Soluble guanylate cyclase gene expression and localization in rat lung after exposure to hypoxia

Dechun Li, Nan Zhou, and Roger A. Johns

Department of Anesthesiology, University of Virginia Health System, Charlottesville, Virginia 22906


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The nitric oxide (NO)-cGMP signal transduction pathway plays an important role in the regulation of pulmonary vascular tone and resistance in pulmonary hypertension. A number of studies have demonstrated that endothelial (e) and inducible nitric oxide synthases (NOS) are upregulated in hypoxia-exposed rat lung. These changes in NOS expression have been found to correlate with the process of pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension, and remodeling is increased in the absence of eNOS. In this study, we examined the expression and localization of soluble guanylate cyclase (sGC), the primary receptor for NO, in hypoxia- and normoxia-treated rat lungs. Male Sprague-Dawley rats were exposed to hypoxia (10% O2, normobaric) or normoxia for 1, 3, 5, and 21 days. The lungs were used for Western analysis of sGC protein, sGC enzyme activity, immunohistochemistry using antiserum against sGC alpha 1- and beta 1-subunits, and nonradioactive in situ hybridization (NRISH) using a digoxigenin-labeled sGC alpha 1-subunit cRNA probe. Western blot analysis revealed a more than twofold increase of sGC protein alpha 1-subunit in rat lungs exposed to 3, 5, and 21 days of hypoxia, correlating well with sGC enzyme activity. Immunohistochemistry and NRISH demonstrated increased expression of sGC in the smooth muscle cells of the pulmonary arteries and arterioles in the hypoxic rat lungs when compared with normoxic controls. Based on our results, the upregulation of sGC may play an important role in the regulation of smooth muscle tone and pressure in the pulmonary circulation during chronic hypoxia.

nitric oxide


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE NITRIC OXIDE (NO)-cGMP signal transduction pathway plays an important role in the regulation of pulmonary vascular tone and resistance in hypertension (2-4, 8, 52, 54, 56, 57). NO binds to the heme component of the soluble guanylate cyclase (sGC), which catalyzes the conversion of GTP to cGMP. An increase in cGMP triggers cellular events that lead ultimately to a decrease in intracellular Ca2+ and smooth muscle relaxation through several mechanisms. These include increased uptake of Ca2+ in intracellular stores, inhibition of Ca2+ influx through plasmalemma Ca2+ channels, increased Ca2+ extrusion through plasmalemma Ca2+-ATPases, membrane hyperpolarization through activation of K+ channels, and inhibition of contractile machinery through activation of phosphatases to dephosphorylate myosin light chain and inhibition of myosin light chain phosphorylation (9, 32, 55). Several isoforms of sGC have been cloned and characterized (20, 22, 40, 41). Each consists of one alpha - and one beta -subunit, both of which are obligatory for catalytic activity (10). The sGC from bovine and rat lung consists of an 82 (rat)- or 73 (bovine)-kDa subunit and a 70-kDa subunit, termed alpha 1 and beta 1, respectively.

Reports from our laboratory and others have demonstrated that endothelial (e) and inducible (i) nitric oxide synthase (NOS) are upregulated in the lungs of hypoxia-exposed rats (30, 46). In addition, NOS expression correlates with the process of pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension (47, 56). Furthermore, a recent report by Steudel and colleagues (52) demonstrates that congenital disruption of the eNOS gene induces mild pulmonary hypertension in mice. Although these studies examined the role of NOS and NO in various models of pulmonary hypertension, the expression and localization of sGC, the primary receptor for NO, has not been investigated. In this study, we examined the expression and localization of sGC in the chronic hypoxia-treated rat lung.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hypoxia exposure of rats and measurement of pulmonary arterial pressure. All of the procedures followed in the care and killing of the animals were approved by the Animal Research Committee of the University of Virginia. Male Sprague-Dawley rats (250-300 g) were exposed to hypoxia according to the methods of Xue and Johns (56) and Le Cras et al. (30). A total of 44 rats were used in the experiment, with rats randomly assigned to hypoxia or normoxia in groups of three or four animals, respectively. The rats were exposed to 10% O2 in a normobaric hypoxic chamber (hypoxic group) or in a Plexiglas chamber open to room air (normoxic group) for 1, 3, 5, and 21 days. Animals were maintained at 20-24°C in a room with a 12:12-h light-dark cycle. Hypoxia was maintained using a Pro: ox model 350 unit (Reming Bioinstruments, Redfield, NY) that controlled fractional concentration of O2 in inspired gas by solenoid-controlled infusion of N2 (Roberts Oxygen, Rockville, MD) balanced against an inward leak of air through holes in the chamber. After exposure, the rats were anesthetized with halothane, and a trachea cannula was inserted. The animals were artificially ventilated with a Harvard rodent ventilator model 683 (Harvard Apparatus, South Natick, MA) set at a rate of 60 breaths/min and a tidal volume of 6-8 ml/kg body wt. To measure pulmonary arterial pressure, the chest of the rat was opened via a midline incision. An 18-gauge catheter filled with heparinized saline was inserted through the wall of the right ventricle and advanced into the pulmonary artery. Pressure in the pulmonary artery was measured with Datascope 2001A (Datascope, Paramus, NJ), and then the animals were killed by exsanguination.

Western blot analysis. Crude lung extracts were prepared from rat lung tissues homogenized in ice-cold 50 mM Tris · HCl, pH 7.4, containing 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 2 µM leupeptin, 1 µM pepstatin, and 0.1% 2-mercaptoethanol. The homogenate was centrifuged at 15,000 g for 30 min at 4°C, and the pellet was discarded. Protein present in the supernatant was measured using a Bio-Rad kit according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Lung homogenates (100 µg each) were separated by 7.5% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). Each blot was blocked with TBS buffer (50 mM Tris · HCl, pH 7.4, 0.15 M NaCl, 2% nonfat milk, 2% BSA, and 0.1% Tween 20) for 1 h at room temperature and then incubated with polyclonal rabbit anti-sGC alpha 1- and beta 1-subunit antiserum (1:500 dilution; Cayman Chemical, Ann Arbor, MI) for 1 h at room temperature, followed by a secondary goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP; Bio-Rad). Proteins were detected with enhanced chemiluminescence (Amersham, Buckinghamshire, UK). The relative amount of sGC alpha 1-subunit was quantitated with a densitometer using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

sGC enzyme activity. sGC enzyme activity was measured as described by Mittal (36). Lung tissue was homogenized in buffer containing 50 mM Tris · HCl (pH 7.6), 1 mM EDTA, 1 mM dithiothreitol, and 2 mM PMSF. Extracts were centrifuged for 10 min at 15,000 g for 30 min at 4°C. Supernatants (50 µg) were incubated for 10 min at 37°C in a reaction mixture containing 50 mM Tris · HCl (pH 7.5), 4 mM MgCl2, 0.5 mM 3-isobutyl-1-methylxanthine, 7.5 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase, and 1 mM GTP. The reaction was terminated by the addition of HCl to a final concentration of 0.1 N. cGMP in the reaction mixture was measured using an RIA described previously. sGC enzyme activity is expressed as picomoles of cGMP produced per minute per milligram of lung protein.

Immunohistochemistry and morphological analysis. For immunohistochemistry and in situ hybridization (ISH), the rat lungs were flushed with heparinized normal saline followed by inflation with 12 ml of paraformaldehyde (4% wt/vol) in 0.1 M PBS (pH 7.3). The lungs were fixed for 4 h and embedded in paraffin. Six-micrometer-thick sections were cut and mounted on precleaned glass slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA). After being dewaxed, the slides were incubated with rabbit anti-sGC antiserum (1:200 dilution; Cayman Chemical) for 1 h at room temperature. After being washed in TBS, the secondary goat anti-rabbit IgG antibody conjugated with alkaline phosphatase (1:200 dilution; Sigma Chemical, St. Louis, MO) was applied and incubated at 4°C overnight. A New Fuchsin substrate kit (DAKO, Carprinteria, CA) was used to obtain a red precipitate staining according to the manufacturer's instructions. Negative controls were carried out by omitting the first antibody and by using normal rabbit serum diluted at the same concentration (1:200).

ISH. A 388-bp fragment of sGC 82-kDa cDNA was subcloned into pGEM3z (Promega, Madison, WI) at the EcoR I and Acc I sites and was propagated in JM 109 cells (Promega). The plasmid was purified using a Wizard Maxipreps kit (Promega) and then was linearized with Acc I and EcoR I. To obtain sense and antisense RNA probes, the RNA probes were labeled with digoxigenin-11-UTP (Boehringer Mannheim, Indianapolis, IN) using T7 (sense) and SP6 (antisense) RNA polymerase, respectively. ISH was carried out as previously described with slight modifications (31, 33). Briefly, after deparaffinization, the slides were permeabilized with 1 µg/ml proteinase K (Sigma) in 100 mM Tris, pH 8.0, and 50 mM EDTA for 15 min and were fixed in 2% paraformaldehyde for 5 min at room temperature. RNA probes (100 ng/ml) were added in the hybridization buffer [50% formamide, 4× saline-sodium citrate (SSC), 100 µg/ml salmon sperm DNA, and 1× Denhardt's solution] and were incubated overnight at 42°C utilizing both the sense and antisense probes. A stringent washing was carried out in 2× SSC and 0.1% SDS at 37°C two times for 10 min each. The slides were then treated with 20 µg/ml RNase A (Sigma) in 2× SSC for 10 min followed by two washes in 0.1× SSC and 0.1% SDS at 45°C for 10 min. The signal was detected with monoclonal anti-digoxigenin antibody (dilution 1:200; Boehringer Mannheim) in PBS containing 0.5 g/100 g BSA for 1 h at room temperature followed by rabbit anti-mouse IgG (dilution 1:100; DAKO) and mouse HRP (dilution 1:200; DAKO). The DAB substrate kit (Vector Laboratories, Burlingame, CA) was used according to the manufacturer's instructions. Slides were conterstained with hematoxylin and mounted with Permount (Fisher) and coverslips.

Analysis and statistics. Values are expressed as means ± SE. For multigroup comparisons, one-way ANOVA (Dunnett's method) was used. A value of P < 0.05 was considered statistically significant compared with the normoxic control.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pulmonary arterial pressure changes. After exposure to 10% O2, the rat pulmonary arterial pressure was increased significantly at 1, 3, 5, and 21 days of hypoxia compared with normoxic controls (P < 0.05, one-way ANOVA, Dunnett's method; Fig. 1).


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Fig. 1.   Pulmonary arterial pressure in normoxia (N)- and hypoxia (H)-treated rats. There is a significant increase of pulmonary arterial pressure in hypoxic rats starting from day 1 of exposure (n = 3 for each group; * P < 0.05 compared with normoxic rat). d, Day.

Western blot analysis. Western analysis using polyclonal rabbit anti-sGC antiserum detected a significant increase of the 82-kDa sGC alpha 1-subunit protein in the crude rat lung homogenates obtained from rats exposed to 3, 5, and 21 days of hypoxia compared with normoxic controls (Fig. 2). No differences were seen in the crude homogenates obtained from rat lungs exposed to 1 day of hypoxia compared with normoxic controls. Quantitation using densitometry revealed that lungs from 3, 5, and 21 days of hypoxia contain about twofold greater alpha 1-subunit protein than those from normoxia-treated rat lungs (P < 0.05 for 3, 5, and 21 days of hypoxia treatment compared with normoxic rat lungs).


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Fig. 2.   Western blot analysis of soluble guanylate cyclase (sGC) alpha 1-subunit expression in normoxic (n = 3) and hypoxic rat lungs at different time points. H1d (n = 3), H3d (n = 3), H5d (n = 4), and H21d (n = 4), hypoxia for 1, 3, 5, and 21 days, respectively. There is a significant increase of sGC alpha 1-subunit after 3, 5, and 21 days of hypoxia exposure (* P < 0.05 compared with normoxic control).

sGC enzyme activity. To correlate rat lung sGC alpha 1-subunit protein levels with enzyme activity, the level of sGC activity was measured at different time points (n = 3 for normoxic and 1-day hypoxic group; n = 4 for 3, 5, and 21 days of hypoxia groups). A significant (2- to 4-fold) increase in sGC activity in rat lungs exposed to 10% hypoxia at 3, 5, and 21 days was observed. There was a trend of increase of sGC activity in 1-day hypoxic rat lungs (Fig. 3).


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Fig. 3.   sGC enzyme activity in rat lungs. There is a significant increase of sGC activity after 3, 5, and 21 days of hypoxia treatment (** P < 0.001 and * P < 0.02 compared with normoxic control).

Immunohistochemistry and ISH. To localize and identify the cell types expressing sGC protein and RNA, immunohistochemistry using polyclonal anti-sGC antiserum and ISH using digoxigenin-labeled sGC RNA probe were used. In the normoxic rat lungs, strong sGC immunopositive staining was found in the smooth muscle cells of large- and medium-sized pulmonary veins, bronchial smooth muscle cells, type II cells of the alveoli, Clara cells of the bronchioles, and macrophages in the alveoli (Fig. 4a). In contrast, smooth muscle cells of the elastic pulmonary artery and arterioles only showed very weak or negative staining. In contrast, in the lungs of rats exposed to hypoxia for 3, 5, and 21 days, the sGC-positive immunostaining signal also was prominent in the smooth muscle cells of pulmonary arteries and arterioles, particularly in the arterioles in the periphery of the lungs (Fig. 4b). ISH of normoxic rat lung demonstrated intensive sGC alpha 1-subunit mRNA staining in smooth muscle of the pulmonary vein, macrophages, type II cells of the alveoli, and Clara cells of the airway epithelium (Fig. 4, c and d). The smooth muscle of the small pulmonary arteries only showed weak to negative staining (Fig. 4c). However, ISH of hypoxia-treated rat lungs demonstrated that sGC alpha 1-subunit mRNA expression was present in the positive cells mentioned above and was strongly positive in the smooth muscle cells of the pulmonary arteries and arterioles but was absent in the endothelium (Fig. 4e). Control hybridization using sGC sense RNA probe showed little background staining (Fig. 4f).


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Fig. 4.   Immunohistochemical staining in rat lungs exposed to 21 days of normoxia (a) and hypoxia (b). In normoxic rat lung (a), the sGC positive signals are strongly positive in type II cells (arrows) and alveolar macrophages (arrowheads) but not in the artery (A). In hypoxic rat lung (b), sGC positive signal is not only present in the cells mentioned above but is also strongly positive in the smooth muscle of small muscularized artery (arrows). Consistent with the immunohistochemistry, in situ hybridization using sGC antisense probe demonstrates sGC expression in smooth muscle of the pulmonary vein (c, arrowheads), macrophages (c, arrows), and Clara cells of the airway epithelium (d, arrowheads) in normoxic rat lung. In 21 days of hypoxia-treated rat lung, sGC mRNA is strongly expressed in smooth muscle of small pulmonary artery (e, arrows). The sense probe exhibited no background staining (f). Original magnification, ×250.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chronic exposure of human and laboratory animals to hypoxia can induce pulmonary hypertension (23, 24, 28, 39). This rise in pulmonary vascular resistance is the result of a combination of hypoxic vasoconstriction and vascular remodeling, including endothelial cell and medial smooth muscle hypertrophy and hyperplasia of normal muscular arteries, abnormal extension of smooth muscle into peripheral arteries where muscularization is not normally evident, and a reduction of lumen diameter in pulmonary arterioles (4, 28, 43-45).

In this study, we demonstrate that sGC mRNA, protein, and enzyme activity are upregulated during the development of hypoxia-induced pulmonary hypertension. Immunohistochemistry and ISH reveal that the upregulation of sGC protein and mRNA occurs primarily in smooth muscle cells of the pulmonary vasculature in lungs of rats exposed to 3, 5, and 21 days of hypoxia.

The NO-cGMP signaling pathway plays an important role in the regulation of vascular tone and resistance. Recent evidence suggests that this pathway also plays a role in the development of the vascular remodeling associated with pulmonary hypertension (1, 3, 9, 26, 37, 38). However, the effect of chronic hypoxia-induced pulmonary hypertension on the expression of the NO-cGMP signaling pathway is controversial (16, 27, 30, 56). Congenital disruption of the eNOS gene in mice resulted in elevation of pulmonary arterial pressure and increases in total pulmonary vascular resistance and remodeling (53). Studies from our laboratory and others demonstrated upregulation of brain NOS, eNOS, and iNOS gene and protein in chronic hypoxia-induced pulmonary hypertension in the rat (30, 50). Furthermore, eNOS mRNA and protein were progressively upregulated over 1-7 days of hypoxia, and this upregulation preceded and progressed with the vascular remodeling that occurred in the course of the development of hypoxic pulmonary hypertension. More importantly, a recent study revealed that there is a reduced vascular remodeling in hypoxia-induced pulmonary hypertension in eNOS knockout mice (42; personal communications).

sGC is a key NO receptor protein involved in the regulation of vasodilation in the vascular wall. Many NO-mediated biological and pathological reactions are demonstrated to activate sGC and to increase the intracellular levels of cGMP (26, 37, 38). In this study, we demonstrated that there is increased sGC protein expression and activity in rat lungs exposed to 3, 5, and 21 days of hypoxia. The increased sGC protein and enzyme activity in hypoxia-treated rat lung may be a response to upregulated eNOS and iNOS gene and protein expression and the resultant increase in levels of NO in the pulmonary vasculature (6). The upregulation of sGC protein, mRNA, and enzyme activity induced by hypoxia in the pulmonary vasculature may be of physiological significance as a mechanism for pulmonary adaptation to hypoxic stress. For example, it may be a means of modulating the hypoxia-induced pulmonary hypertension seen in adult respiratory distress syndrome, chronic obstructive lung disease, and other pulmonary disease states related to alveolar hypoxia. Additionally, this phenomenon may be a physiological adaptation to antagonize pulmonary vascular changes such as vasoconstriction and increased resistance resulting in/from muscular hypertrophy and hyperplasia in the vessels of hypoxia-treated rat lungs (35, 45).

The distribution of sGC in the developing rat and lamb has been reported by Bloch et al. (7) and D'Angelis et al. (13). During the perinatal and neonatal period, with the onset of ventilation, pulmonary blood flow increases dramatically, and pulmonary vascular resistance declines. In the rat, there is abundant alpha 1- and beta 1-subunit mRNA in the lungs of the late-gestation fetus and the neonate but very low levels of sGC expression in adults (7). This parallels changes reported for NOS in the developing rat as reported by Xue et al. (58). In the adult rats, sGC was found prominently expressed in large veins and preacinar veins but very weak to absent in large arteries accompanying bronchi. Smooth muscle cells in the walls of arteries at the level of the terminal bronchioles demonstrated the greatest variation in sGC staining intensity, ranging from negative to intensely positive (7, 13). Our results from normoxia-treated rat lungs are in agreement with these observations in the normal adult lung in which minimal sGC expression was observed (7). Recently, Freas et al. (18) and Bina et al. (6) reported that there is a fivefold difference in basal cGMP levels between pulmonary vein and artery of the pig. The high levels of sGC protein and mRNA, as demonstrated by immunohistochemistry and ISH in the pulmonary vein in this study, may explain the reason for the differences. The lack of sGC staining in the small vessels, particularly the arteries and arterioles, correlates with our previous observations of lack of eNOS in the vessels under normoxic conditions in the rat (57). These results suggest that the NO-cGMP signal transduction pathway does not play a major role in regulating vascular tone in the normal lung (21).

In the hypoxic rat lung, sGC staining for both mRNA and protein is intensely positive in the small arteries and arterioles but not in the large- and medium-sized arteries throughout the lungs. This finding suggests a graded expression of sGC between proximal conduit arteries and the more distal resistance arteries, which determine the pulmonary arterial pressure. The increased expression for both sGC protein and mRNA in smaller more distal arteries may explain the more pronounced NO-induced vasodilation after chronic hypoxia (5, 15, 46).

Factors responsible for the regulation of sGC expression in the hypoxia-treated rat lung are not currently known. This paper demonstrates that sGC mRNA, protein, and enzyme activity levels increase during chronic hypoxia exposure in correlation with increases in NOS expression. This suggests that factors such as levels of NOS activity, NO, cGMP, and phosphodiesterases in the vascular wall may all contribute to the regulation of sGC activity (29, 56). Currently, there is no report of sGC gene expression in hypoxia-induced pulmonary hypertension from the published literature. Studies of lung sGC activity in pulmonary hypertension are controversial. In contrast to the study reported here, which measured protein and enzyme activity levels directly, most of the in vitro and ex vivo studies only measured cGMP levels or the relaxation to NO or cGMP analogs in isolated rings of pulmonary arteries (12, 51). D'Angelis et al. (14) reported that sGC expression is decreased in pulmonary arterial smooth muscle cells of fetal lambs in which pulmonary hypertension was induced by prenatal ligation of the ductus arteriosus. Studies performed by Steinhorn et al. (51) showed disruption of cGMP production in pulmonary arteries isolated from fetal lambs with pulmonary hypertension induced by ligation of the ductus arteriosus 11-12 days before birth. The contradictions with our observations in the hypoxic rat lung may have resulted from the differences of animal models and species used in the experiments.

The current study demonstrates for the first time lung sGC mRNA and protein expression, localization, and activity in hypoxia-induced pulmonary hypertension. These results reflect the changes occurring in the intact animal lungs at different time points during the development of pulmonary hypertension. This may be more consistent with the early stage of pulmonary hypertension in humans. In addition, our results strongly support physiological studies suggesting that sGC regulates pulmonary vascular tone and resistance, particularly during hypoxia-induced pulmonary hypertension. A recent study reported by Fouty and colleagues (17) demonstrated that inhibition of sGC by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, a sGC selective inhibitor that is thought to work by competing with NO for the heme site of sGC (19, 49) increased perfusion pressure and augmented vasoconstriction in the hypoxia-induced pulmonary hypertensive rat lungs, but not in the normoxia control rat lungs. This result is consistent with our findings in this report.

In summary, pulmonary sGC levels are regulated during chronic hypoxia-induced pulmonary hypertension. sGC mRNA, protein, and enzyme activity are increased in the hypoxia-treated rat lungs. Immunohistochemistry and ISH using anti-sGC antiserum and a cRNA probe revealed that the upregulated sGC protein and mRNA mainly occurs in vascular smooth muscle cells of the pulmonary arteries and arterioles, demonstrating that the enzyme is optimally located to respond to NO released by endothelial cells during chronic hypoxia. The upregulation of sGC mRNA, protein, and enzyme activity may play a very important role for the regulation of smooth muscle tone and pressure in the pulmonary circulation during chronic hypoxia.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. A. Johns, Dept. of Anesthesia and Critical Care Medicine, Blalock 1415, Johns Hopkins Univ. School of Medicine, 600 N. Wolfe St., Baltimore, MD 211287-4965 (E-mail: rajohns{at}jhmi.edu).

Received 15 December 1998; accepted in final form 21 May 1999.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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