Nitric oxide synthesis in the CNS, endothelium and macrophages differs in its sensitivity to inhibition by arginine analogues

1 The purpose of this study was to characterize the effects of N0-nitro-L-arginine methyl ester (L-NAME) on the perfusion rate/pressure relations, and on the pressor responses induced to cirazoline and KC1 in isolated, perfused mesenteric arterial beds from normotensive and spontaneously hypertensive rats. 2 The basal perfusion pressure of arterial beds perfused with either physiological salt solution (PSS) or PSS containing 1% polyvinylpyrrolidone increased as the perfusion rate increased. L-NAME, in concentrations up to 100 .lM, failed to alter the basal pressure regardless of the perfusion rate and viscosity; however, at 5 gM, it potentiated cirazoline-induced vasoconstriction at each of the perfusion rates. 3 L-NAME but not D-NAME caused a leftward shift of cirazoline concentration-response curves with a marked increase in the maximal response. The potentiating action of L-NAME was abolished in arterial beds perfused with a Ca2"-free physiological salt solution and also in beds denuded of endothelium by an infusion of distilled water for 5 min. 4 In endothelium-intact and -denuded preparations, L-NAME potentiated KCl pressor responses; the endothelium-independent potentiation of KCl pressor activity was stereospecific, time-independent and was not prevented by the presence of dexamethasone (0.511M) in the perfusion medium. However, L-NAME failed to potentiate vasoconstriction obtained to KCl in arterial beds denervated by cold storage (4-5°C) for 2 days. 5 The absence of K+ in the perfusate did not inhibit the ability of L-NAME to potentiate aadrenoceptor-mediated pressor responses, and nor did L-NAME inhibit KCl-induced vasodilatation in preconstricted arteries. It was thus concluded that L-NAME does not affect Na+/K+-ATPase activity. 6 No differences in the potentiating ability of L-NAME on either cirazoline-or KCl-mediated pressor responses were apparent between normotensive Sprague Dawley (SD), Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. 7 Our data thus provide evidence that: the presence of a vasoconstrictor is required for basal nitric oxide (NO) release in the mesenteric arterial bed from either normotensive or spontaneously hypertensive rats; L-NAME causes potentiation of cirazoline-and KCl-induced vasoconstriction respectively by inhibiting endothelial and neuronal NO synthase(s). Furthermore, our data indicate that NO synthase activity is not impaired in the mesenteric arterial bed of spontaneously hypertensive rats.

Section: Discussionmentioning

confidence: 99%

The effects of perfusion rate and N^G‐nitro‐L‐arginine methyl ester on cirazoline‐ and KCl‐induced responses in the perfused mesenteric arterial bed of rats

Adeagbo¹,

Tabrizchi²,

Triggle³

1994

“…However, the most commonly used inhibitors of NOS, namely NG-methyl-L-arginine (L-NMA), NG-nitro-L-arginine (L-NOARG) and its methyl ester, L-NAME, inhibit ecNOS at least as strongly as they inhibit iNOS (Gross et al, 1990;1991;Lambert et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

Spontaneous rearrangement of aminoalkylisothioureas into mercaptoalkylguanidines, a novel class of nitric oxide synthase inhibitors with selectivity towards the inducible isoform

Southan

Zingarelli

O’Connor

et al. 1996

1 The generation of nitric oxide (NO) from L-arginine by NO synthases (NOS) can be inhibited by guanidines, amidines and S-alkylisothioureas. Unlike most L-arginine based inhibitors, however, some guanidines and S-alkylisothioureas, in particular aminoethylisothiourea (AETU), show selectivity towards the inducible isoform (iNOS) over the constitutive isoforms (endothelial, ecNOS and brain isoform, bNOS) and so may be of therapeutic benefit. In the present study we have investigated the effects of AETU and other aminoalkylisothioureas on the activities of iNOS, ecNOS and bNOS. 2 AETU, aminopropylisothiourea (APTU) and their derivatives containing alkyl substituents on one of the amidino nitrogens, potently inhibit nitrite formation by immunostimulated J774 macrophages (a model of iNOS activity) with EC50 values ranging from 6-30 yM (EC" values for N0-methyl-L-arginine (L-NMA) and N0-nitro-L-arginine were 159 and > 1000 yM, respectively). The inhibitory effects of these aminoalkylisothioureas (AATUs) were attentuated by L-arginine in the incubation medium, indicating that these agents may compete with L-arginine for its binding site on NOS. 3 The above AATUs undergo chemical conversion in neutral or basic solution (pH 7 or above) as indicated by (1) the disappearance of AATUs from solution as measured by h.p.l.c., (2) the generation of free thiols not previously present and (3) the isolation of species (as picrate and flavianate salts) from neutral or basic solutions of AATUs that are different from those obtained from acid solutions. 4 Mercaptoalkylguanidines (MAGs) were prepared and shown to be potent inhibitors of iNOS activity with EC50s comparable to those of their isomeric AATUs. 5 These findings suggest that certain AATUs exert their potent inhibitory effects through intramolecular rearrangement to mercaptoalkylguanidines (MAGs) at physiological pH. Those AATUs not capable of such rearrangement do not exhibit the same degree of inhibition of iNOS. 6 In contrast to their potent effects on iNOS, some AATUs and MAGs were 20-100 times weaker than NG-methyl-L-arginine and N0-nitro-L-arginine as inhibitors of ecNOS as assessed by their effects on the conversion of L-arginine to L-citrulline in homogenates of bovine endothelial cells and by their pressor effects in anaesthetized rats. Thus mercaptoalkylguanidines represent a new class of NOS inhibitors with preference towards iNOS. 7 AETU and mercaptoethylguanidine (MEG), when given as infusions, gave slight decreases in MAP in control rats. However, infusions of AETU or MEG to endotoxin-treated rats caused an increase in MAP and restored 80% of the endotoxin-induced fall in MAP.8 High doses of MEG (30-60 mg kg-') caused a decrease in MAP of normal rats. This depressor effect may be a consequence of the in vivo oxidation of MEG to the disulphide, guanidinoethyldisulphide (GED), which caused pronounced, transient hypotensive responses in anaesthetized rats and caused endothelium-independent vasodilator responses in precontracted rat aortic rings in vitro. 9 In som...

“…L-NAME is widely used to inhibit the constitutively expressed endothelial and brain NOS isoforms, and several studies indicate that the potency of these NOS inhibitors varies depending on the biological system (Rees et al, 1990;Lambert et al, 1991). The variability of NOS inhibition has been ascribed to different rates of uptake or degradation of the inhibitors (Lambert et al, 1991;Bogle et al, 1992;Lewis, 1992), but the present results suggest that the pharmacological profile of L-NAME largely depends on the capacity of particular tissues to convert the drug to the active NOS inhibitor, L-NOARG. Our data may, for example, at least partially explain the recently obtained different effects of L-NAME following acute and chronic administration to rats (Bryant et al, 1995) as well as regional differences observed in the distribution of L-NAME-sensitive and -insensitive nonadrenergic non-cholinergic relaxations in cat airway (Takahashi et al, 1995).…”

mentioning

confidence: 99%

Inhibition of nitric oxide synthesis by N^G‐nitro‐L‐arginine methyl ester (L‐NAME): requirement for bioactivation to the free acid, N^G‐nitro‐L‐arginine

Pfeiffer

Leopold

Schmidt

et al. 1996

212

125

1 The L-arginine derivatives N0-nitro-L-arginine (L-NOARG) and NG-nitro-L-arginine methyl ester (L-NAME) have been widely used to inhibit constitutive NO synthase (NOS) in different biological systems. This work was carried out to investigate whether L-NAME is a direct inhibitor of NOS or requires preceding hydrolytic bioactivation to L-NOARG for inhibition of the enzyme. 2 A bolus of L-NAME and L-NOARG (0.25 Mmol) increased coronary perfusion pressure of rat isolated hearts to the same extent (21 +0.8 mmHg; n = 5), but the effect developed more rapidly following addition of L-NOARG than L-NAME (mean half-time: 0.7 vs. 4.2 min). The time-dependent onset of the inhibitory effect of L-NAME was paralleled by the appearance of L-NOARG in the coronary effluent. 3 Freshly dissolved L-NAME was a 50 fold less potent inhibitor of purified brain NOS (mean IC50 = 70 gM) than L-NOARG (IC50= 1.4 giM), but the apparent inhibitory potency of L-NAME approached that of L-NOARG upon prolonged incubation at neutral or alkaline pH. H.p.l.c. analyses revealed that NOS inhibition by L-NAME closely correlated with hydrolysis of the drug to L-NOARG. 4 Freshly dissolved L-NAME contained 2% of L-NOARG and was hydrolyzed with a half-life of 365 + 11.2 min in buffer (pH 7.4), 207 ± 1.7 min in human plasma, and 29 + 2.2 min in whole blood (n = 3 in each case). When L-NAME was preincubated in plasma or buffer, inhibition of NOS was proportional to formation of L-NOARG, but in blood the inhibition was much less than expected from the rates of L-NAME hydrolysis. This was explained by accumulation of L-NOARG in blood cells. 5 These results suggest that L-NAME represents a prodrug lacking NOS inhibitory activity unless it is hydrolyzed to L-NOARG. Bioactivation of L-NAME proceeds at moderate rates in physiological buffers, but is markedly accelerated in tissues such as blood or vascular endothelium.