Inhibition by SK&F96365 of NO-mediated relaxation induced by Ca2(+) -ATPase inhibitors in rat thoracic aorta.

1. We investigated the effect of SK&F96365, a putative inhibitor of receptor-operated Ca2+ entry, on the endothelium-dependent, NO-mediated relaxation and cyclic GMP formation induced by Ca2(+)-ATPase inhibitors in rat thoracic aorta. 2. SK&F96365 inhibited cyclopiazonic acid or thapsigargin-induced relaxation and cyclic GMP formation mediated by a constitutive NO synthase, which is known to be activated by the Ca2+ that enters into the endothelial cells via plasma membrane Ca2+ channels subsequent to depletion of stored Ca2+ by Ca2(+)-ATPase inhibitors. 3. SK&F96365 also inhibited relaxation and cyclic GMP formation induced by acetylcholine, without affecting those induced by nitroprusside and A23187. 4. Ni2+ attenuated relaxation and cyclic GMP formation induced by cyclopiazonic acid and acetylcholine. 5. In contrast, the voltage-dependent Ca2+ channel blocker, nifedipine, did not affect the relaxation caused by Ca2(+)-ATPase inhibitors. 6. These results suggest that endothelium-dependent, NO-mediated relaxation of the arteries induced by Ca2(+)-ATPase inhibitors is triggered by the Ca2+ that enters into endothelial cells via receptor-operated channels (SK&F96365-sensitive channels) subsequent to depletion of stored Ca2+ as a result of inhibition of the Ca2(+)-ATPase (Ca2+ pump) of the stores.     

Role of endothelial cell hyperpolarization in EDHF-mediated responses in the guinea-pig carotid artery

1. Experiments were performed to identify the potassium channels involved in the acetylcholine-induced endothelium-dependent hyperpolarization of the guinea-pig internal carotid artery. Smooth muscle and endothelial cell membrane potentials were recorded in isolated arteries with intracellular microelectrodes. Potassium currents were recorded in freshly-dissociated smooth muscle cells using patch clamp techniques. 2. In single myocytes, iberiotoxin (0.1 microM)-, charybdotoxin (0.1 microM)-, apamin (0.5 microM)- and 4-aminopyridine (5 mM)-sensitive potassium currents were identified indicating the presence of large- and small-conductance calcium-sensitive potassium channels (BK(Ca) and SK(Ca)) as well as voltage-dependent potassium channels (K(V)). Charybdotoxin and iberiotoxin inhibited the same population of BK(Ca) but a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected. 4-aminopyridine (0. 1 - 25 mM) induced a concentration-dependent inhibition of K(V) without affecting the iberiotoxin- or the apamin-sensitive currents. 3. In isolated arteries, both the endothelium-dependent hyperpolarization of smooth muscle and the hyperpolarization of endothelial cells induced by acetylcholine or by substance P were inhibited by 5 mM 4-aminopyridine. 4. These results indicate that in the vascular smooth muscle cells of the guinea-pig carotid artery, a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected, comforting the hypothesis that the combination of these two toxins should act on the endothelial cells. Furthermore, the inhibition by 4-aminopyridine of both smooth muscle and endothelial hyperpolarizations, suggests that in order to observe an endothelium-dependent hyperpolarization of the vascular smooth muscle cells, the activation of endothelial potassium channels is likely to be required.     

Pramanicin,an antifungal agent,raises cytosolic Ca2+ and causes cell death in vascular endothelial cells

The effects of a newly discovered antifungal agent, pramanicin, on cytosolic Ca(2+) and cell viability of cultured bovine pulmonary artery endothelial cells and on endothelium-dependent relaxation of dog carotid arterial rings were investigated by digital dynamic fluorescence ratio imaging and morphological and contractility studies, respectively. Pramanicin 100 microM, previously shown to cause maximal endothelium-dependent and NO-mediated vascular relaxation, induced a small transient elevation of cytosolic Ca(2+) concentration in Ca(2+)-free medium; subsequent introduction of 1 mM Ca(2+) caused a steady, nonsaturating increase of Ca(2+), which could be brought down to the basal level by the addition of EGTA. At the single cell level, the elevation of cytosolic Ca(2+) initiates from the cell periphery and progresses toward the central region. When added to the plateau phase of phenylephrine-induced contraction, pramanicin induced a slow endothelium-dependent relaxation, which could be reversed with the NO synthase inhibitor, L-NOARG. When preincubated with vascular tissue, pramanicin resulted in an irreversible loss of endothelial function characterized by the lack of carbachol-induced relaxation. Pramanicin caused cell injury characterized by plasmalemmal bleb formation, leading to cell death characterized by Trypan blue staining of the nuclei in cultured vascular endothelial cells in a concentration- and time-dependent manner. Such pramanicin-induced cell death was not associated with Ca(2+)-mediated or NO-mediated mechanisms. The time course of Ca(2+) elevation corresponds with that of pramanicin-induced relaxation of precontracted arterial rings, whereas the time course of endothelial cell death corresponds to that of pramanicin-induced loss of endothelial function as assessed by carbachol-induced relaxation. The pramanicin analogue, PMC-A, a by-product of the biosynthesis of pramanicin, in which the epoxy group is replaced by a CC bond, caused little endothelial-dependent relaxation, but it was able to cause endothelial cell dysfunction, albeit to a lesser extent compared to pramanicin, suggesting a role of the epoxy group in pramanicin for its vasorelaxant effect.     

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.     

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。     

Vasorelaxant effects of pramanicin, an anti-fungal agent: selective action on endothelial cells

A newly discovered antifungal agent, pramanicin, within the therapeutically effective concentration range (4-100 microM), inhibits the tone of phenylephrine (PE)-precontracted dog carotid arterial rings in a concentration-dependent manner and leads to gradual development of relaxation. However, pramanicin had no effect on rings precontracted with 100 mM KCl or on endothelium-denuded rings. Thus, inhibition by pramanicin of PE-induced contraction was endothelium-dependent. Preincubation of 100 microM pramanicin with carotid arterial rings for 30 min did not significantly affect the concentration-contraction response to PE, but almost completely inhibited the endothelium-dependent relaxation response to subsequent addition of 3 microM carbachol or 100 microM pramanicin. This irreversible inhibition of endothelium-dependent relaxation, which is independent of extracellular Ca2+, suggests possible endothelial cell damage by pramanicin. Pretreatment of the endothelium-intact vascular rings with L-N(G)-nitro-arginine (100 microM) inhibited the relaxation of PE-precontracted rings induced by 3 microM carbachol or 100 microM pramanicin, suggesting that the generation of nitric oxide (NO) in endothelial cells mediates the slow vascular relaxation induced by pramanicin. We conclude that pramanicin has little direct effect on the contractility of smooth muscle cells, but causes an initial slow endothelium-dependent, NO-mediated vascular relaxation. This is followed by a cytotoxic effect on vascular endothelial cells, eventually resulting in the loss of vasorelaxant function.     

Role of K+ channels and sodium pump in the vasodilation induced by acetylcholine, nitric oxide, and cyclic GMP in the rabbit aorta.

The endothelium-dependent relaxation caused by acetylcholine (ACh) in rabbit aorta segments was reduced by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester and by blockade of: Na+ pump with ouabain, large-conductance Ca2+-activated K+ (BK(Ca)) channels with charybdotoxin (ChTx), or voltage-dependent K+ (Kv) channels with 4-aminopyridine (4-AP). ACh relaxation was unaltered by glibenclamide, apamin, and Ba2+, blockers of ATP-sensitive K+ channels, small-conductance Ca2+-activated K+ channels, and inward rectifier K+ channels, respectively. The relaxation induced by exogenous NO and 8-bromocyclic GMP (8-BrcGMP) was similar in intact and endothelium-denuded segments, and it was reduced or unaltered by the same drugs used in the case of ACh. However, a 4-AP concentration 20-fold higher was necessary to reduce exogenous NO relaxation. These data suggest a resemblance in the mechanisms implicated in the relaxation elicited by ACh, exogenous NO, and 8-BrcGMP. Therefore, the relaxation caused by ACh is mainly mediated by endothelial NO, which in turn, enhances cGMP levels; this messenger appears to be the major one responsible for the smooth muscle cell hyperpolarization in the relaxation elicited by ACh, which is mediated by activation of the Na+ pump and ChTx- and 4-AP-sensitive K+ channels, likely BK(Ca) and Kv channels.     

Characteristics of ACh-induced hyperpolarization and relaxation in rabbit jugular vein

BACKGROUND AND PURPOSE The roles played by endothelium-derived NO and prostacyclin and by endothelial cell hyperpolarization in ACh-induced relaxation have been well characterized in arteries. However, the mechanisms underlying ACh-induced relaxation in veins remain to be fully clarified. EXPERIMENTAL APPROACH ACh-induced smooth muscle cell (SMC) hyperpolarization and relaxation were measured in endothelium-intact and -denuded preparations of rabbit jugular vein. KEY RESULTS In endothelium-intact preparations, ACh (≤10(-8) M) marginally increased the intracellular concentration of Ca(2+) ([Ca(2+) ](i) ) in endothelial cells but did not alter the SMC membrane potential. However, ACh (10(-10) -10(-8) M) induced a concentration-dependent relaxation during the contraction induced by PGF(2α) and this relaxation was blocked by the NO synthase inhibitor N(ω) -nitro-l-arginine. ACh (10(-8) -10(-6) M) concentration-dependently increased endothelial [Ca(2+) ](i) and induced SMC hyperpolarization and relaxation. These SMC responses were blocked in the combined presence of apamin [blocker of small-conductance Ca(2+) -activated K(+) (SK(Ca) , K(Ca) 2.3) channel], TRAM 34 [blocker of intermediate-conductance Ca(2+) -activated K(+) (IK(Ca) , K(Ca) 3.1) channel] and margatoxin [blocker of subfamily of voltage-gated K(+) (K(V) ) channel, K(V) 1]. CONCLUSIONS AND IMPLICATIONS In rabbit jugular vein, NO plays a primary role in endothelium-dependent relaxation at very low concentrations of ACh (10(-10) -10(-8) M). At higher concentrations, ACh (10(-8) -3 × 10(-6) M) induces SMC hyperpolarization through activation of endothelial IK(Ca) , K(V) 1 and (possibly) SK(Ca) channels and produces relaxation. These results imply that ACh regulates rabbit jugular vein tonus through activation of two endothelium-dependent regulatory mechanisms.     

Cl~-通道阻断剂对5-HT和CPA引起的兔脑椎基底动脉平滑肌细胞Ca~(2+)内流的影响

目的　在培养的兔脑椎基底动脉平滑肌细胞上观察5 HT和CPA诱导的Ca2 + 内流的特性 ,电压依赖性Ca2 + 通道 (VDC)抑制药尼莫地平 ,非电压依赖性Ca2 + 通道抑制药SK&F963 65及Cl-通道阻断剂DIDS、NPPB对两种激动剂引起 [Ca2 + ]i 反应的影响 ,以探讨脑血管平滑肌细胞中 5 HT引起Ca2 + 内流的特性、Cl-通道与Ca2 + 内流的关系。方法　采用生物荧光双波长影像分析系统瞬即测定单细胞胞质[Ca2 + ]i 技术。结果　① 5 HT和CPA均能诱导平滑肌细胞[Ca2 + ]i 呈双相升高 ,并且 5 HT诱导的Ca2 + 释放是环匹阿尼酸 (CPA)敏感Ca2 + 池的一部分 ;②尼莫地平对 5 HT和CPA触发的Ca2 + 内流无明显影响 ,而SK&F963 65可阻止二者触发的Ca2 + 内流 ;③Cl-通道阻断剂DIDS、NPPB呈浓度依赖性抑制Ca2 + 内流 ,在SK&F963 65最大限度抑制Ca2 + 内流后 ,DIDS、NPPB可进一步抑制Ca2 + 内流 ;而Ca2 +内流被DIDS、NPPB分别最大抑制后 ,SK&F963 65也可进一步抑制Ca2 + 内流。结论　 5 HT引起的Ca2 + 内流是经SK&F963 65敏感的非VDC ,其中包含Ca2 + 释放引起的Ca2 + 内流 (CRAC)成分与非CRAC成分 ,并且这两部分Ca2 +内流均与DIDS、NPPB敏感的Cl-通道开放有关     

Effect of simvastatin on endothelium-dependent vaso-relaxation and endogenous nitric oxide synthase inhibitor

AIM: To investigate the effect of simvastatin on endothelium-dependent vasorelaxation and endogenous nitric oxide synthesis inhibitor asymmetric dimethylarginine (ADMA) in rats and cultured ECV304 cells. METHODS: Endothe-lial injury was induced by a single injection of low density lipoprotein (LDL) (4 mg/kg, 48 h) in rats or incubation with LDL (300 mg/L) or oxidative-modified LDL (100 mg/L) in cultured ECV304 cells, and vasodilator responses to acetylcholine (ACh) in the aortic rings and the level of ADMA, nitrite/nitrate (NO) and tumor necrosis factor-alpha (TNF-α) in the serum or cultured medium were determined. And the adhesion of the monocytes to endothe-lial cells and the activity of dimethylarginine dimethylaminohydrolase (DDAH) in the cultured ECV304 cells were measured. RESULTS: A single injection of LDL decreased endothelium-dependent relaxation to ACh, markedly increased the serum level of endogenous ADMA and TNF-α, and reduced serum level of NO. Pretreatment with simvastatin (30 or 60 mg/kg) markedly attenuated inhibition of vasodilator responses to ACh, the increased level of TNF-α and the decreased level of NO by LDL, but no effect on serum concentration of endogenous ADMA. In cultured ECV304 cells, LDL or ox-LDL markedly increased the level of ADMA and TNF-α and potentiated the adhesion of monocytes to endothelial cells, concomitantly with a significantly decrease in the activity of DDAH and serum level of NO. Pretreatment with simvastatin (0.1, 0.5, or 2.5 μmol/L) markedly decreased the level of TNF-α and the adhesion of monocytes to endothelial cells, but did not affect the concentration of endogenous ADMA and the activity of DDAH. CONCLUSION: Simvastatin protect the vascular endothelium against the damages induced by LDL or ox-LDL in rats or cultured ECV304 cells, and the beneficial effects of simvastatin may be related to the reduction of inflammatory cytokine TNF-α level.     

Possible involvement of Ca2+ entry and its pharmacological characteristics responsible for endothelium-dependent, NO-mediated relaxation induced by thapsigargin in guinea-pig aorta.

Thapsigargin, a specific inhibitor of Ca(2+)-pump Ca(2+)-ATPase in the sarcoplasmic/endoplasmic reticulum (SR/ER), produces an endothelium-dependent vascular relaxation. In the present study, pharmacological features of thapsigargin-induced endothelium-dependent relaxation were functionally characterized in the isolated guinea-pig aorta especially focusing on the Ca2+ mobilization mechanisms in endothelial cells. Thapsigargin-induced endothelium-dependent vascular relaxation was markedly suppressed by N(G)-nitro-L-arginine (L-NNA) and calmidazolium, suggesting that the vascular relaxation to thapsigargin is largely attributable to endothelium-derived nitric oxide (NO) produced as a result of the activation of Ca2+, calmodulin-dependent NO synthase (NOS). Removal of Ca2+ from the external solution abolished the endothelium-dependent relaxation of guinea-pig aorta in response to thapsigargin. Thapsigargin-induced endothelium-dependent relaxation was inhibited more strongly compared with the endothelium-independent relaxation to an NO donor, SIN-1 (3-(4-morpholinyl)-sydnonimine), when the artery preparation was preconstricted with a high concentration (80 mM) of KCl instead of agonistic stimulation. Endothelium-dependent relaxation induced by thapsigargin was not affected by diltiazem, a blocker of L-type voltage-gated Ca2+ channels. SK&F96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1 H-imidazole) and Ni2+, both of which block capacitative Ca(2+) entry, did not show any appreciable inhibitory effects on the endothelium-dependent relaxation to thapsigargin. These findings suggest that in guinea-pig aorta, endothelium-dependent NO-mediated relaxation induced by thapsigargin is preceded by the increase in the cytosolic free Ca2+ concentrations ([Ca2+]cyt) following the depletion of stored Ca2+ in thapsigargin-sensitive store sites in endothelial cells. Although the increase in [Ca2+]cyt responsible for the activation of endothelium NOS leading to thapsigargin-induced vascular relaxation may be ascribed to the capacitative Ca2+ entry from extracellular space, the Ca2+ entry mechanism stimulated with thapsigargin is deficient in sensitivity to SK&F96365 and Ni2+ in the endothelium of guinea-pig aorta.     

Endothelium-dependent contractions in SHR: a tale of prostanoid TP and IP receptors

In the aorta of spontaneously hypertensive rats (SHR), the endothelial dysfunction is due to the release of endothelium-derived contracting factors (EDCFs) that counteract the vasodilator effect of nitric oxide, with no or minor alteration of its production. The endothelium-dependent contractions elicited by acetylcholine (ACh) involve an increase in endothelial [Ca²⁺]_i, the production of reactive oxygen species, the activation of endothelial cyclooxygenase-1, the diffusion of EDCF and the subsequent stimulation of smooth muscle cell TP receptors. The EDCFs released by ACh have been identified as PGH₂ and paradoxically prostacyclin. Prostacyclin generally acts as an endothelium-derived vasodilator, which, by stimulating IP receptors, produces hyperpolarization and relaxation of the smooth muscle and inhibits platelet aggregation. In the aorta of SHR and Wistar-Kyoto rats, prostacyclin is the principal metabolite of arachidonic acid released by ACh. However, in SHR aorta, prostacyclin does not produce relaxations but activates the TP receptors on vascular smooth muscle cells and produces contraction. The IP receptor is not functional in the aortic smooth muscle cells of SHR as early as 12 weeks of age, but its activity is not reduced in platelets. Therefore, prostacyclin in the rule protects the vascular wall, but in the SHR aorta it can contribute to endothelial dysfunction. Whether or not prostacyclin plays a detrimental role as an EDCF in other animal models or in human remains to be demonstrated. Nevertheless, because EDCFs converge to activate TP receptors, selective antagonists of this receptor, by preventing endothelium-dependent contractions, curtail the endothelial dysfunction in diseases such as hypertension and diabetes.     

Tamoxifen dilates porcine coronary arteries: roles for nitric oxide and ouabain-sensitive mechanisms

BACKGROUND AND PURPOSE: Experiments were designed to determine the mechanism of the relaxation induced by tamoxifen in porcine coronary arteries at the tissue, cellular and molecular levels. EXPERIMENTAL APPROACH: Porcine left circumflex coronary arteries were isolated and isometric tension was measured. [Ca2+]i in native endothelial cells of intact arteries was determined by a calcium fluorescence imaging technique and eNOS ser1177 phosphorylation was assayed by Western blotting. KEY RESULTS: Tamoxifen induced an endothelium-dependent relaxation that was antagonized by ICI 182,780 and abolished by NG-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one (ODQ). L-Arginine reversed the effect of L-NAME while indomethacin was without effect. Tamoxifen-induced relaxation was attenuated by charybdotoxin (CTX) plus apamin, ouabain or by incubation in a K+ -free solution. Moreover, tamoxifen triggered extracellular Ca2+ -dependent increases in endothelial [Ca2+]i and this effect was abolished by ICI 182,780. Endothelium-independent relaxation to sodium nitroprusside was also inhibited by ouabain or in a K+ -free solution. Furthermore, tamoxifen increased endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177 and ICI 182,780 prevented this effect. CONCLUSIONS AND IMPLICATIONS: The present results suggest that tamoxifen mainly induces endothelium-dependent relaxation and that endothelial nitric oxide (NO) is the primary mediator of this effect. NO-dependent responses may result from elevated [Ca2+]i in endothelial cells; an effect abolished by ICI 182,780. NO activates Na+/K+ -ATPase in vascular smooth muscle, leading to relaxation. These results suggest that tamoxifen is able to modulate eNOS phosphorylation directly.     

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.     

ENDOTHELIUM-DERIVED HYPERPOLARIZING FACTOR

1. Not all endothelium-dependent relaxation can be full explained by the release of either nitric oxide (NO) and/or prostacyclin. Another unidentified substance(s) that hyperpolarizes the underlying vascular smooth muscle cells (endothelium-derived hyperpolarizing factor; EDHF) contributes to endothelium-dependent relaxations. 2. In blood vessels from various species these hyperpolarizations are resistant to inhibitors of NO synthase (NOS) and cycl-oxygenase. In canine, porcine and human blood vessels the hyperpolarization cannot be mimicked by nitrovasodilators or exogeneous NO. However, in other species (rat, guinea-pig, rabbit) endothelium-dependent hyperpolarizations resistant to inhibitors of NOS and cyclo-oxygenase and hyperpolarizations to endothelium-derived or exogeneous NO can be obsercved n the same vascular smooth muscle cells. 3. In blood vessels where NO causes hyperpolarization, the response is blocked by glibenclamide, suggesting the involvement of ATP-dependent potassium channels. Hyperpolarizations caused by EDHF are insensitive to glibenclamide but, depending on the tissue, are inhibited by relatively small concentrations of tetraethylammonium (TEA) or by apamin or the combination of charybdotoxing plus apamin, indicating that calcium-dependent potassium channels are likely to be involved. 4. Metabolites of arachidonic acid, through the cytochrome P450 mono-oxygenase pathway (epoxyeicosatrienoic acids), are produced by the endothelial cells, increase the open-state probability of calcium-activated potassium channels sensitive to TEA or charybdotoxin, and induce the hyperpolarization of arterial smooth muscle cells, indicating that epoxyeicosatrienoic acids could be EDHF. However, in blood vessels from various species, cytochrome P450 inhibitors do not affect endothelium-dependent hyperpolarizations, indicating that EDHF is not yet identified with certainty. 5. Endothelium-derived hyperpolarizing factor released from cultured endothelial cells reduces the intracellular calcium concentration in vascular smooth muscle cells and the EDHF component of the relaxation is proportionally more important in smaller than larger arteries. In aging animals and in various models of diseases, endothelium-dependent hyperpolarizations are diminished. 6. The identification of EDHF and/or the discovery of specific inhibitors of its synthesis and its action may allow a better understanding of its physiological and pathophysiological role(s).     

Chronic treatment of male rats with daidzein and 17 beta-oestradiol induces the contribution of EDHF to endothelium-dependent relaxation

1. We investigated the effect of chronic (7 days) treatment of male rats with the isoflavone daidzein (0.2 mg kg(-1) sc per day) or 17beta-oestradiol (0.1 mg kg(-1) sc per day) on the contribution of nitric oxide (NO), prostaglandins and endothelium-derived hyperpolarising factor (EDHF) to endothelium-dependent relaxation of isolated aortic rings. 2. The sensitivity and maximum relaxation to acetylcholine (ACh) were significantly greater in aortic rings from rats treated with daidzein or 17beta-oestradiol, in comparison to vehicle-treated rats. Inhibition of nitric oxide synthase with N-nitro-l-arginine (l-NOARG) abolished ACh-induced relaxation in the aortae from vehicle-treated rats, but only attenuated relaxation in aortae from daidzein or 17beta-oestradiol-treated rats. The presence of haemoglobin in addition to l-NOARG did not cause any further inhibition of relaxation. 3. The cyclooxygenase inhibitor indomethacin had no effect on endothelium-dependent relaxation in aortae from any treatment group. Charybdotoxin (ChTX), which blocks large-conductance calcium-activated potassium channels (BK(Ca)) and intermediate-conductance calcium-activated potassium channels (IK(Ca)), plus apamin, which blocks small-conductance calcium-activated potassium channels (SK(Ca)), but not iberiotoxin, which only blocks BK(Ca), attenuated endothelium-dependent relaxation of aortae from daidzein or 17beta-oestradiol-treated rats. Blockade of K(Ca) channels had no effect on the responses to ACh in aortae from vehicle-treated rats. In aortae from daidzein- or 17beta-oestradiol-treated rats, endothelium-dependent relaxation was also attenuated by inhibition of cytochrome P450 (CYP450) epoxygenase with 6-(2-propargylloxyphenyl)hexanoic acid (PPOH) or inhibition of K(IR) channels and Na(+)/K(+)-ATPase with barium and oubain, respectively. 4. This study demonstrates that endothelium-dependent relaxation of male rat aorta is normally entirely mediated by NO, whereas treatment with daidzein or 17beta-oestradiol stimulates a contribution from a non-NO, nonprostaglandin factor acting through the opening of SK(Ca) and IK(Ca) channels, and involving activation of Na/K-ATPase, K(IR) and CYP450 epoxygenase. This pattern of sensitivity to the tested inhibitors is consistent with the contribution of EDHF to relaxation. Thus, EDHF contributes to the enhanced endothelium-dependent relaxation that is observed after chronic treatment with the phytoestrogen daidzein or with 17beta-oestradiol.     

Endothelium-dependent inhibition of the use of extracellular calcium for the arterial response to vasoconstrictor agents

The responses of rabbit mesenteric or coeliac artery rings to angiotensin II or adrenaline (but not to K+) were enhanced by endothelium destruction (by rubbing). Potentiation by indomethacin of the response to the agonists was observed in rubbed rings but not in intact ones. Both angiotensin II and adrenaline (in the presence of propranolol and prazosin) induced endothelium-dependent relaxation of the arteries. Rubbed rings, but not intact ones, contracted when Ca2+ was added to a previously Ca2+-free medium containing angiotensin II or adrenaline. The vasoconstrictor response appears to be modulated by the regulation of receptor-operated Ca2+ channels through EDRF released by the endothelium and by some cyclo-oxygenase product at the level of the smooth muscle cell membrane.     

An increase in opening of BK(Ca) channels in smooth muscle cells in streptozotocin-induced diabetic mice

INTRODUCTION Diabetes mellitus (DM) is a kind of disease thatmetabolism decompensates with hyperglycemia and re-sults in multi-organ damage[1]. Thus, the risk of coro-nary disease, cerebrovascular disease, and other car-diovascular complications increase. These changes, atleastpartially, due todiabetes functionalchanges inbloodvessels including endothelial cell dysfunction. Simulta-neously, altered ion channelfunction in vascular smoothmuscle are also involved[2,3]. In vascular…     

Thromboxane receptor stimulation associated with loss of SKCa activity and reduced EDHF responses in the rat isolated mesenteric artery

1. The possibility that thromboxane (TXA(2)) receptor stimulation causes differential block of the SK(Ca) and IK(Ca) channels which underlie EDHF-mediated vascular smooth muscle hyperpolarization and relaxation was investigated in the rat isolated mesenteric artery. 2. Acetylcholine (30 nm-3 microm ACh) or cyclopiazonic acid (10 microm CPA, SERCA inhibitor) were used to stimulate EDHF-evoked smooth muscle hyperpolarization. In each case, this led to maximal hyperpolarization of around 20 mV, which was sensitive to block with 50 nm apamin and abolished by repeated stimulation of mesenteric arteries with the thromboxane mimetic, U46619 (30 nm-0.1 microm), but not the alpha(1)-adrenoceptor agonist phenylephrine (PE). 3. The ability of U46619 to abolish EDHF-evoked smooth muscle hyperpolarization was prevented by prior exposure of mesenteric arteries to the TXA(2) receptor antagonist 1 microm SQ29548. 4. Similar-sized smooth muscle hyperpolarization evoked with the SK(Ca) activator 100 microm riluzole was also abolished by prior stimulation with U46619, while direct muscle hyperpolarization in response to either levcromakalim (1 microm, K(ATP) activator) or NS1619 (40 microm, BK(Ca) activator) was unaffected. 5. During smooth muscle contraction and depolarization to either PE or U46619, ACh evoked concentration-dependent hyperpolarization (to -67 mV) and complete relaxation. These responses were well maintained during repeated stimulation with PE, but with U46619 there was a progressive decline, so that during a third exposure to U46619 maximum hyperpolarization only reached -52 mV and relaxation was reduced by 20%. This relaxation could now be blocked with charybdotoxin alone. The latter responses could be mimicked with 300 microm 1-EBIO (IK(Ca) activator), an action not modified by exposure to U46619. 6. An early consequence of TXA(2) receptor stimulation is a reduction in the arterial hyperpolarization and relaxation attributed to EDHF. This effect appears to reflect a loss of SK(Ca) activity.     

Inhibitory effect of ethanol on endothelium-dependent vascular responsiveness

The effect of ethanol was studied on the endothelium-dependent vascular responses in isolated rat aortic strips. Ethanol depressed the endothelium-dependent relaxation induced by acetylcholine and ATP but not that induced by the calcium ionophore, A23187. Endothelium-independent relaxation in response to sodium nitroprusside, a soluble guanylate cyclase activator, was not depressed by ethanol. On the other hand, ethanol significantly enhanced the contractile response to clonidine, an alpha 2-adrenoceptor agonist, in endothelium-intact strips and depressed it in endothelium-denuded strips. These results suggest that ethanol can inhibit endothelium-dependent relaxation by acting on endothelial cells but not on smooth muscle cells, and can also suppress an inhibitory effect of the endothelium on alpha 2-adrenoceptor-mediated vasoconstriction.