Relationship between extra and intracellular sources of calcium and the contractile effect of thiopental in rat aorta

To evaluate the relationship between the vasocontractile effect of thiopental and the extra and intracellular sources of Ca2+, we analyzed both the contractile effect of the barbiturate on rat aortic rings and its ability to modify the intracellular calcium concentration in cultured rat aorta smooth muscle cells. Thiopental (10-310 microg/mL) contracted aortic rings only in the presence of extracellular Ca2+, and this effect was not blocked by verapamil or diltiazem. On the contrary, Ca2+ (0.1-3.1 mM) evoked contractions only when thiopental (100 microg/mL) was present. Although in calcium-free solution thiopental (100 microg/mL) did not contract aortic rings, it abolished the contractile effect of either phenylephrine (10(-6) M) or caffeine (10 mM). Finally, thiopental augmented the intracellular calcium concentration in cultured smooth muscle cells incubated either in the presence or absence of calcium. In conclusion, thiopental's vasocontractile effect depends on extracellular calcium influx, which is independent of L-calcium channels. The increase in intracellular Ca2+ concentration elicited by thiopental in Ca2+-free solution and its ability to block the effect of phenylephrine and caffeine suggest that this barbiturate can deplete intracellular pools of calcium. Therefore, the calcium entry pathway associated with the contractile effect of thiopental may correspond to the capacitative calcium entry model.     

Endothelial dysfunction is induced by proinflammatory oxidant hypochlorous acid

The myeloperoxidase (MPO)-derived oxidant hypochlorous acid (HOCl) plays a role in tissue injury under inflammatory conditions. The present study tests the hypothesis that HOCl decreases nitric oxide (NO) bioavailability in the vasculature of Sprague-Dawley rats. Aortic ring segments were pretreated with HOCl (1-50 microM) followed by extensive washing. Endothelium-dependent relaxation was then assessed by cumulative addition of acetylcholine (ACh) or the calcium ionophore A23187. HOCl treatment significantly impaired both ACh- and A23187-mediated relaxation. In contrast, endothelium-independent relaxation induced by sodium nitroprusside was unaffected. The inhibitory effect of HOCl on ACh-induced relaxation was reversed by exposure of ring segments to L-arginine but not D-arginine. In cellular studies, HOCl did not alter endothelial NO synthase (NOS III) protein or activity, but inhibited formation of the NO metabolites nitrate (NO3(-) and nitrite (NO2(-). The reduction in total NO metabolite production in bovine aortic endothelial cells was also reversed by addition of L-arginine. These data suggest that HOCl induces endothelial dysfunction via modification of L-arginine.     

Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation

Nitric oxide (NO) is synthesized from l-arginine by the Ca(2+)/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca(2+) ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline. Three CaMK II inhibitors, polypeptide 281-302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca(2+)-dependent activation of eNOS.     

Alteration of p66shc is associated with endothelial dysfunction in the abdominal aortic coarctation of rats

To examine the role of p66shc in endothelial dysfunction, we investigated the endothelium-dependent relaxation, protein expression and superoxide production in abdominal aortic coarctation rats. Endothelium-dependent relaxation to acetylcholine was impaired only in the aortic segments above the aortic coarctation (35.0+/-7.1% vs. 86.6+/-6.0% for sham control at 1 microM Ach). The aortic segments exposed to increased blood pressure showed a decreased phosphorylation of endothelial nitric oxide synthase, an increased phosphorylation of p66shc, and an increased superoxide production. Angiotensin II elicited a significantly increased phosphorylation of p66shc in the endothelial cells. Taken together, these findings suggest that the increased phosphorylation of p66shc is one of the important mediators in the impaired endothelium-dependent relaxation of aortic coarctation rats.     

The role of nitric oxide synthase-derived reactive oxygen species in the altered relaxation of pulmonary arteries from lambs with increased pulmonary blood flow

Congenital cardiac defects associated with increased pulmonary blood flow (Q(p)) produce pulmonary hypertension. We have previously reported attenuated endothelium-dependent relaxations in pulmonary arteries (PA) isolated from lambs with increased Q(p) and pulmonary hypertension. To better characterize the vascular alterations in the nitric oxide-superoxide system, 12 fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). Twin lambs served as controls. PA were isolated from these lambs at 4-6 wk of age. Electron paramagnetic resonance spectroscopy on fourth-generation PA showed significantly increased superoxide anion generation in shunt PA that were decreased to control levels following inhibition of nitric oxide synthase (NOS) with 2-ethyl-2-thiopseudourea. Preconstricted fifth-generation PA rings were relaxed with a NOS agonist (A-23187), a nitric oxide donor [S-nitrosyl amino penicillamine (SNAP)], polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), or H(2)O(2). A-23187-, PEG-SOD-, and H(2)O(2)-mediated relaxations were impaired in shunt PA compared with controls. Pretreatment with PEG-SOD significantly enhanced the relaxation response to A-23187 and SNAP in shunt but not control PA. Inhibition of NOS with nitro-L-arginine or scavenging superoxide anions with tiron enhanced relaxation to SNAP and inhibited relaxation to PEG-SOD in shunt PA. Pretreatment with catalase inhibited relaxation of shunt PA to A-23187, SOD, and H(2)O(2). We conclude that NOS catalyzes the production of superoxide anions in shunt PA. PEG-SOD relaxes shunt PA by converting these anions to H(2)O(2), a pulmonary vasodilator. The redox environment, influenced by the balance between production and scavenging of ROS, may have important consequences on pulmonary vascular reactivity in the setting of increased Q(p).     

Reducing arginase activity via dietary manganese deficiency enhances endothelium-dependent vasorelaxation of rat aorta

L-Arginine is a common substrate for the enzymes arginase and nitric oxide synthase (NOS). Acute inhibition of arginase enzyme activity improves endothelium-dependent vasorelaxation, presumably by increasing availability of substrate for NOS. Arginase is activated by manganese (Mn), and the consumption of a Mn-deficient (Mn-) diet can result in low arginase activity. We hypothesize that endothelium-dependent vasorelaxation is greater in rats fed Mn- versus Mn sufficient (Mn+) diets. Newly weaned rats fed Mn+ diets (0.5 microg Mn/g; n = 12) versus Mn+ diets (45 microg Mn/g; n = 12) for 44 +/- 3 days had (i) lower liver and kidney Mn and arginase activity (P < or = 0.05), (ii) higher plasma L-arginine (P < or = 0.05), (iii) similar plasma and urine nitrate + nitrite, and (iv) similar staining for endothelial nitric oxide synthase in thoracic aorta. Vascular reactivity of thoracic aorta (approximately 720 microm i.d.) and small coronary arteries (approximately 110 microm i.d.) was evaluated using wire myographs. Acetylcholine (ACh; 10(-8)-10(-4) M) produced greater (P < or = 0.05) vasorelaxation in thoracic aorta from Mn- rats (e.g., maximal percent relaxation, 79 +/- 7%) versus Mn + rats (e.g., maximal percent relaxation, 54 +/- 9%) at 5 of 7 evaluated doses. Tension produced by NOS inhibition using N(G) monomethyl-L-arginine (L-NMMA; 10(-3) M) and vasorelaxation evoked by (i) arginase inhibition using difluoromethylornithine (DFMO; 10(-7) M), (ii) ACh (10(-8)-10(-4) M) in the presence of DFMO, and (iii) sodium nitroprusside (10(-9)-10(-4) M) were unaffected by diet. No differences existed between groups concerning these responses in small coronary arteries. These findings support our hypothesis that endothelium-dependent vasorelaxation is greater in aortic segments from rats that consume Mn- versus Mn+ diets; however, responses from small coronary arteries were unaffected.     

Mechanisms of vascular instability in a transgenic mouse model of sickle cell disease

We investigated a transgenic mouse model of sickle cell disease, homozygous for deletion of mouse beta-globin and containing transgenes for human beta(S) and beta(S-antilles) globins linked to the transgene for human alpha-globin. In these mice, basal cGMP production in aortic rings is increased, whereas relaxation to an endothelium-dependent vasodilator, A-23187, is impaired. In contrast, aortic expression of endothelial nitric oxide synthase (NOS) is unaltered in sickle mice, whereas expression of inducible NOS is not detected in either group; plasma nitrate/nitrite concentrations and NOS activity are similar in both groups. Increased cGMP may reflect the stimulatory effect of peroxides (an activator of guanylate cyclase), because lipid peroxidation is increased in aortae and in plasma in sickle mice. Despite increased vascular cGMP levels in sickle mice, conscious systolic blood pressure is comparable to that of aged-matched controls; sickle mice, however, evince a greater rise in systolic blood pressure in response to nitro-L-arginine methyl ester, an inhibitor of NOS. Systemic concentrations of the vasoconstrictive oxidative product 8-isoprostane are increased in sickle mice. We conclude that vascular responses are altered in this transgenic sickle mouse and are accompanied by increased lipid peroxidation and production of cGMP; we suggest that oxidant-inducible vasoconstrictor systems such as isoprostanes may oppose nitric oxide-dependent and nitric oxide-independent mechanisms of vasodilatation in this transgenic sickle mouse. Destabilization of the vasoactive balance in the sickle vasculature by clinically relevant states may predispose to vasoocclusive disease.     

Chronic ouabain treatment increases the contribution of nitric oxide to endothelium-dependent relaxation

The aim of this study was to analyze the contribution of nitric oxide, prostacyclin and endothelium-dependent hyperpolarizing factor to endothelium-dependent vasodilation induced by acetylcholine in rat aorta from control and ouabain-induced hypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omega-nitro-l-arginine methyl esther (L-NAME) inhibited the vasodilator response to acetylcholine in segments from both groups but to a greater extent in segments from ouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higher in segments from ouabain-treated rats. Preincubation with the prostacyclin synthesis inhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacin inhibited the vasodilator response to acetylcholine in aortic segments from both groups. The Ca²⁺-dependent potassium channel blocker charybdotoxin inhibited the vasodilator response to acetylcholine only in segments from control rats. These results indicate that hypertension induced by chronic ouabain treatment is accompanied by increased endothelial nitric oxide participation and impaired endothelium-dependent hyperpolarizing factor contribution in acetylcholine-induced relaxation. These effects might explain the lack of effect of ouabain treatment on acetylcholine responses in rat aorta.     

Involvement of Na(+)/Ca(2+) exchanger in endothelial NO production and endothelium-dependent relaxation

Endothelial nitric oxide (NO) synthase (eNOS) is controlled by Ca(2+)/calmodulin and caveolin-1 in caveolae. It has been recently suggested that Na(+)/Ca(2+) exchanger (NCX), also expressed in endothelial caveolae, is involved in eNOS activation. To investigate the role played by NCX in NO synthesis, we assessed the effects of Na(+) loading (induced by monensin) on rat aortic rings and cultured porcine aortic endothelial cells. Effect of monensin was evaluated by endothelium-dependent relaxation of rat aortic rings in response to acetylcholine and by real-time measurement of NO release from cultured endothelial cells stimulated by A-23187 and bradykinin. Na(+) loading shifted the acetylcholine concentration-response curve to the left. These effects were prevented by pretreatment with the NCX inhibitors benzamil and KB-R7943. Monensin potentiated Ca(2+)-dependent NO release in cultured cells, whereas benzamil and KB-R7943 totally blocked Na(+) loading-induced NO release. These findings confirm the key role of NCX in reverse mode on Ca(2+)-dependent NO production and endothelium-dependent relaxation.     

Inhibitory effect of valves on endothelium-dependent relaxations to calcium ionophore in canine saphenous vein

The present study was designed to evaluate endothelium-dependent relaxation to the calcium ionophore A-23187 in isolated canine saphenous veins. Isometric force recordings and cGMP measurements using isolated veins with and without valves were performed. During contractions to U-46619 (3 x 10(-7) M), endothelium-dependent relaxations to A-23187 (10(-9)-10(-6) M) were significantly reduced in rings with valves compared with rings without valves. Endothelial removal abolished A-23187-induced relaxation. Relaxations to forskolin (FK; 10(-8)-10(-5) M) and diethylaminodiazen-1-ium-1,2-dionate; DEA-NONOate, 10(-9)-10(-5) M) were identical in rings with and without valves. In rings without valves, a nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-4) M), and a cyclooxygenase inhibitor, indomethacin (10(-5) M), partially reduced A-23187-induced relaxation. However, in rings with valves, L-NAME had no effect, whereas indomethacin abolished the relaxation to A-23187. A selective soluble guanylate cyclase inhibitor, 1H-[1,2,4]-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 3x10(-6) M), had no effect on the relaxation to A-23187 in either group. In contrast, ODQ abolished the A-23187-induced increase in cGMP levels, suggesting that relaxation to nitric oxide released by A-23187 is independent of increases in cGMP. These results demonstrate that endothelium-dependent relaxation to A-23187 is reduced in regions of veins with valves compared with relaxation in the nonvalvular venous wall. Lower production of nitric oxide in endothelial cells of valvular segments appears to be a mechanism responsible for reduced reactivity to A-23187.     

Chronic hypertriglyceridemia in young watanabe heritable hyperlipidemic rabbits impairs endothelial and medial smooth muscle function

Several studies have suggested that hypertriglyceridemia is a common risk factor for coronary heart disease. Although increasing serum levels of triglyceride correlate with hypercoagulability, little is known about the contribution of hypertriglyceridemia to vascular function. We successfully segregated two lines of rabbits with genetically-determined severely high (TGH; 2764 +/- 413 mg/dl) and moderately high (TGL; 191 +/- 12 mg/dl) levels of triglyceride, but with comparable levels of total cholesterol, from Watanabe heritable hyperlipidemic rabbits. To determine whether hypertriglyceridemia was involved in alterations of vascular function, we conducted isometric tension studies and analyzed protein expression on thoracic aortic rings isolated from young (3-4 month) TGH, TGL and Japanese White rabbit (JW). No difference in percentage of plaque area in the thoracic aorta was found between TGH and TGL. Relaxing responses, evoked by sodium nitroprusside were similar in JW, TGL and TGH, but endothelium-dependent relaxation to acetylcholine was impaired in TGH compared with JW or TGL (maximal relaxation in JW; 83.5 +/- 2.7%, TGL; 79.9 +/- 5.3%, TGH; 59.1 +/- 5.7%, p<0.05). Relaxation to A23187 was also attenuated in TGH compared with JW, but not significantly different between TGL and JW. Endothelium-independent relaxation elicited by isoproterenol in TGH was significantly decreased compared with JW or TGL (maximal relaxation in JW; 95.2 +/- 2.6% TGL; 91.0 +/- 4.9%, TGH; 75.1 +/- 5.2%, p<0.05). Protein expression of angiotensin II type-1 receptor was increased in TGH and that of nitric oxide synthases-3 was attenuated in TGH compared with TGL. This is the first study showing that endothelium-dependent and -independent vascular relaxation under the condition of combined hyperlipidemia was severely impaired as compared to that under only hypercholesterolemia. These results suggest that hypertriglyceridemia aggravates functional impairment induced by hypercholesterolemia in endothelial and smooth muscle cells.     

Endothelium-dependent relaxation of rat aorta to a histamine H(3) agonist is reduced by inhibitors of nitric oxide synthase, guanylate cyclase and Na,K-ATPase

The possible involvement of different effector systems (nitric oxide synthase, guanylate cyclase, beta-adrenergic and muscarinic cholinergic receptors, cyclooxygenase and lipoxygenase, and Na(+),K(+)-ATPase) was evaluated in a histamine H(3) receptor agonist-induced ((R)alpha-methylhistamine, (R)alpha-MeHA) endothelium-dependent rat aorta relaxation assay. (R)alpha-MeHA (0.1 nM - 0.01 mM) relaxed endothelium-dependent rat aorta, with a pD(2) value of 8.22 +/- 0.06, compared with a pD(2) value of 7.98 +/- 0.02 caused by histamine (50% and 70% relaxation, respectively). The effect of (R)alpha-MeHA (0.1 nM - 0.01 mM) was competitively antagonized by thioperamide (1, 10 and 30 nM) (pA(2) = 9.21 +/- 0.40; slope = 1.03 +/- 0.35) but it was unaffected by pyrilamine (100 nM), cimetidine (1 muM), atropine (10 muM), propranolol (1 muM), indomethacin (10 muM) or nordthydroguaiaretic acid (0.1 mM). Inhibitors of nitric oxide synthase, L-N(G)-monomethylarginine (L-NMMA, 10 muM) and N(G)-nitro-L-arginine methylester (L-NOARG, 10 muM) inhibited the relaxation effect of (R)alpha-MeHA, by approximately 52% and 70%, respectively). This inhibitory effect of L-NMMA was partially reversed by L-arginine (10 muM). Methylene blue (10 muM) and ouabain (10 muM) inhibited relaxation (R)alpha-MeHA-induced by approximately 50% and 90%, respectively. The products of cyclooxygenase and lipoxygenase are not involved in (R)alpha-MeHA-induced endothelium-dependent rat aorta relaxation nor are the muscarinic cholinergic and beta-adrenergic receptors. The results also suggest the involvement of NO synthase, guanylate cyclase and Na(+),K(+)-ATPase in (R)alpha-MeHA-induced endothelium-dependent rat aorta relaxation.     

Role of hydrogen peroxide in ACh-induced dilation of human submucosal intestinal microvessels

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several mediators of vasodilation, which include prostacyclin (PGI(2)), nitric oxide, and endothelium-derived hyperpolarizing factor (EDHF). We have recently defined the role of nitric oxide and PGI(2) in the dilation of submucosal intestinal arterioles from patients with normal bowel function. However, significant endothelium-dependent dilator capacity to ACh remained after inhibiting both these mediators. The current study was designed to examine the potential role of EDHF in human intestinal submucosal arterioles. ACh elicited endothelium-dependent relaxation in the presence of inhibitors of nitric oxide synthase and cyclooxygenase (23 +/- 10%, n = 6). This ACh-induced relaxation was inhibited and converted to constriction by catalase (-53 +/- 10%, n = 6) or KCl (-30 +/- 3%, n = 7), whereas 17-octadecynoic acid and 6-(2-propargylloxyphenyl) hexanoic acid, two inhibitors of cytochrome P450 monooxygenase, had no significant effect (3 +/- 1% and 20 +/- 8%, n = 5, respectively). Exogenous H(2)O(2) elicited dose-dependent relaxation of intact microvessels (52 +/- 10%, n = 7) but caused frank vasoconstriction in arterioles denuded of endothelium (-73 +/- 8%, n = 7). ACh markedly increased the dichlorofluorescein fluorescence in intact arterioles in the presence of nitric oxide synthase and cyclooxygenase inhibitors compared with control and compared with catalase-treated microvessels (363.6 +/- 49, 218.8 +/- 10.6, 221.9 +/- 27.9, respectively, P < 0.05 ANOVA, n = 5 arbitrary units). No changes in the dichlorofluorescein fluorescence were recorded in vessels treated with ACh alone. These results indicate that endothelial production of H(2)O(2) occurs in response to ACh in human gut mucosal arterioles but that H(2)O(2) is not an EDHF in this tissue. Rather, we speculate that it stimulates the release of a chemically distinct EDHF.     

Epigallocatechin gallate preserves endothelial function by reducing the endogenous nitric oxide synthase inhibitor level

Asymmetric dimethylarginine (ADMA), the endogenous nitric oxide synthase inhibitor, is thought to be a key factor contributing to endothelial dysfunction. Tea catechins can cause an endothelium-dependent vasorelaxation. The present study examined the effect of epigallocatechin gallate (EGCG), the major component of tea catechins, on endothelial dysfunction induced by native low density lipoprotein (LDL) in rats and oxidized LDL (ox-LDL) in cultured endothelial cells, and whether the protective effect of EGCG is related to reduction of ADMA level. A single injection of LDL (4 mg x kg(-1), i.v.) markedly reduced endothelium-dependent relaxation and the serum nitrite/nitrate (NO) level, and increased serum concentrations of ADMA, malondialdehyde (MDA), and tumor necrosis factor-alpha (TNF-alpha). EGCG (10 or 50 mg x kg(-1), i.p.) significantly attenuated the inhibition of vasodilator response to acetylcholine and the decreased serum nitrite/nitrate level, and reduced the elevated levels of ADMA, MDA, and TNF-alpha. Exposure of endothelial cells to ox-LDL (100 microg x mL(-1)) for 24 h markedly increased the medium levels of lactate dehydrogenase (LDH), ADMA, TNF-alpha, and MDA, and decreased the level of nitrite/nitrate in the medium and the activity of dimethylarginine dimethylaminohydrolase (DDAH) in the endothelial cells. EGCG (10 and 100 microg x mL(-1)) significantly decreased the levels of LDH, ADMA, TNF-alpha, and MDA, and increased the level of nitrite/nitrate and the activity of DDAH. These results suggest that EGCG protects endothelial dysfunction induced by native LDL in vivo or by ox-LDL in endothelial cells, and the protective effect of EGCG on the endothelium is related to decrease in ADMA level via increasing of DDAH activity.     

Chronic exercise training improves ACh-induced vasorelaxation in pulmonary arteries of pigs.

We hypothesized that exercise training would lead to enhanced endothelium-dependent vasodilation in porcine pulmonary arteries. Pulmonary artery rings (2- to 3-mm OD) were obtained from female Yucatan miniature swine with surgically induced coronary artery occlusion (ameroid occluder). Exercise training was performed for 16 wk, and vasomotor responses were studied by using standard isometric techniques. Contractile responses to 80 mM KCl, isosmotic KCl (10-100 mM), and norepinephrine (10(-8) to 10(-4) M) did not differ between sedentary (Sed) and exercise-trained (Ex) pigs. Relaxation was assessed to endothelium-dependent and endothelium-independent vasodilators after norepinephrine contraction. Pulmonary arteries of Ex pigs exhibited greater maximal relaxation to ACh (61.9 +/- 3.5%) than did those of Sed pigs (52.3 +/- 3.9%; P < 0.05). Endothelium-independent relaxation to sodium nitroprusside did not differ. Inhibition of nitric oxide synthase significantly decreased acetylcholine-induced relaxation, with greater inhibition in arteries from Ex pigs (P < 0.05). Inhibition of cyclooxygenase enhanced relaxation to acetylcholine in arteries from Sed pigs. We conclude that exercise training enhances endothelium-dependent (ACh-mediated) vasorelaxation in pulmonary arteries by mechanisms of increased reliance on nitric oxide and reduced production of a prostanoid constrictor.     

Sphingomyelinase causes endothelium-dependent vasorelaxation through endothelial nitric oxide production without cytosolic Ca(2+) elevation

Neutral sphingomyelinase (N-SMase) elevated nitric oxide (NO) production without affecting intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells in situ on aortic valves, and induced prominent endothelium-dependent relaxation of coronary arteries, which was blocked by N(omega)-monomethyl-L-arginine, a NO synthase (NOS) inhibitor. N-SMase induced translocation of endothelial NOS (eNOS) from plasma membrane caveolae to intracellular region, eNOS phosphorylation on serine 1179, and an increase of ceramide level in endothelial cells. Membrane-permeable ceramide (C(8)-ceramide) mimicked the responses to N-SMase. We propose the involvement of N-SMase and ceramide in Ca(2+)-independent eNOS activation and NO production in endothelial cells in situ, linking to endothelium-dependent vasorelaxation.     

Inhibition by triptoquinone-A of LPS- and IL-1β-primed induction of no synthase in rat thoracic aorta

We investigated the effect of triptoquinone-A (TQA), an active principal of Triptergium wilfordii, on the induction of nitric oxide synthase (NOS) promoted by endotoxin (LPS) and interleukin-1β (IL-1β). Prophylactic application of TQA selectively prevented LPS-primed initiation of L-arginine (Arg)-induced relaxation, and cGMP formation of rat thoracic aorta, and LPS-stimulated nitrite production by cultured aortic smooth muscle cells, which appear to be mediated by NOS expressed by LPS in vascular smooth muscle. TQA also prevented IL-1β-triggered initiation of Arg-induced relaxation and nitrite accumulation. These results suggest that TQA prevents LPS- or IL-1β-promoted induction of NOS in vascular smooth muscle, thus inhibiting development of Arg-induced vasorelaxation.     

Restoration of endothelin-1-induced impairment in endothelium-dependent relaxation by interleukin-10 in murine aortic rings

Endothelin-1 (ET-1) is implicated in the development of endothelial dysfunction through the generation of reactive oxygen species by NADPH oxidase activation. Interleukin-10 (IL-10) is an antiinflammatory cytokine that stimulates nitric oxide production, decreases superoxide production, and restores endothelial integrity after vascular injury. In this study, we tested whether IL-10 attenuates ET-1-induced endothelial dysfunction by improving acetylcholine (ACh)-induced relaxation of cultured murine aortic rings. Aortic rings (2 mm long) of C57BL/6 mice were incubated in 2 mL DMEM containing 120 U/mL penicillin and 120 mug/mL streptomycin in the presence of one of 4 treatments: vehicle (deionized water), ET-1 (100 nmol/L), recombinant mouse IL-10 (300 ng/mL), or a combination of both ET-1 and IL-10. After incubation at 37 degrees C for either 1 or 6 h (short-term exposure) or 22 h (overnight exposure), rings were mounted in a wire myograph and stretched to a passive force of 5 mN. Endothelium-dependent vasorelaxation was assessed by constructing cumulative concentration-response curves to ACh (0.001-10 mumol/L) during 10 mumol/L phenylephrine (PE)-induced contraction. Short-term exposure of ET-1 did not result in an impairment of ACh-induced relaxation. Overnight exposure of aortic rings to ET-1 resulted in a statistically significant endothelial dysfunction characterized by a reduced maximal relaxation response to ACh compared with that of untreated rings (Emax 57% +/- 3% versus 82% +/- 4%). IL-10 treatment restored ACh-induced relaxation (Emax 77% +/- 3%). Western blotting showed decreased eNOS expression in response to ET-1, whereas vessels treated with a combination of ET-1 and IL-10 showed increased expression of eNOS. Immunohistochemical analysis showed decreased eNOS expression in ET-1-treated vessels compared with those treated with both ET-1 and IL-10. We conclude that, in murine aorta, the antiinflammatory cytokine IL-10 prevents impairment in endothelium-dependent relaxation induced in response to long-term incubation with ET-1 via normalization of eNOS expression.