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     

α-Monofluoromethyldopa (MFMD) in combination withl-DOPA

Summary We have investigated the interaction of -monofluoromethyldopa (MFMD) with the effects of i.p. injectedl-DOPA (200 mg·kg^–1) on blood pressure and tissue catecholamines in normal and spontaneously hypertensive rats (SHR). MFMD 10 mg·kg^–1 (i.p.) effectively antagonizes thel-DOPA induced increase in heart dopamine (DA). This action is also seen after 15 or 50 mg·kg^–1. The accumulation of DA in the brain is very much reduced by MFMD 50 mg ·kg^–1 while after 15 or, especially, 10 mg·kg^–1 more DA is formed in the rrain than afterl-DOPA alone, probably due to the peripheral decarboxylase inhibition which presents morel-DOPA to the brain. We conclude that MFMD 10 mg ·kg^–1 gives a relatively selective peripheral inhibition of the decarboxylation ofl-DOPA and this dose combination was accordingly found to result in a reduction of blood pressure in conscious animals. This hypotensive response tol-DOPA was attenuated after MFMD 15 mg·kg^–1 and was absent after MFMD 50 mg·kg^–1. Interestingly, the hypotensive effect ofl-DOPA after MFMD 10 or 15 mg·kg^–1 was more pronounced in SHR.     

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     

Mode of action of l-DOPA on central noradrenaline mechanisms

Summary Changes in central noradrenaline receptor activity were correlated with changes in dopamine and noradrenaline concentrations in rats after treatment with l-3,4-dihydroxyphenylalanine (l-DOPA) in combination with different drugs. The noradrenaline receptor activity was tested by means of flexor reflex increase and blood pressure reduction.After depletion of the endogenous noradrenaline stores by reserpine, and inhibition of the monoamine oxidase by nialamide, the l-DOPA treatment produced increases in flexor reflex activity and in both dopamine and noradrenaline concentrations. Pretreatment with the dopamine--hydroxylase inhibitor FLA-63 revealed that both catecholamines were of importance for this functional effect. Without monoamine oxidase inhibition, the administration of l-DOPA (after inhibition of the peripheral l-DOPA decarboxylase by l--methyldopa hydrazine) did not cause any functional effect, despite almost the same accumulation of dopamine.Injection of l-DOPA (after inhibition of the peripheral decarboxylase) into rats with intact noradrenaline stores evoked increased flexor reflex activity concomitant with a small but significant disappearance of noradrenaline and reduced the mean arterial blood pressure by 24–35 mm Hg. A second dose of l-DOPA had a much smaller effect on flexor reflex activity and on blood pressure, when given 90 min–12 h after the first one. This tachyphylaxis coincided with a fractional loss of endogenous noradrenaline and was not due to desensitization of the effector cells.In conclusion, the dopamine and noradrenaline formed from l-DOPA after monoamine oxidase inhibition act directly on the central noradrenaline receptors. Without monoamine oxidase inhibition, the l-DOPA treatment elicits a central noradrenaline receptor activation via release of endogenous noradrenaline, presumably by displacement of a small noradrenaline store with dopamine.     

Effects of blood pressure changes on the catecholamine release in the locus coeruleus of cats anaesthetized with pentobarbital or chloralose

Summary Effects of carotid occlusion and drugs applied intravenously on the release of endogenous catecholamines in the locus coeruleus of cats anaesthetized with pentobarbital or chloralose were investigated. The locus coeruleus was superfused bilaterally with artificial cerebrospinal fluid through push-pull cannulae inserted stereotaxically. Dopamine, noradrenaline and in some experiments also adrenaline were determined radioenzymatically in the superfusate.Under pentobarbital anaesthesia, a bilateral carotid occlusion increased the release rate of noradrenaline in the locus coeruleus, while the release of dopamine was decreased. These changes were due to the fall of blood pressure in the carotid sinus caused by the occlusion. Loading of baroreceptors by elevating blood pressure with phenylephrine (10 g·kg^–1·min^–1, i.v. infusion) was accompanied by a decreased release of noradrenaline in the locus coeruleus. This decrease in noradrenaline release was not detected in the caudal aspect of the locus coeruleus. Under chloralose anaesthesia, phenylephrine diminished the release rate of noradrenaline to about the same extent as under pentobarbital anaesthesia. The release rate of adrenaline was also decreased. A prolonged infusion of phenylephrine led to a prolonged pressor response associated with a sustained decrease in the noradrenaline release rate. Intravenous injection of chlorisondamine (3 mg·kg^–1) did not change the release of noradrenaline, while dopamine release was reduced.It is concluded that the release of catecholamines in the locus coeruleus is influenced by signals originating from peripheral baroreceptors. The influences are similar under pentobarbital and chloralose anaesthesia. Noradrenergic neurons responding to haemodynamic signals are not uniformly distributed within the locus coeruleus. It is suggested that noradrenergic and possibly dopaminergic and adrenergic neurons of the locus coeruleus are involved in the baroreceptor reflex, thus contributing to central homeostasis of blood pressure.     

Ramipril prevents the detrimental sequels of chronic NO synthase inhibition in rats: hypertension,cardiac hypertrophy and renal insufficiency

Inhibition of the angiotensin converting enzyme (ACE) with ramipril was studied in male Wistar rats during long-term inhibition of nitric oxide (NO) synthase by N^G-nitro-l-arginine methyl ester (l-NAME). Chronic treatment with l-NAME in a dose of 25 mg/kg per day over 6 weeks caused myocardial hypertrophy and a significant increase in systolic blood pressure (245 ± 16 mmHg) as compared to controls (155+4 mmHg). Animals receiving simultaneously l-NAME and ramipril were protected against blood pressure increase and partially against myocardial hypertrophy. L-NAME caused a significant reduction in glomerular filtration rate (GFR: 2.56+0.73 ml·kg^–1·min^–1) and renal plasma flow (RPF: 6.93±1.70ml·kg^–1·min^–1) as compared to control (GFR: 7.29±0.69, RPF: 21.36±2.33ml·kg^–1·min^–1). Addition of ramipril prevented l-NAME-induced reduction in GFR and renal plasma flow. l-NAME produced an elevation in urinary protein excretion and serum creatinine and a decrease in potassium excretion which was antagonised by ramipril. L-NAME-induced increase in plasma renin activity (PRA) was further elevated with ramipril treatment. Isolated hearts from rats treated with l-NAME showed increased post-ischaemic reperfusion injuries. Compared to controls duration of ventricular fibrillation was increased and coronary flow reduced. During ischaemia the cytosolic enzymes lactate dehydrogenase and creatine kinase, as well as lactate in the venous effluent were increased. Myocardial tissue values of glycogen, ATP, and creatine phosphate were decreased, whereas lactate was increased. Coadministration of ramipril reversed these effects. l-NAME treatment reduced the cyclic GMP content in urine and renal arteries, and was not changed by additional ramipril-treatment. In the kidney hyalinosis of arterioles and of glomerular capillaries, as well as mesangial expansion and tubular atrophies seen after long-term inhibition of NO synthase were reduced by coadministration of ramipril. In conclusion, long-term ACE inhibition by ramipril prevented l-NAME-induced hypertension and cardiac hypertrophy, and attenuated functional and morphological changes in the kidneys. In addition, cardiac-dynamic and -metabolic deterioration induced by L-NAME was normalised by co-treatment with ramipril. Correspondence to: Max Hropot at the above address     

Improved pharmacodynamics of l-asparaginase-loaded in human red blood cells

To evaluate the modification of pharmacodynamic parameters induced by the administration of l-asparaginase loaded into red blood cells, 13 patients received a single dose of l-asparaginase internalised into the carrier. The enzyme was loaded using a reversible lysis-resealing process. The dose per patient ranged from 30 to 200 IU·kg^–1. Considerable heterogeneity occurred between patients: the level of l-asparaginase circulating after 24 h represented 47% of the total injected dose as compared to 74.8% for red blood cells (RBCs). However, the half-life of the enzyme remaining in the circulation was very similar to that of the RBC carrier, i.e. 29 days and 27 days, respectively, compared with 8–24 h for the free enzyme. Sustained elimination of plasma l-asparagine occurred, the duration of which was dependent on the injected dose. A single injection of 30·IU·kg^–1 was sufficient to eliminate plasma l-asparagine over 10 days. With 150–200 IU·kg^–1 the elimination period was extended to 50 days. These data show that the use of RBCs as carriers of l-asparaginase greatly improves the pharmacodynamic parameters of the drug.     

Ethylketocyclazocine decreases noradrenaline release and blood pressure in the rabbit at a peripheral opioid receptor

Summary Rabbits were pithed and their sympathetic outflow was stimulated electrically via the pithing rod. Arterial blood pressure, heart rate, the endogenous plasma noradrenaline level, the plasma ³H-noradrenaline clearance and the noradrenaline release rate (the rate of entry of endogenous noradrenaline into the plasma) were determined. Ethylketocyclazocine 0.1 mg kg^–1+0.02 mg kg^–1 h^–1 and 1 mg kg^–1 + 0.2 mg kg^–1 h^–1 but not 0.01 mg kg^–1+ 0.002 mg kg^–1 h^–1 decreased blood pressure, the endogenous plasma noradrenaline level and the noradrenaline release rate. The effects of ethylketocyclazocine 1 mg kg^–1+ 0.2 mg kg^–1 h^–1 were antagonized by naloxone 1 mg kg^–1 + 0.5 mg kg^–1 h^–1. Given alone, naloxone caused no change. It is concluded that ethylketocyclazocine inhibits action potential-evoked release of noradrenaline from postganglionic sympathetic neurones, and hence can lower blood pressure, by a peripheral effect, possibly mediated by opioid receptors at the terminal axons.     

A dose-response study of nitric oxide synthase inhibition in different vascular beds in man

Background and objective Nitric oxide (NO) is an almost ubiquitous messenger molecule and is implicated in several disorders. N^G monomethyl l-arginine (l-NMMA:546C88) is an inhibitor of all three NO synthases (NOS), the enzymes that catalyse the production of NO. The present study was performed to evaluate the dose-response relation of l-NMMA to improve the design and interpretation of studies in migraine sufferers and other diseases.Methods In a double-blind, placebo-controlled, cross-over design, six healthy volunteers were randomised to receive three different doses of l-NMMA (0.3 mg/kg, 1 mg/kg, 3 mg/kg) or placebo (5% dextrose) intravenously (iv) over 5 min on four different days. On a fifth study day, in an open design, the same subjects received l-NMMA in the dose 6 mg/kg iv over 15 min. The effect of l-NMMA on the maximal mean blood velocity (V_mean) in the middle cerebral artery (MCA) (transcranial Doppler), the luminal diameter of the radial artery (high-frequency ultrasound), mean arterial blood pressure (MAP), heart rate and electrocardiogram were repeatedly followed every 5 min until 60 min after start of the infusion, then every 15 min during the following hour, and at 3 h and 4 h.Results Inhibition of NOS had no effect on V_mean in MCA or on the diameter of the radial artery, but MAP increased and heart rate decreased dose dependently. With a dose of 6-mg/kg l-NMMA infused over a 15-min period, the maximum MAP increase was 20% 20 min after the start of l-NMMA infusion. The maximum decrease of heart rate was 24% 15 min after start of the l-NMMA infusion.Conclusion l-NMMA in a dose that caused marked changes in systemic blood pressure and heart rate had no effect on cerebral and radial arteries in man.     

Control of the release of newly synthetized 3H-5-hydroxytryptamine by nicotinic and muscarinic receptors in rat hypothalamic slices

Summary The effects of various cholinergic agonists and antagonists on the spontaneous release of newly synthetized ³H-5-HT were examined in rat hypothalamic slices. ³H-5-HT was measured in incubating medium at the end of a 30 min incubation carried out with l-³H-tryptophan in the presence of the various durgs tested. ACh (10^–5 M) in the presence of eserine (2×10^–4 M), and carbachol (10^–5 M) stimulated the release of ³H-5-HT. In contrast, oxotremorine (10^–5 M) reduced the ³H-amine release. The effect of carbachol was blocked by two nicotinic blockers, mecamylamine (10^–6 M) and d-tubocurarine (10^–6 M). It was not reduced by the muscarinic antagonists, atropine (10^–6 M) and scopolamine (10^–6 M). In fact, each of two antagonists added alone to the incubating medium enhanced ³H-5-HT release. The scopolamine (10^–6 M) stimulating effect on ³H-5-HT release was suppressed by d-tubocurarine (10^–6 M). Finally, the inhibiting effect of oxotremorine on ³H-5-HT release was not prevented by d-tubocurarine (10^–6 M) but was in the presence of atropine (10^–6 M) or scopolamine (10^–6 M).In the concentrations used in the release study, the cholinergic agonists and antagonists had no effect on the total formation of ³H-5-HT and ³H-5-HIAA from l-³H-tryptophan and on the accumulation of l-³H-tryptophan in tissues. In these concentrations, except for eserine, they did not affect the uptake of exogenous ³H-5-HT in hypothalamic synaptosomes (P₂ fraction).These results suggest that cholinergic receptors of the muscarinic and nicotinic type are involved in the control of ³H-5-HT release; since the stimulation of the muscarinic and nicotonic cholinergic receptors resulted in an inhibition and an activation of ³H-5-HT release, respectively. As in the case of peripheral noradrenergic and central dopaminergic neurons the cholinergic receptors could be localized on serotoninergic terminals.     

Stimulus-evoked release of tritiated monoamines from rat periaqueductal gray slices in vitro and its receptor-mediated modulation

Summary The periaqueductal gray is a brain region of considerable interest. It is innervated by monoamine-containing neurons as well as by a variety of peptidergic fiber systems, and it participates in the regulation of various functions. Virtually nothing is known about monoamine release in the periaqueductal gray and its receptor-mediated modulation. We therefore studied the release of radioactivity from periaqueductal gray slices preloaded with tritriated monoamines, using an in vitro superfusion method.The release of radioactivity from superfused periaqueductal gray slices after preloading of the tissue with [³H]noradrenaline increased upon electrical stimulation in a frequency-dependent manner. The stimulus-evoked release of radioactivity was Ca²⁺-dependent. Clonidine reduced and yohimbine enhanced the release. The inhibition curve for the effect of clonidine was shifted to the right in the presence of 10^–6 M yohimbine. While phenylephrine, isoprenaline, SK&F 38393, quinpirole, carbachol, [Arg⁸]vasopressin, -MSH and ACTH-(1-24), at a concentration of 10^–6 M, did not influence the electrically evoked release of radioactivity, [Leu⁵]enkephalin reduced it. The selective -opioid receptor agonists [d-Ala²,NMePhe⁴,Gly-ol⁵]enkephalin and [d-Arg²,Lys⁴]-dermorphin-(1–4)-amide reduced the release of radioactivity, whereas the selective opioid receptor agonist [d-Pen²,d-Pen⁵]enkephalin and the selective K opioid receptor agonist U-69593 had no effect. In the presence of naloxone, which by itself had no effect on the release of radioactivity, the effect of [d-Arg²,Lys⁴]dermorphin-(1–4)-amide was abolished. These results show that the release of noradrenaline from periaqueductal gray slices is via a Ca²⁺-dependent. exocytotic process, and that it is modulated through ₂-adrenoceptors as well as via -opioid receptors. Though the overflow of radioactivity from slices preloaded with [3H]dopamine in the presence of desipramine was measurable, there are reasons to assume that we are dealing here with the release of tritiated catecholamines from a population of nerve endings consisting of noradrenergic and dopaminergic terminals.The release of radioactivity from periaqueductal gray slices preloaded with [³H]5-hydroxytryptamine upon elevation of the K⁺ concentration in the superfusion medium was much more pronounced than that induced by electrical stimulation. The K⁺-evoked release of radioactivity was almost completely abolished in the absence of Cat²⁺; showing that the release is via a Ca²⁺-dependent process. 5-Hydrotryptamine reduced the K⁺-evoked release of radioactivity in a concentration-dependent manner.Some of these data were presented at the XIth International Congress of Pharmacology, 1–6 July 1990, Amsterdam, The Netherlands (Eur J Pharmacol 183:408) Send offprint requests to D. H. G. Versteeg at the above address     

Analysis of the potentiating action of NG-nitro-l-arginine on the contraction of the dog temporal artery elicited by transmural stimulation of noradrenergic nerves

Summary Dog temporal artery strips without endothelium responded to transmural electrical stimulation with a contraction which was potentiated by N^G-nitro-l-arginine (l-NNA). The noradrenaline-induced contraction and the release of 3H-noradrenaline were not affected. The stimulation-induced contraction was reversed to a relaxation by phentolamine. The relaxation was not influenced by timolol and atropine but inhibited by l-NNA; l-arginine abolished the inhibition. Transmural stimulation released NO_x from the arteries, the release being abolished by l-NNA. Potentiation by l-NNA of the neurally-induced contraction appears to be due to elimination of NO produced by non-adrenergic, non-cholinergic vasodilator nerve activation. Send offprint requests to N. Toda at the above address     

Neurotensin and its amide analogue [Gln4]-neurotensin: effects on brain monoamine turnover

Summary Intracerebroventricularly administered neurotensin and [Gln⁴]-neurotensin (50–200 g) increased the formation of Dopa in different brain regions of rats after inhibition of the aromatic l-amino acid decarboxylase. For both neuropeptides these increases were dose dependent (20–150%). In the corpus striatum [Gln⁴]-neurotensin was twice as active as neurotensin and it tended to be more active also in other brain regions. The brain tyrosine concentrations were also increased. [Gln⁴]-neurotensin (100–200 g) following inhibition of the aromatic l-amino acid decarboxylase, increased the accumulation of 5-hydroxytryptophan in all brain regions by 30–60%. In contrast, neurotensin was completely inactive. In both cases the brain tryptophan concentrations were increased. Both neurotensin and [Gln⁴]-neurotensin also accelerated the disappearance of dopamine, noradrenaline and 5-hydroxytryptamine after inhibition of monoamine synthesis. These results show an increased brain monoamine turnover induced by both neuropeptides.     

Protriptyline induced inhibition of the in vivo 3H-noradrenaline synthesis from 3H-l-Dopa in the rat brain

Summary ³H-l-Dopa was given intraperitoneally, after a peripheral decarboxylase inhibitor Ro 4-4602 (50 mg/kg), to male Wistar rats and the effect of protriptyline pretreatment (10 mg/kg, 30 min) on the formation and metabolism of the brain ³H-catecholamines, dopamine and noradrenaline and all their metabolites was investigated.Protriptyline produced a strong decrease of labelled noradrenaline and its metabolites normetanephrine, free and conjugated 3-methoxy-4-hydroxyphenyl-eneglycol and 3,4-dihydroxyphenyleneglycol 60 and 120 min after ³H-l-Dopa. ³H-noradrenaline was also decreased 30 and 45 min after ³H-l-Dopa. In rats and mice the pretreatment with protriptyline (10 mg/kg, 30 min) induced also a significant decrease in brain ³H-noradrenaline but not ³H-dopamine synthesized from ³H-l-tyrosine. Protriptyline (10 mg/kg) produced no effect on endogenous dopamine and noradrenaline in the rat or mouse brain.The present findings strongly indicate that the acute treatment with protriptyline inhibits the ³H-noradrenaline formation from ³H-l-Dopa. This effect seems most likely to be related to an interaction of protriptyline with the precursor(s) ³H-l-Dopa or ³H-dopamine at sites of ³H-noradrenaline biosynthesis.A preliminary report of the present study was presented at the Scandinavian Pharmacological Society, June 1972, Uppsala, Sweden and the VIII C.I.N.P. Congress, August 1972, Copenhagen, Denmark.     

Search for an endogenous cannabinoid-mediated effect in the sympathetic nervous system

Activation of CB₁ cannabinoid receptors by exogenous agonists causes presynaptic inhibition of neurotransmitter release from axon terminals. In the central nervous system, presynaptic CB₁ receptors can also be activated by endogenous cannabinoids (endocannabinoids) released from postsynaptic neurons. Except in the vas deferens, there is no indication of endocannabinoid-mediated presynaptic inhibition in the sympathetic nervous system. The aim of the present study was to search for such inhibition in pithed rats. Artificial sympathetic tone was established by continuous electrical stimulation of preganglionic sympathetic axons. The CB₁ cannabinoid receptor antagonist rimonabant (0.5 and 2 mg kg^–1 i.v.) did not change blood pressure, heart rate or plasma noradrenaline concentration. Since activation of G_q/11 protein-coupled receptors enhances endocannabinoid synthesis in the central nervous system, we attempted to stimulate endocannabinoid production by infusion of arginine vasopressin and phenylephrine (both activate G_q/11 protein-coupled receptors). Rimonabant (2 mg kg^–1 i.v.) did not change blood pressure, heart rate or plasma noradrenaline concentration during infusion of phenylephrine or vasopressin. In the final series of experiments we verified that an exogenous cannabinoid agonist produces sympathoinhibition. The synthetic CB₁/CB₂ receptor agonist WIN55212-2 (0.1 and 1 mg kg^–1 i.v.) markedly lowered blood pressure and plasma noradrenaline concentration in pithed rats with electrically stimulated sympathetic outflow. In contrast, in pithed rats with a pressor infusion of noradrenaline, WIN55212-2 did not change blood pressure or heart rate. The results verify that activation of peripheral presynaptic CB₁ receptors inhibits noradrenaline release from sympathetic nerve terminals. The lack of effect of the CB₁ receptor antagonist rimonabant indicates that, even under conditions favouring endocannabinoid synthesis, endocannabinoid-mediated presynaptic inhibition is not operating in the sympathetic nervous system of the pithed rat.     

Contraction and relaxation of aortas from diabetic rats: effects of chronic anti-oxidant and aminoguanidine treatments

We examined whether chronic treatment with the free radical scavengers butylated hydroxytoluene (1 g kg^–1 day^–1) and n-acetyl-l-cysteine (250 mg kg^–1 day^–1), or the inhibitor of advanced glycosylation reactions, aminoguanidine (1 g kg^–1 day^–1), could prevent the development of relaxation and contraction abnormalities in aorta from 2 month streptozotocin-diabetic rats.Diabetes caused a 24% deficit in maximal endothelium-dependent relaxation to acetylcholine for phenylephrine precontracted aortas (P < 0.01). This was unaffected by tissue-bath glucose concentration (5.5 or 40 mM), or by addition of 1 MM l-arginine. Butylated hydroxytoluene, n-acetyl-l-cysteine and aminoguanidine treatments gave substantial protection, maximum relaxation remaining in the non-diabetic range. Neither diabetes nor treatment affected endothelium-independent relaxation to glyceryl trinitrate. To test the suggestion that aminoguanidine could act as an inhibitor of constitutive nitric oxide synthase, acute aminoguanidine effects on endothelium-dependent relaxation to acetylcholine were also examined. No inhibition was noted. A modest increase in phenylephrine sensitivity with diabetes (P < 0.05) was unaffected by butylated hydroxytoluene or n-acetyl-l-cysteine, but partially prevented by aminoguanidine (P < 0.05). The data, therefore, provide evidence for the involvement of reactive oxygen species and the advanced glycosylation process particularly for impaired endothelium-dependent relaxation in experimental diabetes.     

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     

Pressor,renal and endocrine effects of l-arginine in essential hypertensives

The pressor, renal and endocrine effect of the physiological precursor of endothelial derived nitric oxide, l-arginine was compared, with a substrate inactive on nitric oxide, hypertonic d-glucose, in hypertensive patients. Ten mild-moderate essential hypertensives were assigned to either l-arginine (n–5) or d-glucose (n–5). Substances were infused over 25 min at equiosmolal rates preceded and followed by saline infusion for 25 min. Blood pressure and heart rate were monitored at 3-min intervals, while hormonal and humoral variables, inulin and paraaminohippurate clearance and electrolyte excretion were measured at the end of each period under conditions of maximal diuresis. l-arginine and d-glucose increased serum osmolality comparably and caused similar haemodilution to that with control saline. During l-arginine infusion, systolic and diastolic blood pressure decreased by 16.6% and 11%, respectively, and recovered in the postinfusion period. Heart rate, plasma renin activity, and plasma noradrenaline did not change significantly. The percent blood pressure decrement induced by l-arginine was significantly greater than that by d-glucose. Glomerular filtration rate was stable and renal plasma flow was increased by both substances. However, natriuresis, kaliuresis and chloruresis were markedly stimulated only by l-arginine, which also promoted the development of systemic acidosis, possibly as a consequence of hydrochloridric acid generated during its metabolism. Circulating insulin, atrial natriuretic peptide, growth hormone and glucagon levels were increased and plasma aldosterone was unchanged during infusion of l-arginine. Insulin was stimulated and the other hormones inhibited during infusion of d-glucose. The greater magnitude and the infusion-related time of the hypotensive action suggests a specific mechanism of action of l-arginine, independent of a changing osmolality. l-arginine-mediated hypotension occurred without evident reflexogenic sympathetic activation and was accompanied by marked natriuresis, kaliuresis and chloruresis without changes in glomerular filtration rate. Both l-arginine and d-glucose increased renal plasma flow comparably.This paper was presented in parts at the Sixth Meeting of the European Society of Hypertension (Milan, Italy, 4–7 June, 1993), the XV Congress of the European Society of Cardiology (Nice, France, 29 August–1 September 1993), and the 15th Meeting of the International Society of Hypertension (Melbourne, Australia, 20–24 March 1994).     

Adenosine and the endothelium-dependent modulation of 3H-noradrenaline release in the canine pulmonary artery

This study aimed at characterizing the influence of endothelium on noradrenaline release from the canine pulmonary artery. Tritium overflow from intact or endothelium-free vessels preloaded with 0.2 mol.1^{–1 3}H-noradrenaline was evoked by electrical stimulation (1 Hz, during 5 min) or potassium (25–100 mmol. 1^–1).The fractional release of tritium evoked by electrical stimulation was increased by removing the endothelium [from 1.7 (1.2; 2.4) to 2.7(2.3; 3.2) × 10^–5. pulse^–1, n = 10; P < 0.05]. Neither N^G-nitro-l-arginine methyl ester (l-NAME) (up to 300 mol.l^–1) nor indomethacin (up to 30 ml.l^–1), nor endothelin-1 (up to 30 nmol.l^–1), nor suramin (up to 300 mol.l^–1) changed tritium release evolved by electrical stimulation. In contrast, the selective A₁-adenosine antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (3.3-33 nmol.l^–1) concentration-dependently increased, and the selective A₁-adenosine agonist N⁶-cyclopentyladenosine (CPA) (3.3–100 nmol. l^–1) concentration-dependently decreased the evoked release of noradrenaline. Since the effects of DPCPX were observed in endothelium-intact tissues only, it may be concluded that adenosine secreted by the endothelium activates prejunctional release-inhibiting A₁-receptors. Tetraethylammonium (TEA) (3.3–33 mmol. l^–1) enhanced tritium overflow evoked by electrical stimulation more in endothelium-free than in endothelium-intact vessels, indicating that some K⁺-channel opener is involved in the inhibitory role of endothelium on noradrenaline release. Since it had been previously shown that A₁-adenosine receptors are coupled to K⁺-channels, it is suggested that adenosine may inhibit noradrenaline release through the opening of K⁺-channels.In conclusion, the results show that in the canine pulmonary artery, adenosine is a good candidate for the endothelium-dependent inhibitory factor which is responsible for the reduction of noradrenaline release evoked by electrical stimulation.     

NG-nitro-L-arginine (N5-[imino(nitroamino)methyl]-L-ornithine) impairs endothelium-dependent dilations by inhibiting cytosolic nitric oxide synthesis from l-arginine

Summary We studied the effects of the l-arginine analogue N^G-nitro-l-arginine (l-NNA), in comparison with its d-isomer (d-NNA), on endothelium-dependent dilations of rabbit femoral arteries (RFA) and on the release of endothelium-derived relaxant factor (EDRF) from native and cultured endothelial cells. In addition, we examined the effects of l- and d-NNA on the l-arginine- and NADPH-dependent synthesis of nitric oxide (NO) in the cytosol of porcine aortic endothelial cells. l-NNA enhanced the noradrenaline-induced contraction of endothelium-intact, but not of endothelium-denuded segments of RFA, indicating an inhibition of basal EDRF release. l-NNA also inhibited significantly the endothelium-dependent dilations to acetylcholine (ACh). Both effects of l-NNA were attenuated by l-arginine. l-NNA rapidly inhibited the release of EDRF from cultured and native endothelial cells stimulated with thimerosal or ACh. l-NNA concentration-dependently and reversibly antagonized the l-arginine- and NADPH-dependent activation of a purified soluble guanylate cyclase (GC) by cytosol from.Sreshly harvested porcine aortic endothelial cells, suggesting a direct competition between l-NNA a l-arginine at the level of endothelial NO-synthesis. d-NNA was ineffective in all instances. These results prove l-NNA to be a stereospecific inhibitor of the cytosolic NO formation from l-arginine in endothelial cells. Therefore, l-NNA will be a useful tool to elucidate the molecular mechanism of mammalian NO synthesis. Send offprint requests to A. Mdlsch at the above address