Mikio Nakashima

Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice

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.

Importance of endothelium-derived hyperpolarizing factor in human arteries.

The endothelium plays an important role in maintaining the vascular homeostasis by releasing vasodilator substances, including prostacyclin (PGI2), nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). Although the former two substances have been investigated extensively, the importance of EDHF still remains unclear, especially in human arteries. Thus we tested our hypothesis that EDHF plays an important role in human arteries, particularly with reference to the effect of vessel size, its vasodilating mechanism, and the influences of risk factors for atherosclerosis. Isometric tension and membrane potentials were recorded in isolated human gastroepiploic arteries and distal microvessels (100-150 microm in diameter). The contribution of PGI2, NO, and EDHF to endothelium-dependent relaxations was analyzed by inhibitory effects of indomethacin, NG-nitro- L-arginine, and KCl, respectively. The nature of and hyperpolarizing mechanism by EDHF were examined by the inhibitory effects of inhibitors of cytochrome P450 pathway and of various K channels. The effects of atherosclerosis risk factors on EDHF-mediated relaxations were also analyzed. The results showed that (a) the contribution of EDHF to endothelium-dependent relaxations is significantly larger in microvessels than in large arteries; (b) the nature of EDHF may not be a product of cytochrome P450 pathway, while EDHF-induced hyperpolarization is partially mediated by calcium-activated K channels; and (c) aging and hypercholesterolemia significantly impair EDHF-mediated relaxations. These results demonstrate that EDHF also plays an important role in human arteries.

Effects of volatile anesthetics on acetylcholine-induced relaxation in the rabbit mesenteric resistance artery

Background Vascular endothelium plays an important role in the regulation of vascular tone. Volatile anesthetics have been shown to attenuate endothelium‐mediated relaxation in conductance arteries, such as aorta. However, significant differences in volatile anesthetic pharmacology between these large vessels and the small vessels that regulate systemic vascular resistance and blood flow have been documented, yet little is known about volatile anesthetic action on endothelial function in resistance arteries. Furthermore, endothelium‐dependent relaxation mediated by factors other than endothelium‐derived relaxing factor (EDRF) has recently been recognized, and there is no information available regarding volatile anesthetic action on non‐EDRF‐mediated endothelium‐dependent relaxation. Methods Employing isometric tension recording and microelectrode methods, the authors first characterized the endothelium‐dependent relaxing and hyperpolarizing actions of acetylcholine (ACh) in rabbit small mesenteric arteries, and tested the sensitivities of these actions to EDRF pathway inhibitors and Potassium sup + channel blockers. They then examined the effects of the volatile anesthetics isoflurane, enflurane, and sevoflurane on ACh‐induced endothelium‐dependent relaxation that was sensitive to EDRF inhibitors and that which was resistant to the EDRF inhibitors but sensitive to blockers of ACh‐induced hyperpolarization. The effects of the volatile anesthetics on endothelium‐independent sodium nitroprusside (SNP)‐induced relaxation were also studied. Results Acetylcholine concentration‐dependently caused both endothelium‐dependent relaxation and hyperpolarization of vascular smooth muscle. The relaxation elicited by low concentrations of ACh (less or equal to 0.1 micro Meter) was almost completely abolished by the EDRF inhibitors NG ‐nitro L‐arginine (LNNA), oxyhemoglobin (HbO sub 2), and methylene blue (MB). The relaxation elicited by higher concentrations of ACh (greater or equal to 0.3 micro Meter) was only attenuated by the EDRF inhibitors. The remaining relaxation, as well as the ACh‐induced hyperpolarization that was also resistant to EDRF inhibitors, were both specifically blocked by tetraethylammonium (TEA greater or equal to 10 mM). Sodium nitroprusside, a NO donor, produced dose‐dependent relaxation, but not hyperpolarization, in the endothelium‐denuded (E[‐]) strips, and the relaxation was inhibited by MB and HbO2, but not TEA (greater or equal to 10 mM). One MAC isoflurane, enflurane, and sevoflurane inhibited both ACh relaxation that was sensitive to the EDRF inhibitors and the ACh relaxation resistant to the EDRF inhibitors and sensitive to TEA, but not SNP relaxation (in the E[‐] strips). An additional finding was that the anesthetics all significantly inhibited norepinephrine (NE) contractions in the presence and absence of the endothelium or after exposure to the EDRF inhibitors. Conclusions The results confirm that ACh has a hyperpolarizing action in rabbit small mesenteric resistance arteries that is independent of EDRF inhibitors but blocked by the Potassium sup + channel blocker TEA. The ACh relaxation in these resistance arteries thus appears to consist of distinct EDRF‐mediated and hyperpolarization‐mediated components. Isoflurane, enflurane, and sevoflurane inhibited both components of the ACh‐induced relaxation in these small arteries, indicating a more global depression of endothelial function or ACh signaling in endothelial cells, rather than a specific effect on the EDRF pathway. All these anesthetics exerted vasodilating action in the presence of NE, the primary neurotransmitter of the sympathetic nervous system, which plays a major role in maintaining vasomotor tone in vivo. This strongly indicates that the vasodilating action of these anesthetics probably dominates over their inhibitory action on the EDRF pathway and, presumably, contributes to their known hypotensive effects in vivo. Finally, the vasodilating action of these anesthetics is, at least in part, independent from endothelium.

Comparison of volatile anesthetic actions on intracellular calcium stores of vascular smooth muscle: Investigation in isolated systemic resistance arteries

Background Volatile anesthetic actions on intracellular Ca2+ stores (i.e., sarcoplasmic reticulum [SR]) of vascular smooth muscle have not been fully elucidated. Methods Using isometric force recording method and fura-2 fluorometry, the actions of four volatile anesthetics on SR were studied in isolated endothelium-denuded rat mesenteric arteries. Results Halothane (≥ 3%) and enflurane (≥ 3%), but not isoflurane and sevoflurane, increased the intracellular Ca2+ concentration ([Ca2+]i) in Ca2+-free solution. These Ca2+-releasing actions were eliminated by procaine. When each anesthetic was applied during Ca2+ loading, halothane (≥ 3%) and enflurane (5%), but not isoflurane and sevoflurane, decreased the amount of Ca2+ in the SR. However, if halothane or enflurane was applied with procaine during Ca2+ loading, both anesthetics increased the amount of Ca2+ in the SR. The caffeine-induced increase in [Ca2+]i was enhanced in the presence of halothane (≥ 1%), enflurane (≥ 1%), and isoflurane (≥ 3%) but was attenuated in the presence of sevoflurane (≥ 3%). The norepinephrine-induced increase in [Ca2+]i was enhanced only in the presence of sevoflurane (≥ 3%). Not all of these anesthetic effects on the [Ca2+]i were parallel with the simultaneously observed anesthetic effects on the force. Conclusions In systemic resistance arteries, the halothane, enflurane, isoflurane, and sevoflurane differentially influence the SR functions. Both halothane and enflurane cause Ca2+ release from the caffeine-sensitive SR. In addition, both anesthetics appear to have a stimulating action on Ca2+ uptake in addition to the Ca2+-releasing action. Halothane, enflurane, and isoflurane all enhance, while sevoflurane attenuates, the Ca2+-induced Ca2+-release mechanism. However, only sevoflurane stimulates the inositol 1,4,5-triphosphate–induced Ca2+ release mechanism. Isoflurane and sevoflurane do not stimulate Ca2+ release or influence Ca2+ uptake.

The action of sevoflurane on vascular smooth muscle of isolated mesenteric resistance arteries (part 2) : mechanisms of endothelium-independent vasorelaxation

Background The precise mechanisms behind the direct inhibitory action of sevoflurane on vascular smooth muscle have not been fully elucidated. Methods Endothelium-denuded smooth muscle strips were prepared from rat small mesenteric arteries. Isometric force and intracellular Ca2+ concentration ([Ca2+]i) were measured simultaneously in the fura-2–loaded strips. In another series of experiments, only isometric force was measured in the &bgr;-escin-membrane–permeabilized strips. Results Sevoflurane (3–5%) inhibited the increases in both the [Ca2+]i and the force induced by either norepinephrine (0.5–10 &mgr;m) or 40 mm K+. Sevoflurane still inhibited the increase in [Ca2+]i induced by norepinephrine after depletion of intracellular Ca2+ stores with ionomycin, although it little influenced the increase in [Ca2+]i induced by norepinephrine after treatment with verapamil. In the fura-2–loaded membrane-intact muscle, sevoflurane caused a rightward shift of Ca2+-force relation during force development to stepwise increment of extracellular Ca2+ concentration during 40-mm K+ depolarization in either the presence or the absence of norepinephrine. In contrast, sevoflurane did not influence Ca2+-activated contraction in the &bgr;-escin–permeabilized muscle, in which &agr;-adrenergic receptor coupling was not retained. Conclusions The inhibitory effects of sevoflurane on both norepinephrine- and potassium chloride (KCl)–induced contractions are caused by reduction of [Ca2+]i in vascular smooth muscle and inhibition of the myofilament Ca2+ sensitivity. The [Ca2+]i-reducing effect of sevoflurane observed in both the norepinephrine- and the K+-stimulated muscle is mainly caused by inhibition of voltage-gated Ca2+ influx. The inhibitory effect of sevoflurane on Ca2+ activation of contractile proteins seems to be mediated by the cell membrane or by some diffusible substances that are lost in the &bgr;-escin–permeabilized cells.