Daryl D. Rees

Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo

1 Three analogues of l‐arginine were characterized as inhibitors of endothelial nitric oxide (NO) synthase by measuring their effect on the endothelial NO synthase from porcine aortae, on the vascular tone of rings of rat aorta and on the blood pressure of the anaesthetized rat. 2 NG‐monomethyl‐l‐arginine (l‐NMMA), N‐iminoethyl‐l‐ornithine (l‐NIO) and NG‐nitro‐l‐arginine methyl ester (l‐NAME; all at 0.1–100 μm) caused concentration‐dependent inhibition of the Ca2+‐dependent endothelial NO synthase from porcine aortae. 3 l‐NMMA, l‐NIO and l‐NAME caused an endothelium‐dependent contraction and an inhibition of the endothelium‐dependent relaxation induced by acetylcholine (ACh) in aortic rings. 4 l‐NMMA, l‐NIO and l‐NAME (0.03–300 mg kg−1, i.v.) induced a dose‐dependent increase in mean systemic arterial blood pressure accompanied by bradycardia. 5 l‐NMMA, l‐NIO and l‐NAME (100 mg kg−1, i.v.) inhibited significantly the hypotensive responses to ACh and bradykinin. 6 The increase in blood pressure and bradycardia produced by these compounds were reversed by l‐arginine (30–100 mg kg−1 i.v.) in a dose‐dependent manner. 7 All of these effects were enantiomer specific. 8 These results indicate that l‐NMMA, l‐NIO and l‐NAME are inhibitors of NO synthase in the vascular endothelium and confirm the important role of NO synthesis in the maintenance of vascular tone and blood pressure.

L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation

The formation of nitric oxide (NO) from L-arginine by vascular endothelial cells and its relationship to endothelium-dependent relaxation of vascular rings was studied. The release of NO, measured by bioassay or chemiluminescence, from porcine aortic endothelial cells stimulated with bradykinin was enhanced by infusions of L-, but not D-arginine. The release of 15NO, determined by high resolution mass spectrometry, from L-guanidino 15N (99%) arginine was also observed, indicating that NO is formed from the terminal guanidino nitrogen atom(s) of L-arginine. L-NG-monomethyl arginine (L-NMMA), but not D-NMMA, inhibited both the generation of NO by endothelial cells in culture and the endothelium-dependent relaxation of rabbit aortic rings. Both these effects were reversed by L-arginine. These data indicate that L-arginine is the physiological precursor for the formation of NO which mediates endothelium-dependent relaxation.

A specific inhibitor of nitric oxide formation from l-arginine attenuates endothelium-dependent relaxation

1 The role of l‐arginine in the basal and stimulated generation of nitric oxide (NO) for endothelium‐dependent relaxation was studied by use of NG‐monomethyl l‐arginine (l‐NMMA), a specific inhibitor of this pathway. 2 l‐Arginine (10–100 μm), but not d‐arginine (100 μm), induced small but significant endothelium‐dependent relaxations of rings of rabbit aorta. In contrast, l‐NMMA (1–300 μm) produced small, endothelium‐dependent contractions, while its enantiomer NG‐monomethyl‐d‐arginine (d‐NMMA; 100 μ) had no effect. 3 l‐NMMA (1–300 μm) inhibited endothelium‐dependent relaxations induced by acetylcholine (ACh), the calcium ionophore A23187, substance P or l‐arginine without affecting the endothelium‐independent relaxations induced by glyceryl trinitrate or sodium nitroprusside. 4 The inhibition of endothelium‐dependent relaxation by l‐NMMA (30 μm) was reversed by l‐arginine (3–300 μm) but not by d‐arginine (300 μm) or a number of close analogues (100 μm). 5 The release of NO induced by ACh from perfused segments of rabbit aorta was also inhibited by l‐NMMA (3–300 μm), but not by d‐NMMA (100 μm) and this effect of l‐NMMA was reversed by l‐arginine (3–300 μm). 6 These results support the proposal that l‐arginine is the physiological precursor for the basal and stimulated generation of NO for endothelium‐dependent relaxation.

S‐nitroso‐glutathione inhibits platelet activation in vitro and in vivo

1 The effect of S‐nitroso‐glutathione (GSNO), a stable nitrosothiol, on platelet activation was examined in vitro and in vivo. 2 The adhesion of human platelets to fibrillar collagen and human endothelial cell monolayers was inhibited by GSNO. 3 GSNO caused a concentration‐dependent inhibition of collagen‐induced platelet aggregation in vitro and decreased ADP‐induced aggregation in the conscious rat. 4 Inhibition of platelet aggregation in vitro correlated with the increase in intraplatelet cyclic GMP levels. 5 The release of NO from GSNO was enhanced by platelet lysate, native glutathione and ascorbate. 6 The results show that GSNO is a carrier of NO and therefore has pharmacological activity as an inhibitor of platelet activation.

Nitric oxide and the haemodynamic profile of endotoxin shock in the conscious mouse

The release of cytokines following administration of endotoxin and the contribution of nitric oxide (NO) to the subsequent haemodynamic profile were investigated in the conscious mouse. Administration of endotoxin (E. Coli, 026:B6, 12.5 mg kg−1, i.v.) elevated the concentration of tumour necrosis factor‐α (TNF‐α) in the plasma within 0.5 h, reaching a maximum at 2 h and returning to control concentrations by 4 h. In addition, the concentration of interleukin‐6 (IL‐6) in the plasma was also elevated within 1 h, reaching a maximum at 3 h and remaining elevated throughout the 12 h of study. Endotoxin (12.5 mg kg−1, i.v.) induced the expression of a Ca2+‐independent (inducible) NO synthase in the mouse heart and elevated the concentrations of nitrite and nitrate in the plasma within 4 h, reaching a maximum at 12 h. This was accompanied by a progressive fall in blood pressure over the same period. The vasopressor effect of noradrenaline (0.5–4 μg kg−1 min−1, i.v.) administered as a continuous infusion was significantly attenuated 7 h after endotoxin (12.5 mg kg−1, i.v). The NO synthase inhibitor NG‐monomethyl‐L‐arginine HCl (L‐NMMA; 1–10 mg kg−1, i.v. bolus) reversed the fall in blood pressure when administered 7 h after endotoxin (12.5 mg kg−1, i.v.). In an attempt to maintain a constant blood concentration, L‐NMMA was administered as a continuous infusion (10 mg kg−1 h−1, i.v.), beginning 4 h after a lower dose of endotoxin (6 mg kg−1, i.v.). Such treatment prevented the fall in blood pressure and the elevation of nitrite and nitrate in the plasma throughout the 18 h of observation. The fall in blood pressure following endotoxin (3 mg kg−1, i.v.) was significantly reduced throughout the 18 h of observation in homozygous mutant mice lacking the inducible NO synthase. In summary, we have developed a model of endotoxin shock in the conscious mouse in which an overproduction of NO by the inducible NO synthase is associated with the haemodynamic disturbances. This model, which exhibits many of the characteristics of septic shock in man, will enable the study of the pathology of this condition in more detail and aid the investigation of potential therapeutic agents both as prophylactics and, more importantly, as treatments.

The L-arginine : nitric oxide pathway

The L-arginine: nitric oxide pathway is widely recognized as an important regulator of cell function and communication in a variety of physiological and pathophysiological situations. Recent advances in the biochemistry and molecular biology of nitric oxide synthases have contributed significantly to our understanding of the regulation of nitric oxide synthesis in health and disease. This pathway has been implicated in the pathogenesis of septic shock, hypertension, and atherosclerosis as well as in the antihypertensive action of converting enzyme inhibitors. Progress in this field, which spans the cardiovascular, immune, and nervous systems, has been rapid, and its full potential is yet to be realized.

Immunological development and cardiovascular function are normal in annexin VI null mutant mice

ABSTRACT Annexins are calcium-binding proteins of unknown function but which are implicated in important cellular processes, including anticoagulation, ion flux regulation, calcium homeostasis, and endocytosis. To gain insight into the function of annexin VI, we performed targeted disruption of its gene in mice. Matings between heterozygous mice produced offspring with a normal Mendelian pattern of inheritance, indicating that the loss of annexin VI did not interfere with viability in utero. Mice lacking annexin VI reached sexual maturity at the same age as their normal littermates, and both males and females were fertile. Because of interest in the role of annexin VI in cardiovascular function, we examined heart rate and blood pressure in knockout and wild-type mice and found these to be identical in the two groups. Similarly, the cardiovascular responses of both sets of mice to septic shock were indistinguishable. We also examined components of the immune system and found no differences in thymic, splenic, or bone marrow lymphocyte levels between knockout and wild-type mice. This is the first study of annexin knockout mice, and the lack of a clear phenotype has broad implications for current views of annexin function.

GW274150 and GW273629 are potent and highly selective inhibitors of inducible nitric oxide synthase in vitro and in vivo

1 GW274150 ([2‐[(1‐iminoethyl) amino]ethyl]‐L‐homocysteine) and GW273629 (3‐[[2‐[(1‐iminoethyl)amino]ethyl]sulphonyl]‐L‐alanine) are potent, time‐dependent, highly selective inhibitors of human inducible nitric oxide synthase (iNOS) vs endothelial NOS (eNOS) (>100‐fold) or neuronal NOS (nNOS) (>80‐fold). GW274150 and GW273629 are arginine competitive, NADPH‐dependent inhibitors of human iNOS with steady state Kd values of <40 and <90 nM, respectively. 2 GW274150 and GW273629 inhibited intracellular iNOS in J774 cells in a time‐dependent manner, reaching IC50 values of 0.2±0.04 and 1.3±0.16 μM, respectively. They were also acutely selective in intact rat tissues: GW274150 was >260‐fold and 219‐fold selective for iNOS against eNOS and nNOS, respectively, while GW273629 was >150‐fold and 365‐fold selective for iNOS against eNOS and nNOS, respectively. 3 The pharmacokinetic profile of GW274150 was biphasic in healthy rats and mice with a terminal half‐life of ∼6 h. That of GW273629 was also biphasic in rats, producing a terminal half‐life of ∼3 h. In mice however, elimination of GW273629 appeared monophasic and more rapid (∼10 min). Both compounds show a high oral bioavailability (>90%) in rats and mice. 4 GW274150 was effective in inhibiting LPS‐induced plasma NOx levels in mice with an ED50 of 3.2±0.7 mg kg−1 after 14 h intraperitoneally (i.p.) and 3.8±1.5 mg kg−1 after 14 h when administered orally. GW273629 showed shorter‐lived effects on plasma NOx and an ED50 of 9±2 mg kg−1 after 2 h when administered i.p. 5 The effects of GW274150 and GW273629 in vivo were consistent with high selectivity for iNOS, as these inhibitors were of low potency against nNOS in the rat cerebellum and did not cause significant effects on blood pressure in instrumented mice.

Nitric Oxide and the Regulation of Blood Pressure in the Hypertension-Prone and Hypertension-Resistant Sabra Rat

We examined the role of nitric oxide (NO) in the inherited resistance or susceptibility to hypertension in the Sabra hypertension-prone (SBH) and hypertension-resistant (SBN) rat. Basal mean arterial blood pressure was significantly greater in SBH than in SBN rats. Phenylephrine elevated blood pressure to a similar extent in both substrains, whereas the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) had a greater pressor effect in SBN rats. The vasoconstrictor potency of phenylephrine was significantly higher in endothelium-intact aortic rings from the SBH rat, whereas the vasoconstrictor potency of L-NMMA was higher in those from the SBN substrain. Acetylcholine-induced endothelium-dependent relaxation was greater in aortic rings from SBN rats. The vasodilator potency of glyceryl trinitrate was significantly higher in aortic rings from SBH rats and was enhanced after endothelium removal. Both the activity of calcium-dependent NO synthase from aortic endothelial cells and the basal concentration of nitrite/nitrate in plasma were significantly greater in SBN than in SBH rats. In normotensive Wistar rats, basal mean arterial blood pressure, the pressor effect of L-NMMA, endothelial NO synthase activity, and plasma nitrite/ nitrate concentrations were all between the values in SBH and SBN rats. These results indicate that a decrease in NO generation plays a role in the susceptibility of SBH rats to hypertension. Furthermore, the resistance to hypertension in the SBN strain may be related to increased NO generation.

Lack of correlation between the observed stability and pharmacological properties of S-nitroso derivatives of glutathione and cysteine-related peptides.

S-Nitrosothiols (RSNOs) have been widely studied as donors of nitric oxide. In general, RSNOs are considered to be somewhat unstable; however, they are both potent vasodilators and inhibitors of platelet aggregation. In order to improve our understanding of the factors that determine the biological activity of RSNOs, the chemical stability and pharmacological activity of a series of RSNOs was determined. Results show that millimolar solutions of S-nitrosocysteine (SNOCys) and S-nitroso-L-cysteinylglycine (SNOCysGly) were the least stable, whereas S-nitroso-3-mercaptopropionic acid (SNOPROPA) and S-nitroso-N-acetyl-L-cysteine (SNONAC) were the most stable of the compounds tested. Recent evidence suggests that RSNOs, such as SNONAC, are as unstable as SNOCys at micromolar concentrations. The decomposition of certain RSNOs is catalysed by trace amounts of copper (II) ions, with this phenomenon being particularly evident for SNOCys and SNOCysGly. The decomposition of the more stable RSNOs, including S-nitroso-L-glutathione (SNOGSH) and L-gamma-glutamyl-L-cysteine (SNOGluCys), were not as sensitive to copper ions. The decomposition of the stable RSNO, SNOGSH, was more rapid in the presence of excess thiol, whereas the decay of the unstable RSNO, SNOCys, was reduced with added thiol. All RSNOs tested inhibited platelet aggregation, relaxed vascular smooth muscle, and inhibited cell growth in the nanomolar range, but their order of potency did not correlate with their chemical stability of millimolar solutions. It is apparent that the potency of an RSNO in a physiological situation will depend on the concentration of the compound present, the presence of trace metal ions such as copper, and the occurrence of transnitrosation reactions.