Eiichiro Okabe

Oxygen radical-mediated lipid peroxidation and inhibition of Ca2+-ATPase activity of cardiac sarcoplasmic reticulum

Oxygen radicals have been implicated as important mediators of myocardial ischemic and reperfusion injury. A major product of oxygen radical formation is the highly reactive hydroxyl radical via a biological Fenton reaction. The sarcoplasmic reticulum is one of the major target organelles injured by this process. Using a oxygen radical generating system consisting of dihydroxyfumarate and Fe3+-ADP, we studied lipid peroxidation and Ca2+-ATPase of cardiac sarcoplasmic reticulum. Incubation of sarcoplasmic reticulum with dihydroxyfumarate plus Fe3+-ADP significantly inhibited enzyme activity. Addition of superoxide dismutase, superoxide dismutase plus catalase (15 micrograms/ml) or iron chelator, deferoxamine (1.25-1000 microM) protected Ca2+-ATPase activity. Time course studies showed that this system inhibited enzyme activity in 7.5 to 10 min. Similar exposure of sarcoplasmic reticulum to dihydroxyfumarate plus Fe3+-ADP stimulated malondialdehyde formation. This effect was inhibited by superoxide dismutase, catalase, singlet oxygen, and hydroxyl radical scavengers. EPR spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide verified production of the hydroxyl radical. The combination of dihydroxyfumarate and Fe3+-ADP resulted in a spectrum of hydroxyl radical spin trap adduct, which was abolished by ethanol, catalase, mannitol, and superoxide dismutase. The results demonstrate the role of oxygen radicals in causing inactivation of Ca2+-ATPase and inhibition of lipid peroxidation of the sarcoplasmic reticulum which could possibly be one of the important mechanisms of oxygen radical-mediated myocardial injury.

Selective impairment of endothelium-dependent relaxation by sevoflurane : oxygen free radicals participation

To determine whether sevoflurane alters endothelium-mediated vasodilation of vascular smooth muscle, isolated ring preparations of canine mesenteric arteries were suspended for isometric tension recordings in modified Krebs-Ringer bicarbonate solution at 37 degrees C. Following contraction with norepinephrine, cumulative concentration-response curves were generated using endothelium-dependent vasodilators (acetylcholine, bradykinin, and calcium ionophore A23187) or nitroglycerin. The relaxation produced by acetylcholine, bradykinin, or A23187 was impaired by sevoflurane (2.3 and 4.6 vol%); sevoflurane did not affect relaxation caused by nitroglycerin, which, in these vessels, acts by an endothelium-independent mechanism. Under the same experimental conditions as those used for the concentration-response relationship, electron spin resonance spin-trapping with 5,5-dimethyl-1-pyrroline N-oxide verified generation of hydroxyl radical from the sevoflurane-delivered bathing media; the generation of hydroxyl radical was inhibited by superoxide dismutase, a scavenger of superoxide anion radical, or by the powerful iron chelator deferoxamine. Furthermore, sevoflurane-induced impairment of the relaxation caused by the endothelium-dependent vasodilators used was significantly decreased by superoxide dismutase. These results indicate that superoxide anion radical and/or closely related species of oxygen free radicals, possibly hydroxyl radical, are involved in the observed effect of sevoflurane. We propose that sevoflurane selectively impairs endothelium-dependent relaxation in canine mesenteric arteries by an oxygen free radical mechanism, mainly due to inactivation of endothelium-derived relaxing factor.

Direct pharmacological action of vasoactive substances on pulpal blood flow: An analysis and critique*†

An adequate blood supply to the dental pulp is essential to the health of the tooth; therefore, there have been a number of efforts to study pulpal blood flow and the factors which influence it. However, blood flow to the dental pulp is relatively inaccessible and apparently quite low. Consequently, it is difficult to obtain accurate flow measurements, partly owing to methodological difficulties with the small size of the tissue and its enclosure within rigid walls. In this study, the effects of locally applied vasoactive substances and their specific antagonists on pulpal blood flow have been examined by laser Doppler flowmetry. It is the purpose of this article to examine, in-depth, the involvement of endogenous vasoactive substances in the regulatory mechanism of blood flow within the dental pulp and expand our knowledge of pulpal microcirculatory hemodynamics.

Inhibition by free radical scavengers and by cyclooxygenase inhibitors of the effect of acidosis on calcium transport by masseter muscle sarcoplasmic reticulum

In vitro, arachidonic acid depressed calcium transport by sarcoplasmic reticulum (SR) in the homogenate of canine masseter muscle. This effect was inhibited by superoxide dismutase (SOD), a scavenger of the superoxide anion radial ( . O-2), at pH 7.0, and by SOD plus d-mannitol, a scavenger of hydroxyl free radical ( . OH), at pH 5.5. Indomethacin and 2-aminomethyl-4-tert-butyl-6-propionyl phenol (ONO-3144), a compound known to accelerate the conversion of prostaglandin G2 (PGG2) to PGH2 and scavenge free radicals, inhibited the effect of arachidonic acid at both pH 7.0 and pH 5.5. PGG2, but not PGH2, duplicated the effect of arachidonic acid. The effect of PGG2 on SR function was similar to that of exogenous free radicals generated from the xanthine-xanthine oxidase system. Incubation at pH 5.5, in the absence of an exogenous free-radical generating system, depressed SR calcium transport in the homogenate and in isolated SR. This effect in the homogenate was inhibited by indomethacin or by ONO-3144. At 10-min incubation at pH 5.5, SOD partially and temporarily reversed the depressant effect of acidosis. The addition of SOD plus d-mannitol completely reversed the system. d-Mannitol alone was ineffective. Arachidonic acid was able to mimic these effects of acidosis, except that arachidonic acid further depressed isolated SR calcium transport. These results demonstrate that acidosis can depress SR calcium transport in the homogenate of masseter muscle by an oxygen-free radical mechanism by the generation of . O-2 and . OH. Our results also demonstrate that significant oxygen radical generation can occur through the cyclooxygenase pathway of arachidonic acid metabolism at an acidotic pH in the cellular environment outside of the SR of the muscle cell, and seems to be responsible for the generation of the . OH derived from . O-2.

Effect of Enflurane on Contractile Reactivity in Isolated Canine Mesenteric Arteries and Veins

The effects of enflurane on responses of isolated canine mesenteric arteries and veins to transmural nerve stimulation and to exogenously administered norepinephrine (a mixed α1-and α2-adrenoceptor agonist), phenylephrine (a selective α1-adrenoceptor agonist), and tyramine were studied. The contractile responses of the arteries and the veins to transmural nerve stimulation and to norepinephrine were attenuated by exposure to enflurane; the responses to phenylephrine were decreased more than those to norepinephrine. When compared with the effect of enflurane on transmural nerve stimulation-induced responses, exposure to enflurane resulted in slight attenuation of the contractile responses caused by tyramine, suggesting that enflurane may inhibit the responses to tyramine by interfering with an interaction between released norepinephrine and postjunctional α1-adrenoceptors rather than with tyramine-induced norepinephrine release. The data are also consistent with the view that enflurane acts on sympathetic nerve endings to inhibit release of norepinephrine associated with electrical stimulation-induced nerve membrane depolarization.

Differential sensitivity to hydroxyl radicals of pre- and postjunctional neurovascular transmission in the isolated canine mesenteric vein.

In some pathophysiological conditions, the first target of reactive oxygen intermediates is the vascular system. Superoxide anions, when generated in the vascular circulation, may then escape into the extracellular space via an anion channel and, following dismutation to hydrogen peroxide (H(2)O(2)), form hydroxyl radicals (HO(*)). In an attempt to understand the role of HO(*) in the regulation of transmission at the sympathetic neurovascular junction, the effect of HO(*) at nerve terminals was examined by measuring the amount of noradrenaline (NA) released from isolated, spirally cut, superfused canine mesenteric vein during basal and electrical stimulation (ES; 5Hz, 2ms, 9V); tension development evoked by ES was also recorded simultaneously. HO(*) was generated from Fentons reagent (1. 5x10(-4)M H(2)O(2) plus 10(-4)M FeSO(4)); generation of HO(*) from H(2)O(2)/FeSO(4) in the superfusate was monitored by electron spin resonance spectroscopy using the spin-trap 5, 5-dimethyl-1-pyrroline-N-oxide throughout the experimental time course. Exposure to HO(*) of the helical strips produced an irreversible decrease in tension development evoked by ES with no effect on NA release, suggesting that the observed effect is elicited postjunctionally. The susceptibility of the processes of NA-mediated contraction to HO(*) may differ greatly from that of the NA release mechanism at the prejunctional site. Exposure of the strip preparation to HO(*) leads to a substantial stimulation of basal release of NA without affecting ES-evoked NA release, possibly due to enhanced non-exocytotic Ca(2+)-independent release elicited by HO(*). A direct demonstration of this concept was obtained by showing a significant increase in the basal response of NA release in Ca(2+)-free solution. The major conclusion of the present study is that HO(*) can damage NA-mediated contraction of the vascular preparations at the postjunctional site, and may selectively induce a non-exocytotic release of NA from the prejunctional site of sympathetic neurotransmission.

Susceptibility of caffeine- and Ins(1,4,5)P3-induced contractions to oxidants in permeabilized vascular smooth muscle.

Two principal pathways of Ca2+ release from the sarcoplasmic reticulum of excitable and non-excitable cells have been described: one pathway dependent on the second messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), and a second pathway sensitive to Ca2+ and regulated by caffeine and ryanodine. It was found that the Ca(2+)-pump activity of vascular smooth muscle sarcoplasmic reticulum is inhibited by superoxide anion radicals (O2.-); however, the effects of reactive oxygen intermediates on sarcoplasmic reticulum Ca2+ release in vascular muscle cells are not well defined. The purpose of the present study was to evaluate the effects of reactive oxygen intermediates generated from the hypoxanthine/xanthine oxidase reaction system on contractions induced by caffeine, Ins(1,4,5)P3 and norepinephrine in staphylococcal alpha-toxin-permeabilized rabbit mesenteric arteries. This system generates O2.-, H2O2, and hydroxyl radicals. We wished to identify which class of reactive oxygen intermediates is responsible for the associated loss of vascular smooth muscle contractile function. Caffeine and Ins(1,4,5)P3 produced a transient contraction when the sarcoplasmic reticulum of the permeabilized, preparations was preloaded with pCa 7.0 solution for 5 min before washing with 0.5 mM EGTA solution; norepinephrine also produced a transient contraction. Exposure of the preparations to hypoxanthine/xanthine oxidase (for 30 min) attenuated caffeine-induced contraction, but was without effect on Ins(1,4,5)P3-induced contraction. The observed effect of hypoxanthine/xanthine oxidase exposure was superoxide dismutase-inhibitable, suggesting O2.- involvement. Hypoxanthine/xanthine oxidase also inhibited norepinephrine-induced contraction. The effect of hypoxanthine/xanthine oxidase on norepinephrine contraction was protected by catalase, but not by superoxide dismutase and dimethyl sulfoxide; exogenously added H2O2 mimicked the effect of hypoxanthine/xanthine oxidase exposure. H2O2, added exogenously, was without effect on Ins(1,4,5)P3-induced contraction. It is suggested that the pathway of Ca2+ release from the sarcoplasmic reticulum dependent on Ins(1,4,5)P3 is insensitive to O2.-. Instead, caffeine-induced Ca2+ release mechanisms may be susceptible to O2.- and H2O2, rather than O2.- and hydroxyl radicals, may be the active agent in the norepinephrine-induced contraction. Our results are also consistent with the view that the attenuation by H2O2 of the norepinephrine-induced contraction may be linked to the receptor-associated pathway of Ins(1,4,5)P3 formation, but not to degradation processes of Ins(1,4,5)P3.

Inhibition by singlet molecular oxygen of the vascular reactivity in rabbit mesenteric artery.

The effects of reactive oxygen intermediates derived from photoactivated rose bengal on the vascular reactivity have been evaluated in rabbit mesenteric artery ring preparations. The artery rings were exposed to xanthene dye rose bengal (50 nM) illuminated (6,000 lux) at 560 nm for 30 min. Spin trapping studies with 2,2,6,6‐tetramethylpiperidine (TEMP) and 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) with electron spin resonance spectrometry were also conducted in solution (and not within tissues) to determine quantitatively the reactive oxygen species generated from photoactivated rose bengal. Contraction of the ring preparations induced by noradrenaline (10−8 to 10−4 M) was attenuated by previous exposure to photolysed rose bengal; the observation that the pD2 decreased without a significant reduction in maximum tension generation is consistent with the view that receptor dysfunction may be involved in the effect of photolysed rose bengal. Prior exposure to photolysed rose bengal of the ring preparations inhibited the endothelium‐dependent relaxation evoked by acetylcholine (10−6 M) and calcium ionophore A23187 (10−7 M), but not the endothelium‐independent relaxation evoked by nitroglycerin (10−6 M). A variety of scavengers, superoxide dismutase (33 units ml−1), catalase (32 units ml−1) and 1,3‐dimethyl‐2‐thiourea (DMTU, 10 mM), which should eliminate the superoxide anion radical, H2O2 and the hydroxyl radical, had no effect on the attenuated responses to noradrenaline and acetylcholine induced by photolysed rose bengal. In contrast, the inhibition of the observed effect of photolysed rose bengal was obtained with addition of histidine (25 mM), a singlet molecular oxygen quencher. It was found that photolysis of rose bengal from a 1:2:2:1 quartet, characteristic of the hydroxyl radical‐DMPO spin adduct, which was effectively blunted by DMTU, superoxide dismutase and catalase whereas histidine was ineffective. The results of the electron spin resonance study also showed that a singlet molecular oxygen was produced by photoactivation of rose bengal; this was detected as singlet oxygen‐TEMP product (TEMPO; 2,2,6,6‐tetramethylpiperidine‐N‐oxyl). The formation of the TEMPO signal was strongly inhibited by histidine, but not by DMTU, superoxide dismutase and catalase. It is suggested that the superoxide anion radical, H2O2 and hydroxyl radical are formed in addition to singlet molecular oxygen, and the data obtained from the present study indicate that singlet molecular oxygen is one of the most destructive oxygen species. Endothelium‐dependent relaxation is quite vulnerable to singlet molecular oxygen. Singlet oxygen also depresses noradrenaline‐induced contraction possibly via α‐adrenoceptor dysfunction. This, in turn, may lead to vascular incompetence.

Free Radical Damage to Sarcoplasmic Reticulum of Masseter Muscle by Arachidonic Acid and Prostaglandin G2

In vitro generation of free radicals by xanthine oxidase acting on hypoxanthine as a substrate induced a decreased calcium uptake velocity and reduced calcium-dependent ATPase activity of isolated sarcoplasmic reticulum (SR) vesicles from canine masseter muscle at pH 7.0. At pH 5.5 calcium uptake velocity was also reduced but ATPase activity was unaffected. Application of arachidonic acid or prostaglandin G2 induced the depression of both calcium uptake velocity and ATPase activity. The effect of arachidonic acid and prostaglandin G2 on ATPase activity depended on the pH. At pH 7.0, ATPase activity was decreased, but at pH 5.5 it was unchanged. These effects were reversed by superoxide dismutase (SOD) at pH 7.0, and by SOD plus mannitol at pH 5.5. Prostaglandin H2, prostaglandin E2 and 11,14,17-eicosatrienoic acid had no effect on calcium uptake velocity and ATPase activity at both pH 7.0 and pH 5.5. These results suggest that damage to the masseter muscle is caused by a free radical superoxide anion generated as a result of increased prostaglandins synthesis, and by the production of more lethal hydroxyl radical switched from the production of superoxide anion at low pH.

Calmodulin participation in oxygen radical-induced cardiac sarcoplasmic reticulum calcium uptake reduction☆

The effect of scavengers of oxygen radicals on canine cardiac sarcoplasmic reticulum (SR) Ca2+ uptake velocity was investigated at pH 6.4, the intracellular pH of the ischemic myocardium. With the generation of oxygen radicals from a xanthine-xanthine oxidase reaction, there was a significant depression of SR Ca2+ uptake velocity. Xanthine alone or xanthine plus denatured xanthine oxidase had no effect on this system. Superoxide dismutase (SOD), a scavenger of .O2-, or denatured SOD had no effect on the depression of Ca2+ uptake velocity induced by the xanthine-xanthine oxidase reaction. However, catalase, which can impair hydroxyl radical (.OH) formation by destroying the precursor H2O2, significantly inhibited the effect of the xanthine-xanthine oxidase reaction. This effect of catalase was enhanced by SOD, but not by denatured SOD. Dimethyl sulfoxide (Me2SO), a known .OH scavenger, completely inhibited the effect of the xanthine-xanthine oxidase reaction. The observed effect of oxygen radicals and radical scavengers was not seen in the calmodulin-depleted SR vesicles. Addition of exogenous calmodulin, however, reproduced the effect of oxygen radicals and the scavengers. The effect of oxygen radicals was enhanced by the calmodulin antagonists (compounds 48/80 and W-7) at concentrations which showed no effect alone on Ca2+ uptake velocity. Taken together, these findings strongly suggest that .OH, but not .O2-, is involved in a mechanism that may cause SR dysfunction, and that the effect of oxygen radicals is calmodulin dependent.