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

Sodium N-benzyl-D-glucamine dithiocarbamate (BGD), sodium N-p-methylbenzyl-D-glucamine dithiocarbamate (MBGD), and sodium N-p-isopropylbenzyl-D-glucamine dithiocarbamate (PBGD), which were recently synthesized, were evaluated for their efficacy in the distribution and excretion of cadmium in rats and mice exposed to cadmium. Rats and mice were injected i.p. with 109CdCl2 (1 mg Cd/kg and 2 microCi 109Cd/one animal) and 3 days later, they were treated with the dithiocarbamates (400 mumol/kg) every other day for 2 weeks. These dithiocarbamates were effective in removing cadmium from the body without increasing the cadmium content in the kidney. After treatment with BGD, MBGD, and PBGD, cadmium was excreted mainly in the feces and the effect of MBGD and PBGD on the fecal excretion of cadmium was much larger than that of BGD. The treatment with these dithiocarbamates did not cause the redistribution of cadmium to brain, testes, and heart in rats and mice. The treatment of mice with PBGD decreased the concentrations of essential metals in liver, kidney, and brain. The extent of acute toxicity of the dithiocarbamates in mice was in the order PBGD greater than MBGD greater than BGD.     

Comparison of effectiveness of 3 dithiocarbamates on excretion and distribution of cadmium in rats and mice

Sodium N-benzyl-D-glucamine dithiocarbamate (BGD), sodium N-p-methylbenzyl-D-glucamine dithiocarbamate (MBGD), and sodium N-p-isopropylbenzyl-D-glucamine dithiocarbamate (PBGD), which were recently synthesized, were evaluated for their efficacy in the distribution and excretion of cadmium in rats and mice exposed to cadmium. Rats and mice were injected i.p. with 109CdCl2 (1 mg Cd/kg and 2 microCi 109Cd/one animal) and 3 days later, they were treated with the dithiocarbamates (400 mumol/kg) every other day for 2 weeks. These dithiocarbamates were effective in removing cadmium from the body without increasing the cadmium content in the kidney. After treatment with BGD, MBGD, and PBGD, cadmium was excreted mainly in the feces and the effect of MBGD and PBGD on the fecal excretion of cadmium was much larger than that of BGD. The treatment with these dithiocarbamates did not cause the redistribution of cadmium to brain, testes, and heart in rats and mice. The treatment of mice with PBGD decreased the concentrations of essential metals in liver, kidney, and brain. The extent of acute toxicity of the dithiocarbamates in mice was in the order PBGD greater than MBGD greater than BGD.     

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.     

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.     

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

Cyclosporin A is known to decrease nitric oxide (NO) production in nervous tissues. The effects of systemic cyclosporine A on the induction and expression of morphine tolerance and dependence, acute morphine-induced antinociception, and the probable involvement of the L-arginine/nitric oxide pathway in these effects were assessed in mice. Cyclosporin A (20 mg/kg), N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) and a combination of the two at lower and per se non-effective doses (5 and 3 mg/kg, respectively) showed a similar pattern of action, inhibiting the induction of tolerance to morphine-induced antinociception and increasing the antinociception threshold in the expression phase of morphine tolerance. These agents also inhibited the expression of morphine dependence as assessed by naloxone-precipitated withdrawal signs, while having no effect on the induction of morphine dependence. L-Arginine, at a per se non-effective dose (60 mg/kg), inhibited the effects of Cyclosporin A. Moreover, acute administration of Cyclosporin A (20 mg/kg) or L-NAME (10 mg/kg) enhanced the antinociception induced by acute administration of morphine (5 mg/kg), while chronic pretreatment with Cyclosporin A (20 mg/kg) or L-NAME (10 mg/kg) for 2 days (twice daily) did not affect morphine-induced antinociception. The inducible nitric oxide synthase inhibitor, aminoguanidine (100 mg/kg), did not alter morphine antinociception, tolerance or dependence. In conclusion, decreasing NO production through constitutive nitric oxide synthase may be a mechanism through which cyclosporin A differentially modulates morphine tolerance, dependence and antinociception.     

The effect of cyclosporin A on morphine tolerance and dependence: involvement of L-arginine/nitric oxide pathway

Cyclosporin A is known to decrease nitric oxide (NO) production in nervous tissues. The effects of systemic cyclosporine A on the induction and expression of morphine tolerance and dependence, acute morphine-induced antinociception, and the probable involvement of the L-arginine/nitric oxide pathway in these effects were assessed in mice. Cyclosporin A (20 mg/kg), N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) and a combination of the two at lower and per se non-effective doses (5 and 3 mg/kg, respectively) showed a similar pattern of action, inhibiting the induction of tolerance to morphine-induced antinociception and increasing the antinociception threshold in the expression phase of morphine tolerance. These agents also inhibited the expression of morphine dependence as assessed by naloxone-precipitated withdrawal signs, while having no effect on the induction of morphine dependence. L-Arginine, at a per se non-effective dose (60 mg/kg), inhibited the effects of Cyclosporin A. Moreover, acute administration of Cyclosporin A (20 mg/kg) or L-NAME (10 mg/kg) enhanced the antinociception induced by acute administration of morphine (5 mg/kg), while chronic pretreatment with Cyclosporin A (20 mg/kg) or L-NAME (10 mg/kg) for 2 days (twice daily) did not affect morphine-induced antinociception. The inducible nitric oxide synthase inhibitor, aminoguanidine (100 mg/kg), did not alter morphine antinociception, tolerance or dependence. In conclusion, decreasing NO production through constitutive nitric oxide synthase may be a mechanism through which cyclosporin A differentially modulates morphine tolerance, dependence and antinociception.     

Influence of nitric oxide on morphine-induced conditioned place preference in the rat central amygdala

Effects of intra-central amygdala injections of L-arginine, a nitric oxide (NO) precursor, and N(G)-nitro-L-arginine methyl ester (L-NAME), a NO synthase (NOS) inhibitor, on morphine-induced conditioned place preference in rats were investigated by using an unbiased 3-day schedule of place conditioning design. Animals receiving once daily injections of morphine (0.5-7.5 mg/kg, subcutaneously, s.c.) or saline (1.0 ml/kg, s.c.) showed a significant place preference in a dose-dependent manner. The maximum response was observed with 5.0 mg/kg of the opioid. Co-administration of morphine (5.0 mg/kg) with L-arginine (0.3, 1.0 and 3.0 microg/rat), but not with L-NAME (0.3, 1.0 and 3.0 microg/rat), during the acquisition of morphine-induced conditioned place preference increased morphine-induced conditioned place preference. The response to L-arginine was blocked by L-NAME preadministration. L-arginine and L-NAME by themselves did not induce conditioned place preference. When L-arginine or L-NAME at 0.3-3.0 microg/rat was administered 1 min before conditioned place preference testing, L-arginine but not L-NAME caused an increase in the expression of morphine-induced conditioned place preference, the effect that was blocked by L-NAME preadministration. A dose of L-arginine (0.3 microg/rat), but not L-NAME, during expression of morphine-induced conditioned place preference produced an increase in locomotion compared with that in the control group. It may be concluded that an increase in the NO levels in the central amygdala may have an effect on the acquisition and expression of morphine-induced conditioned place preference.     

Evidence for the involvement of L-citrulline but not nitric oxide in the proconvulsant action of the precursor L-arginine on picrotoxin-induced convulsions in rats

To determine the role of the metabolites of L-arginine in its actions on picrotoxin-induced convulsions in rats, the concentrations of nitric oxide (NO) and L-citrulline were measured in the brain 30 and 60 min after the administration of L-arginine (1000 and 2000 mg/kg) or of N-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg), an inhibitor of NO synthase. Animals treated similarly were challenged 30 and 60 min later with picrotoxin (5mg/kg), and the time of onset of myoclonus and clonic convulsions and the frequency of convulsions were determined. These parameters were also determined 30 and 60 min after administering L-arginine in L-NAME-pretreated (30 min) animals. Thirty minutes after the administration of L-arginine, the concentrations of both NO and L-citrulline were raised, the onset of myoclonus and clonic convulsions was delayed, and the frequency of convulsions was decreased, indicating the anticonvulsant property of L-arginine. A 60-min treatment of L-arginine produced a further increase in the concentration of L-citrulline but not that of NO and promoted the frequency of picrotoxin-induced convulsions. Pretreatment with L-NAME prevented L-arginine from raising the concentrations of both NO and L-citrulline; it also promoted the anticonvulsant actions and prevented the proconvulsant actions of L-arginine. These results lead to the conclusion that NO has no involvement in the time-dependent anti and proconvulsant actions of L-arginine on the picrotoxin convulsion model, and that L-citrulline seems to have a role in the proconvulsant action of L-arginine.     

Possible involvement of l-arginine-nitric oxide pathway in modulating regional blood flow to brown adipose tissue of rats

To evaluate whether the l-arginine-nitric oxide (NO) pathway is involved in the regulation of regional blood flow to brown adipose tissue (BAT), the effects of two specific NO synthase inhibitors, N^G-nitro-l-arginine methyl ester (l-NAME) and N^G-monomethyl-l-arginine (l-NMMA), on the blood flow to interscapular brown adipose tissue (IBAT) were studied in urethane-anesthetized rats. Regional blood flow in MAT was measured with laser-Doppler flowmetry.An intravenous injection of l-NAME and l-NMMA, but not of either d-enantiomer, caused a transient and dose-dependent increase in IBAT blood flow. Dose-response curves for these NO synthase inhibitors showed that l-NAME was more potent than l-NMMA in increasing IBAT blood flow. We also observed a concomitant pressor effect accompanied by a slight decrease in heart rate following intravenous injection of l-NAME and l-NMMA. An elevation of IBAT blood flow and blood pressure induced by both l-NAME and l-NMMA was reversed by l-arginine in an enantiomerically specific manner. The increase in IBAT blood flow induced by NO synthase inhibitors was of shorter duration and less sensitive to l-arginine than the increase in blood pressure.Our results show that the WAY blood flow is increased by inhibition of NO synthase and that the response of IBAT vasculature to NO synthase inhibitors is different from that of the resistance vessels which regulate blood pressure. The involvement of l-arginine-NO pathways in modulating microcirculation in IBAT is suggested. Correspondence to: Y. Uchida at the above address     

Role of nitric oxide in systemic effect of theophylline on mouse body temperature

In the present study, the interaction of nitric oxide synthase (NOS) inhibitors, L-NAME (N(G)-nitro-L-arginine methyl ester HCl) and L-NA (N(omega)-nitro-L-arginine), and its precursor, L-arginine (2-(S)-2-amino-5-[(aminoiminomethyl)amino] pentatonic acid), with theophylline on mouse body temperature was studied. Intraperitoneal (i.p.) injection of different doses of theophylline altered body temperature. Lower doses of theophylline (12.5 and 25 mg/kg) increased, but a higher dose (100 mg/kg) reduced, the animals' body temperature. The combination of L-arginine (20 and 40 mg/kg) with the highest dose of theophylline potentiated the hypothermic effect induced by the latter drug, while L-arginine by itself did not alter body temperature. L-NAME (10-80 mg/kg) or L-NA (10 mg/kg) plus a lower dose of theophylline (12.5 mg/kg) reduced the theophylline-induced hyperthermic response. L-NA (1, 5, and 10 mg/kg) in combination with the high dose of theophylline (100 mg/kg) also induced greater hypothermia. Both L-NAME and L-NA by themselves reduced body temperature. It is concluded that nitric oxide (NO) may be involved in the effects of theophylline on body temperature in mice.     

Study of in vivo and in vitro resting vasodilator nitric oxide tone in normotensive and genetically hypertensive rats

The effects of N^G-nitro- -arginine ( -NNA) on mean arterial pressure and the effects of both -NNA and methylene blue on isolated aorta tone, were studied in order to elucidate potential alterations in vasodilator resting nitric oxide (NO) tone in genetic hypertension. -NNA produced a significantly greater increase of mean arterial pressure in spontaneously hypertensive rats (SHR) than in Wistar Kyoto (WKY) rats; in both cases, -arginine completely inhibited the -NNA hypertensive effect. Neither ganglion blockade with hexamethonium nor cyclooxygenase inhibition with indomethacin significantly modified the effect of -NNA in both rat strains. In intact aorta rings, after submaximally contraction with KCl (25 mM), both -NNA and methylene blue induced strong dose-dependent contractions. The maximum contractions were, however, significantly greater in WKY rats than in SHR. The mechanical elimination of endothelium markedly inhibited both -NNA and methylene blue maximum contractions. In intact rings, -arginine completely inhibited the -NNA effects in both rat strains; in rubbed rings, the -arginine inhibitory effects were strong in WKY rats but not important and erratic in SHR. -Arginine had no effect on the contractions induced only by KCl in any of the preparations. In WKY rat-rubbed rings, sodium nitroprusside was significantly more effective in relaxing the contractions in response to 25 mM KCl than the contractions in response to methylene blue. These results indicate that contractions induced by -NNA and methylene blue in isolated aorta are principally due to the inhibition of an important endothelial resting vasodilator NO tone. They also show that hypertension reduces the resting vasodilator NO tone in isolated rat aorta, in spite of enhancing the total vasodilator NO tone in anaesthetized rat.     

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.     

Lack of involvement of extracellular signal-regulated kinase (ERK) in the agonist-induced endothelial nitric oxide synthesis

In a recent paper, it was shown that stimulation of endothelial cells with bradykinin (BK) leads to phosphorylation of endothelial nitric oxide synthase (eNOS) mediated by extracellular signal-regulated kinase (ERK) (J. Biol. Chem. 275 (2000) 30707). Since in vitro phosphorylation by ERK reduced the catalytic activity of eNOS, it was suggested that this mechanism may be an important determinant of nitric oxide signalling in endothelial cells. To explore the physiological role of ERK as regulator of nitric oxide synthesis in intact cells, we measured the effects of the kinase inhibitor PD 98059 on BK- and ATP-induced nitric oxide formation in cultured endothelial cells and isolated vascular smooth muscle strips. PD 98059 completely inhibited ERK activation by BK and ATP in porcine aortic endothelial cells without affecting eNOS activation. Moreover, PD 98059 did not potentiate relaxation of isolated porcine pulmonary arteries to BK or ATP, indicating that ERK-catalysed eNOS phosphorylation does not contribute to the regulation of nitric oxide formation in intact cells or tissues.     

Evidence for monoaminergic involvement in triadimefon-induced hyperactivity

Triadimefon is a triazole fungicide that produces hyperactivity in both mice and rats similar to that seen following administration of compounds with catecholaminergic activity (e.g., d-amphetamine). To determine whether the triadimefon-induced hyperactivity is due to an action on CNS catecholaminergic systems, we evaluated the effects of combined treatment of triadimefon with either the tyrosine hydroxlase inhibitor d,l--methyl-p-tyrosine methyl ester HCl (MPT) or the amine depletor reserpine. Adult male Long-Evans hooded rats, approximately 70 days of age were used. Dosage-effect functions were determined for MPT (0–200 mg/kg IP), reserpine (0–2.5 mg/kg IP), d-amphetamine (0–3 mg/kg IP), and methylphenidate (0–40 mg/kg IP). Motor activity was measured as photocell interruptions in figure-eight mazes. The interaction between triadimefon and MPT was determined with the following groups: 1) vehicle control; 2) 200 mg/kg triadimefon PO; 3) 100 mg/kg MPT; and 4) both MPT and triadimefon. A similar design was used to determine the interaction between triadimefon and reserpine (0.62 mg/kg), MPT and d-amphetamine (1.5 mg/kg), and reserpine and methylphenidate (5.0 mg/kg). In the first experiment MPT did not block the increased motor activity produced by triadimefon (i.e., both triadimefon alone and MPT in combination with triadimefon produced significant increases in motor activity). MPT did, however, block d-amphetamine-induced hyperactivity. Since MPT did not antagonize the effect of triadimefon, these data suggest that increased motor activity produced by triadimefon is not mediated through release of newly synthesized catecholamines. In contrast, pretreatment with reserpine blocked the hyperactivity induced by both triadimefon and methylphenidate, which suggests that triadimefon-induced hyperactivity may be due to an interaction with CNS catecholamines stored in reserpine-sensitive pools.The research described in this article has been reviewed by the Health Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Presented in part at the Annual Meeting of the Society for Neuroscience, New Orleans, LA, November, 1987     

Participation of the GABA/benzodiazepine receptor and the NO-cyclicGMP pathway in the "antinociceptive-like effects" of diazepam

The mechanism of action underlying the "analgesic activity" of diazepam remains unclear. In this study, the possible participation of the GABA/benzodiazepine receptor and the nitric oxide-cyclic GMP (NO-cGMP) pathway was assessed utilizing the pain-induced functional impairment model in the rat (PIFIR). Nociception was induced by an intra-articular injection of 15% uric acid. Diazepam (1 and 2 mg/kg, i.p.) reversed the dysfunction induced by uric acid. Flumazenil (10 mg/kg, i.p.), a GABA/benzodiazepine receptor antagonist, abolished the "antinociceptive-like effect" of diazepam (at 2 mg/kg). The "antinociceptive-like effect" of diazepam (at 2 mg/kg) was antagonized by the non-selective nitric oxide synthase (NOS) inhibitor, N(omega)-l-nitro-arginine methyl ester hydrochloride (l-NAME, 5 mg/kg, s.c.) (but not by its inactive isomer), and by the selective neuronal NOS inhibitor, 7-nitroindazole (7-NI, 1 mg/kg, i.p). While, the NO precursor, l-arginine (125 mg/kg, s.c.), but not d-arginine (125 mg/kg, s.c.), increased the "antinociceptive-like effect" of a non-effective dose of diazepam (1 mg/kg). Methylene blue (10 mg/kg, i.p.), a guanylate cyclase inhibitor, also prevented the "antinociceptive-like effect" of diazepam (at 2 mg/kg). These results suggest that the GABA/benzodiazepine receptor and the NO-cGMP pathway participate in the "antinociceptive-like effect" of diazepam.     

The role of nitric oxide (NO) in 5-HT-induced relaxations of the guinea-pig stomach

Summary In a previous study we showed that the relaxations induced after vagal stimulation of the guinea-pig stomach are mediated via nitric oxide (NO) or a NO-related substance. Intra-arterial injection (i.a.) of 5-hydroxytryptamine (5-HT) also induced relaxations in the guinea-pig stomach. Since it has been shown that in the guinea-pig colon 5-HT-induced relaxations are mediated via NO the aim of this study was to establish whether NO is involved in the 5-HT-induced relaxations in the guinea-pig stomach. Intra-arterial injection of 5-HT induced dose-dependent relaxations of the stomach. Since atropine and - and -adrenoceptor blocking agents did not influence the relaxation and since tetrodotoxin (TTX) blocked the relaxations, this effect is mediated via NANC-neurons. Administration of a NO-synthase-inhibitor N^G-nitro-l-arginine (L-NNA) concentration-dependently reduced the 5-HT-induced relaxations. Haemoglobin (a NO-scavanger) did not affect the relaxations to 5-HT, while addition of methylene blue, an inhibitor of soluble guanylate cyclase, reduced the relaxations by 50%. Addition of an opioid receptor agonist (loperamide), a 5-HT₁ antagonist (methiothepin or metergoline) or a 5-HT₄ receptor agonist (cisapride) or-antagonist (tropisetron in micromolar concentrations) inhibited the 5-HT-induced relaxations. Neither the 5-HT₄ receptor agonist renzapride, nor the novel 5-HT₄ receptor antagonist SDZ 205-557, affected the relaxations to 5-HT. These data indicate that 5-HT-induced relaxations of the guinea-pig stomach are mediated via NANC-inhibitory nerves on which inhibitory opioid-receptors are present. The use of selective agonists and antagonists indicates that 5-HT does not act via 5-HT₂, 5-HT₃ or 5-HT₄ receptors. 5-HT may act via 5-HT₁ receptors but the subtype involved, if any, has not yet been identified. The inhibitory neurotransmitter which is involved is NO or a NO-related substance. Correspondence to A. L. Meulemans at the above address     

Functional relation between nitric oxide and noradrenaline for the modulation of vascular tone in rat mesenteric vasculature

As previously reported, N-nitro-l-arginine (l-NNA), an inhibitor of nitric oxide (NO) synthesis, decreased transmural field stimulation (TFS)-induced noradrenaline overflow from the isolated perfused rat mesenteric vasculature attached to the intestine. The decrease was attenuated by l-arginine. This suggests that NO may increase noradrenaline release (Yamamoto et al. 1993).The present experiments with this preparation were done in order to monitor changes in vascular perfusion pressure caused by TFS or by noradrenaline infusion in parallel with those in the noradrenaline outflow caused by TFS in the presence of atropine (0.1 mol/l) (to block acetylcholine-induced release of endothelial NO) and of indomethacin (3 mol/l) (to inhibit l-NNA-induced production of vasoconstrictor prostanoids). (1) TFS (2–10 Hz) caused a frequency-dependent increase in noradrenaline overflow and perfusion pressure. (2) l-NNA (10 and 30 mol/l) caused a concentration-dependent inhibition of TFS-induced noradrenaline overflow, whereas the TFS-induced pressure increase was augmented by l-NNA in a concentration-dependent manner. At any given concentration of l-NNA, the potentiation of vasoconstriction by l-NNA became greater in magnitude as the frequency of the TFS was raised. (3) Infusion of noradrenaline (0.38–6 nmol) caused a dose-dependent increase in perfusion pressure up to a value comparable with that caused by TITS. The pressure increase in response to noradrenaline infusion was also enhanced by l-NNA, relatively, to a greater extent than the enhancement, by l-NNA, of the pressure response to TFS. (4) These effects of l-NNA were significantly attenuated by l-arginine (0.3 mmol/l) or sodium nitroprusside (1 mol/l). Our results suggest that NO, presumably originating from several sites, may stimulate the release of noradrenaline in the mesenteric vasculature and that the consequent rise in circulating noradrenaline, in turn, causes the liberation of endothelial NO. Correspondence to: R. Yamamoto at the above address     

Retardation of cerebral dopamine turnover after morphine withdrawal and its enhanced acceleration by acute morphine administration in rats

Summary To clarify the effects of withdrawal from chronic morphine treatment on cerebral dopamine (DA) turnover, we have measured the -methyl-p-tyrosine (MT)-induced depletion of DA in five brain areas of male Wistar rats given morphine twice daily for 40 or 60 days. After the last morphine dose (50 or 70 mg/kg) the rats were withdrawn for 1, 2 or 4 days. In order to study the development of tolerance some of the rats were challenged with 10 mg/kg of morphine.Withdrawal of morphine retarded the MT-induced DA depletion in the limbic forebrain and after long enough chronic treatment in the striatum, too. The challenge dose of morphine accelerated the cerebral DA depletion slightly less in rats withdrawn for 1 day from 60-day chronic morphine treatment than in rats treated chronically with saline, but it enhanced the DA depletion more in rats withdrawn from morphine for 2 and 4 days than in chronic saline rats. This enhancement was clearest in rats withdrawn for 4 days from 60-day treatment. Thus withdrawal from morphine seems to sensitize the rats to the DA depletion accelerating effect of morphine.Our results show that repeated administration of morphine creates no marked tolerance to the DA depletion accelerating effect of morphine. In contrast, the dopaminergic neurones of the chronically treated rats seem to depend on continuous morphine administration for their normal functioning. Furthermore, the retarded DA turnover after discontinuation of morphine treatment seems to sensitize the dopaminergic neurones to the DA depletion accelerating effect of morphine. The limbic dopaminergic neurones are more easily affected by both acute and chronic morphine treatment than the striatal ones.     

A comparative study of the effects of reserpine and p-chlorophenylalanine on morphine analgesia in mice

The potentiating effect of reserpine on morphine analgesia in mice was studied by means of the hot plate method and compared with the anti-analgesic action produced by p-chlorophenylalanine (p-Clphe) in the same strain of mice. The administration of DOPA or 5-hydroxytryptophan did not alter the potentiation, while the combination of both monoamine precursors markedly increased the morphine effect in reserpine-treated mice. p-Clphe showed no significant ability to antagonize the potentiating effect of reserpine. -Methyl tyrosine attenuated the analgesic effect of morphine in both untreated and reserpinized mice. These results suggest that the enhanced effect of morphine observed in reserpinized mice is not mediated by the depletion of brain monoamines produced by reserpine.