Jesús Marín

Role of vascular nitric oxide in physiological and pathological conditions

This review describes the ability of certain diseases, such as essential hypertension, atherosclerosis, angina, and vasospasm, to reduce vascular nitric oxide (NO) formation or to increase its metabolism. In contrast, others, such as hypotension, sepsis, stroke, myocardial depression, and inflammatory responses, increase NO synthesis. The mechanism implicated in the changes in the formation and metabolism of NO are described. To prevent or treat these pathological processes, in which a deficiency in vascular NO formation plays a causative role, NO may be provided through methods such as direct NO administration or indirect NO supply through either NO donors or L-arginine, which facilitates NO formation.

Contractile responses elicited by hydrogen peroxide in aorta from normotensive and hypertensive rats. Endothelial modulation and mechanism involved.

1 The present study analyses the influence of hypertension and endothelium on the effect induced by hydrogen peroxide (H2O2) on basal tone in aortic segments from normotensive Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHR) of 6‐month‐old, as well as the possible mechanisms involved. 2 Single (1 mm) or cumulative (100 nm–10 mm) concentrations of H2O2 produced a transient contraction or a concentration‐dependent increase of basal tone, respectively, in segments from WKY and SHR. In both cases, the contractions were higher in intact segments from hypertensive than from normotensive rats, and increased by endothelium removal in both strains. Catalase (1000 u ml−1, a H2O2 scavenger) abolished the contraction elicited by 1 mm H2O2 in both strains. 3 Superoxide dismutase (SOD, 150 u ml−1) and dimethylsulphoxide (DMSO, 7 mm), scavengers of superoxide anions and hydroxyl radicals, respectively, did not alter H2O2‐induced contractions in intact segments from both strains. However, l‐NG‐nitroarginine methyl ester (l‐NAME, 100 μm, a nitric oxide synthase inhibitor) increased the response to H2O2 in normotensive rats, although the increase was less than that produced by endothelium removal. 4 Incubation of segments with 1 mm H2O2 for 15 min and subsequent washout reduced the contractile responses induced by 75 mm KCl in intact segments from SHR and in endothelium‐denuded segments from both strains; this effect being prevented by catalase (1000 u ml−1). 5 Indomethacin (10 μm, a cyclo‐oxygenase inhibitor) and SQ 29,548 (10 μm, a prostaglandin H2/thromboxane A2 receptor antagonist) practically abolished the contractions elicited by H2O2 in normotensive and hypertensive rats. 6 We conclude that: (1) the oxidant stress induced by H2O2 produces contractions mediated by generation of a product of the cyclo‐oxygenase pathway, prostaglandin H2 or more probably thromboxane A2, in normotensive and hypertensive rats; (2) oxygen‐derived free radicals are not involved in the effect of H2O2; (3) in normotensive rats, endothelium protects against H2O2‐mediated injury to contractile machinery, determined by the impairment of KCl‐induced contractions; and (4) endothelial nitric oxide has a protective role on the contractile effect induced by H2O2, that is lost in hypertension.

Mechanisms involved in the cellular calcium homeostasis in vascular smooth muscle: calcium pumps.

The regulation of cytosolic Ca2+ homeostasis is essential for cells, and particularly for vascular smooth muscle cells. In this regulation, there is a participation of different factors and mechanisms situated at different levels in the cell, among them Ca2+ pumps play an important role. Thus, Ca2+ pump, to extrude Ca2+; Na+/Ca2+ exchanger; and different Ca2+ channels for Ca2+ entry are placed in the plasma membrane. In addition, the inner and outer surfaces of the plasmalemma possess the ability to bind Ca2+ that can be released by different agonists. The sarcoplasmic reticulum has an active role in this Ca2+ regulation; its membrane has a Ca2+ pump that facilitates luminal Ca2+ accumulation, thus reducing the cytosolic free Ca2+ concentration. This pump can be inhibited by different agents. Physiologically, its activity is regulated by the protein phospholamban; thus, when it is in its unphosphorylated state such a Ca2+ pump is inhibited. The sarcoplasmic reticulum membrane also possesses receptors for 1,4,5-inositol trisphosphate and ryanodine, which upon activation facilitates Ca2+ release from this store. The sarcoplasmic reticulum and the plasmalemma form the superficial buffer barrier that is considered as an effective barrier for Ca2+ influx. The cytosol possesses different proteins and several inorganic compounds with a Ca2+ buffering capacity. The hypothesis of capacitative Ca2+ entry into smooth muscle across the plasma membrane after intracellular store depletion and its mechanisms of inhibition and activation is also commented.

Impairment of Endothelium-Dependent Relaxation by Increasing Percentages of Glycosylated Human Hemoglobin: Possible Mechanisms Involved

High levels of glycosylated human hemoglobin impair nitric oxide-mediated responses. However, the percentage of glycosylation for which this effect is observed and the mechanisms involved are unknown. We tested endothelium-dependent relaxations caused by acetylcholine in rat aortic segments either in control conditions or after preincubation with increasing percentages of glycosylated human hemoglobin. Human hemoglobin (1 and 10 nmol/L) inhibited endothelium-dependent relaxations only when glycosylated at 9% or higher. We evaluated the effect of 14% glycosylated human hemoglobin on acetylcholine-evoked responses in vessels preincubated with scavengers of superoxide anions, hydroxyl radical, or hydrogen peroxide (superoxide dismutase, deferoxamine, and catalase, respectively); with inhibitors of xanthine oxidase, cyclooxygenase, or thromboxane synthase (allopurinol, indomethacin, and dazoxiben, respectively); with blockers of thromboxane A2/prostaglandin H2 or endothelin receptors (SQ 30741 and BQ-123); and with the precursor of nitric oxide synthesis L-arginine. Superoxide dismutase abolished the effect of glycosylated hemoglobin, and the other substances did not have any effect. Glycosylated hemoglobin at 14% did not modify either the vasoconstrictions induced by the blocker of nitric oxide synthase NG-nitro-L-arginine methyl ester or the relaxations evoked in deendothelialized vessels by sodium nitroprusside and 8-bromo-cGMP. However, it inhibited the vasodilations evoked by exogenous nitric oxide. Superoxide dismutase abolished this latter effect. We conclude that the threshold for glycosylated human hemoglobin (Hb A1) to inhibit endothelium-dependent relaxation is 9%. This effect is due to interference with endothelial nitric oxide by means of superoxide anion production.

Role of endothelium-formed nitric oxide on vascular responses

1. Endothelial cells of blood vessels generate factors which can modulate underlying smooth muscle tone, inducing vasorelaxation, (endothelium-derived relaxing factor, EDRF, and endothelium-derived hyperpolarizing factor) and/or vasoconstriction (endothelium-derived contracting factors, EDCFs, including the peptide endothelin). 2. EDRF is nitric oxide (NO) or a RNO compound from which this oxide is released. Its half-life is very short (6-50 sec), and it produces rapid vasodilations and inhibits platelet aggregation. 3. NO is formed from the terminal guanidino of L-arginine, but not of D-arginine. NO effects and NO formation are inhibited by NG-monomethyl-L-arginine (L-NMMA), but not by D-NMMA. These inhibitory effects are blocked by L-arginine. 4. Removal of endothelium or pathological situations that can induce endothelial dysfunction (atherosclerosis, diabetes, hypertension or subarachnoid hemorrhage) cause increases on the vascular contractility elicited by agonists (noradrenaline, serotonin, EDCFs, etc.). These findings suggest that EDRF produces a physiological inhibitory modulation of vascular smooth muscle tone and its alteration produces or facilitates the development of diseases such as hypertension or coronary and cerebral vasospasm.

Alterations in phenylephrine-induced contractions and the vascular expression of Na+,K+-ATPase in ouabain-induced hypertension

Hypertension development, phenylephrine‐induced contraction and Na+,K+‐ATPase functional activity and protein expression in aorta (AO), tail (TA) and superior mesenteric (SMA) arteries from ouabain‐ (25 μg day−1, s.c., 5 weeks) and vehicle‐treated rats were evaluated. Ouabain treatment increased systolic blood pressure (127±1 vs 160±2 mmHg, n=24, 35; P<0.001) while the maximum response to phenylephrine was reduced (P<0.01) in AO (102.8±3.9 vs 67.1±10.1% of KCl response, n=12, 9) and SMA (82.5±7.5 vs 52.2±5.8%, n=12, 9). Endothelium removal potentiated the phenylephrine response to a greater extent in segments from ouabain‐treated rats. Thus, differences of area under the concentration‐response curves (dAUC) in endothelium‐denuded and intact segments for control and ouabain‐treated rats were, respectively: AO, 56.6±9.6 vs 198.3±18.3 (n=9, 7); SMA, 85.5±15.4 vs 165.4±24.8 (n=6, 6); TA, 13.0±6.1 vs 39.5±10.4% of the corresponding control AUC (n=6, 6); P<0.05. The relaxation to KCl (1 – 10 mM) was similar in segments from both groups. Compared to controls, the inhibition of 0.1 mM ouabain on KCl relaxation was greater in AO (dAUC: 64.8±4.6 vs 84.0±5.1%, n=11, 14; P<0.05), similar in SMA (dAUC: 39.1±3.9 vs 43.3±7.8%, n=6, 7; P>0.05) and smaller in TA (dAUC: 62.1±5.5 vs 41.4±8.2%, n=12, 13; P<0.05) in ouabain‐treated rats. Protein expression of both α1 and α2 isoforms of Na+,K+‐ATPase was augmented in AO, unmodified in SMA and reduced in TA from ouabain‐treated rats. These results suggest that chronic administration of ouabain induces hypertension and regional vascular alterations, the latter possibly as a consequence of the hypertension.

Role of lipid peroxidation and the glutathione‐dependent antioxidant system in the impairment of endothelium‐dependent relaxations with age

Age‐related changes in the blood prooxidant‐antioxidant state, as well as its influence on the relaxant responses to acetylcholine (ACh) were studied in the tail artery from 6‐, 24‐ and 30‐month‐old Sprague‐Dawley (SD) rats. Malondialdehyde (MDA) plasma levels increased 2 and 3 times in 24‐ and 30‐month‐old rats, respectively, when compared with 6‐month‐old rats (0.43±0.09 μM). This increase was accompanied by an induction of 6‐phosphogluconate dehydrogenase (6PG‐DH) and glutathione reductase (GR) activities in red blood cells from 24‐month‐old rats. In 30‐month‐old rats, a further induction of these enzymatic activities, as well as glucose‐6‐phosphate dehydrogenase (G6P‐DH) and glutathione peroxidase (GPx) activities was observed. No differences with age were found in the concentration‐response curves to ACh in isolated tail artery segments from 6‐ and 24‐month‐old rats precontracted with 0.3 μM noradrenaline (NA). However, a decrease in sensitivity to ACh‐induced relaxation was observed in 30‐month‐old rats; EC30 values were 3.5 (1.3–8.0)×10−7 M and 18.1 (8.9–30.1)×10−7 M for 6‐ and 30‐month‐old rats, respectively. Moreover, a decrease in maximum ACh relaxation (10 μM) was found in 30‐month‐old rats in comparison with that obtained in 6‐month‐old rats (58.5±3.9% and 42.5±3.4% of previous NA contraction, respectively). Incubation of tail artery segments with MDA (0.5, 1 or 10 μM) caused a reduction of ACh‐induced relaxations that was different in the three ages. Thus, the reduction of ACh‐induced relaxations became significant with 0.5 μM MDA in 6‐, with 1 μM MDA in 24‐, and with 10 μM MDA in 30‐month‐old rats. In addition, MDA did not cause a shift in the concentration‐response curve to ACh, but a decrease in the maximum response. Superoxide dismutase (SOD; 150 u ml−1, a superoxide anion scavenger) reversed the inhibitory effect of MDA on ACh‐induced relaxations at all ages studied. We conclude that: (1) ageing produces an increase in lipid peroxidation process, as indicated by the increase in MDA plasma levels, that is accompanied by an induction of lipid peroxide detoxification enzymes; (2) the changes in prooxidant‐antioxidant equilibrium with age contribute, at least partially, to the impairment of the relaxant responses evoked by ACh; and (3) the effect of MDA appears to be mediated by superoxide anion at all ages studied.

Influence of hypertension on nitric oxide synthase expression and vascular effects of lipopolysaccharide in rat mesenteric arteries

Experiments were designed to investigate the effects of the inducible nitric oxide synthase (iNOS) stimulator, lipopolysaccharide (LPS), on noradrenaline (NA) responses and on NOS activity and its expression in intact mesenteric resistance arteries (MRAs) from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. In MRAs from WKY, LPS (10 μg ml−1; 1–5 h) reduced the vasoconstrictor responses to NA (0.1–30 μM) in the presence, but not in the absence of L‐arginine (L‐Arg, 10 μM). However, in SHR arteries, LPS induced an incubation‐time dependent reduction of NA responses in the absence, as well as the presence, of L‐Arg. The LPS inhibitory effect was reduced by the non‐specific NOS inhibitor L‐NG‐nitroarginine methyl ester (L‐NAME, 100 μM) and the selective iNOS inhibitor, aminoguanidine (100 μM). L‐NAME alone similarly shifted the concentration‐response curve to NA leftward in arteries from both strains, while aminoguanidine had no effect. L‐Arg shifted the curve to NA rightward only in SHR MRAs. Basal activity of both iNOS and constitutive NOS (conversion of [3H]‐L‐Arg to [3H]‐L‐citrulline) was similar in arteries from both strains. After 5 h incubation with LPS, only iNOS activity in arteries from SHR was increased. Basal iNOS protein expression was undetectable; basal endothelial (eNOS) protein expression was similar in arteries from both strains, while neuronal (nNOS) was greater in arteries from SHR. LPS induced iNOS protein expression, that was higher in arteries from SHR than in those from WKY. These results indicate that NO production, via iNOS induction, is greater than in those from MRAs from SHR to WKY.

Vascular sodium pump: endothelial modulation and alterations in some pathological processes and aging

The vascular Na+ pump maintains intracellular ionic concentration and controls membrane potential. Its inhibition by cardiac glycosides enhances the intracellular Na+ concentration. This in turn activates the Na+-Ca2+ exchange mechanism, which induces intracellular Ca2+ increase, membrane depolarization, and noradrenaline release from perivascular adrenergic nerve endings; mechanisms that promote vasoconstriction. This article reviews the relevance of the Na+ pump in vascular tone regulation and the modulation of its activity by the endothelium. The endothelium negatively modulates the vasoconstriction elicited by Na+ pump inhibition by the release of nitric oxide, according to some authors, or an unknown factor, as suggested by others. The possible existence of endogenous digitalis-like factors is also reviewed, as is the involvement of the vascular Na+ pump in some cardiovascular disorders and aging.

Effect of clenbuterol on non‐endothelial nitric oxide release in rat mesenteric arteries and the involvement of β‐adrenoceptors

The aim of the present study was to explore the contribution of adrenergic, sensory and nitrergic innervations to the inhibitory effects of the β2‐adrenoceptor agonist clenbuterol on responses to electrical field stimulation (EFS, 200 mA, 0.3 ms, 1–16 Hz, for 30 s, at 1 min interval) in rat mesenteric artery segments without endothelium and the possible involvement of adrenergic, sensory and nitrergic innervations. Clenbuterol (1 μM) reduced EFS‐induced contractile responses, and this effect was reversed by the β‐antagonist propranolol (1 μM) (contraction at 16 Hz expressed as % of 75 mM K+‐induced contraction was: control, 69±9, clenbuterol, 31±6, n=13, P<0.001; control, 83±5, clenbuterol+propranolol 70±7, n=11, P>0.05). In arteries preincubated with [3H]‐noradrenaline (NA), clenbuterol did not modify the tritium overflow evoked by EFS (200 mA, 0.3 ms, 4 Hz, for 60 s; ratio between tritium release in the second and first stimuli was: control, 0.80±0.05 and clenbuterol added before second stimulus, 0.91±0.11, n=5, P>0.05). The nitric oxide (NO) synthase inhibitors NG‐monomethyl‐L‐arginine (L‐NMMA) and NG‐nitro‐L‐arginine methyl ester (L‐NAME) (10 and 100 μM), and the guanylate cyclase inhibitor methylene blue (10 μM) increased the contractions caused by EFS (% contraction at 16 Hz, control, 81±7, n=26; 10 μM L‐NMMA, 109±12, n=8, P<0.05; methylene blue, 119±6, n=6, P<0.05). However, these contractions were decreased by the NO synthase substrate L‐arginine 10 μM (14±6%, n=6, P<0.001), but not modified by either the sensory neurones toxin capsaicin (0.5 μM, 75±6%, n=6, P>0.05) or the protein synthesis inhibitor cycloheximide (10 μM, 83±6%, n=8, P>0.05). None of these drugs altered the concentration‐response curves to exogenous NA (n=7). Pretreatment with capsaicin or cycloheximide did not modify the reduction of the EFS‐evoked contraction provoked by clenbuterol. However the presence of L‐NMMA (or L‐NAME) or methylene blue did decrease the effect of clenbuterol (% contraction at 16 Hz, clenbuterol, 31±6, n=13; clenbuterol+10 μM L‐NMMA, 93±11, n=8, P<0.05; clenbuterol+methylene blue, 90±7, n=6, P<0.05). These results suggest that the reduction caused by clenbuterol in the contraction induced by EFS in rat mesenteric arteries seems to be mediated by NO release, through the activation of β2‐adrenoceptors probably present on nitrergic nerves.