The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several endothelium-derived relaxing factors, such as prostacyclin, nitric oxide (NO), and the previously unidentified endothelium-derived hyperpolarizing factor (EDHF). In this study, we examined our hypothesis that hydrogen peroxide (H 2 O 2 ) derived from endothelial NO synthase (eNOS) is an EDHF. EDHF-mediated relaxation and hyperpolarization in response to acetylcholine (ACh) were markedly attenuated in small mesenteric arteries from eNOS knockout (eNOS-KO) mice. In the eNOS-KO mice, vasodilating and hyperpolarizing responses of vascular smooth muscle per se were fairly well preserved, as was the increase in intracellular calcium in endothelial cells in response to ACh. Antihypertensive treatment with hydralazine failed to improve the EDHF-mediated relaxation. Catalase, which dismutates H 2 O 2 to form water and oxygen, inhibited EDHF-mediated relaxation and hyperpolarization, but it did not affect endothelium-independent relaxation following treatment with the K + channel opener levcromakalim. Exogenous H 2 O 2 elicited similar relaxation and hyperpolarization in endothelium-stripped arteries. Finally, laser confocal microscopic examination with peroxide-sensitive fluorescence dye demonstrated that the endothelium produced H 2 O 2 upon stimulation by ACh and that the H 2 O 2 production was markedly reduced in eNOS-KO mice. These results indicate that H 2 O 2 is an EDHF in mouse small mesenteric arteries and that eNOS is a major source of the reactive oxygen species.J. Clin. Invest. 106:1521-1530. cular smooth muscle (21,22). In this study, we thus tested our hypothesis that H 2 O 2 is an EDHF and also examined a possible role of eNOS as a major source of the reactive oxygen species, using control and eNOS knockout (eNOS-KO) mice (23). MethodsThis study was reviewed by the Committee on Ethics in Animal Experiments of the Kyushu University and was carried out according to the Guidelines for Animal Experiments of the Kyushu University and of the Japanese government.Animals and tissue preparation. Male eNOS-KO mice and C57BL/6 mice, 10-16 weeks of age, were used. The eNOS-KO mice were originally provided by P. Huang and M. Fishman (Harvard Medical School, Boston, Massachusetts, USA) (23) and maintained in the Laboratory of Animal Experiments in the Kyushu University. The eNOS-KO mice were derived from a cross between SV129J and C57BL/6 mice and were backcrossed to C57BL/6 mice over ten generations. Thus, C57BL/6 mice were used as a wild-type control; they were also maintained in the Laboratory of Animal Experiments in the Kyushu University. Some eNOS-KO mice were treated with hydralazine in order to examine the effect of mildly elevated blood pressure on the EDHF-mediated responses. The treatment with hydralazine hydrochloride (20 mg/kg/d) was performed for 6 weeks from 10-16 weeks of age. Systolic blood pressure was measured by tail-cuff method under conscious conditions before the animals were killed. Th...

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Depletion of intracellular Ca2؉ stores activates capacitative Ca 2؉ influx in smooth muscle cells, but the native storeoperated channels that mediate such influx remain unidentified. Recently we demonstrated that calcium influx factor produced by yeast and human platelets with depleted Ca 2؉ stores activates small conductance cation channels in excised membrane patches from vascular smooth muscle cells (SMC). Here we characterize these channels in intact cells and present evidence that they belong to the class of storeoperated channels, which are activated upon passive depletion of Ca 2؉ stores. Application of thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca 2؉ ATPase, to individual SMC activated single 3-pS cation channels in cell-attached membrane patches. Channels remained active when inside-out membrane patches were excised from the cells. Excision of membrane patches from resting SMC did not by itself activate the channels. Load-ing SMC with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid), which slowly depletes Ca 2؉ stores without a rise in intracellular Ca (4, 8) and certain members of the diverse family of TRP channels (9, 10) are thought to be responsible for CCE. However, to date the existence of neither of those has been established in SMC. Importantly, depletion of Ca 2ϩ stores was shown to trigger not only Ca 2ϩ , but also Na ϩ influx in arterial myocytes (11), which implies that store-operated channels in SMC are poorly selective for cations. In freshly isolated mouse anococcygeus SMC there are strong indications that CCE results from activation of a whole cell nonselective cation current (12, 13), although in rat aortic SMC line A7r5 no currents were detected which could be associated with CCE (14, 15). It is totally unclear if the same or different storeoperated channels mediate CCE in SMC from different preparations.Here for the first time we characterize 3-pS cation channels that are activated by Ca 2ϩ store depletion in intact SMC from mouse and rabbit aorta. These channels, contrary to highly Ca 2ϩ -selective CRAC channels, are poorly selective for monoand divalent cations, and under physiological conditions they will allow both Ca 2ϩ and Na ϩ to enter SMC. Recently we found that these channels can also be activated in excised membrane patches by calcium influx factor (CIF) partially purified from human platelets or yeast with depleted Ca 2ϩ stores (16). Taken together, these data strongly support the idea that the native 3-pS channels, which we found in SMC, belong to the class of store-operated ion channels. Preliminary data have been reported in abstract form (17, 18). EXPERIMENTAL PROCEDURES SMC PreparationFour different preparations of aortic SMC were used in our experiments, and the 3-pS channel described in this paper was found to be the same in acutely dispersed and cultured SMC from mouse and rabbit aorta. Most of the experiments on characterization of the single channels and whole cell currents were done on mSMC in short term culture because they pr...

Fasudil ameliorated myocardial ischemia in patients who were most likely having coronary microvascular spasm. The inhibition of Rho-kinase may be a novel therapeutic strategy for this group of patients with microvascular angina.

The expression of L- and T-type Ca2+ channels has been reported to change during various biological events, including cellular differentiation and proliferation. The present study aimed to examine whether or not the expression of L- and T-type Ca2+ channels depends on the cell cycle in rat aortic smooth muscle cells in primary culture. Both the phase of the cell cycle and the functional expression of Ca2+ channels were determined in the same single cell, using an immunocytochemical analysis of cell cycle-specific nuclear antigens and a whole-cell voltage-clamp method, respectively. In the G0 (n = 130) and M (n = 75) phases, all cells showed only L-type Ca2+ currents. The cells showing a T-type Ca2+ current appeared in the G1 phase (37%, n = 85) and increased in the S phase (90%, n = 21). For L-type Ca2+ channels, the current density was significantly greater in the G1 phase than in the G0 and M phases. However, either the voltage-dependent properties or the dose-response relationships of Bay K 8644- and second messenger-induced modulations of L-type Ca2+ current did not differ in the four phases of the cell cycle. These findings thus indicate that the expression of L- and T-type Ca2+ channels depends on the cell cycle, whereas the characteristics of L-type Ca2+ channels do not differ between the phases of the cell cycle.

Objective-Endothelium-derived hyperpolarizing factor (EDHF) plays an important role in modulating vascular tone, especially in microvessels, although its nature has yet to be elucidated. This study was designed to examine whether hydrogen peroxide (H 2 O 2 ) is an EDHF in porcine coronary microvessels with use of an electron spin resonance (ESR) method to directly detect H 2 O 2 production from the endothelium. Methods and Results-Isometric tension and membrane-potential recordings demonstrated that bradykinin and substance P caused EDHF-mediated relaxations and hyperpolarizations of porcine coronary microvessels in the presence of indomethacin and N -nitro-L-arginine. Key Words: endothelium Ⅲ endothelium-derived hyperpolarizing factor Ⅲ hydrogen peroxide Ⅲ membrane potential T he endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several vasodilating factors, including prostacyclin, nitric oxide (NO), and the still-unidentified endothelium-derived hyperpolarizing factor (EDHF). Since the first reports of the existence of EDHF, 1,2 several candidates for EDHF have been proposed, including cytochrome P-450 metabolites, 3,4 endothelium-derived K ϩ , 5,6 and gap-junctional electrical communication between endothelial cells and smooth muscle cells. 7,8 We and others have recently demonstrated that endotheliumderived hydrogen peroxide (H 2 O 2 ) is an EDHF of mouse and human mesenteric arteries, 9,10 piglet pial arterioles, 11 and human and canine coronary microvessels (in flow-induced and autoregulatory relaxation, respectively). 12,13 However, it remains to be determined whether H 2 O 2 is also an EDHF in agonist-induced relaxation of the coronary microvessels, in which EDHF plays an important role in modulating vascular tone. Moreover, endothelial production of H 2 O 2 /EDHF remains to be demonstrated by methods other than conventional tension or membrane-potential recordings. The present study was thus designed to examine whether H 2 O 2 plays a role as an EDHF in porcine coronary microvessels by using an electron spin resonance (ESR) method to directly detect endothelial production of H 2 O 2 . MethodsThis study was reviewed by the Committee on Ethics in Animal Experiments of the Kyushu University and was carried out according to the Guidelines for Animal Experiments of the Kyushu University and of the Japanese Government. Animals and Tissue PreparationWe used a total of 36 domestic male pigs (Nihon Crea, Tokyo, Japan; 2 to 3 months old and weighing 25 to 30 kg). Animals were humanely killed with a lethal dose of pentobarbital sodium, and then the right ventricular free wall was carefully removed. Epicardial right coronary arteries and distal coronary microvessels (250 to 300 m in diameter) were excised from the right ventricular free wall and

Oxygen is required to sustain aerobic organisms. Reactive oxygen species (ROS) are constantly released during mitochondrial oxygen consumption for energy production. Any imbalance between ROS production and its scavenger system induces oxidative stress. Oxidative stress, a critical contributor to tissue damage, is well-known to be associated with various diseases. The kidney is susceptible to hypoxia, and renal hypoxia is a common final pathway to end stage kidney disease, regardless of the underlying cause. Renal hypoxia aggravates oxidative stress, and elevated oxidative stress, in turn, exacerbates renal hypoxia. Oxidative stress is also enhanced in chronic kidney disease, especially diabetic kidney disease, through various mechanisms. Thus, the vicious cycle between oxidative stress and renal hypoxia critically contributes to the progression of renal injury. This review examines recent evidence connecting chronic hypoxia and oxidative stress in renal disease and subsequently describes several promising therapeutic approaches against oxidative stress.

Chronic kidney disease (CKD) is a major public health problem. Accumulating evidence suggests that CKD aggravates renal hypoxia, and in turn, renal hypoxia accelerates CKD progression. To eliminate this vicious cycle, hypoxia-related therapies, such as hypoxia-inducible factor (HIF) activation (prolyl hydroxylase domain inhibition) or NF-E2-related factor 2 activation, are currently under investigation. Clinical studies have revealed heterogeneity in renal oxygenation; therefore, the detection of patients with more hypoxic kidneys can be used to identify likely responders to hypoxia-oriented therapies. In this review, we provide a detailed description of current hypoxia detection methods. HIF degradation correlates with the intracellular oxygen concentration; thus, methods that can detect intracellular oxygen tension changes are desirable. The use of a microelectrode is a classical technique that is superior in quantitative performance; however, its high invasiveness and the fact that it reflects the extracellular oxygen tension are disadvantages. Pimonidazole protein adduct immunohistochemistry and HIF activation detection reflect intracellular oxygen tension, but these techniques yield qualitative data. Blood oxygen level-dependent magnetic resonance imaging has the advantage of low invasiveness, high quantitative performance, and application in clinical use, but its biggest disadvantage is that it measures only deoxyhemoglobin concentrations. Phosphorescence lifetime measurement is a relatively novel in vivo oxygen sensing technique that has the advantage of being quantitative; however, it has several disadvantages, such as toxicity of the phosphorescent dye and the inability to assess deeper tissues. Understanding the advantages and disadvantages of these hypoxia detection methods will help researchers precisely assess renal hypoxia and develop new therapeutics against renal hypoxia-associated CKD.