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Regulation of Cardiovascular Functions by Eicosanoids and Other Lipid Mediators

Mechanism of rat mesenteric arterial K_ATP channel activation by 14,15-epoxyeicosatrienoic acid

¹Division of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota; and ²Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas

Submitted 31 March 2005 ; accepted in final form 5 December 2005

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Recently, we reported that 11,12-epoxyeicosatrienoic acid (11,12-EET) potently activates rat mesenteric arterial ATP-sensitive K⁺ (K_ATP) channels and produces significant vasodilation through protein kinase A-dependent mechanisms. In this study, we tried to further delineate the signaling steps involved in the activation of vascular K_ATP channels by EETs. Whole cell patch-clamp recordings [0.1 mM ATP in the pipette, holding potential (HP) = 0 mV and testing potential (TP) = –100 mV] in freshly isolated rat mesenteric smooth muscle cells showed small glibenclamide-sensitive K_ATP currents (19.0 ± 7.9 pA, n = 5) that increased 6.9-fold on exposure to 5 µM 14,15-EET (132.0 ± 29.0 pA, n = 7, P < 0.05 vs. control). With 1 mM ATP in the pipette solution, K_ATP currents (HP = 0 mV and TP = –100 mV) were increased 3.5-fold on exposure to 1 µM 14,15-EET (57.5 ± 14.3 pA, n = 9, P < 0.05 vs. baseline). In the presence of 100 nM iberiotoxin, 1 µM 14,15-EET hyperpolarized the membrane potential from –20.5 ± 0.9 mV at baseline to –27.1 ± 3.0 mV (n = 6 for both, P < 0.05 vs. baseline), and the EET effects were significantly reversed by 10 µM glibenclamide (–21.8 ± 1.4 mV, n = 6, P < 0.05 vs. EET). Incubation with 5 µM 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), a 14,15-EET antagonist, abolished the 14,15-EET effects (31.0 ± 11.8 pA, n = 5, P < 0.05 vs. 14,15-EET, P = not significant vs. control). The 14,15-EET effects were inhibited by inclusion of anti-G_s antibody (1:500 dilution) but not by control IgG in the pipette solution. The effects of 14,15-EET were mimicked by cholera toxin (100 ng/ml), an exogenous ADP-ribosyltransferase. Treatment with the ADP-ribosyltransferase inhibitors 3-aminobenzamide (1 mM) or m-iodobenzylguanidine (100 µM) abrogated the effects of 14,15-EET on K_ATP currents. These results were corroborated by vasodilation studies. 14,15-EET dose-dependently dilated isolated small mesenteric arteries, and this was significantly attenuated by treatment with 14,15-EEZE or 3-aminobenzamide. These results suggest that 14,15-EET activates vascular K_ATP channels through ADP-ribosylation of G_s.

ATP-sensitive potassium channel; mesenteric artery; G_s; ADP-ribosylation

ATP-sensitive potassium (K_ATP) channels are ubiquitous, linking cellular energy metabolic state to membrane excitability (28, 30, 38). The cardiovascular system is richly endowed with K_ATP channels, and vascular K_ATP channels are thought to play an important role in the regulation of vascular tone (2). Recently, we reported that rat mesenteric arterial K_ATP channels are potently activated by all four EET regioisomers with an EC₅₀ of 87 nM for 11,12-EET (37). Activation of K_ATP channels accounts for 50% of the mesenteric vasodilation by 11,12-EET, and these effects were dependent on protein kinase A (PKA) activities (37). However, the signaling steps associated with the activation of vascular K_ATP channels by EET are unclear. In this study, we hypothesized that ADP-ribosylation of G_s is involved in the activation of mesenteric arterial K_ATP channels by EETs. We found that activation of mesenteric arterial K_ATP channels by 14,15-EET is blocked by 14,15-EEZE, anti-G_s antibodies, and ADP-ribosyltransferase inhibitors. In addition, the effects of 14,15-EET on K_ATP channels were mimicked by cholera toxin (CTX), an ADP-ribosyltransferase. These results were corroborated by vasoreactivity studies showing that 14,15-EET produced potent vasodilation in isolated small mesenteric arteries, and the effects of 14,15-EET were significantly attenuated by 14,15-EEZE and ADP-ribosyltransferase inhibitors. In addition, pinacidil-mediated K_ATP channel activation and vasodilation were not inhibited by preincubation with 14,15-EEZE and ADP-ribosyltransferase inhibitors or by inclusion of anti-G_s antibody in the patch pipette solution, suggesting that the effects of these compounds are specific, inhibiting steps in the EET signaling pathway that results in K_ATP channel activation. These findings suggest that activation of vascular K_ATP channels by EET involves ADP-ribosylation of G_s.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Isolation of arterial smooth muscle cells from rat small mesenteric arteries. The use of animals and procedures involved with tissue isolation were approved by the Animal Care and Use Committee at the Mayo Clinic (Rochester, MN). Single vascular smooth muscle cells were prepared as previously described (37). Briefly, male Sprague-Dawley rats (200–250 g) were anesthetized with pentobarbital sodium (100 mg/kg ip). Bowels and mesenteries were rapidly excised and placed in Krebs solution that contained (in mM) 118.3 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 11.1 dextrose, equilibrated with 5% CO₂-95% room air. The third- and fourth-ordered branches (100–250 µm in intraluminal diameter) of mesenteric arteries were isolated and placed in 1 ml of physiological saline solution that contained (in mM) 145 NaCl, 4.0 KCl, 0.05 CaCl₂, 1.0 MgCl₂, 10.0 HEPES, 10.0 glucose, pH 7.2, with 0.1% BSA. After incubation at room temperature for 10 min, vessels were enzymatically dissociated as previously described (37). The resulting smooth muscle cell suspension was stored at 4°C and used within 8 h.

Whole cell patch-clamp recordings. Whole cell currents were recorded from single smooth muscle cells by using patch-clamp techniques as previously described (21, 37). Isolated smooth muscle cells were placed in a recording chamber on the stage of an inverted microscope and were superfused with an extracellular bath solution that contained (in mM) 140 KCl, 1 MgCl₂, 0.1 CaCl₂, 10 HEPES, 10 glucose, pH 7.4, at 1–2 ml/min, and solution exchanges were complete within 30–60 s. Whole cell K_ATP currents were recorded with a pipette solution that contained (in mM) 110 KCl, 30 KOH, 10 HEPES, 10 EGTA, 1 MgCl₂, 1 CaCl₂, 0.1 or 1 Na₂ATP, 0.1 Na₂ADP, 0.5 Na₂GTP, pH 7.4, by using an Axopatch 200 integrating amplifier (Axon Instruments, Foster City, CA); filtered at 1 kHz; and sampled at 5 kHz. Pipette resistance ranged from 3 to 5 M, and seal resistance was >10 G. pCLAMP 8.0 software (Axon) was used for generating voltage-clamp protocols and for acquisition and analysis of K_ATP currents. Currents were elicited with a holding potential (HP) of 0 mV, and pulsing from –100 to 40 mV in increments of 10 mV. K_ATP currents were obtained by digital subtraction of the residual currents after exposure to glibenclamide (10 µM). The glibenclamide-sensitive K⁺ currents were determined and referred to as K_ATP currents. All cellular electrophysiology experiments were performed at room temperature (21–23°C). To minimize the activity of BK channels, intracellular Ca²⁺ was buffered by EGTA to low levels (20 nM). In addition, 100 nM of iberiotoxin (IBTX) was present in the bath solution to block BK channel activities that could be activated by EETs (21, 37).

Whole cell current-clamp recordings. Membrane potentials in isolated single smooth muscle cells from rat mesenteric arteries were recorded at 37°C using current-clamp technique with a pipette solution that contained (in mM) 140 KCl, 1.0 EGTA, 0.046 CaCl₂ (200 nM free Ca²⁺), 0.5 GTP, 5.0 ATP, 10.0 HEPES, and 1.0 MgCl₂, pH 7.35. The extracellular solution for whole cell current-clamp recordings contained (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 1.0 Na₂HPO₄, 5.0 glucose, 0.0001 IBTX, and 5.0 HEPES, pH 7.4. After baseline membrane potentials were obtained, cells were exposed to 14,15-EET (1 µM), and changes in membrane potential were recorded continuously. After the membrane potentials had reached steady state, 10 µM glibenclamide was added to the 14,15-EET to determine the contribution of K_ATP channels.

Vasoreactivity measurements. Vasoreactivity of isolated small mesenteric arteries (100–250 µm in diameter) was determined by videomicroscopy as previously described (37, 40). Isolated small mesenteric arteries (1–2 mm in length) were transferred to a vessel chamber filled with Krebs solution, cannulated with micropipettes, and pressurized at 60 mmHg for at least 30 min at 37°C before starting the experiment. In some experiments, vessels were incubated with 14,15-EEZE (10 µM), 14,15-EEZE + glibenclamide (1 µM), or 3-aminobenzamide (3-AB, 1 mM) for 30–45 min during the equilibration period. 14,15-EET or 14,15-EEZE was added abluminally, and the cumulative dose response was determined at 3- to 5-min intervals between doses. Vessels were constricted to 30–60% of baseline diameter using endothelin-1, and the effects of 14,15-EET in dilating these vessels were examined over the concentration range of 1 x 10^–10 to 1 x 10^–6 M. In some control experiments, pinacidil (10^–7 to 10^–5 M) was used as the vasodilator to determine the specificity of the 14,15-EEZE and 3-AB effects. Vessels were discarded if they failed to produce an 85% relaxation with 1 x 10^–4 M sodium nitroprusside or failed to produce a 30% constriction with 60 mM KCl.

Materials. 14,15-EET was obtained from Cayman Chemical (Ann Arbor, MI), solubilized in ethanol as a 5 mM stock, and stored at –80°C. 14,15-EEZE was solubilized in ethanol as a 10 mM stock, and stored at –20°C. Glibenclamide and 3-AB were solubilized in DMSO, while m-iodobenzylguanidine (MIBG) was solubilized in methanol. The rest of the chemicals were solubilized in water. CTX and anti-G_s antibody were obtained from Calbiochem (San Diego, CA). Control IgG was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). 3-AB, MIBG, glibenclamide, and IBTX were obtained from Sigma Chemical (St. Louis, MO).

Statistical analysis. All data are expressed as means ± SE. One-way ANOVA was used to analyze data from multiple groups, and pairwise comparisons among groups were performed using a post hoc Tukey test. Student's paired t-test was used to compare data before and after each intervention. Statistically significant difference is defined as P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Activation of vascular K_ATP currents by 14,15-EET is inhibited by 14,15-EEZE. Rat mesenteric artery smooth muscle cells exhibited very little glibenclamide-sensitive K⁺ currents (K_ATP currents) under baseline condition. With 0.1 mM ATP in the pipette, 140 mM symmetrical K⁺, holding at 0 mV, and pulsing to –100 mV, the K_ATP current amplitude was 19.0 ± 7.9 pA (n = 5, Fig. 1A). 14,15-EET at 5 µM produced a 6.9-fold activation of K_ATP currents (132.0 ± 29.0 pA, n = 7, P < 0.05 vs. control, Fig. 1B). However, after incubation with 5 µM 14,15-EEZE, an EET-specific antagonist, the effects of 14,15-EET were significantly attenuated [31.0 ± 11.8 pA, n = 5, P < 0.05 vs. 14,15-EET and P = nonsignificant (NS) vs. control, Fig. 1C]. Figure 1D shows the current-voltage (I-V) relationships of the effects of 14,15-EET and 14,15-EEZE on K_ATP currents. 14,15-EET activated K_ATP currents over the full voltage range examined. Likewise, the inhibitory effects of 14,15-EEZE were independent of membrane potential. Figure 1E shows group data in bar graphs on K_ATP currents elicited at –100 mV. These results suggest that 14,15-EET as a vascular K_ATP channel activator may have specific cellular binding sites, where its structural analog competes for binding and antagonizes its activities.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Activation of ATP-sensitive K+ (KATP) currents in mesenteric arterial smooth muscle cells by 14,15-epoxyeicosatrienoic acid (14,15-EET) is inhibited by 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE). Whole cell K+ currents were measured in the presence of symmetrical 140 mM K+ and 100 nM iberiotoxin (IBTX), with 0.1 mM ATP in the pipette solution, from a holding potential of 0 mV to testing pulses of –100 mV to +40 mV in 10 mV increments. The KATP currents in this and subsequent figures were obtained by digital subtraction of K+ currents that were sensitive to inhibition by 10 µM glibenclamide. Representative current tracings: control with ethanol as vehicle (A), effect of 5 µM 14,15-EET (B), effect of 5 µM 14,15-EET + 5 µM 14,15-EEZE (C). D: current-voltage (I-V) relationship of KATP currents in controls (n = 5), with 5 µM 14,15-EET (n = 7), and with 5 µM 14,15-EET + 5 µM 14,15-EEZE (n = 5). E: bar graphs showing group data on KATP currents elicited with a holding potential of 0 mV and testing potential of –100 mV in controls (n = 5), with 5 µM 14,15-EET (n = 7), and with 5 µM 14,15-EET + 5 µM 14,15-EEZE (n = 5). *P < 0.05 vs. control; + P < 0.05 vs. 14,15-EET.

G_s is required for 14,15-EET-induced activation of K_ATP channels.

View larger version (45K): [in this window] [in a new window]   Fig. 2. Activation of KATP currents by 14,15-EET is inhibited by anti-Gs antibodies. Whole cell KATP currents were elicited in the presence of symmetrical 140 mM K+ and 100 nM IBTX, with 0.1 mM ATP in the pipette solution, from a holding potential of 0 mV to testing pulses of –100 mV to +40 mV in 10-mV steps. Recordings from typical cells showing KATP currents at baseline (A), in the presence of 5 µM 14,15-EET (B), with 5 µM 14,15-EET + anti-Gs antibodies in the pipette solution (1:500 dilution) (C), and with 5 µM 14,15-EET + control IgG in the pipette solution (1:500 dilution) (D). E: bar graphs showing group data on KATP currents elicited from a holding potential of 0 mV and a testing potential of –100 mV at baseline (n = 5), with 5 µM 14,15-EET (n = 7), with 5 µM 14,15-EET + anti-Gs antibodies (n = 6), and with 5 µM 14,15-EET + control IgG (n = 5). F: I-V relationships of KATP (glibenclamide sensitive) currents at baseline (n = 5), with 5 µM 14,15-EET (n = 7), with 5 µM 14,15-EET + anti-Gs antibodies (n = 6), and with 5 µM 14,15-EET + control IgG (n = 5). *P < 0.05 vs. control; + P < 0.05 vs. 14,15-EET.

To study the role of ADP-ribosylation in the activation of vascular K_ATP channels by 14,15-EET, we examined the effects of CTX, an exogenous ADP-ribosyltransferase, on K_ATP channel activation. With 100 ng/ml CTX in the pipette solution, K_ATP currents were significantly activated after pipette breakthrough in the whole cell configuration. At baseline, mesenteric smooth muscle cells had only a small amount of K_ATP currents (11.4 ± 3.4 pA, n = 5) that showed a 10.2-fold increase after treatment with CTX (116.2 ± 19.6 pA, n = 5, P < 0.05 vs. baseline) and that achieved a similar level of current activation by 5 µM 14,15-EET (105.5 ± 15.9 pA, n = 5, P < 0.05 vs. baseline, P = NS vs. CTX). After CTX effects had reached steady state, subsequent exposure to 5 µM 14,15-EET did not produce further K_ATP current increase (112.8 ± 29.7 pA, n = 5, P < 0.05 vs. baseline, P = NS vs. CTX). Figure 3, A–D, shows typical current tracings at baseline, with 5 µM 14,15-EET, CTX, and CTX + 14,15-EET. Group data are shown in Fig. 3E. These results suggest that ADP-ribosylation of G_s may be involved in the activation of vascular K_ATP channels by 14,15-EET.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Activation of KATP currents by 14,15-EET is mimicked by cholera toxin (CTX). Whole cell K+ currents were elicited from a holding potential of 0 mV to testing potentials from –100 mV to 40 mV in 10-mV increments, in the presence of symmetrical 140 mM K+ and 100 nM IBTX with 0.1 mM ATP in the pipette solution. Recordings of KATP (glibenclamide sensitive) currents from a typical cell at baseline (A), on exposure to 5 µM 14,15-EET (B), with CTX (100 ng/ml) in the pipette solution (C), and on exposure to 5 µM 14,15-EET with CTX in the pipette solution (D). E: bar graphs showing group data on KATP currents elicited from 0 mV to –100 mV in control (n = 5), with 5 µM 14,15-EET (n = 5), with 100 ng/ml CTX in the pipette (n = 5), and with 5 µM 14,15-EET + 100 ng/ml CTX in the pipette (n = 5). *P < 0.05 vs. control.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Activation of KATP currents by 14,15-EET is inhibited by inhibitors of endogenous mono-ADP-ribosyltransferases. Representative KATP current tracings elicited with a holding potential of 0 mV to testing pulses of –100 mV to +40 mV in 10-mV increments, with 0.1 mM ATP in the pipette solution, in a control cell (A), on exposure to 5 µM 14,15-EET (B), with 5 µM 14,15-EET after incubation with 1 mM 3-aminobenzamide (3-AB) (C), and with 5 µM 14,15-EET after incubation with 100 µM m-iodobenzylguanidine (MIBG) (D). E: bar graphs showing group data on KATP currents elicited from 0 mV to –100 mV in control (n = 5), with 5 µM 14,15-EET (n = 7), with 1 mM 3-AB (n = 6), with 5 µM 14,15-EET + 1 mM 3-AB (n = 6), with 100 µM MIBG (n = 5), and with 5 µM 14,15-EET + 100 µM MIBG (n = 5). *P < 0.05 vs. control; +P < 0.05 vs. 14,15-EET.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Vascular KATP currents are activated by 14,15-EET in the presence of millimolar cytoplasmic ATP. Whole cell KATP currents were elicited in the presence of symmetrical 140 mM K+ and 100 nM IBTX, with 1 mM ATP in the pipette solution, from a holding potential of 0 mV to testing pulses of –100 mV to +40 mV in 10-mV steps. Recordings from a typical experiment showing KATP currents at baseline (A) and in the presence of 1 µM 14,15-EET (B). C: I-V relationships of KATP (glibenclamide sensitive) currents at baseline (n = 9) and with 1 µM 14,15-EET (n = 9). D: bar graphs showing group data on KATP currents elicited from a holding potential of 0 mV and a testing potential of –100 mV at baseline (n = 9), with 1 µM 14,15-EET (n = 9). *P < 0.05 vs. baseline.

View larger version (22K): [in this window] [in a new window]   Fig. 6. 14,15-EET hyperpolarizes the membrane potentials of vascular smooth muscle cells. Membrane potentials in isolated mesenteric arterial smooth muscle cells were recorded using whole cell current-clamp technique in the presence of 100 nM IBTX in the bath solution with 5 mM ATP and 200 nM free Ca2+ in the pipette solution. A: representative tracing showing the effects of 1 µM 14,15-EET on the membrane potentials of a mesenteric smooth muscle cells. 14,15-EET and glibenclamide were applied as indicated by the bars. B: bar graphs showing group data on the membrane potentials at baseline (n = 6), with 1 µM 14,15-EET (n = 6), and with 1 µM 14,15-EET + 10 µM glibenclamide (n = 6). *P < 0.05 vs. baseline; P < 0.05 vs. 14.15 – EET.

Vasodilation of small mesenteric arteries by 14,15-EET is mediated through K_ATP channel activation and ADP-ribosylation of G_s.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Vasodilation of small mesenteric arteries by 14,15-EET is 14,15-EEZE and 3-AB sensitive. Isolated mesenteric arteries were constricted to 30–60% baseline diameter with endothelin-1, and the vasodilation effect of various concentration of 14,15-EET (1 x 10–10 to 1 x 10–6 M) was determined after incubation with no drug (; control, n = 5), with 10 µM 14,15-EEZE (, n = 5), 10 µM 14,15-EEZE + 1 µM glibenclamide (, n = 5), and 1 mM 3-AB (, n = 6). 14,15-EEZE alone (, n = 5, 1 x 10–10 to 1 x 10–6 M) had very little dilatory effect on small mesenteric arteries. Data are presented as means ± SE; P < 0.001 for all groups vs. control.

Activation of K_ATP channels by pinacidil is not inhibited by 14,15-EEZE, 3-AB, MIBG, or anti-G_s antibody.

View larger version (32K): [in this window] [in a new window]   Fig. 8. Activation of KATP channels by pinacidil is not inhibited by 14,15-EEZE, 3-AB, MIBG, or anti-Gs antibody. A: bar graphs showing activation of KATP currents in isolated rat mesenteric smooth muscle cells (holding potential = 0 mV and testing potential = –100 mV) in the presence of symmetrical 140 mM K+ by pinacidil (10 µM) and after preincubation with 5 µM 14,15-EEZE, 1 mM 3-AB, or 100 µM MIBG, or with anti-Gs antibody (1:500 dilution) in the pipette; n = 4 for all groups. There is no statistical significance between the groups. B: vasodilation of small mesenteric arteries by pinacidil. Isolated mesenteric arteries were constricted to 30–60% baseline diameter with endothelin-1, and the vasodilation effect of pinacidil (1 x 10–7 to 1 x 10–5 M) was determined after incubation with no drug (closed bars, control), with 1 mM 3-AB (open bars), or with 10 µM 14,15-EEZE (hatched bars). There is no significance between the groups, n = 4 for all groups.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

We have previously reported that EETs are potent activators of cardiac and vascular K_ATP channels with EC₅₀ in the range of 1 x 10^–8 M (20, 23, 37). However, there are important differences between the mechanisms by which EETs activate cardiac and vascular K_ATP channels. EETs directly activate cardiac K_ATP channels, without the presence of any diffusible second messengers, by reducing the channel sensitivity to ATP (20, 23). Interaction between EET and cardiac K_ATP channels is stereospecific with 11(S),12(R)-EET being the active enantiomer while 11(R),12(S)-EET is totally inactive (23). Recently, we have identified R192/R195 on Kir6.2, which encodes cardiac K_ATP channels, as the critical EET sensitivity site (19). In contrast, we found that activation of vascular K_ATP channels by EET is mediated by PKA-dependent mechanisms (37). The signaling steps through which extracellular EET activates PKA is not clear, because the presence of an EET-specific cell surface receptor in vascular smooth muscle cells has not been directly identified.

In this study, we found that 14,15-EEZE completely abolished the activation of vascular K_ATP channels by 14,15-EET, suggesting that the action of EET is mediated through a specific binding site and not through nonspecific fatty acid or lipid effects. The presence of a cell surface EET receptor site was first proposed by Wong et al., who demonstrated high affinity binding sites for 14,15-EET in the plasma membrane of human U-937 transformed monocytes (35) and in guinea pig mononuclear cells (34). In both cases, the putative receptor was linked to a cAMP and PKA signal transduction pathway that subsequently downregulates the receptor. In addition, structural analogs of EETs, such as 14,15-EEZE and 14,15-EEZE-mSI, showed regioisomer-specific antagonist activities in coronary arteries, indicating precise structural requirements for 14,15-EET functions, including K⁺ channel activation and vasodilation (6, 10, 12, 13). Recently, cAMP-induced aromatase activities in cultured aortic smooth muscle cells were found to be inhibited by 14,15-EET, by the nonmetabolized N-methylsulfanilamide derivative of 14,15-EET (14,15-EET-SI), and by the membrane impermeable 14,15-EET-SI that was covalently tethered to silica beads. These results suggest the presence of a specific EET binding site, possibly a receptor, in the plasma membrane (31). The results of our study lend support to the growing evidence that an EET receptor might be present in the plasma membrane of smooth muscle cells and is involved in the activation of K_ATP channels in vascular smooth muscle cells (10, 31, 32).

EETs have also been shown to affect cell membrane signaling mechanisms, including ADP-ribosylation (18), G_s-mediated responses (9, 18), activation of PKA (15), and tyrosine phosphorylation (5). The effects of EET on ADP-ribosylation and G_s, in particular, are thought to underlie the activation of BK channels (9, 17, 18). In this study, we found that the effects of 14,15-EET on K_ATP channels were inhibited by the inclusion of anti-G_s antibody but not by control IgG in the pipette solution. We have used similar approaches to determine the direct G_s effects on cardiac Na⁺ channel activation (22, 24). Our results indicate that activation of vascular K_ATP channels requires the integrity of G_s activities. In addition, CTX, an exogenous ADP-ribosyltransferase that is well known to target G_s, produces remarkable activation of the K_ATP currents in the mesenteric smooth muscle cells, enhancing the currents to a similar level attained by 14,15-EET. Exposure to 14,15-EET in the presence of CTX did not produce any further K_ATP current stimulation, suggesting that CTX and EET might share a similar mechanism of action in K_ATP channel activation. These results are similar to those reported on BK channel activation by EET in vascular smooth muscle cells (9, 17) and suggest that ADP-ribosylation of G_s might be responsible for the activation of vascular K_ATP channels by EETs. This contention is supported by the finding that the effects of 14,15-EET were inhibited by two different mono-ADP-ribosyltransferase inhibitors, 3-AB and MIBG, suggesting that ADP-ribosylation is involved in the signaling mechanism through which 14,15-EET activates vascular K_ATP channels. Because the G protein signaling cascades are typically activated through interaction with G protein-coupled receptors that characteristically have a structural motif of seven transmembrane domains, it is plausible that EETs also exert their effects in vascular smooth muscle through similar receptor-dependent interactions (12). EETs have been shown to enhance the GTP-binding activities of G_s by 3.5-fold and to increase intracellular cAMP concentration in cultured endothelial cells (26), cardiac myocytes (36), U-937 cells (35), guinea pig mononuclear cells (34), cerebral microvascular cells (16), and vascular smooth muscle cells (33). The increase in cAMP would then activate PKA, enhancing K_ATP channel activities.

Physiological relevance of the observation in single smooth muscle cells is corroborated by vasoreactivity studies. 14,15-EEZE produced very little vasodilation but was an ardent inhibitor to that produced by 14,15-EET. These results are similar to studies using small bovine coronary arteries (10). We have previously shown that activation of K_ATP channels accounts for 50% of the vasodilation effects of EETs (37). In the present study, addition of glibenclamide to 14,15-EEZE had no additional inhibitory effect, suggesting 14,15-EEZE was effective in totally inhibiting the K_ATP channel activation component of vasodilation induced by 14,15-EET. In addition, incubation with 3-AB also produced significant inhibition of the 14,15-EET vasodilatory effects, indicating that ADP-ribosylation is a critical step in mediating the effects of 14,15-EET. The effects of the inhibitors 14,15-EEZE, 3-AB, MIBG, and anti-G_s antibody are specific in inhibiting the EET signaling steps, and they do not inhibit the effects of pinacidil on K_ATP channels or on vasodilation (Fig. 8). Evidently, activation of K_ATP channels through EET-induced ADP-ribosylation of G_s is an integral mechanism for regulating vasoreactivity.

Previous studies from several laboratories have shown that EETs serve as EDHF in coronary and other circulations (1, 4, 7, 11). EETs are potent activators of vascular BK channels, producing membrane hyperpolarization, leading to vasorelaxation (3, 14, 17), suggesting that BK channels play a critical role in EETs-induced vasodilation. Fukao et al. (8) reported that 11,12-EET-induced smooth muscle hyperpolarization in isolated rat mesenteric arteries was blocked by glibenclamide, suggesting that K_ATP channels were involved. Recently, we have reported that 11,12-EET potently activates rat mesenteric arterial K_ATP channels and produces vasorelaxation (37). In the present study, we found that under physiological conditions, 14,15-EET produces membrane hyperpolarization in rat mesenteric smooth muscle cells through activation of K_ATP channels. These findings confirm that activation of vascular K_ATP channels is an important component in mediating the vasodilatory effects of EETs.

In summary, a specific scheme on the signaling mechanism with which EETs activate vascular K_ATP channels has emerged. On the basis of the results from our current and previous studies, we propose that EETs interact with specific binding sites or receptors in vascular smooth muscle cells, leading to activation of G_s through ADP-ribosylation. This results in activation of adenylyl cyclase activities, promoting the formation of cAMP, which in turn activates PKA. PKA phosphorylates and activates vascular K_ATP channels, producing membrane hyperpolarization and vasodilation.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
These studies were supported by National Institutes of Health Grants HL-63754, HL-74180, and DK-38226.

	FOOTNOTES

 

Address for reprint requests and other correspondence: H. Lee, Div. of Cardiovascular Diseases, Dept. of Internal Medicine, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (e-mail: lee.honchi{at}mayo.edu)

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	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

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