Evidence for the involvement of the nitric oxide–cGMP pathway in the antinociception of morphine in the formalin test

The effect of inhibition of nitric oxide synthesis and guanylate cyclase on the peripheral antinociceptive effect of morphine was assessed by using the formalin test in the rat. Saline, N^G-monomethyl-

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-arginine, a nitric oxide synthesis inhibitor (50 μg) and methylene blue, a guanylate cyclase inhibitor (500 μg), did not exhibit any antinociceptive activity. However, morphine (10 μg) produced a significant antinociceptive effect in phases 2a and 2b, which was reduced by pretreatment with either N^G-monomethyl-

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-arginine or methylene blue. These results suggest that the local administration of morphine induces antinociception by the activation of the

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-arginine–nitric oxide–cGMP pathway.     

Effects of nitric oxide donors on basal and K-evoked release of

The effects of 10 antiallergic drugs (astemizole, azelastine, ebastine, emedastine, epinastine, ketotifen, oxatomide, terfenadine, pemirolast and tranilast) on neuronal dopamine uptake were examined. Some drugs examined showed a concentration-dependent inhibition of

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uptake into synaptosomal preparations of the rat striatum. The inhibition constant (K_i) values were 231–876 nM for ebastine, terfenadine, oxatomide and astemizole. The specific binding of

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(1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) (GBR12935) to the rat striatal membranes was also inhibited by these antiallergic drugs. There was a good correlation between the degrees of inhibition of

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uptake and

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binding. Then, the behavioral excitement induced by

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-DOPA (100 mg/kg, s.c.) plus pargyline hydrochloride (80 mg/kg, i.p.) in mice was significantly enhanced by i.p. treatment with ebastine (10 mg/kg) and astemizole (5 mg/kg). These results suggest that the neuronal dopamine uptake is inhibited by some antiallergic drugs, especially ebastine.     

Inhibition of neuronal dopamine uptake by some antiallergic drugs

The effects of 10 antiallergic drugs (astemizole, azelastine, ebastine, emedastine, epinastine, ketotifen, oxatomide, terfenadine, pemirolast and tranilast) on neuronal dopamine uptake were examined. Some drugs examined showed a concentration-dependent inhibition of

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uptake into synaptosomal preparations of the rat striatum. The inhibition constant (K_i) values were 231–876 nM for ebastine, terfenadine, oxatomide and astemizole. The specific binding of

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(1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) (GBR12935) to the rat striatal membranes was also inhibited by these antiallergic drugs. There was a good correlation between the degrees of inhibition of

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uptake and

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binding. Then, the behavioral excitement induced by

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-DOPA (100 mg/kg, s.c.) plus pargyline hydrochloride (80 mg/kg, i.p.) in mice was significantly enhanced by i.p. treatment with ebastine (10 mg/kg) and astemizole (5 mg/kg). These results suggest that the neuronal dopamine uptake is inhibited by some antiallergic drugs, especially ebastine.     

DAMGO recognizes four residues in the third extracellular loop to discriminate between μ- and κ-opioid receptors

Previously, we reported that replacement of the region from the fifth transmembrane domain to the C-terminus of κ-opioid receptor with the corresponding region of μ-opioid receptor gives high affinity for [

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-Ala², N-MePhe⁴, Gly-ol⁵]enkephalin (DAMGO), a μ-opioid receptor-selective ligand, to the resultant chimeric receptor, suggesting that the difference in the amino acid sequence within this region is critical for the discrimination between μ- and κ-opioid receptors by DAMGO. In the present study, we constructed further six μ/κ-chimeric receptors and revealed that at least two separate regions around the third extracellular loop are critical for the discrimination between μ- and κ-opioid receptors by DAMGO. Furthermore, we constructed several mutant receptors by a site-directed mutagenesis technique and found that the difference between Glu²⁹⁷ of κ-opioid receptor and Lys³⁰³ of μ-opioid receptor in one region, and the difference between Ser³¹⁰, Tyr³¹² and Tyr³¹³ of κ-opioid receptor and Val³¹⁶, Trp³¹⁸ and His³¹⁹ of μ-opioid receptor in the other region, are critical for the discrimination between these receptors by DAMGO. The mutant receptor, κ (E297K+Y313H+Y312W+S310V), in which the Glu²⁹⁷, Ser³¹⁰, Tyr³¹² and Tyr³¹³ of κ-opioid receptor were changed to Lys, Val, Trp and His, respectively, bound to DAMGO with high affinity (K_d=8.7±1.2 nM) and efficiently mediated the inhibitory effect of DAMGO on intracellular cAMP accumulation. The present results showed that these four amino acid residues act as determinants for the discrimination between μ- and κ-opioid receptors by DAMGO.     

Inhibitory effect of N-nitro-

Plasma protein extravasation has been measured in guinea pig skin using

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-albumin and blood flow using

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enon (

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e) clearance. The nitric oxide (NO) synthase inhibitors N^G-nitro-

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-arginine methyl ester (

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-NAME), N^G-monomethyl-

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-arginine (l

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NMMA) and N^G-nitro-

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-arginine (

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-NOArg) and the α-adrenoceptor agonist, phenylephrine, inhibited bradykinin induced plasma protein extravasation when co-injected with the peptide. The inhibitory effects of

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-NAME and

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-NOArg lasted for up to 8 and 4 h, respectively, whereas phenylephrine and

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-NMMA had no persistent inhibitory effects. When co-injected with

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e,

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-NAME,

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-NMMA,

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-NOArg and phenylephrine, but not

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-NAME, produced significant reductions in skin blood flow. When injected prior to

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e,

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-NAME and

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-NOArg, but not phenylephrine or

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-NMMA, significantly reduced flow. The effect of

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-NAME on flow was not significant at 8 h. Thus, although the inhibitory effects of the NO synthase inhibitors on mediator induced plasma protein extravasation show correlations with their effects on blood flow, the persistent effect of

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-NAME on exudation appears to extend beyond its effect on flow.     

Persistence of effects of nitric oxide synthase inhibitors: Comparisons on blood flow and plasma exudation in guinea pig skin

Plasma protein extravasation has been measured in guinea pig skin using

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-albumin and blood flow using

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enon (

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e) clearance. The nitric oxide (NO) synthase inhibitors N^G-nitro-

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-arginine methyl ester (

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-NAME), N^G-monomethyl-

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-arginine (l

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NMMA) and N^G-nitro-

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-arginine (

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-NOArg) and the α-adrenoceptor agonist, phenylephrine, inhibited bradykinin induced plasma protein extravasation when co-injected with the peptide. The inhibitory effects of

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-NAME and

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-NOArg lasted for up to 8 and 4 h, respectively, whereas phenylephrine and

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-NMMA had no persistent inhibitory effects. When co-injected with

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e,

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-NAME,

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-NMMA,

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-NOArg and phenylephrine, but not

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-NAME, produced significant reductions in skin blood flow. When injected prior to

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e,

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-NAME and

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-NOArg, but not phenylephrine or

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-NMMA, significantly reduced flow. The effect of

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-NAME on flow was not significant at 8 h. Thus, although the inhibitory effects of the NO synthase inhibitors on mediator induced plasma protein extravasation show correlations with their effects on blood flow, the persistent effect of

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-NAME on exudation appears to extend beyond its effect on flow.     

Effects of chronic nimodipine on working memory of old rats in relation to defects in synaptosomal calcium homeostasis

The present study was designed to investigate whether chronic (from 12 to 23 months of age) dietary treatment with the L-type Ca²⁺ channel blocker nimodipine (30 mg/kg body weight) enhances the cognitive behavior of aged animals and whether such a treatment would have long-term effects on the mechanisms of Ca²⁺ regulation in synaptic terminals from the aged rat brain. Cognitive behavior was evaluated in an 8-arm radial maze in 6 test series comprising a total of 105 test sessions, with intervals of no training between series. Nimodipine-treated rats performed better than vehicle-treated, aged-matched controls in all the test series, making more correct choices every time a new series was initiated. However, differences between nimodipine- and vehicle-treated rats were most remarkable in the last three test series, when the rats were 19 to 22 months. In these series 74% of the nimodipine-treated rats were able to perform the task in 4 to 9 test sessions whereas only 12%, 14% or none of the control rats learned the task. To study Ca²⁺ regulation in synaptosomes derived from cerebral cortex and hippocampus, we analyzed

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accumulation as well as the levels of the Ca²⁺-binding proteins calbindin-D28K and calreticulin by Western blotting. Nimodipine administration had no effect on hippocampal synaptosomes but increased the levels of calbindin-D28K and calreticulin in cerebral cortex preparations. These results indicate that chronic nimodipine treatment from 12 to 23 months of age prevents age-induced learning deficits without showing any signs of toxicity, and that these effects are associated with a small increase in the levels of synaptosomal Ca²⁺-binding proteins from cerebral cortex. The up-regulation of these proteins might provide a link between the long-term effects of nimodipine on gene expression and learning ability in old rats.     

Role of nitric oxide in the effects of hypoglycemia on the cerebral circulation in awake goats

This study was performed to examine the role of nitric oxide in the effects of hypoglycemia on the cerebral circulation. Hypoglycemia was induced with insulin and its effects on cerebral blood flow (measured with an electromagnetic flow transducer placed on the internal maxillary artery) were studied in awake goats under control conditions and after administration of the nitric oxide synthesis inhibitor N^G-nitro-

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-arginine methyl ester (

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-NAME, 47 mg/kg). Also, cerebrovascular reactivity to vasodilator stimuli was examined during insulin-induced severe hypoglycemia, before and after

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-NAME treatment. In five animals under control conditions (glycemia=90±7 mg/dl, cerebral blood flow=64±4 ml/min, mean systemic arterial pressure=102±4 mmHg, cerebrovascular resistance=1.62±0.11 mmHg/ml per min and heart rate =73±6 beats/min), insulin decreased glycemia: when hypoglycemia was moderate (glycemia=46±2 mg/dl) or severe (glycemia=26±1 mg/dl) cerebral blood flow increased by 25±4% and 47±6%, and cerebrovascular resistance decreased by 18±3% and 34±4%, respectively. Under basal conditions,

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-NAME did not affect glycemia but reduced resting cerebral blood flow by 37±2%, increased mean arterial pressure by 33±2% and decreased heart rate by 28±3%; after

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-NAME, both moderate and severe hypoglycemia did not alter significantly resting cerebral blood flow and cerebrovascular resistance. In five other goats,

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-NAME, administered during severe hypoglycemia, abolished the increase in cerebral blood flow, and increased cerebrovascular resistance and mean arterial pressure over the control (normoglycemic) values. In these animals with severe hypoglycemia, acetylcholine (0.01–1 μg), isoproterenol (0.03–3 μg) and diazoxide (0.3–9 mg), injected into the internal maxillary artery, decreased cerebrovascular resistance in a dose-dependent manner, and this decrease was similar before and after

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-NAME. Therefore, insulin-induced hypoglycemia may produce cerebral vasodilatation by releasing nitric oxide and may diminish the capacity of the cerebral vasculature to release nitric oxide in response to acetylcholine.     

Mechanism of prostaglandin E2-, F2α- and latanoprost acid-induced relaxation of submental veins

The mechanism of prostaglandin E₂-, prostaglandin F_2α- and latanoprost acid (13,14-dihydro-17-phenyl-18,19,20-trinor-prostaglandin F_2α)-induced relaxation of the rabbit submental vein was studied. Prostaglandin E₂ caused maximum relaxation of endothelin-1 precontracted vessels (EC₅₀: 1.8×10⁻⁸ M). Much of the relaxation could be abolished by denuding the endothelium with the nitric oxide synthase inhibitor,

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-NAME (N^G-Nitro-

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-arginine methylester). CGRP-(8–37) (calcitonin gene-related peptide fragment (8–37)), a calcitonin gene-related peptide receptor antagonist, exhibited a partial blocking effect, whereas the tachykinin NK₁ receptor blocker, GR 82334 ([

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-Pro⁹[Spiro-γ-Lactam]Leu¹⁰,Trp¹¹]physalaemin (1–11)), markedly attenuated the response. Both prostaglandin F_2α and the relatively selective FP receptor agonist, latanoprost acid, caused relaxation of the veins to about 50% of the precontracted state in the presence of GR 32191B ([1R-[1α(Z),2β,3β,5α]]-(+)-7-[5-([1,1′-biphenyl]-4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid), a thromboxane receptor antagonist (EC₅₀: for prostaglandin F_2α 7.9×10⁻⁹ M, and for latanoprost acid 4.9×10⁻⁹ M).

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-NAME, as well as denuding the endothelium, completely abolished the effect. In addition, most or at least a large part of the relaxation was also blocked by CGRP-(8–37) as well as GR 82334. These results indicate that the FP receptor-mediated relaxation of veins is based on release of nitric oxide in addition to involvement of calcitonin gene-related peptide and substance P, or some other tachykinin, probably released from perivascular sensory nerves. The more pronounced relaxation induced by prostaglandin E₂ could be due to vasodilator EP receptors in the smooth muscle layer of the veins.     

Age-related differences and roles of endothelial nitric oxide and prostanoids in angiotensin II responses of isolated, perfused mesenteric arteries and veins of rats

We examined whether or not cyclo-oxygenase products of arachidonic acid and endothelium-derived relaxing factor (nitric oxide, NO) regulate the vascular response to angiotensin II differently with aging or development. For this purpose angiotensin II responses of isolated, perfused rat mesenteric vascular beds were compared between rats aged 4 weeks and 32 weeks. Angiotensin II increased perfusion pressure in arteries and veins of both rats aged 4 weeks and 32 weeks. In the arteries of rats aged 32 weeks the increase was slight, and less than that in rats aged 4 weeks. In contrast, the veins showed similar increases in perfusion pressure in rats aged 4 weeks and 32 weeks. Indomethacin, an inhibitor of cyclo-oxygenase, at 5×10⁻⁶ M depressed the increase in perfusion pressure only in the arteries of rats aged 32 weeks. N^G-nitro-

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-arginine methyl ester (

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-NAME), an inhibitor of nitric oxide (NO) synthase, applied at 5×10⁻⁶ M in the presence of indomethacin enlarged the perfusion pressure increase in the arteries of both rats aged 4 weeks and 32 weeks, while it failed to modify that in the veins. After removal of the endothelium from the blood vessels, the perfusion pressure responses in arteries were increased in both rats aged 4 weeks and 32 weeks, whereas those in veins were not affected. Regardless of the endothelium being intact or removed, the increase in arterial perfusion pressure of rats aged 32 weeks all but disappeared with 5×10⁻⁶ M furegrelate, an inhibitor of thromboxane A₂ synthase, and with a combined application of furegrelate and 10⁻⁶ M SQ29,548, a blocker of thromboxane A₂/prostaglandin H₂ receptors. These results indicate the following: in rat mesenteric vascular beds the angiotensin II response in the arteries appears to diminish with aging or development, whereas that in the veins does not change. The NO released from the endothelium regulates the arterial response but vasodilating prostanoids have no role in the response. Moreover, in the arteries of rats aged 32 weeks, vasoconstricting prostanoids, such as prostaglandin H₂ and thromboxane A₂, seem to play a role in angiotensin II-induced vasoconstriction. With aging or development, and depending on the type of blood vessel, NO and prostanoids appear to modify the angiotensin II response differently.     

Interleukin-1β-induced, nitric oxide-dependent and -independent inhibition of vascular smooth muscle contraction

Stimulation of vascular smooth muscle by bacterial lipopolysaccharide has been shown to produce interleukin-1β and to induce vasodilation in septic shock. To understand the mechanisms of interleukin-1β-induced relaxation, we examined the effects of interleukin-1β on contractility and cyclic GMP contents of vascular smooth muscle. After treatment of the rat aorta with interleukin-1β (20 ng/ml) for 6 h, the cyclic GMP content increased and the contraction induced by phenylephrine (1 μM) was partially inhibited. An inhibitor of nitric oxide (NO) synthase, N^G-monomethyl-

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-arginine (

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-NMMA, 100 μM), prevented the inhibitory effect of interleukin-1β. After treatment with interleukin-1β for 24 h, the phenylephrine-induced contraction was inhibited more strongly. Neither

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-NMMA (100 μM) nor aminoguanidine (100 μM) reversed the inhibition, whereas methylene blue (10 μM) partially reversed the inhibition. After treatment with interleukin-1β for 12 or 24 h, the cyclic GMP content increased but to a level lower than that obtained with a 6-h treatment. The effects of sodium nitroprusside (1 μM) to inhibit the phenylephrine-induced contraction and to increase the cyclic GMP content were markedly suppressed by the 24-h interleukin-1β treatment. In contrast, the 24-h interleukin-1β treatment did not change the ability of 8-bromo-cGMP to relax the phenylephrine-stimulated aorta. Addition of

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-NMMA (1 mM) during the 24 h treatment prevented NO production and preserved the sodium nitroprusside-induced cGMP generation by interleukin-1β. The 24 h interleukin-1β treatment increased the threshold concentration of KCl needed to induce contraction without changing the maximum contraction. In the presence of 25.4 mM KCl or the non-selective K⁺ channel inhibitor, tetraethylammonium, the inhibitory effect of the 24-h interleukin-1β treatment on phenylephrine-induced contraction was restored. These results suggest that interleukin-1β inhibits vascular smooth muscle contraction by a time-dependent, dual mechanism. After a 6-h treatment with interleukin-1β, the NO/cyclic GMP system is activated. After a 24-h interleukin-1β treatment, in contrast, the NO/cyclic GMP system may be desensitized and the contraction of vascular smooth muscle is inhibited by another mechanism, possibly membrane hyperpolarization.     

Platelets enhance contractility in perfused rat mesenteric arteries: Involvement of endothelin-1

We investigated the effects of platelet supernatant on pressor responses to norepinephrine in isolated perfused rat mesenteric arteries. Perfusion of the arteries with platelet supernatant for 2 h markedly enhanced the pressor responses to norepinephrine (10⁻⁶ and 3×10⁻⁶ M). This enhancement was significantly inhibited by phosphoramidon (10⁻⁴ M), an endothelin converting enzyme inhibitor. Both BQ788 [N-cis-2,6-dimethylpiperidinocarbonyl-

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-γ-methylleucyl-

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-1-methoxycarbonyltryptophanyl-

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-norleucine] (10⁻⁶ M), an endothelin ET_B receptor antagonist, and bosentan (Ro47-0203, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2´-bipyrimidin-4-yl]-benzenesulfonamide) (10⁻⁵ M), a nonselective endothelin receptor antagonist, also prevented the potentiation of responses to norepinephrine evoked by platelet supernatant, but FR139317 ((R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-methyl-pentanoyl] amino-3-[3-(1-methyl-1H-indoyl)]propionyl]amino-3-(2-pyridyl) propionic acid) (10⁻⁶ M), an endothelin ET_A receptor antagonist, had little effect. Suppressor doses of endothelin-1 (3×10⁻¹⁰ M) or sarafotoxin S6c (S6c) (3×10⁻¹⁰ M) potentiated significantly the norepinephrine-induced vasoconstriction, in the same preparation. Moreover, supernatant-induced enhancement of pressor responses to norepinephrine was markedly suppressed by TGF-β1 neutralizing antibody. Transforming growth factor-β1 (TGF-β1) (40 pM) also significantly enhanced the pressor responses to norepinephrine (10⁻⁶ M) and this enhancement was significantly inhibited by phosphoramidon. These results suggest that platelet-derived TGF-β1 stimulates the vascular production of endothelin-1 and thereby enhances vasoconstrictor responses to norepinephrine. Platelet-induced enhancement of vasoconstrictor responses to norepinephrine seems to be mainly mediated by endothelin ET_B receptor, in rat mesenteric arteries.     

N-Nitro-L-arginine protects against ischaemia-induced increases in nitric oxide and hippocampal neuro-degeneration in the gerbil

To assess the effects of the nitric oxide synthase inhibitor N^G-Nitro-L-arginine on behavioural, biochemical and histological changes following global ischaemia, the Mongolian gerbil was used. Ischaemia was induced by bilateral carotid occlusion for 5 min. N^G-Nitro-L-arginine was administered i.p. at either 1 or 10 mg/kg 30 min, 6, 24, and 48 h after surgery. 5 min bilateral carotid occluded animals were hyperactive 24, 48 and 72 h after surgery. N^G-Nitro-L-arginine caused some attenuation in this hyperactivity. The activity of nitric oxide synthase was increased in the cerebellum, brain stem, striatum, cerebral cortex and hippocampus of 5 min bilateral carotoid occluded animals. N^G-Nitro-L-arginine reversed the increase in nitric oxide synthase activity in all brain regions. Extensive neuronal death was observed in the CA₁ layer of the hippocampus in 5 min bilateral carotid occluded animals 96 h after surgery. N^G-Nitro-L-arginine significantly protected against the neuronal death of cells in the CA₁ layer.     

Effect of N-nitro-l-arginine on shock induced by endotoxin and by platelet activating factor in dogs

When N^G-nitro-l-arginine, a nitric oxide synthase inhibitor, administration was started 5 min prior to shock induction in anesthetized dogs, a partial restoration was observed in endotoxin-induced shock and a complete recovery in platelet activating factor (PAF)-induced shock. When N^G-nitro-l-arginine infusion was started 5 min after shock induction, no significant recovery was observed in endotoxin-induced shock and a complete recovery in PAF-induced shock. These data indicate that enhanced production of nitric oxide by vascular endothelial cells may contribute to endotoxin- or PAF-induced shock and also that some mediators including inducible nitric oxide synthase and/or cellular damage might be involved in endotoxin-induced shock.     

Attenuation of some signs of opioid withdrawal by inhibitors of nitric oxide synthase

Effects of nitric oxide synthase (NOS) inhibitors (l-N ^G-nitroarginine,l-N ^G-nitroarginine methyl ester) on precipitated opioid withdrawal were studied in morphine-dependent rats given naloxone, in order to assess the involvement of nitric oxide (NO) in opioid dependence.l-N ^G-Nitroarginine (7.5 mg/kg, IP, 1 h before naloxone or b.i.d. on days 4–7 of an 8-day morphine treatment) reduced wet dog shakes and weight loss; when given by osmotic pumps (15 mg/kg per day), the drug reduced wet dog shakes but not weight loss.l-N ^G-Nitroarginine methyl ester (60 mg/kg, 1 h before naloxone) also reduced wet dog shakes and weight loss. The results indicate that NOS inhibitors warrant further study as potential treatment of the opioid withdrawal syndrome.Abstracts were presented at meetings of the Society for Neuroscience in New Orleans, La., November 10–15, 1991 and of the American Society for Pharmacology and Experimental Therapeutics in Orlando, Fla., August 10–18, 1992     

Constitutive nitric oxide modulates the injurious actions of vasopressin on rat intestinal microcirculation in acute endotoxaemia

The administration of the nitric oxide (NO) synthase inhibitor, N^G-nitro-l-arginine methyl ester (L-NAME, 5 mg/kg s.c.) concurrently with Escherichia coli endotoxin (3 mg/kg i.v.) increased vascular permeability and caused mucosal damage in the rat intestine 1 h later. The vasopressin V₁ receptor antagonist, [Mca¹,Tyr(Me)²,Arg⁸]vasopressin (0.01–0.2 μg/kg s.c., 15 min before endotoxin) dose-dependently reduced this damage. These results suggest a beneficial role of NO, counteracting the injurious vascular actions of endogenous vasopressin, in maintaining intestinal mucosal integrity in acute endotoxaemic states.     

Endothelium-derived nitric oxide partially mediates salbutamol-induced vasodilatations

This study examined the ability of salbutamol (selective β₂-adrenoceptor agonist) to cause endothelium-dependent relaxation in rat aortic rings and depressor response in conscious rats. Salbutamol (0.01–100 μM) concentration dependently relaxed preconstricted aortic rings. The relaxant response was partially attenuated by either mechanical removal of the endothelium or treatment with N^G-nitro-l-arginine methyl ester (L-NAME, 100 μM). In conscious rats, either i.v. infused phenylephrine (5 μg/kg per min) or i.v. bolus injected L-NAME (12.8 mg/kg), but not the vehicle, caused similar sustained increases in mean arterial pressure (MAP). I.v. infused salbutamol (2–128 μg/kg per min, each dose for 5 min) dose dependently decreased MAP in vehicle-treated rats; the depressor responses were potentiated by hypertension induced by phenylephrine. In contrast, the magnitudes of the depressor response to salbutamol in L-NAME-treated rats were less than those in rats pretreated with phenylephrine or the vehicle. I.v. bolus injections of salbutamol (0.25–16 μg/kg) also caused dose-dependent and transient decreases in MAP in vehicle-treated rats. The magnitude but not the duration of the depressor response to salbutamol was less in rats treated with L-NAME, compared to those in rats given phenylephrine or the vehicle. These results suggest that endothelium-derived nitric oxide is partially involved in β₂-adrenoceptor-mediated vasodilatation.     

Comparison of 7-nitroindazole with other nitric oxide synthase inhibitors as attenuators of opioid withdrawal

Previously, we demonstrated that two nonselective inhibitors of nitric oxide synthase (NOS),l-N ^G-nitroarginine (l-NNA) andl-N ^G-nitroarginine methyl ester (l-NAME), reduced some signs of morphine withdrawal in rats. The present work extended these studies to include 7-nitroindazole (7-NI), an inhibitor specific for cerebral NOS, andN(5)-(1-iminoethyl)-l-ornithine (l-NIO), a potent inhibitor of endothelial NOS. Behavioral effects of these four NOS inhibitors and clonidine, an ₂-adrenoceptor, agonist, on morphine withdrawal in rats were assessed. Rats received one 75-mg morphine pellet subcutaneously (SC). Three days later, NOS inhibitors were administered IP 1 h before withdrawal was precipitated with naloxone (0.5 mg/kg, SC) and scored. 7-NI,l-NIO,l-NAME andl-NNA produced dose-related decreases in weight loss, diarrhea, wet dog shakes and grooming. 7-NI also reduced mastication, salivation and genital effects. Clonidine produced effects similar to 7-NI. In awake, morphine-naive and morphine-dependent rats not subjected to withdrawal, 7-NI was the only NOS inhibitor that did not increase blood pressure. Because 7-NI attenuated more signs of opioid withdrawal thanl-NNA,l-NAME orl-NIO without causing hypertension, 7-NI appears to warrant further testing as a potential candidate for human use.Abstracts were presented at the annual meetings of the College on Problems of Drug Dependence, West Palm Beach, Fla., 18–23, June 1994; International Narcotics Research Conference, North Falmouth, Cape Cod, Mass., 16–21, July 1994; and a Satellite Symposium to IUPHAR, Montreal, Canada, 22–24, July 1994.     

(1→3)-β-d-Glucan stimulates nitric oxide generation and cytokine mRNA expression in macrophages

Beta-glucans are known for their potent ability to induce nonspecific inflammatory reactions and are believed to play a role in bioaerosol-induced respiratory symptoms seen in both occupational and residential environments. Here, the ability of a (1→3)-β- -glucan (Curdlan) to stimulate nitric oxide generation and cytokine mRNA expression in rat alveolar macrophages (AMs) and the murine monocyte/macrophage cell line, RAW 264.7 was investigated. Exposure to (1→3)-β- -glucan (20, 100 and 500 μg/ml) induced a dose-dependent increase in the expression of inducible nitric oxide synthase mRNA and a release of nitric oxide into the culture medium in both rat AMs and RAW 264.7 cells. The mRNA expression of a number of other inflammatory mediators such as interleukin-1β, interleukin-6, tumor necrosis factor-α and cyclooxygenase-2 was also increased by the exposure to β-glucan. The capability of (1→3)-β- -glucan (500 μg/ml) to induce mRNA synthesis of these various mediators were comparable to that of endotoxin (1 μg/ml). These results imply that (1→3)-β- -glucan stimulates the generation of nitric oxide, cytokines and prostaglandins in macrophages and suggest the possibility that this may contribute to bioaerosol-induced respiratory symptoms seen in exposed individuals.     

Purification and characterization of a new l-amino acid oxidase from Daboia russellii siamensis venom

A new l-amino acid oxidase (designated as DRS-LAAO) was purified from Daboia russellii siamensis venom by ion-exchange, gel filtration and affinity chromatographies. DRS-LAAO is a homodimeric enzyme with a molecular weight of 120.0 kDa as measured by size exclusion chromatography and the monomeric molecular weight of 58.0 kDa as measured by SDS-PAGE under both non-reducing and reducing conditions. The N-terminal amino acid sequence (ADDKNPLEECFREDD) of DRS-LAAO shares high identity with other snake venom l-amino acid oxidases, especially with those isolated from viperid venoms. The enzyme displayed high specificity towards hydrophobic l-amino acids. The best substrate of DRS-LAAO was L-Leu followed by L-Phe and L-Ile, while five substrates — L-Pro, L-Asn, L-Gly, L-Ser and L-Cys were not oxidized. Optimal pH of DRS-LAAO was 8.8. The enzyme showed no hemorrhagic activity even at a dosage of 55.0 μg. DRS-LAAO dose-dependently inhibited platelet aggregation induced by ADP (83.33 μM) and TMVA (55.0 nM) with an IC₅₀ value of 32.8 μg/ml and 32.3 μg/ml, respectively. The minimum inhibitory concentrations (MICs) of DRS-LAAO against Staphylococci aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were 9.0, 144.0 and 288.0 μg/ml, respectively. The minimum bactericidal concentrations (MBCs) of the enzyme for these strains were twice of the MIC values. These results showed that DRS-LAAO had the strongest antimicrobial activity against S. aureus among these three international standard stains. Antibacterial-activities of DRS-LAAO against eight clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were also tested. The MICs of DRS-LAAO against these isolates ranged from 4.5 to 36.0 μg/ml. And the MBCs of the enzyme against these isolates ranged from 9.0 to 72.0 μg/ml.