Enhanced Nitric Oxide Release Research Articles

Nitric oxide-associated relaxing effects of adrenomedullin in rat aorta

The relaxing effect of adrenomedullin (AM) and that associated with nitric oxide (NO) were investigated in rat isolated aorta. The relaxing effect produced by AM was almost completely inhibited by the removal of endothelium and by treatment with the NOS inhibitor NG-nitro-L-arginine methyester (L-NAME) and hemoglobin but not by indomethacin. Thus the relaxing effect by AM was entirely dependent of endothelial NO synthesis but not of prostaglandins. The cGMP content in the AM-treated relaxed aorta did not change while the cAMP content significantly increased. These findings suggest that the relaxing effects produced by AM in rat aorta primarily relate to an increase in cAMP as well as cGMP maintained by the stimulation of NO spontaneously released from endothelium but not by cGMP resulting from enhanced NO release. There may be some synergistic mechanism between cAMP and NO-cGMP systems in the relaxing effect of AM in rat isolated aorta. © 1996 Wiley-Liss, Inc.

In vivo and in vitro pressor effects of erythropoietin in rats.

Hypertension (HTN) is a common complication of recombinant erythropoietin (EPO) therapy, but the mechanism of the EPO-associated HTN is uncertain. In the present study we examined the effects of EPO and the vehicle alone on rat caudal artery contractile response and basal and thrombin-stimulated platelet cytosolic Ca2+ concentration ([Ca2+]i) in vitro and on blood pressure (BP) and heart rate in vivo. At high concentrations (200 U/ml) EPO caused a small but consistent contraction in the caudal artery rings (P < 0.01) without affecting the response to either angiotensin II (ANG II) or the alpha 1-agonist methoxamine. Incubation with EPO significantly increased basal platelet [Ca2+]i (P < 0.01) and augmented the thrombin-induced rise of [Ca2+]i in Ca(2+)-free medium (P < 0.05). Long-term EPO administration led to a significant elevation of BP within 2 wk regardless of whether the hematocrit was allowed to rise or was kept constant by dietary iron deficiency. In contrast, single intravenous administration of high-dose EPO (400 and 5,000 U/kg), estimated to yield plasma concentrations comparable with those employed in vitro, failed to either alter BP or modify the BP response to ANG II during a 60-min observation period. This was associated with a significant rise in plasma guanosine 3',5'-cyclic monophosphate but no discernible change in plasma atrial natriuretic peptide, suggesting enhanced nitric oxide (NO) release. Thus, at high concentrations, EPO appears to possess a fast-acting pressor effect in vitro but not in vivo. The observed discrepancy may be due to enhanced NO release with EPO administration in vivo. However, HTN does occur with repeated EPO administration in a time-dependent and hematocrit-independent manner. This suggest that expression of the hypertensive effect of EPO in vivo involves a gradual conditioning process.

Nitric oxide: physiology and pharmacology.

Nitric oxide: physiology and pharmacology.

Nitric Oxide

Nitric Oxide

Role of nitric oxide in endotoxin-induced metabolic and vascular dysregulation of the canine diaphragm.

We assessed the role of nitric oxide (NO) in the regulation of diaphragmatic O2 uptake (Vo2di) and phrenic vascular resistance during endotoxemia in anesthetized, mechanically ventilated dogs. Left diaphragmatic vasculature was isolated and briefly pump perfused with arterial blood at a normal flow rate, at a high rate (50% higher than normal), and at low rat (60 to 70% lower than normal). At each rate, Vo2di and phrenic perfusion pressure (Pphr) were measured. Escherichia coli endotoxin (100 mg) was infused intravenously over 90 min in several groups of animals, whereas normal saline was infused into the other. Endotoxin infusion increased Vo2di and reduced Pphr at a given flow rate. These parameters remained unchanged in the saline-infused animals. Infusion of NG-nitro-L-arginine methyl ester (LNAME 6 x 10(-4) M) into the phrenic artery of the endotoxin group reversed the decline in Pphr with no effect on Vo2di. LNAME infusion in the saline group increased Pphr at normal and high flow rates only. Single intravenous injections of LNAME increased arterial pressure and reduced cardiac output in endotoxemic animals, whereas only an increase in arterial pressure was observed in saline-infused animals. Serum arterial and phrenic venous NO concentrations measured in separate groups of animals increased significantly after endotoxin infusion, whereas saline infusion had no effect on these parameters. These results indicate that enhanced NO release plays a significant role in endotoxin-induced phrenic and systemic vasodilation. However, the increase in Vo2di in the endotoxin group does not seem to be mediated by NO release.

Interactions between L-arginine and L-glutamine change endothelial NO production. An effect independent of NO synthase substrate availability.

The effect of extracellular L-arginine and L-glutamine on nitric oxide (NO) release was studied in cultured bovine aortic endothelial cells and in rabbit aortic rings. Increasing L-arginine (0.01 to 10 mM) did not alter NO release from cultured endothelial cells or modify endothelium-dependent relaxation to acetylcholine in isolated vessels. L-Glutamine (0.6 and 2 mM) inhibited NO release from cultured cells (in response to bradykinin) and from aortic rings (in response to acetylcholine or ADP). L-Arginine (0.1-10 mM) dose-dependently reversed the L-glutamine inhibition of receptor-stimulated NO release in both models. In contrast to its inhibitory response to receptor-mediated stimuli, glutamine alone slightly potentiated NO release in both models when the calcium ionophore, A23187, was added. Furthermore, cultured cells incubated with L-arginine (0.01-10 mM), in the presence or absence of glutamine, released similar amounts of NO in response to A23187. L-Glutamine did not affect intracellular L-arginine levels. Neither D-glutamine nor D-arginine affected NO release or endothelium-dependent vascular relaxation. L-Glutamine had no effect on the activity of endothelial NOS assessed by L-arginine to L-citrulline conversion. These findings show that in the absence of L-glutamine, manipulating intracellular L-arginine levels over a wide range does not affect NO release. L-Glutamine in concentrations circulating in vivo may tonically inhibit receptor-mediated NO release by interfering with signal transduction. One mechanism by which L-arginine may enhance NO release is via reversal of the inhibitory effect of L-glutamine, but apparently independently of enhancing NO synthase substrate.

Ginsenosides‐induced nitric oxide‐mediated relaxation of the rabbit corpus cavernosum

1. Ginsenosides, the active ingredients extracted from Panax ginseng, have been shown to promote nitric oxide (NO) release in bovine aortic endothelial cells. Since the endothelial cells and the perivascular nerves in penile corpus cavernosum contain NO synthase and an NO-like substance has been shown to be released from these cells which relaxes corpus cavernosum, the possibility that ginsenosides may relax corpus cavernosum by releasing endogenous NO was examined. 2. With an in vitro tissue superfusion technique, ginsenosides (250, 500 and 750 micrograms ml-1) relaxed corpus cavernosum, concentration-dependently. 3. Using an in vitro tissue bath technique, acetylcholine (ACh)-induced relaxations were increased in the presence of ginsenosides (250 micrograms ml-1). 4. Ginsenosides at 100 micrograms ml-1 significantly enhanced the tetrodotoxin (TTX)-sensitive relaxation of corpus cavernosum elicited by transmural nerve stimulation. 5. The ginsenosides-induced, ACh-induced and ginsenosides-enhanced transmural nerve stimulation-elicited relaxations were significantly attenuated by NG-nitro-L-arginine (100 microM) and oxyhaemoglobin (oxyHb; 5-10 microM), and were enhanced by superoxide dismutase (SOD; 50 u ml-1). 6. The relaxations and their attenuation by NG-nitro-L-arginine and TTX were associated with increase and decrease in tissue cyclic GMP levels, respectively. 7. It is concluded that ginsenosides may release NO from endothelial cells, and enhance NO release from endothelial cells elicited by other vasoactive substances and from perivascular nitrergic nerves in the corpus cavernosum. These endothelial and neurogenic effects of ginsenosides in inducing relaxation of the corpus cavernosum may account for the aphrodisiac effect of Panax ginseng.

Nitric Oxide Released by Platelets Inhibits Neutrophil B 2 Integrin Function Following Acute Carbon Monoxide Poisoning

Carbon monoxide (CO) poisoning has been reported to temporarily inhibit B2 integrin adherence molecules on leukocytes in previous studies in a rat model. The aim of this study was to investigate the mechanism for this effect. Studies were conducted using blood obtained from rats after they were exposed to CO and also with blood cells exposed to CO in vitro. Initial investigations indicated that inhibition of neutrophil (polymorphonuclear leukocyte, PMN) B2 integrin function was linked to the platelets in blood, as the effect was resolved by decreasing platelet number before PMN adherence was tested. The platelet effect could also be shown by incubating either platelet-rich plasma or whole blood with CO in vitro. The effect of platelets was blocked by superoxide radicals and by NG-nitro-L-arginine methyl ester, an inhibitor of nitric oxide (NO) synthase. These observations suggested that CO caused platelets to release NO, an agent known to inhibit the function of B2 integrins. The concentration of NO measured in suspensions of platelets from rats poisoned with CO according to the established model (exposure to 1000 ppm CO for 40 min and 3000 ppm CO for 20 min) was 47 nmol/10(8) platelets, in contrast to only 0.3 nmol NO/10(8) platelets from control rats. Enhanced NO release occurred despite a 60% inhibition of NO synthase activity, assessed by measuring conversion of [14C]L-arginine to citrulline. Exposure to only 1000 ppm CO for 1 hr caused platelets to release 74 nmol NO/10(8) platelets, and no inhibition of NO synthase occurred. Enhanced NO release, and inhibition of PMN adherence, did not occur after platelets were exposed to light from a quartz lamp to photodissociate CO from heme proteins. The data suggest that the NO flux from platelets increased when CO became bound to heme-containing platelet proteins, which normally scavage intraplatelet NO and thus prevent diffusion beyond the platelet membrane.

Nitric oxide synthase does not contribute to cerebral autoregulatory phenomenon in anesthetized dogs

Nitric oxide (NO) is a potent vasodilator produced by nitric oxide synthase (NOS). We tested the following hypotheses: (1) cerebral blood flow (CBF) is NO dependent, (2) NO contributes to CBF autoregulation, and (3) NO participates in the neurohypophysial vasodilator response to hypotension. Three groups of sodium pentobarbital anesthetized dogs were studied using microspheres. In 7 dogs, N omega-nitro-L-arginine methyl ester (L-NAME; 40 mg/kg, i.v.) increased mean arterial pressure (MAP) by 12%. Cerebrovascular resistance (CVR) increased more than MAP, resulting in a 20 +/- 4% reduction (range 12-67%) in baseline CBF. In unblocked conditions, actively autoregulated regions (e.g. cortex, white matter, median eminence) demonstrated a correlation between CVR and MAP whereas passive regions (neural lobe) did not. NOS block did not effect the relationship between MAP and CVR in most brain regions. However, a significant relationship between CVR and MAP developed in neural lobe after NOS block. Abrupt hypotension increased neural lobe blood flow to 239 +/- 37% control at 3 min, despite NOS block. These results show that baseline cerebral vessel tone depends upon NOS activity. Enhanced NO release cannot explain either cerebral autoregulation or the transient hyperperfusion seen in neural lobe immediately following rapid hemorrhage.

Reduced endothelium-dependent relaxation at enhanced NO release in hearts of hypercholesterolaemic rabbits.

1. Langendorff hearts, perfused at constant volume, were prepared from rabbits fed a cholesterol-enriched diet for 4 months. Coronary perfusion pressure and nitric oxide (NO) release (oxyhaemoglobin technique) into the coronary effluent were measured continuously. Prostacyclin (PGI2) in the effluents was determined by radioimmunoassay (6-oxo-PGF1 alpha). 2. Basal NO release was not different between control and hypercholesterolaemic rabbits. However, the coronary vasculature of hypercholesterolaemic rabbits showed a considerably (> 50%) reduced endothelium-dependent relaxation in response to short-term (3 min) infusion of bradykinin (50 nM) and substance P (50 nM) (P < 0.05, n = 8-9). Under these conditions, NO release into the vessel lumen was increased, by 26%, in hypercholesterolaemic hearts (P < 0.05, n = 8-9). NG-nitro-L-arginine (L-NOARG, 30 microM) significantly attenuated both bradykinin-induced NO formation and vessel relaxation in control hearts but only NO release in hypercholesterolaemia. L-Arginine (200 microM) restored the response to that before L-NOARG but did not improve the reduced endothelium-dependent relaxation in cholesterol-fed rabbits. 3. Superoxide dismutase (10 u ml-1) significantly improved vessel relaxation without changing the hypercholesterolaemia-related coronary dysfunction. Vasodilatation in response to exogenous NO donors (linsidomine) was diminished in hypercholesterolaemia as compared to controls. 4. Basal PGI2 release was unchanged in hypercholesterolaemic hearts. There was a tendency in these hearts for greater PGI2 formation after stimulation by substance P and bradykinin (P > or = 0.05). The coronary relaxation to iloprost was unchanged. 5. The data demonstrate impaired endothelium-dependent relaxation of coronary arterial resistance vessels in hypercholesterolaemia. This diminished vascular response was not due to reduced NO generation but probably a reduced action of released NO, either by accelerated degradation and/or disturbed signal transduction pathways to vascular smooth muscle cells. There was no significant change in PGI2 related pathways of vasomotor control in hypercholesterolaemia.

Effects of nitric oxide-containing compounds on increases in cytosolic ionized Ca 2+ and on aggregation of human platelets

The present study was undertaken to determine the modulatory effects of nitric oxide (NO)-releasing compounds on increases in cytosolic ionized calcium ([Ca2+]i) and on aggregation of gel-filtered human platelets induced via diverse agonists. We used various sydnonimines and organic nitrates as donors of NO. Gel-filtered and fura-2-loaded platelets were stimulated with ADP (4-8 microM), collagen (2-10 micrograms/ml) or thrombin (0.02-0.05 IU/ml), respectively. Half-maximal inhibiting effects of sydnonimines on agonist-evoked increases in [Ca2+]i were observed between 30 and 1000 nM, while half-maximal inhibiting effects of the compounds on aggregation were between 3 and 500 nM. The compound C 87-3754, which is the bioactive metabolite of pirsidomine, was a much stronger inhibitor of increases in [Ca2+]i than of platelet aggregation. This was due to an enhanced NO release from this compound exposed to ultraviolet light during Ca2+ measurement. The organic nitrates isosorbide 5-mono-nitrate and nicorandil inhibited both aggregation and increase of cytosolic ionized calcium in stimulated platelets at half-maximal concentrations of approximately 200 microM. The present results suggest that some of the effects of NO on platelets are independent of cytosolic ionized calcium. The results also suggest that some of the inhibitory effects of NO-releasing compounds correspond rather to the presence of the A forms (NO-containing intermediates) than to the presence of free NO.