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In this work we describe a region of mouse intestine, the caecum, in which inhibitory transmission to the longitudinal muscle is predominantly due to nitric oxide. In the presence of muscarinic receptor blockade, electrical stimulation of intramural nerves in the longitudinal muscle of the mouse caecum evoked a relaxation. The relaxation was reduced to about 25% of the control amplitude by the nitric oxide synthase inhibitor L-NMMA (NG-methyl-L-arginine), but was unaffected by D-NMMA. In the presence of the nitric oxide scavenger oxyhaemoglobin, the relaxation was reduced to less than 10% of the control amplitude. In the circular muscle of the caecum and the longitudinal muscle of the ileum, colon and rectum, electrical field stimulation either evoked only small relaxations, or relaxations that were unaffected by L-NMMA. Nitric oxide synthase-containing neurons in the caecum were localized immunohistochemically using an antibody to neuronal nitric oxide synthase or with NADPH diaphorase histochemistry. Reactive nerve cell bodies were observed in the myenteric plexus, and varicose nerve fibres were present in the longitudinal and circular muscle layers of the caecum. The transduction mechanism of the nitric oxide-mediated relaxation in the longitudinal muscle of the caecum was examined using ODQ (1 H-[1,2,4]oxadiazolo[4,3,-alpha-]quinoxalin-1-one), a selective inhibitor of soluble guanylyl cyclase. ODQ abolished the relaxations induced by applied sodium nitroprusside (0.01-1 mM) and reduced the relaxation induced by electrical stimulation to about 40% of control values. However, ODQ reduced the relaxations induced by electrical stimulation to a lesser extent than L-NMMA. Hence, although the relaxation in this tissue mediated by NO (or an NO-related substance) is largely via soluble guanylyl cyclase, an action of NO on other targets cannot be ruled out.

After subarachnoid hemorrhage (SAH), cerebral arteries display impaired vasomotor control, resulting in decreased regional cerebral blood flow. Recently, propagation of vasomotor responses has been recognized as an important regulatory mechanism in microcirculation. In this study, the authors tested the hypothesis that oxyhemoglobin (OxyHb) inhibits the vasodilatory effect of chemical mediators such as adenosine and adenine nucleotides at a local and/or propagated site. Penetrating intracerebral arterioles were surgically isolated from the middle cerebral arteries of rat brains, cannulated, and observed videomicroscopically in an organ bath under an inverted microscope. The effects of 10(-5) M OxyHb on vasoactive responses to adenosine, adenosine diphosphate (ADP), and adenosine triphosphate (ATP) were examined. The drugs were extraluminally applied either to the bath (10(-10)-10(-3) M) or, using pressure microejection (pipette concentration 10(-2) M), locally. The ATP and ADP initially constricted and then significantly dilated the vessels after both extraluminal application and microapplication. Furthermore, local microstimulation by these drugs produced conducted vasodilation. Adenosine elicited significant vasodilation after both extraluminal and local stimulation. Again, conducted vasodilation was observed. The vasomotor responses that were induced by a maximum local stimulation corresponded in magnitude to those observed at bath concentrations of 10(-5) to 10(-4) M of the same drug. Pretreatment with OxyHb constricted arterioles to an average of 87% of control and blunted extraluminally induced dilation at low concentrations (10(-10)-10(-8)) of ATP and ADP, but did not affect vasodilation induced by 10(-4) M or greater concentrations of ATP, ADP, or adenosine. Although the local response to local microstimulation was unaltered, propagated vasodilation as a response to ATP, ADP, and adenosine was significantly attenuated by OxyHb. These findings indicate that vasodilatory propagation plays an important role in the regulation of brain microcirculation and that its impairment by OxyHb could, in part, explain the cerebral hypoperfusion that is observed after SAH.

To clarify the involvement of nitric oxide (NO) derived from nitrosothiols (RSNO) in 5-hydroxytryptamine (5-HT)-induced Ca(2+)-independent cGMP formation (CIGF) in NG108-15 cells, we investigated the effects of 5-HT on intracellular contents of RSNO as well as of NO metabolites. 5-HT stimulation resulted in an increase in the intracellular contents of nitrate and cGMP. RSNO was detected in NG108-15 cells and was decreased by 5-HT stimulation. Furthermore, the time course of nitrate increase was coincident with that of RSNO decrease. CarboxyPTIO inhibited 5-HT-induced CIGF, whereas oxyhemoglobin failed to inhibit it. The data suggest that NO is stored in a stable form as RSNO and that 5-HT stimulates NO generation from endogenous RSNO, which is followed by elevation of cGMP via activation of cytosolic guanylyl cyclase by NO in NG108-15 cells. We suggest the existence of a novel 5-HT signal transduction pathway involved in NO generation in NG108-15 cells.

Purpose: To determine whether the monkey central retinal artery is innervated by vasodilator nerves and to analyze the mechanism underlying the neurogenic response., Methods: Changes in isometric tension were recorded in helical strips of the arteries, which were stimulated by transmurally applied electrical pulses or nicotine. The presence of perivascular nerve fibers containing nitric oxide (NO) synthase immunoreactivity was determined histologically., Results: Transmural electrical stimulation (5 Hz) and nicotine produced a relaxation of the arterial strips denuded of the endothelium, treated with prazosin, and contracted with prostaglandin F2 alpha. The response was not influenced by timolol, atropine, and indomethacin, but it was abolished by methylene blue and oxyhemoglobin. NG-nitro-L-arginine, a NO synthase inhibitor, abolished the neurogenic relaxation, and L-arginine restored the response. Antagonists of calcitonin gene-related peptide and vasoactive intestinal polypeptide in sufficient concentrations did not influence the response to nerve stimulation by nicotine. There were abundant nerve fibers and bundles containing NO synthase immunoreactivity in the adventitia., Conclusions: Monkey central retinal arteries are innervated by NO synthase-containing nerves that liberate NO possibly as a neurotransmitter on excitation to produce muscular relaxation.

In this study, the effect of tetrahydrobiopterin deficiency on the nitric oxide/cGMP pathway has been investigated in cerebellar slices derived from the hph-1 mouse. This animal displays a partial deficiency of tetrahydrobiopterin. Basal levels of cGMP were significantly reduced (-29.5%) in the hph-1 mouse cerebellum compared to controls. Following kainate stimulation (500 microM) cGMP levels increased in both control and hph-1 preparations but were again significantly lower (-29.1%) in the hph-1 mouse. Exposure of slices to the nitric oxide donors, S-nitroso-N-acetylpenicillamine and S-nitroso-glutathione, revealed no difference in cGMP accumulation between the two groups. These findings suggest that the cerebellar nitric oxide/cGMP pathway may be impaired in partial tetrahydrobiopterin deficiency states due to diminished nitric oxide formation.

It is becoming apparent that nitrogen monoxide (NO) such as nitric oxide has regulatory roles for neuronal cell functions. We examined the role of NO using NO donors on [3H]noradrenaline (NA) release from prelabeled rat hippocampal slices. Sodium nitroprusside (SNP), which had no effect by itself, stimulated [3H]NA release in a dose-dependent manner (ED50 = 0.5 mM) in the presence of dithiothreitol (DTT). The stimulatory effect of SNP with DTT, but not high K+, was observed in an extracellular Ca(2+)-free buffer. The maximal effect of SNP was obtained after incubation for 1-2 h with DTT in buffer at physiological pH (7.4). The simultaneous addition of SNP and DTT to the slices induced a small effect, and the effect of SNP declined after 3.5 h. SNP stimulated cyclic GMP accumulation in the slices without DTT. NaNO2 and 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (a generator of nitric oxide), which stimulated cyclic GMP accumulation by themselves, did not stimulate [3H]NA release in the presence and absence of DTT. 3-Morpholinosydnonimine HC1 (a generator of peroxynitrite) had no effect on the release. The stimulatory effect of SNP and DTT on NA release was inhibited 40% by nitric oxide scavengers such as oxyhemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide, although cyclic GMP accumulation induced by NO donors was completely inhibited. These findings suggest that SNP reacts with DTT to produce unknown active species, and that cyclic GMP is not a mediator for SNP-stimulated NA release.

The sarcoplasmic reticulum-bound creatine kinase from rabbit skeletal muscle was inhibited by the nitric oxide donor S-nitrosoglutathione (GSNO). This led to a decrease in Ca2+ uptake in sarcoplasmic reticulum vesicles when the transport was driven by ATP generated from phosphocreatine and ADP. In contrast, the Ca 2+ transport measured using 2 mM ATP as substrate was unaffected by GSNO up to 200 microM. GSNO (5-20 microM) inhibited the activity of both soluble and membrane-bound creatine kinase. Oxyhemoglobin (15-40 microM) protected creatine kinase against inactivation by GSNO. The inhibition by 10 microM GSNO was reversed by the addition of dithiothreitol (2 mM). The results indicate that nitric oxide (NO, including NO+, NO and NO-) inactivates creatine kinase in vitro by promoting nitrosylation of critical sulphydryl groups of the enzyme.

Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by nitric oxide (NO) and by the NO generators sodium nitroprusside, diethylamine/NO, S-nitroso-N-acetylpenicillamine, and S-nitrosocysteine. The inactivation occurs in an oxygen-free environment and is enhanced by dithiothreitol and ascorbic acid. Protection against the effect of NO on tryptophan hydroxylase is afforded by oxyhemoglobin, reduced glutathione, and exogenous Fe(II). Catalase partially protects the enzyme from NO-induced inactivation, whereas both superoxide dismutase and uric acid are without effect. These findings indicate that tryptophan hydroxylase is a target for NO and suggest that critical iron-sulfur groups in this enzyme serve as the substrate for NO-induced nitrosylation of the protein, resulting in enzyme inactivation.

The involvement of NOergic nerve in vagal reflex-mediated tracheal dilatation during bronchoconstriction was investigated using the guinea pig in vivo tracheo-bronchi separated preparation. Inhalation of 0.01% histamine to the bronchial site induced a biphasic, vagal reflex tracheal response, i.e., constriction followed by dilatation slightly after bronchial constriction. The reflex tracheal dilatation was inhibited by 49% by local treatment of the tracheal site with 1% propranolol. The residual dilatation in the presence of atropine and propranolol was significantly inhibited by 1% oxyhemoglobin or 1% carboxy-PTIO, NO scavengers. Cyclic GMP content in the tissue of the tracheal site during the reflex tracheal dilatation significantly increased. The increase in cyclic GMP was reduced by treatment of the tracheal site with 1% N omega-nitro-L-arginine methyl ester. These results support a hypothesis that reflex tracheal dilatation during bronchoconstriction may be mediated by not only adrenergic but also NOergic nerves in guinea pigs.

Nitric oxide (NO) synthase is a hemoprotein containing several cysteinyl residues including thiolate as its proximal heme ligand. Exposure to NO is known to induce S-nitrosylation of protein thiols and modulation of enzyme activities, including the catalytic activity of NO synthase. Because S-nitrosylation of vicinal thiols promotes disulfide formation, we determined whether exposure to NO results in modulation of the catalytic activity of NO synthase and whether disulfide reduction catalyzed by thioredoxin/thioredoxin reductase (T/TR) and/or by glutaredoxin restores the catalytic activity of NO synthase in pulmonary artery endothelial cells (PAEC). Exposure of intact PAEC, isolated total membranes, plasma membranes, or purified NO synthase to NO significantly reduced NO synthase catalytic activity. Similarly, exposure of isolated total membranes or purified NO synthase to potassium ferricyanide (FeCN) also reduced catalytic activity of NO synthase in a concentration-dependent fashion. Although the catalytic activity of NO synthase was significantly reduced following exposure of intact cells to NO, the expression of NO synthase mRNA was unchanged. NO synthase activity in intact cells or isolated membranes exposed to nitrate, nitrite, or 10 ppm nitrogen dioxide gas was comparable to controls. Incubation in the presence of oxyhemoglobin prevented but did not reverse NO-induced inhibition of NO synthase. Incubation in the presence of T/TR but not glutaredoxin reversed NO-induced reduction of NO synthase activity and a purified enzyme preparation exposed directly to NO. Similarly, FeCN-induced reduction of NO synthase activity was also reversed in the presence of T/TR but not by glutaredoxin. These results demonstrate that the interaction of NO with the regulatory domain of NO synthase protein is responsible for post-translational reduction of its catalytic activity. Thioredoxin-regulated reversal of NO-induced modulation of NO synthase protein suggests that an oxidative conformational change in vicinal thiols, resulting in the formation of intramolecular or intermolecular disulfides or both, is involved.

Vasoactive intestinal peptide (VIP) release, nitric oxide (NO) formation, and relaxation induced by nerve stimulation were examined in rabbit and rat gastric muscle. VIP stimulated NO formation in muscle strips, whereas NO stimulated VIP release. Nerve stimulation (0.025-16 Hz or 2-940 pulses) elicited frequency-dependent stimulation of VIP release, NO formation, and relaxation, all significant at two to three pulses. NG-nitro-L-arginine (L-NMA) abolished NO formation, abolished VIP release and relaxation at low frequencies, and partly inhibited them at higher frequencies. Oxyhemoglobin (oxy-Hb) inhibited VIP release and relaxation by 80% at low frequencies and 20-30% at higher frequencies. VIP-(10-28) abolished NO formation and relaxation at lower frequencies and partly inhibited them at higher frequencies; in contrast, VIP-(10-28) augmented VIP release in both species. The pattern of inhibition was similar in both species. Inhibition of maximal NO formation by VIP-(10-28) (82% in rabbit; 48% in rat) implied that a major component of NO is formed in muscle cells by the action of VIP. Thus 1) inhibition of relaxation by L-NNA reflects suppression of NO and VIP release from nerve terminals and NO formation in muscle cells, 2) inhibition by VIP-(10-28) partly reflects suppression of NO formation in muscle cells, and 3) inhibition by oxy-Hb reflects neutralization of extracellular NO and suppression of VIP release. The study demonstrates the dual origin of NO from nerves and muscle and its interplay with VIP in regulating relaxation.

1. Inhibitory junction potentials (IJPs) and relaxations evoked in response to field stimulation (supramaximal voltage, 0.1 ms, single stimulus and 5 stimuli at 5-40 Hz) of non-adrenergic non-cholinergic (NANC) nerves with atropine and phentolamine (each 1 microM) were measured in the guinea-pig internal anal sphincter (gpIAS). The mean resting membrane potential was -44.2 +/- 0.2 mV (n = 1119 cells from 260 preparations). 2. NANC nerve stimulation evoked frequency-dependent IJPs (19.7 +/- 1.1 mV, n = 165, 33 tissues to a single stimulus) and relaxations. IJPs consisted of two tetrodotoxin (1 microM)-sensitive components: one was abolished by apamin (0.3 microM) and the P2-purinoceptor antagonist suramin (100 microM); the other, smaller in amplitude, was sensitive to inhibitors of nitric oxide synthase (NOS, e.g. L-NAME, 100 microM) and the nitric oxide (NO) scavenger oxyhaemoglobin (HbO, 10 microM). 3. ATP (1 mM), vasoactive intestinal polypeptide (VIP, 0.01-0.25 microM) and pituitary adenylate cyclase-activating peptide (PACAP(1-27), 0.84 microM) each hyperpolarized and relaxed the gpIAS; only ATP responses resembled the evoked IJPs in time course. 4. The guanylyl cyclase inhibitor LY83583 (10 microM) abolished apamin-insensitive IJPs and relaxations. The cGMP phosphodiesterase inhibitor M&B 22948 (30 microM) and 8-Br-cGMP (100 microM) each hyperpolarized the gpIAS. 5. Two components comprise the IJP and relaxation evoked in response to NANC nerve stimulation in the gpIAS. One, sensitive to apamin, resembles the response to ATP and is modulated by purinoceptor antagonists; the other, apamin and suramin insensitive, is inhibited by NO antagonists.

1. Inhibition of nitric oxide synthase by NG-nitro-L-arginine (L-NNA) reduced the neurogenic relaxation of precontracted taenia coli only in the absence of atropine. The membrane hyperpolarization associated with the neurogenic relaxation was also reduced by inhibition of NOS only when atropine was absent. 2. The membrane hyperpolarization associated with the neurogenic relaxation of the taenia coli was inhibited by oxyhaemoglobin only in the absence of atropine. In the presence of atropine, oxyhaemoglobin did not reduce the i.j.p. or nerve evoked relaxation. 3. Inhibition of NOS by L-NNA did not affect the overflow of [3H]-ACh in response to electrical field stimulation (EFS), suggesting that, under the conditions of our experiments, endogenous NO did not modulate release of ACh. Sodium nitroprusside also had no effect on the neurogenic overflow of [3H]-ACh; however, noradrenaline significantly reduced [3H]-ACh overflow. 4. In summary, the postjunctional effects of neurally-released NO are not apparent in guinea-pig taenia coli when atropine is present. This implies muscarinic regulation of NO release or muscarinic regulation of another excitatory substance, such as tachykinin(s), that, when blocked, masks the postjunctional effects of NO. These data, together with previous studies, suggest a possible regulatory role for NO in enteric neurotransmission that may be more prominent in some species or tissues than others.

Effects of nitric oxide (NO) on oxygen uptake of Ehrlich ascites tumor cells (EATC) were examined in a study of the biological actions of NO on respiration and energy metabolism at the cellular level. Endogenous respiration of EATC was inhibited reversibly by NO in a dose dependent manner. Oxyhemoglobin, an NO trapping agent, restored the respiration promptly. The inhibitory action of NO also depended on oxygen-concentration, and the duration of suppression was prolonged remarkably at low oxygen tension. Similar inhibition was also observed in the presence of glucose. In this case, both lactate production and glucose consumption were promoted by NOC 18, an NO generating agent, and the activation was enhanced by lowering the oxygen-concentration. Furthermore, the membrane potential of EATC was depolarized transiently by adding NO, and the degree of depolarization was decreased in the presence of glucose. These results suggest that at physiologically low oxygen tension in ascites fluid, NO acts not only as a cytotoxic respiratory inhibitor but also as a regulatory factor in the energy metabolism of EATC.

1. The vasodilator properties and photochemical decomposition of two synthetic iron-sulphur-nitrosyl clusters (cluster A: [Fe4S4(NO)4], tetranitrosyl-tetra-mu 3-sulphido-tetrahedro-tetrairon; and B:[Fe4S3 (NO)7]-1, heptanitrosyl-tri-mu 3-thioxotetraferrate(-1)) have been investigated. Experiments were carried out on isolated, internally-perfused segments of rat tail artery. 2. Bolus injections (10 microliters) of A or B ( > 0.25 mM) delivered into the internal perfusate generated sustained (or S-type) vasodilator responses, characterized by a persistent plateau of reduced tone due to NO released from clusters which enter and become trapped within endothelial cells. Clusters were therefore irradiated with visible laser light (lambda = 457.9 or 514.5 nm) either (a) in solution, while passing through a glass tube en route to the artery; or (b) when retained within the endothelium, by illuminating the artery directly during the plateau of an S-type response. Irradiation produced an additional vasodilator response, the magnitude of which depended upon wavelength and laser beam energy. 3. The nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (100 microM), had no effect on light-induced vasodilator responses. However, they were (a) blocked entirely by adding oxyhaemoglobin (5 microM) to the internal perfusate; and (b) greatly enhanced by the enzyme superoxide dismutase (150 u ml-1). 4. Photolysis of cluster B was measured by absorption spectroscopy and by detecting NO released with an electrochemical sensor. The photochemical reaction was found to be oxygen-dependent. The half-time for inactivation of cluster-derived NO was measured by interposing different lengths of tubing (i.e. time delays) between the photolysis tube and NO sensor. The steady-state probe current decayed exponentially with increasing delay time, with a t 1/2 of 21 s. The amplitudes of vasodilator responses of the tail artery also decreased exponentially by increasing the time delay (t 1/2 = 58 s). Superoxide dismutase (150 u ml-1) prevented this from happening, showing that "inactivation' of cluster-derived NO was caused by reaction with superoxide anions formed during photolysis. 5. We conclude that potentiation of vasodilator responses to iron-sulphur-nitrosyl clusters by visible light is due to an oxygen-dependent photochemical reaction which accelerates the release of ligated nitrosyl groups as free NO. Based on our measurements, we estimate that ca 100 pM NO is sufficient to produce a just-detectable additional vasodilatation and that the ED50 dose is ca 3.7 nM.

1. The effects of ranges of concentrations of oxyhaemoglobin (0.01-30 microM) and hydroxocobalamin (1-100 microM) were compared for their abilities to reduce relaxant responses to EDRF released by acetylcholine in endothelium-intact rat aortic rings, the nitergic transmitter in rat anococcygeus muscles, and NO in aqueous solution in both tissues (aortic rings were denuded of endothelium). 2. The concentrations of oxyhaemoglobin producing 50% reduction of responses to EDRF and NO in rat aorta correspond closely, the IC50 values being 0.13 +/- 0.02 microM and 0.11 +/- 0.02 microM respectively. 3. Oxyhaemoglobin was equally effective in inhibiting responses to NO in anococcygeus muscles and in aortic rings with an IC50 of 0.14 +/- 0.05 microM. However, responses to the nitrergic transmitter were considerably less sensitive to inhibition by oxyhaemoglobin, the IC50 being 19.7 +/- 5.1 microM. 4. The IC50 values for hydroxocobalamin in inhibiting responses to EDRF and NO in aorta were 3.4 +/- 0.2 microM and 8.4 +/- 0.63 microM, respectively, but it was less effective against responses to NO in anococcygeus muscles the IC50 being 46 +/- 9.6 microM. However, even in the highest concentration used (100 microM), it did not reduce responses to the nitrergic transmitter. 5. The findings are compatible with the views that EDRF is NO, but suggest that the nitergic transmitter in the rat anococcygeus muscle does not behave like free NO.

Effects of nitric oxide (NO) generated from sodium nitroprusside (SNP) on neurons of mouse superior mesenteric ganglion (SMG) were studied in vitro using intracellular recording techniques. SNP solutions caused a membrane hyperpolarization in the majority (64%) of the neurons tested or a hyperpolarization followed by a depolarization in 8% of the neurons tested. The SNP-induced hyperpolarization persisted in a low-Ca2+ high-Mg2+ solution, indicating a direct effect of NO on the postsynaptic membrane. The hyperpolarizing effect of SNP was reduced or abolished by oxyhemoglobin. Electrical stimulation of the colonic nerves evoked a late slow excitatory postsynaptic potential (late sEPSP) in a population of neurons in normal Krebs solution. The amplitude of the late sEPSP was significantly enhanced in the presence of NG-nitroL-arginine, a NO synthase inhibitor. The results, particularly those observed with the NO synthase inhibitor, suggest that endogenous NO was released in the mouse SMG by repetitive nerve stimulation and that it modulated slow synaptic transmission, presumably by a direct action on ganglionic neurons.

The design of new vasodilator derivatives in which two different pharmacophoric groups are present in a single molecule has been pursued by substitution of NO-prodrug furoxan moieties for the furanylcarbonyl function in Prazosin, a well-known alpha 1-receptor antagonist. The aim was to obtain new antihypertensive agents in which two vasodilation mechanisms, alpha 1-antagonist and NO-mediated, can operate in an appropriate balance. The alpha 1-antagonist activity was assessed on rat aortic strips in the presence and in the absence of oxyhemoglobin (HbO2), a well-known scavenger of nitric oxide. The resulting hybrids displayed different pharmacological behaviors. When the 4-furoxanylcarbonyl system, bearing an ester or an amide function at the 3-position, was present (derivatives 7a,b), hybrids with predominant alpha 1-antagonist activity were obtained. By contrast, in the derivative 7c, in which the nitrile function is linked to the 3-position of the furoxan ring, the NO-mediated vasodilating properties are predominant. Finally, the (furoxanylsulfonyl)piperidine derivatives 13a,b showed NO vasodilation and alpha 1-antagonist activities in an appropriate balance. For the furoxan derivatives, the NO-dependent vasodilating ability, assessed on the K(+)-depolarized aortic strip, and the NO release features under the action of thiol cofactors are also discussed.

The present study was undertaken to determine whether oxyhemoglobin (OxyHb) is responsible for the functional alterations in the cerebral arteries observed during chronic vasospasm after subarachnoid hemorrhage. Vascular strips of canine basilar arteries were kept in organ culture for 3 days with or without repetitive exposure to OxyHb (OxyHb-treated and control strips). Contractions elicited by high levels of potassium (80 mM) and uridine 5'-triphosphate (3 x 10(-4) M) were reduced in the OxyHb-treated group in a concentration-dependent manner. The relaxations evoked by nitric oxide and 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP) were not affected. Relaxations elicited by the calcium channel blocker, diltiazem, were attenuated in the OxyHb-treated rings. When the extracellular calcium concentration ([Ca2+]e) was changed from a concentration in the external solution of 10(-8) M to 10(-3) M, myogenic tension developed. Myogenic tension, expressed as a percentage of the maximum contraction in each segment, was augmented in the OxyHb-treated group at [Ca2+]e of 10(-5) M and 10(-4) M. There were no significant differences in passive compliance of the arterial wall between the two groups. These results demonstrated that prolonged exposure to OxyHb in vitro results in a decrease in contractile capacity and an increase in sensitivity to [Ca2+]e, in agreement with previous findings in spastic arteries. By contrast, impairment of the 8-bromo-cGMP-mediated relaxation pathway and increased stiffness of the arterial wall, which have been reported to occur in spastic arteries, were not induced by prolonged exposure to OxyHb in vitro.

1. The involvement of nitric oxide (NO) and the signal transduction mechanisms mediating neurogenic relaxations were investigated in deep intracavernous penile arteries with an internal lumen diameter of 600-900 microns, isolated from the corpus cavernosum of young horses. 2. The presence of nitric oxide synthase (NOS)-positive nerves was examined in cross and longitudinal sections of isolated penile arteries processed for NADPH-diaphorase (NADPH-d) histochemistry. NADPH-d-positive nerve fibres were observed in the adventitia-media junction of deep penile arteries and in relation to the trabecular smooth muscle. 3. Electrical field stimulation (EFS) evoked frequency-dependent relaxations of both endothelium-intact and denuded arterial preparations treated with guanethidine (10(-5) M) and atropine (10(-7) M), and contracted with 10(-6) M phenylephrine. These EFS-induced relaxations were tetrodotoxin-sensitive indicating their non-adrenergic non-cholinergic (NANC) neurogenic origin. 4. EFS-evoked relaxations were abolished at the lowest frequency (0.5-2 Hz) and attenuated at higher frequencies (4-32 Hz) by the NOS inhibitor, NG-nitro-L-arginine (L-NOARG, 3 x 10(-3) M). This inhibitory effect was antagonized by the NO precursor, L-arginine (3 x 10(-3) M). NG-nitro-D-arginine (10(-4) M) did not affect the relaxations to EFS. 5. Incubation with either the NO scavenger, oxyhaemoglobin (10(-5) M), or methylene blue (10(-5) M), an inhibitor of guanylate cyclase activation by NO, caused significant inhibitions of the EFS-evoked relaxations, and while oxyhaemoglobin abolished the relaxations to exogenously added NO (acidified sodium nitrite, 10(-6) - 10(-3) M), there still persisted a relaxation to NO of 24.4 +/- 5.1% (n = 6) in the presence of methylene blue. 6. Glibenclamide (3 x 10(-6) M), an inhibitor of ATP-activated K(+)-channels, did not alter the relaxations to either EFS-stimulation or NO, while the blocker of Ca(2+)-activated K(+)-channels, charybdotoxin (3 x 10(-8) M), caused a significant inhibition of both the electrically-induced relaxations and the relaxations to exogenously added NO. Furthermore, charybdotoxin blocked relaxations induced by the cell permeable analogue of cyclic GMP, 8-bromo cyclic GMP (8 Br-cyclic GMP). 7. These results suggest that relaxations of horse deep penile arteries induced by NANC nerve stimulation involve mainly NO or a NO-like substance from nitrergic nerves. NO would stimulate the accumulation of cyclic GMP followed by increases in the open probability of Ca(2+)-activated K(+)-channels and hyperpolarization leading to relaxation of horse penile arteries.

The role of the L-arginine-NO pathway on the formation of PGE2 by human cultured astroglial cells incubated with NMDA has been investigated. Preincubation of T 67 astroglial cell line with NMDA (10-600 microM) produced a significant dose-dependent increase of both nitrite (the breakdown product of NO), PGE2 and cGMP levels in cell supernatant. This effect was inhibited by coincubation of cells with L-NAME (20-300 microM), an inhibitor of NO synthase showing that the release of PGE2 subsequent to NMDA receptor stimulation was driven by NO. The release of PGE2 but not elevation of nitrite and cGMP levels was affected by indomethacin (10 microM), an inhibitor of cyclooxygenase. The inhibitory effect of L-NAME on PGE2 release by NMDA-pretreated astroglial cells was reverted by arachidonic acid, showing that the effect of NO on PGE2 release occurred at the cyclo-oxygenase level. Thus, the present experiments demonstrate that the release of PGE2 by astroglial cells pretreated with NMDA is driven by activation of the L-arginine-NO pathway, and this may be relevant in the pathophysiological mechanisms where glutamatergic neurotransmission is involved.

1. Administration of peroxynitrite (ONOO-, 30-300 microM) caused relaxation of rabbit aortic strips superfused in series in a cascade. The compound responsible for this effect had a half-life greater than 20 s and could not therefore be either nitric oxide (NO) or ONOO- which have half-lives in the order of 1-2 s under these conditions. However the relaxation was inhibited by oxyhaemoglobin, suggesting the compound could be converted to NO in the vascular tissues or in the superfusate. 2. The products of the reactions between ONOO- and Krebs buffer containing 11 mM glucose, but not glucose-free Krebs buffer, caused relaxation of the bioassay tissues. These data suggest that stable NO donor(s) were formed from the reaction of ONOO- with glucose. We therefore prepared these NO donor(s) by the reaction of glucose solutions with ONOO- in order to characterize their ability to release NO. 3. These reaction product(s) caused relaxation in the cascade and inhibition of platelet aggregation. Both effects were dependent on the concentration of D-glucose, were equally effective if L-glucose was used as a reactant and were reversed by oxyhaemoglobin. 3. The products of the reaction between ONOO- and glucose or other biological molecules containing an alcohol functional group, such as fructose, glycerol, or glyceraldehyde, released NO in the presence of Cu2+and L-cysteine. 5. These results indicate that ONOO- reacts with sugars or other compounds containing an alcohol functional group(s) to form NO donors with the characteristics of organic nitrate/nitrites. This may represent a further detoxification pathway for ONOO- in vivo.

Objectives: While it is probable that the cerebrovascular spasm which occurs after subarachnoid haemorrhage results from the action of haemoglobin, the mechanism of that process remains unclear. These studies were thus designed to test the hypothesis that the action of oxyhaemoglobin results from the iron-catalyzed formation of free radicals and subsequent lipid peroxidation resulting in intracellular changes in the second messengers for contraction., Methods: Levels of intracellular calcium and of inositol (1,4,5)-trisphosphate were measured in cultured vascular smooth muscle cells derived from primate cerebral arteries. Contractility of rings of canine cerebral vessels were examined in vitro using standard pharmacological techniques. Vessels in spasm were obtained from the "two haemorrhage" canine model and the presence of vasospasm was confirmed angiographically. In each case, the effects of oxyhaemoglobin and sometimes of free radicals generated from iron salts were examined in the presence and in the absence of free-radical scavenging agents or the iron chelating agent, deferoxamine., Results: Oxyhaemoglobin produces a slowly-developing sustained contraction of arterial rings which is accompanied by a sustained elevation of intracellular calcium. It also produces a transient but significant elevation of inositol (1,4,5)-trisphosphate, but this is not correlated with the development of sustained constriction. Deferoxamine and the lazaroid compounds U-74389G and U-83836E were effective in preventing the effects of oxyhaemoglobin and free radicals in the models tested, although in vessels in spasm, all effects were smaller., Conclusions: The present study provides results which are consistent with the hypothesis that the actions of haemoglobin on vascular smooth muscle are mediated by the formation of free radicals which subsequently affect intracellular calcium concentrations. This also implies that agents which impair free radical production or other processes leading to iron-catalyzed lipid peroxidation, are of potential value in cerebrovascular spasm.

Neuronal NO synthase (NOS) is a flavin-containing hemeprotein that generates NO from L-arginine, NADPH, and O2. NO has recently been proposed to autoinhibit NOS. We have investigated whether a NOS heme-NO complex forms during aerobic steady-state catalysis. Visible and resonance Raman spectra recorded during steady-state NO synthesis by NOS showed that the majority of enzyme (70-90%) was present as its ferrous-nitrosyl complex. Ferrous-nitrosyl NOS formed only in the coincident presence of NADPH, L-arginine, and O2. Its level remained constant during NO synthesis until the NADPH was exhausted, after which the complex decayed to regenerate ferric resting NOS. Stopped-flow measurements revealed that the buildup of the ferrous-NO complex was rapid (< 2 s) and caused a 10-fold decrease in the rate of NADPH consumption by NOS. Complex formation and decay could occur several times with no adverse affect on its subsequent formation or on NOS catalytic activity. Neither enzyme dilution nor NO scavengers (superoxide and oxyhemoglobin) diminished formation of ferrous-nitrosyl NOS or prevented the catalytic inhibition attributed to its formation. The ferrous-nitrosyl complex also formed in unfractionated cell cytosol containing neuronal NOS upon initiating NO synthesis. We conclude that a majority of neuronal NOS is converted quickly to a catalytically inactive ferrous-nitrosyl complex during NO synthesis independent of the external NO concentration. Thus, NO binding to the NOS heme may be a fundamental feature of catalysis and functions to down-regulate NO synthesis by neuronal NOS.

The therapeutic use of cell-free haemoglobin as a blood substitute has been hampered by toxicological effects. A model asolectin (phosphatidylcholine/phosphatidylethanolamine) liposome system was utilized to study the pro-oxidant efficiency of several chemically modified haemoglobins on biological membranes. Lipid peroxidation, resulting from the interactions between haemoglobin and liposomes, was measured by conjugated diene formation and the maximal rates of oxygen uptake. Spectral changes gave insight into the occurrence of the ferryl iron species. The residual reactivity of oxidatively damaged haemoglobins with ligands during incubation with liposomes was assessed from rapid kinetic carbon monoxide-binding experiments. Liposomes in which cytochrome c oxidase was embedded show both haemoglobin and the enzyme to be oxidatively damaged during incubation. The functional state of cytochrome c oxidase was monitored in the presence and absence of a free radical scavenger. Once in contact, both unmodified and modified haemoglobins triggered and maintained severe radical-mediated membrane damage. Differences in the pro-oxidant activities among haemoglobins may be explained by either the differential population of their ferryl intermediates or disparate dimerization and transfer of haem into the membrane with subsequent haem degradation. This study may contribute to a better understanding of the molecular determinants of haemoglobin interactions with a variety of biological membranes.

The objective of this study was to investigate the role of the L-arginine pathway in the regulation of lymphatic pumping. Bovine mesenteric lymphatic vessels (8 to 12 cm in length containing four to six lymphangions) were immersed in an organ bath with input provided by a reservoir filled with Krebs solution. The vessels were stimulated to pump by applying a 6 cm H2O transmural pressure. The addition of 10(-7)-10(-4) M oxyhemoglobin, NG-monomethyl-L-arginine (L-NMMA), or methylene blue to the reservoir resulted in a reduction in lymphatic pumping. L-Arginine (10(-7)-10(-4) M) had no effect on spontaneous pumping activity. However, L-arginine reversed the inhibition caused by oxyhemoglobin and L-NMMA. When tested between 10(-7) and 10(-6) M, sodium nitroprusside (sNP) had variable effects on lymphatics. sNP depressed pumping in approximately 2/3 of the vessels and increased pumping in the remaining 1/3 of ducts. When the results were meaned, sNP caused a significant depression in activity. However, the lower concentration of sNP (10(-7) M) was able to reverse the inhibitory effects of oxyhemoglobin, L-NMMA, and methylene blue whereas the higher concentration (10(-6) M) caused further reductions in pumping activity. These results suggest that bovine lymphatic vessels produce nitric oxide or a related compound. L-Arginine metabolites appear to facilitate the pumping response by an as yet undefined mechanism.

To examine the basis of pulmonary constriction during chronic hypoxia, rat pulmonary artery rings were precontracted and exposed to multiple episodes of hypoxia. The first hypoxic episode resulted in a transient contraction, followed by potent relaxation, and then a slow sustained contraction. Repeated hypoxic exposure resulted in stronger initial contraction and attenuated relaxation. Prolongation of the normoxic interval between hypoxic episodes reversed the attenuation of hypoxic relaxation. Pulmonary artery rings that were deendothelialized or treated with the nitric oxide synthesis inhibitor N omega-nitro-L-arginine methyl ester or oxyhemoglobin displayed only hypoxic relaxation without initial or late contractions and no attenuation of relaxation during repeated hypoxia. Pretreatment of rings with indomethacin or adenosine or endothelin receptor antagonists had no effect on the hypoxia-mediated alterations. Thus repetitive exposure to hypoxia results in an increase in initial contraction and a decrease in relaxation of pulmonary artery rings. Frequency of hypoxic episodes and endothelial integrity determine pulmonary tone during repeated hypoxia. However, cyclooxygenase products, adenosine, or endothelin do not play a role in hypoxia-mediated changes in rat pulmonary artery tone.

The role of nitric oxide (NO) in the induction of long-term depression (LTD) in the cerebellum was explored using a new, organic, membrane-impermeant form of caged NO. NO photolytically released inside Purkinje neurons mimicked parallel fiber (PF) activity in synergizing with brief postsynaptic depolarization to induce LTD. Such LTD required a delay of < 50 ms between the end of photolysis and the onset of depolarization, was prevented by intracellular Ca2+ chelation, and was mutually occlusive with LTD conventionally produced by PF activation plus depolarization. Bath application of NO synthase inhibitor or of myoglobin, a NO trap, prevent LTD induction via PF stimulation, but not that from intracellular uncaged NO, whereas intracellular myoglobin blocked both protocols. NO is therefore an anterograde transmitter in LTD induction. A biochemical requirement for simultaneous NO and elevation of intracellular free Ca2+ would explain why PF activity must coincide with postsynaptic action potentials.

We examined the mechanism of leukotoxin, 9,10-epoxy-12-octadecenoate (Lx)-induced lung injury in blood-free, physiological salt solution-perfused rat lungs under constant flow conditions. Mean pulmonary arterial (Ppa) and pulmonary capillary pressure (Pcap, estimated by the double-occlusion method), wet lung weight (WLW), pulmonary capillary filtration coefficient (Kfc), lung perfusate lactate dehydrogenase (LDH) activity, and nitrite levels were assessed. Bolus injection of Lx (200 microM) caused insidious and significant lung weight gain, which was not associated with remarkable elevation of Ppa or Pcap but was associated with an increase of perfusate LDH activity and nitrite levels. Lx (20 microM) elevated Kfc, indicating that Lx had affected pulmonary vascular permeability. Because Lx causes endothelium dependent pulmonary vasodilation, we studied the effect of NG-monomethyl-L-arginine (L-NMMA), NG-monomethyl-D-arginine (D-NMMA), superoxide dismutase (SOD), human oxyhemoglobin (oxyHb), and methylene blue (MB) on Lx-induced lung injury. L-NMMA, SOD, and oxyHb, but not MB or D-NMMA, protected the lungs against Lx (200 microM)-induced injury. Lx increased pulmonary vascular permeability and caused lung injury. Because both nitric oxide synthase inhibitors and SOD inhibited the Lx-induced lung injury, it is possible that peroxynitrite is involved in the mechanism whereby Lx causes lung injury.

We examined the responses to intraluminally applied acetylcholine (ACh) in isolated and perfused canine basilar arteries by the stainless-steel cannula-inserting method. In control vessels with intact endothelium, ACh produced a slight vasodilatation followed by a long-lasting vasoconstriction in the absence of induced tone. Propranolol, a beta-adrenoceptor blocker, had no effect on the response to ACh. After endothelial removal with intraluminal saponin, the constrictor component of the response to ACh was significantly enhanced, and the constrictions to prostaglandin F2 alpha (PGF2 alpha) and potassium chloride (KCl) were significantly potentiated. After exposure to extraluminal oxyhemoglobin, the ACh-induced constriction was significantly augmented, whereas the dilator component of the response to calcium ionophore A23187 was significantly attenuated. The enhanced constriction to ACh after endothelial removal was significantly blocked by OKY-046 (a thromboxane synthetase inhibitor) and nimodipine (a dihydropyridine calcium antagonist), but was slightly though not significantly suppressed by AA-861 (a lipoxygenase inhibitor). The response to PGF2 alpha was not altered by OKY-046 or AA-861. These results suggest that the potentiation of ACh-induced constriction after endothelial removal may be mediated in part by thromboxane A2 (TXA2), linked with extracellular calcium entry in smooth muscle cells (SMC). This mechanism might be involved in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH).

The objective of this study was to investigate the regulation of endothelial nitric oxide (NO) synthase by NO. Partially purified endothelial NO synthase was exposed to authentic NO (10-200 microM) and to the nitrovasodilators sodium nitroprusside (SNP; 10-1,000 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 100-1,000 microM), and enzyme activity was assayed by measuring the conversion of L-[3H]arginine to L-[3H]citrulline in the presence of added cofactors. NO, SNP, and SNAP inhibited NO synthase activity in a dose-dependent manner, NO being the most potent inhibitor. The Michaelis constant for L-arginine was not altered (4.87 microM) by NO (50 microM), whereas the maximal velocity of the enzyme decreased from 784 to 633 pmol.mg-1.min-1. Oxyhemoglobin (10 microM) partially prevented the inhibition of NO synthase by NO (50 microM). The data also suggest that NO inhibits endothelial NO synthase activity by directly interacting with the NO synthase and not by an indirect mechanism such as limitation of cofactor or oxygen availability. Dialysis of NO synthase treated with NO (50 microM) partially restored the enzyme activity. This study demonstrates a direct and reversible inhibition of NO synthase by NO, suggesting a feedback mechanism in vivo.

Peroxynitrite stimulated the synthesis of cyclic GMP by rat aortic smooth muscle in a time- and dose-dependent manner. Peak formation of cyclic GMP occurred at 1 min with 100 microM peroxynitrite and was inhibited by oxyhemoglobin. Peroxynitrite was less potent than nitric oxide in stimulating cyclic GMP synthesis. Peroxynitrite also enhanced endothelial-dependent cyclic GMP synthesis, via generation of a long-lived substance, which was prevented by inhibition of glutathione synthesis. These data show that peroxynitrite stimulates cyclic GMP synthesis, inferring production of low yields of nitric oxide or associated derivatives. Additionally, vascular exposure to peroxynitrite potentiates endothelial-dependent activation of guanylate cyclase.

LLC-PK1 epithelial cells and RFL-6 fibroblasts secreted both cyclic AMP (cAMP) and cyclic GMP (cGMP) when costimulated with forskolin and 3-morpholinosydnonimine (a chemical nitric oxide generator). Intracellular cAMP levels as high as 1100 and 12,000 pmol/10(6) cells were achieved for the two cell types, respectively. These levels were high enough to reach approximately 50% saturation of the cAMP transporter and inhibited transport of cGMP to an equal extent, suggesting that the two cyclic nucleotides compete for a common transport system. The rates of secretion of cGMP and cAMP from LLC-PK1 cells increased in proportion to their rates of synthesis as concentrations of stimulant were varied, but increased only 25% relative to intracellular concentrations in response to inhibition of phosphodiesterases by 3-isobutylmethylxanthine. It is proposed that secretion of cyclic nucleotides is not simply proportional to the total intracellular pool in these cells, but rather is coupled to synthesis. In support of this model, oxyhemoglobin was used to trap nitric oxide and block activity of guanylate cyclase in cells treated with 3-morpholinosydnonimine. As a result, secretion of cGMP ceased within 1 min, whereas intracellular levels decreased slowly over 60 min. Probenecid [p-(dipropylsulfamoyl)benzoic acid] is a nonselective antagonist of anion transport that inhibited secretion of cAMP in both cell types but, unexpectedly, blocked synthesis of cGMP, and this was reflected in direct inhibition of soluble guanylate cyclase in cell lysates. Two heat-stable, high molecular weight factors that confer sensitivity to probenecid were identified, and these factors increased the sensitivity of guanylate cyclase to nitric acid by an order of magnitude.

Acute anoxia or severe hypoxia causes an initial transient contraction followed by marked relaxation of vascular tissues. We observed a spontaneous gradual sustained contraction of rat aortic rings following relaxation when hypoxia was prolonged. Deendothelialization as well as treatment of the endothelium-intact rings with nitric oxide synthase inhibitors or oxyhemoglobin abolished the late hypoxic contraction despite prolonged hypoxia. The prolonged hypoxia-induced sustained contraction was not affected by adenosine receptor blockade, cyclooxygenase inhibition, free radical scavengers, or the endothelin receptor antagonists. The ATP-sensitive K+ channel blocker glibenclamide abbreviated the duration of hypoxic relaxation and potentiated the magnitude of late hypoxic contraction. These data suggest that the late-sustained hypoxic contraction of arterial tissues is dependent on the presence of intact functional endothelium. Activation of ATP-sensitive K+ channels may participate in the genesis of hypoxic relaxation. However, cyclooxygenase products, free oxygen radicals, adenosine, and endothelin are not involved in the regulation of hypoxia-mediated events in rat aortic rings.

1. The effects of nitrosocysteine (cys-NO), L-N omega-nitroarginine (L-NNA) and L-N omega-nitro-L-arginine methylester (L-NAME), oxyhaemoglobin and Methylene Blue were observed on the resting membrane potential, muscle tone and excitatory junction potentials (EJPs) of cat tracheal smooth muscle tissue. 2. Cys-NO (10(-9) to 10(-6) M) showed no effect on the resting membrane potential of smooth muscle cells of the cat trachea but it dose-dependently relaxed the tracheal tissue in the presence of 5-HT, atropine and guanethidine. 3. Electrical field stimulation (EFS) applied during contraction evoked by 5-HT in the presence of atropine and guanethidine evoked non-adrenergic, non-cholinergic (NANC) muscle relaxation. L-NNA (10(-4) M) and L-NAME (10(-4) M) completely suppressed the relaxation when single or short repetitive stimuli were applied, but suppression was incomplete with repetitive stimuli of 4 ms pulse duration applied at 20 Hz. A substantial part of the L-NNA- or L-NAME-insensitive relaxation was abolished by tetrodotoxin. 4. Cys-NO dose-dependently suppressed the EJPs without changing the resting membrane potential, and L-NNA, L-NAME, Methylene Blue and oxyhaemoglobin enhanced the amplitude of the EJP to 1.2-1.5 times the control value. 5. EJPs showed some summation when repetitive field stimulation was applied at 20 Hz. L-NNA or L-NAME enhanced the summation, and the mean slopes were increased from 0.61 +/- 0.22 to 2.0 +/- 0.3, or 1.9 +/- 0.2 mV per stimulus. Vasoactive intestinal polypeptide (VIP) antiserum and VIP antagonists further enhanced the summation in the presence of L-NNA. 6. These results indicate that NANC relaxation can be classified into two different components according to the threshold for activation, and nitric oxide is involved in one. The present results also suggest that endogenous or exogenous nitric oxide has a prejunctional action in inhibiting excitatory neuroeffector transmission in addition to a direct action on the smooth muscle cells, presumably by suppressing transmitter release from the vagus nerve.

Experiments were designed to determine whether cyclic GMP-independent relaxation is involved in the endothelium-dependent vascular relaxation response of rat aortic strip to acetylcholine. The relaxation response to acetylcholine in the presence of 3 x 10(-4) M NG-nitro-L-arginine was apparent when the precontraction was induced by norepinephrine at 5 x 10(-9) M or 10(-8) M. The relaxation response to acetylcholine resistant to NG-nitro-L-arginine was abolished by 10(-6) M atropine, 10 mM tetraethylammonium, or endothelium removal, but was not inhibited by 10(-5) M indomethacin, 3 x 10(-6) M oxyhemoglobin or 10(-5) M glibenclamide. The response was virtually abolished when the vascular strips had been preconstricted with 20 mM KCl. The increase in vascular cyclic GMP levels induced by 10(-5) M acetylcholine was completely abolished by 3 x 10(-4) M NG-nitro-L-arginine. These results suggest that acetylcholine-induced endothelium-dependent relaxation resistant to NG-nitro-L-arginine in rat aorta is unmasked when the precontractile force is caused by lower concentrations of norepinephrine and the relaxation is mediated by a cyclic GMP-independent mechanism, possibly an endothelium-derived hyperpolarizing factor.

To investigate a possible link between subretinal hemorrhage and macular degeneration, oxyhemoglobin (HbO2) or methemoglobin (metHb) was incubated with retinal homogenate and unsaturated phospholipid peroxidation was monitored by (a) assay of thiobarbituric acid-reactive substances (TBARS), (b) luminescence originating from an energy transfer of lipid-degraded products to rose bengal, and (c) the decrease in composition of highly unsaturated fatty acids of phospholipids. TBARS formation and rose bengal luminescence in the case of metHb-induced lipid peroxidation were about 1.5 times greater than those in HbO2-induced lipid peroxidation. alpha-Tocopherol, a lipid-soluble antioxidant, and docosahexaenoic acid, a major unsaturated fatty acid, were slightly more rapidly decomposed after a 60-min incubation with metHb than with HbO2 at the same concentration. Atomic absorption analysis revealed that an equal concentration of iron was released from both HbO2 and metHb during incubation with retinal homogenates. The released iron may promote microsomal phospholipid peroxidation in the presence of endogenous ascorbate or NADPH-dependent cytochrome P-450 reductase because ascorbate oxidase and p-chloromercuribenzoic acid (an inhibitor of sulfhydryl enzymes) inhibited metHb- or HbO2-induced lipid peroxidation. MetHb-induced lipid peroxidation in retina was inhibited by KCN or NaN3, which binds to FeIII of metHb. KCN or NaN3 had no effect on HbO2-induced lipid peroxidation, because conversion of HbO2 to metHb, which can proceed in HbO2 incubated with phospholipid liposome, did not occur in retinal homogenates. It is concluded that metHb induces peroxidation of retinal unsaturated phospholipids (1) directly and (2) by releasing iron.

The relaxation to bradykinin in canine coronary arteries is mediated by endothelium-derived nitric oxide (NO) and hyperpolarizing factor (EDHF). Desensitization to the kinin was induced by incubation of canine coronary arteries with endothelium with 10(-8) M bradykinin for 30 min. After washout, tissues were contracted with prostaglandin F2 alpha, and concentration-relaxation curves to bradykinin were obtained in control and desensitized arteries treated with indomethacin. After desensitization, there was a shift to the right of the concentration-relaxation curves to bradykinin. However, the elevation in guanosine 3',5'-cyclic monophosphate (cGMP) levels evoked by bradykinin was similar in both groups of tissues. The curves to bradykinin obtained in the presence of NG-nitro-L-arginine (an NO synthase inhibitor) were depressed, whereas those obtained in arteries contracted with potassium (to eliminate the EDHF-mediated relaxation) were not affected by the desensitization. Addition of NG-nitro-L-arginine, oxyhemoglobin, or methylene blue before the desensitization procedure preserved, whereas 3-morpholinosydnonimine (SIN-1, a donor of NO) and 8-bromoguanosine 3',5'-cyclic monophosphate impaired, the EDHF-mediated relaxation to bradykinin. Thus the selective impairment of the EDHF-dependent relaxation to bradykinin may be mediated by NO, acting mainly through increased production of cGMP.

Iron-derived reactive oxygen species play an important role in the pathogenesis of various vascular disorders including vasculitis, atherosclerosis, and capillary leak syndromes such as the adult respiratory distress syndrome (ARDS). We have suggested that acute incorporation of the heme moiety of hemoglobin released from red blood cells into endothelium could provide catalytically active iron to the vasculature. Adaptation to chronic heme stress involves the induction of heme oxygenase and ferritin; the latter provides cytoprotection against free radicals in vitro. The present studies examine the bioavailability of heme, derived from hemoglobin, to induce heme oxygenase and ferritin in rat lungs in vivo. Intravenous injection of methemoglobin, but not oxyhemoglobin, increases total lung heme oxygenase mRNA approximately fivefold after 16 h. Accompanying this mRNA induction, expression of total lung heme oxygenase enzyme activity is also markedly enhanced. In situ hybridization for heme oxygenase reveals mRNA accumulation in the lung microvascular endothelium, implying incorporation of heme into endothelial cells. Similarly, methemoglobin significantly increases the ferritin protein content of rat lungs and in parallel, ferritin light-chain mRNA increases approximately 1.6-fold, whereas heavy-chain mRNA is upregulated by approximately 1.9-fold. Immunoreactive ferritin is present in lung microvascular endothelium after methemoglobin treatment, suggesting incorporation of heme iron into pulmonary vasculature. Subcutaneous injection of Sn-protoporphyrin IX, a competitive inhibitor of heme oxygenase, does not affect methemoglobin-induced ferritin synthesis in lungs. We speculate that methemoglobin, which might be generated by activated leukocytes in ARDS associated with disseminated interavascular coagulation, can provide heme iron to lung microvascular endothelium to induce heme oxygenase and ferritin.

In the present study, utilizing anti-phosphotyrosine monoclonal antibodies, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) as sources of NO and murine fibroblasts expressing the human epidermal growth factor (EGF) receptor (HER14 cells), we showed that tyrosine phosphorylation of a set of proteins (126, 56 and 43 kDa) was stimulated when cells were incubated with either SNP or SNAP and abolished by Methylene Blue and oxyhaemoglobin. Inhibition by Methylene Blue suggested an involvement of cyclic GMP in the process, which was evidenced by the effects of 8-bromo cyclic GMP. This analogue of cyclic GMP stimulated tyrosine phosphorylation of the same set of proteins phosphorylated after incubation with the NO source. Tyrosine phosphorylation of the same set of proteins was stimulated when cells were incubated simultaneously with SNP and EGF, showing that NO also potentiates EGF-evoked tyrosine kinase activity in HER14 cells. However, stimulation of the autophosphorylation of the EGF receptor, above the levels obtained for EGF alone, was not observed under those conditions. Additionally, we investigated the effects of NO on EGF-receptor tyrosine phosphatase activities in HER14 cells. Increasing concentrations of NO correlate with a gradual inhibition of these activities in HER14 cells, either in intact cells or in cell lysates. Taken together, these observations suggest that NO modulates tyrosine phosphorylation in HER14 cells.

The reactivity and toxicity of nitric oxide is modest in comparison to oxidants derived from nitric oxide. Exposure of Escherichia coli to 1 mM nitric oxide under aerobic or anaerobic conditions did not decrease viability of the bacteria. Peroxynitrite (1 mM), the reaction product of superoxide and nitric oxide, was completely bactericidal after 5 s. The nitrovasodilator, 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), slowly decomposes to release both nitric oxide and superoxide and thereby produces peroxynitrite. SIN-1 killed E. coli in direct proportion to its concentration with an LD50 of 0.5 mM. Copper, zinc superoxide dismutase (50-400 units/ml) provided substantial but not complete protection against SIN-1 killing. Catalase (500-10,000 units/ml) partially protected in direct proportion to its concentration, while inactivated catalase was not protective. Superoxide dismutase and catalase together completely protected E. coli against SIN-1 toxicity. Oxy-hemoglobin eliminated both SIN-1 and peroxynitrite toxicity. The bactericidal activity of SIN-1 was further enhanced by pterin plus xanthine oxidase. Pterin plus xanthine oxidase alone or together with Fe3+ ethylenediamine tetraacetate produced no significant decrease in E. coli viability. Hydrogen peroxide was not directly toxic to the bacteria, but E. coli pretreated with hydrogen peroxide were more susceptible to peroxynitrite, SIN-1, and the aerobic oxidation products of nitric oxide. Hydrogen peroxide pretreatment did not increase significantly the toxicity of nitric oxide under anaerobic conditions. Our results suggest that peroxynitrite is far more toxic to E. coli than nitric oxide or its by products from aerobic oxidation.

This study was undertaken to determine the effects of oxyhemoglobin on vasopressin-induced responses in cerebral arterioles. Rat intracerebral arterioles about 60 microns in diameter were isolated and cannulated using pipettes. Changes in diameter secondary to the extraluminal application of drugs were monitored through a video micro-scaler. Vasopressin produced a triphasic, dose-dependent response consisting of vasodilation (10(-11) M), vasoconstriction (10(-9)-10(-8) M) and a decrease in vasoconstriction (10(-7)-10(-6) M). Pretreatment with oxyhemoglobin (10(-5) M) abolished the vasodilation induced by the lower dose vasopressin and doubled the vasoconstriction induced by the higher dose. The combination of L-arginine (10(-4) M) and superoxide dismutase (600 U) restored low-dose vasopressin vasodilation and suppressed high-dose vasoconstriction in oxyhemoglobin-pretreated arterioles, while they showed little effect when used singly. This study indicates that oxyhemoglobin enhances vasopressin-induced constriction of intracerebral arterioles and these effects can be inhibited by the combination of L-arginine and superoxide dismutase.

Renin secretion may be modulated by nitric oxide (NO). We studied whether interleukin-1 beta (IL-1 beta) induces endogenous NO synthesis in mouse juxtaglomerular cells (JGC) in primary culture, and whether endogenous NO or NO applied exogenously via sodium nitroprusside (SNP) influences renin secretion. JGC seeded on culture plates were stimulated by IL-1 beta or by SNP. Cyclic guanosine 3,5'-monophosphate (cGMP) in the cell supernatant was determined as indicator for NO effects. Stimulation of JGC with IL-1 beta or SNP increased cGMP in the supernatant significantly. The NO synthase inhibitors NG-nitro-L-arginine or NG-monomethyl-L-arginine, or the NO scavenger oxyhaemoglobin prevented the IL-1 beta-induced increase of cGMP. The biological activity of NO was shown in a bioassay by the vasodilatory effect of the effluent from an IL-1 beta-stimulated JGC column on a precontracted rat aortic ring and was prevented by oxyhaemoglobin and methylene blue. Renin activity of JGC was detected in the culture supernatants and the cells. Spontaneous renin secretion into the cell supernatant was 26 +/- 1% of total activity. Melittin or forskolin concentration dependently increased renin secretion up to 90 +/- 2%. Incubation of JGC with IL-1 beta in the absence or presence of NO inhibitors did not alter spontaneous or stimulated renin secretion. SNP (30 microM) had a dual effect on renin secretion. After 1 h of incubation, it inhibited basal renin secretion, whilst it had a stimulatory effect after 20 h of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

The reported effects of nitric oxide (NO) on dopamine release from the striatum are variable and its precise effect on striatal nerve terminals is unclear. In the present study a novel method of applying NO to brain tissue in situ was employed. Photo-activation of Roussin's Black Salt (RBS), retained in isolated perfused brain tissue, was used to release NO at will upon illumination. Basal and electrically-stimulated dopamine efflux from the rat striatum in vitro was measured in real time using fast cyclic voltammetry. Illumination of an RBS pre-treated brain slice elicited a light intensity-related increase in basal dopamine efflux. Concomitantly there was a decrease in the level of electrically-stimulated dopamine efflux. Illumination in the absence of RBS pre-treatment had no effect on basal or stimulated dopamine efflux. The increase in basal dopamine efflux upon photo-activation of RBS was reduced by the presence of 10 microM oxyhaemoglobin, but was insensitive to the removal of extracellular calcium or the addition of 1 microM sulpiride. The decrease in electrically-stimulated dopamine efflux following illumination was not affected by the presence of either oxyhaemoglobin or sulpiride. It is concluded that NO, produced by photo-activation of RBS, releases dopamine from the rat striatum in vitro by a mechanism independent of extracellular calcium entry.

Acetylcholine (ACh) induces an N omega-nitro-L-arginine (L-NOARG)-resistant relaxation and hyperpolarization in the rat isolated hepatic artery. The possibility that carbon monoxide (CO) produced by haem oxygenase (HO) is an endogenous mediator of this response was investigated. Exogenously applied CO evoked a concentration-dependent relaxation, and the CO 'scavenger' oxyhaemoglobin (10 microM) reduced the maximum ACh-induced relaxation by 25%. The HO inhibitor zinc protoporphyrin IX (ZnPP, 10 microM) virtually abolished the ACh-induced relaxation in experiments carried out under ordinary light conditions. However, ZnPP did not affect the ACh-induced relaxation under dark conditions, even after exposure of ZnPP to intense light before the preincubation period. Biliverdin (0.1 mM), a feedback inhibitor of HO, was also inactive under dark conditions, and the HO substrate haematin (0.1 mM) did not facilitate the ACh-induced relaxation. The relaxation induced by the nitric oxide (NO) donor 3-morpholino-sydnonimin was not affected by ZnPP in the presence of light. However, ZnPP inhibited the relaxation evoked by the potassium channel opener levcromakalim and the tonic component of the contractile response to 60 mM potassium, indicating that ZnPP has effects distinct from HO inhibition in the presence of light. ZnPP should therefore be protected from light when used to inhibit HO-mediated CO formation. The results do not suggest that CO generated by HO mediates the endothelium-dependent, L-NOARG-resistant relaxation induced by ACh in the rat hepatic artery.

Interactions between human glomerular endothelial cells and atrial natriuretic peptide (ANP) were studied with 125I-alpha-human-ANP binding and intracellular accumulation of cGMP. Uptake for alpha-hANP (1-28 or 5-28) by homogeneous cultures of human glomerular endothelial cells was dose and time dependent with optimal uptake occurring after 30 min of incubation at 37 degrees C. Scatchard analysis of the specific binding data with a two-compartmental model identified both high (Kd = 0.3 nM)- and low (Kd = 10 nM)-affinity receptors, with a binding site density of 12,000 and 18,060 receptors per cell, respectively. alpha-hANP markedly stimulated glomerular endothelial cell-associated cGMP. After a 2-min incubation, cGMP increased 1.3-fold (from 17.88 +/- 1.29 to 23.33 +/- 3 pmol/mg of protein), in the presence of 1 nM ANP, to more than threefold (from 21 +/- .1 to 80.5 +/- 14.5 pmol/mg of protein) with 1 microM ANP (P < 0.05). In contrast, a 10 microM concentration of the clearance receptor C-ANP4-23 increased cGMP by 1.6 +/- 0.6 fold. ANP stimulation of intracellular cGMP was 100 times more sensitive in human glomerular endothelial than in mesangial cells. In comparison, higher doses of bradykinin were necessary to evoke similar responses in glomerular endothelial cells. In the presence of 10 microM bradykinin, cellular cGMP increased by 1.75 +/- 0.6-fold versus control cells. However, unlike ANP, bradykinin-stimulated cGMP synthesis was significantly inhibited by prior treatment with oxyhemoglobin (10(-5) M), an inhibitor of soluble guanylate cyclase, and NG-nitro-L-arginine (NO2Arg), a specific inhibitor of endothelial-derived relaxing factor (EDRF).(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the effect of nitric oxide (NO) on the interaction of washed human platelets with fibrinogen or fibronectin adsorbed on glass. Platelet contacts were visualized by interference reflection microscopy at 37 degrees C in Tyrode's solution. The areas of spread platelets were measured, using digital video image processing techniques, at times up to 30 minutes after initial surface platelet contact. On fibrinogen spreading was inhibited by NO donors in the order of potency: S-nitroso-acetylpenicillamine > sodium nitroprusside > S-nitroso-glutathione. The inhibitory action of NO donors was prevented by Oxy-haemoglobin, confirming that this inhibition was due to the release of NO. In contrast, NO donors had virtually no effect on platelet spreading on fibronectin. Platelet adhesion to fibrinogen is mediated by GP IIb IIIa and the vitronectin receptor (VNR), while that to fibronectin is via GP IIb IIIa, VNR, Ic IIa and VLA-6. Our results thus indicate that NO inhibits adhesion mediated by GP IIb IIIa and/or VNR but not by the two other receptors. The mechanism of this receptor-specific inhibition of platelet adhesion remains to be elucidated.

The present study was undertaken to determine the role of nitric oxide (NO) in the regulation of vasomotor tone in rat cerebral parenchymal arterioles under physiological and pathological conditions. Cerebral arterioles, about 60 microns in diameter, were isolated from rats and cannulated with glass pipettes. Changes in the arteriolar diameter secondary to extraluminal application of drugs were monitored continuously through a videodimension analysis system. The arterioles were dilated by L-arginine (12.2 +/- 2.2% at 10(-3) M) and were constricted by NG-monomethyl-L-arginine (L-NMMA) (13.8 +/- 1.5% at 10(-4) M) in a dose-dependent manner. Pretreatment with superoxide dismutase (600 U) increased sensitivity to L-arginine. These results suggest that NO plays an important role in regulation of basal vasomotor tone of cerebral parenchymal arterioles under physiological conditions. Next, NO-modification of vasomotor responses to an endogenous vasoactive substance, arginine vasopressin (AVP) was studied in cerebral parenchymal arterioles. Increasing concentrations of AVP produced biphasic responses of vasodilation (10(-11) M) and vasoconstriction (10(-10)-10(-8) M). Inhibition of NO by pretreatment with L-NMMA (10(-4) M) or oxyhemoglobin (10(-5) M) abolished the vasodilation and enhanced the vasoconstriction by AVP. Therefore, under pathological conditions in which NO function was suppressed, such endogenous substances may produce contraction in parenchymal arterioles instead of dilation with aggravation of cerebral ischemia.

The stainless steel cannula method was applied to isolated and perfused canine basilar arteries to examine the role of endothelium in the responses to intraluminal vasoactive substances. After intraluminal treatment with saponin to remove the endothelium, the monophasic constrictions to potassium chloride and prostaglandin F2 alpha were potentiated, while those to phenylephrine (alpha 1-adrenoceptor agonist) and 5-hydroxytryptamine were not changed. Xylazine (alpha 2-adrenoceptor agonist) and acetylcholine induced a constriction preceded by a small dilation in controls. The response to xylazine was not modified, while the constriction to acetylcholine was augmented after endothelium removal. Bradykinin, substance P and vasopressin caused a dilation in lower doses, and a dilation followed by a secondary constriction in higher doses in controls. The dilations to these peptides were reduced and the constrictions were enhanced after endothelial removal. Adenosine triphosphate produced a biphasic response, i.e., a dilation followed by a constriction, which was occasionally preceded by a small constriction in higher doses, and only the dilation in lower doses was attenuated. The monophasic dilation to adenosine was potentiated, while the papaverine-induced dilation was not influenced by endothelial removal. After extraluminal treatment with oxyhemoglobin, the dilations to calcium ionophore A23187 and thimerosal were attenuated, while the constriction to acetylcholine was enhanced. The dilations to substance P and vasopressin were depressed, and the constrictions were potentiated. The monophasic dilation to sodium nitroprusside was augmented, while that to papaverine was not changed. These results suggest that the endothelium may play important roles not only in producing endothelium-derived relaxing factors but also in modulating the calcium influx into the smooth muscle cells. The mechanisms of altered responsiveness might be implicated in cerebral vasospasm following subarachnoid hemorrhage.

The in vitro effect of nifedipine, a calcium channel blocker of the dihydropyridine (DHP) type, on platelet aggregation was reinvestigated considering especially the capability of platelets to form endogenous nitric oxide (NO). We studied the dose-dependent antiaggregatory property of nifedipine in porcine platelet rich plasma. Aggregation was stimulated by collagen (7.5 micrograms/ml). Nifedipine inhibited collagen-induced platelet aggregation with an IC50 of 380 nmol/l. The antiaggregatory effect of nifedipine could be significantly diminished by N-nitro-L-arginine (NNA) in a concentration dependent manner, whereas oxy haemoglobin (4 microM), a NO scavenger, totally abolished the effect of nifedipine. L-Arginine, the precursor of NO, dose-dependently inhibited the collagen-induced platelet aggregation but did not potentiate the effects of nifedipine. Therefore, we propose that in platelet rich plasma the nifedipine induced inhibition of platelet aggregation is mediated by NO, a potent endogenous inhibitor of aggregation. We could confirm this hypothesis by measuring NO directly with a specific electrode.