Philip K. Moore

l-NG-nitro arginine (l-NOARG), a novel, l-arginine-reversible inhibitor of endothelium-dependent vasodilatation in vitro

1 The effect of l‐NG‐nitro arginine (l‐NOARG) was compared with that of l‐NG‐monomethyl arginine (l‐NMMA) on vasodilatation of the isolated aorta of the rabbit and perfused mesentery of the rat in response to acetylcholine (ACh) and sodium nitroprusside (NP). 2 l‐NOARG (1.5–100 μm) and l‐NMMA (3–100 μm) produced concentration‐related contraction of the rabbit aorta precontracted with phenylephrine (700–900 nm). Similarly, l‐NOARG (10–200 μm) and l‐NMMA (30–100 μm) elevated perfusion pressure of the noradrenaline (NA, 0.6–2.5 mm)‐preconstricted rat mesentery preparation. 3 l‐NOARG (1.5–100 μm) and l‐NMMA (3–100 μm) caused concentration‐related inhibition of the vasodilator effect of ACh (0.01–1.0 μm) on the rabbit aorta without influencing responses to NP (0.03–0.5 μm). l‐NOARG methyl ester (30 μm) also inhibited ACh‐induced vasorelaxation with similar potency to NOARG. l‐arginine (30–150 μm) but not d‐arginine (100 μm) caused graded reversal of the inhibitory effect of both l‐NOARG (15 μm) and l‐NMMA (30 μm). Complete reversal of the effect of both inhibitors was achieved with 150 μm l‐arginine. l‐Alanine (50 μm), l‐arginosuccinic acid (5 μm), l‐citrulline (50 μm), l‐methionine (50 μm) and l‐ornithine (50 μm) failed to reverse the inhibitory effect of l‐NOARG (15 μm). 4 l‐NOARG (10–200 μm) and l‐NMMA (30–100 μm) inhibited the vasodilator effect of ACh (0.006–18.0 nmol) in the rat mesentery without affecting vasodilatation due to NP (1.1–11.1 nmol). l‐Arginine (100 μm) but not d‐arginine (100 μm) produced partial reversal of the effect of l‐NOARG (30 μm) and l‐NMMA (30 μm). 5 l‐ and d‐Nα‐butyloxycarbonyl NG‐nitro arginine (100 μm) produced modest (approximately 20%) inhibition of the effect of ACh on the rabbit aorta; this effect was not reversible with l‐arginine (100 μm). l‐Nα‐monocarbobenzoxy arginine (l‐NMCA, 50 μm), l‐Nα‐NG‐dicarbobenzoxy arginine (l‐NDCA, 5 μm) and l‐NG‐tosyl arginine (50 μm) were inactive. 6 These results identify l‐NOARG as a potent, l‐arginine reversible inhibitor of endothelium‐dependent vasodilatation. The available data suggests that l‐NOARG, like l‐NMMA, inhibits endothelial nitric oxide (NO) biosynthesis.

Hydrogen Sulfide and Cell Signaling

Hydrogen sulfide (H₂S) is a gaseous mediator synthesized from cysteine by cystathionine γ lyase (CSE) and other naturally occurring enzymes. Pharmacological experiments using H₂S donors and genetic experiments using CSE knockout mice suggest important roles for this vasodilator gas in the regulation of blood vessel caliber, cardiac response to ischemia/reperfusion injury, and inflammation. That H₂S inhibits cytochrome c oxidase and reduces cell energy production has been known for many decades, but more recently, a number of additional pharmacological targets for this gas have been identified. H₂S activates K(ATP) and transient receptor potential (TRP) channels but usually inhibits big conductance Ca²(+)-sensitive K(+) (BK(Ca)) channels, T-type calcium channels, and M-type calcium channels. H₂S may inhibit or activate NF-κB nuclear translocation while affecting the activity of numerous kinases including p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), and Akt. These disparate effects may be secondary to the well-known reducing activity of H₂S and/or its ability to promote sulfhydration of protein cysteine moieties within the cell.

Hydrogen sulfide is a novel mediator of lipopolysaccharide-induced inflammation in the mouse

Hydrogen sulfide (H2S) is synthesized in the body from l‐cysteine by several enzymes including cystathionine‐γ‐lyase (CSE). To date, there is little information about the potential role of H2S in inflammation. We have now investigated the part played by H2S in endotoxin‐induced inflammation in the mouse. E. coli lipopolysaccharide (LPS) administration produced a dose (10 and 20 mg/kg ip)‐ and time (6 and 24 h)‐dependent increase in plasma H2S concentration. LPS (10 mg/kg ip, 6 h) increased plasma H2S concentration from 34.1 ± 0.7 µM to 40.9 ± 0.6 µM (n=6, P<0.05) while H2S formation from added l‐cysteine was increased in both liver and kidney. CSE gene expression was also increased in both liver (94.2±2.7%, n=6, P<0.05) and kidney (77.5±3.2%, n=6, P<0.05). LPS injection also elevated lung (148.2±2.6%, n=6, P<0.05) and kidney (78.8±8.2%, n=6, P<0.05) myeloperoxidase (MPO, a marker of tissue neutrophil infiltration) activity alongside histological evidence of lung, liver, and kidney tissue inflammatory damage. Plasma nitrate/nitrite (NOx) concentration was additionally elevated in a time‐ and dose‐dependent manner in LPS‐injected animals. To examine directly the possible proinflammatory effect of H2S, mice were administered sodium hydrosulfide (H2S donor drug, 14 µmol/kg ip) that resulted in marked histological signs of lung inflammation, increased lung and liver MPO activity, and raised plasma TNF‐α concentration (4.6±1.4 ng/ml, n=6). In contrast, dl‐propargylglycine (CSE inhibitor, 50 mg/kg ip), exhibited marked anti‐inflammatory activity as evidenced by reduced lung and liver MPO activity, and ameliorated lung and liver tissue damage. In separate experiments, we also detected significantly higher (150.5±43.7 µM c.f. 43.8±5.1 µM, n=5, P<0.05) plasma H2S levels in humans with septic shock. These findings suggest that H2S exhibits proinflammatory activity in endotoxic shock and suggest a new approach to the development of novel drugs for this condition.

Characterization of the novel nitric oxide synthase inhibitor 7-nitro indazole and related indazoles: antinociceptive and cardiovascular effects.

1 7‐Nitro indazole (7‐NI, 10–50 mg kg−1), 6‐nitro indazole and indazole (25–100 mg kg−1) administered i.p. in the mouse produce dose‐related antinociception in the late phase of the formalin‐induced hindpaw licking and acetic acid‐induced abdominal constriction assays. The ED50 values (mg kg−1) were as follows: 7‐NI (27.5 and 22.5), 6‐nitro indazole (62.5 and 44.0) and indazole (41.0 and 48.5) in the two assays respectively. 3‐Indazolinone, 6 amino indazole and 6‐sulphanilimido indazole (all 50 mg kg−1) were without effect. With the exception of 5‐nitro indazole (50 mg kg−1) which produced sedation, none of the other indazole derivates examined caused overt behavioural changes. 2 The antinociceptive effect of 7‐NI (25 mg kg−1, i.p.) in the late phase of the formalin‐induced hindpaw licking assay was partially (46.7 ± 16.2%, n = 18) reversed by pretreatment with l‐ but not d‐arginine (both 50 mg kg−1, i.p.). 3 The time course of 7‐NI induced antinociception in the mouse was correlated with inhibition of brain (cerebellum) nitric oxide synthase (NOS) activity. Maximum antinociceptive activity and NOS inhibition were detected 18–30 min following i.p. administration. In contrast, no antinociceptive effect or inhibition of cerebellar NOS was detected 75 min post‐injection. 4 7‐NI, 6‐nitro indazole, indazole, 3‐indazolinone and 6‐amino indazole (all 50 mg kg−1) failed to influence mean arterial pressure (MAP) over the 45 min after i.p. administration in the anaesthetized mouse. Similarly, 7‐NI (25 mg kg−1) administered i.v. in the anaesthetized rat did not increase MAP or influence the vasodepressor effect of i.v. injected acetylcholine (ACh) over the same period. 5 7‐NI (100 μm) did not influence the vasorelaxant effect of ACh (IC50, 0.2 ± 0.04 μm, cf. 0.16 ± 0.06 μm, n = 6) in phenylephrine‐precontracted rabbit aortic rings. 6 These data provide further evidence that antinociception following administration of 7‐NI in the mouse results from inhibition of central NOS activity and is not associated with inhibition of in vivo vascular endothelial cells NOS. Accordingly, 7‐NI (or a derivative thereof) may provide an alternative approach to the development of novel antinociceptive drugs.

L-NG-nitro arginine methyl ester exhibits antinociceptive activity in the mouse.

1 l‐NG‐nitro arginine methyl ester (l‐NAME, 1–75 mg kg−1) administered intraperitoneally (i.p.) elicits dose‐related antinociception in the mouse assessed by the formalin‐induced paw licking procedure. Antinociceptive activity is still present 24 h after injection. l‐NAME (75 mg kg−1, i.p.) is also antinociceptive in the acetic acid‐induced abdominal constriction and hot plate procedures. 2 l‐NAME additionally produces a dose‐related inhibition of formalin‐induced paw licking following intracerebroventricular (i.c.v., 0.1–100 μg per mouse) and oral (p.o., 75–150 mg kg−1) administration. 3 l‐Arginine (600 mg kg−1, i.p.) but not d‐arginine (600 mg kg−1) or naloxone (5 mg kg−1) reverses the antinociceptive effect of l‐NAME in the formalin test. 4 High doses of l‐NAME (37.5–600 mg kg−1) but not d‐NAME (75 mg kg−1) administered i.p. produce dose‐related increases in blood pressure of the urethane‐anaesthetized mouse whilst i.c.v. injected l‐NAME (0.1 and 100 μg per mouse) is inactive. 5 l‐NAME (75 mg kg−1, i.p.) did not inhibit oedema formation in the formalin‐injected mouse hindpaw. 6 l‐NAME (75 mg kg−1) did not produce any overt behavioural changes in treated mice and failed to influence locomotor activity or the incidence of dipping, crossing, rearing or circling behaviour assessed by a modified ‘head‐dipping’ board procedure. A high dose of l‐NAME (600 mg kg−1) reduced dipping behaviour and locomotor activity suggesting a possible sedative effect. d‐NAME (600 mg kg−1) was inactive. 7 These results suggest that l‐NAME produces an opioid‐independent and long‐lasting antinociception in the mouse most probably by a direct effect within the central nervous system.

The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite ‘scavenger’?

Hydrogen sulfide (H2S) is a well‐known cytotoxic gas. Recently it has been shown to stimulate N‐methyl‐d‐aspartate (NMDA) receptors to enhance long‐term potentiation suggesting a novel neuromodulatory role in vivo. Endogenous levels of H2S in the brain are reported to range between 10 and 160 µm. Considerably lower H2S levels are reported in the brains of Alzheimers disease (AD) patients, where levels of brain protein nitration (probably mediated by peroxynitrite) are markedly increased. Activation of NMDA receptors leads to intracellular tyrosine nitration by peroxynitrite. Because H2S and peroxynitrite are important mediators in brain function and disease, we investigated the effects of the H2S ‘donor’, sodium hydrogen sulfide (NaSH) on peroxynitrite‐mediated damage to biomolecules and to cultured human SH‐SY5Y cells. H2S significantly inhibited peroxynitrite‐mediated tyrosine nitration and inactivation of α1‐antiproteinase to a similar extent to reduced glutathione at each concentration tested (30–250 µm). H2S also inhibited peroxynitrite‐induced cytotoxicity, intracellular protein nitration and protein oxidation in human neuroblastoma SH‐SY5Y cells. These data suggest that H2S has the potential to act as an inhibitor of peroxynitrite‐mediated processes in vivo and that the potential antioxidant action of H2S deserves further study, given that extracellular GSH levels in the brain are very low.

7‐Nitro indazole, an inhibitor of nitric oxide synthase, exhibits anti‐nociceptive activity in the mouse without increasing blood pressure

7‐Nitro indazole (7‐NI) inhibits mouse cerebellar nitric oxide synthase (NOS) in vitro with an IC50 of 0.47 μm. Following i.p. administration in mice, 7‐NI (10–50 mg kg−1) produces dose‐related anti‐nociception as evidenced by an inhibition of late phase (15–30 min) but not early phase (0–5 min) hindpaw licking time following subplantar injection of formalin (10 μl, 5% v/v). The ED50 for this effect was 26 mg kg−1 (equivalent to 159.5 μmol kg−1). Similar i.p. administration of 7‐NI (20 and 80 mg kg−1) in urethane‐anaesthetized mice failed to increase MAP. Thus, 7‐NI is a novel inhibitor of NOS which exhibits selectivity for the brain enzyme. Accordingly, 7‐NI may be a useful starting point for the development of selective, centrally acting NOS inhibitors devoid of cardiovascular side effects and as a tool to study the central pharmacological effects of nitric oxide (NO).

Characterization of a Novel, Water-Soluble Hydrogen Sulfide–Releasing Molecule (GYY4137) New Insights Into the Biology of Hydrogen Sulfide

Background— The potential biological significance of hydrogen sulfide (H2S) has attracted growing interest in recent years. The aim of this study was to characterize a novel, water-soluble, slow-releasing H2S compound [morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate (GYY4137)] and evaluate its use as a tool to investigate the cardiovascular biology of this gas. Methods and Results— The acute vasorelaxant effect of drugs was assessed in rat aortic rings and perfused rat kidney in vitro and in the anesthetized rat in vivo. The chronic effect of GYY4137 on blood pressure in normotensive and spontaneously hypertensive rats was determined by tail-cuff plethysmography. GYY4137 released H2S slowly both in aqueous solution in vitro and after intravenous or intraperitoneal administration in anesthetized rats in vivo. GYY4137 caused a slow relaxation of rat aortic rings and dilated the perfused rat renal vasculature by opening vascular smooth muscle KATP channels. GYY4137 did not affect rat heart rate or force of contraction in vitro. GYY4137 exhibited antihypertensive activity as evidenced by ability to reduce NG-nitro-l-arginine methyl ester–evoked hypertension in the anesthetized rat and after chronic (14-day) administration in spontaneously hypertensive rats. Conclusions— These results identify GYY4137 as a slow-releasing H2S compound with vasodilator and antihypertensive activity. GYY4137 is likely to prove useful in the study of the many and varied biological effects of H2S. GYY4137 may also prove of therapeutic value in cardiovascular disease.

Inhibition of rat cerebellar nitric oxide synthase by 7‐nitro indazole and related substituted indazoles

1 7‐Nitro indazole (7‐NI) produces potent inhibition of rat cerebellar nitric oxide synthase (NOS) with an IC50 of 0.9 ± 0.1 μm (n = 6). NOS activity is dependent on the presence of both exogenous CaCl2 and NADPH. The inhibitory potency of 7‐NI remained unaltered in the presence of different concentrations of either CaCl2 (0.75–7.5 mm) or NADPH (0.05–5.0 mm). 2 Kinetic (Lineweaver‐Burke) analysis of the effect of 7‐NI on rat cerebellar NOS revealed that inhibition was of a competitive nature with a Ki value of 5.6 μm. The Km of cerebellar NOS with respect to L‐arginine was 2.5 μm. 3 The following indazole derivatives (IC50 values shown in parentheses, all n = 6) caused concentration‐related inhibition of rat cerebellar NOS in vitro: 6‐nitro indazole (31.6 ± 3.4 μm), 5‐nitro indazole (47.3 ± 2.3 μm), 3‐chloro indazole (100.0 ± 5.5 μm), 3‐chloro 5‐nitro indazole (158.4 ± 2.1 μm) and indazole (177.8 ± 2.1 μm). The IC50 values for 5‐amino indazole, 6‐amino indazole and 6‐sulphanilimido indazole were in excess of 1 mm; 3‐indazolinone was inactive. 4 7‐NI (10 mg kg−1) administered i.p. to rats produced 60 min thereafter a significant inhibition of NOS activity in cerebellum (31.1 ± 3.2%, n = 6), cerebral cortex (38.2 ± 5.6%, n = 6), hippocampus (37.0 ± 2.8%, n = 6) and adrenal gland (23.7 ± 3.0%, n = 6). NOS activity in olfactory bulb and stomach fundus were unchanged. 5 These results indicate that 7‐NI is a potent and competitive inhibitor of rat brain NOS in vitro and also inhibits NOS in different brain regions and in the adrenal gland in vivo. Inhibition of NOS is a characteristic property of the indazole nucleus. Nitration of the indazole ring at positions 5, 6 and 7 results in a graded increase in inhibitory potency. Indazole‐based inhibitors of NOS may prove useful tools with which to evaluate the biological roles of nitric oxide in the central nervous system.

Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide?

The aim of these experiments was to evaluate the significance of the chemical reaction between hydrogen sulphide (H2S) and nitric oxide (NO) for the control of vascular tone.