Botulinolysin, a thiol-activated hemolysin produced by Clostridium botulinum, inhibits endothelium-dependent relaxation of rat aortic ring

The effects of botulinolysin (Blyn), a thiol-activated hemolysin produced by Clostridium botulinum, on contractility of rat aortic ring were studied in order to clarify an underlying mechanism of vasoconstriction by the toxin observed previously as an increase in perfusion pressure in isolated rat organs. Blyn (30 hemolytic units/ml; HU/ml) itself did not elicit any apparent change in resting tension of the ring. Contractile tension elicited by a high concentration of phenylephrine in endothelium-intact rings increased significantly after treatment with Blyn (30 HU/ml), while phenylephrine-induced contraction of endothelium-denuded rings was not influenced by toxin treatment. In rings with intact endothelium, acetylcholine (ACh)-induced relaxation was significantly inhibited after treatment with Blyn (30, 10, 1 HU/ml). In contrast, relaxation of denuded rings by sodium nitroprusside was not affected by toxin treatment (30 HU/ml). Arginine (10(-4) M) partly reversed the inhibition of ACh-induced relaxation by the toxin (1 HU/ml). Endothelium-dependent relaxation by histamine or adenosine triphosphate was also inhibited by Blyn (1 HU/ml), but the relaxation elicited by calcium ionophore A23187 was not influenced by the toxin. The results indicate that Blyn acts on endothelium and inhibits agonist-induced endothelium-dependent relaxation of blood vessels.     

NG-monomethyl-L-arginine causes nitric oxide synthesis in isolated arterial rings: trouble in paradise

Arginine analogs are commonly used as inhibitors of the synthesis of endothelium-derived relaxing factor, nitric oxide. However, their effect on nitric oxide levels is rarely measured. Using a chemiluminescence assay for nitric oxide, we found that NG-monomethyl-L-arginine enhanced, rather than reduced, nitric oxide synthesis in pulmonary arterial and aortic rings. NG-monomethyl-L-arginine inhibited relaxation to the endothelium-dependent vasodilator A23187 in aortic but not pulmonary arterial rings. In contrast, N omega-nitro-L-arginine did not stimulate nitric oxide synthesis and it inhibited relaxation to A23187 in all rings. We conclude that NG-monomethyl-L-arginine is a partial agonist for nitric oxide synthesis.     

Endothelial dysfunction in cerebral vessels following carotid artery infusion of phorbol ester in rabbits: the role of polymorphonuclear leukocytes

The effect of 4 beta-phorbol-12 beta-myristate-13 alpha-acetate (PMA) on endothelium-dependent and endothelium-independent vasoconstriction and vasodilation was studied in isolated segments of rabbit middle cerebral artery (MCA). Concentration-dependent responses of the left and right MCA to the constrictors KCl, noradrenaline, uridine 5'-triphosphate, serotonin, and histamine, as well as to the dilators acetylcholine, bradykinin, sodium nitroprusside, and calcium ionophore (A23187), were compared in control animals and after PMA injection into the left common carotid artery. In the control animals there was no significant difference in the responses of the left and right MCA to either the constrictors or the dilators studied. After PMA injection the endothelium-dependent relaxation in response to acetylcholine, bradykinin, and A23187 was reduced in the left MCA (PMA-injected side), whereas the effect of the endothelium-independent dilator sodium nitroprusside remained unchanged. Simultaneously greater contractile responses of the left MCA to serotonin and histamine were obtained. Neither infusion of L-arginine in vivo before the PMA injection nor incubation of the isolated MCA segments with L-arginine affected this difference in MCA reactivity. Platelet depletion did not change the PMA-induced reduction in the endothelium-dependent relaxation, whereas after leukocyte depletion this reduction practically disappeared. These results suggest that the PMA-induced brain microembolia causes acute endothelial dysfunction, which is possibly mediated by intravascular activation of leukocytes and is independent of nitric oxide synthesis from L-arginine. This phenomenon might play an important role in cerebral angiospastic disorders after intravascular activation of leukocytes in cerebral ischemia and reperfusion.     

Non-anticoagulant heparin increases endothelial nitric oxide synthase activity: role of inhibitory guanine nucleotide proteins

Heparin, which is widely used clinically, has recently been shown to have specific properties affecting the vascular endothelium. We hypothesized that heparin stimulates endothelial nitric oxide synthase (eNOS) activity by a mechanism independent of its anticoagulant properties and dependent on an inhibitory guanine nucleotide regulatory protein (Gi). We determined the effect of both heparin and N-acetyl heparin (Non-Hep), a heparin derivative without anticoagulant properties, on eNOS activity in cultured bovine aortic endothelial cells and on endothelium-dependent relaxation in isolated vascular rings. The eNOS activity was determined by measuring both citrulline and nitric oxide (NO) metabolite formation. Heparin and Non-Hep dose-dependently increased basal eNOS activity (ED50 1.0 microgram/ml or 0.15 U/ml), an effect that was significantly inhibited by pertussis toxin (100 ng/ml), a Gi-protein inhibitor. Agonist-stimulated (acetylcholine, 10 microM) eNOS activity was potentiated following pre-treatment with both heparin and Non-Hep and reversed by pertussis toxin. Heparin and Non-Hep induced a dose-dependent relaxation in preconstricted thoracic aortic rings, an effect that was significantly inhibited by pertussis toxin, endothelial inactivation (following treatment with sodium deoxycholate) and NG-nitro-L-arginine-methyl ester (L-NAME). We conclude that heparin and non-anticoagulant heparin induce endothelium-dependent relaxation following activation of eNOS by a mechanism involving a Gi-protein. Administration of heparin derivatives without anticoagulant properties may have therapeutic implications for the preservation of eNOS in conditions characterized by endothelial dysfunction.     

Constitutive nitric oxide synthase is expressed and nitric oxide-mediated relaxation is preserved in retrieved human aortocoronary vein grafts

Although little is known about the endothelial cell function of human saphenous vein coronary artery bypass grafts, there is evidence to suggest that receptor-activated, endothelial-dependent relaxation mediated by nitric oxide is impaired. This study examines the expression and function of endothelial cell constitutive nitric oxide synthase (cNOS) of aortocoronary vein bypass grafts and human saphenous veins obtained from 10 patients undergoing repeat coronary artery bypass grafting for recurrent ischemic symptoms. Following precontraction with norepinephrine (10(-5) M), responses to acetylcholine (receptor-mediated, endothelium-dependent), calcium ionophore (A23187; receptor-independent, endothelium-dependent), and sodium nitroprusside (endothelium-independent) were assessed. Following total RNA extraction using phenol/guanidinium isothiocyanate from specimens of human saphenous vein and vein graft, a quantitative RNase Protection Assay (RPA) was performed using a cRNA riboprobe corresponding to a fragment of the human endothelial cell cNOS gene. Histologically, the vein grafts showed both intimal hyperplasia development and focal atherosclerosis formation compared to the saphenous veins. Scanning electron microscopy of the saphenous veins and the vein grafts showed an intact endothelium. Precontracted vein grafts did not relax in response to acetylcholine; in contrast, the saphenous vein relaxed in a dose-dependent manner to reach a maximal relaxation of 19 +/- 4% precontracted tension. Saphenous veins and vein grafts relaxed in response to A23187 with maximal relaxation of 92 +/- 5 and 73 +/- 13%, respectively. Both vessels relaxed in a dose dependent manner to sodium nitroprusside. RPA normalized to beta-actin showed similar levels of expression of endothelial cell cNOS equivalent to 1 pg of sense RNA in both the saphenous vein and vein graft.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional differences in endothelial function in horse lungs: possible role in blood flow distribution?

We investigated regional differences of in vitro responses of pulmonary arteries (6-mm OD) from the dorsocaudal (top) and cranioventral (bottom) lung regions to endothelium-dependent vasodilators (methacholine, bradykinin, and calcium ionophore A-23187). Methacholine relaxed endothelium-intact top vessels; however, in bottom vessels, a small relaxation preceded a profound contraction. In top vessels, removal of endothelial cells converted relaxation to contraction, and in bottom vessels it abolished relaxation and enhanced contraction. Bradykinin and A-23187 were more potent and caused greater endothelium-mediated relaxation in top than in bottom arteries. The endothelium-independent vasodilator sodium nitroprusside caused similar relaxations in all rings. Nomega-nitro-L-arginine and NG-monomethyl-L-arginine and methylene blue abolished relaxation of top and bottom arteries to methacholine; meclofenamate had little effect. We conclude that regional differences in endothelium-mediated relaxation are caused by differences in the magnitude of the endothelial release of nitric oxide. Similar differences in endothelium-dependent flow-mediated vasodilation and endothelial nitric oxide release may result in preferential perfusion of caudodorsal lung regions.     

Differential effects of low- and high-dose estrogen treatments on vascular responses in female rats

In an attempt to study the mechanisms by which estrogens affect vascular responses, we utilized aortic preparations from intact and ovariectomized female rats receiving low- and high-dose subcutaneous estrogen treatments. Oil-treated, as well as male rats, served as controls. In ovariectomized females, low-dose 17-beta-estradiol injections (5 microg/kg daily for two days) affected the basal release of nitric oxide, as evaluated by concentration-related curves to superoxide dismutase and N(G)-Methyl-L-arginine acetate, which was found to be greater in 17-beta-estradiol-treated females compared to oil-treated females or males. Conversely, the nitric oxide-related vascular relaxation evoked by acetylcholine and sodium nitroprusside was unchanged. Prostacyclin production was also evaluated. Aortic rings from ovariectomized 17-beta-estradiol-treated females released significantly more prostacyclin than those from oil-treated females. These results point out a possible role for nitric oxide and prostacyclin in the vascular protection brought about by physiological levels of estrogens. When intact females were treated with high doses of ethynilestradiol (100 microg/Kg daily for one month), a component of contraceptive pills, either the basal release of nitric oxide, or acetylcholine-induced relaxation underwent a significant decrease. Likewise, the relaxant responses to sodium nitroprusside were impaired in the aortic rings obtained from ethynilestradiol-treated animals when compared to controls. Similarly, the amount of prostacyclin released from aortic tissues obtained from ethynilestradiol-treated animals was significantly reduced. These results may provide a possible explanation for the higher incidence of cardiovascular disease in women who take contraceptive preparations containing high doses of estrogens.     

Vascular hyporesponsiveness in aortic rings from cirrhotic rats: role of nitric oxide and endothelium

1. The role of nitric oxide as mediator of the vascular alterations present in different models of experimental liver cirrhosis is controversial. In the present study, we evaluated the role of nitric oxide and that of the endothelium in the response to phenylephrine and acetylcholine of isolated aortic rings from chronic bile duct-ligated (29 days) rats and their corresponding controls. Experiments were performed in rings with or without endothelium, in rings pretreated with N-omega-nitro-L-arginine methyl ester (10(-4) mol/l) to inhibit nitric oxide synthesis and in rings pretreated with aminoguanidine (10(-4) mol/l) to inhibit inducible nitric oxide synthesis. 2. Under basal conditions, the maximum absolute tension developed in response to cumulative addition of phenylephrine was significantly decreased in rings from bile duct-ligated animals (1.62 +/- 0.06 g) compared with the control rings (2.15 +/- 0.099). This hyporesponsiveness to phenylephrine of rings from bile duct-ligated animals was corrected after treatment with N-omega-nitro-L-arginine methyl ester and reduced, but not completely eliminated, in rings without endothelium. In contrast, aminoguanidine did not modify the lower response to phenylephrine rings from bile duct-ligated animals. ED50 values were not different between groups under any experimental conditions. 3. The endothelium-dependent vasodilatation to acetylcholine in phenylephrine-constricted rings was similar in both groups of animals, control and bile duct ligated, under all experimental conditions. N-omega-nitro-L-arginine methyl ester pretreatment and removal of the endothelium completely abolished the response to acetylcholine in cirrhotic and control rings. 4. These results demonstrate that in aortic rings from cirrhotic, bile duct-ligated rats, increased production of nitric oxide, mainly of endothelial origin, is responsible for the lower contractile response to phenylephrine. Our data, however, do not support the involvement of the inducible nitric oxide synthase isoform in this alteration. In contrast, endothelial vasodilatory response to acetylcholine is not altered in this model of cirrhosis, which indicates that not all mechanisms of nitric oxide release are abnormal.     

Smoking impairs the activity of endothelial nitric oxide synthase in saphenous vein

Smoking impairs the endothelium-dependent relaxation of arteries and veins, with the maximum relaxation in response to the calcium ionophore A23187 of saphenous vein rings being reduced from 53 +/- 4% in nonsmokers to 27 +/- 5% in smokers. We have investigated whether this endothelial dysfunction was attributable to altered activity or concentration of nitric oxide synthase (NOS). The concentration of NOS in saphenous vein endothelium, determined by Western blotting and immunohistochemistry, was not different in nonsmokers and smokers. Nitrite production from vein strips stimulated with A23187 was higher in nonsmokers (median 23.6 nmol.cm-2.h-1) than smokers (median 3.3 nmol.cm-2.h-1), P=.001, this difference being abolished when vein strips were preincubated in the presence of NG-monomethyl-L-arginine. Organ chamber studies to monitor the endothelium-dependent relaxation of vein rings in response to A23187 showed that preincubation of rings from smokers with either L-arginine (3mmol/L) or superoxide dismutase (250 U/mL) did not improve the maximum relaxation. In contrast, preincubation of vein rings from smokers with 20 micromol/L tetrahydrobiopterin increased the maximum relaxation from 27 +/- 5% to 51 +/- 6%, P=.01. Preincubation of vein from smokers with tetrahydrobiopterin also significantly increased nitrite and cGMP production in response to stimulation with A23187. The impaired endothelium-dependent relaxation of saphenous vein rings from smokers appears to be caused by a reduction in the activity of endothelial NOS that is attributable to an inadequate supply of the coenzyme tetrahydrobiopterin.     

Atherosclerosis, vascular remodeling, and impairment of endothelium-dependent relaxation in genetically altered hyperlipidemic mice

We examined the vascular structure and endothelium-dependent relaxation in two genetic models of hypercholesterolemia: apolipoprotein E (apoE)-knockout mice and combined apoE/LDL receptor-double-knockout mice. Intimal area was increased markedly in proximal segments of thoracic aortas from apoE/LDL receptor-knockout mice [0.13 +/- 0.03 (mean +/- SE) mm2] compared with normal (C57BL/6J) mice (0.002 +/- 0.002 mm2, P < .05). Despite intimal thickening, the vascular lumen was not smaller in the aortas of apoE/LDL receptor-knockout mice (0.52 +/- 0.03 mm2) than in normal mice (0.50 +/- 0.03 mm2). In apoE-deficient mice, intimal thickening was minimal or absent, even though the concentration of plasma cholesterol was only modestly less than that in the double-knockout mouse (14.9 +/- 1.1 vs 18.0 +/- 1.2 mmol/L, respectively, P < .05). Relaxation of the aorta was examined in vitro in vascular rings precontracted with U46619. In normal mice, acetylcholine produced relaxation, which was markedly attenuated by the nitric oxide synthase inhibitor NG-nitro-L-arginine (100 microM). Relaxation to acetylcholine and the calcium ionophore A23187 was normal in apoE-deficient mice (in which lesions were minimal) but greatly impaired in the proximal segments of thoracic aortas of apoE/LDL receptor-deficient mice, which contained atherosclerotic lesions. Vasorelaxation to nitroprusside was similar in normal and apoE-knockout mice, with modest but statistically significant impairment in atherosclerotic segments of apoE/LDL receptor-knockout mice. In distal segments of the thoracic aorta of apoE/LDL receptor-deficient mice, atherosclerotic lesions were minimal or absent, and the endothelium-dependent relaxation to acetylcholine and calcium ionophore was normal. Thus, in apoE/LDL receptor-knockout mice (a genetic model of hyperlipidemia), there is vascular remodeling with preservation of the aortic lumen despite marked intimal thickening, with impairment of endothelium-dependent relaxation to receptor- and nonreceptor-mediated agonists. Atherosclerosis may be accelerated in the apoE/LDL receptor-double-knockout mouse compared with the apoE-knockout strain alone. We speculate that other factors, such as the absence of LDL receptors, may contribute to the differences in the extent of atherosclerosis in these two models of hyperlipidemia.     

Involvement of nitric oxide and potassium channels in the reduction of basal tone produced by blockade of thromboxane A2/prostaglandin H2 receptors in aortic rings of hypertensive rats

This study was designed to investigate involvement of potassium channels in the action of nitric oxide facilitating reduction of basal tone by thromboxane A2/prostaglandin H2 receptor blockade with ifetroban in rings of thoracic aorta taken from rats with aortic coarctation-induced hypertension. Ifetroban-induced reduction of basal tone in aortic rings without drug pretreatment was attenuated (P<0.05) in rings pretreated with the nitric oxide synthesis inhibitor N(omega-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-4) mol/L; 0.55+/-0.09 g versus 0.23+/-0.07 g). The vasorelaxing effect of ifetroban also was decreased (P<0.05) in preparations pretreated with a potassium channel blocker, either tetraethylammonium (TEA; 10(-2) mol/L) or 4-aminopyridine (4-AP; 3 x 10(-3) mol/L). Ifetroban-induced reduction of basal tone was not attenuated in preparations pretreated first with L-NAME and then with sodium nitroprusside (SNP; 6+/-1 nmol/L) to compensate for the loss of endogenous nitric oxide. However, the facilitatory effect of SNP on ifetroban-induced relaxation of aortic rings pretreated with L-NAME alone was not demonstrable in rings pretreated with L-NAME plus TEA or 4-AP. These observations suggest that a mechanism involving nitric oxide and potassium channels facilitates the reduction in basal tone produced by ifetroban in aortic rings of rats with aortic coarctation-induced hypertension.     

Impaired endothelium-dependent relaxation after coronary reperfusion injury: evidence for G-protein dysfunction

This study was done to determine whether abnormal receptor-dependent release of endothelium-derived relaxing factor (EDRF) might be caused by G-protein dysfunction. Dogs were exposed to global myocardial ischemia (45 minutes, induced by aortic cross-clamping) followed by reperfusion (60 minutes) while on cardiopulmonary bypass, and coronary arteries were then studied in vitro in organ chamber experiments. After reperfusion, endothelium-dependent relaxation to the receptor-dependent agonists adenosine diphosphate and acetyl-choline was significantly impaired as well as to sodium fluoride, which acts on a pertussis toxin-sensitive G-protein. In contrast, endothelium-dependent relaxations to the receptor-independent agonists A23187 and phospholipase C were normal. Furthermore, endothelium-dependent relaxation to poly-L-arginine (molecular weight, 139,200), which appears to induce endothelium-dependent relaxation of the canine coronary artery by a nonnitric oxide pathway, was unaffected by ischemia and reperfusion. These experiments suggest that global myocardial ischemia and reperfusion selectively impair receptor-mediated release of EDRF (nitric oxide) but that the ability of the endothelial cell to produce EDRF or generate endothelium-dependent relaxation to nonnitric oxide-dependent agonists remains intact. We hypothesize that coronary reperfusion injury leads to G-protein dysfunction in the endothelium.     

The use and abuse of routine stool microbiology: a College of American Pathologists Q-probes study of 601 institutions

BACKGROUND/AIMS: In the present study we have evaluated the role of nitric oxide and prostaglandins in the renal vascular response to a vasoconstrictor (methoxamine) and to endothelium-dependent (acetylcholine) and independent (sodium nitroprusside) vasodilators. METHODS: The experiments were performed in isolated and perfused kidneys of portal vein ligated and sham rats under various treatments. RESULTS: Baseline renal perfusion pressure was lower in the portal vein ligated than in the sham group (37.2 +/- 2.6 vs 48.4 +/- 2.5 mmHg). Indomethacin (10(-5)M) did not modify baseline renal perfusion pressure in any group, but the nitric oxide inhibitor N(W)-Nitro-L-Arginine (10(-4) M) increased it in both sham and portal vein ligated kidneys, but without abolishing the differences between them. The vasoconstrictor renal response to methoxamine was blunted in portal vein ligated rats compared to controls. Indomethacin did not modify this renal hyporesponsiveness, but N(W)-Nitro-L-Arginine completely abolished it. In another set of experiments, both acetylcholine and nitroprusside caused dose-dependent vasodilation in kidneys, preconstricted with methoxamine, from sham and portal vein ligated rats, and there were no significant differences between them. N(W)-Nitro-L-Arginine reduced acetylcholine-induced vasodilation and did not modify the vasodilation evoked by nitroprusside. CONCLUSIONS: These results indicate that the renal vasculature of portal vein ligated rats shows a basal reduction in perfusion resistance that is not related to nitric oxide or prostaglandins. However, increased nitric oxide production interferes with the effects of the alfa-agonist methoxamine. This suggests that nitric oxide plays an important role in the modulation of the renal vascular responses to vasoconstrictors in portal hypertension.     

Acute and chronic inhibition of nitric oxide synthase in conscious rabbits: role of nitric oxide in the control of vascular tone

Acute and chronic effects of Nw-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase, were examined on the hindquarter hemodynamics of conscious rabbits. After pharmacological autonomic reflex blockade on four experimental days (days 0, 1, 2, and 7), responses to aortic occlusion (balloon cuff, 5-80 s inflation), intra-aortic infusion of acetylcholine, adenosine, and sodium nitroprusside (SNP) were measured before and after vehicle (day 0) or L-NNA (16 mg/kg/h i.v., days 1, 2, and 7). On day 1, L-NNA raised the mean arterial pressure (MAP), and lowered the heart rate (HR) and hindquarter vascular conductance (HVC = abdominal aortic Doppler blood flow/MAP). On days 2 and 7, L-NNA only slowly raised the MAP. The dilator response to acetylcholine was inhibited by L-NNA on day 1 and before and after L-NNA on days 2 and 7. The responses to aortic occlusion, adenosine, or SNP infusion were unaffected by L-NNA treatment on any day. Thus, if nitric oxide synthase inhibition by L-NNA abolishes NO release, then (i) reactive hyperaemia is independent of NO, (ii) basal NO release normalises the arterial pressure in the short term but other factors become important in the long term, and (iii) the blockade by L-NNA of receptor-stimulated NO release by acetylcholine is only very slowly reversible.     

Development of endothelium-dependent relaxation in canine coronary collateral arteries

BACKGROUND: Little information exists regarding development of vasomotor control mechanisms during coronary collateral artery maturation. Therefore, we studied endothelium-dependent relaxation of canine collateral arteries isolated 2, 4, and 9 months after placement of an ameroid occluder around the proximal left circumflex coronary artery. RESULTS: Collateral arteries isolated after 2 months exhibited markedly reduced endothelium-dependent relaxation in response to acetylcholine (ACh; 10(-10) to 10(-4) mol/L) and bradykinin (BK; 10(-11) to 10(-6) mol/L) compared with relaxation of noncollateral coronary arteries (P<0.01). In contrast, endothelium-independent relaxation of collateral arteries to nitroprusside was only slightly reduced compared with relaxation of noncollateral arteries (P<0.05). Endothelium-dependent relaxation of collateral arteries isolated after 4 and 9 months was increased significantly, to the extent that relaxation to ACh and BK was not significantly different between collateral and noncollateral arteries at these periods. Inhibition of nitric oxide synthesis with NT-nitro-L-arginine methyl ester (L-NAME; 100 micromol/L) markedly inhibited ACh-induced relaxation in all noncollateral arteries and in collateral arteries isolated after 9 months. However, neither L-NAME nor indomethacin (5 micromol/L) alone inhibited ACh-mediated relaxation of collateral arteries isolated after 4 months. ACh-induced relaxation of these collateral arteries was only inhibited when arteries were preconstricted with 30 mmol/L K+ and pretreated with L-NAME and indomethacin (ie, when synthesis/effects of nitric oxide, prostaglandins, and endothelium-derived hyperpolarizing factor were inhibited). CONCLUSIONS: Development of endothelium-dependent relaxation in canine coronary collateral arteries is not complete after 2 months. After 4 months, endothelium-dependent relaxation of collateral arteries is similar to relaxation of noncollateral arteries, but the relaxation exhibits decreased dependence on synthesis of nitric oxide and increased involvement of prostaglandins and endothelium-derived hyperpolarizing factor(s). After 9 months of development, collateral arteries exhibit normal nitric oxide-dependent relaxation, similar to noncollateral arteries.     

Chronic treatment with losartan ameliorates vascular dysfunction induced by aging in spontaneously hypertensive rats

OBJECTIVE: To evaluate the effects of prolonged treatment with losartan on endothelium-dependent and endothelium-independent relaxations of aortic rings from adult and senescent spontaneously hypertensive rats, and to clarify whether these effects were due to specific mechanisms of the drug or a consequence of its blood-pressure-lowering action. MATERIALS AND METHODS: Adult (aged 5 months) and senescent (aged 20 months) male spontaneously hypertensive rats were treated for 12 consecutive weeks with 10 mg/kg per day losartan. Systolic blood pressure and plasma concentration of nitrates were evaluated. We studied endothelium-dependent and endothelium-independent relaxations and response to angiotensin II of aortic rings from rats of each group. The direct effects of angiotensin II type 1 receptor antagonism on vascular reactivity of aortic rings from untreated adult and senescent rats that had been incubated beforehand with losartan were also studied. RESULTS: Losartan treatment comparably reduced blood pressure and increased plasma concentration of nitrates in rats of both age groups. Responses to acetylcholine and sodium nitroprusside were lower for rings from senescent than they were for rings from adult rats. Constrictor responses to angiotensin II were higher for rings from senescent than they were for rings from adult rats. Treatment with losartan increased the magnitude of relaxations in response to acetylcholine for rings from rats in both groups, but increased the magnitude of relaxations in response to nitroprusside only for rings from senescent spontaneously hypertensive rats. Incubation beforehand of aortic rings from untreated rats with losartan enhanced magnitude of relaxations in response both to acetylcholine and to nitroprusside only for rings from senescent spontaneously hypertensive rats. CONCLUSIONS: The consequences of aging for endothelium-dependent and endothelium-independent relaxations of rings from spontaneously hypertensive rats are ameliorated by losartan treatment, suggesting that angiotensin II plays a role via type 1 receptors. The effects of losartan on senescent spontaneously hypertensive rats were due not only to its blood-pressure-lowering action but also to the blockade of specific mechanisms derived from angiotensin II type 1 receptor antagonism, which might involve an increase in availability of NO.     

Decreased flow-induced dilation and increased production of cGMP in spontaneously hypertensive rats

-We investigated flow (shear stress)- and agonist-induced cGMP release in mesenteric vascular beds of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). The mesenteric vascular bed was perfused in situ with Tyrode's solution. Vascular relaxation and cGMP release in the perfusate were determined on stimulation by flow or by acetylcholine (0.1 micromol/L) or sodium nitroprusside (0.1 mmol/L). Flow-induced release of cGMP was significantly greater in SHR than in WKY (P<0.01), despite a lower flow-induced dilation in SHR. In both strains, NG-nitro-L-arginine methyl ester (L-NAME) completely inhibited cGMP release in response to flow (P<0.001), although flow-induced dilation was not affected by L-NAME in SHR. Moreover, the activity of the constitutive nitric oxide synthase (NOS) was significantly greater in SHR (82+/-3.5 fmol/min) than in WKY (66+/-3.5 fmol/min; P<0.05) and was associated with increased expression of endothelial NOS mRNA in SHR. Sodium nitroprusside induced larger increases in cGMP release in SHR (3593+/-304 fmol/min) than in WKY (2467+/-302 fmol/min; P<0.05). The release of cGMP in response to acetylcholine was significantly lower in SHR (292+/-80 fmol/min) than in WKY (798+/-218 fmol/min; P<0.05) in parallel with smaller acetylcholine-induced relaxation in SHR. Despite increased cGMP production in response to flow and NOS activity, flow-induced dilation was decreased in SHR, suggesting an upregulation of the NO/cGMP pathway to compensate for the increased vascular tone in SHR.     

Impairment of endothelial function induced by glyc-oxidized lipoprotein a [Lp(a)]

Diabetic patients develop endothelial dysfunction early in the course of the disease. Atherogenic lipoproteins such as LDL and Lp(a) are important risk factors for endothelial dysfunction and undergo nonenzymatic glycation in hyperglycaemia. Here we assessed whether glycation of Lp(a) potentiates its damaging influence on endothelial function. Human Lp(a) was glycated by dialyzation for 7 days against buffer containing 200 mmol/l glucose, or sham-treated without glucose and oxidized by incubation with Cu++. The degree of glycation accounted to 32 +/- 4%, and glycation rendered Lp(a) more susceptible to oxidative modification when exposed to Cu++. Isolated rings of rabbit aorta were superfused with physiological salt solution, and isometric tension was recorded. Incubation of the aortic rings with sham-treated or with 30 microg/ml glycated Lp(a), not oxidized, had no influence on acetylcholine-induced, endothelium-dependent relaxation. Exposure of the aortic rings to 30 microg/ml oxidized non-glycated (ox) Lp(a) caused a significant inhibition (19% at 1 microM acetylcholine) of the endothelium-dependent relaxation. Incubation of aortic rings with 30 microg/ml oxidized glycated (glyc-ox) Lp(a) attenuated endothelium-dependent relaxation more potently than oxLp(a) (by 34% at 1 microM acetylcholine). The presence of diethyl-dithio-carbamate (DDC), an inhibitor of the endogenous superoxide dismutase (SOD), potentiated the inhibition of relaxation induced by oxLp(a) and by glyc-oxLp(a) [38% inhibition at 1 microM acetylcholine for oxLp(a), and 49% inhibition at 1 microM acetylcholine for glyc-oxLp(a)]. Co-incubation with the O2- scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid disodium salt (TIRON) prevented the inhibition of relaxation by the oxidized lipoproteins, suggesting that enhanced NO-inactivation by O2- could be the underlying mechanism for the impairment of endothelium-dependent dilations by ox- and glyc-oxLp(a). The concentration of lysophosphatidycholine, a lipoprotein oxidation product and stimulus for O2- formation, was significantly enhanced in oxLp(a) and in glyc-oxLp(a) compared to native lipoproteins. Conclusion: Glycation enhances the endothelium-damaging influence of oxLp(a), presumably by enhancing oxidative stress. The likely mechanism for attenuation of endothelium-dependent dilations is increased formation of O2-, resulting in inactivation of nitric oxide. This mechanism may play an important role in diabetic patients and may contribute to disturbed organ perfusion.     

Diabetes-induced endothelial dysfunction is prevented by long-term treatment with the modified iron chelator, hydroxyethyl starch conjugated-deferoxamine

Oxygen radicals are believed to play a role in vascular complications of diabetes mellitus. In this study, we evaluated whether long-term treatment with an iron chelator and inhibitor of metal-catalyzed hydroxyl radicals (.OH) could prevent diabetes-induced defects in endothelium-dependent relaxation. Diabetes was induced in Sprague-Dawley rats by injection of streptozotocin. At 48 h after streptozotocin, a subgroup of diabetic rats received daily injections of 50 mg/kg hydroxyethyl starch conjugated-deferoxamine (HES-DFO) for a total of 8 weeks. Long-term treatment with HES-DFO did not modify serum insulin or blood glucose taken at the end of the study; however, a modest reduction in glycosylated hemoglobin was present. In precontracted aortic rings suspended in tissue baths, endothelium-dependent relaxation to acetylcholine was impaired in diabetic rings compared with control rings in the presence or absence of indomethacin. Endothelium-independent relaxation to nitroglycerin was unaltered. Long-term treatment with HES-DFO had no effect on relaxation to nitroglycerin but completely prevented the impaired relaxation to acetylcholine in diabetic rings in either the presence or absence of indomethacin. These data suggest that iron-catalyzed .OH formation contributes to the development of diabetes-associated endothelial dysfunction.     

Nitric oxide synthase inhibitor and lipopolysaccharide effects on reactivity of guinea pig airways

The in vivo and in vitro effects of nitric oxide (NO) synthase inhibitors and lipopolysaccharide (LPS) on reactivity of guinea pig airways were examined. In isolated, perfused tracheas from untreated animals, the NO synthase inhibitors, N omega-nitro-L-arginine methyl ester (L-NAME; 10(-4)M), NG-methyl-L-arginine (L-NMMA; 10(-4) M) and aminoguanidine (10(-4) M) had no effect or inhibited reactivity to extraluminally (EL) or intraluminally (IL) applied methacholine and histamine. L-NMMA (10(-4) M) did not appreciably contract resting or metacholine-contracted preparations (+/- 3 x 10(-4) M L-arginine) and L-arginine only weakly relaxed contracted tracheas (+/- L-NMMA). Sodium nitroprusside and S-nitroso-N-penicillamine elicited relaxant responses and were more potent extraluminally than intraluminally. Methylene blue (10(-5) M) antagonized relaxation to sodium nitroprusside. Incubation with Escherichia coli LPS (10 micrograms/ml; 30 min incubation) alone in the EL and IL baths depressed methacholine and histamine concentration-response curves. In the presence of LPS, L-NAME potentiated responses to intraluminally applied methacholine but did not affect responses to extraluminally added methacholine. Four days after i.p. injection of animals with LPS (4 mg/kg), L-NAME potentiated responses to IL methacholine, and L-arginine acquired greater relaxant activity. LPS injection increased sensitivity to intraluminally added but not extraluminally added isoproterenol. LPS given by i.p. injection or by inhalation did not affect basal specific airway resistance of conscious animals or reactivity to methacholine aerosol during a postexposure period of 6 to 72 h. NO seems to have little role in regulating reactivity of guinea pig airways to bronchoconstrictor agonists, except after in vitro or in vivo exposure to LPS. After LPS injection the in vitro changes suggestive of NO synthase induction are not associated with altered airway reactivity to inhaled methacholine.