c. injection caused a 18.7, 33.0 and 66.5% increase of glomerular filtration rate and 54.8, 61.2 and 84.1% decrease of blood urea nitrogen, respectively, compared with the values in the saline-treated group 2 days after ischemia. Other renal parameters tested such as fractional excretion of sodium, N-acetyl-beta-D-glucosaminidase and tubular reabsorptance of phosphorus which are thought to represent renal function of proximal and distal tubules, respectively, were also improved by IGF-I treatment. A histochemical study also supported these observations. Severe epithelial necrosis of proximal tubules and decrease of brush borders were observed 2 days after transient ischemia in the saline-treated group, whereas marked histochemical alterations were not observed in the IGF-I-treated group. L-NG-nitroarginine, an inhibitor of nitric oxide synthetase, prevented the improvement of glomerular filtration rate and blood urea nitrogen by IGF-I at 1 mg/kg, suggesting that the ameliorative action on renal function by IGF-I is mediated via nitric oxide, possibly its vasodilating action. These findings provide the first evidence for the efficacy of IGF-I in the model of acute renal failure, suggesting that IGF-I may be useful for the treatment of acute renal failure.

Topics: Acute Kidney Injury; Animals; Arginine; Blood Urea Nitrogen; Drug Synergism; Glomerular Filtration Rate; Insulin-Like Growth Factor I; Ischemia; Kidney; Male; Nitric Oxide; Nitroarginine; Rats; Rats, Sprague-Dawley; Renal Artery; Renal Veins