Irisin Lowers Blood Pressure by Improvement of Endothelial Dysfunction via AMPK-Akt-eNOS-NO Pathway in the Spontaneously Hypertensive Rat.

BACKGROUND: Exercise is a major nonpharmacological treatment for hypertension, but its underlying mechanisms are still not completely elucidated. Irisin, a polypeptide containing 112 amino acids, which is secreted mainly by skeletal muscle cells during exercise, exerts a protective role in metabolic diseases, such as diabetes mellitus and obesity. Because of the close relationship between irisin and metabolic diseases, we hypothesized that irisin may play a role in the regulation of blood pressure. METHODS AND RESULTS: Blood pressures of male Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were monitored through the carotid artery. Our study found that acute intravenous injection of irisin reduced blood pressure in SHRs, but not WKY rats. Irisin, by itself, had no direct vasorelaxing effect in phenylephrine-preconstricted mesenteric arteries from SHRs. However, irisin augmented the acetylcholine-induced vasorelaxation in mesenteric arteries from SHRs that could be reversed by Nω-nitro-l-arginine-methyl ester (L-NAME; 100 µmol/L), indicating a role of nitric oxide (NO) in this action. Indeed, irisin increased NO production and phosphorylation of endothelial nirtic oxide synthase (eNOS) in endothelial cells. 5'-AMP-activated protein kinase (AMPK) was involved in the vasorelaxing effect of irisin because compound C (20 µmol/L), an AMPK inhibitor, blocked the irisin-mediated increase in phosphorylation of eNOS and protein kinase B (Akt) in endothelial cells and vasodilation in mesenteric arteries. CONCLUSIONS: We conclude that acute administration of irisin lowers blood pressure of SHRs by amelioration of endothelial dysfunction of the mesenteric artery through the AMPK-Akt-eNOS-NO signaling pathway.

Inhibition of nitric oxide synthases, but not inducible nitric oxide synthase, selectively worsens left ventricular function after successful resuscitation from cardiac arrest in swine.

OBJECTIVES: Nitric oxide (NO) is a critical regulator of vascular tone and signal transduction in the cardiovascular system. NO is synthesized by three unique enzymes (nitric oxide synthases [NOS]): endothelial and neuronal NOS, both constitutively expressed, and inducible NOS (iNOS), which is induced by proinflammatory stimuli and subsequently produces a burst of NO. NO has been implicated as both an injurious and a beneficial mediator after cardiac arrest and resuscitation. A previous study in swine found that iNOS expression is absent in the myocardium prior to cardiac arrest and that it increases after 10 minutes of untreated ventricular fibrillation (VF), decreases somewhat during the early postresuscitation period, and then steadily increases up to 6 hours postresuscitation. Because this time course of iNOS expression mirrors that of postresuscitation myocardial dysfunction, this study was designed to test the hypothesis that selective inhibition of iNOS improves postresuscitation outcomes in swine. METHODS: Thirty-two domestic swine of either sex were randomly assigned to receive one of the following treatments 15 minutes after return of spontaneous circulation (ROSC): (1) N(G) -nitro-l-arginine methyl ester (l-NAME), a global NO inhibitor; (2) aminoguanidine (AG), a selective iNOS inhibitor; or (3) saline as control. After 10 minutes of untreated VF, swine received a standard resuscitation protocol. Twenty-four-hour survival, neurological status, left ventricular (LV) function, and hemodynamic measurements were obtained. RESULTS: Return of spontaneous circulation occurred in 28 of 32 animals (88%). Only successfully resuscitated animals were assigned to treatment groups and completed the study. There were no differences in survival or neurological outcomes between groups. There were also no differences in LV function or hemodynamic variables found between the control group and the AG group. Global inhibition of NOS with l-NAME post-ROSC increased aortic pressure and transiently decreased pulse pressure. Treatment with l-NAME also increased LV end diastolic pressure and decreased cardiac output within 30 minutes post-ROSC, which was sustained throughout the 4-hour measurements, compared to both the control and the AG groups. In addition, LV ejection fraction recovered to baseline measurements in both the control and AG groups, but failed to recover in the l-NAME group. CONCLUSIONS: Global inhibition of NOS after cardiac arrest and resuscitation markedly worsens hemodynamic variables. Selective inhibition of iNOS after cardiac arrest and resuscitation does not prevent postresuscitation myocardial stunning. There were no significant differences in neurological outcome or survival between treatment groups.

[Involvement of NO-dependent mechanisms of apelin action in myocardial protection against ischemia/reperfusion damage].

Apelin 12 (A-12) was synthesized by the automatic solid phase method with the use of Fmoc technology. The synthesized peptide was purified by preparative HPLC and identified by 1H-NMR spectroscopy and mass spectrometry. Acute myocardial infarction was induced by 40-min LAD occlusion followed by 60-min reperfusion in narcotized Wistar rats. A-12 was administrated at the onset of the reperfusion at doses of 0.07, 0.35 and 0.70 micromole/kg; N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, was applied at a dose of 10 mg/kg 10 min prior to reperfusion alone or before A-12 administration (0.35 micromole/kg); saline was used in control. The indicated A-12 doses induced a transient reduction of the arterial systolic blood pressure (ASBP) to 85, 58, and 56% of the initial level, respectively, which was accompanied by its recovery by the end of reperfusion. All A-12 doses significantly limited myocardial infarct size by 26, 40 and 33%, respectively, compared to the value in control. After administration of A-12 at dose of 0.35 micromol/kg, this effect was combined with reduction of MB-creatine kinase (MB-CK) and lactate dehydrogenase (LDH) activities in plasma at the end of reperfusion by 56 and 47%, respectively, compared to the values in control. Inhibition of NO formation by L-NAME increased SABP but did not affect myocardial infarct size compared with that in control. Coadministration of L-NAME and A-12 resulted in lesser reduction of ASBP during reperfusion than injection of A-12 alone. This intervention led to an increase in infarct size by 26% with concomitant 1.8- and 1.5-times elevation of MB-CK and LDH activities, respectively, compared to the values in the A-12 group. The results indicate that NO is involved as a mediator of the effects of A-12 on the overall protection consisting in a limitation of infarct size and reduction of postischemic cardiomyocyte membrane damage. Cardioprotective mechanisms of apelin action are discussed.

Different mechanisms underlie the effects of acute and long-term inhibition of nitric oxide synthases in antigen-induced pulmonary eosinophil recruitment in BALB/C mice.

Nitric oxide synthase (NOS) inhibitors are largely used to evaluate the NO contribution to pulmonary allergy, but contrasting data have been reported. In this study, pharmacological, biochemical and pharmacokinetic assays were performed to compare the effects of acute and long-term treatment of BALB/C mice with the non-selective NOS inhibitor L-NAME in ovalbumin (OVA)-challenged mice. Acute L-NAME treatment (50 mg/kg, gavage) significantly reduced the eosinophil number in bronchoalveolar lavage fluid (BALF). The inducible NOS (iNOS) inhibitor aminoguanidine (20 mg/kg/day in the drinking water) also significantly reduced the eosinophil number in BALF. In contrast, 3-week L-NAME treatment (50 and 150 mg/kg/day in the drinking water) significantly increased the pulmonary eosinophil influx. The constitutive NOS (cNOS) activity in brain and lungs was reduced by both acute and 3-week L-NAME treatments. The pulmonary iNOS activity was reduced by acute L-NAME (or aminoguanidine), but unaffected by 3-week L-NAME treatment. Acute L-NAME (or aminoguanidine) treatment was more efficient to reduce the NOx- levels compared with 3-week L-NAME treatment. The pharmacokinetic study revealed that L-NAME is not bioavailable when given orally. After acute L-NAME intake, serum concentrations of the metabolite Nomega-nitro-L-arginine decreased from 30 min to 24 h. In the 3-week L-NAME treatment, the Nomega-nitro-L-arginine concentration was close to the detection limit. In conclusion, 3-week treatment with l-NAME yields low serum Nomega-nitro-L-arginine concentrations, causing preferential inhibition of cNOS activity. Therefore, eosinophil influx potentiation by 3-week L-NAME treatment may reflect removal of protective cNOS-derived NO, with no interference on the ongoing inflammation due to iNOS-derived NO.

Sounding airflow enhances aerosol delivery into the paranasal sinuses.

BACKGROUND: The use of aerosol therapy is commonly suggested in the treatment of paranasal disorders but it is difficult to achieve an effective penetration of drugs into the sinuses. The authors have recently shown that an oscillating airflow produced by phonation (nasal humming) causes a large increase in the gas exchange between the nose and the paranasal sinuses. This is reflected by a high peak in nasally exhaled nitric oxide (NO) levels because NO accumulated in the sinuses is rapidly washed-out via the sinus ostia. OBJECTIVE: This study was designed to test whether the increase in sinus gas exchange caused by an oscillating airflow could be used to enhance penetration of a drug into the sinuses. MATERIALS AND METHODS: In six healthy subjects a nitric oxide-synthase inhibitor L-NAME was administrated into the nostrils by a jet nebulizer connected to a duck call, which could be modified to generate either a sounding airflow or a non-sounding airflow. The degree of L-NAME penetration into the sinuses was judged from the reduction in nasal NO during humming exhalations. Sinus drug deposition was also studied in a model of the nose and sinus. RESULTS: In humans the delivery of L-NAME with the non-sounding airflow had no effect on the NO levels achieved during humming, whereas L-NAME administration with sound caused a significant 22-35% reduction in nasal NO. In the model the aerosol delivery with the sounding airflow caused a fourfold increase in sinus drug deposition as compared with an aerosol without sound. CONCLUSION: A sounding airflow increases the delivery of an aerosolized drug into the paranasal sinuses.

Effects of nitric oxide syntase inhibitor on exercise-mediated pro- and anti-oxidative balance in rat blood plasma.

Studies showed that nitric oxide synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME), enhanced anti-oxidative shifts in the blood plasma of rats subjected to exhaustive running exercise. A type of running training (continuous endurance and intermittent) before the exhaustive exercise was found to differentiate L-NAME-induced effects in rats.

Evidence for the role of nitric oxide in nicotine-induced locomotor sensitization in mice.

RATIONALE: Nitric oxide (NO) is implicated in both acute effects of addictive drugs and development of dependence to them. We investigated the role of NO in nicotine-induced locomotor sensitization. OBJECTIVES: The effects of Nomega-nitro-L-arginine methyl ester (L-NAME), a NO synthase inhibitor, and a combination of a NO precursor L-arginine and L-NAME on nicotine-induced locomotor sensitization were investigated in Swiss Webster mice. METHODS: Sensitization to psychomotor stimulating effect of nicotine was rendered by seven injections of nicotine (1 mg/kg) on every other day. To investigate their effect on the development of sensitization to nicotine, L-NAME (15-60 mg/kg) and L-arginine (1 g/kg) were given before nicotine administration during the first seven sessions. To investigate the effect of these compounds on the expression of nicotine sensitization, after a 4-day drug-free period another group of mice received a challenge injection of nicotine on day 18. RESULTS: Nicotine (1 mg/kg) produced a robust locomotor sensitization in mice. The doses of 30 mg/kg and 60 mg/kg of L-NAME blocked the development of sensitization to nicotine; and, L-arginine (1 g/kg) pretreatment reversed this effect of L-NAME. Likewise, the doses of 30 mg/kg and 60 mg/kg of L-NAME inhibited the expression of sensitization to nicotine on day 18; and, L-arginine (1 g/kg) pretreatment reversed this inhibitory effect of L-NAME. CONCLUSIONS: Our results suggest that NO is implicated in the development and expression of nicotine-induced locomotor sensitization in mice.

Ultra-stable zeolites--a tool for in-cell chemistry.

Ultrastable zeolite particles were used as vehicles to carry low molecular bio-active substances and macromolecules as proteins into viable cells. Zeolite particles that can be used for internalisation by phagocytosis were obtained from the non-sedimenting fraction of a commercially available zeolite preparation after 1 x g sedimentation. Protein adsorbed on the zeolite surface was shown to enter the endosomal pathway after phagocytosis and could be cleaved by the endosomal proteases. As a model of a low molecular weight bio-active molecule, the inhibitor of the cellular synthesis of nitrogen oxide, N-nitro-L-arginine methyl ester (L-NAME), was used. A partial inhibition of the cellular NO production was shown after utilizing zeolites as vehicles to introduce the inhibitor into the cells. A targeting of the intra-cellular enzymes that was at least 10 times more efficient was obtained by the use of zeolites as a carrier of the inhibitor, as opposed to addition of the inhibitor to the culture medium.

Central blockade of nitric oxide synthesis reduces moxonidine-induced hypotension.

1. Nitric oxide (NO) and alpha(2)-adrenoceptor and imidazoline agonists such as moxonidine may act centrally to inhibit sympathetic activity and decrease arterial pressure. 2. In the present study, we investigated the effects of pretreatment with l-NAME (NO synthesis inhibitor), injected into the 4th ventricle (4th V) or intravenously (i.v.), on the hypotension, bradycardia and vasodilatation induced by moxonidine injected into the 4th V in normotensive rats. 3. Male Wistar rats with a stainless steel cannula implanted into the 4th V and anaesthetized with urethane were used. Blood flows were recorded by use of miniature pulsed Doppler flow probes implanted around the renal, superior mesenteric and low abdominal aorta. 4. Moxonidine (20 nmol), injected into the 4th V, reduced the mean arterial pressure (-42+/-3 mmHg), heart rate (-22+/-7 bpm) and renal (-62+/-15%), mesenteric (-41+/-8%) and hindquarter (-50+/-8%) vascular resistances. 5. Pretreatment with l-NAME (10 nmol into the 4th V) almost abolished central moxonidine-induced hypotension (-10+/-3 mmHg) and renal (-10+/-4%), mesenteric (-11+/-4%) and hindquarter (-13+/-6%) vascular resistance reduction, but did not affect the bradycardia (-18+/-8 bpm). 6. The results indicate that central NO mechanisms are involved in the vasodilatation and hypotension, but not in the bradycardia, induced by central moxonidine in normotensive rats.

Febrigenic signaling to the brain does not involve nitric oxide.

1. The involvement of peripheral nitric oxide (NO) in febrigenic signaling to the brain has been proposed because peripherally administered NO synthase (NOS) inhibitors attenuate lipopolysaccharide (LPS)-induced fever in rodents. However, how the unstable molecule of NO can reach the brain to trigger fever is unclear. It is also unclear whether NOS inhibitors attenuate fever by blocking febrigenic signaling or, alternatively, by suppressing thermogenesis in brown fat. 2. Male Wistar rats were chronically implanted with jugular catheters; their colonic and tail skin temperatures (T(c) and T(sk)) were monitored. 3. Study 1 was designed to determine whether the relatively stable, physiologically relevant forms of NO, that is, S-nitrosoalbumin (SNA) and S-nitrosoglutathione (SNG), are pyrogenic and whether they enhance LPS fever. At a neutral ambient temperature (T(a)) of 31 degrees C, afebrile or LPS (1 microg kg(-1), i.v.)-treated rats were infused i.v. with SNA (0.34 or 4.1 micromol kg(-1); the controls received NaNO(2) and albumin) or SNG (10 or 60 micromol kg(-1); the controls received glutathione). T(c) of SNA- or SNG-treated rats never exceeded that of the controls. 4. In Study 2, we tested whether the known fever-attenuating effect of the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) at a subneutral T(a) (when fever is brought about by thermogenesis) also occurs at a neutral T(a) (when fever is brought about by skin vasoconstriction). At a subneutral T(a) of 24 degrees C, L-NAME (2.5 mg kg(-1), i.v.) attenuated LPS (10 microg kg(-1), i.v.) fever, presumably by inhibiting thermogenesis. At 31 degrees C, L-NAME enhanced LPS fever by augmenting skin vasoconstriction (T(sk) fall). 5. In summary, both SNA and SNG had no pyrogenic effect of their own and failed to enhance LPS fever; peripheral L-NAME attenuated only fever brought about by increased thermogenesis. It is concluded that NO is uninvolved in febrigenic signaling to the brain.

Involvement of nitric oxide during in vitro fertilization and early embryonic development in mice.

Nitric oxide (NO) has emerged as an important intracellular and intercellular messenger, controlling many physiological processes and participating in the fertilization process via the autocrine and paracrine mechanisms. This study investigated whether nitric oxide synthase (NOS) inhibitior (L-NAME) and L-arginine could regulate in vitro fertilization and early embryonic development in mice. Mouse epididymal spermatozoa, oocytes, and embryos were incubated in mediums of variable conditions with and without L-NAME or L-arginine (0.5, 1, 5 and 10 mM). Fertilization rate and early embryonic development were significantly inhibited by treating sperms or oocytes with L-NAME (93. 8% vs 66.3%, 92.1% vs 60.3%), but not with L-arginine. In contrast, fertilization rate and early embryonic development were conspicuously reduced when L-NAME or L-arginine was added to the culture media for embryos. Early embryonic development was inhibited by microinjection of L-NAME into the fertilized embryos in a dose-dependent manner, but only by high concentrations of L-arginine. These results suggest that a moderate amount of NO production is essential for fertilization and early embryo development in mice.

Multiple cedar pollen challenge diminishes involvement of histamine in allergic conjunctivitis of Guinea pigs.

It has been reported that antihistamines do not fully modify symptoms of allergic conjunctivitis in clinical settings, suggesting that histamine is not the only contributor to symptom generation in the disease. However, in the majority of experimental allergic conjunctivitis models, antihistamines are very effective in the reduction of symptoms. In the present study, we used our recently developed guinea pig model of allergic conjunctivitis and evaluated whether involvement of histamine in the induction of symptoms of allergic conjunctivitis is altered by multiple antigen challenges. Guinea pigs were sensitized by intraperitoneal injection of Japanese cedar pollen extracts adsorbed on aluminum hydroxide gel, and then challenged by dropping a pollen suspension without the adjuvant on each eye once a week until the 15th challenge. The magnitude of the conjunctivitis intensity score (CIS), itch-associated scratching response and albumin leakage were found to increase with repeated challenges. At the 1st-3rd challenges, histamine H(1) receptor antagonist, mepyramine (10 mg/kg, p.o.), strongly reduced all these symptoms. However, symptoms at the 5th-15th challenges were not inhibited by mepyramine. On the other hand, a nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (10 mg/kg, i.v.), potently inhibited the increase of CIS and albumin leakage at the 15th challenge. In conclusion, histamine involvement in the induction of conjunctivitis symptoms in our model was diminished by multiple antigen challenges. The allergic conjunctivitis at the chronic stage is partly mediated by nitric oxide (NO) derived from NOSs that may be activated by mediators other than histamine. The histamine-independent allergic conjunctivitis may be useful for analyzing mechanisms underlying chronic conjunctivitis.

Simvastatin ameliorates injury in an experimental model of lung ischemia-reperfusion.

OBJECTIVES: Statins are lipid-lowering drugs with anti-inflammatory and antioxidant properties. This study explores the potential of these commonly prescribed agents to ameliorate lung ischemia-reperfusion injury. METHODS: Left lungs of Long-Evans rats were rendered ischemic for 90 minutes and reperfused for up to 4 hours. Treated animals received simvastatin orally (0.5 mg/kg) for 5 days before the experiment. Injury was quantitated in terms of tissue myeloperoxidase content, vascular permeability ((125)I bovine serum albumin extravasation), and bronchoalveolar lavage leukocyte and cytokine content. Changes in nuclear translocation of transcription factors were evaluated by electromobility shift assay. Additional animals received N(G)-nitro-L-arginine methyl ester before ischemia-reperfusion to assess whether inhibition of nitric oxide synthase could reverse simvastatin's protective effects. The presence of nicotinamide adenine dinucleotide phosphate oxidase was also evaluated using enzyme staining both histologically and in native electrophoresis. RESULTS: Lung vascular permeability was reduced in treated animals by 71% compared with positive controls (P <.001). Administration of N(G)-nitro-L-arginine methyl ester reversed this protection. The protective effects of statin pretreatment correlated with a 68% reduction in tissue myeloperoxidase content (P <.01), marked reductions in bronchoalveolar lavage leukocyte accumulation, and decreased expression of proinflammatory cytokines. Nicotinamide adenine dinucleotide phosphate oxidase expression also decreased with statin treatment. CONCLUSION: In addition to its antioxidant properties, the protective effects of simvastatin are likely mediated by modulation of endothelial nitric oxide synthase. The potential to pretreat recipients of lung transplantation with statins to ameliorate reperfusion injury is promising.

Involvement of cholinoceptors in cadmium-induced endothelial dysfunction.

Cadmium (Cd) toxicity was produced in male rats to study the role of cholinoceptors in Cd-induced endothelial dysfunction. The changes in the tension of the aortic rings to constrictor and dilator agonists were compared with those of controls. A Cd-induced significant increase in phenylephrine response was associated with a decrease in basal dilator prostanoid release. In Cd-exposed rings, despite an obvious depression in the acetylcholine (ACh) response, the receptor-independent dilation to the calcium ionophore A23187, which elicits a receptor-independent endothelial relaxation, was slightly elevated (p<0.01), but the smooth muscle cell response to the NO donor, sodium nitroprusside (SNP) remained unaltered. Cadmium decreased both the maximal response to ACh (10(-5) M) and its pirenzepine (Prz) sensitive component. The M1 type cholinoceptor-mediated response to ACh decreased in Cd-exposed rings to 10.30 +/- 5.00% from 38.40 +/- 6.90% (p<0.001). Cadmium also reduced the share of indomethacin 1.64% to 13.92 +/- 2.89% (p<0.01), which correlated well with the changes in the M1-mediated response (r=0.991, p<0.0001). Most of the deleterious effect of Cd appears to be restricted to the M1-dependent ACh response. These findings suggest that Cd produces an endothelial dysfunction by impairing the M1 type cholinoceptor mediated response, which seems to be involved in prostanoid release.

Behavioral effects of NMDA receptor agonists and antagonists in combination with nitric oxide-related compounds.

Responding of rats was maintained under a 120-response fixed ratio (FR) schedule of food delivery, and animals received individual and combined injections of N-methyl-D-aspartic acid (NMDA), phencyclidine hydrochloride, (+)-MK-801 hydrogen maleate (MK-801), (+/-)-2-amino-5-phosphonopentanoic acid (AP5), 7-chlorokynurenic acid (7CK), ifenprodil tartrate, N(G)-nitro-L-arginine methyl ester hydorchloride (L-NAME), 7-nitroindazole, aminoguanidine hemisulfate, L-arginine, molsidomine, sodium nitroprusside, and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8). Behavioral suppression after NMDA was completely and dose-dependently reversed by MK-801, phencyclidine, AP5, and aminoguanidine; partially and dose-dependently attenuated by molsidomine, ifenprodil, and 7CK; and not attenuated at all by L-NAME, 7-nitroindazole, or TMB-8. These findings suggested that behavioral suppression after NMDA was associated with nitric oxide from the inducible synthase. In a second series of experiments, comparable behavioral suppression by 0.1 mg/kg MK-801, but not 3 mg/kg phencyclidine, was attenuated by nitroprusside, molsidomine, and L-arginine, suggesting that suppressions from MK-801 and phencyclidine were mediated by different final common pathways, and that behavioral suppression from MK-801, but not phencyclidine, may be associated with Ca(2+)-dependent nitric oxide.

Gastroprotective effects of phenylpropanoids from the rhizomes of Alpinia galanga in rats: structural requirements and mode of action.

The effects of 1'S-1'-acetoxychavicol acetate and related phenylpropanoids isolated from the rhizomes of Alpinia galanga on ethanol-induced gastric lesions in rats were examined. Among them, 1'S-1'-acetoxychavicol acetate and 1'S-1'-acetoxyeugenol acetate markedly inhibited the ethanol-induced gastric mucosal lesions (ED(50)=0.61 and ca. 0.90 mg/kg). In addition, 1'S-1'-acetoxychavicol acetate inhibited the lesions induced by 0.6 M HCl (ED(50)=0.73 mg/kg) and aspirin (ED(50)=0.69 mg/kg) but it did not show a significant effect on indomethacin-induced gastric lesions and acid output in pylorus-ligated rats at doses of 0.5-5.0 mg/kg. From the gastroprotective effects of various related compounds, the 1'-acetoxyl group of 1'S-1'-acetoxychavicol acetate and 1'S-1'-acetoxyeugenol acetate was found to be essential for their strong activity. With regard to the mode of action, the gastroprotective effects of 1'S-1'-acetoxychavicol acetate were attenuated by pretreatment with indomethacin and N-ethylmaleimide, and 1'S-1'-acetoxychavicol acetate significantly increased the glutathione levels of gastric mucosa in rats. These findings suggest that endogenous prostaglandins and sulfhydryl compounds are involved in the protective effect of 1'S-1'-acetoxychavicol acetate.

Cutaneous venous dysfunction studied in vivo in the LPS-treated rabbit: implication of NO in saphenous vein hyporeactivity.

Superficial vein pathology involves both mechanical (hyperpressure and distension) and inflammatory mechanisms. Conflicting results exist about the role of NO in the venous hyporeactivity induced by inflammation. In order to clarify this point, we aimed to investigate the effects of sepsis on cutaneous vein responsiveness in vivo and the possible contributions of constitutive and inducible NOS to the changes of venous contractility. Saphenous vein diameter was recorded by an ultrasonic echo-tracking device in pentobarbital-anaesthetised rabbits. Bacterial lipopolysaccharide (LPS) was administered i.v. at 20 mg/kg/15 min, inducing a progressive fall in mean arterial blood pressure after 2-3 h. The effects of LPS on saphenous vein responsiveness to noradrenaline (2 microg/kg i.v.) were measured simultaneously. In some rabbits, veins were removed for immunochemistry to detect iNOS staining. The venoconstriction to noradrenaline was already significantly reduced at 30 min after LPS (6+/-1% instead of 19+/-1% before LPS) and was completely abolished 3 h after LPS. A reduction of the venoconstriction induced by sumatriptan, a 5-HT(1B/D) agonist, (100 microg/kg, 11+/-1% after saline n=5) was also observed 180 min after LPS infusion (3+/-1%, n=4). The venodilatations induced by acetylcholine or sodium nitroprusside injected locally into the vein were not altered by LPS. When administered 90 min after LPS infusion, the NOS inhibitor L-NAME but not the selective iNOS inhibitor L-NIL (10 mg/kg) induced a recovery of the venoconstriction. Preventive perfusion with L-NAME (10 mg/kg/2 h) reduced the initial hyporeactivity to noradrenaline (30 to 60 min), but accelerated the lethal fall in MAP. L-NIL (10 mg/kg/2 h), to a lesser extent than L-NAME, also reduced the initial hyporeactivity to noradrenaline; in contrast to L-NAME, L-NIL also delayed the complete loss of noradrenaline constriction and improved animal survival. In control animals, neither L-NAME nor L-NIL modified the venoconstriction induced by noradrenaline. iNOS staining was observed in the saphenous vein endothelium after LPS. The experimental model developed in these experiments allows the study of venous responsiveness during sepsis in vivo. Our results show that LPS administration reduces saphenous vein contractility to both adrenergic and serotoninergic constrictor agents. The data suggest that both endothelial and inducible NO are involved in the loss of venous reactivity but these enzymes exert contrasting effects on blood pressure changes.

Noradrenaline-induced contraction mediated by endothelial COX-1 metabolites in the rat coronary artery.

Noradrenaline-induced contraction of the rat coronary arteries was significantly augmented by the presence of NG-nitro-L-arginine methyl ester (L-NAME) and arachidonic acid. The experiments in the study presented here were undertaken to characterize pharmacologically the augmented noradrenaline-induced contraction in ring preparations of rat coronary arteries. The contraction was stopped by a chemical remover of endothelium (saponin). Oxygen radical scavengers, superoxide dismutase and catalase, significantly attenuated the contraction. Cyclooxygenase-1 inhibitors (flurbiprofen, 10(-7) M) attenuated the noradrenaline-induced contraction and cyclooxygenase-2 (nimesulide, 10(-7) M) slightly attenuated the contraction. A thromboxane A2 (TXA2) synthetase inhibitor (OKY-046) and a TXA2 receptor antagonist (S-1452) did not affect the contraction. Based on these results, it was suggested that the contraction induced by noradrenaline in the rat coronary artery in the presence of L-NAME and arachidonic acid is endothelium-dependent, and that it involves reactive oxygen species and endothelial cyclooxygenase-1 metabolites of arachidonic acid.

Exercise-induced hyperaemia and leg oxygen uptake are not altered during effective inhibition of nitric oxide synthase with N(G)-nitro-L-arginine methyl ester in humans.

1. In the present study the highly potent nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was intravenously infused and examined for its efficacy in inhibiting NOS activity and in altering blood flow and oxygen uptake in human skeletal muscle. 2. The plasma concentrations of L-NAME and its active metabolite NG-nitro-L-arginine (L-NA), and the activity of NOS in skeletal muscle were measured in healthy male subjects (n = 6) before (control) and after 60 min of intravenous infusion of L-NAME (4 mg kg(-1)). In another group of healthy males (n = 8), the physiological effects of L-NAME were studied at rest, and during submaximal and exhaustive knee extensor exercise before (control) and 30 min after L-NAME infusion (4 mg kg(-1)). 3. The plasma concentrations of L-NAME and L-NA were highest (8.4 +/- 1.6 and 8.3 +/- 0.8 micromol l(-1)) after 60 min of L-NAME infusion. Ninety minutes later mainly L-NA remained in plasma (5.1 +/- 0.4 micromol l(-1)). Thirty minutes after L-NAME infusion, the muscle L-NA content was 38 +/- 4 micromol (kg dry wt)-1 and muscle NOS activity was reduced by 67 +/- 8 % (P < 0.05). 4. Leg blood flow and leg oxygen uptake during submaximal and exhaustive exercise were similar (P > 0.05) following L-NAME infusion and in control. Blood flow during recovery was lower in the L-NAME condition (P < 0.05). 5. In conclusion, the present study shows for the first time that systemic infusion of L-NAME in humans causes a marked reduction in skeletal muscle NOS activity. Despite this attenuated NOS activity, exercise-induced hyperaemia and oxygen uptake were unaltered. Thus, the data strongly suggest that NO is not essential for the regulation of blood flow or oxygen uptake in contracting human skeletal muscle.

Distribution and metabolism of N(G)-nitro-L-arginine methyl ester in patients with septic shock.

OBJECTIVE: The pharmacokinetics of N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, was investigated in patients with septic shock. METHODS: Blood was sampled at intervals before, during and after 12-h infusion of L-NAME 1 mg x kg(-1) x h(-1) in nine septic shock patients for determination of plasma concentrations by high-performance liquid chromatography (HPLC). In three patients the renal clearance of the drug was determined. RESULTS: Incubation of L-NAME with plasma and blood in vitro revealed hydrolysis to N(G)-nitro-L-arginine (L-NOARG), the active inhibitor of NO synthesis. L-NOARG did not undergo further degradation. Continuous intravenous infusion of 1 mg x kg(-1) x h(-1) of L-NAME for 12 h in patients with septic shock increased blood pressure and resulted in increasing plasma concentrations of L-NOARG (Cmax 6.2 microg x ml(-1) at 12 h) whereas L-NAME concentrations reached a plateau within 1.5 h (Cmax 1.0 microg x ml(-1)). After the infusion was stopped L-NAME disappeared from the plasma rapidly (half-life 19.2 min) whereas L-NOARG concentration declined slowly (half-life 22.9 h). The calculated volume of distribution for L-NAME was 0.451 x kg(-1) body weight and 1.961 x kg(-1) for L-NOARG. The renal clearance for L-NOARG was 3.5% of total body clearance for L-NOARG, whereas L-NAME could not be detected in urine. CONCLUSION: We conclude that vasoconstriction with L-NAME in septic patients may result from hydrolysis to L-NOARG, the active inhibitor of NO synthesis. The long plasma half-life and large volume of distribution for L-NOARG suggests extensive distribution to extravascular tissues. Since renal excretion is minimal, elimination of the metabolite L-NOARG follows other pathways.