Loss of endothelium-derived nitric oxide in rabbit aorta by oxidant stress: restoration by superoxide dismutase mimetics

1. Structurally distinct superoxide dismutase (SOD) mimetics were examined for their ability to protect nitric oxide (NO) from destruction by oxidant stress in rabbit aorta.
2. These were the spin traps, PTIYO (4-phenyl-2,2,5,5-tetramethyl imidazolin-1-yloxy-5-oxide), tempol (4-hydroxy 2,2,6,6,-tetramethylpiperidine-1-oxyl) and tiron (4,5-dihydroxy-1,3-benzene-disulphonic acid), the metal salts, CuSO₄ and MnCl₂, and the metal-based agents CuDIPS (Cu (II)-[diisopropylsalicylate]₂) and MnTMPyP (Mn (III) tetrakis [1-methyl-4-pyridyl]porphyrin).
3. Oxidant stress was generated in isolated aortic rings by inactivating endogenous Cu/Zn SOD with diethyldithiocarbamate (DETCA; 60 min) either alone at 3 mM or at 0.3 mM in combination with superoxide generation using xanthine oxidase (XO; 4.8 mu ml⁻¹) and hypoxanthine (HX; 0.1 mM).
4. Acetylcholine (ACh)-induced relaxation was inhibited by DETCA (3 mM, 60 min) and was not restored by exogenous SOD (250 u ml⁻¹), suggesting the oxidant stress was intracellular. MnTMPyP (600 μM and 1 mM) and MnCl₂ (100 μM) were the only agents to reverse the blockade of ACh-induced relaxation.
5. Addition of XO/HX to DETCA (0.3 mM)-treated tissues powerfully impaired ACh-induced relaxation and exogneous SOD (250 u ml⁻¹) fully reversed the blockade, suggesting the oxidant stress was extracellular. CuDIPS (0.1–3 μM), CuSO₄ (0.3–3 μM), MnCl₂ (1–100 μM) and MnTMPyP (100–600 μM) also reversed blockade powerfully, tempol (30 μM–1 mM) and tiron (0.3–10 mM) reversed blockade weakly and PTIYO (10–300 μM) enhanced the blockade.
6. Thus, MnTMPyP was the only SOD mimetic to restore NO-dependent relaxation in conditions of both extracellular and intracellular oxidant stress. This agent may, therefore, provide a lead in the development of SOD mimetics for the treatment of pathologies associated with oxidant stress.

     

Interaction between superoxide anion and nitric oxide in the regulation of vascular endothelial function

1. Nitric oxide (NO)-mediated, endothelium-dependent vasodilator function in rat aortic smooth muscle was investigated in an in vitro model of endogenous vascular superoxide anion stress, generated by pretreatment with the Cu/Zn superoxide dismutase (SOD, EC 1.15.1.1) inhibitor, diethyldithiocarbamate (DETCA).
2. Contraction to noradrenaline (NA, 1 nM–1 μM) in endothelium-intact vessels was augmented after a 30 min pretreatment with DETCA (10 mM) followed by 30 min washout. This effect was abolished by N^G-nitro-L-arginine methyl ester (L-NAME, 0.3 mM) and removal of the endothelium and partially reversed by exogenous Cu/Zn SOD (200 u ml⁻¹).
3. Endothelium- and basal NO-dependent vasorelaxation to the phosphodiesterase (PDE) type V inhibitor ONO-1505 (4-[2-(2-hydroxyethoxy)ethylamino]-2-(1H-imidazol-1-yl)-6-methoxyquinazoline methanesulphonate) (0.1–10 μM) was inhibited after DETCA (10 mM) pretreatment. In addition, the ability of L-NAME (0.3 mM) to enhance established contractile tone was effectively absent.
4. In contrast, DETCA pretreatment did not significantly affect vasorelaxation to acetylcholine (ACh, 1 nM–3 μM) or S-nitroso-N-acetyl penicillamine (SNAP, 0.03–30 μM). However, L-NAME (0.3 mM) unmasked an inhibitory effect of DETCA pretreatment on vasorelaxation to SNAP in endothelium-intact vessels while markedly potentiating vasorelaxation to SNAP in control tissue.
5. L-NAME (0.3 mM)- and exogenous catalase (200 u ml⁻¹)-sensitive vasorelaxation to exogenous Cu/Zn SOD (200 u ml⁻¹) was greater after DETCA (10 mM) pretreatment in endothelium-intact aortic rings. This difference was abolished by catalase (200 u ml⁻¹).
6. In conclusion, tissue Cu/Zn SOD inhibition elicited a selective lesion in basal endothelial function in rat isolated aortic smooth muscle, consistent with the inactivation of basal NO by superoxide anion. The resulting leftward shift in nitrovasodilator reactivity, due to the loss of the tonic depression by basal NO, is likely to mask the inhibitory effect of superoxide anion on agonist-stimulated endothelial function and nitrovasodilator-derived NO, thereby accounting for the differential pattern of endothelial dysfunction after DETCA pretreatment.

     

Inhibition of nitrergic neurotransmission in the bovine retractor penis muscle by an oxidant stress: effects of superoxide dismutase mimetics

1. A number of superoxide dismutase (SOD) mimetics were examined both biochemically for their ability to inhibit the superoxide-catalyzed reduction of cytochrome c and nitro blue tetrazolium, and functionally for their ability to mimic authentic Cu/Zn SOD in restoring nitrergic neurotransmission in bovine retractor penis (BRP) muscle following its inhibition by oxidant stress.
2. The SOD mimetics investigated were CuSO₄, MnCl₂, CuDIPS (copper [II] [diisopropylsalicylate]₂), MnTBAP (manganese [III] tetrakis 4-benzoic acid porphyrin), MnTMPyP (manganese [III] tetrakis 1-methyl-4-pyridyl porphyrin pentachloride), tiron (4,5-dihydroxy-1,3-benzene disulphonic acid), PTIYO (4-phenyl,2,2,5,5,-tetramethyl-3-imidazolin-1-yloxy-3-oxide) and tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl).
3. The rank order of potency in inhibiting the reduction of cytochrome c was: CuSO₄⩾MnCl₂⩾CuDIPS⩾MnTMPyP>MnTBAP>tempol⩾tiron>PTIYO.
4. The requirement for EDTA (0.1 mM) prevented assessment of the activity of CuSO₄, MnCl₂ and CuDIPS in the assay involving inhibition of reduction of nitro blue tetrazolium. However, the rank order of potency for those agents which could be examined (MnTMPyP>MnTBAP>tiron⩾tempol>PTIYO) was essentially similar to that seen in the cytochrome c assay.
5. Inhibition of endogenous Cu/Zn SOD with diethyldithiocarbamate (DETCA, 3 mM, 120 min) in BRP muscle strips, followed by addition of the superoxide anion generator, LY 83583 (1 μM), resulted in almost complete abolition of nitrergic relaxation (4 Hz, 10 s).
6. Authentic Cu/Zn SOD (1–300 u ml⁻¹), CuSO₄ (0.1–300 μM), MnCl₂ (0.1–100 μM) and MnTMPyP (10–300 μM) each restored nitrergic transmission by around 50%. However, CuDIPS (0.1–30 μM), MnTBAP (0.1–100 μM), tempol (10 μM–3 mM), PTIYO (1–300 μM) and tiron (10 μM–10 mM) all failed to restore nitrergic transmission.
7. The ability of MnTMPyP to restore nitrergic neurotransmission may therefore provide a lead in the development of SOD mimetics as therapeutic agents in the treatment of neuropathies associated with oxidant stress.

     

Homocysteine as a modulator of platelet-derived growth factor action in vascular smooth muscle cells: a possible role for hydrogen peroxide

1. Homocysteine is an independent risk factor for cardiovascular disease. The mechanisms by which elevated plasma concentrations of homocysteine are related to the pathogenesis of atherosclerosis are not fully understood. Therefore, we examined the effect of homocysteine on cell replication of rat cultured vascular smooth muscle cells (VSMCs) at concentrations similar to those observed in clinical studies.
2. The incorporation of [³H]-thymidine was used as a marker of mitosis. Homocysteine (250–500 μM) was a weak mitogen as compared to platelet-derived growth factor-BB (PDGF-BB, 1 nM) and serum (10%), but it potentiated the mitogenic effect of PDGF-BB four fold at 500 μM. This enhancement of mitogenesis was blunted by the addition of the scavenging enzyme catalase or the antioxidant N-acetyl-L-cysteine.
3. Furthermore, stimulation of VSMC with homocysteine (25–500 μM) decreased the glutathione peroxidase activity of the cells to 50% of control at 500 μM. Inversely, homocysteine enhanced the superoxide dismutase (SOD) activity to 137% of control at 500 μM, but it had no effect on the catalase activity.
4. Homocysteine decreased the activity of bovine purified liver cytosolic glutathione peroxidase in a time- and dose-dependent manner. The maximum decrease was 50%.
5. In summary, homocysteine has a weak mitogenic effect on VSMC, but it dramatically enhances the mitogenic response of PDGF-BB, presumably by disturbing the activity of antioxidant enzymes.

     

Evidence that ATP acts at two sites to evoke contraction in the rat isolated tail artery

1. The site(s) at which P2-receptor agonists act to evoke contractions of the rat isolated tail artery was studied by use of P2-receptor antagonists and the extracellular ATPase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156).
2. Suramin (1 μM–1 mM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (0.3–300 μM) inhibited contractions evoked by equi-effective concentrations of α,β-methyleneATP (α,β-meATP) (5 μM), 2-methylthioATP (2-meSATP) (100 μM) and adenosine 5′-triphosphate (ATP) (1 mM) in a concentration-dependent manner. Responses to α,β-meATP and 2-meSATP were abolished, but approximately one third of the peak response to ATP was resistant to suramin and PPADS.
3. Contractions evoked by uridine 5′-triphosphate (UTP) (1 mM) were slightly inhibited by suramin (100 and 300 μM) and potentiated by PPADS (300 μM).
4. Desensitization of the P2X₁-receptor by α,β-meATP abolished contractions evoked by 2-meSATP (100 μM) and reduced those to ATP (1 mM) and UTP (1 mM) to 15±3% and 68±4% of control.
5. Responses to α,β-meATP (5 μM) and 2-meSATP (100 μM) were abolished when tissues were bathed in nominally calcium-free solution, while the peak contractions to ATP (1 mM) and UTP (1 mM) were reduced to 24±6% and 61±13%, respectively, of their control response.
6. ARL 67156 (3–100 μM) potentiated contractions elicited by UTP (1 mM), but inhibited responses to α,β-meATP (5 μM), 2-meSATP (100 μM) and ATP (1 mM) in a concentration-dependent manner.
7. These results suggest that two populations of P2-receptors are present in the rat tail artery; ligand-gated P2X₁-receptors and G-protein-coupled P2Y-receptors.

     

Evidence that both nitric oxide (NO) and a non-NO hyperpolarizing factor elicit NANC nerve-mediated relaxation in the rat isolated anococcygeus

1. Responses to electrical field stimulation (EFS; 0.5–10 Hz, 0.2 ms duration, supramaximal voltage for 20 s) of non-adrenergic, non-cholinergic, (NANC) nerves were obtained in preparations of rat anococcygeus pre-contracted with titrated concentrations of phenylephrine (0.1–1 μM) to ∼40% of their maximum contraction to phenylephrine (F_max) regardless of drug treatment.
2. With this set level of active force, NANC nerve stimulation resulted in relaxations that were maximal (peak relaxation) at 0.5–1 Hz, abolished by tetrodotoxin (1 μM) but only minimally blocked by the nitric oxide synthase (NOS) inhibitor, N^G-nitro-L-arginine, (L-NOARG; 100 μM). Furthermore, the nitric oxide (NO) scavenger, oxyhaemoglobin (HbO; 30 μM) gave no further block alone or in combination with L-NOARG (100 μM). By comparison, in preparations contracted with phenylephrine to ∼70% F_max, relaxations to NANC nerve stimulation were markedly reduced or abolished by combined treatment with L-NOARG (100 μM) and HbO (30 μM).
3. Nifedipine (0.3 μM) significantly inhibited NANC nerve-mediated relaxations, which became frequency-dependent and abolished those resistant to L-NOARG (100 μM) and HbO (30 μM).
4. These data suggest that a non-NO, hyperpolarizing factor and NO both contribute to NANC nerve-mediated inhibitory responses in the rat anococcygeus. However, responses to the non-NO factor were observed only in preparations contracted sub-maximally by a nifedipine-sensitive mechanism.

     

S-nitrosothiols and the nitrergic neurotransmitter in the rat gastric fundus: effect of antioxidants and metal chelation

1. The effects of the antioxidants ascorbic acid and α-tocopherol and of the metal chelator ethylenediaminetetraacetic acid (EDTA) were studied on relaxations in response to S-nitrosothiols, authentic nitric oxide (NO) and nitrergic non-adrenergic non-cholinergic stimulation of the rat gastric fundus.
2. The S-nitrosothiols S-nitrosocysteine (1–100 nM), S-nitrosoglutathione (0.01–3 μM) and S-nitroso-N-acetylpenicillamine (0.01–3 μM) induced concentration-dependent relaxations of the rat gastric fundus muscle strips, which were precontracted with prostaglandin F_2α. The relaxations to all S-nitrosothiols were concentration-dependently enhanced by the antioxidants ascorbic acid (0.1–3 μM) and α-tocopherol (3–30 μM) and inhibited by the metal chelator EDTA (26 μM).
3. Ascorbic acid and α-tocopherol alone did not induce a relaxation of the precontracted rat gastric fundus muscle strip. However, when ascorbic acid (1 μM) or α-tocopherol (1 μM) were injected in the organ bath 1 minute after S-nitrosoglutathione (0.1 μM) or after S-nitroso-N-acetylpenicillamine (0.1 μM), they induced an immediate, sharp and transient relaxation. This relaxation was inhibited by the superoxide generator pyrogallol (2 μM). Such a relaxation to ascorbic acid or α-tocopherol was not observed in the presence of S-nitrosocysteine (10 nM).
4. Electrical field stimulation (0.5–4 Hz) of the precontracted rat gastric fundus strips induced frequency-dependent nitrergic relaxations which were mimicked by authentic NO (3–300 nM) and by acidified sodium nitrite NaNO₂ (0.3–10 μM). Ascorbic acid (0.3–3 μM), α-tocopherol (3–30 μM) or EDTA (26 μM) did not affect the relaxations to nitrergic stimulation, NO or NaNO₂.
5. In summary, relaxations to S-nitrosothiols in the rat gastric fundus are enhanced by the antioxidants ascorbic acid and α-tocopherol and inhibited by the metal chelator EDTA. However, relaxations to nitrergic stimulation of the rat gastric fundus or those to authentic NO were not affected by the antioxidants or by the metal chelator. These results indicate that antioxidants and metal chelators have a different effect on the biological activity of S-nitrosothiols and on that of the nitrergic neurotransmitter. Therefore, our results suggest that S-nitrosothiols do not act as intermediate compounds in nitrergic neurotransmission in the rat gastric fundus.

     

Early effects of acute γ-radiation on vascular arterial tone

1. To determine the acute effects of irradiation on the functionality of vessel, rat aortic rings were mounted in an organ bath for isometric tension measurements and irradiated (⁶⁰Co, 1 Gy min⁻¹, 15 min).
2. Irradiation, which is without effect on non-contracted or endothelium-denuded vessels, led to an immediate and reversible increase in vascular tone on (−)-phenylephrine (1 μM)-precontracted aortic rings. The tension reached a plateau about 5 min after the beginning of irradiation.
3. The maximal radiation-induced contraction occurred on aortic rings relaxed by acetylcholine (ACh) (1 μM). In this condition, the addition of catalase (1000 u ml⁻¹), which reduces hydrogen peroxide, and DMSO (0.1% v/v), which scavenges hydroxyl radical, had no influence on tension level while superoxide dismutase (SOD) (100 u ml⁻¹), a superoxide anion scavenger, reduced the observed contraction. A similar result was obtained in the presence of indomethacin (10 μM), a cyclo-oxygenase blocker.
4. Pretreatment of rings with the nitric oxide synthase inhibitor, N_ω-nitro-L-arginine methyl ester (L-NAME) (10–100 μM) inhibited the radiation-induced contraction.
5. This effect was dose rate-dependent and even occurred for a very low dose rate (0.06 Gy min⁻¹).
6. The present results indicate that γ-radiation induces an instantaneous vascular tone increase that is endothelium and dose rate-dependent. This effect is (i) maximal when nitric oxide (NO) is produced, (ii) greatly reduced by SOD and (iii) inhibited by L-NAME, suggesting a major involvement of complexes between NO and superoxide anion.

     

Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells

1. We have examined whether modulation of the polyamine biosynthetic pathway, through inhibition by α-difluoromethylornithine (DFMO) of the rate limiting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J774 macrophages.
2. DFMO potentiated LPS-stimulated nitrite production in both a concentration- and time-dependent manner, increasing nitrite levels by 48±5% at 10 mM. This effect was observed in cells pre-treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS-induced nitrite production.
3. Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS-induced nitrite production or in the ability of DFMO (10  mM) to potentiate LPS-induced NO synthesis.
4. Metabolism of L-[³H]arginine to L-[³H]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by either DFMO (1–10 mM) or by putrescine (0.001–1 mM), spermidine (0.001–1 mM) or spermine (0.001–1 mM). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70±0.07%) by L-NMMA (100 μM).
5. Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with LPS resulted in enhanced (∼2 fold) iNOS protein expression.
6. These results show that DFMO potentiates LPS-induced nitrite production in the murine macrophage cell line J774. Since the only known mechanism of action of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we conclude that expression of iNOS can be critically regulated by endogenous polyamines.

     

Release of the antioxidants ascorbate and urate from a nitrergically-innervated smooth muscle

1. The main object of the present study was to determine whether ascorbate, an antioxidant which has been shown to protect nitric oxide (NO) from attack by scavenger molecules, might be released from nitrergically-innervated smooth muscle; ascorbate release from the rat anococcygeus was measured by use of h.p.l.c. with electrochemical detection.
2. Incubation of rat anococcygeus muscles in normal physiological salt solution (PSS; 30 min) resulted in release of ascorbate into the bathing medium (7.7±0.9 nmol g⁻¹ tissue). This release was increased by 96% when muscles were incubated in high K⁺ (70 mM) PSS. The resting release of ascorbate was unaffected by tetrodotoxin (TTX; 1 μM), ω-conotoxin GVIA (10 nM) or omission of calcium ions from the PSS (with addition of 0.2 mM EGTA), but all three procedures attenuated the increased release observed under depolarizing conditions. Resting release of ascorbate was unaffected by glutamate (100 μM), aspartate (100 μM), γ-aminobutyric acid (100 μM) or carbachol (50 μM).
3. A second h.p.l.c. peak, which always preceded the ascorbate peak, was identified as urate. Urate release from the anococcygeus, following 30 min incubation in normal PSS, was 64.6±12.7 nmol g⁻¹ tissue but, unlike ascorbate, urate release was unchanged in high K⁺ PSS. In functional experiments, urate (100–400 μM) partially protected NO (15 μM)-induced relaxations of the rat anococcygeus from inhibition by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; 50 μM), but not from inhibition by hydroquinone or duroquinone (both 100 μM).
4. Muscles chemically sympathectomized with 6-hydroxydopamine (6-OHDA, 500 μM; 2 h) still exhibited release of ascorbate (2.5±0.4 nmol g⁻¹ tissue) and urate (22.2±2.9 nmol g⁻¹ tissue); in both cases the release was similar to that observed in time-matched control tissues not exposed to 6-OHDA. High K⁺ PSS produced a TTX-sensitive increase in release of ascorbate, but not urate, from 6-OHDA-treated muscles.
5. The results demonstrate that significant amounts of ascorbate and urate are released from the rat anococcygeus muscle. Ascorbate, but not urate, release appears to be enhanced by activation of nerves which are resistant to 6-OHDA pretreatment. Since both antioxidants can protect NO from attack by scavenger molecules, their release in nitrergically-innervated tissues may be important for the provision of the correct redox environment to allow NO to fulfill its proposed neurotransmitter role.

     

Selective antagonism of the GABAA receptor by ciprofloxacin and biphenylacetic acid

1. Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit γ-aminobutyric acid (GABA)_A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand-gated ion channels.
2. Agonist-evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique.
3. GABA (50 μM)-evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3–10 μM) and picrotoxin (0.3–10 μM), with IC₅₀ values and 95% confidence intervals (CI) of 1.2 μM (1.1–1.4) and 3.6 μM (3.0–4.3), respectively, and were potentiated by sodium pentobarbitone (30 μM) and diazepam (1 μM) to (mean±s.e.mean) 168±18% and 117±4% of control, respectively. 5-Hydroxytryptamine (5-HT; 0.5 μM)-evoked responses were inhibited by MDL 72222 (1 μM) to 10±4% of control; DMPP (10 μM)-evoked responses were inhibited by hexamethonium (100 μM) to 12±5% of control, and αbMeATP (30 μM)-evoked responses were inhibited by PPADS (10 μM) to 21±5% of control. Together, these data are consistent with activation of GABA_A, 5-HT₃, nicotinic ACh and P2_X receptors, respectively.
4. Ciprofloxacin (10–3000 μM) inhibited GABA_A-mediated responses in the vagus nerve with an IC₅₀ (and 95% CI) of 202 μM (148–275). BPAA (1–1000 μM) had little or no effect on the GABA_A-mediated response but concentration-dependently potentiated the effects of ciprofloxacin by up to 33,000 times.
5. Responses mediated by 5-HT₃, nicotinic ACh and P2_X receptors in the vagus nerve and strychnine-sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 μM), BPAA (100 μM) or the combination of these drugs (both at 100 μM).
6. GABA (1 mM)-evoked responses in the optic nerve were inhibited by bicuculline with an IC₅₀ of 3.6 μM (2.8–4.5), a value not significantly different from that determined in the vagus nerve. Ciprofloxacin also inhibited the GABA-evoked response with an IC₅₀ of 334 μM (256–437) and BPAA (100 μM) potentiated these antagonist effects. However, the magnitude of the synergy was 48 times less than that seen in the vagus nerve.
7. These data indicate that ciprofloxacin and BPAA are selective antagonists of GABA_A receptors, an action that may contribute to their excitatory effects in vivo. Additionally, our data suggest that the molecular properties of GABA_A receptors in different regions of the CNS influence the extent to which these drugs synergistically inhibit the GABA_A receptor.

     

Pharmacological characterization of endothelin-induced rat pulmonary arterial dilatation

1. The aim of study was to characterize endothelin (ET)-induced vasodilatation in isolated extrapulmonary rat arteries (EPA) and in intrapulmonary arteries (IPA) preconstricted with 1 μM phenylephrine.
2. The ET-3 (1 nM–100 nM)- and ET-1 (10 nM–100 nM)-induced transient vasodilatations in EPA were more potent than those in IPA. The vasodilatation induced by ET-3 (100 nM) was larger than that induced by ET-1 (100 nM).
3. Both the ET_B antagonist, BQ788 (3 μM) and or endothelium denudation, but not the ET_A antagonist, BQ123 (3 μM), abolished the vasodilatation induced by ET-1 or ET-3 (100 nM each) in EPA and in IPA. The ATP-sensitive K+channel blocker, glibenclamide (20 μM) and the nitric oxide synthase inhibitor, N^G-monomethyl-L-arginine (L-NMMA, 1 mM) suppressed the ET-induced vasodilatation in EPA and in IPA.
4. We conclude that the vasodilatation induced by endothelins is markedly reduced in rat isolated IPA, and suggest that the endothelial ET_B-mediated vasodilatation varies depending on rat pulmonary arterial regions. Furthermore, ET_B-mediated vasodilatation involves activation of ATP-sensitive K⁺ channels and of nitric oxide synthase in rat isolated EPA and IPA.

     

Nitric oxide-related cyclic GMP-independent relaxing effect of N-acetylcysteine in lipopolysaccharide-treated rat aorta

1. We have recently demonstrated the formation of protein-bound dinitrosyl-iron complexes (DNIC) in rat aortic rings exposed to lipopolysaccharide (LPS) and shown that N-acetylcysteine (NAC) can promote vasorelaxation in these arteries, possibly via the release of nitric oxide (NO) as low molecular weight DNIC from these storage sites. The aim of the present study was to investigate further the mechanism of the relaxation induced by NAC in LPS-treated vessels.
2. In rings incubated with LPS (10 μg ml⁻¹ for 18 h) and precontracted with noradrenaline (NA, 3 μM) plus N^ω-nitro-L-arginine methylester (L-NAME, 3 mM), the relaxation evoked by NAC (0.1 to 10 mM) was abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 μM, a selective inhibitor of soluble guanylyl cyclase) but not affected by Rp-8-bromoguanosine 3′5′-cyclic monophosphorothioate (Rp-8BrcGMPS, 60 μM a selective inhibitor of cyclic GMP-dependent protein kinase). Tetrabutylammonium (TBA, 3 mM, as a non selective K⁺ channels blocker) or elevated concentration of external KCl (25 or 50 mM) significantly attenuated the NAC-induced relaxation. Selective K⁺ channels blockers (10 μM glibenclamide, 0.1 μM charybdotoxin, 0.5 μM apamin or 3 mM 4-aminopyridine) did not affect the NAC-induced relaxation. The relaxing effect of NAC (10 mM) was not associated with an elevation of guanosine 3′ : 5′ cyclic monophosphate (cyclic GMP) in LPS-treated rings.
3. In aortic rings precontracted with NA (0.1 μM), low molecular weight DNIC (with thiosulphate as ligand, 1 nM to 10 μM) evoked a concentration-dependent relaxation which was antagonized by ODQ (1 μM) and Rp-8BrcGMPS (150 μM) but not significantly affected by TBA (3 mM) or by the use of KCl (50 mM) as preconstricting agent. The relaxation produced by DNIC (0.1 μM) was associated with an 11 fold increase in aortic cyclic GMP content, which was completely abolished by ODQ (1 μM).
4. Taken together with our previous data, the main finding of the present study is that the vascular relaxation induced by NAC in LPS-treated aorta, although probably related to NO through an interaction via preformed NO stores, was not mediated by activation of the cyclic GMP pathway. It may involve the activation of TBA-sensitive K⁺ channels. The differences in the mechanism of relaxation induced by NAC and by exogenous DNIC suggest that the generation of low molecular weight DNIC from protein-bound species does not play a major role in the NAC-induced relaxation observed in LPS-treated rat aorta. In addition, it is suggested that ODQ may display other properties than the inhibition of soluble guanylyl cyclase.

     

Developmental changes in endothelium-dependent vasodilation and the influence of superoxide anions in perinatal rabbit pulmonary arteries

1. ACh-induced vasodilation was investigated in pulmonary arteries from 8 and 2 day pre-term foetal, neonatal (0–12 h and 4 day old) and adult rabbits. The effects of superoxide anion generation [with hypoxanthine (HX, 0.1 mM)/xanthine oxidase (XO, 15 mu ml⁻¹)], endogenous superoxide dismutase (SOD) inhibition [with the Cu-Zn SOD inhibitor triethylenetetramine (TETA, 1 mM)], endogenous superoxide anion scavenging [by superoxide dismutase (SOD, 50 u ml⁻¹)] and inhibition of endothelial nitric oxide synthase (eNOS) [with, N^ω-nitro-L-arginine methylester (L-NAME, 0.1 mM)], on basal and ACh-induced NO activity were studied by examining phenylephrine-induced contraction and ACh-induced vasodilation respectively.
2. L-NAME and endothelium removal abolished all ACh-induced vasodilation and 1 μM sodium nitroprusside fully dilated all vessels. ACh-induced vasodilation was absent in the 8 day pre-term foetus and 0–12 h neonate but present at all other ages. L-NAME itself contracted 2 day pre-term foetal vessels. At 0–12 h, SOD, but not the phosphodiesterase 5 inhibitor zaprinast (1 μM), uncovered ACh-induced vasodilation. At this age SOD reduced phenylephrine-induced contraction which was not influenced by TETA, L-NAME or HX/XO, and L-NAME itself did not cause contraction. This suggests both ACh-induced and basal NO activity are compromise in these vessels by endogenous superoxide anion production and deficiencies in endogenous SOD activity.
3. In 4 day vessels, but not adult vessels, L-NAME, TETA and HX/XO augmented contractions to phenylephrine, and L-NAME itself induced vasoconstriction, suggesting that basal NO and SOD activities were present by 4 days but were not evident in the adult. ACh-induced NO activity, and the influence of endogenous SOD on this, were present in the adult (and 4 day) vessels as superoxide generation with HX/XO significantly reduced ACh-induced vasodilation and this effect was inhibited by SOD and augmented by TETA.
4. Increased oxygen tensions >500 mmHg attenuated ACh-induced vasodilation in the foetal but not neonatal rabbits. Raising the oxygen tension from ∼20 to ∼120 mmHg revealed ACh-induced vasodilation in the 8 day pre-term vessels.
5. In summary, superoxide anion accumulation combined with deficiencies in SOD activity may transiently compromise basal and ACh-induced NO activity at birth. Experimental oxygen tensions markedly influence ACh-induced vasodilation in foetal rabbit pulmonary arteries.

     

Effect of sodium rhein on electrically-evoked and agonist-induced contractions of the guinea-pig isolated ileal circular muscle

1. This study examined the effects of sodium rhein (0.03–30 μM) on the contractions of the isolated circular muscle of guinea-pig ileum induced by acetylcholine (100 nM), substance P (3 nM) and electrical stimulation (10 Hz for 0.3 s, 100 mA, 0.5 ms pulse duration). The effect of sodium rhein was also evaluated on the ascending excitatory reflex using a partitioned bath (oral and anal compartments). Ascending excitatory enteric nerve pathways were activated by electrical field stimulation (10 Hz for 2 s, 20 mA, 0.5 pulse duration) in the anal compartment and the resulting contraction of the guinea-pig intestinal circular muscle in the oral compartment was recorded.
2. Sodium rhein (0.3, 3 and 30 μM) significantly potentiated (52±11% at 30 μM) acetylcholine-induced contractions. In the presence of tetrodotoxin (0.6 μM) or ω-conotoxin GVIA (10 nM) sodium rhein (3 and 30 μM) did not enhance, but significantly reduced (49±10% and 44±8%, respectively, at 30 μM) acetylcholine-induced contractions.
3. Sodium rhein (0.3, 3 and 30 μM) significantly increased (65±11% at 30 μM) substance P-induced contractions. In the presence of tetrodotoxin (0.6 μM), ω-conotoxin GVIA (10 nM) or atropine (0.1 μM), sodium rhein (3 and 30 μM) significantly reduced (50±10%, 55±8% and 46±10%, respectively, at 30 μM) substance P-induced contractions.
4. N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) abolished the potentiating effect of sodium rhein on acetylcholine and substance P-induced contractions. At the highest concentration (30 μM), sodium rhein, in presence of L-NAME, reduced the acetylcholine (30±6%)- or substance P (36±6%)-induced contractions.
5. Sodium rhein (30 μM) significantly potentiated (29±9%) the electrically-evoked contractions. L-NAME (100 μM), but not phentolamine, enhanced the effect of sodium rhein. Sodium rhein (30 μM) significantly increased (32±9%) the ascending excitatory reflex when applied in the oral, but not in the anal compartment.
6. These results indicate that sodium rhein (i) activates excitatory cholinergic nerves on circular smooth muscle presumably through a facilitation of Ca²⁺ entry through the N-type Ca²⁺ channel, (ii) has a direct inhibitory effect on circular smooth muscle and (iii) does not affect enteric ascending neuroneural transmission. Nitric oxide could have a modulatory excitatory role on sodium rhein-induced changes of agonist-induced contractions and an inhibitory modulator role on sodium rhein-induced changes of electrically-induced contractions.

     

Modulation by general anaesthetics of rat GABAA receptors comprised of α1β3 and β3 subunits expressed in human embryonic kidney 293 cells

1. Radioligand binding and patch-clamp techniques were used to study the actions of γ-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5α-pregnan-3α-ol-20-one on rat α1 and β3 GABA_A receptor subunits, expressed either alone or in combination.
2. Membranes from HEK293 cells after transfection with α1 cDNA did not bind significant levels of [³⁵S]-tert-butyl bicyclophosphorothionate ([³⁵S]-TBPS) (<0.03 pmol mg⁻¹ protein). GABA (100 μM) applied to whole-cells transfected with α1 cDNA and clamped at −60 mV, also failed to activate discernible currents.
3. The membranes of cells expressing β3 cDNAs bound [³⁵S]-TBPS (∼1 pmol mg⁻¹ protein). However, the binding was not influenced by GABA (10 nM–100 μM). Neither GABA (100 μM) nor picrotoxin (10 μM) affected currents recorded from cells expressing β3 cDNA, suggesting that β3 subunits do not form functional GABA^A receptors or spontaneously active ion channels.
4. GABA (10 nM–100 μM) modulated [³⁵S]-TBPS binding to the membranes of cells transfected with both α1 and β3 cDNAs. GABA (0.1 μM–1 mM) also dose-dependently activated inward currents with an EC₅₀ of 9 μM recorded from cells transfected with α1 and β3 cDNAs, clamped at −60 mV.
5. Propofol (10 nM–100 μM), pentobarbitone (10 nM–100 μM) and 5α-pregnan-3α-ol-20-one (1 nM–30 μM) modulated [³⁵S]-TBPS binding to the membranes of cells expressing either α1β3 or β3 receptors. Propofol (100 μM), pentobarbitone (1 mM) and 5α-pregnan-3α-ol-20-one (10 μM) also activated currents recorded from cells expressing α1β3 receptors.
6. Propofol (1 μM–1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing β3 homomers. In contrast, application of 5α-pregnan-3α-ol-20-one (10 μM) failed to activate detectable currents.
7. Propofol (100 μM)-activated currents recorded from cells expressing either α1β3 or β3 receptors reversed at the C1⁻ equilibrium potential and were inhibited to 34±13% and 39±10% of control, respectively, by picrotoxin (10 μM). 5α-Pregnan-3α-ol-20-one (100 nM) enhanced propofol (100 μM)-evoked currents mediated by α1β3 receptors to 1101±299% of control. In contrast, even at high concentration 5α-pregnan-3α-ol-20-one (10 μM) caused only a modest facilitation (to 128±12% of control) of propofol (100 μM)-evoked currents mediated by β3 homomers.
8. Propofol (3–100 μM) activated α1β3 and β3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 μM and 1 mM) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a ‘surge'' current on cessation of propofol (300 μM and 1 mM) application. Surge currents were also evident following pentobarbitone (1 mM) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5α-pregnan-3α-ol-20-one (10 μM) to cells expressing α1β3 receptors.
9. These observations demonstrate that rat β3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABA_A receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.

     

Effect of 17β-oestradiol on cytokine-induced nitric oxide production in rat isolated aorta

1. Studies were performed on isolated aortic rings without endothelium to investigate the effect of 17β-oestradiol on cytokine-induced nitric oxide production by the inducible nitric oxide synthase (iNOS).
2. Treatment of the isolated aortic rings with interleukin-1β (IL-1β, 20 μ ml⁻¹) led to the expression of iNOS mRNA and protein, as well as significant nitrite accumulation in the incubation media and suppression of phenylephrine (1 nM–10 μM)-evoked contraction.
3. Cycloheximide (1 μM), a protein synthesis inhibitor, prevented iNOS protein expression, nitrite accumulation and the suppression of contractility by IL-1β on the isolated aortic rings. 17β-oestradiol (1 nM–10 μM) and the partial oestrogen receptor agonist 4-OH-tamoxifen (1 nM–10 μM) produced concentration-dependent inhibition of IL-1β-induced nitrite accumulation and restored vasoconstrictor responsiveness to phenylephrine, similar to the iNOS inhibitor aminoguanidine (100 μM).
4. Semiquantitative PCR demonstrated decreased iNOS mRNA in the IL-1β-induced and 17β-oestradiol-treated rings. Western blot analysis of rat aorta homogenates revealed that 17β-oestradiol treatment resulted in a reduction in IL-1ß-induced iNOS protein level.
5. Incubation with tumour necrosis factor α (TNFα, 1 ng ml⁻¹) resulted in significant nitrite accumulation in the incubation media and suppression of the smooth muscle contractile response to phenylephrine, similar to IL-1β. The effects of TNFα were also inhibited by co-incubation of the rings with 17β-oestradiol and 4-OH-tamoxifen (1 μM).
6. The anti-transforming growth factor-β1 (TGF-β1) antibody, which inhibited TGF-β1-induced suppression of nitrite production from IL-1β-treated vascular rings, did not affect the inhibitory action of 17β-oestradiol, suggesting that the effect of oestrogen on iNOS inhibition was not mediated by TGF-β1.
7. These results show that the ovarian sex steroid, 17β-oestradiol is a modulator of cytokine-induced iNOS activity in rat vascular smooth muscle and its mechanism of action involves decrease of iNOS mRNA and protein.

     

Modulation of angiotensin-converting enzyme by nitric oxide

1. The aim of the present study was to determine the effect of nitric oxide (NO) on angiotensin-converting enzyme (ACE) activity.
2. A biochemical study was performed in order to analyse the effect of the NO-donors, SIN-1 and diethylamine/NO (DEA/NO), and of an aqueous solution of nitric oxide on the ACE activity in plasma from 3-month old male Sprague-Dawley rats and on ACE purified from rabbit lung. SIN-1 significantly inhibited the activity of both enzymes in a concentration-dependent way between 1 and 100 μM. DEA/NO inhibited the activity of purified ACE from 0.1 μM to 10 μM and plasma ACE, with a lower potency, between 1 and 100 μM. An aqueous solution of NO (100 and 150 μM) also inhibited significantly the activity of both enzymes. Lineweaver-Burk plots indicated an apparent competitive inhibition of Hip-His-Leu hydrolysis by NO-donors.
3. Modulation of ACE activity by NO was also assessed in the rat carotid artery by comparing contractions elicited by angiotensin I (AI) and AII. Concentration-response curves to both peptides were performed in arteries with endothelium in the presence of the guanylyl cyclase inhibitor, ODQ (10 μM), and the inhibitor of NO formation, L-NAME (0.1 mM). NO, which is still released from endothelium in the presence of 10 μM ODQ, elicited a significant inhibition of AI contractions at low concentrations (1 and 5 nM). In the absence of endothelium, 1 μM SIN-1 plus 10 μM ODQ, as well as 10 μM DEA/NO plus 10 μM ODQ induced a significant inhibition on AI-induced contractions at 1 and 5 nM and at 1–100 nM, respectively.
4. In conclusion, we demonstrated that (i) NO and NO-releasing compounds inhibit ACE activity in a concentration-dependent and competitive way and that (ii) NO release from endothelium physiologically reduces conversion of AI to AII.

     

No evidence for a significant non-nitrergic,hyperpolarising factor contribution to field stimulation-induced relaxation of the mouse anococcygeus

1. The aim of the study was to determine whether a nerve-derived hyperpolarizing factor (NDHF) might contribute to non-adrenergic, non-cholinergic (NANC) relaxations of the mouse anococcygeus when low concentrations of contractile agent are used to raise tone and low frequencies of field stimulation applied; such a non-nitrergic NDHF has been proposed to contribute to NANC relaxations of the rat anococcygeus and guinea-pig taenia coli.
2. Phenylephrine (0.1–100 μM) produced concentration-related contractions of the mouse isolated anococcygeus muscle; 0.2 μM phenylephrine (EC₂₆) was used to raise tone in subsequent experiments.
3. Field stimulation (0.5, 1.0 and 5.0 Hz) produced frequency-dependent relaxations of phenylephrine-induced tone. In the presence of the nitric oxide synthase inhibitor L-N^G-nitro-arginine (L-NOARG; 100 μM), the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 μM), or a combination of these two drugs, relaxations to field stimulation were abolished at all frequencies studied. Relaxations to sodium nitroprusside (0.01–5 μM) were unaffected by L-NOARG but strongly inhibited by ODQ; neither enzyme inhibitor affected relaxations to 8-Br-cyclic GMP (10 μM).
4. Nifedipine (1 μM) reduced the contractile response to 0.2 μM phenylephrine by 38%; however, it had no effect on NANC relaxations.
5. It is concluded that NANC relaxations of the mouse anococcygeus are purely nitrergic and that there is no significant contribution from a putative NDHF.

     

Central role of intracellular calcium stores in acute flow- and agonist-evoked endothelial nitric oxide release

1. We have used a cascade bioassay system and isolated arterial ring preparations to investigate the contribution of Ca²⁺ release from endothelial intracellular stores to nitric oxide (NO) production evoked by increases in shear stress and by acetylcholine in rabbit aorta.
2. Experiments were performed before and following incubation with either the endoplasmic reticulum Ca²⁺-ATPase inhibitors cyclopiazonic acid (CPA, 10 μM) and thapsigargin (TSG, 1 μM) or ryanodine (30, 100 μM) which binds to a specific endoplasmic reticulum Ca²⁺-release channel.
3. In cascade bioassay all three agents induced relaxations of the recipient ring (CPA, 24.4±3.8%; TSG, 51.5±10.6%; ryanodine, 17.4±1.6%) which were significantly attenuated by preincubation of the donor with 100 μM N^G-nitro-L-arginine methyl ester (L-NAME). However, in isolated rings, only CPA and TSG induced L-NAME-sensitive relaxations (CPA 52.7±6.5%; TSG 61.3±7%).
4. Addition of superoxide dismutase (SOD) to the donor perfusate evoked relaxations of the recipient ring in cascade bioassay (13.3±1.4%, n=22). Prior administration of SOD attenuated relaxations to TSG (23.2±3.8%, n=4) and ryanodine (1.7±0.8%, n=4), and pre-incubation with TSG and ryanodine blunted SOD-induced responses (4±1.5%, n=4 and 8.9±1.1%, n=4, respectively). By contrast, no interaction was observed between the relaxations evoked by SOD and CPA. In isolated rings, SOD exerted no direct relaxant action and did not modulate relaxations to CPA, TSG or ryanodine.
5. In cascade bioassay studies time-averaged shear stress was manipulated with dextran (1–4% w/v, 80000 MW) to increase perfusate viscosity. NO-dependent relaxation of the recipient ring induced by increased perfusate viscosity was significantly attenuated by CPA (P<0.01; n=6) and TSG (P<0.05; n=7), but not by ryanodine (n=6).
6. Endothelium-dependent relaxations to acetylcholine (0.1–30 μM) in cascade bioassay and in isolated aortic ring preparations were markedly attenuated by pretreatment with CPA and TSG, but were unaffected by ryanodine. Ryanodine and CPA caused only a small attenuation of endothelium-independent relaxations to sodium nitroprusside (0.001–10 μM), whereas TSG had no effect.
7. We conclude that release of Ca²⁺ from CPA- and TSG-sensitive endothelial stores is necessary for NO release evoked by acute flow changes and agonists in rabbit abdominal aorta. Ca²⁺-induced Ca²⁺ release via the ryanodine-sensitive release channel plays no direct role in these responses. Free radical interactions may complicate the interpretation of findings in cascade bioassay compared with isolated ring preparations.