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Journal of Physiology (2001), 537.3, pp. 811-827
© Copyright 2001 The Physiological Society
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ABSTRACT |
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INTRODUCTION |
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The KATP channels link cellular energy metabolism to membrane excitability (Noma, 1983), and are widely distributed in a variety of tissues and cell types, including cardiac myocytes and vascular smooth muscle cells (Noma, 1983; Edwards & Weston, 1993; Yokoshiki et al. 1998). The heart is richly endowed with KATP channels; the channel density in cardiac myocytes is estimated to be 1-10 µm-2 compared with only 0.1-0.5 µm-2 in vascular smooth muscle cells (Noma, 1983; Kakei et al. 1985; Nichols & Lederer, 1990; Yokoshiki et al. 1998). KATP channels are inhibited by cytoplasmic ATP and sulfonylurea drugs. However, the mechanism whereby ATP inhibits the KATP channel has not been fully delineated.
Cardiac KATP channels are activated during ischaemia, acidosis and anoxia, when intracellular ATP is depleted and ischaemic metabolites accumulate (Nichols & Lederer, 1990; Nakaya et al. 1991; Koyano et al. 1993; Shigematsu & Arita, 1997). Activation of KATP channels leads to the shortening of the cardiac action potential and could reduce Ca2+ entry, thereby minimizing Ca2+ overload during cardiac ischaemia. However, the role of the cardiac KATP channels under physiological conditions is unclear. Besides the inhibition by ATP and sulfonylurea drugs, cardiac KATP channels are activated by intracellular nucleotide diphosphates (Tung & Kurachi, 1991), intracellular protons (Koyano et al. 1993), G proteins (Terzic et al. 1994), and protein kinase A (Lin et al. 2000). Recent studies demonstrated that membrane phospholipids including phosphatidylinositol phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) (Fan & Makielski, 1997, 1999; Shyng & Nichols, 1998; Baukrowitz et al. 1998; Enkvetchakul et al. 2000), increase the cardiac KATP channel activity by reducing the channel sensitivity to ATP.
Arachidonic acid (AA) is an endogenous constituent of membrane phospholipids and a precursor of many bioactive lipids, such as prostaglandins, thromboxanes, and leukotrienes, that are involved in signal transduction and cellular regulatory mechanisms. More recently, an AA metabolism pathway involving cytochrome P450-dependent mono-oxygenases has been identified (McGiff, 1991). The cytochrome P450 AA epoxygenases CYP2J2 and CYP2J3 were recently cloned from human and rat hearts, respectively (Wu et al. 1996, 1997). These enzymes convert AA into four biologically active epoxyeicosatrienoic acid (EET) regioisomers: 5,6-, 8,9-, 11,12- and 14,15-EET, which can be further converted by epoxide hydrolases to dihydroxyeicosatrienoic acids (DHETs) (McGiff, 1991). EETs and DHETs are potent endothelium-derived vasodilators that modulate vascular tone by acting on ion channels (Campbell et al. 1996; Weintraub et al. 1997; Oltman et al. 1998). Na+, Ca2+ and K+ channels have all been shown to be modulated by EETs in cardiac and vascular smooth muscle cells (Campbell et al. 1996; Xiao et al. 1998; Lee et al. 1999). In the present study, we found that 8,9- and 11,12-EET are potent activators of the KATP channels with an EC50 of 10-8 M. Our results also show that 11,12-EET markedly reduces the KATP channel sensitivity to ATP, and activation of the KATP channels by 11,12-EET leads to resting potential hyperpolarization in cardiac myocytes. Hence, EETs are endogenous activators of the cardiac KATP channels and may play an important role in modulating cardiac electrophysiology.
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METHODS |
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All animal procedures were approved by the Animal Care and Use Committee of the University of Iowa.
Preparation of single ventricular myocytes
Single ventricular myocytes were prepared as previously described (Lee et al. 1999). Briefly, Sprague-Dawley rats (male, 200-250 g) were anaesthetized with methoxyflurane. The hearts were exposed by mid-sternotomy and the animals were killed by rapid excision of the hearts, which were immediately placed in ice-cold nominally Ca2+-free Tyrode solution that contained (mM): NaCl 138.0, KCl 4.5, MgCl2 0.5, Na2HPO4 0.33, glucose 5.5 and Hepes 10.0, pH to 7.38 with NaOH. The aortas were cannulated, and the hearts were perfused using a modified Langendorff apparatus with nominally Ca2+-free Tyrode solution containing 0.1 (w/v) bovine serum albumin (BSA) for 5 min at 37 °C. The perfusate was then changed to nominally Ca2+-free Tyrode solution containing 0.6 mg ml-1 collagenase (Worthington, CLS-2, 347 units mg-1) and 0.1 % (w/v) BSA for 10 min at 37 °C. The ventricles were removed and placed into 25 ml of fresh 0.6 mg ml-1 collagenase solution for 5 min. The hearts were cut into small pieces (1-2 mm3) and filtered through a medium mesh. After washing twice with nominally Ca2+-free Tyrode solution, single cells were maintained in KB solution which contained (mM): KOH 70.0, KCl 40.0, L-glutamic acid 50.0, taurine 20.0, MgCl2 0.5, K2HPO4 1.0, EGTA 0.5, Hepes 10.0, creatine 5.0, pyruvic acid 5.0 and Na2ATP 5.0, pH to 7.38 with KOH. All solutions were vigorously oxygenated for 30 min before use.
Electrophysiological recordings
Unitary currents of KATP channels in rat ventricular myocytes were recorded with the inside-out configuration of the patch-clamp method (Hamill et al. 1981). Isolated myocytes were placed in a chamber (volume, 0.5 ml) on the stage of an inverted microscope, and were superfused at 1-2 ml min-1 with a bath solution that contained (mM): KCl 70.0, L-aspartic acid monopotassium salt 70.0, EGTA 2.0, Hepes 5.0, N-methy-D-glucamine (NMDG) 7.0 and Mg2ATP 0.001, pH to 7.35 with NMDG. This concentration (1 µM) of ATP was included to prevent rundown of the channel upon patch excision (Terzic et al. 1995). In some experiments, various concentrations of ATP were used in the bath solution to determine the effects of ATP on the cytoplasmic side of the membrane, and the bath ATP concentrations are referred to as cytoplasmic ATP concentrations. Solution exchanges were complete within 1-2 min. Patch pipettes (Corning 7056, Warner Instrument, Hamden, CT, USA) were coated with Sylgard 184 (Dow Corning, Midland, MI, USA) and had a typical tip resistance between 2 and 5 M
when filled with the pipette solution, which contained (mM): KCl 140.0, EGTA 1.0, Hepes 5.0, CaCl2 1.0 and MgCl2 1.0, pH to 7.4 with KOH. Single KATP channel current was recorded with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA) and the output of the amplifier was filtered through an 8-pole Bessel filter unit at 5 kHz (Frequency Devices, Haverhill, MA, USA) and digitized at 40 kHz (Digidata, Axon Instruments). Data were acquired using pCLAMP 6.05 (Axon Instruments).
KATP channel currents were identified by the unitary current amplitude (i) and by inhibition with 5-10 mM ATP. Unitary KATP current amplitudes were estimated from amplitude histograms fitted with a Gaussian function. The block of KATP outward currents by internal Mg2+ results in an inward rectification property in the unitary current amplitude and voltage (i-V) relationship (Horie et al. 1987). To determine the unitary conductance of KATP channels in the presence of cytoplasmic Mg2+, we used the equations described by Standen & Stanfield (1978):
(V - Vk),
)VF/RT)}{1 + ([K+]R2/Kk)},
where, Vk is the K+ equilibrium potential, is channel unitary conductance, y is the fraction of channels that are open in the presence of cytoplasmic Mg2+, [Mg2+]i is the intracellular Mg2+ concentration (1 mM), Km is the Mg2+ dissociation constant, Kk is the K+ dissociation constant, is the electrical distance, measured from the inner surface of the membrane, z is the valency of Mg2+, F is the Faraday constant, R is the universal gas constant and T is the absolute temperature. [K+]R represents the concentration of K+ bound to the channel, and [K+]R = [K+]oexp(-VkF/RT), where [K+]o is the extracellular K+ concentration and Vk = 0 (in symmetric K+ concentration, 140 mM).
Single KATP channel kinetics analysis
Kinetics analysis was performed using recordings obtained from patches that contained only one active channel as judged by the maximum number of simultaneous openings observed in the presence of 0.1-1 µM ATP. The opening and closing transitions of KATP channels were detected by the half-amplitude threshold method using TAC 
4.0.9 software (Bruxton, Seattle, WA, USA). Data were filtered at 2.0 kHz bandwidth with a digital Gaussian filter to achieve the appropriate signal-to-noise ratio. The open- and closed-dwell times were fitted with the sums of exponential probability density functions using the maximum likelihood algorithm with simplex optimization as implemented in TAC. The fit was corrected for the dead time of the recording system, estimated as 0.253/f where f represents the filter cut-off frequency, and it was typically 185 µs. Only dwell times longer than the dead time were fitted. The number of exponential components was determined using the likelihood ratio test, and an additional exponential component was included only when the probability was > 0.95. Dose-response curves were fitted using a Hill equation of the following form:
where Po,max represents the maximal channel activity, S represents concentration of chemical, EC50 represents the concentration at half-maximal effect, and nH is the Hill coefficient.
Whole-cell current-clamp recordings
Resting membrane potentials and action potentials were recorded at 37 °C in isolated rat cardiac ventricular myocytes using current-clamp techniques. Action potentials were elicited by 1 nA stimuli at 0.5 Hz. The signals were filtered at 5 kHz and digitized at 20 kHz. After the resting and action potentials had reached steady state, the effects of 11,12-EET (5 µM) followed by 11,12-EET plus glyburide (2 µM) were measured. The bath solution was Tyrode solution containing 1.8 mM CaCl2, whereas the pipette solution contained (mM): KCl 130, EGTA 1, Hepes 10, MgCl2 1, ATP 5, GTP 1 and CaCl2 0.018 (200 nM free Ca2+).
Materials
8,9-, 11,12-EET and 11,12-DHET were obtained from Cayman Chemical (Ann Arbor, MI, USA). AA was purchased from Nu-Chek-Prep (Elysian, MN, USA). Glyburide, ATP and all other chemicals were obtained from Sigma (St Louis, MO, USA). AA, EETs and 11,12-DHET were prepared in 100 % ethanol (5 mM) and stored under nitrogen at -20 °C. Glyburide was dissolved in DMSO as a stock solution and stored at -20 °C. The chemical stocks were used at greater than or equal to 1:106 dilutions in the experiment, and the final concentrations of ethanol or DMSO were less than 0.0001 %.
Statistical methods
Data are presented as means ± S.E.M. Student's paired t test was used to compare data obtained before and after an intervention. One-way ANOVA followed by contrast testing was used to compare data from multiple groups. Statistical significance was assumed at P < 0.05.
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RESULTS |
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Effects of 11,12-EET on KATP channels in rat ventricular myocytes
In the presence of symmetrical K+ (140 mM) and low cytoplasmic ATP concentrations (1 µM), inside-out recordings from rat ventricular myocytes showed robust KATP channel openings with single channel amplitudes of about 5 pA at -60 mV (Fig. 1). Increasing the concentration of cytoplasmic ATP decreased the frequency of channel openings without affecting the current amplitude (Fig. 1B, left). Channel activity was totally suppressed by 1 mM ATP and this inhibition was readily reversible upon washout of ATP (Fig. 1A and B). Glyburide (5 µM) also reversibly inhibited the channel activity (data not shown). However, in the presence of 5 µM 11,12-EET, 1 mM ATP was unable to completely suppress the KATP channels, with typically about 25 % of the channels remaining active (Fig. 1A and B). Even in the presence of 5 mM ATP, which represents the typical physiological intracellular ATP concentration, 5 µM 11,12-EET was able to activate KATP channels. These results suggest that 11,12-EET antagonizes the inhibition by ATP and modulates the gating mechanism of the KATP channel.
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Figure 1. Activation of cardiac KATP channels by 11,12-EET A, macroscopic KATP currents of rat ventricular myocytes were recorded continuously from a membrane potential of -60 mV in an inside-out patch. B, dose-dependent inhibition of KATP activity by ATP (1-5 mM), in the presence of 0 (left) or 5 µM (right) 11,12-EET. Dotted lines in A and B represent the closed (C) channel level. C, relationship of the normalized open probability (Po) plotted against the ATP concentration in the presence of 0 ( | ||
The dose-dependent effects of 11,12-EET on the KATP channel are illustrated in Fig. 1C. Fractional block of the KATP channel was plotted as a function of ATP concentration, in the absence and in the presence of 0.1 and 5 µM 11,12-EET. Under control conditions, the KATP channel activity was inhibited by ATP with an IC50 = 21.2 ± 2.0 µM (n = 5) and a Hill coefficient of 0.96 ± 0.06 (Fig. 1C), consistent with results previously reported (Edwards & Weston, 1993; Yokoshiki et al. 1998). 11,12-EET shifted the ATP dose-response curve to the right, increasing the ATP IC50 to 240 ± 60 µM with 0.1 µM 11,12-EET (n = 5, P < 0.05 vs. control) and to 780 ± 30 µM with 5 µM 11,12-EET (n = 10, P < 0.05 vs. control), without altering the Hill coefficient (nH = 0.96 ± 0.04, n = 5, P = n.s. vs. control). Thus, the sensitivity of cardiac KATP channels to ATP was decreased 11.4-fold with 0.1 µM 11,12-EET and 37-fold with 5 µM 11,12-EET.
Effects of 8,9-EET, 11,12-EET, 11,12-DHET and AA on cardiac KATP channels
We examined and compared the effects of 8,9- and 11,12-EET, which are the major EET regioisomers in the rat heart, accounting for 67 % of the total (Wu et al. 1997). Figure 2A shows results from a typical experiment illustrating the effects of different concentrations of 11,12-EET on KATP channel activities in the presence of 100 µM cytoplasmic ATP. The half-maximum activation concentration (EC50) for 11,12-EET was 18.9 ± 2.6 nM (n = 5) with a maximal channel Po of about 0.6 (Fig. 2B). The EC50 for 8,9-EET was 19.1 ± 4.8 nM (n = 5, P = n.s. vs. 11,12-EET; Fig. 2B), and the dose-response curves for the two regioisomers were virtually indistinguishable. Our results suggest that both 8,9- and 11,12-EET are potent activators of the cardiac KATP channels.
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Figure 2. Comparing the effects of AA, 8,9-EET, 11,12-EET and 11,12-DHET on cardiac KATP channels A, raw current tracings showing the dose-dependent effect of 11,12-EET (0.001-1 µM) on the KATP channel activity. Single channel KATP currents were recorded at -60 mV in the presence of 100 µM cytoplasmic ATP. B, dose-response relationships of 8,9-EET ( | ||
We also compared the effects of 11,12-EET with those of its precursor (AA) and its metabolite (11,12-DHET) on KATP channel Po at -60 mV in the presence of 100 µM ATP. The chemical structures of 11,12-EET (epoxide) and 11,12-DHET (diol) are similar to AA except they have one less double bond and an oxygen-containing group substituted in its place. Figure 2C shows that the KATP channel Po was increased by 400 ± 54 % with 5 µM 11,12-EET (n = 10, P < 0.05 vs. control), whereas neither 5 µM AA (n = 7) nor 5 µM 11,12-DHET (n = 8) affected the channel Po. These results indicate that the epoxide group of 11,12-EET is an important structural determinant for the enhancement of KATP channel activity.
11,12-EET did not alter the inhibition of KATP channels by glyburide
ATP and glyburide are both inhibitors of the KATP channel, although their mechanisms of action are different (Edwards & Weston, 1993; Aguilar-Bryan et al. 1998). We examined whether 11,12-EET modulates the inhibition of the cardiac KATP channels by glyburide. In the presence of 1 µM cytoplasmic ATP, the glyburide IC50 was 20.3 ± 7.1 nM (n = 5, control) and it was 15.4 ± 1.4 nM (n = 5, P = n.s. vs. control) when 0.1 µM 11,12-EET was added. The Hill coefficient was also unchanged at 1.2 ± 0.1 for control (n = 5) and 1.0 ± 0.1 with 0.1 µM 11,12-EET (n = 5, P = n.s. vs. control). These results indicate that 11,12-EET does not activate the KATP channels when the channels are inhibited by sulfonylurea drugs.
Effects of ATP on KATP channel kinetics
The kinetics of the KATP channel are characterized by bursts of openings separated by long closures, and involving many kinetically distiguishable states, depending on the ATP concentration (Qin et al. 1989; Alekseev et al. 1998; Trapp et al. 1998; Fan & Makielski, 1999; Lin et al. 2000; Enkvetchaul et al. 2000). To gain insight into the biophysical mechanisms whereby 11,12-EET antagonizes the inhibitory effect of ATP on the KATP channel, we first examined the effects of ATP on KATP channel kinetics.
At low cytoplasmic ATP concentrations (< 10 µM), the KATP channel openings were interrupted mainly by short flicker closures, and long closures that define bursts were rare (Fig. 3A and B). With higher concentrations of ATP (
100 µM), long closures became much more evident and the apparent burst durations decreased (Fig. 3C and D). The channel open durations at all ATP concentrations examined were adequately described by a single exponential component, and the mean open time constant (
o) decreased with increasing concentrations of ATP (Fig. 3, open time histograms). The channel closed durations at low cytoplasmic ATP concentrations were well described by the sum of two exponential components (Fig. 3A and B). The time constant of the fast closed time component (c1) typically had a value of 0.7-0.8 ms and it represented the short flicker closures within bursts. The long closed time component (c2) represented the interburst intervals (Fig. 3A and B). At higher concentrations of ATP, additional longer closed duration components (c3 and c4) emerged (Fig. 3C and D). Interestingly, the mean duration of the intraburst flicker closures (c1) was independent of the ATP concentration and remained at 0.7-0.8 ms accounting for 54-98 % of all the closed events.
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Figure 3. Effect of ATP on cardiac KATP channel kinetics Single KATP channel activity was recorded at -60 mV in the presence of 1 µM (A), 10 µM (B), 100 µM (C) and 1000 µM (D) cytoplasmic ATP. Representative KATP channel tracings are shown on the left with selected segments expanded to show more details. The corresponding channel open dwell-time histograms and the closed dwell-time histograms are displayed to the right of the tracings. The dashed lines represent the distribution of the exponential components determined by the likelihood ratio test. The continuous line represents the best fit of data using TAC software. The values of the open ( | ||
The KATP channel kinetics in the presence of ATP can be described using a simple three-state model (Scheme 1; see Discussion):
where C1 
O represents the transitions between open and intraburst channel closings and O C2* represents the transitions between open and interburst channel closings. The ATP association step(s) occurs during the O C2* transitions. In addition, C2* represents a group state, which contains multiple closed states that contribute to the multiple interburst components (c2, c3 and c4). Using this model, the transition rate constants between the sojourn states can be calculated using the following equations (Sakmann & Trube, 1984): kc1o = 1/c1, kB = 1/c2, koc1 = f1/o and kA = f2/o, where f1 and f2 represent the closed time fractions of c1 and c2, respectively. Here, the mean intraburst closed time is given by 1/c1, the mean open time by 1/o, assuming that the C1 O transitions are fast and at equilibrium, the mean burst duration by 1/((kc1o/(kc1o + koc1))kA), and the mean interburst closed time by 1/kB. The dependence of these rate constants in the three-state model on ATP is illustrated in Fig. 4. The model above predicts that the reciprocal of o is a linear function of [ATP]. However, we found that the reciprocal of o plotted against ATP concentration, as shown in Fig. 4A, was not linear, indicating that a complete description of the KATP channel kinetics requires additional states (Qin et al. 1989; Alekseev et al. 1998; Trapp et al. 1998; Fan & Makielski, 1999; Lin et al. 2000; Enkvetchaul et al. 2000). The observed non-linear dependence of the mean open time on [ATP] suggests that multiple ATP molecules may interact with the open state and/or that multiple channel closing pathways, each with different ATP dependence, could exist. The rate constant kc1o, which is the reciproal of the fast closed time constant c1, was essentially ATP independent at about 1400 s-1 (Fig. 4B). The relationship between the reciprocal of the mean interburst duration and ATP concentration was complex, suggesting that additional ATP interactions may occur (Fig. 4C). The single channel results indicate that a complete description of the channel kinetics requires additional states; however, our dwell time results alone do not sufficiently constrain the model/parameter selection. Thus, we opted to use the three-state model above as the framework to compare the effects of ATP and 11,12-EET on the KATP channel.
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Figure 4. Effects of ATP on the mean open time, the intraburst closed time and the mean interburst duration of the KATP channels Reciprocals of the KATP channel mean open time, | ||
11,12-EET reduces the apparent association rate of ATP to the KATP channel
Our results showed that ATP decreases the KATP channel open time (Fig. 3 and Fig. 4A), suggesting ATP interacts with the open state of the channel. We also found that the relationship between ATP and the reciprocal of the mean burst durations was roughly linear (Fig. 4D). This observation is consistent with the idea that binding of one ATP molecule terminates the KATP channel burst openings. From the slopes of the curves in Fig. 4D, the apparent ATP association rate constant (kA) for the burst termination step can be estimated to be 6.24 105 M-1 s-1, which is similar to the reported value of 6 105 M-1 s-1 by Davies et al. (1992) and to that of 8 105 M-1 s-1 by Fan & Makielski (1999). Application of 1 µM 11,12-EET reduced kA 135-fold to 4.64 103 M-1 s-1.
Voltage-dependent effects of 11,12-EET on KATP channel kinetics
Figure 5A shows representative recordings of single KATP channels at membrane potentials from -80 to +80 mV, in the presence of 100 µM ATP and 0.1 µM 11,12-EET. The unitary conductance () of the KATP channel was 80.9 ± 0.5 pS (n = 4), similar to values reported by other laboratories (Noma, 1983; Yokoshiki et al. 1998), and application of 11,12-EET did not change (79.6 ± 0.6 pS, n = 4, P = n.s.; Fig. 5B). 11,12-EET (0.1 µM) enhanced the Po 7-fold at all negative membrane potentials examined but had no effect on the KATP channel Po at positive membrane potentials, suggesting that the effects of 11,12-EET as an activator of the KATP channel are highly voltage dependent (Fig. 5C). In contrast, the channel Po in the presence of ATP alone was unaffected by voltage (Fig. 5C). These results suggest that the overall interaction between 11,12-EET and the KATP channel is voltage dependent.
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Figure 5. Voltage-dependent effects of 11,12-EET on the cardiac KATP channels A, single KATP channel currents were recorded at membrane potentials from -80 to +80 mV in the presence of 100 µM cytoplasmic ATP and 0.1 µM 11,12-EET. B, the KATP channel current-voltage relationships are plotted, showing the characteristic weak inward rectification at strong depolarizations. Continuous lines represent the best fits using equations described in Methods. Single channel conductance ( | ||
Dose-dependent effect of 11,12-EET on single KATP channel kinetics
We examined the dose-dependent effects of 11,12-EET on the KATP channel activity in the presence of 100 µM ATP, which inhibits the KATP channel by 80 % (Fig. 1C). Representative single channel openings and the corresponding open and closed durations are shown in Fig. 6. Without 11,12-EET, the KATP channel openings were interrupted by long closures and the overall Po was 0.12. With increasing concentrations of 11,12-EET, the long closures became less frequent, dramatically increasing Po (5-fold or more). The inhibitory effect of 100 µM ATP on Po was almost completely abolished by 0.1 µM 11,12-EET. As in Fig. 3, the open time histograms showed a single exponential component and the mean open as well as the mean burst durations were prolonged by increasing concentrations of 11,12-EET (Fig. 6). The long closed events became less common with higher concentrations of 11,12-EET. With ATP (100 µM) alone, the closed time histograms showed three distinct components (0.62, 26.8 and 1200 ms). With the addition of 1 µM 11,12-EET, the long closed durations virtually disappeared, and channel closure was dominated by the fast intraburst flicker closure (c1 = 0.7 ms), accounting for 95 % of all the closed events. These results show that 11,12-EET lengthens the open time in a dose-dependent manner (Fig. 7A) and shortens the mean interburst duration (Fig. 7C) of the KATP channels. As found with ATP alone, 11,12-EET did not alter the fast closed time constant, c1 (Fig. 7B). Thus, ATP and 11,12-EET have opposite effects on the overall KATP channel activity; 11,12-EET reverses the inhibitory effects of ATP on the channel.
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Figure 6. Dose-dependent effects of 11,12-EET on cardiac KATP channel kinetics Single KATP channel activities were recorded at -60 mV in the presence of 100 µM ATP and 0 µM (A), 0.01 µM (B), 0.1 µM (C) and 1 µM (D) 11,12-EET. Representative KATP channel tracings are shown on the left with selected segments enlarged to show more details. The corresponding channel open time histograms and the closed time histograms are shown to the right of the tracings. The values of the open and closed time constants are displayed above each histogram. The relative weight of each closed time constant is given in parentheses and the sum of all weights is equal to 100 %. | ||
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Figure 7. Effects of 11,12-EET on the mean open time, the intraburst closed time and the mean interburst duration of the KATP channels Reciprocals of the KATP channel mean open time, | ||
The antagonism between ATP and EET can be further delineated by comparing the KATP single channel kinetic behaviour in the presence of 10 µM ATP and 100 µM ATP plus 1 µM 11,12-EET. Under these conditions, the channel has a similar overall Po, and the open and closed durations are adequately described by one and two exponential components, respectively. Therefore, we used the simple three-state model presented earlier as a tool to compare the dwell times in the two conditions. Table 1 shows the open time constant (o) and the fast closed time constant (c1) at membrane potentials from -80 to -20 mV. Depolarization enhanced o but shortened c1, and 11,12-EET did not change the voltage-dependent effects on o and c1. 11,12-EET, however, exerts its effects almost exclusively on the interburst closed time constant (c2). The voltage dependence of these rate constants is shown in Fig. 8. kc1o increased but koc1 and kA decreased with membrane depolarization and 11,12-EET did not change the voltage dependence of these rate constants (Fig. 8A-C). The principal effect of 11,12-EET was on kB (Fig. 8D). In the presence of 10 µM ATP alone, kB slightly increased with membrane depolarization. However, in the presence of 1 µM 11,12-EET and 100 µM ATP, kB showed steep voltage dependence and decreased with membrane depolarization. These findings suggest that the sites of action of ATP and 11,12-EET are different and kinetically distinguishable.
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Figure 8. Comparison of the transition rate constants between 10 µM ATP alone and 100 µM ATP plus 1 µM 11,12-EET The transition rates between the channel open and closed states were calculated and compared for 10 µM ATP alone (Control; open symbols and dotted lines) and 100 µM ATP plus 1 µM 11,12-EET (filled symbols and continuous lines). The transition rate constants from the intraburst closed state to the open state, kc1o (A), the open state to the intraburst closed state, koc1 (B), the open state to the interburst closed state, kA (C) and the interburst closed state to the open state, kB (D) (n = 3) are plotted against membrane potential (-20 to -80 mV). The data were fitted using a single exponential equation. The charge movement associated with each rate constant was estimated by fitting the voltage dependence of each transition rate to the following exponential function: k = koexp(z | ||
11,12-EET hyperpolarizes the resting membrane potential in cardiac myocytes through activation of the KATP channels
To determine whether the activation of the cardiac KATP channels by 11,12-EET is physiologically relevant, we investigated the effects of 11,12-EET on the action potentials in isolated rat ventricular myocytes using current-clamp techniques with the pipette solution containing 5 mM ATP. Recordings from a representative experiment are shown in Fig. 9A and results are summarized in Table 2. The resting membrane potential was -73.1 ± 2.0 mV (n = 7) under control conditions and exposure to 5 µM 11,12-EET resulted in significant hyperpolarization (-77.0 ± 2.3 mV, n = 7, P < 0.05 vs. control). The effects of 11,12-EET on the resting potential were totally reversed by simultaneous exposure to 2 µM glyburide (-71.3 ± 0.8 mV, n = 7, P = n.s. vs. control; Fig. 9B), suggesting that the 11,12-EET effects were mediated through the KATP channels. While the effect of 11,12-EET on the resting membrane potential was observed in every myocyte examined, the effect of 11,12-EET on the action potential duration was variable: it could shorten or lengthen the action potential duration at 90 % repolarization (APD90). These results indicate that 11,12-EET could modulate the electrophysiological properties of cardiac myocytes through activation of the KATP channels and may be important in the regulation of cardiac excitability.
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Figure 9. Effect of 11,12-EET on the resting membrane potential in cardiac myocytes A, recordings of action potential from an isolated cardiac myocyte at 37 °C using current-clamp techniques. The patch-clamp pipette contained 5 mM ATP and action potentials were elicited by 1 nA stimuli at 0.5 Hz. Tracings at baseline (Control), with 11,12-EET (5 µM), and with 11,12-EET (5 µM) plus glyburide (2 µM) are superimposed for comparison. B, bar graph comparing the resting membrane potentials of isolated cardiac myocytes under control conditions, and in the presence of 11,12-EET (5 µM) and 11,12-EET (5 µM) plus glyburide (2 µM). The data are presented as means ± S.E.M., n = 7, *P < 0.05 vs. Control. | ||
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DISCUSSION |
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The major conclusions that can be drawn from this study are as follows. First, we have demonstrated that 8,9- and 11,12-EET are potent activators of the cardiac KATP channels with an EC50 in the 10-8 M range. Second, we have demonstrated that EETs can activate the cardiac KATP channels in the presence of physiological concentrations of ATP. Third, EETs reduce the sensitivity of the cardiac KATP channels to cytoplasmic ATP by more than 10-fold. Fourth, 11,12-EET modulates the gating behaviour of the cardiac KATP channels by prolonging channel opening times, lengthening channel burst durations, while shortening the interval of channel closure between bursts. Fifth, EET and ATP have different sites of action on the KATP channel and their mechanisms are kinetically distinguishable. Sixth, 11,12-EET hyperpolarizes the resting membrane potentials of cardiac myocytes through activation of the KATP channels. Therefore, EETs could play an important role in the regulation of cardiac electrical excitability.
Effects of 11,12-EET on the cardiac KATP channel
In the present study, we showed that EETs are potent activators of the cardiac KATP channels. Although the cytoplasmic concentration of EETs in heart has not been directly measured, it has been suggested that EETs are present in nanomolar concentrations in plasma (Karara et al. 1992), and the tissue concentration could reach the micromolar range with activation of phospholipase A2 (Zhu et al. 1995). Studies in cultured vascular endothelial and smooth muscle cells have demonstrated that EETs are avidly incorporated into cellular phospholipids (Fang et al. 1996; Weintraub et al. 1997). Once incorporated, EETs can be readily released from cultured endothelial cells in response to the calcium ionophore A23187 (Weintraub et al. 1999). Hence, an EC50 of 10-8 M for 11,12-EET in cardiac KATP channel activation should be highly physiologically relevant.
It is also important to note that the cellular concentration of EETs could be significantly increased during conditions such as cardiac ischaemia. Ischaemia-induced hydrolysis of phospholipids occurs in many tissues, including the heart (Hazen & Gross, 1992), leading to the release of AA and other esterified fatty acids. The increase in AA may promote the in situ formation of EETs through the cytochrome P450 mono-oxygenase pathway. At the same time, significant amounts of esterified EETs can be released through hydrolysis of phospholipids (Zhu et al. 1995). Moreover, the amount of esterified EETs in phospholipids can be dramatically increased (59-fold increase) by ischaemia, which induces free radical oxidation of membrane phospholipids (Nakamura et al. 1997). Hence, during cardiac ischaemia, free EETs could increase many fold through several mechanisms, and their role in the modulation of the cardiac KATP channels could be even more important under these conditions.
Activation of the KATP channels by the AA metabolites demonstrates unique structural specificity. 8,9- and 11,12-EET activate the KATP channels with equal potency. However, neither the parent compound, AA, nor the metabolite, 11,12-DHET, was effective in activating the KATP channels. These results suggest that the epoxide substitution at the double bond is an important structural determinant for activation of the channels. Thus, non-specfic fatty acid effects, such as altering membrane fluidity, are not responsible for the observed effects of EETs on cardiac KATP channels.
Mechanism of the effects of EET on the KATP channel
The major mechanism through which 11,12-EET activates the cardiac KATP channels is by decreasing the overall sensitivity of the channel to ATP inhibition. 11,12-EET at 0.1 µM produced an 11.4-fold increase and at 5 µM a 37-fold increase in the ATP IC50. This apparent decrease in ATP sensitivity allowed 25 % of the channels to remain active in the presence of 1 mM cytoplasmic ATP, while almost 10 % of the channels remained active even in the presence of 5 mM ATP. These findings are highly provocative. Because of the high density and conductance of the KATP channels in heart, it has been estimated that the opening of 1 % of these channels would result in a 50 % reduction in the cardiac action potential duration (Nichols & Lederer, 1991). Further analysis of the channel kinetics allowed us to gain insight into the mechanism of the 11,12-EET effects. 11,12-EET at 1 µM reduced the apparent ATP association rate 135-fold, from ~6.24 105 M-1 s-1 at baseline to 4.64 103 M-1 s-1. Hence, our results suggest that EETs are endogenous activators of the KATP channels through modulation of ATP sensitivity.
Each cardiac KATP channel consists of two types of subunit: an inwardly rectifying K+ channel, Kir6.2, and a sulfonylurea receptor protein, SUR2A, forming an octameric complex of 4:4 stoichiometry (Aguilar-Bryan et al. 1998; Ashcroft & Gribble, 1998). The site that determines ATP sensitivity on the KATP channel remains to be established. There is no consensus sequence in Kir6.2 for ATP binding, but recent evidence suggests that the cytoplasmic segments of both the N- and C-termini in Kir6.2 are important in mediating ATP sensitivity of the channel, based on mutagenesis and photoaffinity labelling studies (Drain et al. 1998; Takano et al. 1998; Tucker et al. 1998; Tanabe et al. 1999). The site on the KATP channel that interacts with EETs is unknown. Given the difference in structure between ATP and EETs and the requirement of the epoxide substitution for EETs to activate the KATP channels, it is unlikely that ATP and EETs bind to the same sites on the channel. The effects of 11,12-EET are dramatically voltage dependent, with potent activation of the KATP channels at all negative potentials but no effect at all positive membrane potentials. It is plausible that a crucial site mediating the EET effects involves a charged residue in the membrane. Positive potentials would induce a conformational change at that site, uncoupling the EET interaction. In contrast, the effects of ATP were not voltage dependent. Further delineation of the sites and mechanisms of EET interaction will require molecular approaches to correlate structure-function relationships.
Modelling the effects of 11,12-EET on KATP channel kinetics
The kinetics of the cardiac KATP channel are complex. ATP did not alter the mean fast intraburst closed time. However, ATP reduced the mean open time in native cardiac myocytes and in heterologously expressed Kir6.2/SUR2A channels (Nichols et al. 1991; Trapp et al. 1998; Fan & Makielski, 1999). Thus, ATP is probably not involved with the fast intraburst flicker closures, but it interacts with the open channel. We identified at least one closed and one open state within the bursts and at least three closed states that separately contribute to the interburst intervals. This finding is generally consistent with previous reports (Nichols et al. 1991; Alekseev et al. 1998; Trapp et al. 1998; Enkvetchaul et al. 2000). A general kinetic scheme consistent with our dwell time results is shown in Scheme 2:
The data presented in this study do not precisely establish how these kinetically distinguishable states communicate with each other. Based on our results, we tried to explain the KATP channel behaviour using the C1 O C2* three-state model presented earlier. In this model, the interburst closed states are grouped into C2*. A full description of the intraburst kinetics requires additional kinetics states as the model fails to account for the non-linear dependence of the mean open time on ATP (Fig. 4A) and ATP may also influence the transitions within C2* (Fig. 3). However, this model is useful in comparing the effects of ATP and 11,12-EET. We found that 11,12-EET exerts its effects on both kA and kB, hence it not only reduces the exit rate from the open to the long closed state, but also increases the transition rate from the long closed state to the open state. In addition, the effects of 11,12-EET on kB are voltage dependent, with greater effects at resting potentials. These findings suggest that 11,12-EET modulates the KATP channel not only by reducing the apparent ATP association rate constant, but also by accelerating the rate from closed state to open state. Hence, 11,12-EET shifts the equilibrium between O C2* (equilibrium constant K = kB/kA) towards O.
Physiological implications of the modulation of cardiac KATP channels by EETs
Despite the considerable wealth of information on KATP channel modulation, activation of these channels under physiological conditions remains controversial. Nevertheless, using perforated patch techniques, the KATP channels in freshly dissociated atrial myocytes can be activated upon repeated exposure to acetylcholine (Wang & Lipsius, 1995). Ashcroft & Ashcroft (1990) have also reported that about 10 % of the KATP channels could be activated despite the relatively high levels of intracellular ATP. Single KATP channel measurements in saponin-permeabilized cardiac myocytes showed an IC50 for ATP of 0.5 mM (Kakei et al. 1985). The discrepancy between the IC50 values for ATP in excised membrane patches vs. intact cells implicates the presence of endogenous cellular KATP channel activators. With the high density and large conductance of the cardiac KATP channels, they can generate considerable amounts of repolarizing currents at plateau potentials. There are indications that KATP channels could potentially modulate the electrophysiology of the normal heart, yet the identity of endogenous KATP channel activators has been elusive. In this study, we have demonstrated that 11,12-EET hyperpolarizes the resting potential in cardiac myocytes through activation of the KATP channels, indicating that EETs are capable of modulating cardiac excitability under physiological conditions. This effect could be particularly important in restoring proper diastolic membrane potentials under conditions of cardiac ischaemia. The variable effect of 11,12-EET on the APD90 is probably due to the fact that the effects of EETs on the cardiac action potential are complex. EETs are known to modulate various voltage-dependent cardiac ion channels, including inhibition of Na+ channels (Lee et al. 1999) and activation of L-type Ca2+ channels (Xiao et al. 1998). Nevertheless, the effect of 11,12-EET on the resting membrane potential was observed in every myocyte examined and is consistent with our observation that the effect of 11,12-EET is voltage dependent, enhancing the KATP channel activity only at negative potentials.
Recent reports have demonstrated that PIP and PIP2, like EETs, can dramatically reduce the sensitivity of the KATP channel to ATP (Fan & Makielski, 1997, 1999; Baukrowitz et al. 1998; Shyng & Nichols, 1998). PIP2 is capable of activating KATP channels in the presence of millimolar concentrations of ATP. There is an apparent requirement of a hydrophobic tail and a charged hydrophilic head for the anionic phospholipids to activate KATP channels (Fan & Makielski, 1997). It has been proposed that the positively charged residues at the beginning of the C-terminus of Kir6.2 are anchored or tethered by the electrostatic force of the anionic groups of the phospholipids at the cytoplasmic face of the membrane, allowing the channel to be active (Fan & Makielski, 1997), and this may also distort the ATP binding site to prevent binding (Ashcroft, 1998). Although EETs are structurally very different from PIP and PIP2, they all contain an anionic group attached to a large hydrophobic tail. Hence, it would be of interest to compare the effects of EETs and phosphoinositides on the KATP channel, to determine whether they produce activation through a similar mechanism.
In summary, we have demonstrated here that EETs potently and selectively activate KATP channels in cardiac myocytes. The mechanism involves decreasing the overall sensitivity of the channel to ATP inhibition, although EETs and ATP appear to work through different effector sites on the channel. These lipid metabolites, nevertheless, could be important endogenous activators of the KATP channel under normal conditions and in disease states such as ischaemia.
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REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| AGUILAR-BRYAN, L., CLEMENT, J. P. IV, GONZALEZ, G., KUNJILWAR, K., BABENKO, A. & BRYAN, J. (1998). Toward understanding the assembly and structure of KATP channels. Physiological Reviews 78, 227-245 | |
| ALEKSEEV, A. E., BRADY, P. A. & TERZIC, A. (1998). Ligand-insensitive state of cardiac ATP-sensitive K+ channels: basis for channel opening. Journal of General Physiology 111, 381-394 | [Abstract/Full Text] |
| ASHCROFT, F. M. (1998). Exciting times for PIP2. Science 282, 1159-1160 | |
| ASHCROFT, F. M. & GRIBBLE, F. M. (1998). Correlating structure and function in ATP-sensitive K+ channels. Trends in Neurosciences 21, 288-294 | [Medline] |
| ASHCROFT, S. J. & ASHCROFT, F. M. (1990). Properties and function of ATP-sensitive K-channels. Cellular Signaling 2, 197-214 | |
| BAUKROWITZ, T., SCHULTE, U., OLIVER, D., HERLITZE, S., KRAUTER, T., TUCKER, S. J., RUPPERSBERG, J. P. & FAKLER. B. (1998). PIP2 and PIP as determinants for ATP inhibition of KATP channels. Science 282, 1141-1144 | |
| CAMPBELL, W. B., GEBREMEDHIN, D., PRATT, P. F. & HARDER, D. R. (1996). Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circulation Research 78, 415-423 | [Abstract/Full Text] |
| DAVIES, N. W., STANDEN, N. B. & STANFIELD, P. R. (1992). The effect of intracellular pH on ATP-dependent potassium channels of frog skeletal muscle. Journal of Physiology 455, 549-568 | |
| DRAIN, P., LI, L. & WANG, J. (1998). KATP channel inhibition by ATP requires distinct functional domains of the cytoplasmic C terminus of the pore-forming subunit. Proceedings of the National Academy of Sciences of the USA 95, 13953-13958 | [Abstract/Full Text] |
| EDWARDS, G. & WESTON, A. H. (1993). The pharmacology of ATP-sensitive potassium channels. Annual Review of Pharmacology and Toxicology 33, 597-637 | [Medline] |
| ENKVETCHAKUL, D., LOUSSOUARN, G., MAKHINA, E., SHYNG, S. L. & NICHOLS, C. G. (2000). The kinetic and physical basis of KATP channel gating: toward a unified molecular understanding. Biophysical Journal 78, 2334-2348 | [Abstract/Full Text] |
| FAN, Z. & MAKIELSKI, J. C. (1997). Anionic phospholipids activate ATP-sensitive potassium channels. Journal of Biological Chemistry 272, 5388-5395 | [Abstract/Full Text] |
| FAN, Z. & MAKIELSKI, J. C. (1999). Phosphoinositides decrease ATP sensitivity of the cardiac ATP-sensitive K+ channel. A molecular probe for the mechanism of ATP-sensitive inhibition. Journal of General Physiology 114, 251-269 | [Abstract/Full Text] |
| FANG, X., KADUCE, T. L., WEINTRAUB, N. L., VANROLLINS, M. & SPECTOR, A. A. (1996). Functional implications of a newly characterized pathway of 11,12-epoxyeicosatrienoic acid metabolism in arterial smooth muscle. Circulation Research 79, 784-793 | [Abstract/Full Text] |
| HAMILL, O. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflügers Archiv 391, 85-100 | [Medline] |
| HAZEN, S. L. & GROSS, R. W. (1992). Principles of membrane biochemistry and their application to the pathophysiology of cardiovascular disease. In The Heart and Cardiovascular System, second edn, ed. FOZZARD, H. A., HABER, E., JENNINGS, R. B., KATZ, A. M. & MORGAN, H. E., pp. 839-860. Raven Press, New York | |
| HORIE, M., IRISAWA, H. & NOMA, A. (1987). Voltage-dependent magnesium block of adenosine-triphosphate-sensitive potassium channel in guinea-pig ventricular cells. Journal of Physiology 387, 251-272 | [Abstract] |
| KAKEI, M., NOMA, A. & SHIBASAKI, T. (1985). Properties of adenosine-triphosphate-regulated potassium channels in guinea-pig ventricular cells. Journal of Physiology 363, 441-462 | [Abstract] |
| KARARA, A., WEI, S., SPADY, D., SWIFT, L., CAPDEVILA, J. H. & FALCK, J. R. (1992). Arachidonic acid epoxygenase: structural characterization and quantification of epoxyeicosatrienoates in plasma. Biochemical and Biophysical Research Communications 182, 1320-1325 | [Medline] |
| KOYANO, T., KAKEI, M., NAKASHIMA, H., YOSHINAGA, M., MATSUOKA, T. & TANAKA, H. (1993). ATP-regulated K+ channels are modulated by intracellular H+ in guinea-pig ventricular cells. Journal of Physiology 463, 747-766 | [Abstract] |
| LEE, H., LU, T., WEINTRAUB, N. L., VANROLLINS, M., SPECTOR, A. A. & SHIBATA, E. F. (1999). Effects of epoxyeicosatrienoic acids on the cardiac sodium channels in isolated rat ventricular myocytes. Journal of Physiology 519, 153-168 | [Abstract/Full Text] |
| LIN, Y. F., JAN, Y. N. & JAN, L. Y. (2000). Regulation of ATP-sensitive potassium channel function by protein kinase A-mediated phosphorylation in transfected HEK293 cells. EMBO Journal 19, 942-955 | [Abstract/Full Text] |
| MCGIFF, J. C. (1991). Cytochrome P-450 metabolism of arachidonic acid. Annual Review of Pharmacolgy and Toxicology 31, 339-369 | |
| NAKAMURA, T., BRATTON, D. L. & MURPHY, R. C. (1997). Analysis of epoxyeicosatrienoic and monohydroxyeicosatetraenoic acids esterified to phospholipids in human red blood cells by electrospray tandem mass spectrometry. Journal of Mass Spectrometry 32, 888-896 | [Medline] |
| NAKAYA, H., TAKEDA, Y., TOHSE, N. & KANNO, M. (1991). Effects of ATP-sensitive K+ channel blockers on the action potential shortening in hypoxic and ischemic myocardium. British Journal of Pharmacology 103, 1019-1026 | [Abstract] |
| NICHOLS, C. G. & LEDERER, W. J. (1990). The regulation of ATP-sensitive K+ channel activity in intact and permeabilized rat ventricular myocytes. Journal of Physiology 423, 91-110 | [Abstract] |
| NICHOLS, C. G. & LEDERER, W. J. (1991). Adenosine triphosphate-sensitive potassium channels in the cardiovascular system. American Journal of Physiology 261, H1675-1686 | [Medline] |
| NICHOLS, C. G. & LEDERER, W. J. & CANNELL, M. B. (1991). ATP-dependence of KATP channel kinetics in isolated membrane patches from rat ventricle. Biophysical Journal 60, 1164-1177 | [Abstract] |
| NOMA, A. (1983). ATP-regulated K+ channels in cardiac muscle. Nature 305, 147-148 | [Medline] |
| OLTMAN, C. L., WEINTRAUB, N. L., VANROLLINS, M. & DELLSPERGER, K. C. (1998). Epoxyeicosatrienoic acids and dihydroxyeicosatrienoic acids are potent vasodilators in the canine coronary microcirculation. Circulation Research 83, 932-939 | [Abstract/Full Text] |
| QIN, D. Y., TAKANO, M. & NOMA, A. (1989). Kinetics of ATP-sensitive K+ channel revealed with oil-gate concentration jump method. American Journal of Physiology 257, H1624-1633 | [Medline] |
| SAKMANN, B. & TRUBE, G. (1984). Voltage-dependent inactivation of inward-rectifying single-channel currents in the guinea-pig heart cell membrane. Journal of Physiology. 347, 659-683 | [Abstract] |
| SHIGEMATSU, S. & ARITA, M. (1997). Anoxia-induced activation of ATP-sensitive K+ channels in guinea pig ventricular cells and its modulation by glycolysis. Cardiovascular Research 35, 273-282 | [Medline] |
| SHYNG, S. L. & NICHOLS, C. G. (1998). Membrane phospholipid control of nucleotide sensitivity of KATP channels. Science 282, 1138-1141 | |
| STANDEN, N. B. & STANFIELD, P. R. (1978). Inward rectification in skeletal muscle: a blocking particle model. Pflügers Archiv 378, 173-176 | [Medline] |
| TAKANO, M., XIE, L. H., OTANI, H. & HORIE, M. (1998). Cytoplasmic terminus domains of Kir6. x confer different nucleotide-dependent gating on the ATP-sensitive K+ channel. Journal of Physiology 512, 395-406 | [Abstract/Full Text] |
| TANABE, K., TUCKER, S. J., MATSUO, M., PROKS, P., ASHCROFT, F. M., SEINO, S., AMACHI, T. & UEDA, K. (1999). Direct photoaffinity labeling of the Kir6. 2 subunit of the ATP-sensitive K+ channel by 8-azido-ATP. Journal of Biological Chemistry 274, 3931-3933 | [Abstract/Full Text] |
| TERZIC, A., JAHANGIR, A. & KURACHI, Y. (1995). Cardiac ATP-sensitive K+ channels: regulation by intracellular nucleotides and K+ channel-opening drugs. American Journal of Physiology 269, C525-545 | [Medline] |
| TERZIC, A., TUNG, R. T., INANOBE, A., KATADA, T. & KURACHI, Y. (1994). G proteins activate ATP-sensitive K+ channels by antagonizing ATP-dependent gating. Neuron 12, 885-893 | [Medline] |
| TRAPP, S., PROKS, P., TUCKER, S. J. & ASHCROFT, F. M. (1998). Molecular analysis of ATP-sensitive K channel gating and implications for channel inhibition by ATP. Journal of General Physiology 112, 333-349 | [Abstract/Full Text] |
| TUCKER, S. J., GRIBBLE, F. M., PROKS, P., TRAPP, S., RYDER, T. J., HAUG, T., REIMANN, F. & ASHCROFT., F. M. (1998). Molecular determinants of KATP channel inhibition by ATP. EMBO Journal 17, 3290-3296 | [Abstract/Full Text] |
| TUNG, R. T. & KURACHI, Y. (1991). On the mechanism of nucleotide diphosphate activation of the ATP-sensitive K+ channel in ventricular cell of guinea-pig. Journal of Physiology 437, 239-256 | [Abstract] |
| WANG, Y. G. & LIPSIUS, S. L. (1995). Acetylcholine activates a glibenclamide-sensitive K+ current in cat atrial myocytes. American Journal of Physiology 268, H1322-1334 | [Medline] |
| WEINTRAUB, N. L., FANG, X., KADUCE, T. L., VANROLLINS, M., CHATTERJEE, P. & SPECTOR, A. A. (1997). Potentiation of endothelium-dependent relaxation by epoxyeicosatrienoic acids. Circulation Research 81, 258-267 | [Abstract/Full Text] |
| WEINTRAUB, N. L., FANG, X., KADUCE, T. L., VANROLLINS, M., CHATTERJEE, P. & SPECTOR, A. A. (1999). Epoxide hydrolases regulate epoxyeicosatrienoic acid incorporation into coronary endothelial phospholipids. American Journal Physiology 277, H2098-2108 | |
| WU, S., MOOMAW, C. R., TOMER, K. B., FALCK, J. R. & ZELDIN, D. C. (1996). Molecular cloning and expression of CYP2J2, a human heart cytochrome P450 arachidonic acid epoxygenase highly expressed in heart. Journal of Biological Chemistry 271, 3460-3468 | [Abstract/Full Text] |
| WU, S., CHEN, W., MURPHY, E., GABEL, S., TOMER, K. B., FOLEY, J., STEENBERGEN, C., FALCK, J. R., MOOMAW, C. R. & ZELDIN, D. C. (1997). Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes. Journal of Biological Chemistry 272, 12551-12559 | [Abstract/Full Text] |
| XIAO, Y.-F., HUANG, L. & MORGAN, J. P. (1998). Cytochrome P450: a novel system modulating Ca2+ channels and contraction in mammalian heart cells. Journal of Physiology 508, 777-792 | [Abstract/Full Text] |
| YOKOSHIKI, H., SUNAGAWA, M., SEKI, T. & SPERELAKIS, N. (1998). ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells. American Journal of Physiology 274, C25-37 | [Medline] |
| ZHU, Y., SCHIEBER, E. B., MCGIFF, J. C. & BALAZY, M. (1995). Identification of arachidonate P-450 metabolites in human platelet phospholipids. Hypertension 25, 854-859 | [Abstract/Full Text] |
Acknowledgements
This work was supported by a Merit Review Award from the Department of Veterans Affairs, Program Project Grants from the National Institutes of Health HL-49264 and HL-62984, an RO1 Award from NIH HL-63754, and a Grant-in-Aid award (0051311Z) from the American Heart Association. T.H. was supported in part by the NIH (HL-61645). N.L.W. is a Clinician-Scientist awardee of the American Heart Association.
Corresponding author
H.-C. Lee: Room E 318-2 GH, Division of Cardiovascular Diseases, Department of Internal Medicine, The University of Iowa Hospitals and Clinics, 200 Hawkins Drive, Iowa City, IA 52242, USA.
Email: hon-chi-lee{at}uiowa.edu
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, n = 5), 0.1 µM (
, n = 5), or 5 µM (
, n = 10) 11,12-EET. Each point represents the mean ± S.E.M., and the continuous lines represent the best fits by a Hill equation.
) and 11,12-EET (
) on the reciprocal of the mean burst duration were compared with controls (
) that contained no added EET. Data were fitted using the equation 1/
= (kc1o/(kc1o + koc1))kA[ATP]. Since the flickering transitions are fast and reach equilibrium, then koc1/kc1o should be a constant, independent of [ATP]. Moreover, to allow long burst durations, kc1o should be faster or greater than koc1. Hence, our data suggest the following relationship: 0 < koc1 < kc1o 
1.5 ms-1, which suggests 0 < kc1o/(kc1o + koc1)