INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

THERE IS INCREASING evidence that many cellular effects of reactive oxygen species are mediated by changes in membrane ionic conductance. Studies suggest that H₂O₂ modulates currents carried via Na⁺ (36), Cl (27), or Ca²⁺ (14, 18) channels in various cell types. There is also substantial evidence that K⁺ channel activity is regulated by oxidation/reduction, and a majority of studies report that H₂O₂ increases K⁺ conductance. For example, H₂O₂ increases K⁺ currents in lung adenocarcinoma cells (17), renal epithelial cells (10), pyramidal neurons (28), and pancreatic -cells (16). In addition, H₂O₂ relaxes arterial smooth muscle by stimulating K⁺ conductance (30), but no single-channel studies have identified a specific K⁺ channel opened by H₂O₂ in arterial myocytes. On the other hand, H₂O₂ can also contract smooth muscle, including some blood vessels (21, 24); however, the signaling pathway(s) mediating the effects of H₂O₂ on vascular smooth muscle has not been fully characterized.

H₂O₂ is proposed to stimulate a variety of cellular transduction mechanisms, including protein kinase C (4, 11), phospholipase A₂ (PLA₂) (5, 23), nitric oxide (NO)/L-arginine (6), hydroxytryptamine (33), arachidonic acid (AA) (22), cyclooxygenase (13, 25), and guanylyl cyclase (3). Other studies have suggested that oxidation might directly affect ion channel proteins (26) or that a change in cellular redox status might modulate K⁺ channel gating (38); however, there is no general agreement on the mechanism of how H₂O₂ may induce contraction or relaxation of smooth muscle (2, 13, 24, 31). In light of these apparently contradictory findings, the purpose of the present study was to identify a specific molecular effector of H₂O₂ action in vascular smooth muscle and further characterize the signal transduction mechanism(s) stimulated by H₂O₂ in these cells. We present direct evidence that H₂O₂ opens the large-conductance, Ca²⁺- and voltage-activated K⁺ (BK_Ca) channel in myocytes from porcine coronary arteries. Subsequent pharmacological characterization employing tissue and cellular studies suggested that this response is mediated via lipoxygenase metabolites of AA, whereas cyclooxygenase metabolites appeared to mediate H₂O₂-induced arterial contraction. These findings now identify a specific K⁺ channel and single transduction mechanism (AA metabolism) that could underlie many of the vascular effects of H₂O₂ and other reactive oxygen species.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Arterial tension studies. Fresh porcine hearts were obtained from a local abattoir. The left anterior descending coronary artery was excised and placed into ice-cold dissociation medium of the following composition (mM): 110.0 NaCl, 5.0 KCl, 2.0 MgCl₂, 0.16 CaCl₂, 10.0 HEPES, 10.0 NaHCO₃, 0.5 KH₂PO₄, 0.5 NaH₂PO₄, 0.49 EDTA, 10.0 taurine, and 10.0 glucose (pH 6.9). Arteries were kept on ice during transport to the laboratory. Arterial rings (4-5 mm long, 2-4 mm diameter) were obtained from each left anterior descending coronary artery and prepared for isometric contractile force recordings as described previously (39). To control for possible indirect effects of endothelium-derived vasoactive factors, the endothelium was removed by rubbing the intimal surface. Rings were mounted between two triangular tissue supports, with one fixed to the bottom of the tissue bath and the other attached to a force-displacement transducer, and contractile force (grams) was recorded on a computer every 3 s. The tissue-bathing solution was a modified Krebs-Henseleit buffer of the following composition (mM): 118.0 NaCl, 4.8 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 25.0 NaHCO₃, 1.2 KH₂PO₄, and 11.0 glucose (pH 7.4). This solution was continuously oxygenated (97% O₂-3% CO₂) and heated to 37°C. Coronary ring preparations were equilibrated for 90 min under an optimal resting tension of 2.0 g, and fresh solution was added every 30 min. After equilibration, preparations were exposed to contractile agents [e.g., 10 µM histamine, 5 µM prostaglandin F₂ (PGF₂)], which maximally contracted the arteries and ensured stabilization of the muscle. After agonist removal and reequilibration (30 min), the contractile agent was reapplied to the tissue bath, and when the tissue reached a stable maximum contraction, H₂O₂ was added to the bathing medium. All drug solutions were prepared fresh daily, and for solutions with high K⁺ concentration ([K⁺]), NaCl was reduced to maintain normal osmolarity and Cl concentration.

Cell isolation. Myocytes were isolated as described previously (39). Fat and connective tissue were removed, and the adventitia was carefully dissected away. Each artery was then cut into 1-mm strips and placed in test tubes containing dissociation medium as described above. Media strips were incubated at 37°C in 5 ml of the dissociation solution with 5.0 mg of papain, 2.3 mM dithiothreitol, and 0.2% BSA. After 30 min of shaking in a water bath at 37°C, the tissue was triturated and the enzyme activity was diluted by addition of excess enzyme-free solution. The solution was removed and centrifuged at 500 g for 6 min at 4°C. The pellet was then resuspended in fresh medium and kept at 4°C. Experiments were performed within 6-8 h after cell dissociation.

Patch-clamp studies. For cell-attached patches, several drops of cell suspension were placed in a recording chamber (Warner Instruments) containing a solution of the following composition (mM): 140 KCl, 10 MgCl₂, 0.1 CaCl₂, 10 HEPES, and 30 glucose (pH 7.4, 22-25°C). Single K⁺ channels were measured in cell-attached patches by filling the patch pipette (2-5 M) with Ringer solution composed of (mM) 140 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, and 10 HEPES and making a gigaohm seal on a single myocyte. Voltage across the patch was controlled by clamping the cell at 0 mV with the high-[K⁺] extracellular solution. In experiments recording K⁺ channel activity of inside-out patches, the bathing solution exposed to the cytoplasmic surface of the membrane consisted of the following (in mM): 60 K₂SO₄, 30 KCl, 2 MgCl₂, 0.16 CaCl₂, 10 HEPES, 5 ATP, 1 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, and 10 glucose (pH 7.4, 22-25°C). The pipette solution was the same Ringer solution described above. Currents were filtered at 2 kHz and digitized at 10 kHz. Average channel activity [open channel probability (NP_o)] in patches with multiple BK_Ca channels was determined as described previously (39).

Drugs. Eicosatriynoic acid (ETI), papain, indomethacin, dithiothreitol, PGF₂, tetraethylammonium (TEA), and histamine were purchased from Sigma Chemical (St. Louis, MO). ODQ {1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one} and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) were purchased from Calbiochem (La Jolla, CA). H₂O₂ was purchased from Fisher Scientific (Pittsburgh, PA).

Statistical analysis. Data from tissue studies are expressed as the percentage of maximum relaxation, and all other data are expressed as means ± SE. Statistical significance between two groups was evaluated by Student's t-test for paired data. Comparison between multiple groups was carried out by one-way ANOVA, with a post hoc Tukey's test to determine significant differences among the data groups. P < 0.05 was considered to indicate a significant difference.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

H₂O₂ relaxes porcine coronary arteries. H₂O₂ produced concentration-dependent relaxation of endothelium-denuded coronary artery ring preparations precontracted with histamine or PGF₂ (Fig. 1A). On average, 300 µM H₂O₂ induced a 56 ± 4% relaxation (n = 16) within 5 min of administration. To further characterize this effect, a complete concentration-response relationship for H₂O₂-induced relaxation was determined (Fig. 1B). H₂O₂ at >1 mM induced a maximal relaxation of ~80%, and the EC₅₀ was calculated to be 252 ± 22 µM (n = 4-11). Interestingly, 300 µM is a physiological concentration of H₂O₂ achieved in the vasculature (32).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   H2O2 relaxes porcine coronary arteries. A: representative trace illustrating relaxant effect of 300 µM H2O2 (5 min) on endothelium-denuded arteries precontracted by 10 µM histamine. Arrows, point of drug application; dotted line, baseline tension. B: complete concentration-response relationship for H2O2-induced relaxation of porcine coronary arteries (n = 4-11).

H₂O₂-induced vascular relaxation involves K⁺ efflux. To begin characterizing the molecular mechanism through which H₂O₂ relaxes arteries, tension studies were performed under different ionic conditions. Exposure to 80 mM KCl contracted coronary artery ring preparations via depolarization and impaired K⁺ channel function by reducing the gradient for K⁺ efflux. In contrast to our previous studies using a physiological (4.8 mM) extracellular [K⁺], H₂O₂ did not relax arteries precontracted with 80 mM KCl, even after exposures >10 min, as illustrated by a typical recording in Fig. 2A. The results of these relaxation studies are summarized in Fig. 2B. In these studies, agonist-contracted arteries were relaxed 75 ± 3% by 300 µM H₂O₂, but when these same preparations were contracted by 80 mM KCl, H₂O₂ produced only a 2 ± 1% relaxation (P = 0.0001, n = 4). These studies suggested involvement of K⁺ channels in H₂O₂-induced relaxations. Therefore, single-channel patch-clamp experiments were performed to directly determine the potential involvement of these channels in the vascular response to H₂O₂.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   H2O2-induced relaxation of coronary arteries requires physiological K+ gradients. A: H2O2 (300 µM) did not relax arteries precontracted with 80 mM KCl (n = 4). These same preparations had previously demonstrated H2O2-induced relaxation when precontracted with 10 µM histamine in physiological extracellular K+ concentration. Dashed line, baseline tension; w/o, washout of all drugs. B: comparison of 300 µM H2O2-induced relaxation responses in agonist-contracted (histamine) arteries and in arteries contracted with 80 mM KCl. * Significant depression of relaxation effect.

H₂O₂ stimulates BK_Ca channel activity. Cell-attached patch-clamp studies revealed that H₂O₂ stimulated K⁺ channel activity (Fig. 3A). Addition of 300 µM H₂O₂ to the extracellular bathing solution increased NP_o (+40 mV) by >200-fold within 30 min (from 0.003 ± 0.001 to 0.722 ± 0.060, P < 10⁶, n = 11). Direct evidence identifying this protein as the BK_Ca channel was obtained from studies of inside-out patches (Fig. 3B). After a stimulatory effect of 300 µM H₂O₂ pipette solution on K⁺ channels was demonstrated in cell-attached experiments (NP_o = 0.583 ± 1.09), the patch was excised into an inside-out configuration. K⁺ channel activity persisted (NP_o = 0.254 ± 1.03), but subsequent addition of 1 mM TEA almost completely abolished channel activity (NP_o = 0.017 ± .01; P = 0.0002, n = 5). At this concentration, TEA is a selective blocker of BK_Ca channels. Previous experiments from our laboratory measured the single-channel conductance of this same channel to be 119 ± 14 pS and also demonstrated its sensitivity to intracellular Ca²⁺, 1 mM TEA, and charybdotoxin (39). Therefore, this pharmacological and biophysical analysis clearly identifies the BK_Ca channel as an important target of H₂O₂ action in porcine coronary arteries. In contrast to our previous studies with estrogen and NO (7, 39), the stimulatory effect of H₂O₂ on BK_Ca channels did not involve production of cGMP, inasmuch as pretreating myocytes with 10 µM ODQ, a selective inhibitor of guanylyl cyclase activity (IC₅₀ = 20 nM), had no effect on H₂O₂-induced channel activity but attenuated stimulation by 10 µM nitroprusside (NP_o = 0.0, 0.112, and 0.975 for control, nitroprusside, and H₂O₂, respectively, n = 3; data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   H2O2 stimulates large-conductance Ca2+- and voltage-activated K+ (BKCa) channel activity in myocytes from porcine coronary arteries. A: consecutive recordings from a cell-attached patch (+40 mV) before and 30 min after administration of 300 µM H2O2. Upward deflections, channel openings; dashed line, channel closed state. B: summary of single-channel studies identifying BKCa channel as a target of H2O2. H2O2 (300 µM, within the patch pipette) stimulated channel activity in cell-attached (C-A) patches (+40 mV). Patches were then excised into inside-out (I-O) configuration, and effects of 1 mM tetraethylammonium (TEA) on channel activity were determined (n = 5). * Significant increase in BKCa channel open probability compared with control (C-A and I-O) or significant inhibition of channel activity produced by TEA.

H₂O₂ activation of BK_Ca channels involves the lipoxygenase pathway. Previous studies suggested that some of the vascular effects of H₂O₂ might be mediated by metabolites of the PLA₂/AA cascade (5, 11), and the importance of this mechanism was tested on cell-attached patches. Addition of 10 µM AA to the bath solution stimulated BK_Ca channel activity by ~200-fold (NP_o from 0.0017 to 0.2857, +40 mV, n = 3, P = 0.003) in cell-attached patches (Fig. 4). This increase was very similar to the ~200-fold increase in NP_o produced by H₂O₂. Interestingly, AA-stimulated channel activity was reversed ~80% by 5 µM ETI (30 min, NP_o = 0.0657), an inhibitor of 5-, 12-, and 15-lipoxygenase activity. The inhibitory effect of ETI was subsequently reversed by 10 µM indomethacin, an inhibitor of cyclooxygenase activity. Because AA mimicked the stimulatory effect of H₂O₂ and appeared to enhance lipoxygenase metabolism, the next experiments tested whether these metabolites might also mediate the effects of H₂O₂ on BK_Ca channels. Once again, H₂O₂ enhanced single-channel activity within 30 min of exposure (NP_o from 0.00 to 0.825; Fig. 5A), but subsequent addition of 5 µM ETI (30 min) almost completely reversed H₂O₂-induced BK_Ca channel activity (NP_o = 0.067). On average, 5 µM ETI inhibited the response to H₂O₂ by 84 ± 13% (P = 0.005, n = 4). This reversal was similar to the inhibitor effect of ETI on AA-induced channel activity (80%) and suggested that lipoxygenase metabolites were involved in the vascular response to H₂O₂. A complete time-course plot of channel activity before and after H₂O₂, with subsequent inhibition by ETI, is illustrated in Fig. 5B.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Arachidonic acid (AA) stimulates BKCa channel activity via lipoxygenase metabolites. Cumulative data from 3 cell-attached patch experiments (+40 mV) are shown. AA (10 µM, 30 min) stimulated BKCa channel activity, and this effect was reversed by 5 µM eicosatriynoic acid (ETI; 30 min). Subsequent addition of indomethacin (Indo; 10 µM) restored AA-stimulated channel activity. All drugs were added cumulatively. * Significant increase in channel open probability vs. control.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Activation of BKCa channels by H2O2 involves the lipoxygenase pathway. A: consecutive traces of single-channel recordings from cell-attached patches (+40 mV) before and 30 min after 300 µM H2O2 and then after subsequent administration of 5 µM ETI (30 min). Dashed line, channel closed state. B: time course of BKCa channel activity of cell-attached patch illustrated in A. Each bar represents single-channel open probability in a 100-ms trace at +40 mV. Breaks in time axis are periods when channel activity was not recorded.

H₂O₂ induces vasoconstriction when lipoxygenase activity is inhibited. To further characterize the potential involvement of AA metabolites, tension studies were performed to test whether lipoxygenase metabolites were also involved in the relaxation effect of H₂O₂. Arteries were initially contracted with histamine or PGF₂, and exposure to 300 µM H₂O₂ induced the expected relaxation. After washout, preparations were incubated with 5 µM ETI for 30 min, and after precontraction, subsequent addition of H₂O₂ (5 min) never induced significant relaxation (Fig. 6A; n = 5). In contrast, when arteries were preincubated with ETI and then exposed to 300 µM H₂O₂ without precontraction, the oxidant contracted the arteries (Fig. 6B). This vasoconstrictor effect was observed in 11 arteries pretreated with ETI (P = 0.0003), whereas only a vasodilator effect was observed in these same arteries in the absence of lipoxygenase inhibition. Interestingly, this contractile response to H₂O₂ was reversed by further treatment with 10 µM indomethacin (Fig. 6C). Indomethacin reversed H₂O₂-induced contraction by an average of 85 ± 4% (n = 4). These findings demonstrated that H₂O₂ can act as a vasodilator or a vasoconstrictor, depending on the disposition of AA metabolism. Additional control experiments demonstrated that the inhibitory effect of ETI did not involve inhibition of guanylyl cyclase activity. Pretreating arteries with 5 µM ETI did not inhibit relaxation produced by 10 µM sodium nitroprusside, which induced 100% relaxation in the absence or presence of ETI (n = 4; data not shown).

View larger version (8K): [in this window] [in a new window]   Fig. 6.   H2O2 induces vasoconstriction and vasodilation of porcine coronary arteries. Ability of 300 µM H2O2 to relax all (histamine-precontracted) preparations was confirmed before 30-min incubation with 5 µM ETI. Dashed line, baseline tension. A: pretreating endothelium-denuded arterial rings with ETI inhibits relaxation effect of 300 µM H2O2. Rings were precontracted with 10 µM histamine. B: H2O2 (300 µM) induces contraction in preparations pretreated with ETI. C: indomethacin (10 µM) reverses contractile effect of 300 µM H2O2.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

It is clear that H₂O₂ is a vasoactive substance; however, our understanding of how this oxidant affects vascular smooth muscle cells is far from complete. For example, H₂O₂ may act as a vasoconstrictor (24) or a vasodilator (6, 33), depending on the specific artery, species, or experimental conditions. There is even less certainty regarding the signal transduction and/or effector mechanism(s) that may be involved in the vascular response to H₂O₂, e.g., protein kinase C (4, 11), PLA₂ (5, 23), guanylyl cyclase (3), or NO (6). The present study helps resolve some of this controversy by providing direct evidence for an effector molecule that can mediate H₂O₂-induced vasodilation, the BK_Ca channel, and also proposes that a single signaling pathway, AA metabolism, can mediate the vasodilatory and vasoconstrictor effects of H₂O₂ on porcine coronary arteries.

Twenty years ago, Olson and Boerth (19) found that H₂O₂ exerted beneficial effects on the coronary circulation. More recent studies have demonstrated that H₂O₂ increases coronary blood flow (29) and relaxes coronary arteries in vitro by a mechanism that is specific to H₂O₂ and does not involve superoxide anion or hydroxyl radical (2). In the present study, physiological concentrations of H₂O₂ relaxed porcine coronary arteries precontracted with agonists. In contrast, H₂O₂ failed to relax arteries precontracted by elevating extracellular [K⁺] to 80 mM. Raising external [K⁺] reduces the driving force for K⁺ efflux, thereby functionally limiting the influence of K⁺ channels on vascular reactivity. The inability of H₂O₂ to relax arteries precontracted with high extracellular [K⁺] suggested that the relaxation response to H₂O₂ involved stimulation of K⁺ conductance, a mechanism that has also been suggested from pharmacological studies of arterial smooth muscle (30, 36) and microelectrode/whole cell studies employing other cell types (10, 28). In contrast, the present study has measured the effects of H₂O₂ on single-channel activity directly in cell-attached patches. H₂O₂ increased the open probability of a large-conductance (>100 pS), Ca²⁺- and 1 mM TEA-sensitive K⁺ channel, which we and others previously identified as the BK_Ca channel in these cells (34, 39). These channels are expressed in high density in myocytes from porcine or human (12) coronary arteries and are important effector molecules that mediate vasodilation produced by a number of agents by inducing membrane repolarization. Further support for BK_Ca channel involvement is gained from previous studies indicating that H₂O₂ hyperpolarizes myocytes from rat carotid arteries by a mechanism that was partially sensitive to charybdotoxin (15). Moreover, H₂O₂-induced relaxation of cerebral arteries is inhibited by 1 mM TEA, further implicating a role for BK_Ca channels (30).

Although there is increasing evidence that ion channels mediate the effects of H₂O₂ and other oxidants on cell excitability, we have found no studies examining the effects of H₂O₂ on single ion channels expressed in their native cells. Microelectrode and/or whole cell studies revealed that H₂O₂ increases a Ca²⁺-activated Cl current in oocytes (27) and slows inactivation of tetrodotoxin-sensitive Na⁺ currents in rat ventricular myocytes (36). In addition, H₂O₂ opens or closes Ca²⁺ channels incorporated into artificial lipid bilayers, depending on the concentration employed (14, 18). Several studies also indicate that H₂O₂ increases K⁺ currents. For example, H₂O₂ induces a K⁺ conductance in CA1 pyramidal neurons (28), activates a glibenclamide-sensitive K⁺ conductance in LLC-PK₁ cells (10), and inhibits inactivation of K⁺ currents in oocytes expressing cloned K⁺ channels (35). Studies utilizing oxidizing agents other than H₂O₂ have also demonstrated stimulation of BK_Ca channel activity in cell-attached patches on myocytes from pulmonary or ear artery (20). In contrast, H₂O₂ decreases open probability of cloned hslo Ca²⁺-activated K⁺ channels expressed in oocytes or HEK-293 cells (8). The rationale for the variance between this finding and those of the present and other studies (20) is not apparent but may be related to differences in cell or channel properties. For example, studying the regulation of cloned channels expressed in transfected cells can be limited by the fact that signaling systems and/or regulatory proteins that influence channel activity under normal conditions may not be present in transfected cells.

Previous studies have proposed a variety of molecular pathways that can be stimulated by H₂O₂. For example, activation of guanylyl cyclase may underlie H₂O₂ relaxation of pulmonary arteries (3). Because we showed previously that stimulation of the NO/cGMP signaling cascade opened BK_Ca channels in myocytes from porcine coronary arteries (7, 39), it was possible that H₂O₂ was also acting via this mechanism; however, inhibition of guanylyl cyclase did not affect the response to H₂O₂. Furthermore, ETI did not inhibit relaxation induced by nitroprusside, nor does cGMP induce vascular contraction as H₂O₂ does when lipoxygenase is blocked. These findings make it unlikely that the effect of H₂O₂ on BK_Ca channels in porcine coronary arteries involves cGMP. On the other hand, H₂O₂ increases protein kinase C production in A7r5 smooth muscle cells (11) and pulmonary arterial smooth muscle, with subsequent increases in PLA₂ activity and AA release (5). Because other studies had demonstrated a link between AA and K⁺ channels in neurons (1) and neuroendocrine cells (9), we considered that AA metabolites might mediate the stimulatory effect of H₂O₂ on BK_Ca channels in coronary smooth muscle. Addition of exogenous AA stimulated BK_Ca channel activity in cell-attached patches dramatically, and this increased activity was significantly reversed by ETI, an inhibitor of 5-, 12-, and 15-lipoxygenase activity. Moreover, the inhibitory effect of ETI was completely reversed by indomethacin, probably because of shunting of AA metabolism from cyclooxygenase toward the lipoxygenase pathway. These experiments suggested that lipoxygenase metabolites of AA stimulate the activity of BK_Ca channels, and subsequent experiments further implicated AA metabolism as an important signaling mechanism in the response to H₂O₂. Not only did AA mimic the effect of H₂O₂ on BK_Ca channels, but ETI also inhibited the effect of H₂O₂ on intact tissues and single myocytes. In fact, ETI unmasked a contractile effect of H₂O₂ (Fig. 6B). As in single-channel recordings, indomethacin reversed the effect of ETI by inhibiting H₂O₂-induced contraction. The ability of ETI and indomethacin to inhibit and disinhibit responses to H₂O₂, respectively, casts doubt on the possibility that H₂O₂ is acting directly on BK_Ca channel proteins. Instead, these findings provide evidence that AA metabolism may account for H₂O₂-induced contraction and relaxation of coronary arteries.

We propose that under normal conditions H₂O₂ increases AA metabolism, probably via stimulation of PLA₂ activity (11), and lipoxygenase derivatives of AA then promote vasorelaxation because of their powerful stimulation of BK_Ca channel activity; however, if lipoxygenase activity is inhibited, then AA metabolism is shunted toward cyclooxygenase, and vasoconstriction ensues because of production of contractile prostanoids (e.g., thromboxane). If this hypothesis is correct, then inhibition of cyclooxygenase should reverse the contractile effect of H₂O₂. Experiments with indomethacin revealed that this was indeed the case (Fig. 6C). Therefore, H₂O₂ appears to stimulate PLA₂/AA metabolism, and, depending on which pathway is dominant, lipoxygenase or cyclooxygenase, H₂O₂ could act as a vasodilator or a vasoconstrictor. This mechanism is consistent with studies demonstrating that H₂O₂ stimulates PLA₂ activity in arterial smooth muscle cells (23) and others demonstrating that indomethacin attenuates H₂O₂-induced contraction of tracheal smooth muscle (25, 33). Furthermore, Duerson et al. (9) demonstrated that the neurohormone somatostatin inhibits excitability of pituitary cells by opening BK_Ca channels via lipoxygenase metabolites. In this and the present study, ETI reversed agonist-induced stimulation of BK_Ca channel activity but had no direct inhibitory effect on channel proteins. Therefore, stimulation of BK_Ca channel activity by lipoxygenase metabolites may be a ubiquitous mechanism to depress activity of excitable cells; however, further studies are needed to identify the specific leukotrienes and/or AA metabolites involved. Identification of these products, coupled with a more complete understanding of their effects on cellular physiology, should help shed more light on our understanding of diseases such as ischemiareperfusion injury, myocardial stunning, and obstructive airway diseases, where oxidative damage has been implicated.