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Am J Physiol Heart Circ Physiol 288: H1580-H1585, 2005. First published December 9, 2004; doi:10.1152/ajpheart.00807.2004
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Erythrocytes of humans with cystic fibrosis fail to stimulate nitric oxide synthesis in isolated rabbit lungs

Griffith Liang,2 Alan H. Stephenson,1 Andrew J. Lonigro,1 and Randy S. Sprague1

Departments of 1Pharmacological and Physiological Science and 2Internal Medicine, Saint Louis University School of Medicine, St. Louis, Missouri

Submitted 9 August 2004 ; accepted in final form 2 December 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Erythrocytes (red blood cells) of either rabbits or healthy humans are required to demonstrate the participation of nitric oxide (NO) in the regulation of pulmonary vascular resistance in the isolated rabbit lung. The property of the erythrocyte that is responsible for the stimulation of NO synthesis was reported to be the ability to release ATP in response to physiological stimuli, including deformation. Moreover, a signal transduction pathway that relates mechanical deformation of erythrocytes to ATP release has been described, and the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a component, i.e., erythrocytes of individuals with CF do not release ATP in response to deformation. Here, we investigated the hypothesis that, in contrast to those of healthy humans, erythrocytes of humans with CF fail to stimulate endogenous NO synthesis in the isolated rabbit lung. We report that CFTR is a component of the membranes of both rabbit and human erythrocytes. The addition of the NO synthase inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME, 100 µM) produced increases in vascular resistance in isolated rabbit lungs perfused with physiological salt solution (PSS) containing erythrocytes of healthy humans, but L-NAME was without effect when the lungs were perfused with PSS alone or PSS containing erythrocytes of CF patients. These results provide support for the hypothesis that, in CF, a defect in ATP release from erythrocytes could lead to decreased endogenous pulmonary NO synthesis and contribute to pulmonary hypertension.

adenosine triphosphate; red blood cells; pulmonary circulation

IT IS WELL ESTABLISHED that the endothelium-derived relaxing factor nitric oxide (NO) is produced by endothelial cells and, when released, relaxes vascular smooth muscle (10, 14, 19). Physiologically, an alteration in shear applied to the endothelium of blood vessels has been suggested to be a major stimulus for NO release (3, 22). However, in the pulmonary circulation, a reappraisal of this viewpoint was required when it was reported that, in isolated rabbit (31) and rat (36, 38) lungs perfused with a physiological salt solution (PSS), alterations in shear stress alone did not evoke release of NO. In contrast, when lungs of these species were perfused with either blood (36) or PSS containing washed erythrocytes (30, 38), inhibition of endogenous NO synthesis produced a shift in the slope of the pressure-flow relationship consistent with a decrease in vascular caliber, i.e., NO was a determinant of vascular resistance. The ability of the erythrocyte to stimulate NO synthesis in the pulmonary circulation of the rabbit and rat was reported to be independent of effects on viscosity or pressure (31, 36). The property of the erythrocyte that was associated with the stimulation of endogenous NO synthesis was shown to be the release of ATP (26, 28, 30). In support of the hypothesis that erythrocyte-derived ATP is a stimulus for endogenous NO synthesis in the lung, the addition of ATP to the perfusate of isolated rabbit lungs produces vasodilation that is, in large part, dependent on the stimulation of endogenous NO synthesis (30). Importantly, in addition to rabbit erythrocytes, human erythrocytes were reported both to release ATP in response to physiological stimuli, such as mechanical deformation, and to stimulate endogenous NO synthesis in the isolated perfused rabbit lung (26).

Recently, a signal transduction pathway that relates mechanical deformation to ATP release from human and rabbit erythrocytes has been described (17, 18, 27, 29). This pathway includes the heterotrimeric G proteins Gs (17) and Gi (18), adenylyl cyclase (29), PKA (29), and the cystic fibrosis transmembrane conductance regulator (CFTR) (27). The activity of CFTR was shown to be required for deformation-induced ATP release from erythrocytes of both rabbits and humans (27). Thus erythrocytes of humans with cystic fibrosis, a condition in which CFTR activity is absent or markedly reduced, and rabbit erythrocytes incubated with inhibitors of CFTR activity failed to release ATP in response to mechanical deformation (27). Here, we demonstrate that CFTR is expressed in the membrane of both rabbit and human erythrocytes and that, in contrast to those of healthy humans, erythrocytes from humans with cystic fibrosis fail to stimulate endogenous NO synthesis in the isolated rabbit pulmonary circulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of rabbit and human erythrocytes. Rabbit erythrocytes were obtained from New Zealand White rabbits (male, 2–3 kg body wt). The animals were anesthetized with ketamine (12.5 ml/kg im) and xylazine (1 mg/kg im) followed by pentobarbital sodium (10 mg/kg iv). After tracheal intubation, the animals were mechanically ventilated with room air (tidal volume 10 ml/kg, rate 20–25 breaths/min). A catheter was placed into the carotid artery for the administration of heparin (500 units) and for a phlebotomy. Ten minutes after heparin, the animals were exsanguinated. Human erythrocytes were obtained by venipuncture performed in an antecubital vein without the use of a tourniquet. Sixty milliliters of blood were collected into a syringe containing 50 units of heparin. Protocols for removal of blood from rabbits and humans were approved by the Institutional Animal Care and Use Committee and the Institutional Review Board of Saint Louis University, respectively.

Rabbit and human blood was centrifuged at 500 g for 10 min at 4°C. The plasma, buffy coat, and uppermost erythrocytes were removed by aspiration and discarded. The remaining erythrocytes were washed three times in buffer solution containing 140.5 mM NaCl, 21.0 mM Tris, 4.7 mM KCl, 2.0 mM CaCl2, 1.2 mM MgSO4, 0.1% dextrose, and 0.5% BSA fraction V (ICN Biomedicals; Aurora, OH), with a final pH of 7.4. The hematocrit of the washed erythrocytes was determined using an Autocrit Ultra 3 centrifuge (Becton Dickinson; Bedford, MA).

Preparation of erythrocyte membranes. Washed rabbit or human erythrocytes were diluted 1:100 with ice-cold lysis buffer (5 mM Tris·HCl and 2 mM EDTA, pH 7.4) and stirred at 4°C for 20 min. The resulting mixture was centrifuged at 23,000 g for 15 min at 4°C. The supernatant was removed and discarded. The membranes were resuspended in cold lysis buffer and centrifuged a second time. The pellets were pooled, resuspended in ice-cold lysis buffer, and centrifuged at 23,000 g for 15 min at 4°C a final time. The supernatant was discarded, and the protein concentration was determined (BCA Protein Assay, Pierce; Rockford, IL). Aliquots were frozen at –80°C. Immediately before electrophoresis, membranes were thawed on ice, sonicated briefly (W-220F sonicator, Heat Systems-Ultrasonics; Farmingdale, NY), vortexed with 5 µl of 10% SDS per 200 ml of sample, and heated to 95°C for 90 s.

Preparation of T84 human colon carcinoma cells. T84 cells (American Type Culture Collection), which express large amounts of CFTR (4, 33), were grown to confluence in a 1:1 mixture of DMEM and Ham’s F12 medium on a 70-mm culture dish. The dishes were then washed for 5 min with PBS (pH 7.4) containing a cocktail of protease inhibitors (Complete, Roche Diagnostics; Mannheim, Germany). Cells were removed by mechanical scraping and centrifuged at 8,000 g for 90 s. The resulting cell pellet was resolubilized in ice-cold 250 mM sucrose, 1 mM EDTA, and 20 mM imidazole (pH 7.2) with the protease inhibitor cocktail. After a 10-min incubation on ice, the suspension was briefly sonicated and centrifuged at 16,000 g for 30 s at 4°C. The supernatant (200 ml) was vortexed with 10% SDS (5 ml) and heated at 95°C for 90 s. Samples were stored at –80°C for Western blot analysis.

Western blot analysis. Membrane proteins were mixed in sample buffer [1.5% SDS, 6% (vol/vol) glycerol, 0.6% dithiothreitol, 10 mM Tris (pH 6.8), and 0.01% bromophenol blue], heated to 95°C for 2 min, and resolved by electrophoresis in 5% SDS-polyacrylamide precast gels (Bio-Rad; Hercules, CA). After electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore; Bedford, MA) in transfer buffer (25 mM Tris base, 192 mM glycine, and 10% methanol). The PVDF membranes were blocked for 1 h in a buffer solution containing 5% nonfat dry milk, 10 mM sodium phosphate, 150 mM NaCl, and 0.1% Tween 20 at pH 7.4. The PVDF membranes were then immunoblotted overnight with a mouse monoclonal anti-CFTR antibody (R&D Systems) in wash buffer solution containing 1% nonfat dry milk, 10 mM sodium phosphate, 150 mM NaCl, and 0.1% Tween 20 at pH 7.4. The PVDF membranes were washed three times with 10 mM sodium phosphate, 150 mM NaCl, and 0.1% Tween 20 at pH 7.4 and immunoblotted with anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (Amersham; Piscataway, NJ). After the membranes were washed, protein was visualized using enhanced chemiluminescence (Amersham).

Isolated perfused rabbit lungs. Rabbits were anesthetized, ventilated, and exsanguinated as described above. The heart and lungs were removed en block during continuous ventilation. A catheter was placed in the main pulmonary artery through the right ventricle, and a second catheter was placed in the left atrium. The heart and lungs were suspended in a humidified chamber maintained at ~37°C and ventilated with gas containing 15% O2-6% CO2-balance N2. Residual blood present in the lungs was removed by perfusion with 200 ml PSS [118.3 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25.0 mM NaHCO3, 0.026 mM Na-EDTA, 11.1 mM glucose, and 5% hetastarch (Baxter Healthcare; Deerfield, IL)] without recirculation. The lungs were then perfused with PSS at 80 ml/min with a recirculating volume of 100 ml. Pulmonary arterial pressure (PPA), left atrial pressure (PLA), and airway pressure (PAW) were recorded continuously. PLA was set to a pressure of 2–3 mmHg with a screw clamp, and lungs were perfused under zone III conditions (PPA > PLA > PAW). Pulmonary microvascular pressure (PMV) was determined by the double occlusion method (15). The arterial and venous cannulae were simultaneously occluded with electronically controlled solenoid pinch valves that cause minimal fluid displacement. Occlusions were maintained for 3–5 s, and the resulting equilibration pressures in the pulmonary arterial and venous catheters were recorded as an estimate of PMV. Samples of perfusate were analyzed for pH, PO2, and PCO2 under control and experimental conditions (pHOx blood gas analyzer, Nova Biomedical; Waltham, MA). After a minimum of 15 min of perfusion at 80 ml/min, the pressure-flow relationship was determined by measuring vascular pressures (PPA and PMV) at flow rates of 50, 100, 200, and 300 ml/min. Within 20 s of a change in flow rate, PLA was adjusted to 2–3 mmHg and PMV was determined. Finally, the flow rate was returned to 80 ml/min (recovery). N{omega}-nitro-L-arginine methyl ester (L-NAME; Calbiochem) was then added to the perfusate to achieve a final concentration of either 30, 100, or 300 µM. After 30 min, a second pressure-flow relationship was determined. In separate studies, the isolated lungs were perfused with either PSS alone or PSS to which washed erythrocytes obtained from healthy humans or humans with cystic fibrosis were added. Erythrocytes were obtained on the day of use and were added to the perfusate within 15 min of being washed.

Statistical methods. Statistical significance between experimental periods was determined with ANOVA. In the event that the F-ratio indicated that changes had occurred, a least-significant difference test was used to identify individual differences. The slope of the pressure-flow relationships was determined by least-squares linear regression analysis. P values of 0.05 or less were considered statistically significant. Results are reported as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of CFTR in erythrocyte membranes. If CFTR is a component of the signal transduction pathway for ATP release from rabbit and human erythrocytes, then it must be present in these cell membranes. Membranes prepared from a human colon carcinoma cell line that has been shown to express CFTR (T84 cells) were used as a positive control (4, 33). Membranes of both human and rabbit erythrocytes were found to contain CFTR protein when examined by Western blot analysis (Fig. 1).



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  		Fig. 1. Identification of cystic fibrosis transmembrane conductance regulator (CFTR) protein in the membranes of humans and rabbit erythrocytes and T-84 colon carcinoma cells. Cell membranes were prepared as described, and protein was resolved using a 5% SDS-PAGE gel. Protein was transferred to a polyvinylidene difluoride membrane and incubated with mouse monoclonal anti-CFTR antibody. Lane 1, rabbit erythrocyte membranes (15 µg protein, representative of 3 studies); lane 2, T-84 cell membranes (15 µg protein, representative of 3 studies); lane 3, human erythrocyte membranes (50 µg protein, representative of 5 studies).

 
Effect of L-NAME on pressure-flow relationships in isolated rabbit lungs perfused with PSS in the absence of erythrocytes. The pressure-flow relationship was determined in isolated rabbit lungs perfused with PSS in the absence and presence of L-NAME at concentrations of either 30, 100, or 300 µM. In the absence of erythrocytes, L-NAME had no effect on the pressure-flow relationship (Fig. 2A).



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  		Fig. 2. A: effect of N{omega}-nitro-L-arginine methyl ester (L-NAME) at perfusate concentrations of either 30, 100, or 300 µM on the relationship between flow rate and the pressure gradient across the pulmonary circulation [pulmonary artial pressure (PPA) – left arterial pressure (PLA)] in lungs perfused with physiological salt solution (PSS) in the absence of erythrocytes (n = 3 in all groups). B: effect of L-NAME on the pressure gradient across the pulmonary circulation (PPA – PLA) in lungs perfused with PSS containing erythrocytes of healthy humans (n = 5) or humans with cystic fibrosis (n = 4). *P < 0.05 compared with all other groups. Values are means ± SE.

 
Perfusion of isolated rabbit lungs with PSS containing washed human erythrocytes. Initial pH, gas tensions, and hematocrit in studies in which lungs were perfused with PSS containing erythrocytes from healthy human erythrocytes or from humans with cystic fibrosis are depicted in Table 1. Humans with cystic fibrosis, identified as homozygous for the {Delta}F508 genotype, were treated with medications including inhaled {beta}-agonists, ipratropium bromide, corticosteroids, antibiotics, DNAse, and inhibitors of mast cell degranulation as well as oral pancreatic enzyme replacement, antibiotics, proton pump inhibitors, and vitamins. The addition of washed human erythrocytes to the perfusate of isolated rabbit lungs did not result in increased airway pressure or weight gain over the course of the experiment.


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  		Table 1. Perfusate pH and gas tensions in isolated perfused rabbit lungs perfused with PSS containing RBCs of healthy humans or humans with cystic fibrosis

 
Effect of L-NAME on pressure-flow relationships in isolated rabbit lungs perfused with PSS containing erythrocytes from healthy humans. Under control conditions (perfusion at 80 ml/min), the addition of L-NAME to the perfusate resulted in an increase in perfusion pressure (Fig. 2B). Moreover, the addition of L-NAME resulted in a shift in the pressure-flow relationship such that the pressure difference across the pulmonary circulation (PPA – PLA) was increased at each flow rate studied (Fig. 3). Importantly, the slope of this relationship was increased after L-NAME (P < 0.01) consistent with the interpretation that, under these conditions, inhibition of endogenous NO synthesis was associated with an increase in Ohmic resistance (15). Finally, the site of increased resistance was examined by determination of the effect of L-NAME on changes in both upstream (PPA – PMV) and downstream (PMV – PLA) vascular resistance. The increases in vascular resistance were confined to the upstream vascular segment (Fig. 4, A and B).



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  		Fig. 3. Effect of L-NAME (100 µM) on the relationship between flow rate and the pressure gradient across the pulmonary circulation (PPA – PLA) in lungs perfused with PSS containing washed erythrocytes of healthy humans (n = 5). *P < 0.05 compared with control (pre-L-NAME) values. Values are means ± SE.

 


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  		Fig. 4. Effect of L-NAME (100 µM) on the relationship between flow rate and the pressure gradient across the upstream (arterial) segment of the pulmonary circulation (A) [PPA – pulmonary microvascular pressure (PMV)] and the pressure gradient across the downstream (venous) segment of the pulmonary circulation (B; PMV – PLA) in lungs perfused with PSS containing washed erythrocytes of healthy humans (n = 5). *P < 0.05 compared with control (pre-L-NAME) values. Values are means ± SE.

 
Effect of L-NAME on pressure-flow relationships in isolated rabbit lungs perfused with PSS containing erythrocytes from humans with cystic fibrosis. In isolated rabbit lungs perfused with PSS containing erythrocytes from humans with cystic fibrosis, L-NAME administration did not result in any change in perfusion pressure under control conditions (Fig. 2B). Moreover, in the presence of these erythrocytes, L-NAME had no effect on pressure-flow relationships (Fig. 5).



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  		Fig. 5. Effect of L-NAME (100 µM) on the relationship between flow rate and the pressure gradient across the pulmonary circulation (PPA – PLA) in lungs perfused with PSS containing washed erythrocytes of humans with cystic fibrosis (n = 4). Values are means ± SE.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
It was reported previously that erythrocytes from either rabbits (31), rats (36, 38), or healthy humans (26) are required for the demonstration of flow-induced NO synthesis in isolated perfused lungs. These observations, coupled with the findings that physiological stimuli, such as mechanical deformation (18, 26, 27, 29), stimulate ATP release from erythrocytes and that ATP is a stimulus for endogenous NO synthesis in the isolated rabbit lung (30), suggest a novel mechanism for the control of pulmonary vascular caliber. In this construct, as the erythrocyte is deformed by increments in the velocity of blood flow through a vessel and/or by reductions in vascular caliber, it releases ATP, which stimulates endothelial synthesis of NO resulting in relaxation of vascular smooth muscle and, thereby, an increase in vascular caliber. This vasodilation would then result in a decrease in pulmonary vascular resistance as well as a decrease in the stimulus for additional erythrocyte deformation and ATP release. On the basis of this scheme, erythrocyte-derived ATP would contribute to the low vascular pressure in the healthy lung via stimulation of endogenous NO synthesis.

Recently, a signal transduction pathway that relates mechanical deformation to ATP release from erythrocytes of rabbits and humans has been described. Components of this pathway include the heterotrimeric G proteins Gs (17) and Gi (18), adenylyl cyclase (29), PKA (29) and CFTR (27). A role for CFTR in this pathway was suggested by reports that this member of the ATP binding cassette could facilitate the movement of ATP out of cells either by acting as an ATP conduit (21, 22) or by regulating another channel for ATP release (20, 23). The finding that CFTR activity is lost or markedly diminished in humans with cystic fibrosis (5, 6) provided a unique opportunity for investigation of the role of CFTR in the release of ATP from erythrocytes in response to the physiological stimulus of mechanical deformation. It was reported previously that erythrocytes of patients with cystic fibrosis as well as rabbit erythrocytes incubated with two chemically dissimilar inhibitors of CFTR, namely, glibenclamide (24, 25) and niflumic acid (7), failed to release ATP in response to mechanical deformation (27).

A necessary step in establishing that CFTR is a component of a signal transduction pathway for ATP release from erythrocytes is the demonstration of its presence in erythrocyte membranes. Recently, it was shown that CFTR protein is present in erythrocyte membranes of both healthy humans and humans with cystic fibrosis (1, 32). In addition, it was reported that human erythroblasts contain CFTR mRNA (37). However, it must be noted that neither CFTR protein in human erythrocytes nor mRNA in human erythroid progenitor cells were detected in another study (13). In the present work, we confirm the finding that CFTR protein is a component of human erythrocyte membranes (Fig. 1). In addition, we show that the membranes of rabbit erythrocytes also possess CFTR (Fig. 1). Thus CFTR, a necessary component of a proposed signal transduction pathway for ATP release from erythrocytes, is present in the membranes of both rabbit and human erythrocytes, cells that release ATP in response to mechanical deformation (26, 27, 29).

Although CFTR activity is required for deformation-induced ATP release from erythrocytes of humans (27), the loss of CFTR activity may have a different effect on basal ATP release from these cells. It was reported that the concentration of ATP was increased in the blood of CFTR knockout mice as well as humans with cystic fibrosis compared with either wild-type controls or healthy humans, respectively (1). It is important to note that, in these studies, ATP release was studied under quiescent conditions, i.e., erythrocytes were not subjected to mechanical deformation. Thus it is possible that, although CFTR is required for deformation-induced ATP release from erythrocytes, another member of the ATP binding cassette, such as the multidrug resistance-associated protein 1, is involved in basal ATP release from these cells (1).

The hypothesis that ATP, released from erythrocytes of healthy humans in response to deformation, stimulates endogenous NO synthesis in the isolated rabbit lung is supported by previous studies. Erythrocytes of healthy humans release ATP in response to mechanical deformation as would be encountered as they pass through the pulmonary circulation. The amount of ATP released in response to deformation was shown to be stimulus dependent (26, 27, 29). In addition, the inclusion of human erythrocytes in the perfusate of isolated rabbit lungs resulted in the stimulation of endogenous NO synthesis (26). Finally, the addition of ATP to the perfusate of isolated rabbit lungs resulted in concentration-dependent decreases in total pulmonary vascular resistance that were inhibited by pretreatment with the NO synthase inhibitor L-NAME (30).

In view of the findings that CFTR is expressed in human erythrocyte membranes, that it is a component of a signal transduction pathway that relates erythrocyte deformation to ATP release (27), and that ATP release from erythrocytes is a stimulus for endogenous NO synthesis in the rabbit pulmonary circulation (30), we hypothesized that erythrocytes of humans with cystic fibrosis, a condition in which the activity of CFTR is defective, would fail to stimulate NO synthesis in the isolated perfused rabbit lung. In the presence of washed erythrocytes from healthy humans, the administration of the NO synthase inhibitor L-NAME to the perfusate of isolated lungs resulted in a shift in the pressure-flow relationship in a manner consistent with an increase in Ohmic resistance (Fig. 3) (15). This increase in vascular resistance was confined to the upstream vascular segment (PPA – PMV as a function of flow rate; Fig. 4A). In contrast, in the presence of erythrocytes of patients with cystic fibrosis, L-NAME was without effect on the pressure-flow relationship, i.e., NO was not a determinant of pulmonary vascular resistance (Fig. 5).

Several studies suggest that endogenous NO synthesis in the lung may be decreased in humans with cystic fibrosis. It was reported that amounts of NO detected in exhaled gas are decreased in humans with cystic fibrosis compared with healthy humans (2, 15, 35). It must be recognized that the source of NO that is measured in exhaled gas is unknown and could derive from the endothelium of blood vessels, the airways, or other resident cells. However, it was reported that exhaled NO is reduced in neonates with cystic fibrosis before the development of detectable respiratory symptoms, suggesting that the decrease in exhaled NO is not the result of increased mucus accumulation and/or inflammation (9). Thus the decrease in measurable NO in exhaled gas in cystic fibrosis patients compared with healthy controls could reflect, in part, the failure of erythrocyte-derived ATP to stimulate endogenous NO synthesis.

Importantly, it was reported that isolated blood vessels obtained from lungs of humans with cystic fibrosis, when precontracted with phenylephrine, relaxed in response to the application of ADP, the first degradation product of ATP (8). This response was shown to be endothelium dependent, although the nature of the endothelium-derived relaxing factor was not determined. Thus it is reasonable to hypothesize that if erythrocyte-derived ATP were released into the pulmonary circulation of humans with cystic fibrosis, endogenous NO synthesis could be stimulated.

Failure of deformation-induced ATP release from erythrocytes and, thereby, the loss of a physiological stimulus for endogenous NO synthesis could be expected to lead, ultimately, to the development of pulmonary hypertension. Pulmonary hypertension is seen in humans with cystic fibrosis; however, this condition has most often been attributed to the destructive lung disease and the subsequent loss of pulmonary capillaries and/or hypoxic pulmonary vasoconstriction (11, 34). The results presented here suggest that a defect in ATP release from erythrocytes of cystic fibrosis patients as they traverse the pulmonary circulation could be an additional factor leading to the development of pulmonary hypertension in these individuals.

In summary, we demonstrate that CFTR is a component of the erythrocyte membrane of rabbits and humans. Erythrocytes from humans in whom this protein is functional stimulate endogenous NO synthesis in the isolated rabbit lung. In contrast, erythrocytes of humans with cystic fibrosis, a condition in which CFTR activity is impaired, do not stimulate NO synthesis in this model. These findings provide support for the hypothesis that erythrocyte-derived ATP, released in response to a physiological stimulus, such as mechanical deformation, could serve to aid in the maintenance of normal pulmonary vascular resistance via the stimulation of endogenous NO synthesis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-51298, HL-52675, and HL-39226.


   ACKNOWLEDGMENTS
 
The authors thank Elizabeth Bowles for technical assistance and J. Sprague and B. Dugdale for inspiration.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. S. Sprague, Dept. of Pharmacological and Physiological Science, Saint Louis Univ. School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 (E-mail: spraguer{at}slu.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.


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 DISCUSSION
 GRANTS
 REFERENCES
 

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