Cyclic stretch of airway epithelium inhibits prostanoid synthesis

Ushma Savla1, Peter H. S. Sporn2, and Christopher M. Waters1,3

Departments of 1 Biomedical Engineering, 2 Medicine, and 3 Anesthesiology, Northwestern University and Medical Service, Lakeside Division, Veterans Affairs Chicago Health Care System, Chicago, Illinois 60611

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Airway epithelial cells (AEC) metabolize arachidonic acid (AA) to biologically active eicosanoids, which contribute to regulation of airway smooth muscle tone and inflammatory responses. Although in vivo the airways undergo cyclical stretching during ventilation, the effect of cyclic stretch on airway epithelial AA metabolism is unknown. In this study, cat and human AEC were grown on flexible membranes and were subjected to cyclic stretch using the Flexercell strain unit. Cyclic stretch downregulated synthesis of prostaglandin (PG) E2, PGI2, and thromboxane A2 by both cell types in a frequency-dependent manner. The percent inhibition of prostanoid synthesis in both cell types ranged from 53 ± 7 to 75 ± 8% (SE; n = 4 and 5, respectively). Treatment of cat AEC with exogenous AA (10 µg/ml) had no effect on the stretch-induced inhibition of PGE2 synthesis, whereas treatment with exogenous PGH2 (10 µg/ml) overcame the stretch-induced decrease in PGE2 production. These results indicate that stretch inhibits prostanoid synthesis by inactivating cyclooxygenase. When cells were pretreated with the antioxidants catalase (100 µg/ml, 150 U/ml) and N-acetylcysteine (1 mM), there was a partial recovery of eicosanoid production, suggesting that cyclic stretch-induced inactivation of cyclooxygenase is oxidant mediated. These results may have important implications for inflammatory diseases in which airway mechanics are altered.

arachidonic acid; cyclooxygenase; antioxidants

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE AIRWAY EPITHELIUM provides the first line of defense against inhaled toxins, carcinogens, and infectious agents and is involved in the regulation of airway smooth muscle tone and inflammatory responses (7). Airway epithelial cells (AEC) play a key role in regulating inflammatory airway diseases through their ability to synthesize and release lipid mediators derived from arachidonic acid (AA) as well as by synthesizing multiple inflammatory cytokines (7, 8). AEC can also alter cell adhesion molecule expression, thereby influencing leukocyte influx (8), and can modulate immune responses through expression of antigens such as HLA-DR (16). In vitro models of the airway epithelium have facilitated the investigation of some of these specific functions, which may be important in airway diseases such as asthma.

Nearly all in vitro studies of AEC function have been performed using cells cultured in a static environment. However, in vivo, the movement of air into and out of the lungs during normal respiration leads to variations in the distension of the airways, thus subjecting AEC to circumferential stretch with changes in airway diameter (strain = Delta D/D) and longitudinal stretch depending upon increases in lung volume, which lead to airway lengthening (strain = Delta L/L; see Ref. 22). Using high-resolution computed tomography, Brown et al. (6) reported canine airway cross-sectional area constriction up to 49% with breath holding and dilation up to 133% after deep inspiration. Under the assumption that changes in airway dimension were not due to folding of the airway wall, the circumferential wall strain would range from -42% in compression to +32% in distension.

Accumulating evidence, primarily within the field of vascular biology, indicates that physical forces and changes in the mechanical structure of tissues influence cellular physiology (26, 31). Although it is recognized that significant mechanical distension takes place in the lungs and that several airway diseases, including asthma, involve significant changes in airway mechanics, little is known about the influence of mechanical stimulation on the function of AEC. Airway distension is partially regulated by epithelial-derived mediators of smooth muscle tone such as AA metabolites (11). AEC actively metabolize AA via cyclooxygenase (COX) to prostanoids (prostaglandins and thromboxanes) and via 5-, 12-, or 15-lipoxygenase to leukotrienes and/or hydroxyeicosatetraenoic acids depending on species and culture conditions (7, 15). Because previous studies have demonstrated that stimulation by physical forces can regulate eicosanoid production in other cell types (17, 25, 26, 33), we hypothesized that cyclic stretching of AEC would alter the synthesis and release of bronchoactive mediators derived from AA. We subjected monolayer cultures of primary cat AEC and a human AEC line (Calu-3) to cyclic stretch using the Flexercell strain unit. In this study, cyclic stretch resulted in a rapid, frequency-dependent downregulation of eicosanoid synthesis by both cat and human AEC.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Phosphate-buffered saline (PBS), Dulbecco's phosphate-buffered saline (DPBS), Dulbecco's minimum essential medium (DMEM), minimum essential medium (MEM), Ham's F-12 medium (F-12), fetal bovine serum (FBS), gentamicin, trypsin-EDTA, and nonessential amino acids (NEAA) were obtained from GIBCO (Grand Island, NY). Antibiotic/ antimycotic solution, deoxyribonuclease (DNase), dithiothreitol, trypan blue, type I rat tail collagen, sodium pyruvate, catalase, N-acetylcysteine (NAC), and AA were purchased from Sigma (St. Louis, MO). PGH2 was from Cayman Chemical (Ann Arbor, MI). Collagenase was obtained from Worthington Biochemical (Freehold, NJ). NG-monomethyl-L-arginine (L-NMMA) was purchased from ICN Biomedicals (Aurora, OH). The Flexercell system and type I collagen-coated Flex-I six-well culture plates were purchased from Flexercell International (McKeesport, PA).

Cell culture. Cat tracheal epithelial (CTE) cells were isolated from healthy cats (courtesy of Dr. Robert TenEick, Department of Molecular Pharmacology, Northwestern University) euthanized with an intravenous injection of pentobarbital sodium (Abbott Laboratories, North Chicago, IL). The trachea was dissected from the surrounding tissue, cut from the larynx to the first bifurcation of the bronchi, placed on a gauze-covered sheet of styrofoam, cut longitudinally, and pinned open. Superficial parallel incisions were made longitudinally with a razor blade jig. Strips of epithelium pulled away from the underlying tissue were rinsed two to three times in sterile PBS containing antibiotics and then were placed in a solution of 0.02% (wt/vol) type II collagenase, 5 mM dithiothreitol, and 200 U/ml DNase in Ca2+/Mg2+-free PBS containing antibiotics at 37°C. The mixture was placed in a heated shaker or was agitated by inversion every 10-15 min. After 60 and 120 min, the cell suspension was removed and washed two times in DMEM-F-12 (1:1) with antibiotics. Flex-I six-well culture plates were coated with type I rat tail collagen (50 µg/ml in 2.5% acetic acid). Cells were seeded at 2.5-3 × 105 cells/well and were cultured in DMEM-F-12 with 10% FBS and 1% antibiotic/antimycotic solution. The medium was changed every 2 days, and the cells were used in experiments on day 6 or 7 of culture.

Calu-3 cells (American Type Culture Collection, Rockville, MD), derived from a human lung adenocarcinoma, have been previously shown to resemble tracheal epithelial cells and retain constant properties over repeated passages (28). Calu-3 cells were maintained in T-150 culture flasks in MEM, 1 mM sodium pyruvate, 1 mM NEAA, 0.1% gentamicin, and 10% FBS. Cells between passages 18 and 24 were used on day 4 or 5 of culture when plated into six-well Flex-I plates at 3-3.5 × 105 cells/well; the medium was replaced every 2 days. Prostanoid production by Calu-3 cells was found to be independent of passage number in this range.

Cyclic strain. The Flexercell strain unit has been described in detail previously (1, 12, 31). The system utilizes vacuum pressure regulated by a solenoid valve to deform a silicone rubber substrate on which the cells have been cultured. When the vacuum is applied, the culture plate bottoms deform downward to a known percent elongation, which is translated to the cultured cells. Upon release of the vacuum, the Silastic substrate returns to its original conformation. The frequency, duration, and magnitude of applied strain can be varied in this system. When cells were stretched at 10 cycles/min, the membrane was distended for 1 s and relaxed for 5 s; for 30 cycles/min, the membrane was stretched for 1 s and relaxed for 1 s. Finite-element analysis of the levels of strain in the Flex plate have shown that, upon deformation, a nonuniform radial strain profile results that is maximum at the periphery and minimal at the center (12). There is minimal circumferential strain in the wells (12).

Experimental protocol. In each experiment, duplicate paired plates were either subjected to cyclic stretch using the Flexercell apparatus or maintained unstretched as a control. The frequency of stretch was varied on the Flexercell from 1 to 30 cycles/min, and the maximum percent elongation was 20-22% for all experiments (mean strain 9%; see Ref. 12). Before initiation of stretch, wells were washed one time with DPBS, and 1 ml/well fresh medium (DMEM-F-12 + 10% FBS for CTE or MEM + 10% FBS for Calu-3) was added. Aliquots (200 µl) of media were removed at various times and were immediately frozen at -20°C in sterile Eppendorf tubes for subsequent eicosanoid analysis. When wells were to be sampled at subsequent time points, fresh medium was added to replace the aliquots removed. At the end of each experiment, monolayers were trypsinized, and cells were counted on a Coulter Counter (Coulter Electronics, Hialeah, FL). As a measure of cell injury, lactate dehydrogenase levels were measured in one set of experiments (Sigma, St. Louis, MO). In some experiments, regions of each well were isolated for cell culture using cloning cylinders (either 9.5 mm ID and 12.5 mm OD or 6.5 mm ID and 9.7 mm OD; Bel-Art Products, Pequannock, NJ).

Eicosanoid measurement. Prostaglandin (PG) E2, 6-keto-PGF1alpha [stable metabolite of prostacyclin (PGI2)], and thromboxane (Tx) B2 (stable metabolite of TxA2) were analyzed by sensitive and highly specific competitive enzyme immunoassays from Cayman Chemical (Ann Arbor, MI). Briefly, 96-well plates precoated with a specific mouse monoclonal antibody for the specific eicosanoid were incubated with a 50-µl aliquot of standard or unknown sample, 50 µl of tracer, and 50 µl of a specific rabbit anti-analyte antiserum. After an overnight incubation and addition of a colorimetric compound, the concentration of the sample was determined through the intensity of its absorbance read at 405 nm on a plate reader compared with a standard curve. Quantities of eicosanoids measured were normalized to cell number.

Incubations with exogenous AA or PGH2. To determine whether eicosanoid synthesis was limited by AA or PGH2 availability, cells were stretched in the presence of either exogenous AA (10 µg/ml) or exogenous PGH2 (10 µg/ml) added to culture media (with 10% FBS). In these experiments, 200-µl aliquots were sampled for eicosanoid analysis after 10 min of 10 cycles/min cyclic stretch.

Antioxidants and nitric oxide synthase inhibition. In selected experiments, Calu-3 cells were subjected to cyclic stretch (10 cycles/min) in the presence and absence of the antioxidants catalase or NAC or the nitric oxide (NO) synthase inhibitor L-NMMA. Because catalase from Sigma contains the preservative thimerosal, which has been shown to interfere with AA reacylation (30), the catalase was washed three times with distilled water before being dissolved in media. In each case, cells were preincubated with catalase (100 µg/ml = 150 U/ml, 1 h), NAC (1 mM, overnight), or L-NMMA (10 µM, 1 h) before and during cyclic stretch. Aliquots of media were removed after 10 or 20 min for eicosanoid analysis.

Statistical analysis. Results from duplicate wells in each experiment were averaged, and the mean was treated as a single data point. Data are presented as means ± SE for the indicated number of experiments (n). Comparisons between mean values were made using repeated-measures analysis of variance and Tukey's modified t-test (the Bonferroni criterion). P < 0.05 was considered significant. Statistical analysis was performed with the SigmaStat program (Jandel Scientific, San Rafael, CA).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Eicosanoid synthesis is inhibited by cyclic stretch. To determine whether epithelial cell synthesis of AA metabolites was influenced by cyclic strain, we measured the production of PGE2, TxB2, and 6-keto-PGF1alpha in cells cultured on Flex-I plates and stretched at 10 cycles/min, 20% maximal elongation using the Flexercell apparatus. The production of all three prostanoids by CTE cells was significantly lower in stretched cells compared with unstretched cells, as shown in Fig. 1, A-C. Figure 1, D-F, shows a similar attenuation of eicosanoid production in Calu-3 cells stretched at 10 cycles/min. The production of eicosanoids reached a plateau relatively quickly in stretched cells, achieving maximum values in 2.5-20 min. The effect was not dependent on serum components, since the same stretch-induced inhibition was observed when PGE2 was measured in Calu-3 cells in media without serum [5.39 (static) vs. 1.26 (10 cycles/min) ng/106 cells after 20 min, n = 2]. CTE cells produced more TxB2 than PGE2 (Fig. 1, A and C), whereas the reverse was true for Calu-3 cells (Fig. 1, D and F). Both cell types produced much less 6-keto-PGF1alpha (Fig. 1, B and E), but inhibition was still apparent. To detect 6-keto-PGF1alpha production by Calu-3 cells, it was necessary to supplement the medium with 10 µg/ml AA (Fig. 1E). Inhibition of PGE2, TxB2, and 6-keto-PGF1alpha production by stretch was sustained for 2 h in both cell types (data not shown), and inhibition of PGE2 was sustained for 6 days in Calu-3 cells (static, 9.91 ng/106 cells; 10 cycles/min, 5.09 ng/106 cells; n = 2). The difference in eicosanoid levels between static and stretched cultures was not due to enhanced mixing or low shear; in one experiment, the unstretched plates were gently rocked, and no difference in eicosanoid production was observed compared with unstretched, stationary plates. Lactate dehydrogenase levels in the media were below the detection limit of the assay (Sigma) for all treatments, indicating that no significant cell injury took place within the 20-min time course.


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Fig. 1.   Cyclic stretch decreases eicosanoid release in cat tracheal epithelial (CTE) cells (A-C) and Calu-3 cells (D-F). Filled symbols represent unstretched cells; open symbols denote cells stretched at 10 cycles/min, 20% maximal elongation. Data are expressed as ng/106 cells; error bars represent SE. * Significant difference from static cells [P < 0.05, n = 4 experiments for prostaglandin (PG) E2, 6-keto-PGF1alpha , and thromboxane (Tx) B2 in CTE; n = 5 for PGE2 and TxB2 in Calu-3; n = 2 for 6-keto-PGF1alpha in Calu-3]. Note differences in scales in A-F. In E, 10 µg/ml arachidonic acid were added to produce a detectable signal.

It has previously been shown that the strain field in Flex-I plates is radially dependent, with maximal elongation in the periphery and a small degree of compression in the center (12). According to Gilbert et al. (12), in the central region of the well (within 4 mm from the center), the strain is either zero or there is minimal (1%) compression depending on the vacuum pressure. In the rest of the well (~90% of the area), the strain varies from 0 to 20% (at a vacuum pressure of 15.3 kPa), with the highest strain present 2.5 mm from the wall. To determine whether prostanoid production was dependent on the strain field, cloning cylinders were used to isolate different regions of the well. We cultured cells within the central regions of the well (6.5 or 14.5% by area) or in the outer regions (75 or 85% by area) and measured PGE2 production. As shown in Table 1, PGE2 production after 10 min by unstretched Calu-3 cells was not significantly different over all regions of the well. In wells stretched at 10 cycles/min, however, PGE2 production was decreased in all regions of the wells.

                              
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Table 1.   PGE2 released from Calu-3 cells after 10 min

Eicosanoid production is frequency dependent. To investigate whether inhibition of eicosanoid synthesis was dependent on the frequency of stretch applied to the cells, the frequency was varied from 1 to 30 cycles/min, with 20% maximal elongation for all experiments. As shown in Fig. 2, the stretch-induced inhibition of PGE2 and TxB2 synthesis was dependent on the frequency of stretch in both CTE and Calu-3 cells. Maximum inhibition of PGE2 and TxB2 synthesis occurred at 10 cycles/min in both cell types. Although the production of PGE2 in CTE cells stretched at 1 cycle/min (n = 1) was not markedly different from unstretched CTE cells, PGE2 production at 10 cycles/min (n = 4) was significantly attenuated in both cell types. PGE2 release was less attenuated at 20 cycles/min (n = 1) and 30 cycles/min (n = 3) in both cell types. TxB2 release showed a similar trend, with the most marked inhibition in eicosanoid production at 10 cycles/min.


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Fig. 2.   Eicosanoid production is dependent on the frequency of stretch as measured for both PGE2 (open symbols) and TxB2 (filled symbols) after 20 min in CTE cells (A) and Calu-3 cells (B). A: static (n = 9), 1 cycle/min (n = 1), 10 cycles/min (n = 5), 20 cycles/min (n = 1), and 30 cycles/min (n = 4). B: static (n = 9), 10 cycles/min (n = 5), 20 cycles/min (n = 1), and 30 cycles/min (n = 4). * Significant difference from static (P < 0.05). # Significant difference from static and 10 cycles/min (P < 0.05). Error bars may be smaller than symbols.

Inhibition occurs at the level of COX. Several enzymatic steps are required for the production of these eicosanoids, including liberation of AA from phospholipids by phospholipase A2 (PLA2), conversion of AA to PGH2 by COX, and conversion of PGH2 to specific prostanoids by specific isomerases or synthases. The fact that formation of PGE2, TxB2, and 6-keto-PGF1alpha was each inhibited to a similar degree suggested that stretch interfered with synthesis at the level of either PLA2 or COX, the common steps in the prostanoid synthetic pathway. To determine whether inhibition of prostanoid synthesis by stretch resulted from an effect on PLA2 or COX, cells were subjected to 10 cycles/min stretch in medium supplemented with either exogenous AA or exogenous PGH2. As shown in Fig. 3, supplementation with exogenous AA (10 µg/ml) failed to abrogate the inhibitory effect of stretch on synthesis of PGE2 or TxB2 by CTE cells. On the other hand, supplementation with exogenous PGH2 (10 µg/ml) completely overcame the stretch-induced inhibition. These results indicate that stretch inhibits prostanoid synthesis primarily by interfering with the activity of COX rather than by limiting AA availability through PLA2.


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Fig. 3.   Inhibition of PGE2 (A) and TxB2 (B) due to stretch occurs at the level of cyclooxygenase. Bars at top show stretch-induced inhibition of prostanoid production seen in untreated cells. Bars in middle show same stretch-induced inhibition in cells treated with 10 µg/ml arachidonic acid (AA). Bars on bottom show that, in cells treated with 10 µg/ml PGH2, stretch inhibition is abrogated. Filled bars, unstretched cells; open bars, stretched (10 cycles/min) cells. Data are expressed as ng/106 CTE cells after 10 min. Length of bars represents mean eicosanoid production, and error bars represent SE (n = 4). * Significant difference from corresponding unstretched condition (P < 0.05).

Antioxidants influence PGE2 production. Because fluid shear stress increases free radical production in endothelial cells (20) and because oxidants can inactivate COX (9, 14), we hypothesized that the inhibition of COX by cyclic stretch may be oxidant mediated. We therefore tested the ability of the antioxidants catalase (100 µg/ml = 150 U/ml) and NAC (1 mM) to prevent the inhibition of PGE2 synthesis by cyclic stretch. As shown in Fig. 4, neither antioxidant affected PGE2 synthesis by unstretched Calu-3 cells after 10 min. However, Calu-3 cells stretched at 10 cycles/min for 10 min produced significantly more PGE2 in the presence of catalase and NAC than in the absence of the antioxidants. Attenuation of stretch-induced inhibition of PGE2 synthesis by the antioxidants was incomplete but suggests that impairment of COX activity by stretch is at least partially oxidant mediated.


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Fig. 4.   Oxidant-induced inactivation of cyclooxygenase (COX) in stretched cells is partially responsible for decrease in PGE2 production in Calu-3 cells. PGE2 production in Calu-3 cells stretched at 10 cycles/min after 10 min was significantly increased in the presence of the antioxidants catalase (100 µg/ml, 1-h pretreatment) and N-acetylcysteine (NAC, 1 mM, overnight pretreatment). PGE2 production by stretched cells was not altered in the presence of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 10-5 M, 1-h pretreatment). Filled bars, unstretched cells; open bars, cells stretched at 10 cycles/min. Length of bars represents mean PGE2 production (expressed as ng PGE2/106 cells), and error bars represent SE (n = 9 for the untreated conditions, n = 3 for all others). * Significant difference from corresponding unstretched condition (P < 0.05). # Significant increase from untreated stretched condition (P < 0.05).

Another potential stretch-induced free radical species that might inactivate COX is NO (32). We therefore tested the ability of the NO synthase inhibitor L-NMMA to prevent the inhibition of prostanoid synthesis by stretch in Calu-3 cells. As shown in Fig. 4, L-NMMA (10 µM) had no effect on synthesis of PGE2 by either stretched (10 cycles/min) or unstretched cells, suggesting that stretch-induced inhibition of prostanoid synthesis is not mediated by NO. Because NO has also been reported to increase COX activity in some systems (27), we hypothesized that NO synthesis might contribute to the higher levels of prostanoid formation seen in cells stretched at 20 and 30 cycles/min compared with 10 cycles/min (see Fig. 2). L-NMMA had no effect on PGE2 synthesis in Calu-3 cells stretched at either 20 cycles/min (2.47 ng/106 cells, untreated; 2.55 ng/106 cells, L-NMMA; n = 2) or 30 cycles/min (3.23 ng/106 cells, untreated; 3.12 ng/106 cells, L-NMMA; n = 2). Thus we could not demonstrate a role for NO in regulating PGE2 synthesis at the higher frequencies of stretch.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the last ten years, it has become apparent that cells derived from diverse tissues can sense their mechanical environment and respond to changes in shear stress, pressure, and stretch. For example, cyclic stretch has been shown to alter cytoskeletal fiber orientation in skeletal muscle (33), induce protooncogenes, and upregulate TGF-beta isoforms in rat glomerular mesangial cells (25); increase DNA synthesis in human amnion cells (17); increase levels of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol in endothelial cells (26); and increase levels of IP3 and intracellular Ca2+ in rat tracheal epithelial cells (3, 10).

In vivo, the airways are subjected to continuous cyclic distension and elongation during ventilation, yet there is little information about the effects of cyclic stretch on AEC function (3, 10). We investigated the effects of cyclic stretch on AEC metabolism of AA to prostanoids, which are important mediators in the regulation of smooth muscle tone (11), vascular permeability (23), mucous secretion (21), and immune function (7). Initiation of cyclic stretch resulted in a rapid, frequency-dependent downregulation of PGE2, TxB2, and 6-keto-PGF1alpha synthesis by both primary cultures of cat AEC and a line of human AEC. Our results show that inhibition of prostanoid production caused by cyclic stretch occurs at the level of COX and that oxidants are at least partially responsible for the inactivation of the enzyme. This stretch-induced depression of prostanoid production was sustained over 6 days and was not dependent on the presence of serum or NO. To our knowledge, this is the first report to demonstrate that physiological levels of cyclic strain regulate the synthesis of AA metabolites by AEC.

Using the Flexercell apparatus, Sumpio and Banes (31) observed that cyclic strain of endothelial cells increased PGI2 (but not TxA2) production when cells were supplemented with AA. Skinner et al. (29) demonstrated an increase in PGI2 production by fetal rat lung epithelial cells cultured in a gelatin foam and stretched for 15 min. In human amnion cells (17), human periodontal ligament fibroblasts (24), and skeletal muscle cells (33), PGE2 production was stimulated by a single constant stretch that was applied with a cone pushing upward on a flexible membrane. Thus the effect of stretch on eicosanoid production is variable and depends on the type of cell, the duration, magnitude, and frequency of the stretch, and potentially on the type of device used to strain the cells.

In this study, production of PGE2, TxB2, and 6-keto- PGF1alpha was decreased in both CTE cells and Calu-3 cells exposed to cyclic strain at a frequency of 10 cycles/min, 20% maximal elongation (Fig. 1). The reduction in both PGE2 and TxB2 production was observed both in regions with variable elongation strain as well as in regions of low compression (Table 1). These results suggest that cyclic compression may be just as important as cyclic elongation. The response was also frequency dependent, with the largest decreases occurring at 10 cycles/min (Fig. 2). The frequency dependence of inhibition shows that the response to cyclic strain is not simply an on/off mechanism; one regulatory mechanism may act to depress prostanoid synthesis upon initiation of low-frequency cyclic stretch, and another may exist that increases prostanoid synthesis with higher frequencies of stretch.

Adding exogenous AA failed to abrogate the difference in PGE2 and TxB2 levels produced when cells were cyclically stretched, but, after bypassing the COX enzyme by adding exogenous PGH2, the difference between static and strained cells was abolished (Fig. 3). This indicates that inhibition of prostanoid synthesis in stretched cells is not due to reduced availability of free AA but rather to impaired conversion of AA to PGH2 by COX. Isomerases or synthases that convert PGH2 to specific prostanoids are not significantly affected by stretch, since exogenous PGH2 completely reversed stretch-induced inhibition of PGE2 and TxB2 synthesis. Potentially, AA reincorporation and release may be affected by cyclic stretch, which may alter the production of leukotrienes or other AA metabolites not measured here. [In one experiment, leukotriene B4 (LTB4) synthesis from stretched and unstretched Calu-3 cells was undetectable.]

COX may be inactivated by oxidants, including those generated through catalytic action of the enzyme itself (9, 14). Because it has been previously shown that fluid shear stress increases oxygen free radical production by endothelial cells (20), we hypothesized that stretch-induced inhibition of prostanoid synthesis in AEC might be oxidant mediated. Antioxidant treatment (catalase or NAC) attenuated the stretch-induced inhibition of PGE2 synthesis, indicating that the effect of stretch on COX activity is at least partially oxidant mediated (Fig. 4). If generation of oxygen radicals in stretched cells were directly proportional to the frequency of stretch, a larger degree of inactivation of COX and hence a greater inhibition of prostanoid synthesis might be expected at higher frequencies of stretch. However, inhibition of prostanoid synthesis was greatest at 10 cycles/min and decreased at 20 and 30 cycles/min (Fig. 2). These results suggest either that oxidant production is greatest at 10 cycles/min and decreased at higher frequencies of stretch or, perhaps more likely, that a separate mechanism exists at higher frequencies which opposes oxidant inactivation of COX or somehow serves to overcome it.

An inhibitor of NO synthase, L-NMMA, was also used to determine whether NO production might impair or enhance COX activity, since NO has been shown to be a source of free radical species (32) and has also been shown to activate COX (27). Inhibition of NO production had no effect on PGE2 production in static or stretched cells at any frequency, suggesting that NO does not play a role in the stretch-induced inhibition of COX (Fig. 4).

Many of the mediators that have been cited as potential epithelium-derived modulators of airway function and inflammation are products of AA metabolism (27). Although there has been much research concerning regulation of inflammation in airway diseases, there has been little examination of the influence of physical forces on the synthesis of these inflammatory mediators by AEC. COX products are released during lung inflation and have been shown to modulate airway contraction and reactivity (11). Berend et al. (2) have provided evidence that the increase in lung volume in response to positive end-expiratory pressure is dependent on endogenous prostanoid synthesis. Gray et al. (13) showed decreased PGE2 production by AEC in a spontaneously occurring asthma-like condition in horses. It is conceivable that altered airway mechanics may have been responsible for decreased prostanoid synthesis in their study. Depressed eicosanoid production due to altered airway mechanics could alter inflammatory responses; for example, PGE2 can inhibit polymorphonuclear cell chemotaxis and release of other lipid mediators (such as LTB4; see Ref. 5) and may reduce production of inflammatory mediators such as tumor necrosis factor (19) and interleukin-1 (18). The relationship between stretch-induced inhibition of airway epithelial prostanoid synthesis in vitro and respiratory disease states in vivo remains uncertain. The impact of changes in airway mechanics and alterations in airway distension and compression in asthma and other airway diseases will require further study in animal models and in human subjects.

In summary, we have shown that cyclic stretch causes rapid, frequency-dependent inhibition of prostanoid synthesis by cat and human AEC. Our results demonstrate for the first time that stretch-induced inhibition of prostanoid production in AEC results from inactivation of COX and that oxidants are partially responsible for this effect. These results may have important implications for the pathogenesis of inflammatory diseases, including bronchial asthma, in which airway mechanics are altered.

    ACKNOWLEDGEMENTS

This work was supported in part by a Whitaker Foundation Special Opportunity Award, the American Lung Association of Metropolitan Chicago, the Research Service of the Department of Veterans Affairs, and the Cornelius Crane Asthma Center of Northwestern University. U. Savla was supported by a National Science Foundation graduate fellowship.

    FOOTNOTES

Address for reprint requests: C. M. Waters, Dept. of Anesthesiology, Northwestern University Medical School, Ward Bldg. 12-189 W139, 303 E. Chicago Ave., Chicago, IL 60611.

Received 3 January 1997; accepted in final form 11 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 273(5):L1013-L1019
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