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Am J Physiol Heart Circ Physiol 284: H1865-H1871, 2003. First published February 6, 2003; doi:10.1152/ajpheart.01031.2002
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Vol. 284, Issue 5, H1865-H1871, May 2003

Isoflurane activates rat mitochondrial ATP-sensitive K+ channels reconstituted in lipid bilayers

Yuri Nakae1, Wai-Meng Kwok1,2, Zeljko J. Bosnjak1,3, and Ming Tao Jiang1

Departments of 1 Anesthesiology, 2 Pharmacology and Toxicology, and 3 Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of mitochondrial ATP-sensitive K+ (mitoKATP) channels is critical in myocardial protection induced by preconditioning with volatile anesthetics or brief periods of ischemia. In this study, we characterized rat mitoKATP channels reconstituted in lipid bilayers and examined their direct regulation by isoflurane. Mitochondria and the inner membrane fraction were isolated from rat ventricles and fused into lipid bilayers. On the basis of their inhibition by 5-hydroxydecanoate (5-HD)/ATP or activation by diazoxide, mitoKATP channels of several conductance states were observed in symmetrical (150 mM) potassium glutamate (26, 47, 66, 83, and 105 pS). Isoflurane (0.8 mM) increased the cumulative open probability from 0.09 ± 0.02 at baseline to 0.50 ± 0.09 (P < 0.05, n = 5), which was inhibited by 5-HD. Isoflurane caused a dose-dependent rightward shift in ATP inhibition of mitoKATP channels, which increased the IC50 for ATP from 335 ± 4 to 940 ± 34 µM at 0.8 mM (P < 0.05, n = 5~8). We conclude that direct activation of the mitoKATP channel by isoflurane is likely to contribute to volatile anesthetic-induced myocardial preconditioning.

heart; mitochondria; potassium channel; volatile anesthetics


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MITOCHONDRIAL ATP-sensitive K+ (mitoKATP) channels are considered critical in myocardial protection induced by ischemic preconditioning (6, 20) or by brief exposure to volatile anesthetics, known as anesthetic preconditioning (APC) (3). MitoKATP channels are located in the mitochondrial inner membranes, with a reported conductance ranging between 9.7 and 56 pS (10, 22, 30).

Garlid et al. (5) have shown that diazoxide selectively opens the mitoKATP channel, whereas 5-hydroxydecanoate (5-HD) selectively blocks them in mitoKATP proteoliposomes or native mitochondria. Liu et al. (16) demonstrated that diazoxide increases flavoprotein oxidation in rabbit cardiac myocytes. Moreover, because diazoxide protects the heart from ischemia-reperfusion injury and 5-HD inhibits its effect (4, 7), mitoKATP channels have been implicated as critical effectors/mediators of cardioprotection.

Previous studies (3, 11, 13, 14, 23, 25, 27) have shown that preconditioning by brief exposure to volatile anesthetic isoflurane (Iso) followed by washout protects the myocardium against subsequent ischemic injury, which mimics the ischemic preconditioning phenomenon. Experimental evidence suggests that the cardioprotective effect of Iso is mediated through adenosine receptors, protein kinase C (PKC), and KATP channels in hearts from humans (25) or animals (11, 13), which is blocked by 5-HD (23). Our previous study has shown that Iso increases mitochondrial flavoprotein fluorescence in guinea pig ventricular myocytes (14). Iso also induces delayed myocardial protection in isolated rabbit hearts (27). However, the proposed role of mitoKATP channels in APC in previous studies is all based on pharmacological or indirect evidence (i.e., flavoprotein fluorescence). In this study, we investigated the effect of Iso on native mitoKATP channels reconstituted in lipid bilayers. Our results are the first to indicate that Iso directly activates mitoKATP channels, which likely contributes to its myocardial protective effect in APC. Preliminary work has been previously presented elsewhere (12, 17).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was conducted according to National Institutes of Health standards (NIH Pub. No. 95-23, Revised 1996) and was approved by the Institutional Animal Use and Care Committee.

Isolation of mitochondria. Cardiac mitochondria were isolated according to the procedure of Solem and Wallace with modifications (26). Briefly, ventricles from fresh rat hearts were cut into small pieces in 6 volumes of ice-cold isolation buffer containing 30 mM MOPS (pH 7.2), 200 mM mannitol, 50 mM sucrose, 5 mM KH2PO4, 0.1% BSA, and 1 mM EGTA in the presence of protease inhibitor cocktails (Sigma). The tissues were then homogenized with a PT10 Polytron (Brinkman Instruments; Westbury, NY) for 15 s for three times at 30-s intervals. The homogenates were centrifuged at 3,000 g for 10 min. The supernatants were then centrifuged at 8,000 g for 20 min. The pellet was resuspended in the isolation medium without EGTA and centrifuged again at 8,000 g. The final pellet enriched in mitochondria was suspended in the isolation medium without EGTA and BSA and stored on ice for preparation of mitochondrial inner membranes.

Preparation of mitochondrial inner membranes. Submitochondrial fraction enriched in inner membranes was prepared as reported (2, 18). The mitochondria obtained from above were osmotically shocked by incubating in 10 mM phosphate buffer (pH 7.4) for 20 min and then in 20% sucrose for another 15 min. The membrane was sonicated with a sonicator (Dual Horn for model 550, Fisher Scientific; Hanover Park, IL) for 30 s for three times and centrifuged at 8,000 g for 10 min. The supernatants containing submitochondrial particles were fractionated using a continuous sucrose gradient (30-60%) and centrifuged at 80,000 g overnight in a SW28 rotor (Beckman). The inner mitochondrial membrane (which is enriched in the heavy fraction) was suspended with the isolation medium without EGTA and centrifuged at 184,000 g for 30 min. The final pellet-enriched inner mitochondrial membranes were suspended in the isolation medium without EGTA and BSA and stored at -80°C in small aliquots until use.

Reconstitution of mitoKATP channels into lipid bilayers. Inner mitochondrial membranes were fused into lipid bilayers as reported previously for ryanodine receptors (12) with modifications. Briefly, L-alpha -phosphatidylethanolamine (from the heart) and L-alpha -phophatidylserine (from the brain) in chloroform were mixed in a 1:1 (wt/wt) ratio, dried under nitrogen, and suspended in n-decane for a final concentration of 20 mg/ml. A delrin cup (with an aperture of 250 µm between the cis and trans chambers, 0.8 ml volume) was placed into a holder. Both chambers contained a symmetrical solution of 30 mM MOPS (pH 7.4), 150 mM potassium glutamate, 1 mM EGTA, 50 µM K2ATP, and 0.2 mM MgCl2. Ag/AgCl electrodes were placed into each chamber via agar salt bridges and connected to the headstage of the bilayer clamp amplifier (BC-525C, Warner Instruments; Hamden, CT). The cis chamber was held at virtual ground, whereas the trans chamber was at command. The experiments were performed at room temperature (23~25°C). Lipid bilayers were formed by painting the aperture with the lipids, and the mitochondrial membranes were added into the cis chamber. After the appearance of cation current, indicating successful fusion, single channel currents at a holding potential of +40 mV (trans/cis, -40 mV by convention) were collected using an Axon Digidata 1332 AD/DA interface (Axon Instruments; Union City, CA) with the pCLAMP program (version 8.01, Axon Instruments) on a personal computer (Pentium II). The currents were filtered at 0.5 kHz with an eight-pole Bessel filter and digitized at 2.5 kHz. The channel activity accumulated over 2-min intervals was expressed in cumulative channel open probability (NPo, where N is the apparent number of channels and Po is the mean open state probability). NPo was determined from amplitude histograms after multiple Gaussian curve fitting (Origin 6.0, Microcal Software; Northampton, MA).

Identification of mitoKATP channel reconstituted in lipid bilayers. MitoKATP channels were identified by their inhibition with 5-HD (a selective mitoKATP channel inhibitor) and/or ATP and activation with diazoxide (a mitoKATP channel opener). All modulators were added to the cis chamber during continuous stirring for 1 min. Single-channel current amplitude at +40 mV (trans/cis) was used for determination of chord conductance. The voltage-current relationship was obtained at various holding potentials and used to calculate the slope conductance with linear regression with Origin 6.0.

Effect of Iso on mitoKATP channel reconstituted in lipid bilayers. A stock solution of Iso (14.5 mM) was made by mixing excess Iso with the identical buffer used for channel reconstitution. The Iso concentrations in the stock solution and cis chamber were measured by gas chromatography (GC-8A, Shimazu; Columbia, MO).

After the appearance of K+-conducting current in bilayers, an aliquot of Iso from the stock solution was added to the cis chamber under stirring. The channel activities were monitored for up to 10 min, and the identity of the mitoKATP channels was confirmed by their inhibition with 5-HD and/or ATP in the end. HMR1098, a sarcolemmal KATP channel inhibitor, was used to distinguish the sarcolemmal KATP channel from the mitoKATP channel. To examine the effect of Iso on ATP-dependent inhibition of mitoKATP channels, ATP from 50 µM to 2.5 mM was cumulatively added every 2 min in the presence and absence of Iso. An ATP inhibition curve was obtained from normalized NPo and the logarithm of the ATP concentration using Hill equation. The IC50 and Hill coefficient (nH) were determined (Origin 6.0). NPo was normalized to the NPo at baseline (50 µM ATP).

Chemicals. The following drugs and chemicals were used: Iso (Abbott Laboratories; Chicago, IL); protein inhibitor cocktail, n-decane, MOPS, diazoxide, 5-HD, and ATP (Sigma; St. Louis, MO); BSA (Serologicals; Milwaukee, WI); and L-alpha -phosphatidylethanolamine and L-alpha -phosphatidylserine (Avanti Polar Lipids; Alabaster, AL). Diazoxide was dissolved in DMSO before being added to the experimental solution. The final concentration of DMSO was <0.1%. DMSO (0.1%) did not show any effect on channel current (data not shown).

Statistical analysis. Data are presented as means ± SE. The data were analyzed by ANOVA for repeated measures, followed by Duncan's multiple-range test. A value of P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of rat cardiac mitoKATP channels reconstituted in lipid bilayers. We first identified the mitoKATP channels based on their sensitivity to 5-HD, ATP, and/or diazoxide. Figure 1A shows original single channel recordings of a cluster of mitoKATP channels that were active at baseline (Fig. 1A1). The addition of ATP (0.5 mM; Fig. 1A2) completely inhibited their activities. Diazoxide at 50 µM (Fig. 1A3) reactivated the channels and increased the peak current, which was largely blocked with the subsequent addition of 5-HD (100 µM; Fig. 1A4). As shown in Fig. 1B, the initial mitoKATP openings at baseline (Fig. 1B1) declined gradually to a complete closure (Fig. 1B2), likely due to rundown. The application of diazoxide (100 µM, Fig. 1B3) activated a channel with a current amplitude of 1.8 pA at +40 mV (trans/cis) after 5 min (chord conductance of 46 pS). 5-HD (100 µM, Fig. 1B4) inhibited its opening despite the continuous presence of diazoxide. These effects of diazoxide and 5-HD were similar to those reported in reconstituted bovine KATP channels in bilayers (30). However, in contrast, we found that ATP was effective in inhibiting mitoKATP channel activities when added to the cis chamber. The time course of diazoxide effect is shown in Fig. 1C. Marked activation of KATP channels occurred 6 min after diazoxide application, which was blocked by 5-HD after 5 min of application. Cumulative data from several experiments are summarized in Fig. 1D. Diazoxide (100 µM) increased NPo from 0.01 ± 0.01 at baseline (50 µM ATP) to 0.47 ± 0.12 (P < 0.05, n = 5). 5-HD (100 µM) almost completely inhibited diazoxide-induced activation (P < 0.05). These experiments confirmed the identity of mitoKATP channels in lipid bilayers.


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  		Fig. 1.   Effects of diazoxide and 5-hydroxydecanoate (5-HD) on mitochondrial ATP-sensitive K+ (mitoKATP) channels reconstituted in lipid bilayers. A: original recordings of mitoKATP channel activity in lipid bilayers. Upward deflection represents channel opening at a holding potential of +40 mV (trans/cis); C, channels closed. A cluster of mitoKATP channels in lipid bilayers were active at baseline (1), inhibited by 0.5 mM ATP (2), and then activated by 50 µM diazoxide (3), which was blocked by 5-HD (4). Data are representative of 3 observations. B: original recordings of mitoKATP channel activity in a separate experiment. At baseline (1), channel openings were seen initially and then run down (2); the addition of 50 µM diazoxide to the cis chamber activated a channel with a chord conductance of 46 pS (3), a segment of which is shown with an expanded scale (inset). This channel was subsequently inhibited by 100 µM 5-HD (4). C: time course of mitoKATP channel modulation by diazoxide and 5-HD. D: cumulative data on diazoxide activation and 5-HD inhibition of the cumulative open probability (NPo) of mitoKATP channels. Baseline, 50 µM ATP. Diazoxide (50~100 µM) increased NPo, and 5-HD (100~200 µM) blocked diazoxide-induced activation (n = 5 each). *P < 0.05 vs. baseline; dagger P < 0.05 vs. diazoxide.

Several conductance states were observed in the reconstituted mitoKATP channels. Figure 2 shows representative recordings of a mitoKATP channel opening at various holding potentials (Fig. 2A) and its corresponding current-voltage relationship (Fig. 2B). This channel showed a slope conductance of 58 pS, which exhibited no significant rectification in the presence of low [Mg2+] (0.2 mM). Of 33 channels with clearly defined conductance states observed at +40 mV, 5 peaks of chord conductance were seen at 26 ± 2 pS (n = 9), 47 ± 2 pS (n = 10), 66 ± 2 pS (n = 5), 83 ± 2 pS (n = 4), and 105 ± 5 pS (n = 5).


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  		Fig. 2.   Original recordings and current-voltage relationship of a mitoKATP channel reconstituted in lipid bilayers. A: recordings of mitoKATP channel currents at various holding potentials from -80 to +90 mV (trans/cis). C, closed; O, open. B: current-voltage relationship, which shows a slope conductance of 58 pS. C: distribution of mitoKATP channels in terms of chord conductance at +40 mV (trans/cis). Five separate peaks were observed at 26 ± 2, 47 ± 2, 66 ± 2, 83 ± 2, and 105 ± 5 pS.

The mitoKATP channels constituted ~30% of the total K+-conducting cation channels we observed in the bilayers. Sarcolemmal KATP channels based on their blockade by a specific blocker, HMR1098 (30 µM), were excluded, and the incidence was rare (2 in ~100 successful cation channel incorporations). Fewer than 6% of cation channels were stimulated by ATP and inhibited by ruthenium red or ryanodine, an alkaloid blocking the ryanodine receptors from the sarcoplasmic reticulum (SR). Thus contamination from SR membranes is minimal, and some of those ryanodine receptors may also come from the mitochondria, as shown recently (2).

Effects of isoflurane on reconstituted mitoKATP channels. We then investigated the direct effect of Iso on mitoKATP channels. Aliquots of Iso stock solution (14.5 mM) were added to the cis chamber and stirred for 1 min to obtain a final concentration of 0.4 and 0.8 mM. Its concentration remained stable for at least 10 min (data not shown). Figure 3A shows the effect of Iso on KATP channel activity. At baseline, the channels were active. However, 5 min after the addition of 0.8 mM Iso, the peak channel current was increased, likely due to increased Po as well as the number of channels being open. The effect of Iso was completely blocked by 5-HD, confirming their identity as mitoKATP channels. We also tested a sarcolemmal KATP blocker, HMR1098, in some experiments. As shown in Fig. 3B, a single KATP channel was active with a current of <2 pA at baseline. The addition of Iso at 0.8 mM enhanced the peak current amplitude to ~10 pA, indicating more channels being open, which were silent at baseline. That addition of HMR1098 (25 µM) had no effect on the channel openings, but 5-HD at 100 µM was effective in blocking the channel openings, confirming their identity as mitoKATP channels. Figure 3C shows the time course of the effect of Iso on mitoKATP channels. Its maximum stimulatory effect was seen at 6 min after administration, which was blocked by 5-HD (100 µM). Cumulative data from several experiments are summarized in Fig. 3D. Iso (0.8 mM) increased NPo from 0.09 ± 0.02 at baseline to 0.50 ± 0.09 (n = 5, P < 0.05). 5-HD (100 µM) inhibited the effects of Iso to 0.08 ± 0.02 (n = 5, P < 0.05 compared with Iso).


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  		Fig. 3.   A: original recordings of mitoKATP channel activity in the presence of isoflurane (Iso) and 5-HD. MitoKATP channel activity was increased from baseline by Iso (0.8 mM) and inhibited by 5-HD (100 µM). Upward deflection represents opening. B: effects of HMR1098 and 5-HD on mitoKATP channel activities after activation by Iso. 5-HD (100 µM), but not HMR1098 (30 µM), inhibited the currents induced by Iso (0.8 mM). C: time course of mitoKATP activation by Iso. The peak effect was seen 6 min after Iso addition, followed by 5-HD inhibition. D: cumulative effect of effect of Iso on NPo. Iso (0.8 mM) increased NPo, and 5-HD (100~200 µM) reversed the effects of Iso (n = 5 each). *P < 0.05 vs. baseline; dagger P < 0.05 vs. Iso.

We further investigated whether Iso may affect ATP-dependent inhibition of mitoKATP channels. Figure 4A shows representative recordings of the effect of ATP on the mitoKATP channel activity induced by Iso. Iso (0.4 mM) increased the channel activity from baseline (50 µM ATP). ATP at 0.5 and 1.0 mM inhibited the Iso-induced channel activity dose dependently. Normalized NPo from several experiments are summarized in Fig. 4B. Under control conditions (50 µM ATP), ATP inhibited the mitoKATP activity in a dose-dependent manner. The IC50 was 335 ± 4 µM, and nH was 5.4 ± 0.2 (n = 8). Iso at 0.4 and 0.8 mM caused a rightward shift in the ATP inhibition curve, with an increased IC50 of 485 ± 17 µM (n = 5) and 940 ± 34 µM (n = 5), respectively. nH decreased to 2.1 ± 0.1 and 3.2 ± 0.4 at 0.4 and 0.8 mM Iso, respectively (n = 5 each, P < 0.05 vs. control).


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  		Fig. 4.   A: effects of ATP on mitoKATP channel activity induced by Iso. Iso (0.4 mM) increased the channel activity from baseline (50 µM ATP). ATP was then raised in the cis chamber to 0.5 and 1.0 mM, which dose dependently inhibited mitoKATP channel activities. B: cumulative data on the effects of ATP and Iso on activities of the mitoKATP channels. ATP induced dose-dependent inhibition of the mitoKATP channel under control (baseline) conditions (IC50 = 335 ± 4 µM, Hill coefficient = 5.4 ± 0.2, n = 8). Iso (0.4 and 0.8 mM) caused a rightward shift in the ATP inhibition curve, which increased the IC50 to 485 ± 17 and 940 ± 34 µM and decreased the Hill coefficients to 2.1 ± 0.1 and 3.2 ± 0.4, respectively (n = 5 each).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first study investigating the direct effect of volatile anesthetics on mitoKATP channels reconstituted in lipid bilayers. Our results provide strong evidence that Iso directly activates rat cardiac mitoKATP channels. This finding supports previous assertions that the mitoKATP channels play a critical role in volatile anesthetic-mediated cardiac preconditioning (3, 11, 13, 14, 23, 25). As Iso activates the mitoKATP channel despite the presence of millimolar ATP, its direct effect likely plays an important role in myocardial protection induced by APC. This effect is independent of its potential modulation of other signaling cascades such as adenosine receptors (25). Furthermore, our observation of five peaks for mitoKATP conductance states suggests that native KATP channels may exist in more than one molecular complex of sulfonylurea receptors (SUR) and Kir subunits. This possibility is supported by the recent observation that the cardiac mitoKATP channel may be composed of Kir6.1 and Kir6.2 subunits as well as a novel small-molecular-weight SUR subunit (15).

Regulation of mitoKATP channels by ATP and Iso. Although the ATP regulatory site of the mitoKATP channel has been shown to be unipolar and cytosolic (29), others proposed that it faces the matrix side (10). As the ATP-inhibiting site is cytosolic and resides in the pore-forming Kir subunit of surface KATP channel complex (28), we speculate that the ATP-inhibiting site on the Kir subunit of the mitoKATP channels faces the cytosol. In the present study, ATP (and other modulators) was added to the cis chamber, which likely exerted its inhibitory effect via the cytosolic binding sites on the Kir subunits of mitoKATP channels. Zhang et al. (30) observed that ATP-induced inhibition of mitoKATP channels occurs only in the trans chamber and that K+ flux favors flows from trans to cis (inward rectification). Thus it is possible that the cytosolic side of some mitoKATP channels may be oriented toward the trans chamber after reconstitution in lipid bilayers or that another ATP-binding/inhibiting site is present on the matrix side as originally proposed (10). The orientation of the mitoKATP channels reconstituted in proteoliposomes was shown to be dependent on the presence of Mg2+ (29), which might also have affected their orientation during reconstitution into lipid bilayers.

The results of our current study showed that Iso directly activates KATP channels. This is probably due to more channels being open as well as increased Po of each channel. The effect of Iso is likely due to its disruption of the allosteric interaction between the channel subunits, probably the pore-forming subunit Kir 6.x, and ATP, which reduced the positive cooperativity between ATP-binding sites (i.e., decreased nH). Our observations also showed that Iso was capable of opening KATP channels that were otherwise silent or inactive in the lipid bilayers. These channels were likely in the rundown mode or dephosphorylated. In addition, no other cytosolic factors are required for their activation by Iso. Therefore, the direct activation of mitoKATP channels by Iso in bilayers is likely independent of their phosphorylation status or presence of other signaling components. The mechanisms by which opening of mitoKATP channels protect the heart from ischemic injury are still controversial. Opening of the mitoKATP channels has been shown to trigger generation of oxygen and nitrogen free radicals, which in turn activate other cellular targets and induce myocardial protection (19, 21). Oxygen free radicals/superoxide may also exert a positive feedback effect directly on mitoKATP channel activation (12, 30).

Limitations of the study. In this study, KATP channels were identified based on their sensitivity to 5-HD/ATP inhibition and/or diazoxide activation. Recently, it has been shown that diazoxide and 5-HD may have mitoKATP channel-independent targets. Diazoxide decreased succinate oxidation dose dependently, and 5-HD may be metabolized into its active form (8). Volatile anesthetics including Iso have also been shown to inhibit NADH/complex I of the electron transfer chain in mitochondria (1, 9, 24). In the present study, however, although enzymes involved in electron chain transfer may be part of the inner membrane vesicle fused into the bilayers, we did not add substrates such as NADH and succinate in our system. Thus we consider it unlikely that diazoxide or Iso induced significant production of free radicals that caused activation of the mitoKATP channels reconstituted in lipid bilayers.

In summary, the native rat mitoKATP channels of several conductance states were identified by their inhibition with ATP and 5-HD and activation with diazoxide after reconstitution into lipid bilayers. Iso directly stimulates the openings of mitoKATP channels by reducing their ATP sensitivity. This mechanism likely contributes to volatile anesthetic-induced myocardial preconditioning.


    ACKNOWLEDGEMENTS

We thank Dr. Shey-Shing Sheu of the University of Rochester for sharing the protocol for isolation of mitochondrial subparticles. We thank Dr. Leon Tseng of the Department of Anesthesiology at the Medical College of Wisconsin for access to the equipment for mitochondria preparation. We also thank Dr. David Stowe for many insightful discussions during the course of the study.


    FOOTNOTES

This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-34708 (to Z. J. Bosnjak), by the Department of Anesthesiology at the Medical College of Wisconsin, and by Sapporo Medical University (Sapporo, Japan) through a fellowship support (to Y. Nakae from Prof. Akiyoshi Namiki).

Address for reprint requests and other correspondence: M. T. Jiang, Dept. of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: mtjiang{at}mcw.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 6, 2003;10.1152/ajpheart.01031.2002

Received 2 December 2002; accepted in final form 21 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 284(5):H1865-H1871
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