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(Circulation Research. 1997;80:877-884.)
© 1997 American Heart Association, Inc.

Articles

Epoxyeicosatrienoic Acids Activate K⁺ Channels in Coronary Smooth Muscle Through a Guanine Nucleotide Binding Protein

Pin-Lan Li, , William B. Campbell

From the Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee.

	Abstract

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Abstract Epoxyeicosatrienoic acids (EETs) are endothelium-derived arachidonic acid metabolites of cytochrome P450. They dilate coronary arteries, open K⁺ channels, and hyperpolarize vascular smooth muscles. However, the mechanisms of these smooth muscle actions remain unknown. This study examined the effects of EETs on the large-conductance Ca²⁺-activated K⁺ channel (K_Ca) in smooth muscle cells of small bovine coronary arteries. In cell-attached patch-clamp experiments, 11,12-EET produced a 0.5- to 10-fold increase in the activity of the K_Ca channels when added in concentrations of 1, 10, and 100 nmol/L. In the inside-out excised membrane patch mode, 11,12-EET was without effect on the activity of the K_Ca channel unless GTP (0.5 mmol/L) or GTP and ATP (1 mmol/L) were added to the bath solution. In the presence of GTP and ATP, the increase in the K_Ca channel activity with 11,12-EET in inside-out patches was comparable to that in cell-attached patches. This effect of 11,12-EET in inside-out patches was blocked by the addition of GDP-ß-S (100 µmol/L). In outside-out patches, 11,12-EET also increased the K_Ca channel activity when GTP and ATP were added to the pipette solution. The addition of a specific anti-G_S antibody (100 nmol/L) in the pipette solution completely blocked the activation of the K_Ca channels induced by 11,12-EET. An anti-Gß or anti-G_i antibody was without effect. We conclude that 11,12-EET activates the K_Ca channels by a G_S-mediated mechanism. This mechanism contributes to the effects of EETs as endothelium-derived hyperpolarizing factors to hyperpolarize and relax arterial smooth muscle.

Key Words: endothelium-derived hyperpolarizing factor • patch clamp • K⁺ channel • eicosanoid • G protein

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent studies have indicated that coronary endothelial cells synthesize EETs, a family of cytochrome P450 epoxygenase metabolites of arachidonic acid.^{1 2 3 4 5} Likewise, the intima of coronary blood vessels possesses cytochrome P450 monooxygenase activity.^{6 7} In vitro studies have demonstrated that EETs dilate coronary arteries^{4 5 8 9} as well as renal, cerebral, pial, and caudal arteries.^{10 11 12 13} We have found that all four regioisomeric EETs relax coronary arteries in nanomolar concentrations in vitro.^{4 5 8} EETs activate K⁺ channels and hyperpolarize vascular smooth muscle in similar concentrations.^{8 10 11 14} These studies suggest that EETs are excellent candidates to serve as endothelium-dependent vasodilators that hyperpolarize vascular smooth muscle. In this regard, we and other investigators have reported that inhibition of cytochrome P450 blocks the endothelium-dependent vasorelaxation to arachidonic acid, whereas induction of cytochrome P450 monooxygenase enhances the vasodilation to arachidonic acid.^{3 7 8 9} EETs appear to mediate a portion of the endothelium-dependent relaxation to acetylcholine and bradykinin in coronary arteries.^{7 8 9 15 16} Cytochrome P450 inhibitors blocked acetylcholine-induced endothelium-dependent hyperpolarization and relaxation of coronary vascular smooth muscle.^{8 16 17} In addition, acetylcholine stimulates EET release.⁸ These studies led us to propose that EETs serve as EDHFs in coronary arteries.⁸

The mechanism by which EETs dilate coronary arteries and hyperpolarize vascular smooth muscle remains unknown. Recent studies have indicated that EETs activate a K_Ca channel in vascular smooth muscle cells.^{8 10 14} These results further support the hypothesis that EETs serve as EDHFs, since the K_Ca channels are thought to mediate the effect of EDHF.^{18 19} The purpose of the present study was to examine the effect of 11,12-EET on the activity of large-conductance K_Ca channels in vascular smooth muscle cells isolated from small bovine coronary arteries and to determine the mechanism by which 11,12-EET activates these channels.

	Materials and Methods

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Isolation of Vascular Smooth Muscle Cells From Small Coronary Arteries
Bovine hearts were obtained from a local slaughterhouse. A branch of the coronary artery was cannulated and filled with 10 to 20 mL of ice-cold 3% Evan's blue in 50 mmol/L sodium phosphate containing 0.9% sodium chloride at pH 7.4 (PSS) and 6% albumin. Then the heart was dissected into 2x3x1-cm pieces and sliced into 300-µm-thick tissue sections. Small coronary arteries stained with Evans blue were identified under a dissecting stereomicroscope. These arteries were microdissected, pooled, and stored in ice-cold PSS. The dissected small coronary arteries were first incubated for 30 minutes at 37°C with collagenase type II (340 U/mL) (Worthington), elastase (15 U/mL) (Worthington), dithiothreitol (1 mg/mL), and soybean trypsin inhibitor (1 mg/mL) in HEPES buffer consisting of (mmol/L) NaCl 119, KCl 4.7, CaCl₂ 0.05, MgCl₂ 1, glucose 5, and HEPES 10 (pH 7.4). The digested tissue was then agitated with a glass pipette to free the vascular smooth muscle cells, and the supernatant was collected. Remaining tissue was further digested with fresh enzyme solution, and the supernatant was collected at 5-minute intervals for an additional 15 minutes. The supernatants were pooled and diluted 1:10 with HEPES buffer and stored at 4°C until used.

Current Recordings
Single-channel K⁺ currents were recorded using the patch-clamp technique as described by Hamill et al.²⁰ Cell-attached, inside-out, and outside-out configurations were used to identify the K_Ca channels and to determine the effect of 11,12-EET on the K⁺ currents in vascular smooth muscle cells. Patch pipettes were made from borosilicate glass capillaries that were pulled with a two-stage micropipette puller (PC-87, Sutter) and heat-polished with a microforge (MF-90, Narishige). The pipettes had tip resistances of 8 to 10 M for single-channel recordings when filled with 145 mmol/L KCl solution. Smooth muscle cells were placed in a 1-mL perfusion chamber mounted on the stage of a Nikon inverted microscope. After the tip of the pipette was positioned on a cell, a high-resistance seal (5 to 15 G) was formed between the pipette tip and the cell membrane by applying a light suction. The activity of K⁺ channels in the membrane spanning the pipette tip was recorded. These measurements represented the cell-attached mode. Inside-out membrane patches were excised by lifting the pipette membrane complex to the air/solution interface. Outside-out membrane patches were obtained by withdrawing the pipette tip from the cell after establishment of the whole-cell configuration, in which the membrane within the pipette was disrupted by a large pulse of suction.

An EPC-7 patch-clamp amplifier (List Biological Laboratories, Inc) was used to record single-channel currents. The amplifier output signals were filtered at 1 KHz with an eight-pole Bessel filter (Frequency Devices Inc). Currents were digitized at a sampling rate of 3 kHz and stored on the hard disk of a Gateway 486 DS66 computer for off-line analysis. Data acquisition and analysis were performed with pClamp software (version 5.7.1, Axon Instruments). Average channel activity (NP_o) in patches was determined from recordings of several minutes by the following:

where N is the maximal number of channels observed in conditions producing high levels of P_o, T is the duration of the recording, and t_j is the time with j=1,2 ... N channels opening.

Solutions
For single-channel recordings in the cell-attached mode, the bath solution contained (mmol/L) KCl 145, CaCl₂ 1.8, MgCl₂ 1.1, glucose 10, and HEPES 5 (pH 7.4), and the pipette solution contained (mmol/L) KCl 145, CaCl₂ 1.8, MgCl₂ 1.1, and HEPES 5 (pH 7.4). For single-channel recordings using the inside-out excised membrane patch, the bath solution contained (mmol/L) KCl 145, MgCl₂ 1.1, HEPES 10, and EGTA 2, along with 300 nmol/L ionized Ca²⁺ (pH 7.2). To determine the sensitivity of the channels to cytosolic Ca²⁺, the concentration of ionized Ca²⁺ in the bath solution was varied from 10^-7 to 10^-6 and then to 10^-5 mol/L. Ca²⁺ concentration was estimated by a computer program²¹ and was confirmed by measuring the free Ca²⁺ concentration in the solution using fura 2 (Molecular Probes Co) with a dual-wavelength spectrofluorometer (Perkin-Elmer). The pipette solution contained (mmol/L) KCl 145, CaCl₂ 1.8, MgCl₂ 1.1, and HEPES 10 (pH 7.4). For single-channel recordings in the outside-out configuration, the bath solution contained (mmol/L) KCl 145, CaCl₂ 1.8, MgCl₂ 1.1, glucose 10, and HEPES 10 (pH 7.4), and the pipette solution contained (mmol/L) KCl 145, MgCl₂ 1.1, HEPES 10, and EGTA 2, along with 100 nmol/L ionized Ca²⁺ (pH 7.2). All patch-clamp experiments were performed at room temperature, 20°C.

Identification of the K_Ca Channel in Small Bovine Coronary Arteries
To establish current-voltage relations of the K_Ca channel, inside-out patches were exposed to symmetrical KCl (145 mmol/L) solutions, and single-channel currents were recorded while membrane potential was varied from -60 to +60 mV in steps of 20 mV. K⁺ selectivity of the single-channel current was determined by reducing K⁺ concentration in the pipette solution to 5.4 mmol/L (n=5). By changing the concentration of ionized Ca²⁺ from 10^-7 to 10^-5 mol/L on the cytosolic side of inside-out patches, the sensitivity of this K_Ca channel to intracellular Ca²⁺ concentration was examined (n=8). The effect of TEA (Sigma) (n=4) and IBX (Research Biochemicals Inc) (n=5) on single K⁺ channels was examined using outside-out excised membrane patches. TEA was added to bath solution at concentrations of 0.1, 0.3, and 1 mmol/L. IBX was added to bath solution at a concentration of 100 nmol/L.

Patch-Clamp Studies on the Effect of 11,12-EET
In cell-attached patches, symmetrical KCl (145 mmol/L) solutions were used to null the membrane potential of the single smooth muscle cell to near 0 mV. A 3-minute control recording at a membrane potential of +40 mV was obtained after a tight seal was established. Then the bath solution was rapidly changed by flushing the perfusion chamber with 10 mL of the same solution containing 11,12-EET (1, 10, or 100 nmol/L, n=7), 12-HETE (10 or 100 nmol/L, n=5), or 20-HETE (10 or 100 nmol/L, n=6), and a series of 3-minute recordings was obtained. To examine the interaction of cholera toxin and 11,12-EET on the activity of the K_Ca channel, 100 ng/mL cholera toxin was included in the pipette solution (n=8).

The excised-patch modes were used to further determine the mechanisms for the effect of 11,12-EET on the activity of the K_Ca channels. After inside-out patches were established, a 3-minute control recording was obtained at a membrane potential of +40 mV. Then the bath solution was rapidly changed by flushing the perfusion chamber with 5 to 10 mL of the same solution containing 1, 10, or 100 nmol/L 11,12-EET (n=6) with 0.5 mmol/L GTP and 1 mmol/L ATP, and a second successive 3-minute recording was obtained.

In some experiments, the concentration of ionized Ca²⁺ on the cytosolic side of inside-out patches was changed from 10^-7 to 10^-5 mol/L in the presence and absence of 11,12-EET (100 nmol/L), and the K_Ca channel current was recorded for 3 minutes at each Ca²⁺ concentration (n=5).

The excised inside-out patch mode was used to determine the effect of GDP-ß-S (100 µmol/L) on 11,12-EET–induced activation of the K⁺ channel (n=6). GDP-ß-S (100 µmol/L) and 11,12-EET were added to the GTP/ATP bath solution. The outside-out patch mode was used to examine the effects of anti-G_S (n=7), anti-Gß (n=8), and anti-G_i (n=4) antibody (New England Nuclear and Signal Transduction, Inc) and rabbit IgG (n=4). Antibodies at concentrations of 10 or 100 nmol/L were added to the pipette solution containing GTP/ATP.²²

Western Blots of G_S Protein
The dissected coronary arteries were cut into very small pieces and homogenized with a glass homogenizer in ice-cold HEPES buffer containing 25 mmol/L sodium HEPES, 1 mmol/L EDTA, and 100 µmol/L phenylmethylsulfonyl fluoride. The homogenate containing 30 µg protein was incubated with 11,12-EET at concentrations of 1 nmol/L to 10 µmol/L for 30 minutes and then subjected to 12% SDS-PAGE at 200 V for 65 minutes (Bio-Rad).²³ The proteins were electrophoretically transferred onto a nitrocellulose membrane. The membrane was washed and probed with a 1:1000 dilution of a specific anti-G_S antibody (New England Nuclear). The ECL detection kit (Amersham) was used to detect the specific G_S protein bands as described by the manufacturer.

Statistics
Data are presented as mean±SEM. The significance of the differences in mean values between and within multiple groups was examined using an ANOVA for repeated measures, followed by a Duncan's multiple-range test. Student's t test was used to evaluate statistical significance of differences between two paired observations. Single-channel conductances were fit by least-squares linear regression or by using the Goldman-Hodgkin-Katz constant field equation. A value of P<.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of K_Ca Currents in Small Bovine Coronary Arteries
The K⁺ channel activity was characterized using inside-out and outside-out excised membrane patches that were exposed to symmetrical KCl solutions (145 mmol/L) to enhance single-channel conductances. Unitary K⁺ currents were detected predominantly at membrane potentials from -60 to +60 mV in inside-out patches (Fig 1A). The current-voltage relationship for this channel was linear between -60 to +60 mV, and mean slope conductance was 256.3±5 pS, with a reversal potential of 0 mV. When the K⁺ concentration in the pipette was reduced to 5.4 mmol/L, the reversal potential shifted in a manner predicted by the Nernst equation for K⁺. This shift in reversal potential in response to changes in K⁺ gradient across the membrane suggests that this channel is selective for K⁺ (Fig 1B). This large-conductance K⁺ current was activated by membrane depolarization. At the resting membrane potential, the activity of this K⁺ channel was low, with a mean open probability (NP_o) of 0.002±0.0001. When the membrane patch was depolarized to +60 mV, NP_o was increased to 0.15±0.02.

View larger version (17K): [in this window] [in a new window]   Figure 1. Characterization of the KCa channel in vascular smooth muscle cells isolated from small bovine coronary arteries. A, A representative recording of a large-conductance K+ channel recorded from an inside-out excised membrane patch at membrane potential ranging from -60 to +60 mV. c indicates closed. B, Current-voltage relations for large-conductance K+ channel current recorded from inside-out patches of smooth muscle cells using symmetrical 145 mmol/L KCl solution and after lowering KCl in the pipette to 5.4 mmol/L.

The effects of changes in the cytosolic Ca²⁺ concentration on the activity of this channel were examined using inside-out patches. When the Ca²⁺ concentration on the cytosolic side of the membrane patch was increased from 10^-7 to 10^-5 mol/L, the activity of K⁺ channel increased markedly. At a cytosolic Ca²⁺ concentration of 10^-7 mol/L and membrane potential of +40 mV, the NP_o of this K⁺ channel was 0.02±0.0012. When cytosolic Ca²⁺ concentration was increased to 10^-6 and then to 10^-5 mol/L, the NP_o of this K⁺ channel was increased to 0.04±0.003 and 1.88±0.06, respectively.

TEA and IBX, inhibitors of the K_Ca channel, were studied using the outside-out membrane patch mode. Representative tracings depicting the results of these experiments are presented in Fig 2A. Addition of TEA to the bath produced a concentration-dependent reversible flickery-type blockade of the K⁺ channel. The mean unitary current amplitude of this channel fell from 9.89 pA under control conditions to 6.8, 4.7, and 2.24 pA after 0.1, 0.3, and 1 mmol/L TEA, respectively, was added to bath (Fig 2B). NP_o of this K⁺ channel was not altered by the addition of TEA. Fig 2C presents representative tracings depicting the effect of IBX on this K⁺ channel. IBX (100 nmol/L) decreased NP_o of the channel by 93% when added to the bath (Fig 2D), but it had no effect on the current amplitude of this channel. These data are consistent with this being a large-conductance K_Ca channel.

View larger version (28K): [in this window] [in a new window]   Figure 2. Effect of TEA and iberiotoxin (IBTX) on the KCa channel in outside-out membrane patches of smooth muscle cells isolated from small bovine coronary arteries. TEA was added to the bath so that the external surfaces of outside-out patches were exposed to the indicated concentrations of the drug. A, A representative tracing showing the effect of TEA at various concentrations on the KCa channel current recorded at a membrane potential (Em) of +40 mV. c indicates closed. B, Effect of TEA on current amplitude of the KCa channels. C, A representative recording of the KCa channel before and after IBTX (100 nmol/L) was added to the bath. D, Effect of IBTX on NPo of the KCa channels. *P<.05 vs control (C).

Effect of 11,12-EET on the Activity of the K_Ca Channel in the Cell-Attached Patch Mode
Representative recordings of single-channel K⁺ currents that were recorded in the cell-attached mode before and after the addition of 11,12-EET to the bath are presented in Fig 3A. 11,12-EET caused a concentration-dependent increase of the activity of the K_Ca channel. 11,12-EET at concentrations of 1, 10, and 100 nmol/L produced a 0.5- to 10-fold increase in NP_o of this K_Ca channel (Fig 3B). A significant effect was seen even at the lowest concentration of 11,12-EET studied (1 nmol/L) (P<.05). The amplitude of these channels was unaltered by 11,12-EET even at the highest concentration studied (100 nmol/L) (Fig 3C). When cell membrane potential was changed by adjusting the pipette potential from -20 to -40 and then to -60 mV, the activity of the K_Ca channel was significantly increased as described above, but the effects of 11,12-EET were not altered. 11,12-EET at a concentration of 100 nmol/L produced an 10-fold increase in NP_o of the K_Ca channels in spite of changes in membrane potential from 20 to 60 mV. 12-HETE, a structural analogue of 11,12-EET, had no effect on the activity of the K_Ca channel, and 20-HETE decreased the activity of this K_Ca channel (P<.05) (Table 1).

View larger version (33K): [in this window] [in a new window]   Figure 3. Effect of 11,12-EET on the KCa channel activity in cell-attached patches of smooth muscle cells isolated from small bovine coronary arteries. A, A representative recording of the KCa channel under control conditions and after addition of 1, 10, and 100 nmol/L 11,12-EET to the bath at a membrane potential (Em) of +40 mV. c indicates closed. B, Effect of 11,12-EET on NPo of the KCa channels in smooth muscle cells isolated from small bovine coronary arteries. C, Effect of 11,12-EET on current amplitude of the KCa channels. W indicates washout of the EET. *P<.05 vs control (C).

View this table: [in this window] [in a new window]   Table 1. Effect of 12-HETE and 20-HETE on KCa Channel Activity in Cell-Attached Patches of Coronary Smooth Muscle Cells

Effect of 11,12-EET on the Activity of the K_Ca Channel in the Inside-out Patch Mode
In contrast to the marked effects of 11,12-EET (100 nmol/L) in cell-attached patches, 11,12-EET had no effect on the activity of the K_Ca channel when applied to the internal surface of inside-out excised membrane patches (Table 2). The number of channel openings, mean open time, and the amplitude of these K_Ca channels recorded from inside-out excised membrane patches were not significantly altered when even a high concentration of 11,12-EET (1 µmol/L) was applied to the internal surface of the patch. NP_o was 0.03±0.009 for control and 0.0267±0.01 with 11,12-EET. However, when 0.5 mmol/L GTP and 1 mmol/L ATP were included in the bath solution, 11,12-EET produced a concentration-dependent increase in the K_Ca activity (Fig 4A). 11,12-EET increased the NP_o of these channels to an extent comparable to that in the cell-attached patch mode and in comparable concentrations. The increase in the channel activity was reversed by washing out the 11,12-EET (Fig 4B). In addition, addition of 0.5 mmol/L GTP alone also increased the basal activity of the K_Ca channels by 15% and restored the effect of 11,12-EET in these excised membrane patches to a level comparable to that obtained in the cell-attached patches (Table 2). However, ATP alone had no effect on the basal activity of the K_Ca channels, and 11,12-EET did not stimulate the K_Ca channels in the presence of only ATP.

View this table: [in this window] [in a new window]   Table 2. Effect of 11,12-EET in the Presence and Absence of GTP and ATP on KCa Channel Activity in Inside-out Patches of Coronary Smooth Muscle Cells

View larger version (32K): [in this window] [in a new window]   Figure 4. Effect of 11,12-EET on the activity of the KCa channels in the inside-out patches of smooth muscle cells isolated from small bovine coronary arteries in the presence of GTP and ATP in the bath solution. A, A representative recording of the KCa channel under control conditions and after addition of 1, 10, and 100 nmol/L 11,12-EET to the bath solution containing 0.5 mmol/L GTP and 1 mmol/L ATP (G+A) at a membrane potential (Em) of +40 mV. c indicates closed. B, Effect of 11,12-EET on NPo of the KCa channels in bovine coronary smooth muscle cells. C, Effect of 11,12-EET on current amplitude of the KCa channels. W indicates washout of the EET. *P<.05 vs control (C).

In the presence of GTP and ATP in the bath solution, the activity of the K_Ca channel was also increased in response to the increase in intracellular Ca²⁺ concentration. NP_o of the K_Ca channel was 0.044±0.0013, 0.2995±0.04, and 1.88±0.02 at cytosolic Ca²⁺ concentrations of 10^-7, 10^-6, and 10^-5 mol/L, respectively. When 11,12-EET was added to the bath, Ca²⁺-induced increase in NP_o of the K_Ca channel was not altered. NP_o of the K_Ca channel was 0.275±0.02, 0.832±0.12, and 2.61±0.2 at Ca²⁺ concentrations of 10^-7, 10^-6, and 10^-5 mol/L, respectively. Calculated pCa₅₀ in the presence of 11,12-EET averaged 2.3x10^-6 mol/L, which was not significantly different from 2.6x10^-6 mol/L in the absence of 11,12-EET in the bath solution.

Effect of GDP-ß-S on 11,12-EET–Induced Activation of the K_Ca Channel in the Inside-out Patch Mode
11,12-EET at a concentration of 100 nmol/L produced a 6-fold increase in NP_o of the K_Ca channels in the presence of GTP/ATP. GDP-ß-S reduced NP_o of the K_Ca channels by 16% and completely abolished the effect of 11,12-EET on the activity of these channels (Fig 5A).

View larger version (17K): [in this window] [in a new window]   Figure 5. Effect of GDP-ß-S and antibodies against G protein subunits on the 11,12-EET–induced activation of the KCa channels. A, Effect of 11,12-EET on NPo of the KCa channels in the inside-out patches obtained from bovine coronary smooth muscle cells in the absence and presence of GDP-ß-S. B, Effect of 11,12-EET on NPo of the KCa channels in outside-out patches obtained from bovine coronary smooth muscle cells in the presence of different antibodies. *P<.05 vs control (C).

Effect of Anti-G_S Antibody on 11,12-EET–Induced Increase in the K_Ca Channel Activity in the Outside-out Patch Mode
Addition of 11,12-EET to the bath solution had no effect in the excised outside-out patches in the absence of GTP and ATP in the cytosolic solution: NP_o was 0.15±0.02 in the control condition and 0.167±0.023 with 100 nmol/L EET. When GTP and ATP were included in the pipette solution, 11,12-EET increased markedly the activity of these K_Ca channels. NP_o of the K_Ca channels was increased from 0.15±0.02 to 0.52±0.01, but channel amplitude was not altered when 11,12-EET (100 nmol/L) was added to the bath.

The effects of antibodies against G_S, G_i, or Gß on the response of the K_Ca channel to 11,12-EET were examined using this outside-out patch mode. When an antibody against G_S at a concentration of 100 nmol/L was included in the pipette solution, addition of 11,12-EET (100 nmol/L) to the bath failed to alter the activity of the K_Ca channels. This inhibitory effect of the anti-G_S antibody was lost when the antibody concentration was decreased to 10 nmol/L or when it was boiled for 10 minutes. When an anti-Gß at a concentration of 100 nmol/L, which completely blocks the stimulatory effect of purified Gß subunits on the K_Ca channels (data not shown), was substituted for anti-G_S antibody in the pipette solution, 11,12-EET still increased the activity of the K_Ca channel to a comparable extent. Anti-G_i antibody or rabbit IgG (100 nmol/L) had no effect on 11,12-EET–induced increase in the K_Ca channel activity. Changes in NP_o of the K_Ca channels induced by 11,12-EET in the presence of different antibodies in the pipette solution are summarized in Fig 5B. Only anti-G_S antibody inhibited the increase in NP_o of the K_Ca channel induced by 11,12-EET.

Effect of Cholera Toxin on the Activity of the K_Ca Channel
Cholera toxin at a concentration of 100 ng/mL increased the activity of the K_Ca channels by 6-fold (Fig 6A). In the presence of cholera toxin in the pipette solution, 11,12-EET did not further increase the K_Ca channel activity. NP_o of the K_Ca channel was 0.1033±0.03 and 0.125±0.01 before and after the addition of 11,12-EET in the bath solution, respectively (Fig 6B). Cholera toxin did not alter the current amplitude of the K_Ca channels (Fig 6C).

View larger version (29K): [in this window] [in a new window]   Figure 6. Effect of cholera toxin (CTX) on the activity of the KCa channels in the cell-attached patches of smooth muscle cells isolated from small coronary arteries. A, A representative recording of the KCa channel under control conditions and after addition of 11,12-EET or vehicle in the presence of 100 ng/mL CTX in the pipette solution at a membrane potential (Em) of +40 mV. c indicates closed. B, Effect of CTX on NPo of the KCa channels in smooth muscle cells isolated from small bovine coronary arteries. C, Effect of CTX on current amplitude of the KCa channels. *P<.05 vs control (C).

Presence of G_S in Coronary Smooth Muscle Cells
Based on molecular weight and reaction with a specific anti-G_S antibody, there is a smooth muscle cell protein that appears to be the subunit of G_S. Western blot of the smooth muscle homogenate with anti-G_S antibody gave two protein bands of 52 and 45 kD. Similar results were obtained with the G_S standard. 11,12-EET had no effect on the electrophoretic migration of G_S.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, the K⁺ channel activity was characterized in vascular smooth muscle cells isolated from small bovine coronary arteries using the patch-clamp technique, and the effects of 11,12-EET on the activity of K⁺ channels were examined. Using single-channel recording modes, we found that the dominant K⁺ channel in vascular smooth muscle cells isolated from small bovine coronary arteries was the large-conductance (256.3-pS) K_Ca channel. This finding is consistent with previous reports that the K_Ca channel is the most active channel found in vascular smooth muscle cells isolated from the coronary artery of the rabbit and dog.^{24 25}

Recent studies have indicated that the vasodilatory response to EETs is associated with activation of the K_Ca channels in vascular smooth muscle cells isolated from cat cerebral arteries, rabbit portal vein, rat caudal arteries, and guinea pig aorta.^{10 14} In these studies, vascular smooth muscle cells were isolated from large arteries, and EET concentrations of 0.3 to 10 µmol/L were required to activate these channels. In the present study, using the cell-attached mode for single-channel recording, we found that the addition of 11,12-EET markedly enhanced the frequency of opening of the K_Ca channel in vascular smooth muscle cells in concentrations as low as 1 nmol/L. These data suggest that vascular smooth muscle cells from small coronary arteries are more sensitive to the effect of EET. In this regard, we have recently found that smaller vessels are more sensitive than large vessels to the vasorelaxant effect of EETs in isolated bovine coronary arterial rings.⁸ Why vascular smooth muscle cells isolated from small coronary arteries are more sensitive to 11,12-EET than those obtained from other species and vascular beds remains to be determined. This may reflect species differences or differences in vascular reactivity between vascular beds. Another possibility is that vascular smooth muscle cells from small resistance arteries like those studied in the present study are inherently more sensitive to the stimulatory effect of 11,12-EET on the K_Ca channels.

EETs are a series of metabolites of arachidonic acid synthesized by a cytochrome P450 epoxygenase.²⁶ Although the regiospecific effect of the EETs was not determined in the present study, previous studies have demonstrated that the four regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) have similar stimulatory effects on the activity of the K_Ca channels in vascular smooth muscle cells isolated from coronary and caudal arteries and aorta.^{8 14} As a result, we studied only 11,12-EET as a prototype. However, 12-HETE, a structural analogue of 11,12-EET, and 20-HETE, another cytochrome P450 metabolite of arachidonic acid, did not activate the K_Ca channels. In contrast, 20-HETE markedly reduced the activity of the K_Ca channels. These results indicate that the epoxide group of the EETs is essential for activation of the K_Ca channels.

To determine the mechanism by which 11,12-EET increases the activity of the K_Ca channels in smooth muscle cells, excised-membrane patches were used to examine the effects of 11,12-EET on the activity of the K_Ca channels. In contrast to the results obtained in cell-attached patch mode, 11,12-EET had no effect on the activity of the K_Ca channel when added to the cytoplasmic surface of excised inside-out or outside-out membrane patches. These results suggest that 11,12-EET does not directly act on the K_Ca channels but that some cytosolic component or second messenger system is required to alter the K_Ca channel activity. These results are consistent with previous reports of others.^{11 14}

To further determine the cellular mechanism mediating the effect of 11,12-EET on the K_Ca channels, a series of experiments was performed using excised-membrane patches to examine whether the stimulatory effect of 11,12-EET on the K_Ca channel could be reconstituted. Surprisingly, when GTP alone or GTP and ATP were included in the cytosolic solution in inside-out or outside-out patches, the effect of 11,12-EET on the K_Ca channel activity was completely restored. This finding suggested that 11,12-EET–induced activation of the K_Ca channel is a GTP-dependent mechanism and may involve GTP binding proteins. By using the inside-out patch mode, the effect of a GTP binding protein inhibitor, GDP-ß-S, was examined on 11,12-EET–induced activation of the K_Ca channels. GDP-ß-S abolished the effect of 11,12-EET on the activity of the K_Ca channels. These findings further suggest that GTP and a GTP binding protein may contribute to 11,12-EET-induced activation of the K_Ca channel.

Since the GTP binding protein G_S has been reported to participate in the regulation of the K_Ca channel activity,^{27 28} activation of G_S may be involved in the effect of 11,12-EET on the activity of the K_Ca channel. To test this hypothesis, an anti-G_S antibody was used to block G_S activity. Addition of an anti-G_S antibody to the cytosolic solution blocked the effect of 11,12-EET on the activity of the K_Ca channel. Since 11,12-EET was added to the external cell membrane surface and the antibody to the cytosolic surface, it is unlikely that blockade was due to a direct binding of 11,12-EET to the antibody. In addition, the addition of an anti-G_i antibody, anti-Gß antibody, or rabbit IgG to the cytosolic solution in the pipette had no effect on activation of the K_Ca channels by 11,12-EET. Therefore, we conclude that a specific blockade of G_S abolishes the effect of 11,12-EET on the activity of the K_Ca channels. Our findings support the view that 11,12-EET activates a G protein, likely G_S, and, subsequently, the K_Ca channels. Since only anti-G_S antibody blocked 11,12-EET–induced activation of the K_Ca channels, the data imply that G_S subunit is implicated in the effect of EET.

G_S may regulate the K_Ca channel via two independent mechanisms.²⁹ G_S can activate adenylyl cyclase and promote cAMP accumulation, resulting in PKA-dependent phosphorylation of the channel or some proteins coupled to the channel.^{30 31 32} Alternatively, the G_S may have a direct action on the channel or a closely associated protein.²⁸ With respect to the cAMP-PKA–dependent mechanism, an increase in the production of cAMP in response to 11,12-EET would be required. However, in a recent study, we found that the vasodilator effect of 11,12-EET is not associated with an increase in the tissue content of cAMP and cGMP.⁸ Furthermore, ATP would be required for PKA to phosphorylate the channel. However, GTP alone can restore the effect of 11,12-EET on the channel, so ATP is not required. These data suggest that the cAMP-PKA–dependent mechanism is not involved. Therefore, the direct action of G_S on the K_Ca channel is a likely mechanism for the effect of 11,12-EET. It has been reported that this membrane-delimited action of G_S is a ubiquitous mechanism for regulating K⁺ and Ca²⁺ channels.^{33 34} Although the significance of this action of G_S in the gating of the K_Ca channels has not been characterized, previous studies on the regulation of Ca²⁺ channels have indicated that this membrane-delimited pathway for G_S is far faster (<1 second) than the cytoplasmic cAMP pathway (30 seconds).^{35 36} This membrane action of G_S has been shown to enhance the effect of agonists on ion channels.^{37 38} However, the detailed coupling mechanisms between G_S and the K_Ca channels for this membrane-delimited pathway are not yet known. G_S might act upon the channel proteins either directly, without requiring any other membrane component, or indirectly, via intermediate membrane-associated effectors. Further elucidation of the coupling mechanism between G_S and the K_Ca channel in coronary vascular smooth muscle will require functional reconstitution of the interacting proteins in an artificial membrane system. On the other hand, our results cannot exclude the possibility that second messengers independent of cAMP and cGMP may participate in the regulation of the activity of the K_Ca channel or mediate the effect of 11,12-EET in the intact cells. This dual modulation of the K_Ca channel through membrane-delimited action of G_S and some cytosolic signaling molecules has been reported by others.^{28 29}

There is evidence for a high-affinity binding site for 14(R),15(S)-EET in guinea pig mononuclear cell membranes, suggesting that there may be EET receptors.³⁹ Whether there is a specific receptor for 11,12-EET in the vascular smooth muscle cell membrane remains to be determined. However, our results raise questions about this possibility, since addition of 11,12-EET to the cytosolic solution in excised inside-out membrane patches also increased the activity of the K_Ca channels in the presence of ATP and GTP. It is, however, possible that 11,12-EET could diffuse across the membrane and activate a receptor on the external surface. Nevertheless, the findings that the effects of 11,12-EET on the activity of the K_Ca channels were reversible in the excised patches but not in the cell-attached patches suggest that 11,12-EET may activate G_S on the cytosolic side of the membrane and that G_S may be a target for the effect of lipid-soluble mediators that are slowly washed out of intact cells. This direct effect of 11,12-EET on the G_S activity independent of the receptors on the cell surface may represent a unique mechanism mediating the action of lipid-soluble mediators in vascular smooth muscle cells, by which activation of G_S only produces a membrane-delimited effect but does not stimulate the production of cAMP. Alternatively, 11,12-EET may have a second action in intact cells that is not observed in inside-out patches. This second action must be slowly reversible. A recent study has indicated that EETs stimulated the endogenous ADP-ribosylation of a 52-kD protein in the liver.⁴⁰ It remains to be determined whether this endogenous ADP-ribosylation is activated by 11,12-EET in coronary arterial smooth muscle and what the relationship is between EET-activated endogenous ADP-ribosylation and the activity of G_S.

In summary, 11,12-EET stimulates activation of the K_Ca channel in small bovine coronary arteries. This increase in the activity of the K_Ca channel appears to be due to activation of a GTP binding protein, probably G_S. Activation of the K_Ca channel appears to contribute to the vasodilator effect of 11,12-EET by hyperpolarizing the vascular smooth muscle.⁸ EETs represent EDHFs.

	Selected Abbreviations and Acronyms

 

EDHF = endothelium-derived hyperpolarization factor EET = epoxyeicosatrienoic acid IBX = iberiotoxin KCa channel = Ca2+-activated K+ channel NPo = open-state probability of N channels PKA = protein kinase A TEA = tetraethylammonium chloride

	Acknowledgments

 
This study was supported by grants from the National Heart, Lung, and Blood Institute (HL-51055 and HL-57244) and the American Heart Association, Wisconsin Affiliate, Inc (95-GB-52). The authors thank Gretchen Barg for her secretarial assistance and Phillip F. Pratt and Dr William S. Edgemond for providing the 11,12-EET.

	Footnotes

 
Reprint requests to Pin-Lan Li, MD, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.

Received July 8, 1996; accepted March 27, 1997.

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				D. Ye, D. Zhang, C. Oltman, K. Dellsperger, H.-C. Lee, and M. VanRollins Cytochrome P-450 Epoxygenase Metabolites of Docosahexaenoate Potently Dilate Coronary Arterioles by Activating Large-Conductance Calcium-Activated Potassium Channels J. Pharmacol. Exp. Ther., November 1, 2002; 303(2): 768 - 776. [Abstract] [Full Text] [PDF]

				T. Lu, M. VanRollins, and H.-C. Lee Stereospecific Activation of Cardiac ATP-Sensitive K+ Channels by Epoxyeicosatrienoic Acids: A Structural Determinant Study Mol. Pharmacol., November 1, 2002; 62(5): 1076 - 1083. [Abstract] [Full Text] [PDF]

				M. Fuloria, T. K. Smith, and J. L. Aschner Role of 5,6-epoxyeicosatrienoic acid in the regulation of newborn piglet pulmonary vascular tone Am J Physiol Lung Cell Mol Physiol, August 1, 2002; 283(2): L383 - L389. [Abstract] [Full Text] [PDF]

				K. M. Gauthier, C. Deeter, U. M. Krishna, Y. K. Reddy, M. Bondlela, J.R. Falck, and W. B. Campbell 14,15-Epoxyeicosa-5(Z)-enoic Acid: A Selective Epoxyeicosatrienoic Acid Antagonist That Inhibits Endothelium-Dependent Hyperpolarization and Relaxation in Coronary Arteries Circ. Res., May 17, 2002; 90(9): 1028 - 1036. [Abstract] [Full Text] [PDF]

				M. Potente, U. R. Michaelis, B. Fisslthaler, R. Busse, and I. Fleming Cytochrome P450 2C9-induced Endothelial Cell Proliferation Involves Induction of Mitogen-activated Protein (MAP) Kinase Phosphatase-1, Inhibition of the c-Jun N-terminal Kinase, and Up-regulation of Cyclin D1 J. Biol. Chem., May 3, 2002; 277(18): 15671 - 15676. [Abstract] [Full Text] [PDF]

				J.-K. Chen, J. Capdevila, and R. C. Harris Heparin-binding EGF-like growth factor mediates the biological effects of P450 arachidonate epoxygenase metabolites in epithelial cells PNAS, April 30, 2002; 99(9): 6029 - 6034. [Abstract] [Full Text] [PDF]

				B. Lauterbach, E. Barbosa-Sicard, M.-H. Wang, H. Honeck, E. Kargel, J. Theuer, M. L. Schwartzman, H. Haller, F. C. Luft, M. Gollasch, et al. Cytochrome P450-Dependent Eicosapentaenoic Acid Metabolites Are Novel BK Channel Activators Hypertension, February 1, 2002; 39(2): 609 - 613. [Abstract] [Full Text] [PDF]

				R. J. Roman P-450 Metabolites of Arachidonic Acid in the Control of Cardiovascular Function Physiol Rev, January 1, 2002; 82(1): 131 - 185. [Abstract] [Full Text] [PDF]

				W. Zou, Q. Yang, A. P.C. Yim, and G.-W. He Epoxyeicosatrienoic acids (EET11,12) may partially restore endothelium-derived hyperpolarizing factor-mediated function in coronary microarteries Ann. Thorac. Surg., December 1, 2001; 72(6): 1970 - 1976. [Abstract] [Full Text] [PDF]

				I. Fleming Cytochrome P450 and Vascular Homeostasis Circ. Res., October 26, 2001; 89(9): 753 - 762. [Abstract] [Full Text] [PDF]

				W. M. Chilian and R. Koshida EDHF and NO: Different Pathways for Production--Similar Actions Circ. Res., October 12, 2001; 89(8): 648 - 649. [Full Text] [PDF]

				A. W. Miller, C. Dimitropoulou, G. Han, R. E. White, D. W. Busija, and G. O. Carrier Epoxyeicosatrienoic acid-induced relaxation is impaired in insulin resistance Am J Physiol Heart Circ Physiol, October 1, 2001; 281(4): H1524 - H1531. [Abstract] [Full Text] [PDF]

				T. Lu, P. V G Katakam, M. VanRollins, N. L Weintraub, A. A Spector, and H.-C. Lee Dihydroxyeicosatrienoic acids are potent activators of Ca2+-activated K+ channels in isolated rat coronary arterial myocytes J. Physiol., August 1, 2001; 534(3): 651 - 667. [Abstract] [Full Text] [PDF]

				Y. Zhang, C. L. Oltman, T. Lu, H.-C. Lee, K. C. Dellsperger, and M. VanRollins EET homologs potently dilate coronary microvessels and activate BKCa channels Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2430 - H2440. [Abstract] [Full Text] [PDF]

				C. Benoit, B. Renaudon, D. Salvail, and E. Rousseau EETs relax airway smooth muscle via an EpDHF effect: BKCa channel activation and hyperpolarization Am J Physiol Lung Cell Mol Physiol, May 1, 2001; 280(5): L965 - L973. [Abstract] [Full Text] [PDF]

				P. F. Pratt, P. Li, C. J. Hillard, J. Kurian, and W. B. Campbell Endothelium-independent, ouabain-sensitive relaxation of bovine coronary arteries by EETs Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1113 - H1121. [Abstract] [Full Text] [PDF]

				S. I. Pomposiello, M. A. Carroll, J. R. Falck, and J. C. McGiff Epoxyeicosatrienoic Acid-Mediated Renal Vasodilation to Arachidonic Acid Is Enhanced in SHR Hypertension, March 1, 2001; 37(3): 887 - 893. [Abstract] [Full Text] [PDF]

				M. H. Zink, C. L. Oltman, T. Lu, P. V. G. Katakam, T. L. Kaduce, H.-C. Lee, K. C. Dellsperger, A. A. Spector, P. R. Myers, and N. L. Weintraub 12-Lipoxygenase in porcine coronary microcirculation: implications for coronary vasoregulation Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H693 - H704. [Abstract] [Full Text] [PDF]

				R. Gu, Y. Wei, H. Jiang, M. Balazy, and W. Wang Role of 20-HETE in mediating the effect of dietary K intake on the apical K channels in the mTAL Am J Physiol Renal Physiol, February 1, 2001; 280(2): F223 - F230. [Abstract] [Full Text] [PDF]

				M. Fukao, H. S. Mason, J. L. Kenyon, B. Horowitz, and K. D. Keef Regulation of BKca Channels Expressed in Human Embryonic Kidney 293 Cells by Epoxyeicosatrienoic Acid Mol. Pharmacol., January 1, 2001; 59(1): 16 - 23. [Abstract] [Full Text]

				D. G. Welsh and S. S. Segal Role of EDHF in conduction of vasodilation along hamster cheek pouch arterioles in vivo Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1832 - H1839. [Abstract] [Full Text] [PDF]

				J. H. Capdevila, J. R. Falck, and R. C. Harris Cytochrome P450 and arachidonic acid bioactivation: molecular and functional properties of the arachidonate monooxygenase J. Lipid Res., February 1, 2000; 41(2): 163 - 181. [Abstract] [Full Text]

				S.-S. BOLZ, B. FISSLTHALER, S. PIEPERHOFF, C. DE WIT, I. FLEMING, R. BUSSE, and U. POHL Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca2+ changes and dilation in isolated resistance arteries FASEB J, February 1, 2000; 14(2): 255 - 260. [Abstract] [Full Text]

				J. E. HUNGERFORD, W. C. SESSA, and S. S. SEGAL Vasomotor control in arterioles of the mouse cremaster muscle FASEB J, January 1, 2000; 14(1): 197 - 207. [Abstract] [Full Text]

				N. L. Weintraub, X. Fang, T. L. Kaduce, M. VanRollins, P. Chatterjee, and A. A. Spector Epoxide hydrolases regulate epoxyeicosatrienoic acid incorporation into coronary endothelial phospholipids Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H2098 - H2108. [Abstract] [Full Text] [PDF]

				J. C. McGiff and J. Quilley 20-HETE and the kidney: resolution of old problems and new beginnings Am J Physiol Regulatory Integrative Comp Physiol, September 1, 1999; 277(3): R607 - R623. [Abstract] [Full Text] [PDF]

				M Frieden, M Sollini, and J-L Beny Substance P and bradykinin activate different types of KCa currents to hyperpolarize cultured porcine coronary artery endothelial cells J. Physiol., September 1, 1999; 519(2): 361 - 371. [Abstract] [Full Text] [PDF]

				P.-L. Li, C.-L. Chen, R. Bortell, and W. B. Campbell 11,12-Epoxyeicosatrienoic Acid Stimulates Endogenous Mono-ADP-Ribosylation in Bovine Coronary Arterial Smooth Muscle Circ. Res., August 20, 1999; 85(4): 349 - 356. [Abstract] [Full Text] [PDF]

				H.-C. Lee, T. Lu, N. L Weintraub, M. VanRollins, A. A Spector, and E. F Shibata Effects of epoxyeicosatrienoic acids on the cardiac sodium channels in isolated rat ventricular myocytes J. Physiol., August 15, 1999; 519(1): 153 - 168. [Abstract] [Full Text] [PDF]

				P.-L. Li, D. X. Zhang, A.-P. Zou, and W. B. Campbell Effect of Ceramide on KCa Channel Activity and Vascular Tone in Coronary Arteries Hypertension, June 1, 1999; 33(6): 1441 - 1446. [Abstract] [Full Text] [PDF]

				R. Schubert, T. Noack, and V. N. Serebryakov Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells Am J Physiol Cell Physiol, March 1, 1999; 276(3): C648 - C658. [Abstract] [Full Text] [PDF]

				J.-K. Chen, D.-W. Wang, J. R. Falck, J. Capdevila, and a. R. C. Harris Transfection of an Active Cytochrome P450 Arachidonic Acid Epoxygenase Indicates That 14,15-Epoxyeicosatrienoic Acid Functions as an Intracellular Second Messenger in Response to Epidermal Growth Factor J. Biol. Chem., February 19, 1999; 274(8): 4764 - 4769. [Abstract] [Full Text] [PDF]

				I. R. Hutcheson, A. T. Chaytor, W. H. Evans, and T. M. Griffith Nitric Oxide–Independent Relaxations to Acetylcholine and A23187 Involve Different Routes of Heterocellular Communication : Role of Gap Junctions and Phospholipase A2 Circ. Res., January 22, 1999; 84(1): 53 - 63. [Abstract] [Full Text] [PDF]

				J. D. Imig, E. W. Inscho, P. C. Deichmann, K. M. Reddy, and J. R. Falck Afferent Arteriolar Vasodilation to the Sulfonimide Analog of 11,12-Epoxyeicosatrienoic Acid Involves Protein Kinase A Hypertension, January 1, 1999; 33(1): 408 - 413. [Abstract] [Full Text] [PDF]

				J.-K. Chen, J. R. Falck, K. M. Reddy, J. Capdevila, and R. C. Harris Epoxyeicosatrienoic Acids and Their Sulfonimide Derivatives Stimulate Tyrosine Phosphorylation and Induce Mitogenesis in Renal Epithelial Cells J. Biol. Chem., October 30, 1998; 273(44): 29254 - 29261. [Abstract] [Full Text] [PDF]

				M. Dumoulin, D. Salvail, S. B. Gaudreault, A. Cadieux, and E. Rousseau Epoxyeicosatrienoic acids relax airway smooth muscles and directly activate reconstituted KCa channels Am J Physiol Lung Cell Mol Physiol, September 1, 1998; 275(3): L423 - L431. [Abstract] [Full Text] [PDF]

				D. Salvail, M. Dumoulin, and E. Rousseau Direct modulation of tracheal Cl--channel activity by 5,6- and 11,12-EET Am J Physiol Lung Cell Mol Physiol, September 1, 1998; 275(3): L432 - L441. [Abstract] [Full Text] [PDF]

				P.-L. Li, A.-P. Zou, and W. B. Campbell Regulation of KCa-channel activity by cyclic ADP-ribose and ADP-ribose in coronary arterial smooth muscle Am J Physiol Heart Circ Physiol, September 1, 1998; 275(3): H1002 - H1010. [Abstract] [Full Text] [PDF]

				J. Bylund, T. Kunz, K. Valmsen, and E. H. Oliw Cytochromes P450 with Bisallylic Hydroxylation Activity on Arachidonic and Linoleic Acids Studied with Human Recombinant Enzymes and with Human and Rat Liver Microsomes J. Pharmacol. Exp. Ther., January 1, 1998; 284(1): 51 - 60. [Abstract] [Full Text]

				P.-L. Li, M.-W. Jin, and W. B. Campbell Effect of Selective Inhibition of Soluble Guanylyl Cyclase on the KCa Channel Activity in Coronary Artery Smooth Muscle Hypertension, January 1, 1998; 31(1): 303 - 308. [Abstract] [Full Text] [PDF]

				W. B. Campbell, C. Deeter, K. M. Gauthier, R. H. Ingraham, J. R. Falck, and P.-L. Li 14,15-Dihydroxyeicosatrienoic acid relaxes bovine coronary arteries by activation of KCa channels Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1656 - H1664. [Abstract] [Full Text] [PDF]

				P.-L. Li, D. X. Zhang, Z.-D. Ge, and W. B. Campbell Role of ADP-ribose in 11,12-EET-induced activation of KCa channels in coronary arterial smooth muscle cells Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1229 - H1236. [Abstract] [Full Text] [PDF]

				P. Vequaud and E. Thorin Endothelial G Protein {beta}-Subunits Trigger Nitric Oxide- but not Endothelium-Derived Hyperpolarizing Factor-Dependent Dilation in Rabbit Resistance Arteries Circ. Res., October 12, 2001; 89(8): 716 - 722. [Abstract] [Full Text] [PDF]

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