INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

REACTIVE OXYGEN SPECIES, like H₂O₂, have been implicated in the pathophysiology of a variety of cardiovascular disorders such as ischemia-reperfusion injury, myocardial stunning, stroke, and obstructive airway disease (13). Recent clinical evidence indicates that H₂O₂ is produced in human plasma at micromolar concentrations and that plasma from hypertensive individuals contains higher levels of H₂O₂ than plasma from normotensive individuals (22). These studies suggest that H₂O₂ is a pathophysiological substance that contributes to essential hypertension and its associated sequelae (e.g., atherosclerosis, myocardial infarction, stroke). In contrast, H₂O₂ also produces beneficial effects on the cardiovascular system and other tissues. For example, H₂O₂ was administered intravenously as an anticancer agent over 30 years ago (32). More recently, H₂O₂ has been shown to relax pulmonary (4), cerebral (35, 39), or coronary (2, 26) arteries and to significantly enhance postischemic functional recovery and coronary blood flow in isolated hearts (15, 34). These studies imply that H₂O₂ can act as either a physiological or a pathophysiological substance, but the basis of this interesting conundrum remains obscure. We are only beginning to understand the cellular and/or molecular basis of H₂O₂ action, particularly its effects on the heart and blood vessels.

We recently demonstrated that physiological concentrations of H₂O₂ relax porcine coronary arteries via an endothelium-independent mechanism involving activity of the large-conductance, calcium- and voltage-activated potassium (BK_Ca) channel in coronary smooth muscle cells (2). This study further suggested that H₂O₂-induced coronary relaxation might involve arachidonic acid (AA) production. The goal of the present study was to characterize this potential H₂O₂ transduction mechanism in coronary smooth muscle cells and relate these cellular findings to the effect of H₂O₂ on coronary artery function. We now provide direct biochemical and electrophysiological evidence that H₂O₂ stimulates a cellular transduction cascade, i.e., phospholipase A₂ (PLA₂) activity and AA production, to open BK_Ca channels in coronary smooth muscle and promote relaxation. Evidence is also presented that this effect of H₂O₂ involves stimulation of protein kinase C (PKC) activity and lipoxygenase metabolism but does not require activity of the cyclooxygenase or P-450 cascades. We propose that vascular effects of H₂O₂, and possibly other reactive oxygen species, involve the PLA₂/AA signaling cascade in vascular smooth muscle cells and that stimulation of potassium channel activity can mediate the vasodilatory response to these agents.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell isolation. Myocytes were isolated by a modification of a procedure described previously (2). Briefly, fresh porcine hearts were obtained from a local abattoir. The left anterior descending coronary artery (LAD) was excised and placed in ice-cold dissociation medium of the following composition (in 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. The LAD was cleaned of adherent fat and connective tissue under a dissecting microscope, 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 dissociation medium with 5.0 mg papain, 2.3 mM dithiothreitol (DTT), and 0.2% BSA for 30 min. Afterward, the tissue was triturated, and the enzyme activity was diluted by adding 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 (in mM): 140 KCl, 10 MgCl₂, 0.1 CaCl₂, 10 HEPES, and 30 glucose (pH 7.4; 22-25°C). Single potassium channels were measured in cell-attached patches by filling the patch pipette (2-5 M) with Ringer solution (in 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-concentration extracellular potassium solution. Currents were filtered at 2 kHz and digitized at 10 kHz. Average channel activity (NP_o) in patches with multiple BK_Ca channels was determined as described previously (40). For consistency, statistics on channel activity were reported at a membrane potential of +40 mV. Although the effect of H₂O₂ and other agents was observed at a variety of potentials, BK_Ca channels are very clearly identified at +40 mV thus increasing the accuracy and reliability of NP_o calculations. Previous studies from our laboratory (14, 40) and others (9, 41) commonly record BK_Ca channel activity at such potentials. In experiments recording potassium channel activity of inside-out patches, the bathing solution exposed to the cytoplasmic surface of the membrane consisted of the following low-calcium solution (in mM): 60 K₂SO₄, 30 KCl, 2 MgCl₂, 1 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 0.16 CaCl₂ (pCa 7), 10 HEPES, 5 ATP, and 10 glucose (pH 7.4; 22-25°C).

Arterial tension studies. Arterial rings (4-5 mm in length, 2-4 mm in diameter) were obtained from each LAD and were prepared for isometric contractile force recordings as described previously (40). H₂O₂ induces endothelium-independent relaxation of porcine coronary arteries (2); therefore, 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 (in 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 grams, and fresh solution was added every 30 min. After equilibration, preparations were exposed to contractile agent (5-10 µM PGF₂) that 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. Each day, one tissue was contracted with PGF₂ but was not exposed to H₂O₂ to control for possible "fading" of the contractile response. Tissues were incubated with inhibitors of AA metabolism at least 30 min before H₂O₂ exposure. All drug solutions were prepared fresh daily.

AA assay. Myocytes were isolated by the procedure described above, except that multiple tissue samples were dissociated concurrently, and the final cell suspensions were pooled. An aliquot of each suspension was examined microscopically to assess possible contamination with nonsmooth muscle cells. All cells were incubated at 37°C for 2 h with 20 µCi of tritiated AA ([³H]AA) in dissociation medium, after which the medium was removed and the cells were washed three to four times with cold Krebs-Henseleit buffer. After the final wash, cells were resuspended in 0.2% fatty acid-free BSA and then divided into l-ml aliquots in microfuge tubes. The cells then were incubated in a 37°C water bath for 15 min, after which H₂O₂ was added to each vial to a final concentration of either 100, 300, or 1,000 µM. Tubes were again incubated in a 37°C water bath for 15 min and then were placed on ice for 10 min to stop all reactions. Tubes were then centrifuged at high speed, 900 µl were transferred (remaining 100 µl was saved for protein determination) to labeled glass tubes, and two times the volume (1.8 ml) of a 2:1 vol/vol solution of chloroform-methanol (CHCl₃/CH₃OH) was added. Tubes were vortexed and centrifuged at high speed, and the lower chloroform layer was removed. The chloroform layer was evaporated overnight under vacuum, leaving a thin white residue. Silicic acid columns were prepared by adding 0.5 g silicic acid to glass- and wool-plugged Pasteur pipettes supported in a rack. Each column was washed with 10 ml petroleum ether (60-80°C) before use. The samples were dissolved in 1 ml petroleum ether-diethyl ether (96:4 vol/vol) and then were applied to a column, which was then washed with 3 ml petroleum ether-diethyl ether and 3 ml diethyl ether to elute the [³H]AA from the column. The diethyl ether was collected in a scintillation vial to which was added 10 ml Scintiverse II and then was counted at 5-min counts on a scintillation counter preprogrammed for beta emission. AA generation was normalized for total amount of protein per sample after Lowry (25) protein determination.

Drugs. AA, papain, indomethacin, DTT, proadifen, eicosatrienoic acid (ETI), sodium nitroprusside, BSA, baicalein, and PGF₂ were purchased from Sigma Chemical. H₂O₂ and Scintiverse II were purchased from Fisher Scientific. MK-886 and calphostin C were purchased from Calbiochem. Arachidonyl trifluoromethyl ketone (ATK) and sphingosine were purchased from Research Biochemicals International, and [³H]AA was purchased from Amersham.

Statistical analysis. Data from tissue studies were expressed as the percentage of maximum relaxation, and all other data were expressed as means ± SE. Statistical significance between two groups was evaluated by Student's t-test for paired data. Comparison between multiple groups was made by the one-way ANOVA test, 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 AND METHODS RESULTS DISCUSSION REFERENCES

H₂O₂ opens BK_Ca channels via second messengers. Exposing cells to H₂O₂ stimulated activity of a large-amplitude channel isolated in cell-attached patches (Fig. 1A). On average, single-channel open probability (NP_o) increased from ~0 to 0.532 ± 0.07 (+40 mV, n = 8) after 20-30 min exposure to 300 µM H₂O₂. This concentration of H₂O₂ is near the EC₅₀ value for H₂O₂-induced relaxation of porcine coronary arteries (2) and is a physiological concentration (37). In contrast to its effect on intact cells, H₂O₂ did not affect channel activity when superfused on the cytoplasmic surface of inside-out membrane patches (Fig. 1B; n = 4). In the same excised patch, however, channel activity was stimulated dramatically by increasing "intracellular" calcium concentration from 0.1 to 100 µM (NP_o from 0.11 ± 0.01 to 0.76 ± 0.11 at +40 mV; n = 4). Single-channel current-voltage relationships indicated that the H₂O₂- and calcium-sensitive channel exhibited a conductance of >100 pS in physiological K⁺ gradients and was sensitive to blockade by 1 mM tetraethylammonium (TEA; data not shown). Because of this high conductance and sensitivity to elevated intracellular calcium levels and TEA, we have identified this protein as the BK_Ca channel that we and others have characterized previously in these and other identical cells (e.g., see Refs. 9, 12, 14, 40, 41). In addition, the reducing agents glutathione (1 mM) or DTT (1 mM) completely prevented the stimulatory effect of H₂O₂ on BK_Ca channel activity (n = 5-9; P < 0.0001; data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   H2O2 stimulates large-conductance, calcium-activated (BKCa) channel activity via a second messenger mechanism. A: recordings from the same cell-attached patch (+40 mV) before and 20 min after 300 µM H2O2. Upward deflections are channel openings, and dotted line indicates closed state. B: recordings from the same inside-out patch under control conditions (+40 mV), 30 min after exposure to 300 µM H2O2, and then immediately after increasing concentration of calcium in the bath solution from 0.1 to 100 µM.

2

View larger version (7K): [in this window] [in a new window]   Fig. 2.   Inhibition of BKCa channels attenuates H2O2-induced relaxation of coronary arteries. Representative tracing from the same artery contracted first with 5 µM PGF2 and then with 1 mM tetraethylammonium (TEA). H2O2 (300 µM) was added as indicated by the arrows. w/o, Washout of all agents. Dotted line indicates baseline tension.

H₂O₂ stimulates the PLA₂-AA cascade in coronary smooth muscle. Previous studies have indicated that H₂O₂ can activate PLA₂ in vascular smooth muscle (28). To test this possibility in coronary arteries, we employed ATK, which selectively inhibits cytosolic PLA₂ (36). In four out of four cell-attached patches, ATK reversed the stimulatory effect of H₂O₂ on BK_Ca channel activity by an average of 97 ± 8% (Fig. 3). As expected, 300 µM H₂O₂ stimulated channel NP_o from 0.003 ± 0.003 to 0.498 ± 0.176 (+40 mV), but channel NP_o was reduced to 0.013 ± 0.013 by 15 µM ATK (20 min; P < 0.05). In contrast, ATK had no direct inhibitory effect on channel activity in inside-out patches (n = 3, data not shown). These findings suggested that H₂O₂ opens BK_Ca channels via stimulation of PLA₂ activity, and further experiments were performed to verify this conclusion directly.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Inhibition of PLA2 activity reverses the effect of H2O2 on BKCa channels. Recordings from the same cell-attached patch (+40 mV) before and 20 min after 300 µM H2O2 and then 20 min after addition of 15 µM arachidonyl trifluoromethyl ketone (ATK). Channel openings are upward deflections, and the dotted line indicates channel closed state.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   H2O2 stimulates release of arachidonic acid (AA) and BKCa channel activity in coronary myocytes, possibly through activation of protein kinase C. Each bar represents the mean ± SE. A: treatment of coronary smooth muscle cells with 300 µM H2O2 (15 min) stimulated release of [3H]AA. In contrast, H2O2 had no effect on cells pretreated for 15-20 min with 15 µM sphingosine. * Significant increase in [3H]AA release compared with control levels (P < 0.001; n = 6). B: average values of BKCa channel activity (NPo) in cell-attached patches (+40 mV) before and 15-20 min after addition of 300 µM H2O2 and 30 min after subsequent addition of 15 µM sphingosine. * Significant increase in single-channel NPo above control level. # Significant reduction of channel NPo after sphingosine (P < 0.001; n = 5).

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Inhibitors of protein kinase C (PKC) activity attenuate the relaxation effect of H2O2 on coronary arteries. A: calphostin C inhibits H2O2-induced relaxation of coronary arteries. Coronary arteries were precontracted with 5 µM PGF2. After contraction had reached a plateau level, the relaxation response to 300 µM H2O2 was measured in the absence or presence of 1 µM calphostin C (30 min). Each bar represents the average relaxation response ± SE. * Significant depression of H2O2-induced coronary relaxation (P < 0.002; n = 5). B: mean concentration-response relationship for H2O2-induced relaxation of coronary arteries before (solid line) and after (dashed line) a 45- to 60-min pretreatment with 1 µM phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)]. Each point represents the mean of 6 experiments ± SE.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   AA stimulates BKCa channel activity via lipoxygenase metabolism. Recordings from the same cell-attached patch (+40 mV) before and 30 min after 10 µM AA. Subsequent addition of 5 µM eicosatrienoic acid (ETI; 30 min) reversed the effect of AA (P = 0.003; n = 5). Channel openings are upward deflections from baseline activity (dotted line). B: ETI has no effect on nitrovasodilator-induced coronary relaxation. Typical tracing from the same coronary artery precontracted with 5 µM PGF2. Sodium nitroprusside (SNP, 10 µM) was added as indicated by the arrows. After washout (w/o) of all agents, arteries were allowed to reequilibrate for 30 min, followed by a 30-min exposure to 5 µM ETI, as indicated by bar. The relaxation response to 10 µM SNP was repeated in the continued presence of ETI. Dashed line indicates baseline tension.

Lipoxygenase metabolites mediate the effect of H₂O₂ and AA on coronary arteries. Studies on single myocytes and intact arteries were performed to characterize the downstream events of AA metabolism that mediated the effect of H₂O₂. Studies on cell-attached patches revealed that the stimulatory effect of H₂O₂ on BK_Ca channel activity was reversed by inhibitors of lipoxygenase activity. We employed three different agents that are known to inhibit lipoxygenase: ETI, a general inhibitor of lipoxygenase pathways (27); MK-886, which specifically inhibits the 5-lipoxygenase activating protein (31); and baicalein, an inhibitor of 12-lipoxygenase (27). Each of these inhibitors was effective in reversing the effect of H₂O₂/AA on BK_Ca channels in cell-attached patches. We reported previously that 5 µM ETI reversed the effect of H₂O₂ on channel activity (2) and now demonstrate that ETI produces a similar inhibition of AA-stimulated channel activity (Fig. 6A). On average, BK_Ca channel NP_o stimulated by 10 µM AA was reversed 60% 30 min after treatment with 5 µM ETI (from 0.529 ± 0.04 to 0.217 ± 0.025 at +40 mV; n = 4; P = 0.003). To control for potential nonspecific effects of ETI, the ability of this lipoxygenase inhibitor to inhibit nitrovasodilator-induced coronary relaxation was also examined. In arteries precontracted with 5 µM PGF₂, 10 µM sodium nitroprusside induced a nearly complete (91 ± 2%; n = 4; Fig. 6B) relaxation. After a 30-min incubation with 5 µM ETI, nitroprusside produced an almost identical response (89 ± 2%; n = 4; Fig. 6B). Because ETI neither blocks BK_Ca channels directly (n = 4 out of 4 inside-out patches) nor affects nitrovasodilator-induced coronary relaxation, we conclude that H₂O₂-induced BK_Ca channel activity involves the lipoxygenase signaling pathway.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   H2O2 stimulation of BKCa channels involves lipoxygenase metabolism. Recordings from the same cell-attached patch (+40 mV) before and 20 min after 300 µM H2O2 and 30 min after subsequent exposure to 1 µM MK-886. Channel openings are upward deflections from the baseline levels (dotted line).

View larger version (29K): [in this window] [in a new window]   Fig. 8.   H2O2-induced coronary relaxation does not involve cyclooxygenase or P-450 metabolites. Coronary arteries were precontracted with 5 µM PGF2. After contraction had reached a plateau level, the relaxation response to 300 µM H2O2 was measured in the absence or presence of either 10 µM proadifen (30 min; n = 4) or 10 µM indomethacin (30 min; n = 6). Each bar represents the mean relaxation response ± SE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

H₂O₂ is recognized as an important intracellular signaling trigger that activates a variety of signal transduction systems, including PKC (6), PLA₂ (7, 28), nitric oxide (8), guanylyl cyclase (4), and cyclooxygenase (30), in a variety of cell types. Interestingly, H₂O₂ is also a vasoactive agent that can either relax (35) or contract (29) blood vessels, and recent clinical studies have revealed that plasma levels of H₂O₂ correlate with the incidence of essential hypertension (22). On the other hand, H₂O₂ exerts "beneficial" vasodilatory effects on the coronary (26, 34) and cerebral (35) circulations. Moreover, H₂O₂ can either contract or relax porcine coronary arteries, and these responses do not involve stimulation of guanylyl cyclase or cGMP (2). In light of these somewhat contradictory findings, it is clear that our understanding of the cellular and/or molecular basis of H₂O₂ effects on blood vessels is far from complete. The present study now combines evidence from biochemical, pharmacological, and single-channel patch-clamp experiments to strongly suggest that H₂O₂ opens BK_Ca channels in single coronary myocytes via a mechanism involving PKC stimulation of the PLA₂-AA-lipoxygenase signaling pathway.

Because of their large conductance and high density of expression, BK_Ca channels appear to be the predominant potassium channel of both porcine (40) and human (12) coronary arteries. Opening of these channels produces a powerful repolarizing action that closes voltage-dependent calcium channels to induce vascular relaxation. Previous studies suggested that H₂O₂ increased potassium conductance in various cell types, including renal epithelial cells (11), pyramidal neurons (33), pancreatic -cells (20), and lung adenocarcinoma cells (21). The important role of potassium channels in mediating H₂O₂-induced vasorelaxation has been suggested by observations that the relaxation response to H₂O₂ is abolished in aorta (17) or coronary arteries (2) precontracted by high extracellular potassium concentration, which reduces the driving force for potassium efflux. However, no single-channel studies had identified a specific potassium channel opened by H₂O₂ in vascular smooth muscle cells. Findings from the present study confirm our previous report that H₂O₂ opens BK_Ca channels in coronary smooth muscle (2). Because it can easily cross cell membranes, it was possible that H₂O₂ was interacting directly with channel proteins; however, H₂O₂ did not affect channels in cell-free patches (Fig. 1B). These findings indicate that H₂O₂ stimulates a second messenger process in coronary smooth muscle cells. Identification of the BK_Ca channel as a target of H₂O₂ action allowed us to employ this protein as a sensitive molecular assay to investigate the importance of signaling mechanisms stimulated by H₂O₂ in coronary smooth muscle.

H₂O₂ activates cytosolic PLA₂ in cultured aortic smooth muscle cells (28), but this mechanism has not been investigated in primary cells from coronary arteries. Involvement of PLA₂ in the response to H₂O₂ was investigated by several means. First, patch-clamp studies indicated that inhibition of cytosolic PLA₂ activity by a selective inhibitor, ATK (36), reversed the effect of H₂O₂ on BK_Ca channel activity. In addition, the same stimulatory concentrations of H₂O₂ that opened BK_Ca channels also induced [³H]AA release from coronary myocytes, confirming involvement of PLA₂ in the response to H₂O₂. Furthermore, if AA was indeed a cellular messenger for H₂O₂ effects, then exogenous AA should mimic the stimulatory effect of H₂O₂. Subsequent patch-clamp studies revealed that direct application of AA to coronary myocytes stimulated the activity of BK_Ca channels, as did H₂O₂. Stimulation of BK_Ca channels by AA was reversed by ETI, indicating that AA, like H₂O₂, does not affect channel proteins directly. Because ETI did not affect nitroprusside-induced coronary relaxation, the likelihood that ETI produces significant nonspecific effects (e.g., on calcium levels, contractile proteins, channel blockade) appears remote. In light of this evidence from both functional studies and biochemical measurements, we conclude that H₂O₂-induced stimulation of BK_Ca channel activity involves PLA₂ activity and release of AA from membrane lipids in coronary artery smooth muscle. PLA₂ has been classified as either secretory or cytosolic, and evidence indicates that the cytosolic form is the agonist-stimulated PLA₂ (10), which is also stimulated by H₂O₂ in endothelial (3) or aortic smooth muscle cells (28).

There is increasing evidence that PLA₂ activity is regulated by a number of cellular mechanisms, including PKC, mitogen-activated protein (MAP) kinase, and possibly G proteins (23). The present study provides evidence suggesting that the effect of H₂O₂ on coronary arteries may also involve PKC. The stimulatory effect of H₂O₂ on either BK_Ca channel activity or [³H]AA release from coronary smooth muscle was antagonized by sphingosine, an inhibitor of PKC activity. Furthermore, a more selective inhibitor of PKC activity, calphostin C, was even more effective (94%) at attenuating H₂O₂-induced relaxation of coronary arteries. In addition to these experiments with PKC antagonists, experiments were performed on arteries pretreated with phorbol ester. Such pretreatment is known to downregulate PKC activity, and results from these studies indicated that the relaxant effect of H₂O₂ was significantly depressed in TPA-treated arteries. These findings from four different types of experiments on coronary myocytes and intact arteries are consistent with a H₂O₂ transduction pathway involving PKC stimulation of PLA₂ activity. Previous studies have also reported that PKC inhibitors depress H₂O₂-induced AA release in other cell types (3). PKC phosphorylates PLA₂ in vitro (24), but at present there is limited evidence that PKC phosphorylates PLA₂ directly in vivo (23). An alternative explanation for the present results could be that the immediate substrate for H₂O₂-stimulated PKC is actually MAP kinase, which activates (phosphorylates) cytosolic PLA₂ in other cell types (24). H₂O₂ stimulates MAP kinase activity in perfused rat hearts (18) and pulmonary artery smooth muscle (42) but may (28) or may not stimulate this enzyme in rat aorta (1). Therefore, the action of H₂O₂ on MAP kinase activity might be heterogeneous with respect to artery and/or species. Although the present findings argue for PKC involvement in the response of coronary arteries to H₂O₂, the specific isoform(s) remains to be identified. Also, it is still unclear whether MAP kinase is involved as an intermediary component of this signaling cascade. However, the present findings demonstrate that inhibitors of PKC activity completely reversed H₂O₂-induced release of [³H]AA (Fig. 4) and significantly depressed H₂O₂-stimulated BK_Ca channel activity or relaxation of coronary arteries. Therefore, it seems unlikely that H₂O₂ acts independently of PKC and exclusively through MAP kinase, but further studies are necessary to help resolve this issue.

The present findings indicate that both H₂O₂-induced coronary relaxation and stimulation of BK_Ca channel activity in single myocytes are mediated via lipoxygenase metabolites of AA. The present study provides substantial pharmacological support for this conclusion, but it is recognized that few of the agents employed are completely selective. However, these pharmacological studies are further supported by direct biochemical measurement of H₂O₂-stimulated [³H]AA release from coronary myocytes and the fact that exogenous AA mimics the effect of H₂O₂ on BK_Ca channel activity. Furthermore, we had demonstrated previously that H₂O₂ induces contraction of porcine coronary arteries via cyclooxygenase metabolites of AA (2). Therefore, we conclude that H₂O₂-induced stimulation of BK_Ca channel activity involves AA metabolism, most likely via lipoxygenase activity. Although involvement of lipoxygenase metabolites has not been established unequivocally, it is known that lipoxygenase metabolism underlies AA stimulation of BK_Ca channel activity in neuroendocrine (9) and chromaffin (38) cells. Other studies of vascular smooth muscle indicate that metabolites of cytochrome P-450 epoxygenase, possibly acting as endothelium-derived hyperpolarizing factors, stimulate BK_Ca channel activity in portal vein, caudal artery, aorta, and coronary artery (16); however, such epoxyeicosatrienoic acids are not synthesized by smooth muscle cells (5). Furthermore, we found that inhibitors of the P-450 or cyclooxygenase cascades did not affect H₂O₂-induced coronary relaxation (Fig. 8). Thus the present findings, as well as and those from other studies, indicate that there are pleotropic effects of AA metabolism on vascular smooth muscle excitability, and, in some cases, even divergent responses in the same vessel. For example, we propose that a single signaling pathway, stimulation of AA metabolism, can now account for the interesting observation that H₂O₂ can induce either relaxation or contraction (2) in the same artery. Further studies are required to identify a specific lipoxygenase(s) that mediates the response to H₂O₂.

We propose that H₂O₂ can either contract or relax porcine coronary arteries by stimulating the PLA₂/AA transduction pathway. Coronary relaxation mediated by BK_Ca channel activity occurs when lipoxygenase metabolism of AA predominates, but contraction can be induced when AA is metabolized primarily by cyclooxygenase. Because H₂O₂ affects different arteries in different ways, it may be premature to assume that H₂O₂ must be a pathogenic agent in the etiology of essential hypertension, simply because plasma levels are elevated (22). Elevated H₂O₂ might also be compensatory under certain conditions. In light of our findings (and those of others), we propose that a key to understanding the role of H₂O₂ in regulating vascular smooth muscle tone depends on the disposition of AA metabolism. Because circulating and/or tissue levels of free AA or other fatty acids increase during pathological states (e.g., ischemia, stroke, diabetes), understanding the molecular basis of how these compounds affect vascular smooth muscle and other cells will continue to be an important emphasis of future studies.