Departments of 1 Cellular and Molecular Physiology and 2 Anesthesia, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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Alveolar type II epithelial cells undergo phenotypic changes and establish gap junction intercellular communication as they reach confluence in primary culture. The pattern of gap junction protein (connexin) expression changes in parallel. Although connexin (Cx)43 mRNA and protein increase significantly by culture day 2, Cx26 and Cx32 expression decline. Along with increasing Cx43 expression, the cells assemble fibronectin derived both from serum in the culture medium and from de novo synthesis into the extracellular matrix (ECM). The present studies indicate that this ECM regulates Cx43 expression. Culture of type II cells in DMEM containing 8-10% fetal bovine serum (FBS) promotes assembly of a fibronectin-rich ECM that stimulates expression of both Cx43 mRNA and protein. Although Cx43 protein expression increased in response to FBS in a dose-dependent manner, fibronectin also elevated Cx43 protein in the absence of FBS. Anti-fibronectin antibody significantly reduced the serum-dependent increase in Cx43 expression. These results support the premise that fibronectin in the ECM contributes to the regulation of Cx43 expression by alveolar epithelial cells in primary culture.
gap junctions; lung injury; alveolar type II cells
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INTRODUCTION |
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GAP JUNCTIONS WERE FIRST DEMONSTRATED in alveolar epithelial cells in vivo by electron microscopy (2, 3, 34). Gap junction channels, connexons, are formed by hexameric assemblies of transmembrane protein subunits, connexins. Expression of at least seven connexins has been demonstrated in lung tissue. These include connexin (Cx)45 (16), Cx40 (17, 38), and Cx37 (41). Pulmonary tissue is, however, composed of diverse cell types, and thus inferences concerning cell-specific connexin expression cannot be made from tissue analysis. For example, vascular endothelial cells express Cx37 and Cx40 mRNAs (30), so high levels of these transcripts in lung tissue may reflect expression by this numerous cell population. Conversely, Cx26 and Cx32 are expressed by freshly isolated type II epithelial cells (25), but these connexins are present only at low levels in the whole lung (44). In addition to Cx26 and Cx32, alveolar type II cells express both Cx43 (25) and Cx46 (1) proteins. Although these observations help to characterize patterns of connexin expression in lung tissue, the physiological role of gap junctions in the alveolar epithelium under both normal and pathological conditions remains poorly defined.
Fibronectin derived from cellular sources and from plasma is a major component of the provisional extracellular matrix (ECM) formed after injury to the alveolar epithelium (7, 37). In addition to providing a scaffold for repopulation of the denuded alveolar basement membrane, fibronectin may also contribute to restoration of a low-resistance blood-gas barrier by modulating the pattern of gene expression and the phenotype of alveolar epithelial cells. Fibronectin mediates biological effects through transmembrane glycoprotein heterodimers, integrins, which physically link the ECM to the cytoskeleton (43). Through these interactions, integrins are positioned to affect changes in cell shape (39) as well as to modulate gene expression and cellular phenotype (8, 43). Thus fibronectin may contribute directly to the regulation of type II cell phenotype and therefore to tissue repair subsequent to injury of the alveolar epithelium.
In primary culture, type II alveolar epithelial cells synthesize and assemble a fibronectin-rich ECM (11, 31). Observations that exogenous fibronectin (9, 35, 42) and preformed type II cell-derived matrix (32) determine the alveolar epithelial cell phenotype suggest that ECM fibronectin may contribute to culture time-dependent changes in type II cell morphology and gene expression (36). Although type II cells assemble both newly synthesized and exogenous fibronectin into the ECM, the principal source of fibronectin in vitro is serum in the culture medium (7, 36). As matrix fibronectin deposition increases in early culture, the cells flatten to reach confluence and establish gap junctional intercellular communication (GJIC) (25). The profile of connexin expression changes over a parallel time course. Freshly isolated type II cells express mRNA for several connexins including Cx26, Cx32, Cx43, and Cx46 (1, 25). Over the first several days of primary culture, Cx43 mRNA and protein levels increase substantially as does the extent of cell coupling (25). Although matrix fibronectin levels increase in parallel with changes in Cx43 abundance and GJIC, a direct effect of fibronectin on the latter parameters has not been established.
The present studies were thus designed to extend observations that suggest a role for ECM fibronectin in the regulation of connexin expression by alveolar epithelial cells. Results indicate that fibronectin levels in the matrix correlate with the concentration of fetal bovine serum (FBS) in the culture medium. Serum also promotes Cx43 mRNA and protein expression. The importance of fibronectin in the regulation of connexin protein abundance was investigated further with an anti-fibronectin antibody to prevent type II cell-fibronectin interactions. The data support the premise that fibronectin in the type II cell-derived matrix modulates expression of Cx43 protein. They are relevant to the definition of the relationship between ECM composition, cell-to-cell communication, and alveolar epithelial cell phenotype.
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EXPERIMENTAL PROCEDURES |
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Isolation and culture of type II cells. Type II cells were isolated from the lungs of male Sprague-Dawley rats (200-250 g body weight; Charles River Laboratories) according to methods previously described (33). The rats were anesthetized with pentobarbital sodium (60 mg/kg). After washout of the pulmonary circulation and lavage of the airways, alveolar cells were dispersed by intratracheal instillation of Joklik minimal essential medium (JMEM) containing elastase and BaSO4. After 30 min, proteolysis was terminated with soybean trypsin inhibitor in JMEM containing 50% newborn calf serum and DNase. The lungs were then minced and filtered through Nitex HC160 nylon mesh (Tetko, Elmsford, NY); the cells were collected by centrifugation and resuspended in JMEM containing DNase. Type II cells were purified by density centrifugation on discontinuous Percoll gradients followed by differential adherence. Purity of the cell preparation was monitored by staining with tannic acid and OsO4. On day 2, the cultures contained >90% type II cells.
The final cell preparation was resuspended in Dulbecco's modified Eagle's medium (DMEM). Cells to be used for Northern blot analysis were plated at 1.5 × 105 cells/cm2 on 100-mm tissue culture plates. For Western blot analysis, cells were plated at 2.1 × 105 cells/cm2 on six-well plates. In some experiments, 24-well plates were incubated for 6 h at 37°C with bovine plasma fibronectin (Sigma, St. Louis, MO) with and without affinity-purified rabbit anti-rat fibronectin polyclonal antibody (Calbiochem, San Diego, CA); the cells were then plated at 2.8 × 105 cells/cm2. In all experiments, the day of cell isolation was designated as day 0. The medium was changed on the morning of day 1 to DMEM without and with FBS (2-10%). In experiments designed to prevent type II cell-fibronectin interactions, anti-rat fibronectin antibody diluted 1:500 in culture medium was added on day 1 and/or day 2. Cells were cultured for 48 h (day 2).Preparation of matrix proteins for SDS-PAGE and Western blot analysis. The cells were separated from the matrix with established methods (31). Briefly, the monolayer was washed once with PBS containing 1 mM EDTA and then treated for 30 min at 4°C with 0.25 M NH4OH containing 1 mM phenylmethylsulfonyl fluoride and 1 mM EDTA. The culture surface was then washed twice with PBS containing 1 mM EDTA and extracted for 15 min at 4°C with 1 M NaCl in 50 mM Tris (pH 7.4), 1 mM phenylmethylsulfonyl fluoride, and 1 mM EDTA. The matrix fraction, which remained associated with the culture surface after this extraction protocol, was prepared for SDS-PAGE and Western blot as described in Western blot analysis.
Western blot analysis. Matrix fractions were solubilized in SDS-PAGE sample buffer (10% glycerol, 5% mercaptoethanol, 2% SDS, and 0.00125% bromphenol blue in 62.5 mM Tris, pH 6.8) containing Complete, Mini, EDTA-free protease inhibitor cocktail tablets (Boehringer Mannheim, Indianapolis, IN). Cell proteins were obtained by washing the monolayer with PBS and then solubilizing the cells directly in sample buffer containing protease inhibitors. The samples were boiled for 5 min before the cell and matrix proteins were separated by electrophoresis on gels containing 10 and 6% polyacrylamide, respectively. The cell proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) in Tris-glycine transfer buffer (192 mM glycine, 20% methanol and 0.1% SDS in 25 mM Tris, pH 8.3) at 300 mA for 2 h at 4°C. ECM proteins were transferred to polyvinylidene difluoride membranes in 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) transfer buffer (10% methanol and 0.1% SDS in 10 mM CAPS, pH 11.0) at 80 V for 1 h at 4°C. After incubation in BLOTTO (5% nonfat dry milk and 500 mM NaCl in 20 mM Tris, pH 7.5), the blots were incubated with polyclonal rabbit anti-rat Cx43 (cell fraction; Zymed Laboratories) or anti-fibronectin (matrix fraction; Calbiochem) antibody. The membranes were then washed six times in BLOTTO (cell fraction) or three times in 500 mM NaCl and 0.5% Tween 20 in 20 mM Tris, pH 7.5 (TBS-T; matrix fraction) and incubated with biotinylated anti-rabbit IgG (Vector Laboratories). Membranes were again washed as above and then incubated for 30 min to 1 h in BLOTTO containing preformed avidin-horseradish peroxidase complexes (Vector). Immunoreactive proteins were visualized with enhanced chemiluminescence (Amersham) that allows detection of bands through exposure to X-ray film. The relative levels of immunoreactive proteins were determined by densitometric analysis.
Northern blot analysis.
At the end of the culture interval, the plates were rinsed with PBS and
stored immediately at 70°C. The cells were lysed in TRI Reagent
(Molecular Research Center, Cincinnati, OH). After addition of
chloroform and centrifugation, RNA was collected from the aqueous
phase, precipitated with isopropanol, washed with 75% ethanol,
air-dried at room temperature, and solubilized in Formazol. RNA
concentration was determined spectrophotometrically. RNA samples (20 µg) were applied to 1.2% agarose gels containing 0.4 M formaldehyde
and were electrophoresed for 4 h at 55 V in 20 mM MOPS (pH 7.0)
containing 1 mM EDTA and treated with 0.1% diethyl pyrocarbonate. RNA
was transferred by capillary action to a nylon membrane in 10×
saline-sodium phosphate-EDTA buffer (SSPE; 1.5 M NaCl, 100 mM
NaH2PO4, and 12.5 mM EDTA, pH 7.4) treated with
0.1% diethyl pyrocarbonate. The membranes were baked for 2 h at
80°C and then cross-linked by ultraviolet light.
DNA quantitation. Cellular DNA content was measured with Hoechst dye 33258 (Calbiochem) as previously described (6). The cell monolayers were washed once with PBS and then dissolved in a solution of 0.154 M NaCl and 0.03% SDS in 0.015 M sodium citrate (pH 7.0). Samples and standards (calf thymus DNA) were sonicated with a Branson 250 sonifier before quantitation with a TKO minifluorometer (Hoefer Scientific, San Francisco, CA).
Statistical analysis. Data are means ± SE from at least 2 independent cell isolations, totaling 4-10 observations under each condition. Statistical comparisons were performed with a two-tailed Student's t-test. Multiple comparisons were carried out with one-way analysis of variance followed by an appropriate post hoc test. Values were considered to be statistically different at P < 0.05.
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RESULTS |
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Serum increases ECM fibronectin content.
The effect of FBS on the fibronectin content (36) of type
II cell-derived ECM was examined by Western blot analysis. Cells were
plated in DMEM and allowed to attach overnight. Beginning on day
1, the cultures were exposed to a range of serum concentrations from 0 to 10%; ECM fibronectin content was quantitated on day 2. Figure 1 shows a representative
Western blot of fibronectin from type II cell-derived ECM
(top). Matrix fibronectin was similar when the cells were
cultured in DMEM containing 0-6% FBS (11.8 ± 2.8 and
17.6 ± 2.2 ng/well, respectively; Fig. 1, bottom). In contrast, 8-10% FBS elevated ECM fibronectin approximately
fourfold (44.6 ± 6.1 and 46.9 ± 7.9 ng/well, respectively;
Fig. 1, bottom). These data indicate that the
serum-dependent increase in ECM fibronectin content previously reported
on day 2 (36) occurs in the presence of high
but not low FBS concentrations.
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Serum stimulates Cx43 expression.
During the first 2-3 days of primary culture, levels of Cx43 mRNA
and protein (25) increased in parallel with ECM
fibronectin content (36). Consistent with the premise that
fibronectin regulates connexin expression, previously assembled type II
cell-derived matrix promoted increased Cx43 expression and GJIC
compared with DMEM containing 10% FBS alone (15a). Additional
evidence that fibronectin modulates connexin expression by type II
cells was obtained by investigating the effect of FBS on the expression of Cx43 mRNA. Total RNA was isolated from cells cultured for 2 days
without and with 10% FBS. A representative Northern blot probed with
Cx43 cDNA as well as with a control probe (EFTu) is shown in Fig.
2, inset. Cells cultured with
10% FBS expressed twice as much Cx43 mRNA as cells cultured without
serum. In agreement with the data in Fig. 1, an intermediate
concentration of serum (6%) had no significant effect on Cx43 mRNA
compared with DMEM alone (data not shown). Thus increased expression of
Cx43 mRNA correlates with high levels of serum as well as with elevated matrix fibronectin content (Fig. 1).
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Fibronectin alters expression of Cx43 protein.
The results above demonstrate that ECM fibronectin content and Cx43
expression each respond to FBS in a dose-dependent manner. The
observation that Cx43 protein levels increase gradually over the whole
range of serum concentrations tested suggests that both serum and
fibronectin contribute to the observed effects. To determine whether
fibronectin alone modulates Cx43 levels, cells in DMEM were cultured
for 2 days in wells precoated with graded amounts of bovine plasma
fibronectin (0-28.3 µg/cm2) and were then analyzed
for Cx43 expression. A representative Western blot probed for Cx43 is
shown in Fig. 4, top. Cx43-NP and Cx43-P are shown separately. Although Cx43-NP expression increased nearly 50% in the presence of 2.8 µg/cm2 of fibronectin,
no further effect was evident when fibronectin was increased
10-fold (Fig. 4, bottom). Similarly, an abundance of Cx43-P
increased ~70% when fibronectin was elevated over the same range of
concentrations.
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DISCUSSION |
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In vivo repair after damage to the alveolar region of the lung involves formation of a fibronectin-rich provisional ECM (7, 37). Similarly, pulmonary epithelial cells in primary culture assemble a multicomponent, fibronectin-rich matrix in vitro. In addition to abundant fibronectin, the latter substratum also contains laminin, type IV collagen, and proteoglycans (11). Although the detailed relationship between these responses is not well defined, in both cases, increased matrix fibronectin content is paralleled by transition of cuboidal type II cells to a squamous type I-like phenotype (7, 36, 37).
Effects of fibronectin on alveolar epithelial cell phenotype have been studied under three conditions: on fibronectin-coated plastic, on a fibronectin-rich ECM previously assembled by type II cells in vitro, or on tissue culture plastic with FBS, an exogenous source of fibronectin. In the present experiments, ECM effects on type II cell phenotype were investigated in vitro where the cells assembled a fibronectin-rich matrix over the first 2 days of primary culture. A previous study (36) established that during this interval, both newly synthesized and serum-derived fibronectin are deposited into the type II cell matrix. Those observations, as well as the present results, suggest that relatively high concentrations of serum are required for the assembly of a fibronectin-rich matrix in primary culture. Although soluble factors in serum may affect the type II cell phenotype, culture in the presence and absence of serum provides a straightforward approach to identify potential biological effects of an underlying matrix rich in fibronectin.
Observations that matrix fibronectin content, Cx43 expression, and GJIC increase in parallel over the first several days in primary culture raised the possibility that fibronectin modulates Cx43 expression directly. Type II cells cultured for 2 days in DMEM containing 10% serum express twice as much Cx43 mRNA as cells cultured without serum. High serum levels are apparently required for this result because 6% FBS has little effect on Cx43 mRNA. In contrast, Cx43 protein abundance increases progressively with serum concentration. Thus although growth factors in serum are also likely to contribute to regulation of connexin expression, increased Cx43 mRNA and protein correlate with elevated ECM fibronectin content. The premise that serum-derived fibronectin modulates Cx43 expression is further supported by the observation that levels of Cx43 protein increase in response to fibronectin alone.
Cx43 protein is phosphorylated at multiple sites, and these modifications contribute to regulation of its function (22, 27, 28). To identify potential fibronectin-dependent effects on posttranslational modification of Cx43, levels of Cx43-NP and Cx43-P were measured individually on Western blots. The present data do not indicate that serum regulates Cx43 phosphorylation. This conclusion is consistent with the observation that in type II cells, newly synthesized Cx43 equilibrates rapidly into existing pools of Cx43-NP and Cx43-P protein (15). The observation that the abundance of Cx43-P is sensitive to higher concentrations of fibronectin (Fig. 4) suggests that the glycoprotein may affect posttranslational processing of Cx43 or the stability of its phosphorylated forms. Although Cx43 stability has not been examined in the present context, independent observations suggest that both the native and phosphorylated forms of the protein turn over at similar rates in alveolar epithelial cells (data not shown).
More direct evidence that matrix fibronectin modulates Cx43 expression comes from experiments in which type II cell interactions with fibronectin were prevented. Compared with cells cultured in DMEM alone, type II cells reach confluence more rapidly in the presence of serum or fibronectin. The polyclonal anti-fibronectin antibodies used here, however, did not affect cellular morphology, whereas the antibody significantly reduced both fibronectin- and serum-dependent increases in Cx43 protein abundance. Consistent with the observation that Cx43 protein increases substantially, nearly 30-fold by day 2 (25), the anti-fibronectin antibody was most effective when added on day 2. These results suggest that fibronectin in the type II cell-derived matrix might interfere with the effectiveness of anti-fibronectin antibody added at early culture times (36). The antibody reduced Cx43-NP and Cx43-P abundance to similar extents. This observation, together with the effects of serum and fibronectin discussed above, suggests that matrix fibronectin modulates expression of Cx43 protein. Specific regions of the fibronectin molecule and type II cell surface matrix receptors that interact to regulate Cx43 expression remain to be identified.
The present studies establish that type II cell interactions with
fibronectin contribute to the serum-dependent increase in Cx43 protein
expression. This result provides a basis for future investigations
aimed at identifying specific biochemical and mechanical components of
signal transduction initiated by alveolar epithelial cell-fibronectin
interactions. The small GTPase Rho, for example, is a candidate
effector of fibronectin-mediated changes in intercellular communication. Integrins affect changes in ECM organization (45, 46) and cell-cell adherens junctions (47) through
Rho-dependent mechanisms. Rho signaling is also required for
stimulation of GJIC by the integrin 3
1 in
a human keratinocyte cell line (23).
The observation that anti-fibronectin antibodies only partially inhibit the serum-dependent increase in Cx43 protein expression may reflect a failure of the antibody to interfere with all cell-fibronectin interactions. By morphological criteria, anti-fibronectin antibody was toxic to type II cells at dilutions below 1:500, independent of serum concentration. Alternatively, incomplete effects of the antibody may be due to the effects of other serum-associated factors on Cx43 expression. The observation that serum elicits a dose-dependent increase in Cx43 abundance over and above the maximal effect of fibronectin is consistent with this premise. Cx43-NP and Cx43-P increased in cells cultured with both fibronectin and serum. Thus the possibility that additional factors in serum modulate Cx43 expression warrants further investigation.
Matrix fibronectin content (36), Cx43 protein, and GJIC were previously demonstrated to increase in parallel during alveolar epithelial cell culture (25). The present data extend this correlation and indicate that fibronectin regulates expression of Cx43 protein. On the other hand, the effects of fibronectin on functional GJIC were not addressed in these studies. Recent observations indicate that fibronectin does, in fact, promote GJIC in alveolar epithelial cells (Guo Y and Rannels DE, unpublished observations). The latter data suggest that inhibition of GJIC would parallel decrements in Cx43 protein in cells exposed to anti-fibronectin antibody.
It has not been established directly that Cx43 accounts for the entire
culture time-dependent increase in alveolar epithelial cell coupling.
Inhibition of GJIC by 18-glycyrrhetinic acid parallels decreases in
Cx43 phosphorylation as well as redistribution of Cx43-immunoreactive
gap junction plaques from the plasma membrane to the cytosol
(15). Although this observation is consistent with a role
for Cx43 in alveolar epithelial cell-cell communication, 18
-glycyrrhetinic acid does not act in a connexin-specific manner, and effects of the drug on GJIC may thus be independent of those on
Cx43 distribution. Type II cells express multiple connexins in vitro
(1, 25), and there is ample evidence to indicate that
permeability and conductance characteristics of individual gap
junctions can be regulated by specific connexin expression patterns
(5, 10, 21, 40). Thus despite the fact that Cx43 appears
to be abundant in cultured type II cells, additional connexins are
likely to play important or essential roles in alveolar epithelial cell GJIC.
Similarly, the role of GJIC in the alveolar epithelium during normal and pathological conditions is not well understood. In an in vitro model of endothelial cell wounding, GJIC is decreased but not abolished at the wound edge (24). On the other hand, the relative extent of dye coupling between cells in different regions of wounded alveolar epithelial cell monolayers (20) has not been investigated. Fibronectin accelerates the rate of alveolar wound closure (13), so the effects of the glycoprotein on connexin expression and GJIC in this model system also warrant examination.
The possibility that GJIC plays a role in lung injury is further
supported by observations that Cx43 immunostaining is elevated in
alveolar epithelial cells of rat lungs undergoing repair after irradiation (19). In addition, the protective effect of
taurine against nitrogen dioxide-induced pulmonary inflammation is
associated with increased formation of alveolar epithelial gap
junctions. Taurine also has a protective effect on the integrity of
tight junctions between alveolar epithelial cells in this model of
pulmonary injury (14). On the other hand, GJIC between
primary type II cells is inhibited by airborne particulate matter
(18). In contrast, the inflammatory mediators
lipopolysaccharide and interleukin-1 upregulate Cx43 promoter
activity in vitro (12). Thus there is fragmentary evidence
that GJIC may be involved in the response of the alveolar epithelium to
injury, but the functional role of GJIC in this process is poorly understood.
In summary, the pulmonary alveolar epithelial cell phenotype is modulated by the ECM after injury and in primary cell culture. With alveolar epithelial cell damage, fibronectin derived from local cellular sources and from plasma accumulates in a provisional ECM that is incorporated into a new blood-gas barrier. Although elevated matrix fibronectin correlates with progression of alveolar reepithelialization, specific contributions of the glycoprotein to alveolar cell phenotype after injury are largely unknown. The present studies demonstrate that ECM fibronectin modulates Cx43 expression in primary cultures of alveolar type II cells. These data extend previous observations that fibronectin promotes loss of typical type II cell morphology and identify Cx43 as a specific target of fibronectin-mediated signaling. Fibronectin in the provisional matrix may contribute to formation of intercellular junctions between cells of the newly formed alveolar epithelium and thereby to the repair process. The present studies provide a foundation for future in vitro and in vivo investigations of the role of both cell-matrix and cell-cell interactions in repair of the alveolar blood-gas barrier.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. A. Levine for the EFTu cDNA probe and Dr. S. Lye for the connexin43 cDNA probe.
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FOOTNOTES |
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This research was supported by National Heart, Lung, and Blood Institute Grant R01-HL-31560.
Address for reprint requests and other correspondence: D. E. Rannels, Dept. of Cellular and Molecular Physiology (H166), The Pennsylvania State Univ. College of Medicine, 500 University Dr., Hershey, PA 17033 (E-mail:grannels{at}psu.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 17 May 2000; accepted in final form 9 November 2000.
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