内皮依赖性超极化因子在剪切应力引起的内皮依赖性舒张反应中的作用

目的研究内皮依赖性超极化因子(EDHF)在剪切应力引起的内皮依赖性舒张反应中的作用及机制。方法测定不同流量下的血管内径及各种内皮依赖性舒张因子抑制剂、钾通道抑制剂、细胞色素P450单氧化酶抑制剂作用下的血管内径。结果剪切应力在大鼠肠系膜微动脉引起的舒张反应是内皮依赖性的,且在大的肠系膜动脉明显大于小阻力型肠系膜动脉。EDHF在上述两种动脉的内皮依赖性舒张反应中作用均明显大于NO,起主要作用。剪切应力引起的内皮依赖性舒张反应不受SKF525A的抑制，ChTx加apamin明显抑制了此舒张反应，TBA则几乎完全抑制此舒张反应。结论在剪切应力引起的内皮依赖性舒张反应中EDHF起主要作用，钾通道特别是K_Ca通道的激活为主要机制。     

Evidence against potassium as an endothelium-derived hyperpolarizing factor in rat mesenteric small arteries

1. Endothelium-derived hyperpolarizing factor (EDHF) has recently been identified as potassium released from endothelial cells into the myo-endothelial space. The present study was designed to test this hypothesis. 2. In rat small mesenteric arteries, mounted in a wire myograph, relaxation to acetylcholine or potassium was not significantly changed following incubation with oxadiazolo-quinoxalin-1-one (ODQ, 4 microM) and indomethacin (10 microM, n = 9). 3. Maximal relaxations to acetylcholine occurred in all arteries, were maintained and were significantly greater (P < 0.01, n = 9) than the transient relaxations to potassium, which only occurred in 30-40% of vessels. 4. Removal of the vascular endothelium abolished relaxant responses both to potassium and acetylcholine (P < 0.005, n = 9). 5. Compared with responses in 5.5 mM potassium PSS, relaxation responses to added potassium in arteries maintained in 1.5 mM potassium PSS were more marked and were not dependent on the presence of an intact endothelium (n = 8). 6. Incubation with BaCl2 (50 microM) significantly inhibited the maximal relaxant response to potassium in the presence of an intact endothelium in 5.5 mM potassium PSS (P < 0.05, n = 4), but had no effect on relaxation of de-endothelialized preparations in 1.5 mM potassium PSS (n = 5). 7. Treatment with ouabain (0.1 mM) abolished the relaxant response to potassium in 1.5 mM potassium PSS (P < 0.001, n = 9), but only partly inhibited the maximal relaxant response to acetylcholine in 5.5 mM potassium PSS (P < 0.01, n = 5). 8. These data show that at physiological concentrations of potassium an intact endothelium is necessary for potassium-induced relaxation in rat mesenteric arteries. Furthermore, the response to potassium is clearly different to that from acetylcholine, indicating that potassium does not mimic EDHF released by acetylcholine in these arteries.     

Role of endothelium-derived hyperpolarizing factor in the regulation of radial artery basal diameter and endothelium-dependent dilatation in vivo

1. The role of the balance between nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), synthesized by cytochrome epoxygenase and acting through calcium-activated potassium channels, in the regulation of basal diameter and endothelium-dependent flow-mediated dilatation of conduit arteries has been poorly assessed in humans. 2. Radial artery diameter and flow (echotracking coupled to Doppler) were measured in healthy volunteers under basal conditions and during flow-mediated dilatation induced by hand skin heating, in the presence of saline and inhibitors of NO-synthase, N(G)-monomethyl-L-arginine (L-NMMA), calcium-activated potassium channels, tetraethylammonium (TEA) and cytochrome epoxygenases, fluconazole, infused alone and in combination. Mean wall shear stress, the flow-mediated dilatation stimulus, was calculated and taken as cofactor into statistical analysis. 3. Under basal conditions, the radial artery diameter was not affected by L-NMMA and fluconazole infused alone but was decreased by TEA, the combinations of L-NMMA + fluconazole and, to a greater extent, L-NMMA + TEA. During heating, radial artery diameter increased with temperature in all cases. This increase in diameter, compared with saline, was reduced by L-NMMA, TEA, fluconazole and to a greater extent, by L-NMMA + TEA and L-NMMA + fluconazole. 4. These data show that EDHF is involved in balance with NO in the regulation of basal diameter and endothelium-dependent dilatation of human peripheral conduit arteries. The alteration of this balance could play a major role in the physiopathology of the endothelial dysfunction, in particular during essential hypertension.     

Suppression of K(+)-induced hyperpolarization by phenylephrine in rat mesenteric artery: relevance to studies of endothelium-derived hyperpolarizing factor

In intact mesenteric arteries, increasing [K(+)]o by 5 mM hyperpolarized both endothelial and smooth muscle cells. Subsequent exposure to 10 microM phenylephrine depolarized both cell types which were then repolarized by a 5 mM increase in [K(+)]o. In endothelium-denuded vessels, increasing [K(+)]o by 5 mM hyperpolarized the smooth muscle but K(+) had no effect after depolarization by 10 microM phenylephrine. On subsequent exposure to iberiotoxin plus 4-aminopyridine, the repolarizing action of 5 mM K(+) was restored. In endothelium-intact vessels exposed to phenylephrine, pretreatment with a gap junction inhibitor (gap 27) reduced K(+)-mediated smooth muscle repolarization without affecting the endothelial cell response. It is concluded that phenylephrine-induced efflux of K(+) via smooth muscle K(+) channels produces a local increase in [K(+)]o which impairs repolarization to added K(+). Thus, studies involving vessels precontracted with agonists which increase [K(+)]o maximize the role of gap junctions and minimize any contribution to the EDHF pathway from endothelium-derived K(+).     

Type 1 diabetes and hypercholesterolaemia reveal the contribution of endothelium-derived hyperpolarizing factor to endothelium-dependent relaxation of the rat aorta

1. The present study evaluated the effect of diabetes, hypercholesterolaemia and their combination on the contribution of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to relaxation of rat isolated aortic rings and the potential contribution of oxidant stress to the disturbance of endothelial function. 2. Thoracic aortic rings from control, diabetic, hypercholesterolaemic and diabetic plus hypercholesterolaemic rats were suspended in organ baths for tension recording. Generation of superoxide by the aorta was measured using lucigenin-enhanced chemiluminescence. 3. The maximal response to acetylcholine (ACh) was significantly reduced in diabetic or hypercholesterolaemic rats compared with control rats. In rats with diabetes plus hypercholesterolaemia, both the sensitivity and maximal response to ACh was impaired. In control rats, the response to ACh was abolished by the NO synthase inhibitor N(G)-nitro-L-arginine (L-NNA) or inhibition of soluble guanylate cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In contrast, in rats with diabetes, hypercholesterolaemia or both, relaxation to ACh was resistant to inhibition by L-NNA or ODQ, but abolished by additional inhibition of K(Ca) channels with charybdotoxin plus apamin. 4. The generation of superoxide was not significantly enhanced in aortic rings from either diabetic or hypercholesterolaemic rats, but was significantly increased in aortic rings from rats with diabetes plus hypercholesterolaemia. 5. These results suggest that when diabetes and hypercholesterolaemia impair endothelium-dependent relaxation, due to a diminished contribution from NO, a compensatory contribution of EDHF to endothelium-dependent relaxation of the aorta is revealed. The attenuation of NO-mediated relaxation, at least in the presence of both diabetes and hypercholesterolaemia, is associated with enhanced superoxide generation.     

内皮依赖性超极化因子的研究进展

血管内皮通过释放血管活性物质来调节血管紧张度,包括舒张性和收缩性两类,内皮依赖性超极化因子(endothelium-de-pendent hyperpolarizing factor,EDHF)是继一氧化氮(NO)和前列环素(PGI2)后在血管平滑肌舒张反应过程中发现的内皮释放的第三类舒血管因子,尽管其作用机制有几种不同类型,但都包括了细胞内Ca2 浓度的升高、钙依赖性钾通道(calcium-activatedpotassium channels,KCa)的开放及内皮细胞(endothelial cell)的超极化,最终导致了血管平滑肌细胞(smooth muscle cell,SMC)的超极化。SMC的超极化可以由直接接触机制(包括缝隙连接和细胞间隙的K )和可扩散性化学活性物质机制完成,两种机制不具有排他性,可同时存在于某些血管。     

Ovariectomy attenuates hyperpolarization and relaxation mediated by endothelium-derived hyperpolarizing factor in female rat mesenteric artery: a concomitant decrease in connexin-43 expression

Estrogen status is known to affect the incidence of cardiovascular disease. Experiments were designed to prove the influences of in vivo estrogen manipulations on vascular hyperpolarization and relaxation mediated by endothelium-derived hyperpolarizing factor (EDHF), and to explore the possible mechanism contributing to the altered EDHF responses in estrogen-deficient states. Mesenteric arteries with intact endothelium were isolated from sham-operated (control), ovariectomized (OVX), or OVX with 17beta-estradiol replacement (OVX + E ) female rats. In the presence of apamin and charybdotoxin, there was no difference between groups in relaxations to the Ca ionophore A23187 and the endoplasmic reticulum Ca -adenosine triphosphatase inhibitor cyclopiazonic acid (CPA). However, N -nitro-L-arginine produced a marked decrease in A23187- and CPA-induced relaxations in OVX compared with control and OVX + E arteries. In control arteries, A23187 and CPA elicited membrane hyperpolarization in a sustained manner. In contrast, A23187 produced only a small and transient hyperpolarizing effect in OVX arteries. OVX also greatly attenuated the sustained pattern of hyperpolarization to CPA. Such changes in hyperpolarizations were not seen in OVX + E arteries. The EDHF-mediated relaxant and hyperpolarizing responses of control arteries to A23187 and CPA were significantly inhibited by the gap junction inhibitor 18 alpha-glycyrrhetinic acid. Immunohistochemical examination for connexin-43 showed that the expression was abundant along the endothelial layer in control and OVX + E arteries, while being much less in OVX arteries. It was concluded that estrogen deficiency specifically impairs EDHF-mediated vascular actions. This may be partly explained by the reduced expression of connexin-43, a protein molecule that could form myoendothelial gap junction channels.     

Acetylcholine releases endothelium-derived hyperpolarizing factor and EDRF from rat blood vessels.

1. The effects of haemoglobin and methylene blue on the acetylcholine (ACh)-induced electrical and mechanical responses of smooth muscle cells were investigated in rat aorta and rat main pulmonary artery. 2. When the endothelium was intact, ACh induced a transient hyperpolarization and sustained relaxation of tissues precontracted with noradrenaline. Both hyperpolarization and relaxation were absent in preparations without endothelium. 3. Haemoglobin and methylene blue inhibited the ACh-induced relaxation, but not the transient hyperpolarization. 4. In aorta with an intact endothelium, ACh produced an increase in both the rate of 86Rb efflux and tissue cyclic GMP levels. The changes in ion flux were unaffected by either haemoglobin or methylene blue in concentrations which almost abolished the increase in cyclic GMP concentrations. 5. In arteries with an intact endothelium, indomethacin had no effect on the ACh-induced electrical and mechanical responses or on the increase in 86Rb efflux and tissue cyclic GMP levels. 6. It is concluded that in the rat aorta and rat main pulmonary artery, ACh releases two different substances, an endothelium-derived relaxing factor (EDRF) and a hyperpolarizing factor (EDHF), from the endothelial cells. Neither substance appears to be derived from a pathway dependent on cyclo-oxygenase. EDHF seems to play a minor role in the relaxation of noradrenaline-induced contractions.     

Different role of nitric oxide and endothelium-derived hyperpolarizing factor in endothelium-dependent hyperpolarization and relaxation in porcine coronary arterial and venous system

OBJECTIVE: We compared the basal and bradykinin (BK)-induced NO release and endothelium-derived hyperpolarizing factor-mediated function between coronary arteries and cardiac veins. METHODS AND RESULTS: Isolated coronary vessels (large arteries, cardiac veins as well as microvessels) were studied. An NO-specific electrode and a conventional intracellular glass microelectrode were used to directly measure NO released from endothelial cells and hyperpolarization of smooth muscle cells in conduit coronary vessels. The basal and BK-induced release of NO was 14.2 +/- 2.0 nmol/L and 237.1 +/- 27.2 nmol/L (n = 8) in the artery, significantly greater than in veins (8.0 +/- 1.1 nmol/L and 135.6 +/- 14.5 nmol/L, n = 8, P < 0.01). The BK-induced hyperpolarization was significantly reduced by N-nitro-L-arginine, indomethacin, and hemoglobin in both arteries and veins and was greater in the arteries. The EDHF-mediated relaxation was significantly higher in the arteries than in veins, greater in microveins than in large veins, and almost abolished by charybdotoxin and apamin. CONCLUSIONS: Both NO and EDHF are involved in the regulation of the vascular tone in the coronary arterial and venous systems but the amount of NO release and the EDHF-mediated relaxation and associated hyperpolarization are less significant in the vein than in the artery in the coronary system.     

内皮超极化因子的研究进展

内皮超极化因子（EDHF）是由内皮释放的NO和PGI_2以外的另一种舒张因子,它通过使平滑肌细胞膜超极化而舒张血管,是内皮依赖性血管松驰的第3种重要机制。EDHF可能是花生四烯酸的细胞色素P450代谢产物EET-s,乙酰胆碱、缓激肽等激动剂作用于内皮细胞,使细胞内游离钙浓度升高,合成和（或）释放EDHF,作用于平滑肌细胞膜,激活钙依赖性钾通道,使细胞膜超极化,抑制电压依赖性钙通道的开放,引起血管松弛。在大血管中NO-cGMP松弛机制可能占主导地位,并且抑制EDHF生成;而在阻力小血管,EDHF则可能是引起血管松弛的主要因素。     

Selective inhibition by barbiturates of the synthesis of endothelium-derived hyperpolarizing factor in the rabbit carotid artery.

1. Several lines of evidence suggest that both volatile and intravenous anaesthetics may interfere with the synthesis and release of endothelium-derived vasoactive factors. We have investigated the effects of three different barbiturates on the release of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in phenylephrine (1 microM)-preconstricted, endothelium-intact ring segments of the rabbit carotid artery. The segments were pretreated with the cyclo-oxygenase inhibitor, diclofenac (1 microM), to prevent the formation of vasoactive prostanoids, such as prostacyclin (PGI2). 2. Acetylcholine (ACh) elicited a concentration-dependent relaxation (EC50 0.15 microM) in control segments which was not significantly different from the relaxant responses of segments pretreated with methohexitone (0.03-0.3 mM), phenobarbitone (0.1-0.3 mM) or thiopentone (0.1-0.3 mM). 3. Inhibition of NO synthesis with NG-nitro-L-arginine (0.1 mM) significantly reduced the maximum relaxant response to ACh from 96 to 40%. This NO/PGI2-independent relaxation appeared to be mediated by the release of EDHF, since it was strongly diminished in the presence of the K+Ca inhibitors, tetrabutylammonium (1-3 mM) and charybdotoxin (10 nM), following preconstriction with potassium calcium (40 mM) or removal of the endothelium. Thiopentone or methohexitone markedly attenuated the EDHF-mediated relaxant response to ACh, while phenobarbitone had no effect. The endothelium-independent relaxation elicited by sodium nitroprusside (0.01-10 microM), on the other hand, was only marginally affected by these anaesthetics. 4. The cytochrome P450 inhibitor, clotrimazole (3-100 microM), mimicked the inhibitory effect of thiopentone and methohexitone on the NO/PGI2-independent relaxant response to ACh.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thapsigargin- and cyclopiazonic acid-induced endothelium-dependent hyperpolarization in rat mesenteric artery.

1. The present study was designed to determine whether putative, selective inhibitors of the Ca(2+)-pump ATPase of endoplasmic reticulum, thapsigargin (TSG) and cyclopiazonic acid (CPA), induce endothelium-dependent hyperpolarization in the rat isolated mesenteric artery. The membrane potentials of smooth muscle cells of main superior mesenteric arteries were measured by the microelectrode technique. 2. In tissues with endothelium, TSG (10(-8)-10(-5) M) caused sustained hyperpolarization in a concentration-dependent manner. In tissues without endothelium, TSG did not cause any change in membrane potential. CPA (10(-5) M) also hyperpolarized the smooth muscle membrane, an effect that was endothelium-dependent and long-lasting. 3. The hyperpolarizing responses to these agents were not affected by indomethacin or NG-nitro-L-arginine (L-NOARG). 4. In Ca(2+)-free medium, neither TSG nor CPA elicited hyperpolarization, in contrast to acetylcholine which generated a transient hyperpolarizing response. 5. In rings of mesenteric artery precontracted with phenylephrine, TSG and CPA produced endothelium-dependent relaxations. L-NOARG significantly inhibited the relaxations to these agents, but about 40-60% of the total relaxation was resistant to L-NOARG. The L-NOARG-resistant relaxations were abolished by potassium depolarization. 6. These results indicate that TSG and CPA can cause endothelium-dependent hyperpolarization in rat mesenteric artery possibly by releasing endothelium-derived hyperpolarizing factor and that membrane hyperpolarization can contribute to the endothelium-dependent relaxations to these agents. The mechanism of hyperpolarization may be related to increased Ca2+ influx into endothelial cells triggered by depletion of intracellular Ca2+ stores due to inhibition of endoplasmic reticulum Ca(2+)-pump ATPase activity.     

Role of gap junctions in endothelium-derived hyperpolarizing factor-mediated vasodilatation in rat renal artery

AIM: To investigate the effects of gap junction inhibitors on endothelium-derived but nitric oxide (NO)- and prostacyclin (PGI2)-independent vasodilatations induced by carbachol in the rat isolated renal artery. METHODS: Isolated renal arteries were mounted on a wire myograph apparatus were tissues treated with the nitric oxide synthase inhibitor N^ω-nitro-L-arginine methyl ester hydrochloride (NAME; 100 μmol/L) and indomethacin (10 μmol/L) and precontracted with phenylephrine (0.1 μmol/L). NAME and indomethacin treated Carbachol (0.01-10 μmol/L)- or sodium nitroprusside (SNP; 1-300 nmol/L)-induced mediated relaxations were observed in the presence of gap junction inhibitors. RESULTS: Carbachol produced a concentration-dependent relaxation in tissues treated with NAME (100 μmol/L) and indomethacin (10 μmol/L). This relaxation was not affected by hemoglobin (3 μmol/L), but was inhibited by charybdotoxin (200 nmol/L) and ouabain (30 μmol/L). The putative gap junction inhibitors, GAP 27 peptides with sequence homology to connexins 40 and 43 respectively reduced carbachol- but not SNP-induced relaxations mediated by endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations. The inhibition by the connexin 43 inhibitor was greater than that of the connexin 40 inhibitor. CONCLUSION: The results indicate the presence of gap junctions sensitive to 43^GAP 27 and 43^GAP 27 in the rat renal artery and each of these different types of gap junctions plays a role in the NO- and PGI2-independent relaxations induced by carbachol in this blood vessel. However, connexin 43 appears to play a more predominant role in mediating gap junction communications in the rat renal artery.     

内皮依赖性超极化因子在血管舒张中的作用

目的研究内皮依赖性超极化因子(EDHF)在血管舒张中的作用及机制。方法测定各种内皮依赖性舒张因子抑制剂、钾通道抑制因子、细胞色素P450单氧化酶抑制剂作用下的血管环张力。结果EDHF的血管舒张作用在大鼠肠系膜微动脉明显大于胸主动脉。一氧化氮(NO)合成受到慢性抑制时,胸主动脉的EDHF作用有增加趋势,在肠系膜微动脉投药后3 d和1周的EDHF作用明显增加。ChTx部分抑制、TBA明显抑制EDHF在肠系膜微动脉的舒张作用。结论EDHF在大鼠肠系膜微动脉的内皮依赖性舒张反应中起主要作用;在NO合成受抑制时其作用明显增加;其作用介导于K_Ca通道。     

The significance of endothelium-derived hyperpolarizing factor in the human circulation

Although nitric oxide (NO) is recognized as the primary vasodilator derived from vascular endothelium in regulating the vascular tone, another factor, i.e. the endothelium-derived hyperpolarizing factor (EDHF), has recently gained much attention and has been demonstrated to participate in vasodilatation in various blood vessels from different species, despite its unidentified nature. Most of the studies were conducted in animals and the knowledge of this factor in the human vasculature is relatively limited. This review attempts to address the relevance of EDHF-mediated function in humans with the possible identity of EDHF and mechanisms involved. We consider the human vasculature where EDHF involvement has been documented including the systemic, coronary, and visceral (gastrointestinal, renal and reproductive) circulation. In these vascular systems, EDHF plays a role under physiological conditions either as another mechanism or as the "back-up" for NO. Furthermore, the contribution of EDHF changes under certain physiological conditions, such as ageing and pregnancy. In addition, altered EDHF function has been suggested in various pathological conditions including heart diseases, atherosclerosis, hypertension, diabetes, eclampsia, glaucoma, chronic renal failure, erectile dysfunction and ischemia-reperfusion period during open heart surgery. Pharmacological agents such as potassium channel openers or cytochrome P450 metabolites have been used to either protect or recover EDHF-dependent mechanisms. To further develop new therapeutic strategies that target EDHF, a better understanding is essential with regard to the function of EDHF under pathophysiological conditions in humans. Furthermore, the interaction between NO and EDHF as well as their relative contributions in various conditions are critical.     

Relationship between NaF- and thapsigargin-induced endothelium-dependent hyperpolarization in rat mesenteric artery.

1. In isolated rat mesenteric artery with endothelium, NaF caused slowly developing hyperpolarization. The hyperpolarizing effect was unchanged in the presence of N(G)-nitro-L-arginine (L-NOARG) and indomethacin, but was markedly reduced by high K+. In Ca2+ -free medium or in the presence of Ni2+, NaF failed to produce hyperpolarization. 2. NaF-induced hyperpolarization was substantially unaffected by deferoxamine, an Al3+ chelator, okadaic acid and calyculin A, phosphatase inhibitors, and preincubation with pertussis toxin, suggesting that neither the action of fluoroaluminates as a G protein activator nor inhibition of phosphatase activity contributes to the hyperpolarizing effect. 3. The selective inhibitors of the Ca2+ -pump ATPase of endoplasmic reticulum, thapsigargin and cyclopiazonic acid, elicited hyperpolarization, whose properties were very similar to those of NaF. When intracellular Ca2+ stores had been depleted with these inhibitors, NaF no longer generated hyperpolarization. 4. In Ca2+ -free medium, NaF (or thapsigargin) caused a transient increase in the cytosolic Ca2+ concentration ([Ca2+]i) in cultured porcine aortic endothelial cells, and subsequent application of thapsigargin (or NaF) failed to increase [Ca2+]i. 5. In arterial rings precontracted with phenylephrine, NaF produced endothelium-dependent relaxation followed by sustained contraction even in the presence of L-NOARG and indomethacin. The relaxant response was abolished by high K+ or cyclopiazonic acid. 6. These results indicate that NaF causes endothelium-dependent hyperpolarization, thereby leading to smooth muscle relaxation of rat mesenteric artery. This action appears to be mediated by the promotion of Ca2+ influx into endothelial cells that can be triggered by the emptying of intracellular Ca2+ stores, as proposed for those of thapsigargin and cyclopiazonic acid.     

血管内皮依赖性超极化因子的研究进展

内皮依赖性超极化因子（endothelium-dependent hyperpolarizing factor，EDHF）是血管平滑肌内皮源性舒张反应中通过非环氧合酶、非一氧化氮合酶途径的舒张因子。在不同物种、不同血管，EDHF反应都得到了确认。其在心血管生理学和药理学方面有着重要的作用。     

The role of endothelium-derived nitric oxide in relaxations to levcromakalim in the rat aorta

The present study was designed to examine the role of basally released nitric oxide in relaxations to an ATP-sensitive K+ channel opener. Whether relaxations to levcromakalim are modulated by endothelial removal or the inhibitors of vasodilator effects of endothelium-derived nitric oxide, were investigated in the rat aorta. During contractions to phenylephrine (3 x 10(-7) to 10(-6) M), levcromakalim (10(-8) to 10(-5) M) or a nitric oxide donor, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7, 10(-9) to 10(-5) M), was added in a cumulative fashion. Relaxations to levcromakalim (10(-8) to 10(-5) M) were significantly reduced by the endothelium-removal. In aortas with endothelium, relaxations in response to levcromakalim were decreased by selective inhibitors of nitric oxide synthase (N(G)-nitro-L-arginine methyl ester, 10(-4) M) and soluble guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one; ODQ, 10(-5) M) and a scavenger of nitric oxide (carboxy-PTIO, 10(-3) M). Relaxations to levcromakalim in aortas treated with these inhibitors are comparable to those seen in aortas without endothelium. KCl (30 mM) and an ATP-sensitive K+ channel inhibitor, glibenclamide (10(-5) M), abolished relaxations to levcromakalim in aortas with or without endothelium, whereas glibenclamide did not alter relaxations to NOC-7 (10(-9) to 10(-5) M) in aortas without endothelium. These results suggest that in rat aortas, inhibition of vasodilator effects of basally released nitric oxide can reduce relaxations via ATP-sensitive K+ channels, although these channels do not mediate relaxations to exogenously applied nitric oxide.     

Identifying endothelium-derived hyperpolarizing factor: recent approaches to assay the role of epoxyeicosatrienoic acids

Investigation of endothelial regulation of vascular reactivity and tone has led to the discovery of chemical mediators such as nitric oxide (NO) and prostacyclin (PGI2). Evidence has emerged indicating another as yet unidentified hyperpolarizing agent (endothelium-derived hyperpolarizing factor or EDHF) that is different from NO and PGI2 and exerts it effects through calcium-activated potassium channels (KCa). Previous studies to identify EDHF have been carried out using inhibitors that block NOS and COX before application of KCa channel and/or muscarinic receptor antagonists. Such pharmacological manipulation has complicated interpretation of results, clearly pointing to the need for altered approaches to verify previous studies. Evidence has emerged that potential EDHF candidates vary with vessel size, species and tissue beds, indicating that there may be more than one EDHF. To date, the most commonly described and best characterized of them all are a set of arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs). These compounds are synthesized both intra- and extravascularly. Until recently, methodology to detect EETs in the microvasculature has been tedious and expensive, limiting the experimentation that is necessary to confirm EETs as an EDHF. This review describes state-of-the-art methods for assaying EETs in biological samples, after summarizing evidence for EETs as an EDHF and introducing emerging concepts of the role of extravascular EETs in linking neuronal activity to localized blood flow during functional hyperemia.     

Regulation of vascular tone by endothelium-derived hyperpolarizing factor

1. Endothelium-derived hyperpolarizing factor (EDHF) mediates the nitric oxide (NO)-independent component of the relaxation in rat mesenteric arteries. The relationship between hyperpolarization and vascular tone was studied by simultaneous recording of membrane potential with intracellular microelectrodes and tension in ring segments of rat mesenteric arteries. 2. By depolarizing arteries with high potassium solutions, it was determined that the threshold for contraction is approximately -46 mV. Maximum contraction was attained when the arteries were depolarized to -20 mV. Thus, 1 mV depolarization resulted in an approximate 4% increase in tone. This relationship was not altered in spontaneously hypertensive rats. 3. Noradrenaline (0.3 mumol/L) caused contraction and depolarized arteries by 13 mV. Acetylcholine caused endothelium-dependent relaxation and hyperpolarization up to 14 mV. In the presence of N omega-nitro-L-arginine, the EDHF-mediated relaxation was correlated to hyperpolarization. A hyperpolarization of 1 mV corresponded to a 4.3% decrease of the induced tone. 4. At concentrations (10 mumol/L) causing total relaxation, the maximum hyperpolarization induced by NO was only 7.6 mV. 5. A maximum relaxation of 88% was observed with pinacidil (3 mumol/L), despite a 25 mV hyperpolarization. Relaxations to NO and pinacidil were not correlated with hyperpolarization. At similar levels of hyperpolarization, NO and pinacidil elicited more relaxation than EDHF. 6. These studies show that vascular tone is very sensitive to membrane potential change in the range between -46 and -20 mV in the rat mesenteric artery. The relaxation response to EDHF, unlike that to NO and pinacidil, can be accounted for solely by its effect on the membrane potential.