Epoxyeicosatrienoic Acids Activate K+ Channels in Coronary ...



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.

|[pic]|   Abstract |

|[pic]Top |

|[pic]Abstract |

|[pic]Introduction |

|[pic]Materials and Methods |

|[pic]Results |

|[pic]Discussion |

|[pic]References |

 

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 (KCa) 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 KCa 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 KCa 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 KCa 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 KCa channel activity when GTP and ATP were added to the pipette solution. The addition of a specific anti-GS[pic] antibody (100 nmol/L) in the pipette solution completely blocked the activation of the KCa channels induced by 11,12-EET. An anti-Gß[pic] or anti-Gi[pic] antibody was without effect. We conclude that 11,12-EET activates the KCa channels by a GS[pic]-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

|[pic]|   Introduction |

|[pic]Top |

|[pic]Abstract |

|[pic]Introduction |

|[pic]Materials and Methods |

|[pic]Results |

|[pic]Discussion |

|[pic]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 arteries4 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 KCa channel in vascular smooth muscle cells.8 10 14 These results further support the hypothesis that EETs serve as EDHFs, since the KCa 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 KCa 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.

|[pic]|   Materials and Methods |

|[pic]Top |

|[pic]Abstract |

|[pic]Introduction |

|[pic]Materials and Methods |

|[pic]Results |

|[pic]Discussion |

|[pic]References |

 

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 KCa 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[pic] 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[pic]) 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 (NPo) in patches was determined from recordings of several minutes by the following:

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where N is the maximal number of channels observed in conditions producing high levels of Po, T is the duration of the recording, and tj 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, [pic]20°C.

Identification of the KCa Channel in Small Bovine Coronary Arteries

To establish current-voltage relations of the KCa 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 KCa 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 KCa 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 KCa 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 KCa 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-GS[pic] (n=7), anti-Gß[pic] (n=8), and anti-Gi[pic] (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 GS[pic] 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-GS[pic] antibody (New England Nuclear). The ECL detection kit (Amersham) was used to detect the specific GS[pic] 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 ................
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