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.     

Endothelium-dependent responses in small human mesenteric arteries

The aim of the present study was to investigate the endothelial function in human mesenteric arteries with specific reference to defining the role of endothelium-derived nitric oxide (EDNO) and the endothelium-derived hyperpolarizing factor (EDHF). Isolated segments of small human mesenteric arteries (225-450 microm inner diameter) were mounted in organ baths for recording isometric tension. In arteries precontracted with U46619 (thromboxane A(2) analogue, 10(-7) M), endothelium-dependent relaxations were induced in a concentration-dependent manner by substance P and histamine. In normal Krebs solution the relaxations to substance P (10(-9) M) and histamine (10(-7) M) were not significantly affected by preincubation with N(omega)-nitro-L-arginine (L-NNA, 10(-4) M) or indomethacin (10(-5) M). When the preparations were exposed to a solution containing 60 mM KCl, stable contractions were induced, but relaxations could still be induced by substance P and histamine. When the arteries were further preincubated with L-NNA, the relaxations were almost abolished. A combination of apamin (3 x 10(-7) M) and charybdotoxin (10(-9) M) almost abolished relaxations in normal Krebs solution. It is concluded that isolated human mesenteric arteries respond to substance P and histamine with relaxations that are endothelium-dependent. Synthesis of both EDNO and EDHF seem important for these relaxations, whereas prostaglandins seem to be of minor importance.     

Coronary endothelial dysfunction from ischemia and reperfusion: effect of reactive oxygen metabolite scavengers

Using anesthetized mongrel dogs exposed to 60 min of ligation of the left anterior descending coronary artery followed by 60 min of reperfusion, we examined the effect of superoxide dismutase (SOD) and dimethylthiourea (DMTU) on evidence of endothelial injury in coronary rings studied in vitro. In 13 dogs treated with saline rings from the normal left circumflex coronary artery (LCF) relaxed by 98 +/- 4% when exposed to 10(-5) M acetylcholine whereas rings from the left anterior descending coronary artery (LAD) relaxed by 79 +/- 7% (p less than 0.05). In the same rings maximum relaxation with the ionophore A23187 was 107 +/- 5% versus 87 +/- 8% (p less than 0.05) for the LCF and the LAD, respectively. Comparisons of concentration-response curves through a range of doses of both acetylcholine and A23187 revealed significant differences for both vasodilators between the LCF and the LAD (p less than 0.01 for each). Nine dogs were treated with bovine SOD infused in the left atrium the last 20 min of ligation and throughout reperfusion (140 units/kg/min) and six other dogs were treated with DMTU 500 mg/kg i.v. given the last 30 min of the ligation period. Neither SOD nor DMTU prevented endothelial injury in the LAD. Despite pretreatment with these agents, there were significant reductions in maximum relaxation and in total concentration-response curves in the LAD as compared with the results in rings from the LCF with both acetylcholine and A23187. There were normal responses to nitroprusside in both the LCF and LAD in all three experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of autonomic blockers on linear and nonlinear indexes of blood pressure and heart rate in SHR

Endothelium-derived hyperpolarizing factor (EDHF) is released in response to agonists such as ACh and bradykinin and regulates vascular smooth muscle tone. Several studies have indicated that ouabain blocks agonist-induced, endothelium-dependent hyperpolarization of smooth muscle. We have demonstrated that epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of arachidonic acid, function as EDHFs. To further test the hypothesis that EETs represent EDHFs, we have examined the effects of ouabain on the electrical and mechanical effects of 14,15- and 11,12-EET in bovine coronary arteries. These arteries are relaxed in a concentration-dependent manner to 14,15- and 11,12-EET (EC(50) = 6 x 10(-7) M), bradykinin (EC(50) = 1 x 10(-9) M), sodium nitroprusside (SNP; EC(50) = 2 x 10(-7) M), and bimakalim (BMK; EC(50) = 1 x 10(-7) M). 11,12-EET-induced relaxations were identical in vessels with and without an endothelium. Potassium chloride (1-15 x 10(-3) M) inhibited [(3)H]ouabain binding to smooth muscle cells but failed to relax the arteries. Ouabain (10(-5) to 10(-4) M) increased basal tone and inhibited the relaxations to bradykinin, 11,12-EET, and 14,15-EET, but not to SNP or BMK. Barium (3 x 10(-5) M) did not alter EET-induced relaxations and ouabain plus barium was similar to ouabain alone. Resting membrane potential (E(m)) of isolated smooth muscle cells was -50.2 +/- 0.5 mV. Ouabain (3 x 10(-5) and 1 x 10(-4) M) decreased E(m) (-48.4 +/- 0.2 mV), whereas 11,12-EET (10(-7) M) increased E(m) (-59.2 +/- 2.2 mV). Ouabain inhibited the 11,12-EET-induced increase in E(m). In cell-attached patch clamp studies, 11,12-EET significantly increased the open-state probability (NP(o)) of a calcium-activated potassium channel compared with control cells (0.26 +/- 0.06 vs. 0.02 +/- 0.01). Ouabain did not change NP(o) but blocked the 14,15-EET-induced increase in NP(o). These results indicate that: 1) EETs relax coronary arteries in an endothelium-independent manner, 2) unlike EETs, potassium chloride does not relax the coronary artery, and 3) ouabain inhibits bradykinin- and EET-induced relaxations as has been reported for EDHF. These findings provide further evidence that EETs are EDHFs.     

Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in human mesenteric arteries.

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several vasodilating factors, including prostacyclin, nitric oxide, and endothelium-derived hyperpolarizing factor (EDHF). We have recently identified that endothelium-derived hydrogen peroxide (H(2)O(2)) is an EDHF in mice. The present study was designed to examine whether this is also the case in humans. Bradykinin elicited endothelium-dependent relaxations and hyperpolarizations in the presence of indomethacin and N(omega)-nitro-l-arginine, which thus were attributed to EDHF, in human mesenteric arteries. The EDHF-mediated relaxations were significantly inhibited by catalase, an enzyme that specifically decomposes H(2)O(2), whereas catalase did not affect endothelium-independent hyperpolarizations to levcromakalim. Exogenous H(2)O(2) elicited relaxations and hyperpolarizations in endothelium-stripped arteries. Gap junction inhibitor 18alpha-glycyrrhetinic acid partially inhibited, whereas inhibitors of cytochrome P450 did not affect the EDHF-mediated relaxations. These results indicate that H(2)O(2) is also a primary EDHF in human mesenteric arteries with some contribution of gap junctions.     

Dietary soy modulates endothelium-dependent relaxation in aged male rats: Increased agonist-induced endothelium-derived hyperpolarising factor and basal nitric oxide activity

We examined the effects of dietary soy on the contributions of endothelium-derived hyperpolarising factor (EDHF), nitric oxide (NO), and oxidative stress to vascular tone in isolated aortic rings and small mesenteric and pulmonary arteries in vitro. Male Wistar rats were either continuously fed a soy-deficient diet (SD) or switched from a soy-deficient diet to a soy-rich one for 6 months (SW). Contractile responses were generally smaller in arteries from SW rats. In mesenteric arteries, this difference was blunted by L-NAME, but not by charybdotoxin and apamin. Preconstricted SW mesenteric arteries were more sensitive to acetylcholine (ACh) than SD ones. This difference was unaffected by L-NAME but was abolished by charybdotoxin and apamin. Exogenous superoxide dismutase (SOD) and catalase induced powerful relaxations in aortic rings, which were smaller in those from SW rats. In mesenteric and pulmonary arteries, however, they partially inhibited ACh-mediated relaxation, and enhanced PGF(2alpha)-mediated contraction, respectively. Our results suggest that feeding aging male rats a soy-rich diet results in improved agonist-mediated EDHF production and a generalized reduction in contractile force, which is partly due to elevated basal NO. Our data also suggest a prorelaxant role for endogenous H(2)O(2) in small arteries, which is modulated by a soy diet.     

EDHF mediates the relaxation of stretched canine femoral arteries to acetylcholine.

To test the hypothesis that mechanically stretched arteries relax to endothelium-derived vasodilators, we challenged endothelium-intact dog femoral artery rings stretched from 1 to 16 g total initial tension (active force and passive elastic) with 10(-6) M acetylcholine (ACh), an endothelium-dependent dilator. The relaxation to 10(-6) M sodium nitroprusside (SNP), an endothelium-independent dilator, increased with the total initial tension. The relaxation to ACh averaged approximately 65% of the relaxation to SNP at total initial tensions of 4 to 16 g. To determine the nature of the endothelial-derived products involved, we compared the ACh-induced relaxation of stretched rings (6.5 +/- 0.2 g total initial tension) with rings chemically contracted with phenylephrine (Phe, 10(-7) to 10(-5) M) (6.5 +/- 0.3 g total initial tension). ACh-induced relaxation was evaluated before and after the inhibition of the synthesis of eicosanoids [cyclooxygenase (10(-5) M indomethacin) and lipoxygenase (10(-5) M nordihydroguariaretic acid)] and nitric oxide [nitric oxide synthase (10(-5) M Nw-nitro-L-arginine)]. The contribution of endothelium-derived hyperpolarizing factor (EDHF) was identified by blocking calcium-activated potassium channels (10(-8) M iberiotoxin). SNP (10(-6) M) relaxed stretched rings by 1.7 +/- 0.1 g and chemically-activated rings by 4.8 +/- 0.2 g. ACh relaxed stretched rings to 73 +/- 3% of the SNP relaxation and this was only attenuated in the presence of iberiotoxin. ACh relaxed Phe-activated rings to 60 +/- 3% of the SNP relaxation. This relaxation was attenuated by inhibition of the synthesis of nitric oxide and (or) eicosanoids. Therefore, ACh relaxed stretched rings through the release of EDHF whereas the relaxation of chemically activated rings to ACh involved multiple endothelium-derived vasodilators.     

Is hydrogen peroxide an EDHF in human radial arteries?

In human radial arteries, a nitric oxide/prostanoid-independent mechanism that has the pharmacological characteristics of an EDHF contributes to endothelium-dependent relaxation. H2O2 can act as an EDHF in some vascular beds. We examined the hypothesis that endogenously produced H2O2 mediated the nitric oxide/prostanoid-independent relaxation to carbachol in radial arteries obtained from patients undergoing coronary artery bypass surgery. Superoxide levels, measured by chemiluminescence, were similar in radial and internal mammary arteries, but immunohistochemical staining for Cu/Zn superoxide dismutase (SOD) was lower in endothelium from radial arteries. In organ chamber studies, neither addition of catalase nor addition of SOD to the bathing fluid modified nitric oxide/prostanoid-independent relaxations to carbachol in radial arteries. However, nitric oxide-dependent vasorelaxation was enhanced in the presence of SOD. Thus the nitric oxide/prostanoid-independent relaxation to carbachol is not due to H2O2 and, unlike nitric oxide-mediated vasorelaxation, is not attenuated by superoxide. Blood vessels showing EDHF-mediated relaxations resistant to oxidative stress may provide favorable outcomes in revascularization surgery.     

Chronic nitric oxide exposure alters the balance between endothelium-derived relaxing factors released from rat renal arteries: prevention by treatment with NOX-100, a NO scavenger

We investigated whether nitric oxide (NO) exposure alters the balance between NO and endothelium-derived hyperpolarizing factor (EDHF) released from rat renal arteries. To produce states of acutely or chronically excessive NO, lipopolysaccharide (LPS) was administered intraperitoneally to rats in a single dose of 4 mg/kg (LPS-single group) or in stepwise doses of 0.5, 1.0 and 2.0 mg/kg every other day (LPS-repeated group). On the day after LPS treatment, the protein levels of inducible NO synthase (iNOS) and endothelial NOS (eNOS) were measured, and the relaxation responses were determined in the renal arteries. The protein levels of iNOS markedly increased in both LPS-treated groups, while those of eNOS significantly increased in the LPS-repeated group compared with those in the respective control groups. In both LPS-treated groups, the relaxations in response to acetylcholine (ACh) and sodium nitroprusside remained unchanged. The ACh-induced relaxations in the presence of N(G)-nitro-L-arginine methyl ester, a NOS inhibitor, or by 1H-[1, 2, 4-] oxadiazole [4, 3-a] quinoxalin-1-one, a soluble guanylyl cyclase inhibitor, i.e. EDHF-mediated relaxations were significantly impaired in the LPS-repeated group but not in the LPS-single group, indicating increase in NO-mediated relaxation in the LPS-repeated group. These changes in the protein levels and EDHF-mediated relaxations induced by ACh observed in the LPS-repeated group were restored by treatment with NOX-100, a NO scavenger. These results suggest that persistent but not acute excessive NO exposure in rats impairs EDHF-mediated relaxation in renal arteries, leading to a compensatory upregulation of the eNOS/NO pathway.     

Endothelum-dependent and-independent relaxations in aortic rings obtained from hypertensive hooded (Aguti) rats.

Experimental hypertension studies are few in the hooded (Aguti) rat. The present study was designed to investigate the usefulness of this rat strain for experimental hypertension studies and to test the hypothesis that the hypertension may be associated with a diminution of endothelium dependent and independent relaxations. Hypertension was induced in inbred hooded rats (n=8 each) by administering 8% salt in the diet and /or 100 mg/kg/day Nomega-nitro-L-arginine-methyl-ester (L-NAME) in the drinking water for six and/or four weeks respectively. The rats were anaesthetized using a 25% urethane and 1% chloralose mixture given intraperitoneally at a dose of 5 mg/kg. Their blood pressure was measured invasively. Thereafter, relaxations of rat aortic preparations to acetylcholine, histamine and sodium nitroprusside (SNP) were assessed using standard organ bath conditions. Probabiliity level of 0.05 was taken as statistically significant. The mean arterial pressure (MAP;mm Hg) rose significantly in all test groups (Salt: 148.3 +/- 4.6; L-NAME: 181.7 +/-8.3; Salt+L-NAME:154.9 +/-8.7) compared with control (94.2 +/-6.8; [P < 0.05]. The MAP was significantly [P < 0.05] higher in the L-NAME group than in all the other groups. The heart rate fell significantly in the salt + L-NAME group compared to control [P <0.05].The IC50 of acetylcholine in aortic rings from L-NAME rats (7.9 x 10(-1) +/- 6.0 x 10(-3)) was significantly higher than in rings from control (9.4 x 10(-8) +/- 2.8 x 10(-8)), salt (7.8 x 10(-7) +/- 4.7 x 10(-7)) and salt + L-NAME (3.3 x 10 (-7) +/- 2.1 x 10(-7)) rats [P < 0.05]. The IC50 of histamine and SNP in the rings from the test groups of rats showed no significant difference from control. Also, endothelium dependent and independent relaxations were preserved in the various forms of hypertension studied except in chronic NOS inhibition where the former was attenuated in response to acetylcholine.     

Elucidation of the temporal relationship between endothelial-derived NO and EDHF in mesenteric vessels

Although the endothelium co-generates both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), the relative contribution from each vasodilator is not clear. In studies where the endothelium is stimulated acutely, EDHF responses predominate in small arteries. However, the temporal relationship between endothelial-derived NO and EDHF over more prolonged periods is unclear but of major physiological importance. Here we have used a classical pharmacological approach to show that EDHF is released transiently compared with NO. Acetylcholine (3 x 10(-6) mol/l) dilated second- and/or third-order mesenteric arteries for prolonged periods of up to 1 h, an effect that was reversed fully and immediately by the subsequent addition of L-NAME (10(-3) mol/l) but not TRAM-34 (10(-6) mol/l) plus apamin (5 x 10(-7) mol/l). When vessels were pretreated with L-NAME, acetylcholine induced relatively transient dilator responses (declining over approximately 5 min), and vessels were sensitive to TRAM-34 plus apamin. When measured in parallel, the dilator effects of acetylcholine outlasted the smooth muscle hyperpolarization. However, in the presence of L-NAME, vasodilatation and hyperpolarization followed an identical time course. In vessels from NOSIII(-/-) mice, acetylcholine induced small but detectable dilator responses that were transient in duration and blocked by TRAM-34 plus apamin. EDHF responses in these mouse arteries were inhibited by an intracellular calcium blocker, TMB-8, and the phospholipase A(2) inhibitor AACOCF(3), suggesting a role for lipid metabolites. These data show for the first time that EDHF is released transiently, whereas endothelial-derived NO is released in a sustained manner.     

Identification of 15-hydroxy-11,12-epoxyeicosatrienoic acid as a vasoactive 15-lipoxygenase metabolite in rabbit aorta

Arachidonic acid (AA) causes endothelium-dependent smooth muscle hyperpolarizations and relaxations that are mediated by a 15-lipoxygenase-I (15-LO-I) metabolite, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). We propose that AA is metabolized sequentially by 15-LO-I and hydroperoxide isomerase to an unidentified hydroxyepoxyeicosatrienoic acid (HEETA), which is hydrolyzed by a soluble epoxide hydrolase (sEH) to 11,12,15-THETA. After incubation of aorta with 14C-labeled AA, metabolites were extracted and the HEETAs were resolved by performing HPLC. Mass spectrometric analyses identified 15-Hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Incubation of aortic incubates with methanol and acetic acid trapped the acid-sensitive 15-H-11,12-EETA as methoxydihydroxyeicosatrienoic acids (MDHEs) (367 m/z, M-H). Pretreatment of the aortic tissue with the sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA; 10(-6) M) increased the formation of 15-H-11,12-EETA, measured as MDHEs. Thus 15-H-11,12-EETA is an acid- and sEH-sensitive precursor of 11,12,15-THETA. Aortic homogenates and endothelial cells contain a 57-kDa protein corresponding to the rabbit sEH. In preconstricted aortic rings, AA (10(-7)-10(-4) M) and acetylcholine (10(-9)-10(-6) M) caused concentration-related relaxations that were enhanced by pretreatment with AUDA. These enhanced relaxations were inhibited by increasing extracellular [K(+)] from 4.8 to 20 mM. AA (3 x 10(-6) M) induced cell membrane hyperpolarization (from -31.0 +/- 1 to -46.8 +/- 2 mV) in aortic strips with an intact endothelium, which was enhanced by AUDA. These results indicate that 15-H-11,12-EETA is produced by the aorta, hydrolyzed by sEH to 11,12,15-THETA, and mediates relaxations by membrane hyperpolarization. 15-H-11,12-EETA represents an endothelium-derived hyperpolarizing factor.     

Role of estrogen in modulating EDHF-mediated dilations in the female rat middle cerebral artery

We tested the hypothesis that endothelium-derived hyperpolarizing factor (EDHF) plays a less dominant role in the female cerebrovasculature. The contribution of EDHF to the ATP-mediated dilation was determined in middle cerebral arteries (MCAs) isolated from male and female rats. Four groups of rats were tested: intact male (n = 12), intact female (n = 13), estrogen-treated ovariectomized female (n = 13), and vehicle-treated ovariectomized female (n = 20) rats. Maximal dilation to ATP was similar in all groups. However, in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-5) M) and indomethacin (10(-5) M), the maximal dilation to ATP was significantly reduced in intact female (24 +/- 9%) and estrogen-treated ovariectomized female (29 +/- 9%) rats compared with intact male (95 +/- 4%) and vehicle-treated ovariectomized female (96 +/- 2%) rats. The ATP-mediated dilation in L-NAME- and indomethacin-treated MCAs isolated from male and ovariectomized female rats was inhibited by charybdotoxin (10(-7) M), an inhibitor of large-conductance Ca2+-sensitive K+ channels. We have defined EDHF as the L-NAME- and indomethacin-insensitive component of the ATP-mediated dilation. Our findings indicate that EDHF-mediated dilations are negligible in the female rat MCA; these dilations can be significantly enhanced after ovariectomy, suggesting that this effect is mediated by estrogen.     

Grape-derived polyphenols improve aging-related endothelial dysfunction in rat mesenteric artery: role of oxidative stress and the angiotensin system

Aging is characterized by the development of an endothelial dysfunction, which affects both the nitric oxide (NO)- and the endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations, associated with vascular oxidative stress and the activation of the angiotensin system. This study investigated whether red wine polyphenols (RWPs), antioxidants and potent stimulators of NO- and EDHF-mediated relaxations improve aging-related endothelial dysfunction, and, if so, examined the underlying mechanism. Mesenteric artery reactivity was determined in organ chambers, vascular oxidative stress by dihydroethidine and MitoSOX staining, and expression of target proteins by immunohistochemical staining. Control young rats (16 weeks) received solvent (ethanol, 3% v/v), and middle-aged rats (46 weeks) either solvent or RWPs (100 mg/kg/day) in the drinking water. The acetylcholine-induced endothelium-dependent NO component was slightly reduced whereas the EDHF component was markedly blunted in rings of middle-aged rats compared to young rats. The endothelial dysfunction was associated with oxidative stress, an upregulation of angiotensin II and AT1 receptors and a down-regulation of SK(Ca), IK(Ca), and angiotensin converting enzyme. Intake of RWPs for either one or two weeks improved the NO and the EDHF components of the relaxation, and normalized oxidative stress, the expression of SK(Ca), IK(Ca) and the components of the angiotensin system. The protective effect of the 2-week RWPs treatment persisted for one and two weeks following stopping intake of RWPs. Thus, intake of RWPs caused a persistent improvement of the endothelial function, particularly the EDHF component, in middle-aged rats and this effect seems to involve the normalization of the expression of SK(Ca), IK(Ca) and the angiotensin system.     

Role of endothelial Ni(2+)-sensitive Ca(2+) entry pathway in regulation of EDHF in porcine coronary artery

Elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells is proposed to be required for generation of vascular actions of endothelium-derived hyperpolarizing factor (EDHF). This study was designed to determine the endothelial Ca(2+) source that is important in development of EDHF-mediated vascular actions. In porcine coronary artery precontracted with U-46619, bradykinin (BK) and cyclopiazonic acid (CPA) caused endothelium-dependent relaxations in the presence of N(G)-nitro-L-arginine (L-NNA). The L-NNA-resistant relaxant responses were inhibited by high K(+), indicating an involvement of EDHF. In the presence of Ni(2+), which inhibits Ca(2+) influx through nonselective cation channels, the BK-induced EDHF relaxant response was greatly diminished and the CPA-induced response was abolished. BK and CPA elicited membrane hyperpolarization of smooth muscle cells of porcine coronary artery. Ni(2+) suppressed the hyperpolarizing responses in a manner analogous to removal of extracellular Ca(2+). EDHF-mediated relaxations and hyperpolarizations evoked by BK and CPA in porcine coronary artery showed a temporal correlation with the increases in [Ca(2+)](i) in porcine aortic endothelial cells. The extracellular Ca(2+)-dependent rises in [Ca(2+)](i) in endothelial cells stimulated with BK and CPA were completely blocked by Ni(2+). These results suggest that Ca(2+) influx into endothelial cells through nonselective cation channels plays a crucial role in the regulation of EDHF.     

Endothelium-dependent relaxations to histamine in the rat common carotid, renal and cranial mesenteric artery

The effect of histamine on the isolated rat common carotid, renal and cranial mesenteric arteries was examined. Histamine (10(-8)-10(-4) M) caused concentration-dependent relaxations of the arteries during contractions induced with phenylephrine (10(-8)-10(-7) M). Removal of the vascular endothelium inhibited the histamine-induced relaxations. Pyrilamine (6 X 10(-6) M), but not metiamide (10(-6) M), abolished the relaxant effect of histamine. Moreover, pyrilamine (6 X 10(-6) M) did not affect endothelium-dependent relaxations of the arteries produced with acetylcholine. These results indicate that histamine causes endothelium-dependent relaxations of the rat peripheral large conduit arteries, which appeared to be mediated via H1-histaminergic receptors.     

In eNOS knockout mice skeletal muscle arteriolar dilation to acetylcholine is mediated by EDHF

The mechanisms that account for acetylcholine (ACh)-induced responses of skeletal muscle arterioles of mice lacking endothelial nitric oxide (NO) synthase (eNOS-KO) were investigated. Isolated, cannulated, and pressurized arterioles of gracilis muscle from male eNOS-KO (74.1 +/- 2.3 microm) and wild-type (WT, 87.2 +/- 2.1 microm) mice developed spontaneous tone accounting for 63 and 61% of their passive diameter (116.8 +/- 3.4 vs. 143.2 +/- 2.8 microm, respectively) and dilated dose-dependently to ACh (10(-9)-10(-7) M). These dilations were significantly smaller in vessels of eNOS-KO compared with WT mice (29.2 +/- 2.0 microm vs. 46.3 +/- 2.1 microm, at maximum concentration) but responses to the NO donor, sodium nitrite (NaNO(2), 10(-6)-3 x 10(-5) M), were comparable in the vessels of the two strains. N(G)-nitro-L-arginine (L-NNA, 10(-4) M), an inhibitor of eNOS, inhibited ACh-induced dilations by 60-90% in arterioles of WT mice but did not affect responses in those of eNOS-KO mice. In arterioles of eNOS-KO mice, dilations to ACh were not affected by indomethacin but were essentially abolished by inhibitors of cytochrome P-450, clotrimazole (CTZ, 2 x 10(-6) M) or miconazole (MCZ, 2 x 10(-6) M), as well as by either high K(+) (40 mM) or iberiotoxin [10(-7) M, a blocker of Ca(2+)-dependent K(+) channels (K(Ca) channels)]. On the other hand, in WT arterioles CTZ or MCZ inhibited ACh-induced dilations only by approximately 10% and only in the presence of L-NNA. These results indicate that in arterioles of eNOS-KO mice, endothelium-derived hyperpolarizing factor (EDHF), synthesized via cytochrome P-450, accounts entirely for the mediation of ACh-induced dilation via an increase in K(Ca)-channel activity. In contrast, in arterioles of WT mice, endothelium-derived NO predominantly mediates ACh-induced dilation in which participation of EDHF becomes apparent only after inhibition of NO synthesis.     

Evidence for the involvement of myoendothelial gap junctions in EDHF-mediated relaxation in the rat middle cerebral artery

The mechanisms underlying endothelium-dependent hyperpolarizing factor (EDHF) in the middle cerebral artery (MCA) remain largely unresolved. In particular, very little is known regarding the way in which the signal is transmitted from endothelium to smooth muscle. The present study tested the hypothesis that direct communication via myoendothelial gap junctions contributes to the EDHF response in the male rat MCA. EDHF-mediated dilations were elicited in rat MCAs by luminal application of ATP or UTP in the presence of Nomega-nitro-L-arginine methyl ester and indomethacin. Maximum dilation to luminal ATP (10(-4) M) was reduced significantly after incubation with a gap peptide cocktail (9 +/- 4%, n = 6) compared with a scrambled gap peptide cocktail (99 +/- 1%, n = 6, P < 0.05). A gap peptide cocktail had no effect on amplitude of endothelial cell hyperpolarization in response to 3 x 10(-5) M UTP (22 +/- 3 vs. 22 +/- 1 mV, n = 4), whereas smooth muscle cell hyperpolarization was significantly attenuated (17 +/- 1 vs. 6 +/- 1 mV, n = 4, P = 0.004). Connexin (Cx) 37 was localized to smooth muscle and Cx43 to endothelium, whereas Cx40 was found in endothelium and smooth muscle. Electron microscopy revealed the existence of frequent myoendothelial junctions. The total number of myoendothelial junctions per 5 microm of MCA sectioned was 2.5 +/- 0.5. Our results suggest that myoendothelial communication contributes to smooth muscle cell hyperpolarization and EDHF dilation in male rat MCA.     

Endomorphins restored the endothelium-dependent relaxation of the rabbit aorta rings exposed to high D-glucose condition via NO-cGMP pathway

Rings of rabbit aorta that were both incubated in a high concentration of D-glucose and contracted submaximally by phenylephrine showed significantly decreased endothelium-dependent relaxations induced by acetylcholine. The cGMP production of aorta rings was also reduced. Treatment with endomorphins (1-1000 nmol/L) restored acetylcholine-induced relaxations of aorta rings incubated in high glucose concentrations and increased the cGMP synthesis. Moreover, this effect of endomorphins on endothelium was antagonized by naloxone, and the increase in the production of cGMP was also blocked.     

Nitrergic and purinergic regulation of the rat pylorus

The role of nitric oxide (NO) and ATP in the regulation of nonadrenergic, noncholinergic (NANC) inhibitory transmission in the pylorus remains unclear. In the presence of atropine and guanethidine, electric field stimulation induced NANC relaxations in a frequency-dependent manner (1-20 Hz) in the rat pylorus. NANC relaxations were significantly inhibited by N(G)-nitro-L-arginine methyl ester (L-NAME; 10(-4) M). P(2X) purinoceptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 3 x 10(-5) M) and P(2Y) purinoceptor antagonist reactive blue 2 (2 x 10(-5) M) had no effect on NANC relaxations. However, the combined administration of L-NAME and PPADS, but not reactive blue 2, evoked greater inhibitory effects on NANC relaxation than that evoked by L-NAME alone. alpha-Chymotrypsin and vasoactive intestinal polypeptide antagonist did not affect NANC relaxations. ATP (10(-5)-10(-3) M) and P(2X) purinoceptor agonist alpha, beta-methyleneadenosine 5'-triphosphate (10(-7)-10(-5) M), but not P(2Y) purinoceptor agonist 2-methylthioadenosine 5'-triphosphate (10(-7)-10(-5) M), induced muscle relaxations in a dose-dependent manner, and relaxations were significantly reduced by PPADS and unaffected by TTX. These studies suggest that NO and ATP act in concert to mediate NANC relaxation of the rat pylorus. ATP-induced relaxation appears to be mediated by P(2X) purinoceptors located on smooth muscle cells.