Pharmacological approaches of endothelial nitric oxide-dependent vasorelaxation induced by polyphenols from plant extracts.
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Animals↗
Biomedical subjects
J C Stoclet
Publications and source records attributed to J C Stoclet.
Mechanism of endothelial nitric oxide-dependent vasorelaxation induced by wine polyphenols in rat thoracic aorta.
The mechanisms by which red wine polyphenolic compounds (RWPCs) induced endothelium-dependent relaxation were investigated in rat thoracic aorta rings with endothelium. RWPCs produced relaxation that was prevented by the nitric oxide (NO) synthase inhibitor, N(omega)-nitro-L-arginine-methyl-ester. This relaxation was abolished in the absence of extracellular calcium in the medium or in the presence of the Ca2+ entry blocker, La3+, but it was not affected by the nonselective K+ channels blocker, tetrabutylammonium. N-Ethyl-maleimide (NEM), a sulfhydryl alkylating agent, abolished vasorelaxation produced by RWPCs and acetylcholine but not that produced either by the sarcoendoplasmic reticulum Ca2+-adenosine triphosphatase (ATPase) pump inhibitor, cyclopyazonic acid (CPA) or the calcium ionophore, ionomycin. Neither pertussis toxin (PTX) nor cholera toxin (CTX) inhibited the vasorelaxant effect of RWPC. The effect of RWPC was not affected by the phospholipase C (PLC) blocker, L-alpha-glycerophospho-D-myo-inositol 4-monophosphate (Gro-pip), and the phospholipase A2 pathway blockers, quinacrine and ONO-RS-082. Finally, the protein kinase C (PKC) inhibitor, GF 109203X, and tyrosine kinase inhibitors, tyrphostin A-23 and genistein, did not impair the response to RWPCs. These results suggest that RWPCs produce endothelium-NO-derived vasorelaxation through an extracellular Ca2+-dependent mechanism via an NEM-sensitive pathway. They also show that PTX- or CTX-sensitive G proteins, activation of PLC or PLA2 pathways, PKC, or tyrosine kinase may not be involved.
Animals↗
Mechanism of Ca2+ release and entry during contraction elicited by norepinephrine in rat resistance arteries.
The intracellular Ca2+ stores and the mechanisms of Ca2+ entry produced by norepinephrine (NE) were investigated in small mesenteric resistance arteries of the rat. In Ca2+-free medium, NE (10 microM) elicited a transient increase in both intracellular free Ca2+ concentration ([Ca2+]i) and tension that were both drastically reduced by caffeine and only partially reduced by the two sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) blockers thapsigargin and cyclopiazonic acid, despite the presence of SERCA2a and SERCA2b isoforms in the medial smooth muscle layer of the artery. After depletion of intracellular Ca2+ stores with 10 microM NE, addition of exogenous CaCl2 (2.5 mM) produced large and sustained increases in both [Ca2+]i and contraction of the arteries provided that the agonist was continuously present. In these conditions, the responses to CaCl2 were inhibited by the voltage-dependent Ca2+ entry blocker nitrendipine (1 microM), the putative inhibitor of receptor-operated Ca2+ entry SKF-96365 (30 microM), and NiCl2 (1 mM). The inhibition produced by SKF-96365 and NiCl2 was greater than that of nitrendipine. Also, the responses to CaCl2 were greatly reduced or abolished in the presence of the Na+/Ca2+ exchanger inhibitors 1,3-dimethyl-2-thiourea, 3',4'-dichlorobenzamil, MgCl2, and amiloride or after omission of NaCl in the medium. Also, protein kinase C inhibitors, calphostin C and staurosporine, and tyrosine kinase inhibitors, genistein and tyrphostin 23, both reduced the responses to CaCl2. The inhibitory effect of protein kinase C inhibitor and tyrosine kinase were additive. These results suggest that NE releases Ca2+ from intracellular stores that are caffeine sensitive and partially sensitive to SERCA inhibitors. They indicate that in addition to Ca2+ influx via nitrendipine-sensitive and SKF-96365-sensitive channels, Na+/Ca2+ exchanger participates in the CaCl2-induced contraction produced in NE-exposed vessels. The pathway leading to Ca2+ entry probably involves tyrosine kinase and protein kinase C. All the above mechanisms require ongoing receptor stimulation.
Animals↗
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 microg ml(-1) for 18 h) and precontracted with noradrenaline (NA, 3 microM) plus N(omega)-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 microM, a selective inhibitor of soluble guanylyl cyclase) but not affected by Rp-8-bromoguanosine 3'5'-cyclic monophosphorothioate (Rp-8BrcGMPS, 60 microM 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 microM glibenclamide, 0.1 microM charybdotoxin, 0.5 microM 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 microM), low molecular weight DNIC (with thiosulphate as ligand, 1 nM to 10 microM) evoked a concentration-dependent relaxation which was antagonized by ODQ (1 microM) and Rp-8BrcGMPS (150 microM) 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 microM) was associated with an 11 fold increase in aortic cyclic GMP content, which was completely abolished by ODQ (1 microM). 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.
Acetylcysteine↗
Role of adventitial nitric oxide in vascular hyporeactivity induced by lipopolysaccharide in rat aorta.
This study was designed to elucidate the role of the adventitia in NO-mediated vascular effects of lipopolysaccharide (LPS). After incubation of rat aorta with LPS, the adventitia generated 3.5 times more nitrite plus nitrate than a corresponding segment of media. Control media covered by adventitia from LPS-treated aortic rings exhibited a 4 fold elevated level of cyclic GMP. Medial layers from LPS-treated aortic rings (like LPS-treated adventitia-intact rings) exhibited a decrease in sensitivity to noradrenaline (NA) that was reversed by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (1 microM) or N omega-nitro-L-arginine methylester (0.3 mM). However, in contrast to LPS-treated adventitia-intact rings, medial layers showed no reduction in maximal contraction to NA and virtually no relaxation to L-arginine. These data indicate that in blood vessels exposed to LPS, the adventitia is a more powerful source of NO than the media. The adventitia-derived NO can reach soluble guanylyl cyclase in the medial layer and contribute greatly to vascular hyporeactivity and L-arginine-induced relaxation observed in blood vessels exposed to LPS.
Animals↗
Natural dietary polyphenolic compounds cause endothelium-dependent vasorelaxation in rat thoracic aorta.
This study investigated the possible active principles which support the endothelial nitric oxide-dependent relaxation produced by red wine and other plant polyphenolic compounds in thoracic aorta from male Wistar rats (12-14 wk old). Relaxation experiments were recorded isometrically on vessels precontracted with norepinephrine. Ten different chromatographic fractions (3-18 mg) isolated from red wine polyphenolic compounds (RWPC) and some available defined polyphenols (10-15 mg) were tested. Fractions enriched into either anthocyanins or oligomeric condensed tannins exhibited endothelium-dependent vasorelaxant activity (maximal relaxation in the range of 59-77%) comparable to the original RWPC. However, polymeric condensed tannins elicited a weaker vasorelaxant activity than the original RWPC (maximal relaxation ranged between 20-47%, P < 0.01). Moreover, the representative of either phenolic acid derivatives (benzoic acid, vanillic acid, gallic acid), hydroxycinnamic acid (p-coumaric acid, caffeic acid) or the flavanol [(+)-epicatechin] classes failed to induce this type of response. Among the anthocyanins, delphinidin (maximal relaxation being 89%), but not malvidin or cyanidin, showed endothelium-dependent vasorelaxation. These results show that anthocyanins and oligomeric-condensed tannins exhibited a pharmacological profile comparable to the original RWPC. These compounds may be involved in the reduction of cardiovascular mortality related to the presence of wine, fruits and vegetables in the diet.
Animals↗
Overproduction of nitric oxide in pathophysiology of blood vessels.
Sustained production of large amounts of nitric oxide (NO) is induced in blood vessels by inflammatory stimuli as a result of the expression of the inducible form of NO-synthase (NOS-2). This happens in systemic inflammatory reactions like septic shock and in local reactions produced by endothelium denudation and atherosclerosis. NOS-2 activity in blood vessels may protect tissues by virtue of the vasodilating, anti-thrombotic and leukocyte adhesion inhibitory effects of NO. It may also participate in vascular remodeling as a result of the antiproliferative and pro-apoptotic actions of NO. However excessive production of NO in blood vessels is involved in circulatory failure that takes place in systemic inflammatory reactions and it may be cytotoxic for surrounding tissues. For these reasons, inhibition of NO overproduction has been proposed in the treatment of septic shock. Selective inhibitors of NOS-2 activity or NO trapping agent, or both, might prove to be valuable drugs in the treatment of some inflammatory diseases. The conditions in which NO shifts from a tissue protective to a damaging role are not well elucidated. Recent findings suggest that the interactions with superoxide radicals, thiols, and metals (particularly with Fe2+) may be important not only in buffering excess NO produced by NOS-2, but also in channeling it from physiologically to pathophysiologically relevant targets. It has also been found recently that adventitial cells may play an important part in vascular NO production and generation of NO stores in the media layer. The ultimate effect of NO in blood vessels might depend on its site of production, local concentration, and interactions with other tissue components.
Animals↗
Nitric oxide generation from extracellularly applied NG-hydroxy-L-arginine in LPS-activated RAW 264 macrophages.
Lipopolysaccharide (LPS)-activated but not control RAW 264 macrophages produced nitric oxide (NO) from extracellularly-applied NG-hydroxy-L-arginine (L-NOHA) in a concentration-dependent manner, as measured by EPR spin trapping and assays for NO2- and NO3-. This production was inhibited by NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine, NO-synthase inhibitors, as well as by L-lysine, a competitor for the y+ amino acid carrier system. No significant differences were found between L-NOHA and L-arginine with respect to the rate of NO production and the effects of inhibitors. These results provide evidence that extracellular L-NOHA can enter LPS-activated RAW 264 macrophages via a cationic amino acid carrier system and be metabolized to NO by NO-synthase. The data also suggest that no alternative pathway exists for NO production from L-NOHA in non-activated RAW 264 macrophages.
Animals↗
Nitric oxide production and endothelium-dependent vasorelaxation induced by wine polyphenols in rat aorta.
1. The aim of this work was to investigate the mechanism of vasorelaxation induced by red wine polyphenolic compounds (RWPC) and two defined polyphenols contained in wine, leucocyanidol and catechin. The role of the endothelium, especially endothelium-derived nitric oxide (NO), was also investigated. 2. Relaxation produced by polyphenols was studied in rat aortic rings with and without functional endothelium, pre-contracted to the same extent with noradrenaline (0.3 and 0.1 microM, respectively). RWPC and leucocyanidol, but not catechin, produced complete relaxation of vessels with and without endothelium. However, 1000 fold higher concentrations were needed to relax endothelium-denuded rings compared to those with functional endothelium. 3. High concentrations of catechin (in the range of 10(-1) gl-1) only produced partial relaxation (maximum 30%) and had the same potency in rings with and without endothelium. 4. The NO synthase inhibitor, N omega-nitro-L-arginine-methyl-ester (L-NAME, 300 microM) completely abolished the endothelium-dependent but not the endothelium-independent relaxations produced by all of the polyphenolic compounds. 5. In contrast to superoxide dismutase (SOD, 100 u ml-1), neither RWPC nor leucocyanidol affected the concentration-response curve for the NO donor, SIN-1 (3-morpholino-sydnonimine) which also produces superoxide anion (O2-). 6. In aortic rings with endothelium, RWPC (10(-2) gl-1) produced, a 7 fold increase in the basal production of guanosine 3':5'-cyclic monophosphate (cyclic GMP) which was prevented by L-NAME (300 microM). 7. Electron paramagnetic resonance (e.p.r.) spectroscopy studies with Fe(2+)-diethyldithiocarbamate as an NO spin trap demonstrated that RWPC and leucocyanidol increased NO levels in rat thoracic aorta about 2 fold. This NO production was entirely dependent on the presence of the endothelium and was abolished by L-NAME (300 microM). 8. These results show that RWPC and leucocyanidol, but not the structurally closely related polyphenol catechin, induced endothelium-dependent relaxation in the rat aorta. They indicate that this effect results from enhanced synthesis of NO rather than enhanced biological activity of NO or protection against breakdown by O2. It is concluded that some polyphenols, with specific structure, contained in wine possess potent endothelium-dependent vasorelaxing activity.
Animals↗
Characterization of endothelium-derived relaxing factors released by bradykinin in human resistance arteries.
1. Relaxing factors released by the endothelium and their relative contribution to the endothelium-dependent relaxation produced by bradykinin (BK) in comparison with different vasodilator agents were investigated in human omental resistance arteries. 2. BK produced an endothelium-dependent relaxation of arteries pre-contracted with the thromboxane A2 agonist, U46619. The B2 receptor antagonist, Hoe 140 (0.1, 1 and 10 microM), produced a parallel shift to the right of the concentration-response curve to BK with a pA2 of 7.75. 3. Neither the cyclo-oxygenase inhibitor, indomethacin (10 microM) alone, the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 300 microM) alone, the nitric oxide scavenger, oxyhaemoglobin (Hb, 10 microM) alone, nor the combination of L-NAME plus Hb affected the concentration-response curve to BK. Conversely, the combination of indomethacin with either L-NAME or Hb attenuated but did not abolish the BK-induced relaxation. By contrast, the relaxations produced by the Ca2+ ionophore, calcimycin (A23187), and by the inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, thapsigargin (THAPS), were abolished in the presence of indomethacin plus L-NAME. Also, the presence of indomethacin plus L-NAME produced contraction of arteries with functional endothelium. 4. The indomethacin plus L-NAME resistant component of BK relaxation was abolished in physiological solution (PSS) containing 40 mM KCl and vice versa. However, in the presence of KCl 40 mM, indomethacin plus L-NAME did not affect the nitric oxide donor, S-N-acetylpenicillamine-induced relaxation. 5. The indomethacin plus L-NAME resistant component of the relaxation to BK was significantly attenuated by the K+ channel blocker tetrabutylammonium (TBA, 1 mM). However, it was not affected by other K+ channel blockers such as apamin (10 microM), 4-aminopyridine (100 microM), glibenclamide (10 microM), tetraethylammonium (10 mM) and charybdotoxin (50 nM). 6. In the presence of indomethacin plus L-NAME, the relaxation produced by BK was not affected by the phospholipase A2 inhibitor, quinacrine (10 microM) or by the inhibitor of cytochrome P450, SKF 525a (10 microM). Another cytochrome P450 inhibitor, clotrimazole (10 microM) which also inhibits K+ channels, inhibited the relaxation to BK. 7. These results show that BK induces endothelium-dependent relaxation in human small omental arteries via multiple mechanisms involving nitric oxide, cyclo-oxygenase derived prostanoid(s) and another factor (probably an endothelium-derived hyperpolarizing factor). They indicate that nitric oxide and cyclo-oxygenase derivative(s) can substitute for each other in producing relaxation and that the third component is not a metabolite of arachidonic acid, formed through the cytochrome P-450 pathway, in these arteries.
Arteries↗
Alterations in calcium stores in aortic myocytes from spontaneously hypertensive rats.
The aim of the present work was to further characterize intracellular calcium stores released by angiotensin II (Ang II) in spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat (WKY) vascular smooth muscle cells (VSMCs) and to study their alterations associated with proliferation. Intracellular Ca2+ concentration was monitored by image analysis in aortic myocytes loaded with fura 2. In the presence of extracellular Ca2+, sensitivity to Ang II in proliferating VSMCs was not different in the two strains, but it increased 10-fold in confluent VSMCs from SHR-compared with those from WKY. In Ca(2)+-free medium, Ca2+ release induced by thapsigargin (10 mumol/L) was significantly greater (about twofold) in SHR than WKY, in both proliferating and confluent cultures, with responses during proliferation being 0.7-fold smaller. Responses to Ang II were abolished after exposure of the cells to thapsigargin. In proliferating cultures, ryanodine (10 mumol/L) did not modify the rises in intracellular Ca2+ concentration induced by Ang II in VSMCs from both strains. Conversely, in confluent cultures, ryanodine reduced Ang II (100 nmol/L)-induced Ca2+ release to the same level as in proliferating cultures, and it suppressed the difference between SHR and WKY. These results show that the ryanodine-sensitive Ca2+ release induced by Ang II is enhanced in VSMCs from SHR at confluence and is impaired during proliferation. Thus, they suggest that differences in Ca2+(-)induced Ca2+ release from the sarcoplasmic reticulum may participate in increased responsiveness of VSMCs to Ang II in SHR and in phenotypic modulation of vascular myocytes during proliferation.
Angiotensin II↗
Hyporeactivity of mesenteric vascular bed in endotoxin-treated rats.
Vascular reactivity and activation of the nitric oxide (NO) pathway were investigated in perfused mesenteric vascular bed removed from rats 5 h after i.p. injection of bacterial lipopolysaccharide (E. coli lipopolysaccharide, 30 mg kg -1). Lipopolysaccharide treatment induced hyporesponsiveness to noradrenaline. Maximal noradrenaline-induced vasoconstriction was significantly reduced in lipopolysaccharide-treated vs. untreated preparations. Continuous infusion of L-arginine (L-Arg) (0.2 mM) enhanced noradrenaline hyporeactivity of lipopolysaccharide-treated rats. N omega-Nitro-L-arginine methyl ester (L-NAME) (0.2 mM), a non-selective inhibitor of NO synthase, failed to completely restore the noradrenaline hyporeactivity of lipopolysaccharide-treated + L-Arg-infused mesenteric vascular bed. After L-NAME treatment. Methylene blue (10 microM), a guanylate cyclase inhibitor, produced no additional increase of noradrenaline vasoconstriction in lipopolysaccharide-treated + L-Arg-infused mesenteric vascular bed, suggesting that an NO-independent activation of guanylate cyclase may be excluded. In lipopolysaccharide-treated preparations, L-Arg (0.2 mM) elicited a significant increase in nitrite production, which was antagonized by L-NAME. In conclusion, lipopolysaccharide-induced noradrenaline hyporesponsiveness of rat resistance vessels can only be partially explained by NO overproduction. Other mechanisms, probably related to vasoconstriction, may be involved.
Animals↗
Calcium handling and purinoceptor subtypes involved in ATP-induced contraction in rat small mesenteric arteries.
1. The relationship between the stimulation of ATP receptors, the increase in intracellular free calcium concentration ([Ca2+]i; measured using the fluorescent indicator fura-2), contraction and the subtypes of purinoceptors involved were investigated in the small mesenteric artery of the rat. 2. In normal physiological solution, ATP (0.001-3 mM) caused concentration-dependent increases in both [Ca2+]i and contraction. Both responses produced by ATP (1 mM) were inhibited by 50% in the presence of nitrendipine (1 microM) and were abolished in the presence of nitrendipine plus SK&F 96365 (30 microM). 3. In Ca(2+)-free medium, ATP (3 mM) elicited a transient increase in both [Ca2+]i and tension which were abolished by caffeine and decreased by 65% by thapsigargin (1 microM). Moreover, ATP (1 and 3 mM) produced increases in the [3H]D-myo-inositol 1,4,5-trisphosphate ([3H]IP3) content of vessels in a concentration-dependent manner. 4. Treatment of the vessels with Bordetella pertussis toxin (PTX) inhibited contractions to ATP linked to the influx of calcium through nitrendipine-sensitive mechanisms, but not those linked to the release of Ca2+ from intracellular stores nor the capacity of ATP in increasing IP3 content of the vessels. 5. The order of potency of ATP and its analogues in eliciting contraction was alpha, beta-methylene-ATP (alpha, beta-MeATP) > 2-methylthio-ATP (2-MeSATP) > ATP = ADP. The response to ATP was inhibited by suramin. Reactive Blue 2 (up to 100 microM) did not affect the contractile response to ATP. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (PPADS) and alpha, beta-MeATP abolished the response to low concentrations of ATP and reduced contractions elicited by high concentrations of ATP. 6. After blockade of P2X-purinoceptors with PPADS, the order of potency of ATP and its analogues was 2-MeSATP > ATP = ADP. UTP produced concentration-dependent contractions which were not affected by suramin, Reactive Blue 2, PPADS or alpha, beta-MeATP, suggesting the presence of P2U-purinoceptors. 7. The results suggest that low concentrations of ATP activate P2X-purinoceptors and produce an influx of calcium through both voltage-dependent calcium channels sensitive to nitrendipine and through receptor-operated calcium channels sensitive to SK&F 96365. High concentrations of ATP activate P2Y-purinoceptors which promote firstly a nitrendipine-sensitive calcium influx via a PTX-sensitive G protein and secondly a release of Ca2+ from an internal source via the production of IP3.
Adenosine Triphosphate↗
Use of human vessels and human vascular smooth muscle cells in pharmacology.
Relatively limited information is available regarding the mechanisms controlling vasomotricity in human vessels. Isolated vessels obtained from patients undergoing surgery were used to characterize the role of endothelial factors and to study coupling mechanisms between receptors, intracellular calcium, and contraction. However, these investigations are limited by the availability of tissues and many uncontrolled factors. Cultured human vascular cells were also used, but these cells rapidly lose at least some of their differentiated characters. Recently, a human blood vessel equivalent was constructed in vitro from cultured cells, using tissue engineering. This technique allowed us to obtain vessel equivalents containing intima, media, and adventitia layers or tubular media layer only. Contraction and rises in intracellular calcium produced by agonists were studied, indicating that such human vessel equivalents may provide valuable models for pharmacological studies.
Calcium↗
Possible mechanism of nitric oxide production from N(G)-hydroxy-L-arginine or hydroxylamine by superoxide ion.
It has been speculated the N(G)-hydroxy-L-arginine (OH-L-Arg), which is an intermediate in NO production from L-arginine, may be converted to NO by superoxide ion. However, there is still no direct evidence for this conversion. In the present study this was investigated using superoxide ion generated either in acellular or cellular systems. It was found that OH-L-Arg and hydroxylamine were converted to nitrite and nitrate apparently via NO by superoxide ion in aqueous solution. Arginine remained unaffected. These changes were observed during reaction of chemical substances as well as in a biological system (zymosan-activated macrophages in culture). Superoxide dismutase prevented this transformation. OH-L-Arg was also spontaneously hydrolysed to hydroxylamine and L-citrulline, however this occurred at pH > 9 only. Activated microsomes (containing different isoforms of cytochrome P450) were unable to replace NO-synthase in its ability to produce OH-L-Arg from L-arginine. These data support the hypothesis that a pathway alterative to the well-known synthesis of NO by NO-synthase via OH-L-Arg exists. This pathway may involve the production of OH-L-Arg by NO-synthase and decomposition of OH-L-Arg to NO by the action of superoxide ion. Alternatively, hydrolysis of OH-L-Arg to hydroxylamine may occur followed by its oxidation to NO, again by superoxide ion.
Animals↗
Influence of indomethacin on the haemodynamic effects of lipopolysaccharide in rats.
This study was undertaken to evaluate the influence of the cyclooxygenase inhibitor indomethacin on the time course of the haemodynamic effects of lipopolysaccharide (LPS) intravenous infusion (10 mg.kg-1.h-1) in anaesthetized rats. LPS infusion produced a rapid (within 10 min) and prolonged (over the 90 min observation period) fall in mean arterial blood pressure (MABP) and a decrease in the pressor responses to noradrenaline (NA, 0.1, 0.3 and 1 microgram.kg-1, intravenously [i.v.]) elicited 60 and 90 min after the onset of LPS infusion. Indomethacin (7 mg.kg-1 i.v. 30 min prior to the onset of saline or LPS infusion), which by itself did not affect basal MABP nor reactivity to NA in control rats, significantly attenuated the fall in MABP observed within 20 min after the onset of LPS infusion (but did not significantly modify the hypotension observed between 30 and 90 min). Indomethacin also completely prevented the hyporeactivity to NA observed 60 min after the onset of LPS infusion, but it did so only partially at 90 min. Aortic rings removed from LPS or LPS + indomethacin-treated rats showed an identical profile of contractile reactivity (hyporesponsiveness to NA, relaxation to L-arginine, and restoration of the contractile response by methylene blue). These results suggest that in this model, cyclooxygenase products are involved in the early haemodynamic effects of LPS. However, they do not seem to play an obligatory role in the onset of longer term haemodynamic changes.
Analysis of Variance↗
Alteration by lipopolysaccharide of the relationship between intracellular calcium levels and contraction in rat mesenteric artery.
1. The aim of this work was to investigate the effect of lipopolysaccharide (LPS) treatment on the relationship between the cytosolic Ca2+ ion concentration ([Ca2+]i) and contraction in rat resistance arteries, and the involvement of the L-arginine-nitric oxide (NO)-guanosine 3'-5' cyclic monophosphate (cyclic GMP) pathway in these effects. 2. [Ca2+]i and tension were simultaneously recorded in small mesenteric arteries removed from rats 4 h after intraperitoneal injection of E. coli LPS (30 mg kg-1) or solvent. Cyclic GMP was assayed in vessels submitted to identical treatments. 3. Basal [Ca2+]i was higher in vessels from LPS-treated rats compared to controls. LPS did not modify the concentration-contraction curve of noradrenaline. However, the increase in basal [Ca2+]i produced by LPS resulted in a shift of the noradrenaline [Ca2+]i-contraction curve to higher [Ca2+]i concentrations. 4. L-Arginine (300 microM) relaxed noradrenaline (10 microM) pre-contracted arteries from LPS-treated but not from control rats. This effect of L-arginine was reversed by two inhibitors of NO synthase: N omega-nitro-L-arginine-methyl-ester (L-NAME, 1 mM) and S-methyl-isothiourea (SMT, 0.1 mM). Both the relaxing effect of L-arginine and its reversal by L-NAME or SMT occurred without any change in [Ca2+]i. 5. LPS treatment did not modify the cyclic GMP content of the small mesenteric arteries. In arteries removed from LPS-treated rats but not from controls, addition of L-arginine (300 microM) was associated with a significant increase in cyclic GMP content, an effect which was prevented by both L-NAME (1 mM) and SMT (0.1 mM). 6. L-NAME (1 mM) produced a greater reduction in cyclic GMP content than SMT (0.1 mM) in control vessels exposed to L-arginine (300 microM). Under the same conditions, SMT produced a larger decrease in cyclic GMP level than L-NAME in arteries taken from LPS-treated rats, consistent with selective inhibition by SMT of the inducible NO-synthase after LPS. 7. These results show that LPS produced two effects in small mesenteric arteries: (i) alterations in Ca2+ handling and a decreased sensitivity of myofilaments to Ca2+, (ii) induction of NO-synthase activity resulting in exogenous L-arginine-dependent production of NO and cyclic GMP accumulation. Both effects are likely to be involved in the hyporeactivity induced by LPS in resistance arteries.
Analysis of Variance↗
Evidence for N-acetylcysteine-sensitive nitric oxide storage as dinitrosyl-iron complexes in lipopolysaccharide-treated rat aorta.
1. The aim of this study was to assess whether or not vasoactive nitric oxide (NO) stores exist within vascular tissue after lipopolysaccharide (LPS)-treatment. 2. Rat thoracic aortic rings (for contraction experiments) or whole thoracic aortae (for electron paramagnetic resonance (e.p.r.) spectroscopy) were incubated for 18 h at 37 degrees C in the absence (control) or in the presence of LPS (10 micrograms ml-1), with or without L-arginine (L-Arg, 1 mM), the substrate of NO synthase (NOS) or N omega-nitro-L-arginine methyl ester (L-NAME, 1 mM), an inhibitor of NOS. 3. Incubation of rat aortic rings with LPS and L-Arg resulted in a significant decrease of the maximum contractile response to noradrenaline (NA, 3 microM). Addition of L-NAME (3 mM) enhanced contraction towards control values. After precontraction with NA and L-NAME, addition of N-acetyl-L-cysteine (NAC, 0.1 to 10 mM) evoked a concentration-dependent relaxation in rings incubated with LPS and L-Arg, but not in control rings, rings incubated with LPS in the absence of L-Arg or rings incubated with LPS in the presence of L-Arg and L-NAME. Removal of the endothelium did not significantly modify the relaxation induced by NAC. Methylene blue (3 microM), an inhibitor of the activation of guanylyl cyclase by NO, completely abolished the relaxing effect of NAC. 4. The presence of protein-bound dinitrosyl non-haem iron complexes (DNIC) was detected by e.p.r. spectroscopy in aortae incubated with LPS and L-Arg, but not in control aortae. Furthermore in LPS-treated aortae, addition of NAC (20 mM) gave rise to the appearance of an e.p.r. signal characteristic of low molecular weight DNIC. 5. These results provide evidence that, within vascular tissue, NO generated from L-Arg by LPS-induced NOS activity can be stored as protein-bound DNIC in non-endothelial cells. Upon addition of NAC, low molecular weight DNIC are released from these storage sites and induce vascular relaxation probably through guanylyl cyclase activation.
Acetylcysteine↗