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.     

Inactivation of nitric oxide synthases and cellular nitric oxide formation by N-iminoethyl-

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.     

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.     

Inhibitory effect of N-nitro-

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.     

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.     

Glucose deprivation increases basal and electrically evoked transmitter release from rat striatal slices. Role of NMDA and adenosine A1 receptors

We have investigated how glucose deprivation in vitro influences the basal and electrically evoked release of dopamine and acetylcholine from rat striatal slices and the role of endogenous activation of NMDA receptors and adenosine A₁ receptors in determining the magnitude of this response. Rat striatal slices, preincubated with [

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]dopamine and [

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]choline, were superfused continuously and stimulated electrically. Before and during the second stimulation, some slices were superfused with glucose-free Krebs' solution. Such glucose deprivation caused a 2 to 3-fold increase of the electrically evoked, calcium-dependent release of endogenous adenosine (but not hypoxanthine and inosine) and [

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]dopamine and a 30% increase in release of [

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]acetylcholine. Glucose deprivation also caused a delayed increase in the release of [

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]dopamine, but not of [

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]acetylcholine. The dopamine release was not calcium dependent. The addition of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 μM), a selective adenosine A₁ receptor antagonist, slightly enhanced the glucose deprivation-induced stimulatory effect on the evoked release of these two transmitters, whereas the NMDA receptor antagonist dizocilpine((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine; 3 μM) markedly attenuated the stimulatory effect of glucose deprivation. The change in basal dopamine release was not influenced by DPCPX, but was slightly attenuated by dizocilpine. In summary, the results suggest that lack of substrate induces release of both glutamate, which by actions on presynaptic NMDA receptors causes the release of dopamine, and of adenosine, which via adenosine A₁ receptors reduces the electrically evoked release of both dopamine and acetylcholine.     

Mutual dependence of calcitonin-gene related peptide and acetylcholine release in neuromuscular preparations

To investigate the mutual dependence of calcitonin gene-related peptide (CGRP) and acetylcholine release, we examined the effect of a cholinesterase inhibitor neostigmine on the release of CGRP-like immunoreactivity in rat phrenic nerve-hemidiaphragm muscle preparation, and conversely, the effect of CGRP on [

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]acetylcholine release from motor nerve terminals loaded with [

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]choline in the same preparations of mice. Release of CGRP-like immunoreactivity was increased by electrical nerve stimulation (train of 40 pulses of 200 μs pulse duration and frequency of 50 Hz applied every 10 s) in the whole preparation but not in the segmental preparation containing the endplate region. Neostigmine (0.1–0.3 μM) enhanced the resting release of CGRP-like immunoreactivity in a concentration-dependent manner, whereas it depressed the nerve-evoked release of CGRP-like immunoreactivity. CGRP (1 μM) added to perfusate decreased nerve-evoked [

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]acetylcholine release. These results suggest that CGRP, which is released by electrical nerve stimulation or a cholinesterase inhibitor in intact skeletal muscles, negatively modulates nerve-evoked acetylcholine release.     

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.     

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.     

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.     

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.     

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.     

Influence of fatty acid ethanolamides and Δ-tetrahydrocannabinol on cytokine and arachidonate release by mononuclear cells

The effects of arachidonic acid ethanolamide (anandamide), palmitoylethanolamide and Δ⁹-tetrahydrocannabinol on the production of tumor necrosis factor-α (TNF-α), interleukin-4, interleukin-6, interleukin-8, interleukin-10, interferon-γ, p55 and p75 TNF-α soluble receptors by stimulated human peripheral blood mononuclear cells as well as [

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]arachidonic acid release by non-stimulated and N-formyl-Met–Leu–Phe (fMLP)-stimulated human monocytes were investigated. Anandamide was shown to diminish interleukin-6 and interleukin-8 production at low nanomolar concentrations (3–30 nM) but inhibited the production of TNF-α, interferon-γ, interleukin-4 and p75 TNF-α soluble receptors at higher concentrations (0.3–3 μM). Palmitoylethanolamide inhibited interleukin-4, interleukin-6, interleukin-8 synthesis and the production of p75 TNF-α soluble receptors at concentrations similar to those of anandamide but failed to influence TNF-α and interferon-γ production. The effect of both compounds on interleukin-6 and interleukin-8 production disappeared with an increase in the concentration used. Neither anandamide nor palmitoylethanolamide influenced interleukin-10 synthesis. Δ⁹-Tetrahydrocannabinol exerted a biphasic action on pro-inflammatory cytokine production. TNF-α, interleukin-6 and interleukin-8 synthesis was maximally inhibited by 3 nM Δ⁹-tetrahydrocannabinol but stimulated by 3 μM Δ⁹-tetrahydrocannabinol, as was interleukin-8 and interferon-γ synthesis. The level of interleukin-4, interleukin-10 and p75 TNF-α soluble receptors was diminished by 3 μM Δ⁹-tetrahydrocannabinol. [

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]Arachidonate release was stimulated only by high Δ⁹-tetrahydrocannabinol and anandamide concentrations (30 μM). These results suggest that the inhibitory properties of anandamide, palmitoylethanolamide and Δ⁹-tetrahydrocannabinol are determined by the activation of the peripheral-type cannabinoid receptors, and that various endogenous fatty acid ethanolamides may participate in the regulation of the immune response.     

The cytosol of N1E-115 neuroblastoma cells synthesizes an EDRF-like substance that relaxes rabbit aorta

Summary The cytosolic fraction of N1E-115 neuroblastoma cells catalysed the l-arginine- and NADPH-dependent formation of a substance that relaxed endothelium-denuded strips of rabbit aorta. Relaxations in response to this substance were enhanced in the presence of superoxide dismutase. N-Nitro-l-arginine and N^G-monomethyl-l-arginine, two inhibitors of EDRF synthesis, markedly attenuated the relaxations. Hemoglobin, a scavenger of EDRF, and methylene blue, an inhibitor of soluble guanylate cyclase, completely abolished the relaxation to N1E-115 cytosol. In contrast, the cyclo-oxygenase inhibitor indomethacin did not alter the relaxations. These data demonstrate that the cytosol of a neuronally-derived cell line is able to synthesize a substance with pharmacological properties similar to EDRF.This work was supported in part by Research Grants AM 30787 and HL 28474 from the National Institutes of Health, USA     

NMDA receptor antagonists block stress-induced prolactin release in female rats at estrus

In order to evaluate the role of glutamate in prolactin secretion, we examined the effects of N-methyl-

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,

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-aspartic acid (NMDA) receptor antagonists on serum prolactin levels at both resting and restraint-stress conditions in female rats at estrus. NMDA increased basal serum prolactin levels. Administration of the selective NMDA receptor antagonist, cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS 19755) (5 and 10 mg/kg i.p.), to rats under resting conditions enhanced basal prolactin levels. A low dose of CGS 19755 (3 mg/kg) was unable to modify the hormone serum level. Under stress conditions the pretreatment with CGS 19755 (3 and 5 mg/kg) prevented the increase in serum prolactin levels. This effect was reversed by NMDA (60 mg/kg s.c.). The NMDA receptor antagonist (5 mg/kg) decreased the median eminence concentration of the dopamine metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), without modifying dopamine content. To examine the probable link between serotonin (5-HT) and glutamate in prolactin release, the 5-HT_2A/5-HT_2C receptor antagonist, ritanserin, was used. Under resting conditions, a dose of 5 mg/kg s.c. blocked the NMDA-induced prolactin release. In rats submitted to restraint, ritanserin decreased the prolactin response and NMDA was unable to correct the stress serum prolactin levels. The 5-HT_1A receptor agonist, 8-hidroxypropyl-amino tetralin (8-OH-DPAT) (3 mg/kg s.c.), increased basal serum prolactin levels and restored serum prolactin in stressed animals pretreated with CGS 19755 (5 mg/kg). The present data strongly suggest that the glutamatergic system participates in the regulation of prolactin secretion. A stimulation tone seems to be exerted via the tuberoinfundibular dopaminergic system, and the prolactin release evoked by restraint apparently involves glutamate/NMDA receptors linked to a serotoninergic pathway.     

Nitric oxide synthesis in endothelial cytosol: Evidence for a calcium-dependent and a calcium-independent mechanism

Summary Release of nitric oxide (NO) from endothelial cells critically depends on a sustained increase in intracellular free calcium maintained by a transmembrane calcium influx into the cells. Therefore, we studied whether the free cytosolic calcium concentration directly affects the activity of the NO-forming enzyme(s) present in the cytosol from freshly harvested porcine aortic endothelial cells. NO was quantified by activation of a purified soluble guanylate cyclase coincubated with the cytosol. In the presence of 1 mM L-arginine, 0.1 mM NADPH and 0.1 mM EGTA, endothelial cytosol (0.2 mg of cytosolic protein per ml) stimulated the activity of guanylate cyclase 5.0 + 0.5-fold (from 31 + 9 to 153 + 15 nmol cyclic GMP formed per min per mg guanylate cyclase). Calcium chloride increased this stimulation further in a concentration-dependent fashion by up to 136 + 15% (with 2 M free calcium; EC₅₀ 0.3 M). The calcium-dependent and -independent activation of guanylate cyclase was enhanced by superoxide dismutase (0.3 M) and was inhibited by the stereospecifically acting inhibitor of L-arginine-dependent NO formation N^G-nitro-L-arginine (1 mM) and by LY 83583 (1 M), a generator of superoxide anions. Our findings suggest a calcium-dependent and -independent synthesis of NO from L-arginine by native porcine aortic endothelial cells. Send of fprint requests to A. Mülsch, at the above address     

Possible role of nitric oxide in the antinociceptive action of intraventricular bradykinin in mice

The i.c.v. administration of bradykinin (4, 8 and 16 μg) induced antinociception in mice which was resistant to naloxone; furthermore, the induction of tolerance to morphine by a single s.c. injection (100 mg/kg, 24 h before test doses of the peptide) did not affect antinociception. Since bradykinin is known to increase nitric oxide (NO) in peripheral tissues, we studied the possibility that its antinociceptive action may be related to NO effects in the central nervous system. Bradykinin effects were antagonized by previous treatment with N^G-nitro- -arginine or concomitant i.c.v. administration of bradykinin and methylene blue. The immediate precursor of NO, -arginine, which by itself produces analgesia, also reduced bradykinin effects; moreover, tolerance to -arginine significantly decreased the response to the peptide. These results suggest that NO is involved in antinociception induced by i.c.v. administration of bradykinin.     

Arginine homeostasis in allergic asthma

Allergic asthma is a chronic disease characterized by early and late asthmatic reactions, airway hyperresponsiveness, airway inflammation and airway remodelling. Changes in l-arginine homeostasis may contribute to all these features of asthma by decreased nitric oxide (NO) production and increased formation of peroxynitrite, polyamines and l-proline. Intracellular l-arginine levels are regulated by at least three distinct mechanisms: (i) cellular uptake by cationic amino acid (CAT) transporters, (ii) metabolism by NO-synthase (NOS) and arginase, and (iii) recycling from l-citrulline. Ex vivo studies using animal models of allergic asthma have indicated that attenuated l-arginine bioavailability to NOS causes deficiency of bronchodilating NO and increased production of procontractile peroxynitrite, which importantly contribute to allergen-induced airway hyperresponsiveness after the early and late asthmatic reaction, respectively. Decreased cellular uptake of l-arginine, due to (eosinophil-derived) polycations inhibiting CATs, as well as increased consumption by increased arginase activity are major causes of substrate limitation to NOS. Increasing substrate availability to NOS by administration of l-arginine, l-citrulline, the polycation scavenger heparin, or an arginase inhibitor alleviates allergen-induced airway hyperresponsiveness by restoring the production of bronchodilating NO. In addition, reduced l-arginine levels may contribute to the airway inflammation associated with the development of airway hyperresponsiveness, which similarly may involve decreased NO synthesis and increased peroxynitrite formation. Increased arginase activity could also contribute to airway remodelling and persistent airway hyperresponsiveness in chronic asthma via increased synthesis of l-ornithine, the precursor of polyamines and l-proline. Drugs that increase the bioavailability of l-arginine in the airways – particularly arginase inhibitors – may have therapeutic potential in allergic asthma.