Endothelium-intact Strips Research Articles

Endothelium-dependent vasodilatory effect of Smilax china Linn. water extract via PI3K/Akt signaling

Objective: The objective of this study was to investigate the pharmacological effect of Smilax china Linn. water extract (SCLWE) on vascular relaxation and its underlying biochemical mechanisms.Methodology: Isolated rat aortic rings were pre-constricted with phenylephrine (PE). This was followed by the cumulative addition of SCLWE. The effect of endothelial nitric oxide and PI3K/Akt on the SCLWE-induced vasodilation was investigated by the pretreatment of endothelium-intact aortic strips with or without NG-nitro-L-arginine methyl ester (L-NAME) or wortmanin before constriction with PE.Results: Treatment of PE (1 μM)-pre-contracted aortic strips with SCLWE induced endothelium-dependent relaxation, which was attenuated by L-NAME and wortmanin. Further studies using HUVECs indicated that nitrite production, eNOS and PI3K/PKB (Akt) phosphorylations were increased after exposure to SCLWE but was attenuated by pretreatment with wortmanin.Conclusion: These results suggest that SCLWE induces vasodilation by augmenting NO production in endothelial cells via PI3K/Akt-dependent eNOS phosphorylation.

Heartworm extract induces relaxation of isolated rat thoracic aorta.

Isolated rat thoracic aortic strips undergoing noradrenaline-induced contraction were treated with an adult heartworm (HW) crude extract and then examined for isometric changes in tension. HW extract caused relaxation of endothelium-intact strips, but not endothelium-denuded strips. This effect was inhibited by treatment with NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) and could be reversed by additional treatment with L-arginine. However, HW extract at a high concentration caused slight relaxation of endothelium-denuded strips, and relaxation persisted after L-NAME treatment in endothelium intact-strips. These data suggested that the relaxation induced by HW extract was mainly endothelium-dependent, nitric oxide-mediated, but in part, also endothelium-independent. In addition, a bioassay using isolated rat thoracic aortas may be a useful tool for investigating vasoactive substances in the HW extract.

Endothelial dysfunction of internal thoracic artery graft in patients with chronic kidney disease

ObjectivesThe present study was designed to evaluate the association between chronic kidney disease and the endothelial function of internal thoracic artery (ITA) grafts in patients undergoing coronary bypass surgery. An isometric tension study was performed in ITA strips obtained during surgery. Concentration-response curves for acetylcholine (ACh) and sodium nitroprusside were constructed in ITA strips partially precontracted with phenylephrine under the inhibition of cyclooxygenase. The integrity of the endothelium was verified histologically by en-face staining of the luminal surface with the use of silver nitrate solution. ResultsIn endothelium-intact ITA strips, ACh produced a concentration-dependent relaxation in patients with glomerular filtration rate (GFR, mL/min/1.73 m2) > 60. A concentration-dependent relaxation response also was observed in patients with GFR 30 to 60, but it was reduced significantly compared with those with GFR > 60. In both groups, removal of endothelium or treatment with nitric oxide (NO) synthase inhibitors almost abolished the ACh-induced relaxation. On the other hand, in patients with GFR < 30, mild contraction rather than relaxation was induced at a high concentration of ACh, which was modified neither by treatment with NO synthase inhibitors nor by removal of the endothelium. Vasodilator responses to sodium nitroprusside were comparable among the 3 groups. The relaxation of endothelium-intact strips to a peak ACh concentration correlated positively with GFR. This relationship held true in a multiple linear regression model, and interaction terms between GFR and other risk factors were not statistically significant. ConclusionsEndothelial function of ITA grafts to release NO is impaired at the time of surgery in patients with chronic kidney disease.

Characteristics of the actions by which 5‐HT affects electrical and mechanical activities in rabbit jugular vein

5-HT is known to be a potent vasospasmogenic agonist in various arteries. However, in veins the vasomodulating actions of 5-HT, and the underlying mechanisms, remain to be fully clarified. Here, we characterized the actions by which 5-HT affects electrical and mechanical activities in the rabbit jugular vein. Membrane potential and isometric tension were measured in endothelium-intact and -denuded preparations. Localization of 5-HT receptor subtypes was examined immunohistochemically. 5-HT induced a transient then a small, sustained smooth muscle cell hyperpolarization in endothelium-intact strips. In endothelium-denuded strips, 5-HT induced only a sustained hyperpolarization, and this was changed to a depolarization by the selective 5-HT(7) receptor inhibitor SB269970. This depolarization was inhibited by the 5-HT(2A) receptor blocker sarpogrelate. 5-HT induced a relaxation of PGF(2α) -induced contracted strips that was similar in endothelium-intact and -denuded preparations. The latter relaxation was changed to contraction by SB269970 and this contraction was inhibited by sarpogrelate. Immunoreactive responses against endothelial and smooth muscle 5-HT(2A) receptors and smooth muscle 5-HT(7) receptors were identified in the vein. The 5-HT-induced relaxation of the PGF(2α) contraction was inhibited by the cAMP-dependent protein kinase inhibitor Rp-cAMPS and by the AC inhibitor SQ22536. These results indicate that 5-HT activates both smooth muscle 5-HT(7) receptors (to produce relaxation) and smooth muscle 5-HT(2A) receptors (to produce contraction) in rabbit jugular vein. We suggest that in this particular vein, the 5-HT(2A) receptor-induced depolarization and contraction are masked by the 5-HT(7) receptor-induced responses, possibly via actions mediated by cAMP.

Vazoactive Effects of Oxidative Stress Elicited by Hydrogen Peroxide in the Human Umbilical Artery: An &amp;lt;i&amp;gt;in Vitro&amp;lt;/i&amp;gt; Study

The vasoactive effects of oxidative stress induced by hydrogen peroxide (H2O2) on human umbilical artery strips as well as the possible mechanisms involved are studied. Contraction responses to cumulative H2O2 (10–7 M-3 × 10–2 M) in endothelium intact and denuded umbilical arteries and responses to cumulative H2O2 after incubation with L-NAME (10–4 M) (n = 8), indomethacin (10–5 M) (n = 8) and verapamil (10–6) (n = 8) were recorded. Responses elicited with cumulative H2O2 in Ca2+ free extracellular medium and the responses to cumulative Ca2+ (10–4 M-2 × 10–3 M) after H2O2 (10–3 M) induced contraction were also studied. The Emax for each experiment was calculated. p 2O2 elicited contraction was greater in endothelium denuded artery strips compared to endothelium intact strips (p 2O2 (p 2+ free extracellular medium caused decreases in cumulative H2O2 elicited contractions and cumulative Ca2+ caused concentration dependent increases in the contraction caused by a single bolus of H2O2 (p 2O2 causes concentration-dependent constriction in human umbilical arteries. The presence of the endothelium and NOS enzyme activation influences the H2O2 responses. Removal of the endothelium increases the H2O2 elicited contractions more than incubation with L-NAME suggesting beside NO, other endothelial vasodilators are also involved in vascular tonus of the umbilical arteries. Both intracellular and extracellular Ca2+ ions and constrictor cyclooxygenase metabolites play a role in the contractile responses elicited by H2O2 in human umbilical arteries.

Sarpogrelate hydrochloride reduced intimal hyperplasia in experimental rabbit vein graft

The selective 5-HT(2A) receptor antagonist sarpogrelate has been clinically used for treatment in atherosclerotic diseases. However, it remains unknown whether administration of sarpogrelate inhibits intimal hyperplasia seen in autologous vein grafts. Therefore, we sought to clarify this question using an experimental rabbit vein graft model. Male rabbits were divided into two groups: a control group and a sarpogrelate-treated group. The jugular vein was interposed in the carotid artery in reversed fashion for 4 weeks and intimal hyperplasia of the grafted vein was measured (n = 8, in each group). Acetylcholine (ACh)-induced endothelium-dependent relaxation was tested by precontraction with prostaglandin F(2alpha) (PGF(2alpha), 5 muM) (n = 5, in each). endothelial nitric oxide synthase (eNOS) protein expression and superoxide production of these veins were also assessed. The suppression of intimal hyperplasia was significantly greater in the sarpogrelate-treated group than in the control group. ACh induced an endothelium-dependent relaxation in the sarpogrelate-treated group (but not in the control group). In endothelium-intact strips from the sarpogrelate-treated group, the nitric oxide (NO) synthase inhibitor nitroarginine enhanced the PGF(2alpha)-induced contraction and blocked the ACh-induced relaxation. Immunoreactive eNOS protein expression was similar between the two groups but superoxide production (estimated from ethidium fluorescence) in endothelial cells was significantly smaller in the sarpogrelate-treated group. The present results indicate that in vivo blockade of 5-HT(2A) receptors leads to an inhibition of intimal hyperplasia in rabbit vein graft. It is suggested that an increased function of endothelium-derived NO through a reduction in endothelial superoxide production may be a possible underlying mechanism for this. These novel findings support the clinical usefulness of sarpogrelate for preventing intimal hyperplasia in vein graft after bypass grafting.

The Mechanisms of the Direct Action of Etomidate on Vascular Reactivity in Rat Mesenteric Resistance Arteries

Etomidate minimally influences hemodynamics at a standard induction dose in young healthy patients, but can cause significant systemic hypotension at higher doses for induction or electroencephalographic burst suppression (i.e., cerebral protection) in patients with advanced age or heart disease, and during cardiopulmonary bypass. However, less is known about its action on systemic resistance arteries. Using an isometric force recording method and fura-2-fluorometry, we investigated the action of etomidate on vascular reactivity in small mesenteric arteries from young (7-8 wk old, n = 179) and aged (96-98 wk old, n = 10) rats. In the endothelium-intact strips from young rats, etomidate enhanced the contractile response to norepinephrine or KCl (40 mM) at 3 microM but inhibited it at higher concentrations (>or=10 microM). The enhancement was still observed after treatment with N(G)-nitro l-arginine, tetraethylammonium, diclofenac, nordihydroguaiaretic acid, losartan, ketanserin, BQ-123, or BQ-788, but was not observed in aged rats. In the endothelium-denuded strips from young rats, etomidate (>or=10 microM) consistently inhibited the contractile response to norepinephrine or KCl without enhancement at 3 microM. In the fura-2-loaded, endothelium-denuded strips from young rats, etomidate inhibited norepinephrine- or KCl-induced increases in both intracellular Ca(2+) concentration ([Ca(2+)]i) and force. Etomidate still inhibited the norepinephrine-induced increase in [Ca(2+)]i after depletion of the intracellular Ca(2+) stores by ryanodine, which was sensitive to nifedipine. Etomidate had little effect on norepinephrine- or caffeine-induced Ca(2+) release from the intracellular stores or Ca(2+) uptake into the intracellular stores. During stimulation with norepinephrine or KCl, etomidate had little effect on the [Ca(2+)]i-force relation at low concentrations (<or=30 microM) but caused its downward shift at 100 microM. In small mesenteric arteries, etomidate influences the contractile response to norepinephrine or membrane depolarization through endothelium-dependent enhancing and endothelium-independent inhibitory actions. The enhancement is at least in part independent of nitric oxide, endothelium-derived hyperpolarizing factor, cyclooxygenase products, lipoxygenase products, angiotensin II, serotonin, or endothelin-1, but may involve some signaling pathway that is impaired by aging. The endothelium-independent inhibition is due to decreases in both the [Ca(2+)]i and myofilament Ca(2+) sensitivity in vascular smooth muscle cells. The decrease in [Ca(2+)]i would be due mainly to inhibition of voltage-gated Ca(2+) influx. The observed inability of lower concentrations (1-3 microM) of etomidate to cause significant vasodilation is consistent with minimal changes in hemodynamics during induction of anesthesia with etomidate in young subjects, whereas the observed vasodilator action of higher concentrations of etomidate might underlie systemic hypotension caused by higher doses of etomidate in the clinical setting.

Effects of chronic in vivo administration of nitroglycerine on ACh‐induced endothelium‐dependent relaxation in rabbit cerebral arteries

In the setting of nitrate tolerance, endothelium-dependent relaxation is reduced in several types of peripheral vessels. However, it is unknown whether chronic in vivo administration of nitroglycerine modulates such relaxation in cerebral arteries. Isometric force and smooth muscle cell membrane potential were measured in endothelium-intact strips from rabbit middle cerebral artery (MCA) and posterior cerebral artery (PCA). ACh (0.1-10 microM) concentration-dependently induced endothelium-dependent relaxation during the contraction induced by histamine in both MCA and PCA. Chronic (10 days) in vivo administration of nitroglycerine reduced the ACh-induced relaxation in PCA but not in MCA, in the presence of the cyclooxygenase inhibitor diclofenac (3 microM). In the presence of the NO-synthase inhibitor N (omega)-nitro-L-arginine (L-NNA, 0.1 mM) plus diclofenac, in MCA from both nitroglycerine-untreated control and -treated rabbits, ACh (0.1-10 microM) induced a smooth muscle cell hyperpolarization and relaxation, and these were blocked by the small-conductance Ca(2+)-activated K(+)-channel inhibitor apamin (0.1 microM), but not by the large- and intermediate-conductance Ca(2+)-activated K(+)-channel inhibitor charybdotoxin (0.1 microM). In contrast, in PCA, ACh (<3 microM) induced neither hyperpolarization nor relaxation under these conditions, suggesting that the endothelium-derived relaxing factor is NO in PCA, whereas endothelium-derived hyperpolarizing factor (EDHF) plays a significant role in MCA. It is suggested that in rabbit cerebral arteries, the function of the endothelium-derived relaxing factor NO and that of EDHF may be modulated differently by chronic in vivo administration of nitroglycerine.

Regional difference of endothelium-dependent contractility in human umbilical artery

To investigate whether vascular reactivity of the umbilical artery influences differences in Doppler waveform depending on the sampling site along the cord, we studied a regional difference of endothelium-dependent vascular contractility in human umbilical artery using mechanical responses to the quick stretch of vascular smooth muscle strips. The umbilical artery resistance index (RI) values for the fetal end were significantly higher than those for the placental end. In endothelium-intact strips, stretch-induced contraction of the umbilical artery appears to decrease from the fetal to the placental end of the umbilical artery. No significant differences were noted between strips from the two locations when endothelial cells were rubbed, indicating that this regional difference is related to endothelial cell function. Indo-methacin decreased the stretch-induced contraction of endothelium-intact strips, but tetrodotoxin did not affect this association, suggesting that arachidonic acid metabolism via the cyclooxygenase pathway contributes to modulation of human umbilical artery responsiveness to stretch. These findings suggest that the difference in Doppler waveform of the umbilical artery depending on the sampling site is related to differences in vascular resistance that result from endothelium-related function.

Diminution of angiotensin II-induced contraction of the abdominal aorta isolated from Watanabe heritable hyperlipidemic rabbits

The purpose of this study was to investigate the changes in vasocontractile responses in atherosclerosis, using abdominal aortic strips isolated from Watanabe heritable hyperlipidemic (WHHL) rabbits and Japanese White (control) rabbits. The aortic strips from WHHL rabbits showed a significantly lower contractile response to angiotensin II than that in strips from control rabbits. The contractile responses to phenylephrine and 5-hydroxytryptamine were not different in WHHL and control groups. The contractile response to angiotensin II was higher in endothelium-denuded aortic strips than in endothelium-intact strips, but to a greater extent in the control group than in the WHHL group. The contractile response to angiotensin II in the absence of the endothelium was also lower in the WHHL group than in the control group. Pretreatment with N(G)-nitro-L-arginine significantly increased the contractile response to angiotensin II in the endothelium-intact aortic strips in both the WHHL and control groups, while pretreatment with diclofenac did not affects the aortic contractile response to angiotensin II. The contractile responses to angiotensin II in the presence of N(G)-nitro-L-arginine and diclofenac were lower in the WHHL group than in the control group. The contractile response to angiotensin II in the presence of PD123319 was also lower in the WHHL group than in the control group. Endothelium-dependent relaxation by acetylcholine occurred to the some extent in the WHHL and control groups. These results suggest that the WHHL rabbit abdominal aorta displays attenuated angiotensin II-induced contraction, mainly due to an abnormality in the angiotensin II-specific contractile pathway of the medial smooth muscle.

Interleukin-6 impairs endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of pregnant rats.

IL-6 is elevated in plasma of preeclamptic women, and twofold elevation of plasma IL-6 increases vascular resistance and arterial pressure in pregnant rats, suggesting a role of the cytokine in hypertension of pregnancy. However, whether the hemodynamic effects of IL-6 reflect direct effects of the cytokine on the mechanisms of vascular contraction/relaxation is unclear. The purpose of this study was to test the hypothesis that IL-6 directly impairs endothelium-dependent relaxation and enhances vascular contraction in systemic vessels of pregnant rats. Active stress was measured in aortic strips isolated from virgin and late pregnant Sprague-Dawley rats and then nontreated or treated for 1 h with IL-6 (10 pg/ml to 10 ng/ml). In endothelium-intact vascular strips, phenylephrine (Phe, 10(-5) M) caused an increase in active stress that was smaller in pregnant (4.2 +/- 0.3) than virgin rats (5.1 +/- 0.3 x 10(4) N/m(2)). IL-6 (1,000 pg/ml) caused enhancement of Phe contraction that was greater in pregnant (10.6 +/- 0.7) than virgin rats (7.5 +/- 0.4 x 10(4) N/m(2)). ACh and bradykinin caused relaxation of Phe contraction and increases in vascular nitrite production that were greater in pregnant than virgin rats. IL-6 caused reductions in ACh- and bradykinin-induced vascular relaxation and nitrite production that were more prominent in pregnant than virgin rats. Incubation of endothelium-intact strips in the presence of N(omega)-nitro-L-arginine methyl ester (10(-4) M) to inhibit nitric oxide (NO) synthase, or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 10(-5) M) to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced stress in nontreated but to a lesser extent in IL-6-treated vessels, particularly those of pregnant rats. Removal of the endothelium enhanced Phe-induced stress in nontreated but not IL-6-treated vessels, particularly those of pregnant rats. In endothelium-denuded strips, relaxation of Phe contraction with sodium nitroprusside, an exogenous NO donor, was not different between nontreated and IL-6-treated vessels of virgin or pregnant rats. Thus IL-6 inhibits endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of virgin and pregnant rats. The greater IL-6-induced inhibition of vascular relaxation and enhancement of contraction in systemic vessels of pregnant rats supports a direct role for IL-6 as one possible mediator of the increased vascular resistance associated with hypertension of pregnancy.

Angiotensin II-induced modulation of endothelium-dependent relaxation in rabbit mesenteric resistance arteries

The role of local endogenous angiotensin II (Ang II) in endothelial function in resistance arteries was investigated using rabbit mesenteric resistance arteries. First, the presence of immunoreactive Ang II together with Ang II type-1 receptor (AT1R) and angiotensin converting enzyme (ACE) was confirmed in these arteries. In endothelium-intact strips, the AT1R-blocker olmesartan (1 μm) and the ACE-inhibitor temocaprilat (1 μm) each enhanced the ACh (0.03 μm)-induced relaxation during the contraction induced by noradrenaline (NA, 10 μm). Similar effects were obtained using CV-11974 (another AT1R blocker) and enalaprilat (another ACE inhibitor). The nitric-oxide-synthase inhibitor NG-nitro-l-arginine (l-NNA) abolished the above effect of olmesartan. In endothelium-denuded strips, olmesartan enhanced the relaxation induced by the NO donor NOC-7 (10 nm). Olmesartan had no effect on cGMP production (1) in endothelium-intact strips (in the absence or presence of ACh) or (2) in endothelium-denuded strips (in the absence or presence of NOC-7). In β-escin-skinned strips, 8-bromoguanosine 3′,5′ cyclic monophosphate (8-Br-cGMP, 0.01–1 μm) concentration dependently inhibited the contractions induced (a) by 0.3 μm Ca2+ in the presence of NA+GTP and (b) by 0.2 μm Ca2++GTPγS. Olmesartan significantly enhanced, while Ang II (0.1 nm) significantly inhibited, the 8-Br-cGMP-induced relaxation. We propose the novel hypothesis that in these arteries, Ang II localized within smooth muscle cells activates AT1Rs and inhibits ACh-induced, endothelium-dependent relaxation at least partly by inhibiting the action of cGMP on these cells.

Involvement of H2O2 in superoxide-dismutase-induced enhancement of endothelium-dependent relaxation in rabbit mesenteric resistance artery.

1 The mechanism underlying the enhancement by superoxide dismutase (SOD) of endothelium-dependent relaxation was investigated in rabbit mesenteric resistance arteries. 2 SOD (200 U ml(-1)) increased the production of H(2)O(2) in smooth muscle cells (as indicated by the use of an H(2)O(2)-sensitive fluorescent dye). 3 Neither SOD nor catalase (400 U ml(-1)) modified either the resting membrane potential or the hyperpolarization induced by acetylcholine (ACh, 1 micro M) in smooth muscle cells. 4 In arteries constricted with noradrenaline, the endothelium-dependent relaxation induced by ACh (0.01-1 micro M) was enhanced by SOD (200 U ml(-1)) (P<0.01). This action of SOD was inhibited by L-N(G)-nitroarginine (nitric oxide (NO)-synthase inhibitor) but not by either charybdotoxin+apamin (Ca(2+)-activated-K(+)-channel blockers) or diclofenac (cyclooxygenase inhibitor). 5 Neither ascorbate (50 micro M) nor tiron (0.3 mM), superoxide scavengers, had any effect on the ACh-induced relaxation, but each attenuated the enhancing effect of SOD on the ACh-induced relaxation. Similarly, catalase (400 U ml(-1)) inhibited the effect of SOD without changing the ACh-induced relaxation. 6 In endothelium-denuded strips constricted with noradrenaline, SOD enhanced the relaxation induced by the NO donor 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7) (P<0.05). Ascorbate and catalase each attenuated this effect of SOD. 7 H(2)O(2) (1 micro M) enhanced the relaxation on the noradrenaline contraction induced by NOC-7 and that induced by 8-bromo-cGMP, a membrane-permeable analogue of guanosine 3',5' cyclic monophosphate (cGMP). 8 SOD had no effect on cGMP production, whether measured in endothelium-intact strips following an application of ACh (0.1 micro M) or in endothelium-denuded strips following an application of NOC-7 (0.1 micro M). 9 It is suggested that in rabbit mesenteric resistance arteries, SOD increases the ACh-induced, endothelium-dependent relaxation by enhancing the action of NO in the smooth muscle via its H(2)O(2)-producing action (rather than via a superoxide-scavenging action).

Angiotensin II-induced modulation of endothelium-dependent relaxation in rabbit mesenteric resistance arteries.

The role of local endogenous angiotensin II (Ang II) in endothelial function in resistance arteries was investigated using rabbit mesenteric resistance arteries. First, the presence of immunoreactive Ang II together with Ang II type-1 receptor (AT1R) and angiotensin converting enzyme (ACE) was confirmed in these arteries. In endothelium-intact strips, the AT1R-blocker olmesartan (1 microM) and the ACE-inhibitor temocaprilat (1 microM) each enhanced the ACh (0.03 microM)-induced relaxation during the contraction induced by noradrenaline (NA, 10 microM). Similar effects were obtained using CV-11974 (another AT1R blocker) and enalaprilat (another ACE inhibitor). The nitric-oxide-synthase inhibitor NG-nitro-L-arginine (L-NNA) abolished the above effect of olmesartan. In endothelium-denuded strips, olmesartan enhanced the relaxation induced by the NO donor NOC-7 (10 nM). Olmesartan had no effect on cGMP production (1) in endothelium-intact strips (in the absence or presence of ACh) or (2) in endothelium-denuded strips (in the absence or presence of NOC-7). In beta-escin-skinned strips, 8-bromoguanosine 3',5' cyclic monophosphate (8-Br-cGMP, 0.01-1 microM) concentration dependently inhibited the contractions induced (a) by 0.3 microM Ca2+ in the presence of NA+GTP and (b) by 0.2 microM Ca2++GTPgammaS. Olmesartan significantly enhanced, while Ang II (0.1 nM) significantly inhibited, the 8-Br-cGMP-induced relaxation. We propose the novel hypothesis that in these arteries, Ang II localized within smooth muscle cells activates AT1Rs and inhibits ACh-induced, endothelium-dependent relaxation at least partly by inhibiting the action of cGMP on these cells.

Reduced function of endothelial prostacyclin in human omental resistance arteries in pre-eclampsia.

It remains unclear in pre-eclampsia whether or not a functional change occurs in the role played by prostacyclin in endothelium-dependent relaxation in resistance arteries. We examined this using human omental resistance arteries (obtained from pre-eclamptic or normotensive pregnant women) in the presence of N(G)-nitro-L-arginine (L-NNA, an inhibitor of nitric oxide synthase). In endothelium-intact strips from both groups, 9,11-epithio-11,12-methano-thromboxane A(2) (STA(2), a thromboxane A(2) mimetic) produced a contraction. Diclofenac (an inhibitor of cyclooxygenase) enhanced the STA(2) contraction only in the normotensive pregnant group (1.4 times control, P < 0.01). In the presence of STA(2), bradykinin (0.1 microM) produced an endothelium-dependent relaxation in both groups, the relaxation being significantly smaller for the pre-eclamptic group (P < 0.002). Diclofenac significantly attenuated the bradykinin-induced relaxation only for the normotensive pregnant group (31 % inhibition, P < 0.001). The bradykinin-induced membrane hyperpolarization consisted of diclofenac-sensitive and -insensitive components. The former, but not the latter, was significantly smaller in pre-eclampsia (-4.3 vs. -2.6 mV, P < 0.05). The concentrations of 6-keto-PGF(1alpha) (a stable metabolite of prostacyclin) in these arteries were significantly lower in pre-eclampsia in both the absence and presence of bradykinin (about 0.2-0.4 times the normotensive pregnant value in each case, P < 0.01). By contrast, both the relaxation and the membrane hyperpolarization in response to beraprost (10 nM, a stable analogue of prostacyclin) were similar between the two groups. We conclude that, in pre-eclampsia, a reduced part is played by prostaglandins in the endothelium-dependent relaxation seen in resistance arteries and that this may be due to a reduced production of prostacyclin by the endothelium.

Reduced endothelial NO-cGMP vascular relaxation pathway during TNF-α-induced hypertension in pregnant rats

Placental ischemia during pregnancy is thought to release cytokines such as tumor necrosis factor-alpha (TNF-alpha), which may contribute to the increased vascular resistance associated with pregnancy-induced hypertension. We have reported that a chronic twofold elevation in plasma TNF-alpha increases blood pressure in pregnant but not in virgin rats; however, the vascular mechanisms are unclear. We tested the hypothesis that increasing plasma TNF-alpha during pregnancy impairs endothelium-dependent vascular relaxation and enhances vascular reactivity. Active stress was measured in aortic strips of virgin and late-pregnant Sprague-Dawley rats untreated or infused with TNF-alpha (200 ng x kg(-1) x day(-1) for 5 days) to increase plasma level twofold. Phenylephrine (Phe) increased active stress to a maximum of 4.2 +/- 0.4 x 10(3) and 9.9 +/- 0.7 x 10(3) N/m2 in control pregnant and TNF-alpha-infused pregnant rats, respectively. Removal of the endothelium enhanced Phe-induced stress in control but not in TNF-alpha-infused pregnant rats. In endothelium-intact strips, ACh caused greater relaxation of Phe contraction in control than in TNF-alpha-infused pregnant rats. Basal and ACh-induced nitrite/nitrate production was less in TNF-alpha-infused than in control pregnant rats. Pretreatment of vascular strips with 100 microM N(G)-nitro-L-arginine methyl ester, to inhibit nitric oxide (NO) synthase, or 1 microM 1H-[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one, to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced stress in control but not in TNF-alpha-infused pregnant rats. Phe contraction and ACh relaxation were not significantly different between control and TNF-alpha-infused virgin rats. Thus an endothelium-dependent NO-cGMP-mediated vascular relaxation pathway is inhibited in late-pregnant rats infused with TNF-alpha. The results support a role for TNF-alpha as one possible mediator of the increased vascular resistance associated with pregnancy-induced hypertension.

Role of endothelium in the action of isoflurane on vascular smooth muscle of isolated mesenteric resistance arteries.

It is believed that isoflurane decreases blood pressure predominantly by decreasing systemic vascular resistance with modest myocardial depression. Nevertheless, little information is available regarding the direct action of isoflurane on systemic resistance arteries. With use of the isometric force recording method, the action of isoflurane on contractile response to norepinephrine, a neurotransmitter that plays a central role in sympathetic maintenance of vascular tone in vivo, was investigated in isolated rat small mesenteric arteries. In the endothelium-intact strips, the norepinephrine response was initially enhanced after application of isoflurane (2-5%), but it was subsequently almost normalized to the control level during exposure to isoflurane. However, the norepinephrine response was notably inhibited after washout of isoflurane. In the endothelium-denuded strips, the norepinephrine response was gradually inhibited during exposure to isoflurane (> or = 3%), and the inhibition was prolonged after wash-out of isoflurane. The isoflurane-induced enhancement of norepinephrine response was still observed after inhibitions of the nitric oxide, endothelium-derived hyperpolarizing factor, cyclooxygenase and lipoxygenase pathways, or after blockade of endothelin-1, angiotensin-II, and serotonin receptors; however, it was prevented by superoxide dismutase. In isolated mesenteric resistance artery, the action of isoflurane on contractile response to norepinephrine consists of two distinct components: an endothelium-dependent enhancing component and an endothelium-independent inhibitory component. During exposure to isoflurane, the former counteracted the latter, preventing the norepinephrine response from being strongly inhibited. However, only the endothelium-independent component persists after washout of isoflurane, causing prolonged inhibition of the norepinephrine response. Superoxide anions may be involved in the enhanced response to norepinephrine.

High-salt diet enhances vascular reactivity in pregnant rats with normal and reduced uterine perfusion pressure.

High-salt (HS) diet is often associated with increased vascular resistance and arterial pressure; however, the effects of HS intake on the vascular control mechanisms of arterial pressure during pregnancy are unclear. We investigated whether a HS diet during pregnancy is associated with increases in vascular reactivity. Active stress was measured in aortic strips of virgin and normal pregnant Sprague-Dawley rats and a hypertensive pregnant rat model produced by reduction in uterine perfusion pressure (RUPP), fed either normal-sodium (NS, 1%) or HS diet (8%) for 7 days. In endothelium-intact strips, phenylephrine (Phe) caused a concentration-dependent contraction that was greater in RUPP rats than in normal pregnant or virgin rats and was significantly enhanced in pregnant/HS and RUPP/HS rats compared with pregnant/NS and RUPP/NS rats, respectively. Removal of the endothelium enhanced the Phe-induced stress slightly in virgin rats and significantly in pregnant/NS but not in pregnant/HS, RUPP/NS, or RUPP/HS. In endothelium-intact strips, acetylcholine (ACh) caused a concentration-dependent relaxation that was reduced in RUPP/NS (max, 31%) compared with pregnant/NS rats (max, 75%). ACh relaxation was further reduced in pregnant/HS rats compared with pregnant/NS rats and in RUPP/HS rats compared with RUPP/NS rats. Pretreatment of endothelium-intact strips with N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) mol/L), to inhibit NO synthase, or with 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 10(-6) mol/L), to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced contraction in pregnant/NS rats but not in pregnant/HS, RUPP/NS, or RUPP/HS rats. Basal and ACh-induced nitrite/nitrate production from aortic strips showed significant reduction in pregnant/HS rats compared with pregnant/NS rats but not in RUPP/HS rats compared with RUPP/NS rats. Sodium nitroprusside, an exogenous NO donor, caused relaxation of Phe contraction that was similar in virgin or pregnant rats on an NS or HS diet but was significantly reduced in RUPP/HS rats (ED(50) 6x10(-8) mol/L) compared with RUPP/NS rats (ED(50) 6x10(-9) mol/L). Thus, a HS diet in normal pregnant and RUPP rats is associated with increases in vascular reactivity. The enhanced vascular reactivity with the HS diet is possibly related to abnormalities in NO synthesis/release from the endothelium in normal pregnant rats and an additional decrease in the sensitivity of the smooth muscle to relaxation by NO in pregnant rats with reduced uterine perfusion pressure.

Mechanisms of direct inhibitory action of ketamine on vascular smooth muscle in mesenteric resistance arteries.

Ketamine was previously suggested to relax vascular smooth muscle by reducing the intracellular Ca2+ concentration ([Ca2+]i). However, no direct evidence is available to indicate that ketamine reduces the [Ca2+]i in vascular smooth muscle of systemic resistance arteries. Endothelium-intact or -denuded smooth muscle strips were prepared from rat small mesenteric arteries. Isometric force and [Ca2+]i were measured simultaneously in the fura-2-loaded, endothelium-denuded strips. In some experiments, only isometric force was measured in either the endothelium-intact or beta-escin-treated, endothelium-denuded strips. In the endothelium-intact strips, lower concentrations (< or = 30 microm) of ketamine slightly enhanced norepinephrine-induced contraction, whereas higher concentrations (> or = 100 microM) of ketamine inhibited both norepinephrine- and KCl-induced contractions. In the fura-2-loaded strips, ketamine (> or = 100 microM) inhibited the increases in both [Ca2+]i and force induced by either norepinephrine or KCl. Ketamine also inhibited the norepinephrine-induced increase in [Ca2+]i after treatment with ryanodine. In the absence of extracellular Ca2+, ketamine notably inhibited the norepinephrine-induced increase in [Ca2+]i, whereas it only minimally inhibited caffeine-induced increase in [Ca2+]i. Ketamine had little influence on the [Ca2+]i-force relation during force development to stepwise increment of extracellular Ca2+ concentration during either KCl depolarization or norepinephrine stimulation. Ketamine did not affect Ca2+-activated contractions in the beta-escin membrane-permeabilized strips. The action of ketamine on contractile response to norepinephrine consists of endothelium-dependent vasoconstricting and endothelium-independent vasodilating components. The direct vasorelaxation is largely a result of reduction of[Ca2+]i in vascular smooth muscle cells. The [Ca2+]i-reducing effects are caused by inhibitions of both voltage-gated Ca2+ influx and norepinephrine-induced Ca2+ release from the intracellular stores.

Role of endothelin B receptors in enhancing endothelium-dependent nitric oxide-mediated vascular relaxation during high salt diet.

High salt diet is often associated with increases in blood pressure, and the state of activation of endothelium-dependent vascular relaxation pathways is critical under these conditions. Basal activation of endothelial endothelin B (ET(B)) receptors by endothelin has been suggested to stimulate the release of factors that promote vascular relaxation. However, whether ET(B) receptors play a role in enhancing endothelium-dependent vascular relaxation during high salt diet is unclear. In this study, we investigated whether chronic treatment with an ET(B) receptor antagonist is associated with impaired endothelium-dependent vascular relaxation and enhanced vascular reactivity particularly during high salt diet. Isometric contraction was measured in aortic strips isolated from male Sprague-Dawley rats on normal sodium (NS, 1%) and high sodium diet (HS, 8%) for 7 days and untreated or treated with the ET(B) receptor antagonist A-192621 (30 mg/kg per day) for 5 days. The mean arterial pressure was (in mm Hg) 122+/-3 in NS, 132+/-3 in HS, 144+/-2 in NS/ET(B) antagonist, and 171+/-12 in HS/ET(B) antagonist rats. In endothelium-intact strips, phenylephrine (Phe, 10(-5) mol/L) increased active stress to 7.6+/-1.0x10(3)N/m(2) in NS rats and 8.2+/-0.9x10(3)N/m(2) in HS rats. Phe (10(-5) mol/L) -induced stress was significantly greater in NS/ET(B) antagonist (11.3+/-0.9x10(3)N/m(2)) than NS and far greater in HS/ET(B) antagonist (14.1+/-0.1.2x10(3)N/m(2)) than HS rats. Also, Phe was more potent in NS/ET(B) antagonist and HS/ET(B) antagonist rats (ED(50)=0.3x10(-7) and 0.15x10(-7) mol/L) than in NS and HS rats (ED(50)=0.8x10(-7) and 0.7x10(-7) mol/L). Removal of the endothelium enhanced Phe-induced contraction significantly in NS and to a greater extent in HS, but not in NS/ET(B) antagonist or HS/ET(B) antagonist rats. In endothelium-intact strips, acetylcholine (ACh) caused relaxation of Phe contraction that was less in NS/ET(B) antagonist than NS and far less in HS/ET(B) antagonist than HS rats. Pretreatment of endothelium-intact strips with L-NAME (10(-4) mol/L), to inhibit nitric oxide (NO) synthase, or with methylene blue (10(-5) mol/L) or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 10(-6) mol/L), to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced contraction significantly in NS and HS, slightly in NS/ET(B) antagonist, but not in HS/ET(B) antagonist rats. Measurement of basal and ACh-induced nitrite/nitrate production from aortic strips showed a significant reduction in NS/ET(B) antagonist compared with NS, and a greater reduction in HS/ET(B) antagonist compared with HS rats. Relaxation of Phe contraction with sodium nitroprusside was not significantly different among the different groups of rats. Thus, an endothelial ET(B) receptor-mediated pathway of vascular relaxation involving release of NO seems to be active under basal conditions and may protect against excessive vasoconstriction and increased blood pressure particularly during high salt diet.