Coronary arteriolar dilation to acidosis: role of ATP-sensitive potassium channels and pertussis toxin-sensitive G proteins

BACKGROUND: We previously demonstrated that coronary arteriolar dilation in response to acidosis is mediated by the opening of ATP-sensitive potassium (KATP) channels. However, the signal transduction involved in the KATP-channel activation during acidosis has not been elucidated. A recent study in cardiac myocytes implied that pertussis toxin (PTX)-sensitive G proteins may be involved in the signal transduction for KATP-channel activation. However, it remains unclear whether this transduction process also occurs in the vascular tissue and, in particular, whether it exerts functional dilation in response to acidosis. METHODS AND RESULTS: To examine the signaling pathway for acidosis-induced dilation, porcine coronary arterioles were isolated, cannulated, and pressurized for in vitro study. The GTPase activity in reconstituted G proteins was examined at different levels of pH. Extravascular acidosis (pH 7.3 to 7.0) produced a graded dilation of coronary arterioles. This dilation was not affected by removal of endothelium but was significantly attenuated after inhibition of KATP channels and G proteins by glibenclamide and PTX, respectively. Glibenclamide and PTX attenuated the acidosis-induced arteriolar dilation to the same extent, and combined administration of both inhibitors did not further inhibit the vasodilation. These results indicated that both inhibitors act on the same vasodilatory pathway. Furthermore, vasodilation of coronary arterioles to the KATP-channel opener pinacidil and to the endothelium-independent vasodilator sodium nitroprusside was not affected by PTX. Because PTX inhibited acidosis-induced vasodilation without inhibiting KATP-channel function, it is suggested that PTX inhibits the vasodilatory pathway upstream from KATP channels. GTPase activity in reconstituted G proteins was significantly enhanced by a reduction in pH, indicating that G proteins were directly activated by acidosis. CONCLUSIONS: On the basis of these findings, we conclude that acidosis-induced coronary arteriolar dilation is mediated by the opening of smooth muscle KATP channels through the activation of PTX-sensitive G proteins.     

Biochemical evidence that Saccharomyces cerevisiae YGR262c gene, required for normal growth, encodes a novel Ser/Thr-specific protein kinase

BACKGROUND AND PURPOSE: This study was designed to determine regional differences and age-related changes in the contribution of ATP-sensitive potassium (KATP) channels to vasodilator responses in the brain stem circulation in vivo. METHODS: Changes in diameter of the basilar artery (baseline diameter, 270 +/- 5 microns [mean +/- SEM]), its large branch (112 +/- 5 microns), and its small branch (49 +/- 2 microns) in response to KATP channel openers levcromakalim and Y-26763 were measured through a cranial window in anesthetized adult (4 to 6 months) and aged (24 to 26 months) Sprague-Dawley rats. RESULTS: Topical application of levcromakalim and Y-26763 produced concentration-dependent vasodilation that was similar among the three vessel groups in adult rats. In aged rats, dilator responses of the branches, but not of the basilar artery, to the KATP channel openers were smaller than those in adult rats (P < .05). Glibenclamide, a selective KATP channel blocker, almost abolished this vasodilation in both groups of rats. Vasodilator responses to sodium nitroprusside were preserved in aged rats. CONCLUSIONS: In adult rats, there is no regional heterogeneity in vasodilator response to KATP channel openers in the brain stem circulation in vivo. In aged rats, although KATP channels are also functional in the brain stem circulation, dilator response of the microvessels but not of the large arteries to direct activation of KATP channels is impaired.     

The effects of levcromakalim and pinacidil on the human internal mammary artery

The present study was undertaken to examine the effects of pinacidil and levcromakalim, two potassium, channel openers, on human internal mammary artery (HIMA) obtained from patients undergoing coronary artery bypass surgery, and to clarify the contribution of different K+ channel subtypes in pinacidil and levcromakalim action in this blood vessel. Pinacidil and levcromakalim induced a concentration-dependent relaxation of the precontracted arterial segments (pEC50 = 5.77 +/- 0.05 and 6.89 +/- 0.03, respectively), 4-Aminopyridine (3 mM), a non-selective blocker of K+ channels, induced significant shifts to the right of the concentration-response curves for pinacidil and levcromakalim. Tetraethylammonium (6 mM), charybdotoxin (0.4 microM) and apamin (0.1 microM), blockers of Ca(2+)-sensitive K+ channels, had no effect on the pinacidil- and levcromakalim-evoked relaxation. Glibenclamide (0.1-10 microM), a selective blocker of adenosine triphosphate (ATP)-sensitive K+ channels, competitively antagonized the response to levcromakalim (pKB = 7.92 +/- 0.07). In contrast, glibenclamide, in significantly higher concentrations (3-30 microM), non-competitively antagonized the response to pinacidil. High concentrations of pinacidil (> 10 microM) relaxed arterial rings bathed by a medium containing 100 mM K+ with maximum response 83 +/- 6%. Under the same conditions, the maximum levcromakalim-induced relaxation on HIMA was almost abolished (15 +/- 2%). It is concluded that pinacidil and levcromakalim do not relax the HIMA through the same subtype of K+ channel. ATP-sensitive K+ channels are probably involved in levcromakalim- but not in a pinacidil-induced relaxation in the HIMA. In addition, in pinacidil-induced relaxation of the HIMA, K+ channel-independent mechanisms seem to be involved.     

Cerebral vascular smooth muscle potassium channels and their possible role in the management of vasospasm

One of the promising therapeutic uses of the potassium channel openers is in the management of cerebral vasospasm, a prolonged vasoconstriction of major cerebral arteries which follows aneurysmal subarachnoid haemorrhage. In this review, we first summarize the properties of potassium channels in cerebral vascular smooth muscle. Calcium-activated and ATP-dependent potassium channels are the major potassium channels identified in the cerebrovascular smooth muscle and both are believed to play a role in the regulation of cerebrovascular smooth muscle tone. The calcium-activated potassium channels can be activated by depolarization, by elevation of internal calcium and by some vasodilators. Some neuropeptides and potassium channel openers open the ATP-dependent potassium channels and produce vasodilation. We then review the effects of both synthetic and endogenous potassium channel openers in the cerebrovascular system, discuss their efficacy in the management of models of cerebrovascular spasm, and outline the clinical promise of these agents.     

Temperature-sensitive effects of potassium channel openers on isolated guinea pig myocardium and aorta

The effects of K+ channel openers, NIP-121, cromakalim, and pinacidil, on isolated myocardium and aorta were investigated at two different temperatures, 23 degrees C and 37 degrees C. In right ventricular myocardium, NIP-121 shortened the action-potential duration with little influence on other action-potential parameters at 37 degrees C, but not at 23 degrees C. In whole-cell clamped ventricular myocytes, NIP-121 induced a glibenclamide-sensitive outward current at 37 degrees C but not at 23 degrees C. No difference in tissue adenosine triphosphate (ATP) concentration was detected between ventricular myocardia incubated at 37 degrees C and at 23 degrees C. In aortic preparations precontracted with norepinephrine, NIP-121, cromakalim, and pinacidil produced endothelium-independent relaxation at 37 degrees C, which was antagonized by glibenclamide. The vasorelaxant effects were greatly reduced at 23 degrees C. Thus we demonstrated that the effects of K+ channel openers on the myocardium and vascular smooth muscle are temperature sensitive.     

Increased myogenic tone and diminished responsiveness to ATP-sensitive K+ channel openers in cerebral arteries from diabetic rats

Diabetes mellitus has profound adverse effects on vascular and, in particular, endothelial function. Although pressure-induced constriction ("myogenic tone") is a major contributor to the regulation of blood flow, little is known about the effects of diabetes on this response. Diabetes has been shown to diminish the dilation of cerebral arteries to synthetic ATP-sensitive K+ (KATP) channel openers. In this study, we explored the effects of diabetes induced in rats by streptozotocin on cerebral artery (250 to 300 microns) myogenic tone and on vasodilations to the synthetic KATP channel openers pinacidil and levcromakalim. Elevation of intravascular pressure caused a graded membrane potential depolarization and constriction, which was greater in arteries from diabetic rats compared with normal rats (at 60 mm Hg, 5 mV more depolarized and 22 microns more constricted). Pressurized arteries (at 60 mm Hg) from diabetic rats were 5- to 15-fold less sensitive to pinacidil and levcromakalim than were control arteries (EC50 values for pinacidil and levcromakalim were 1.4 and 0.6 mumol/L, respectively, in diabetic animals and 0.3 and 0.04, respectively, in control animals; P < .05). Removal of the endothelium or addition of a NO synthase inhibitor, NG-nitro-L-arginine (LNNA), in control arteries decreased the sensitivity to KATP channel openers and depolarized and constricted control arteries to levels similar to those observed in arteries from diabetic animals. Sodium nitroprusside caused a membrane potential hyperpolarization and enhanced the response to pinacidil in arteries from diabetic animals. Removal of the endothelium or LNNA had little effect on the apparent KATP channel opener sensitivity, the membrane potential, and pressure-induced constrictions of arteries from diabetic animals. The results are consistent with the hypothesis that this type of diabetes leads to a decrease in tonic NO release from the endothelium, which in turn causes membrane potential depolarization and vasoconstriction, resulting in a diminished response to KATP channel openers.     

Flexor reflex afferents reset the step cycle during fictive locomotion in the cat

It has been reported that antiarrhythmic drugs possessing the property of potassium channel blockade were most effective in preventing halothane-epinephrine induced arrhythmias. Recent attention has focused on ATP-sensitive potassium (K(ATP)) channels because of their contribution to the cardiovascular actions of volatile anesthetics. The present study was designed to evaluate whether K(ATP) channels or transient outward potassium channels (Ito) were involved in the mechanism of halothane-epinephrine arrhythmias in rat. Rats were anesthetized with halothane (1.5%), and the lungs were mechanically ventilated. The arrhythmogenic thresholds of epinephrine during halothane anesthesia were determined in 74 rats receiving saline or one of tested agents. The arrhythmogenic dose of epinephrine (ADE) was significantly increased by a K(ATP) channel opener, JTV506 (P < 0.01), and had a tendency to be increased by other K(ATP) channel openers, cromakalim, nicorandil, KRN2391 and Y 26763, but were not affected by a K(ATP) channel blocker, glibenclamide. The Ito blocker, 4-aminopyridine, also significantly increased the ADE. Epinephrine produced second-degree or complete atrioventricular block in 4 out of 7 rats receiving glibenclamide. These results suggest that Ito but not K(ATP) channels might be involved in the mechanism in producing halothane-epinephrine arrhythmias.     

Optimization of exopolysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus RR grown in a semidefined medium

The role of K+ channels in the nitric oxide-independent renal vasodilator effect of acetylcholine (Ach) was examined to address the hypothesis that the mechanism underlying this response was different from that of bradykinin, because an earlier study indicated the possibility of different mediators. We used the rat isolated, perfused kidney that was constricted with phenylephrine and treated with nitroarginine and indomethacin to inhibit nitric oxide synthase and cyclooxygenase, respectively. The nonspecific K+ channel inhibitors, procaine and tetraethylammonium (TEA), reduced vasodilator responses to Ach and cromakalim, but not those to nitroprusside. Glibenclamide, an inhibitor of ATP-sensitive K+ channels, reduced vasodilator responses to cromakalim but did not affect those to Ach or nitroprusside. Charybdotoxin, an inhibitor of Ca(++)-activated K+ channels, reduced vasodilator responses to Ach without affecting those to cromakalim or nitroprusside. Iberiotoxin and apamin, inhibitors of large- and small-conductance Ca(++)-activated K+ channels, respectively, did not reduce vasodilation induced by Ach, cromakalim or nitroprusside. The inhibitor of cytochrome P450, clotrimazole, reduced the renal vasodilator effects of Ach and bradykinin but not those of nitroprusside or SCA 40, an agonist for Ca(++)-activated K+ channels. These results suggest that in the rat kidney, Ach, like bradykinin, utilizes a charybdotoxin-sensitive Ca(++)-activated K+ channel of intermediate conductance to elicit vasodilation and that this effect may be dependent on cytochrome P450 activity.     

Assessing health in musculoskeletal disorders--the appropriateness of a German version of the Sickness Impact Profile

Intra-coronal bleaching of root-filled teeth has been associated with invasive cervical root resorption. It is considered that during bleaching hydrogen peroxide diffuses through the tooth structure into the cervical periodontium, resulting in periodontal tissue destruction and initiating a resorptive process. Hydrogen peroxide is capable of generating hydroxyl radical, an oxygen-derived free radical, in the presence of ferrous salts. Hydroxyl radicals are extremely reactive and have been shown to degrade components of connective tissue, particularly collagen and hyaluronic acid. The aim of the present study was to determine whether hydroxyl radicals are generated during the bleaching of root-filled teeth which have been discoloured by blood. Forty extracted human premolar teeth were root-filled with gutta-percha and AH26 sealer cement. Twenty of the teeth were experimentally discoloured by blood. All teeth were then thermo-catalytically bleached using 30% hydrogen peroxide while tooth roots were seated in a test solution of sodium salicylate. Hydroxyl radical generation was determined by the detection of reaction products of this radical with salicylate using high performance liquid chromatography with electrochemical detection (HPLC-ECD). The presence of hydroxyl radicals was detected in twenty-five of the teeth. There was a significant association between the production of hydroxyl radicals and the presence of tooth discolouration caused by blood components. Greatest yields of hydroxyl radicals occurred in teeth in which EDTA had been used to clean the pulp chamber prior to bleaching. It was concluded that hydroxyl radicals are generated during the thermo-catalytic bleaching of root-filled teeth. Generation of this toxic chemical species may be one mechanism underlying periodontal tissue destruction and root resorption after intra-coronal bleaching.     

Maintained vasodilatory response to cromakalim after inhibition of nitric oxide synthesis

Activation of vascular smooth-muscle adenosine triphosphate-sensitive potassium channels (KATP channels) causes membrane hyperpolarization, reduced entry of Ca2+ through L-type voltage-gated Ca2+ channels, and subsequent smooth-muscle relaxation. Conversely, opening of endothelial KATP channels elicits hyperpolarization but may induce Ca2+ influx and stimulation of endothelium-derived nitric oxide (EDNO) because these cells appear not to possess L-type Ca2+ channels. We therefore hypothesized that EDNO contributes to KATP channel-mediated vasodilation. To test this hypothesis, we examined vasodilatory responses to the KATP channel opener cromakalim in conscious rats, perfused rat tail artery segments, and isolated perfused rat lungs in the presence or absence of the EDNO synthesis inhibitor Nomega-nitro-L-arginine (L-NNA). Additionally, we compared the effect of cromakalim with the EDNO-dependent dilator bradykinin on NO production and intracellular Ca2+ in cultured rat pulmonary artery endothelial cells. Vasodilatory profiles to cromakalim were unaffected by L-NNA in conscious rats, tail arteries, and isolated lungs. Consistent with these results, cromakalim had no apparent effect on either NO synthesis or Ca2+ levels in cultured endothelial cells. These data suggest a lack of a role for EDNO in contributing to KATP-channel-mediated vasodilation in the rat.     

Sulfonylureas inhibit cytokine-induced eosinophil survival and activation

Eosinophils play a key role in the pathogenesis of asthma and other allergic inflammatory diseases. We have previously shown that treatment of eosinophils with lidocaine preferentially inhibits IL-5-induced survival. This inhibition cannot be overcome by increasing concentrations of IL-5 and is not due to the blocking of Na+ channels by lidocaine. Here we report that one class of K+ channel blockers, the sulfonylureas, inhibits eosinophil survival in a manner similar to lidocaine. The sulfonylurea glyburide inhibits eosinophil survival even at high concentrations of IL-5. In contrast, increasing concentrations of IL-3 or granulocyte-macrophage CSF overcome glyburide inhibition. Glyburide also blocks cytokine-induced eosinophil superoxide production. Similar results were seen with the sulfonylureas tolbutamide and glipizide. Interestingly, the effects of glyburide are not antagonized by the ATP-sensitive K+ channel openers cromakalim, pinacidil, or diazoxide. Although Scatchard analysis of [3H]glyburide binding to eosinophil membranes indicated that the high affinity sulfonylurea receptor (SUR1) is not present on eosinophils, human eosinophils do express mRNA homologous to the sulfonylurea receptor family, in keeping with the presence of a sulfonylurea receptor. Finally, coculture of eosinophils with combinations of glyburide, lidocaine, and dexamethasone resulted in synergistic inhibition of cytokine-mediated eosinophil survival and superoxide production. These results have intriguing clinical implications for the treatment of eosinophil-associated diseases.     

Protective effect of K(ATP) openers in ischemic rat hearts treated with a potassium cardioplegic solution

ATP-sensitive potassium channel (KATP) openers directly protect ischemic myocardium, which may make them useful for treating patients undergoing cardiopulmonary bypass, but whether high-potassium-containing cardioplegic solutions would inhibit their protective effects is not clear. We determined whether additional protection greater than that provided by cardioplegia could be found for KATP openers. We studied the effect of 10 microM cromakalim or BMS-180448 pretreatment (10 min before cardioplegia) on severity of ischemia in isolated rat hearts given normothermic or cold St. Thomas' cardioplegic solution (16 mM K+). After cardioplegic arrest, the hearts were subjected to 30-min (normothermic) or 150-min (hypothermic) global ischemia, each followed by 30-min reperfusion. The cardioplegic solutions significantly protected the hearts, as measured by increased time to onset of contracture, enhanced recovery of function, and reduced lactate dehydrogenase (LDH) release. Cromakalim and BMS-180448 both further significantly increased time to contracture in both normothermic and hypothermic arrested hearts; this was accompanied by enhanced recovery of reperfusion contractile function and reduced cumulative LDH release. This additional protective effect of the K ATP openers was abolished by glyburide. Because administration of the K ATP openers only with the cardioplegic solution (1 min before global ischemia) was not efficacious, >1-min pretreatment apparently is necessary. K ATP openers provide additional protection to that afforded by cold or normothermic potassium cardioplegia in rat heart, although the timing of treatment may be crucial.     

Effects of hydroxyl radicals on KATP channels in guinea-pig ventricular myocytes

We studied the effects of oxygen free radicals on the ATP-sensitive potassium channel (KATP channel) of guinea-pig ventricular myocytes. Single KATP channel currents were recorded from inside-out patches in the presence of symmetrical K+ concentrations (140 mM in both bath and pipette solutions). Reaction of xanthine oxidase (0.1 U/ml) on hypoxanthine (0.5 mM) produced superoxide anions (.O2-) and hydrogen peroxide (H2O2). Exposure of the patch membrane to.O2- and H2O2 increased the opening of KATP channels, but this activation was prevented by adding 1 microM glibenclamide to the bath solution. In the presence of ferric iron (Fe3+: 0.1 mM), the same procedure produced hydroxyl radicals (.OH) via the iron-catalysed Haber-Weiss reaction.OH also activated KATP channels; however, this activation could not be prevented by, even very high concentrations of glibenclamide (10 microM). These different effects of glibenclamide suggest that the mode of action of these oxygen free radicals on KATP channels is different and that.OH is more potent than.O2-/H2O2 in activating KATP channels in the heart.     

Cross tolerance between cromakalim and RP49356 in the uterus of the rat in vivo and in vitro

We investigated tolerance to the potassium (K+) channel openers, cromakalim and RP49356 and cross tolerance between them in the uterus of the rat. Continuous infusion of RP49356 in vivo produced 5.2-fold (200 or 300 micrograms/kg per h) and 7.2-fold (600 micrograms/kg per h) desensitization of the uterus to RP49356. Uterine sensitivity to RP49356 was reduced 7.4-fold in cromakalim-tolerant rats and sensitivity to cromakalim was reduced 7.9-fold in RP49356-tolerant rats. Incubation of isolated uterus with 10 microM cromakalim for 4 h reduced uterine sensitivity to cromakalim by about 45-fold and to RP49356 by 2.1-fold but did not alter sensitivity to salbutamol. Incubation with 10 microM RP49356 for 4 h reduced sensitivity to RP49356 by 2.5-fold and to cromakalim by 3.8-fold. Occurrence of uterine tolerance to K+ channel openers in vitro suggests that tolerance in vivo has a pharmacodynamic basis. Cross tolerance between cromakalim and RP49356 provides evidence that these two K+ channel openers possess common binding sites and/or mechanism(s) of action. However, the greater extent of self-tolerance to cromakalim than to RP49356 suggests a difference in their actions.     

Formation and properties of peroxynitrite as studied by laser flash photolysis, high-pressure stopped-flow technique, and pulse radiolysis

Flash photolysis of alkaline peroxynitrite solutions results in the formation of nitrogen monoxide and superoxide. From the rate of recombination it is concluded that the rate constant of the reaction of nitrogen monoxide with superoxide is (1.9 +/- 0.2) x 10(10) M-1 s-1. The pKa of hydrogen oxoperoxonitrate is dependent on the medium. With the stopped-flow technique a value of 6.5 is found at millimolar phosphate concentrations, while at 0.5 M phosphate the value is 7.5. The kinetics of decay do not follow first-order kinetics when the pH is larger than the pKa, combined with a total peroxynitrite and peroxynitrous acid concentration that exceeds 0.1 mM. An adduct between ONOO- and ONOOH is formed with a stability constant of (1.0 +/- 0.1) x 10(4) M. The kinetics of the decay of hydrogen oxoperoxonitrate are not very pressure-dependent: from stopped-flow experiments up to 152 MPa, an activation volume of 1.7 +/- 1.0 cm3 mol-1 was calculated. This small value is not compatible with homolysis of the O-O bond to yield free nitrogen dioxide and the hydroxyl radical. Pulse radiolysis of alkaline peroxynitrite solutions indicates that the hydroxyl radical reacts with ONOO- to form [(HO)ONOO].- with a rate constant of 5.8 x 10(9) M-1 s-1. This radical absorbs with a maximum at 420 nm (epsilon = 1.8 x 10(3) M-1 cm-1) and decays by second-order kinetics, k = 3.4 x 10(6) M-1 s-1. Improvements to the biomimetic synthesis of peroxynitrite with solid potassium superoxide and gaseous nitrogen monoxide result in higher peroxynitrite to nitrite yields than in most other syntheses.     

Increased 3-nitrotyrosine in brains of Apo E-deficient mice

Apolipoprotein E (Apo-E) is linked to the pathogenesis of Alzheimer's disease. Apo-E deficient mice have increased lipid peroxidation in plasma. In the present study we examined two markers of oxidative stress in brains of Apo-E deficient mice. The ratios of 2,3 and 2,5 dihydroxybenzoic acid (DHBA)/salicylate, an index of hydroxyl radical generation, were unchanged except for an increase in 2.5-DHBA/salicylate in the cerebellum. 3-Nitroxyrosine is a marker for nitration of proteins produced by peroxynitrite. Concentrations of 3-nitrotyrosine were significantly increased in the cerebral cortex, hippocampus, brainstem and cerebellum of Apo-E deficient mice. These results suggest the Apo-E may modulate oxidative stress produced by peroxynitrite.     

Inhibition of contractile responses of human myometrium and intramyometrial arteries by potassium channel openers

OBJECTIVES: The effects of cromakalim and pinacidil on the contractile activity of the isolated human myometrium and intramyometrial arteries were studied. MATERIAL AND METHOD: Myometrial strips and pieces of uterine arteries were obtained from women undergoing hysterectomy. Contractions were recorded under isometric conditions. RESULTS: Both compounds (10(-9) M-5 x 10(-6) M) inhibited the spontaneous activity of the myometrium and vasopressin induced contractions of myometrium and intramyometrial arteries. There was no statistically significant difference in the responses of both tissues to cromakalim or pinacidil. Both compounds reduced responses of myometrium and intramyometrial arteries to low concentration of K+ (< 40 mM) but did not decrease contractions induced by 40 mM and 80 mM. CONCLUSION: The combined effect on the uterus and intrauterine vasculature would suggest a potential in the use of cromakalim or pinacidil for the treatment of dysmenorrhea.     

Removals of hydrogen peroxide and hydroxyl radical by thiol-specific antioxidant protein as a possible role in vivo

Thiol-specific antioxidant protein (Protector Protein; PRP) from Saccharomyces cerevisiae was found to remove hydrogen peroxide and hydroxyl radical in the presence of dithiothreitol (DTT). Without DTT as a reducing equivalent, the antioxidant protein did not show the activities for destroying hydrogen peroxide and hydroxyl radical. N-ethylmaleimide (NEM) was observed to prevent the PRP from both removing hydrogen peroxide and protecting the cleavage of DNA. These observations suggest that the sulfhydryl of cysteine in PRP could function as a strong nucleophile to attack and destroy H2O2 and .OH.     

The origin of the hydroxyl radical oxygen in the Fenton reaction

There is an ongoing discussion in the chemical literature regarding the nature of the highly reactive hydroxyl radical formed from the reaction between ferrous iron and hydrogen peroxide (the Fenton reaction). However, the fundamental experiment of directly determining the source of the hydroxyl radicals formed in the reaction has not yet been carried out. In this study, we have used both hydrogen peroxide and water labeled with 17O, together with ESR spin trapping, to detect the hydroxyl radicals formed in the reaction. ESR experiments were run in phosphate buffer with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, and either H2O2 or H2O labeled with 17O. The hydroxyl radical was generated by addition of Fe2+ ion to H2O2, or as a control, by photolysis of H2O2 in the ESR cavity. Observed ESR spectra were the sum of DMPO/.16OH and DMPO/.17OH radical adduct spectra. Within experimental accuracy, the percentage of 17O-labeled hydroxyl radical trapped by the DMPO was the same as in the original hydrogen peroxide, for either method of hydroxyl radical generation, indicating that the trapped hydroxyl radical was derived exclusively from hydrogen peroxide and that there was no exchange of oxygen atoms between H2O2 and solvent water. Likewise, the complementary reaction with ordinary H2O2 and 17O-labeled water also showed that none of the hydroxyl radical was derived from water. Our results do not preclude the ferryl intermediate, [Fe = O]2+ reacting with DMPO to form DMPO/.OH if the ferryl oxygen is derived from H2O2 rather than from a water ligand.