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Am J Physiol Heart Circ Physiol 281: H191-H197, 2001;
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Vol. 281, Issue 1, H191-H197, July 2001

Role of nitric oxide and protein kinase C in ACh-induced cardioprotection

Huiping Liu1, Bradley C. McPherson1, Xiangdong Zhu1, Mark L. A. Da Costa2, Valluvan Jeevanandam2, and Zhenhai Yao1

1 Department of Anesthesia and Critical Care and 2 Department of Surgery, University of Chicago, Chicago, Illinois 60637


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the roles of nitric oxide and protein kinase C (PKC) in ACh-produced protection of cultured cardiomyocytes during simulated ischemia and reoxygenation. Cell viability was quantified using propidium iodide in chick embryonic ventricular myocytes. O2 radicals were quantified using 2',7'-dichlorofluorescin diacetate. After a 10-min infusion of ACh (0.5 or 1 mM) and a 10-min drug-free period, we simulated ischemia for 1 h and reoxygenation for 3 h. ACh reduced cardiocyte death [32 ± 4%; n = 6 and 23 ± 4%; n = 7 (P < 0.05)] and attenuated oxidant stress during ischemia and reoxygenation in a concentration-dependent manner compared with controls (47 ± 4%; n = 8; P < 0.05). The increase in O2 radicals before simulated ischemia [357 ± 49; n = 4 and 528 ± 52; n = 8 vs. 211 ± 34; n = 8; P < 0.05 (arbitrary units)] was abolished by the specific nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and was markedly attenuated by NG-monomethyl-L-arginine (L-NMMA). L-NAME or L-NMMA blocked the protective effects of ACh, which selectively increased PKC-epsilon isoform activity in the particulate fraction. The PKC inhibitor Gö-6976 had no effect on O2 radical production before simulated ischemia but it abolished the protection; therefore nitric oxide is a large component of ACh-generated O2 radicals. Nitric oxide and O2 radicals activate the PKC-epsilon isoform by which ACh protects against injury.

oxygen radicals; cardiomyocytes; ischemia; reperfusion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACH PROTECTS AGAINST ischemia-reperfusion injury in vivo (35, 36), in isolated perfused hearts (27), and in cultured cardiomyocytes (38). Intravascular administration of ACh affects coronary endothelium and circulating blood elements and activates a series of signal transduction cascades in cardiomyocytes. However, which effect is responsible for cardioprotection remains unclear.

ACh increases nitric oxide production from vascular endothelial cells (28, 32). In studying anesthetized dogs, we found that intracoronary infusion of ACh reduced myocardial infarction (35, 36), and the beneficial effects were abolished by NG-nitro-L-arginine methyl ester (L-NAME) but not by NG-monomethyl-L-arginine (L-NMMA). Both L-NAME and L-NMMA are specific nitric oxide synthase inhibitors, whereas the importance of nitric oxide in ACh-induced cardioprotection is not conclusive. Several recent studies strongly suggest that nitric oxide from vascular endothelium mediates the cardioprotection of early and late preconditioning (1, 32). Because there are many confounding factors present in in vivo settings, we chose to use isolated cultured cardiomyocytes to determine whether nitric oxide (which originates from cardiomyocytes) mediates ACh cardioprotection.

In isolated cultured cardiomyocytes, ACh generated free radicals before simulated ischemia occurred; this correlated with protection during simulated ischemia and reoxygenation (38). The free radicals originated in the mitochondria (38). O2 radicals have been shown (7, 8) to activate protein kinase C (PKC), which may mediate cardioprotection (13, 26). We hypothesized that nitric oxide is a major component of O2 radicals generated in cardiomyocytes with ACh.

An elegant study performed by Ping and coworkers (21) demonstrated that nitric oxide induces translocation of an activated PKC-epsilon isoform and mediates preconditioning in a conscious-rabbit model of cardiac ischemia-reperfusion. We intended to examine whether this signaling pathway mediates the cardioprotection of ACh. Accordingly, we measured the effects of ACh on enzyme activity of total PKC and the PKC-epsilon and PKC-delta isoforms in the cytosol and in particulate fractions.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cardiomyocyte preparation. Ventricular myocytes from 10-day-old chick embryos were prepared according to a method described previously (29, 30). Described briefly, hearts were harvested and placed in Hanks' balanced salt solution (BSS) lacking magnesium and calcium (Life Technologies; Grand Island, NY). Ventricles were minced and myocytes were dissociated via four to six repetitions of trypsin degradation (0.025%; Life Technologies) at 37°C with gentle agitation. Isolated cells were then transferred to a solution containing a trypsin inhibitor for 8 min, filtered through a 100-µm mesh filter, centrifuged for 5 min at 1,200 rpm and 4°C, and finally resuspended in a nutritive medium that was described previously (38). Resuspended cells were placed in a petri dish in a humidified incubator (5% CO2-95% air at 37°C) for 45 min to promote early adherence of fibroblasts. Nonadherent cells were counted with a hemocytometer, and viability was measured using 0.4% trypan blue. Approximately 1 × 106 cells from the nutritive medium were pipetted onto 25-mm coverslips. These were incubated for 3-4 days, after which synchronous contractions of the monolayer were noted. Experiments were performed on spontaneously contracting cells at day 3 or day 4 after isolation.

Perfusion system. Glass coverslips containing spontaneously beating chick myocytes were placed in a stainless steel 1-ml flow-through chamber (Penn Century; Philadelphia, PA). The chamber was sealed with Kynar film (McMaster-Carr; Elmhurst, IL) placed between the coverslip and the metal hypoxic chamber (to minimize O2 exchange between the chamber wall and the perfusate) and then mounted on a temperature-controlled platform (37°C) on an inverted microscope. A water-jacketed glass equilibration column mounted above the microscope stage was used to equilibrate the perfusate to known O2 tensions (PO2). The standard perfusion medium was equilibrated for 1 h before the experiment by bubbling it with a 21% O2-5% CO2-74% N2 gas mixture. A simulated ischemia solution composed of glucose-free BSS with 20 mM 2-deoxyglucose added (to inhibit glycolysis) was bubbled with a gas mixture of 20% CO2-80% N2 for 1 h before the experiments. The pH of the perfusion solution was routinely verified (normoxic BSS, 7.4; simulated ischemic BSS, 6.8). Stainless steel or low-O2-solubility polymer tubing connected the equilibration column to the flow-through chamber to minimize ambient O2 transfer into the perfusate. PO2 in our hypoxic chamber was routinely monitored using the OxySpot system (Medical Systems; Greenvale, NY) under conditions identical to those of experiments using an optical phosphorescence quenching method (14, 24, 33).

Cell viability. Fluorescent cell images were obtained with a ×10 objective lens (Nikon Fluor). Data were acquired and analyzed with MetaMorph software (Universal Imaging). There were ~600 cardiomyocytes under the selected field for each experiment. Multiple fields were examined and compared before each study, and the field with normal synchronous contraction was chosen and monitored throughout the experiments. Cell viability was quantified with 5 µM propidium iodide (PI, Molecular Probes; Eugene, OR), an exclusion fluorescent dye that binds to chromatin on loss of membrane integrity (30). PI is not toxic to cells over a course of 8 h and therefore may be added to the perfusate throughout the experiments. At the completion of each experiment, 300 µM digitonin was added to the perfusate for 1 h. Digitonin disrupted the cell-membrane integrity of all cells and allowed PI to enter. Percent loss of viability (cell death) was then expressed relative to the maximum value after 1 h of digitonin exposure (100%).

Measurement of O2 radicals. O2 radicals generated in cells were assessed using the probe 2',7'-dichlorofluorescin (DCFH). The membrane-permeable diacetate form of DCFH, DCFH-DA, was added to the perfusate at a final concentration of 5 µM. Within the cell, esterases cleave the acetate groups on DCFH-DA, thus trapping DCFH intracellularly (25). O2 radicals in the cells lead to oxidation of DCFH and yield the fluorescent product 2',7'-dichlorofluorescein (DCF) (24). DCFH in cardiomyocytes is readily oxidized by H2O2 or hydroxyl radical but is relatively insensitive to superoxide (29). Fluorescence was measured with an excitation wavelength of 480 nm, a dichroic 505-nm long pass, and an emitter bandpass of 535 nm (Chroma Technology) with neutral-density filters to attenuate the excitation light intensity. Fluorescence intensity was assessed in clusters of several cells identified as regions of interest. The background was identified as an area without cells or with minimal cellular fluorescence. Intensity is reported as the percentage of the initial value after subtraction of the background value.

PKC enzyme assay. Enzyme activity of total PKC, PKC-epsilon , and PKC-delta was measured by a method described previously (22). For each experiment, 5 × 106 cells were collected in sample buffer that contained 50 mM Tris · HCl (pH 7.5), 5 mM EDTA, 10 mM each EGTA and benzamidine, 50 µg/ml phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml each of aprotinin, leupeptin, and pepstatin A, and 0.3% beta -mercaptoethanol (Sigma; St. Louis, MO). The collection was centrifuged at 45,000 g for 30 min and separated into cytosol and particulate fractions. The particulate pellet was dissolved ultrasonically in sample buffer, and protein concentration was determined according to the Bradford method (2). Each 50- to 100-µg fraction was assayed for activity of total PKC, PKC-epsilon , and PKC-delta (assay kit, Amersham Pharmacia; Piscataway, NJ). For PKC-epsilon and PKC-delta assays, proteins were immunoprecipitated overnight by PKC-epsilon and PKC-delta mAb (BD Transduction Laboratories; Franklin Lakes, NJ) in immunoprecipitation buffer that contained (in mM) 150 NaCl, 50 Tris · HCl, 1 EGTA, 1 EDTA, 1 sodium orthovanadate, and 1 PMSF; plus 1% NP-40, 16 µg/ml benzamidine-HCl, and 10 µg/ml each of phenanthroline, aprotinin, leupeptin, and pepstatin A (pH 7.4; Sigma) with protein A/G beads (Santa Cruz Biotech). PKC-epsilon - or PKC-delta -specific substrate, ERMRPRKRQGSVRRRV (BioMol; Plymouth Meeting, PA), was used for the phosphorylation reaction with [32P]ATP (Amersham).

Chemicals. ACh, L-NMMA, and L-NAME were purchased from Sigma. Gö-6976 was purchased from Calbiochem-Novabiochem (San Diego, CA). ACh, L-NMMA, or L-NAME was dissolved in BSS buffer before administration. PI and DCFH-DA were purchased from Molecular Probes.

Experimental design. The experimental protocol is depicted in Fig. 1. Nine groups of cardiomyocytes (control, 0.5 mM ACh, 1 mM ACh, Gö-6976, Gö-6976 + ACh, L-NMMA, L-NMMA + ACh, L-NAME, and L-NAME + ACh) were studied. Cardiocytes were subjected to 1 h of simulated ischemia and then 3 h of reoxygenation. Saline (control series) or ACh (0.5 or 1 mM) was added to the perfusate for 10 min; then a 10-min drug-free period occurred before the cells were subjected to simulated ischemia and reoxygenation. In addition, Gö-6976 (0.2 µM), L-NMMA (100 µM), or L-NAME (100 µM) was added to the perfusate during the 1-h baseline period before 60 min of ischemia for the corresponding series.


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  		Fig. 1.   Schematic of the experimental protocol. L-NAME, NG-nitro-L-arginine methyl ester; L-NMMA, NG-monomethyl-L-arginine.

Nine additional series of experiments were used to determine the role of nitric oxide and the possible correlation between reduction in cell death and attenuated oxidant stress during simulated ischemia and reoxygenation.

For the PKC enzyme activity assay, ACh (1 mM) was administered for 10 min and a 10-min drug-free period ensued; cardiocytes were then collected for the assay. In the control group, vehicle (saline) was given for 10 min instead of ACh.

Statistical analysis. Data are expressed as means ± SE. Differences between groups for cell death and O2 radical production were compared by a two-factor ANOVA with repeated measures and Fisher's least significant difference test. Differences between groups were considered significant at values of P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of ACh on cell death, contraction, and oxidant stress. ACh (0.5 and 1 mM) reduced cell death in a concentration-dependent manner. The pattern and extent of cell death were similar to those previously reported (38). After 3 h of reoxygenation, cardiocyte death was 47.2 ± 4.2% in controls (n = 8), 32.3 ± 3.9% in 0.5 mM ACh-treated cells (n = 6), and 22.5 ± 4.0% in 1 mM ACh-treated cardiocytes (n = 7). Spontaneous contractile activity was noticed in 18 of 25 ACh-treated cells (1 mM; 72.0%) and 3 of 16 ischemic controls (18.8%). ACh decreased oxidant stress (see Fig. 2A) and conferred protection from cell death and contractile dysfunction during simulated ischemia and reoxygenation. The data from DCF fluorescence and percentage cell death for the 1 mM ACh-treated and control groups were repeatedly used in Figs. 2-5 for convenience of comparison.


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  		Fig. 2.   A: effects of ACh on 2',7'-dichlorofluorescin (DCFH) oxidation, an index of O2 radical generation. In control cells (n = 8), intensity of 2',7'-dichlorofluorescin (DCF) fluorescence (measured in arbitrary units, a.u.) increased slightly over 1 h of baseline. Infusion of ACh (0.5 and 1 mM) for 10 min with a subsequent 10-min drug-free period produced O2 radicals before ischemia in a dose-dependent manner (n = 4 and 8, respectively). In contrast, oxidant stress (as measured by free radical production during simulated ischemia and reoxygenation) was attenuated by ACh in a dose-dependent manner. B: ACh dose dependently reduced cardiocyte death [measured as percentage propidium iodide (PI) uptake]. *P < 0.05 vs. corresponding points in controls.



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  		Fig. 3.   A: treatment with the specific nitric oxide synthase inhibitor L-NAME abolished the increase in O2 radicals before simulated ischemia and restored ACh-reduced oxidant stress to control levels. B: reduced cell death by ACh was blocked by L-NAME. L-NAME alone had no effect on cell death. *P < 0.05 vs. corresponding points in controls.



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  		Fig. 4.   A: treatment with the specific nitric oxide synthase inhibitor L-NMMA attenuated the increase in O2 radicals before simulated ischemia and restored ACh-reduced oxidant stress to control levels. B: reduced cell death by ACh was blocked by L-NMMA. L-NMMA alone had no effect on cell death. *P < 0.05 vs. corresponding points in controls.



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  		Fig. 5.   A: treatment with the specific protein kinase C (PKC) inhibitor Gö-6976 had no effect on the increase in O2 radicals by ACh before simulated ischemia and restored ACh-reduced oxidant stress to control levels. B: reduced cell death by ACh was blocked by Gö-6976. Gö-6976 alone had no effect on cell death. *P < 0.05 vs. corresponding points in controls.

Role of nitric oxide synthase. The protection afforded by 1 mM ACh on reduced cell death and attenuated oxidant stress was lost in the presence of the specific nitric oxide synthase inhibitors L-NAME (100 µM) or L-NMMA (100 µM) (52.0 ± 3.5%; n = 7 and 39.3 ± 5.0%; n = 7, respectively) compared with controls (47.2 ± 4.2%; n = 8). The 100 µM dose of L-NAME or L-NMMA had no effect on cardiocyte death (43.6 ± 4.5%; n = 3 and 45.5 ± 4.4%; n = 3) compared with ischemic controls (Figs. 3B and 4B) or on oxidant stress (data not shown).

ACh increased DCFH oxidation (an index of O2 radicals) in a concentration-dependent manner before simulated ischemia (see Fig. 2A). The increase was significantly attenuated by L-NMMA (see Fig. 4A) and abolished by L-NAME (see Fig. 3A).

Role of PKC. The effects of ACh on reduced cell death and oxidant stress were blocked by the specific PKC inhibitor Gö-6976 (0.2 µM; 37.0 ± 2.5%; n = 10) compared with controls (47.2 ± 4.2%; n = 8). Gö-6976 alone had no effect on cell death compared with controls (see Fig. 5). The increase in O2 radicals with ACh before simulated ischemia was not affected by Gö-6976 (see Fig. 5A). These results indicate that PKC activation is a downstream signal of O2 radicals in mediating ACh protection.

ACh markedly increased the enzyme activity of the PKC-epsilon isoform in the particulate fraction but had no effect on the enzyme activity of total PKC and the PKC-delta isoform compared with controls. In the cytosol fraction, no difference was observed in the enzyme activity of total PKC or the PKC-delta and PKC-epsilon isoforms (see Fig. 6).


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  		Fig. 6.   A: ACh selectively increased the enzyme activity of the PKC-epsilon isoform in the particulate fraction but had no effect on the enzyme activity of total PKC or the PKC-delta isoform compared with controls (Cont). B: cytosol enzyme activity values for total PKC, the PKC-epsilon isoform, and the PKC-delta isoform were not different between control and ACh-treated cardiocytes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We made several novel observations in this study. First, ACh generated O2 radicals that were markedly attenuated by inhibition of nitric oxide synthase but not by inhibition of PKC. Second, the protection of ACh was associated with a weakening of oxidant stress. Third, these effects were abolished by inhibition of PKC. Finally, ACh selectively increased enzyme activity of the PKC-epsilon isoform in the particulate fraction. Thus nitric oxide is a major component of ACh-generated O2 radicals. Nitric oxide contributes to activation of the PKC-epsilon isoform. Through this signal transduction, ACh exerts cardioprotection.

Transient intracoronary infusion of ACh mimicked ischemic preconditioning to reduce myocardial infarction in anesthetized dogs (35, 36). Using isolated cultured cardiomyocytes, we previously showed (27, 38) that ACh reduced cardiocyte death during simulated ischemia and reoxygenation. These results are consistent with previous reports in which ACh attenuated ischemia-reperfusion injury in isolated perfused hearts and cultured cardiomyocytes.

Simulated ischemia and reoxygenation generated a large number of free radicals in our simple system. Such oxidant stress contributes to ischemia-reperfusion injury in vivo (11, 16, 40) and in vitro (29). Transient administration of ACh markedly attenuated oxidant stress. Previously we found (34) that monophosphoryl lipid A limited cardiac infarction by decreasing free radicals from neutrophils. Reduced cardiocyte death with ACh correlates with the effect of ACh on attenuating oxidant stress during simulated ischemia and reoxygenation. Because temperature, pH, perfusion rate, and partial pressures of O2 and CO2 were controlled throughout the experiment, our observations indicate that ACh exerts salutary effects via an intracellular signaling mechanism.

The attenuated oxidant stress by ACh during the simulated ischemic period could slow the depletion of endogenous antioxidants from the cardiocytes, which would preserve the cells' ability to reduce oxidant stress at reoxygenation and thereby increase survival; this is as critical as the reduced oxidant stress at reoxygenation for the cardioprotection of ACh. Free radicals generated during simulated ischemia and reoxygenation contribute to the pathogenesis of cardiocyte damage. The mechanism by which free radicals damage cardiocytes is not established. Free radical burst at reoxygenation is transient and only lasted 15 min in our system; however, cell death linearly increased over the 3-h reoxygenation period. Free radicals (superoxide in particular) induce apoptosis (39), which may play a role in progressive cell death with reoxygenation.

ACh increased the generation of O2 radicals before the start of ischemia. This effect correlated with reduced cardiocyte death and attenuated oxidant stress. The O2 radicals were abolished by L-NAME, which by itself had no effects on O2 radical production. L-NAME is a specific inhibitor of nitric oxide synthesis and selectively blocks muscarinic receptors (5). L-NMMA, another potent nitric oxide synthase inhibitor that is not a muscarinic receptor antagonist (4, 5), only partially blocked the increase in O2 radicals with ACh. These results suggest that muscarinic receptors are important in generating O2 radicals. Nitric oxide is significant but is not the sole component in such generation (see Fig. 7). H2O2/hydroxyl radicals also mediate the cardioprotection of preconditioning and flumazenil (30, 37). Inhibition of the mitochondrial electron transport system abolished the increase in O2 radicals with ACh (38); thus mitochondria seem to be the source of nitric oxide and O2 radicals.


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  		Fig. 7.   Signal transduction pathway of ACh-produced cardioprotection.

The cardioprotection provided by ACh was also abolished by specific inhibition of nitric oxide synthase with L-NAME or L-NMMA. Nitric oxide protects against ischemia-reperfusion injury in the myocardium (15, 31). Stimulation of muscarinic receptors with carbachol exerted cardioprotective effects in isolated hearts (17). ACh may produce nitric oxide via stimulation of muscarinic receptors thereby attenuating oxidant stress and cardiocyte death during simulated ischemia and reoxygenation injury. The mechanism, however, is not well understood. ACh activates K+ channels in myocytes via M2 muscarinic receptors (20). Blockade of ATP-sensitive K+ (KATP) channels antagonized the negative chronotropic, inotropic, and cardioprotective effects of ACh in dogs (18, 36). We found that increased O2 radicals and reduced cell death with ACh were abolished with 5-hydroxydecanoate, a selective mitochondrial KATP channel antagonist (10). Therefore, stimulation of muscarinic receptors and activation of mitochondrial KATP channels cause mitochondria to release O2 radicals such as nitric oxide and H2O2, by which ACh produces cardioprotection.

The ACh dosage used for this study was higher than the dosage we previously used in anesthetized dogs (35, 36). It is possible that a subtype of muscarinic receptors exists in chick embryonic cardiomyocytes that is less sensitive to ACh. Alternatively, ACh might activate mitochondrial KATP channels and generate O2 radicals via an effect independent of sarcolemmal muscarinic receptors. There is no evidence that M2 receptors exist in the mitochondrial membrane. In addition, L-NAME, a muscarinic receptor antagonist, blocked the ACh protection. Therefore, stimulated sarcolemmal M2 receptors likely mediate the opening of mitochondrial KATP channels.

That stimulation of M2 receptors opens the mitochondrial KATP channels has not been convincingly demonstrated. The only evidence to suggest such a link was provided by Ito and co-workers (12): using a patch-clamp technique, they demonstrated that stimulation of M2 receptors increased K+ channel activity via G proteins in guinea pig atrial and ventricular myocytes. Such a mechanism is difficult to demonstrate in intact cells and awaits further study via the patch clamping of mitochondrial membranes.

The protective effects of ACh on reducing cardiocyte death and attenuating oxidant stress were blocked but not totally abolished by Gö-6976, a specific PKC inhibitor. Using a similar cardiomyocyte preparation, Liang (13) showed that PKC activation protected cells against injury after simulated ischemia and reoxygenation. Others have also shown that PKC activation mediates cardioprotection in isolated hearts and in vivo models of ischemia-reperfusion (3, 8, 9, 22). Besides PKC activation, other intracellular second messengers may mediate the protection afforded by ACh.

Gö-6976 did not affect the increase in O2 radicals before the simulated ischemia. PKC is a downstream signal of nitric oxide in ACh protection (21, 23). Nitric oxide activated PKC and mediated cardioprotection in isolated rabbit hearts (6, 19). We further observed that ACh selectively increased enzyme activity of the PKC-epsilon isoform in the particulate fraction without affecting the activity of total PKC and the PKC-delta isoform. Ping and co-workers (21) have demonstrated that nitric oxide selectively activates the PKC-epsilon isoform and that translocation of the activated PKC-epsilon isoform to membrane components mimics ischemic preconditioning in conscious rabbits.

We conclude that ACh generates nitric oxide and O2 radicals that originate from mitochondria. Nitric oxide activates the PKC-epsilon isoform, and the activated PKC-epsilon isoform was redistributed to membrane components of cardiomyocytes. Through this signal transduction pathway, ACh attenuated oxidant stress and reduced cell death in cardiomyocytes during simulated ischemia and reoxygenation.


    ACKNOWLEDGEMENTS

The authors thank Sally Kozlik for editorial assistance and Rhonda Judkins for secretarial support.


    FOOTNOTES

This work is supported by National Heart, Lung, and Blood Institute Grant HL-03881-02.

Address for reprint requests and other correspondence: Z. Yao, Dept. of Anesthesia and Critical Care, Univ. of Chicago, 5841 S. Maryland Ave., MC 4028, Chicago, IL 60637 (E-mail: zyao{at}airway.uchicago.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.

Received 8 November 2000; accepted in final form 22 January 2001.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 281(1):H191-H197
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