Intracellular signaling mechanisms of interleukin-1beta in synovial fibroblasts

Oleg V. Kolomytkin1,2, Andrew A. Marino1, Kalia K. Sadasivan1, Robert E. Wolf3, and James A. Albright1

1 Department of Orthopaedic Surgery and 3 Section of Rheumatology, Department of Medicine, Louisiana State University Medical Center, Shreveport, Louisiana 71130-3932; and 2 Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino 142292, Russia

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The possibility that membrane depolarization of synovial fibroblasts caused by interleukin-1beta (IL-1beta ) was mediated by protein kinase C (PKC) and Ca2+ influx was studied using inhibitor and activator analysis. The effect of IL-1beta was blocked by bisindolylmaleimide I, an inhibitor of PKC, and by the Ca2+ channel blockers nifedipine and verapamil. In other experiments, PKC was activated using phorbol 12-myristate 13-acetate, and Ca2+ influx was increased by means of a Ca2+ ionophore. Simultaneous application of phorbol ester and Ca2+ ionophore in the absence of IL-1beta mimicked the depolarization caused by IL-1beta . The results were consistent with the hypothesis that, under the conditions studied, activation of PKC and Ca2+ influx are necessary and sufficient processes in the transduction of IL-1beta by synovial cells leading to membrane depolarization. The essential role of protein phosphorylation and Ca2+ influx in the early electrophysiological response of synovial fibroblasts to IL-1beta was therefore established. The role of IL-1beta -induced depolarization in regulating protein expression by the cells remains to be determined, but the results reported here, taken together with observations that protein phosphorylation and Ca2+ influx also mediate the effect of IL-1beta on protease production (1, 2), suggest that electrophysiological changes are actually part of the pathway for expression of proteases in response to IL-1beta .

membrane potential; nystatin; voltage clamp

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE CYTOKINE interleukin-1beta (IL-1beta ) is an important regulator of synovial cells. Binding of IL-1beta by a cell surface receptor results in signal transduction leading to the expression of many different proteins, including metalloproteinases and IL-1beta itself (5, 8, 19). Dysregulation of this response, especially chronic overproduction of metalloproteinases, can lead to the destruction of joint cartilage and other adverse changes (17, 23).

A spectrum of second messengers, enzymes, G proteins, and transcription factors have been implicated as components of IL-1 signal transduction in various cell types (7, 16). The wide range of reported processes and mechanisms suggests that the consequences of ligation of IL-1beta with its receptor vary with cell type, and perhaps even within a cell type under different study conditions.

Contemporary ideas regarding the effect of IL-1beta in inexcitable cells do not attribute a significant role to electrophysiological factors (7, 16). Nevertheless, using the perforated-patch method, we observed changes in the current-voltage (I-V) characteristics of rabbit synovial fibroblasts during transduction of IL-1beta . When aggregated, the cells exhibited two states having high and low membrane potentials (Vm), respectively. IL-1beta caused cells to transit from the high to the low state, particularly when the cell was voltage clamped under conditions that favored Ca2+ influx (14). Thus it is possible that IL-1beta can affect important cellular processes known to be influenced by Vm, such as transport of amino acids and exocytosis.

The aim of this study was to determine the mechanism responsible for the previously observed joint effect of IL-1beta and voltage clamp on the Vm of synovial cells. We hypothesized that activation of protein kinase C (PKC) and Ca2+ influx were necessary and sufficient processes for the effect of IL-1beta , and this was established using inhibitor and activator analysis.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells. Rabbit synovial fibroblasts (HIG-82, American Type Culture Collection) were grown at 37°C with 5% CO2 in 25-ml polystyrene flasks containing F-12 medium (GIBCO BRL) with 10% fetal bovine serum and without antibiotics. For passage, confluent cultures were trypsinized (1 ml, 0.08%) for 3-5 min, after which 4 ml of medium were added and the suspended cells were centrifuged, resuspended, and then seeded (106 cells) into 4 ml of medium. For electrophysiological measurements, 105 cells were added to 35-mm petri dishes and incubated at 37°C for 24 h, after which the cells were treated for 2 min with 1 ml of 0.01% collagenase and 0.01% hyaluronidase. This step, which was mild by comparison with routine trypsinization of the cells, was necessary to obtain stable gigaseals. The cells were incubated in medium for 40-60 min at 37°C to allow recovery from the enzyme treatment. The medium was then replaced with bath solution, and all measurements were made in bath solution at 25°C, employing the cells (10-20 µm in diameter) that remained adherent to the bottom of the petri dish (most cells remained adherent following enzyme treatment).

Electrodes. The nystatin perforated-patch method (9) was used to measure the transmembrane current under voltage clamp. The perforated-patch method was employed because it permitted use of the whole cell configuration to measure electrical properties of the cell while preventing diffusion of small signaling molecules from the cell into the electrode, thereby preserving intracellular regulation. Glass capillaries 1.0 mm in diameter were pulled in two steps (PB-7, Narishige) and fire polished in a microforge (MF-9, Narishige). The resistance of the electrodes was 7-9 MOmega in bath solution. The pipette salt solution was (in mM) 125 monopotassium aspartate, 30 KCl, 4 NaCl, and 10 HEPES-KOH (pH 7.2; 318 mosmol/l, calculated). The composition of the bath solution was (in mM) 145 NaCl, 5.4 KCl, 1.5 CaCl2, 1.0 MgCl2, 5.0 HEPES-NaOH, and 5.0 glucose (pH 7.3; 328 mosmol/l, calculated). Because nystatin interfered with gigaseal formation, the tip of the pipette was filled with a nystatin-free solution before the addition of pipette solution containing 0.3 µg/ml nystatin. The gigaseal was formed during the time needed for the nystatin to diffuse to the tip of the micropipette (9).

Inhibitors and activators. The dish containing the clamped cell was perfused with bath solution containing the agent under study. The agents used were human recombinant IL-1beta (Sigma no. I 4019), phorbol 12-myristate 13-acetate (PMA; Sigma no. P 8139), bisindolylmaleimide I (Calbiochem no. 203290), A 23187 Ca2+ ionophore (Sigma no. C 7522), verapamil (Sigma no. V 4629), and nifedipine (Sigma no. N 7634). IL-1beta was added into solution in the presence of 0.1% bovine albumin (Sigma no. A 2153) as carrier protein. Control experiments showed that 0.1% bovine albumin did not influence the I-V characteristics of the cells. PMA, bisindolylmaleimide I, A-23187 Ca2+ ionophore, verapamil, and nifedipine were dissolved in DMSO (Sigma no. D 5879) and then added to the bath solution. Final concentration of DMSO did not exceed 0.3%; control experiments showed that it did not influence the I-V characteristics of the cells.

The inhibitors and activators were used at concentrations typically used in previous studies to cause relatively specific reactions and thereby permit inferences regarding particular mechanisms involved in processes under study.

Electrical measurements. Gigaseals (~10 GOmega ) were formed under negative pressure (5-10 cm H2O), typically within 0.5-5 min; the success rate was >50%. After gigaseal formation, the negative pressure was removed and the nystatin channels formed within 5-15 min; the resistance of the perforated-patch membrane was 40 ± 20 MOmega . Gigaseals and nystatin pores usually remained stable for hours.

I-V curves and Vm (measured as the reversal potential at zero-current clamp) were recorded using a patch-clamp amplifier (Axopatch 200B, Axon Instruments) as described earlier (14). The amplifier was connected to a computer (TL-1 DMA interface, Axon Instruments), and commercial software (pCLAMP 6, Axon Instruments) was used to control the amplifier and collect and analyze the experimental data. For measurement of I-V curves, a voltage ramp was applied to the electrode from -80 to 0 mV at 1.67 mV/s, and the current was sampled at 6 kHz. It was shown that I-V curves thus obtained represented steady-state I-V characteristics of HIG-82 cells (14).

Procedure. The influence of different agents on the IL-1beta -induced changes in the I-V curves of synovial fibroblasts was studied, and the responsible mechanisms were evaluated using substances having known physiochemical properties. In each experiment, the I-V curve was measured initially in bath solution and then measured again after perfusion of the dish with bath solution containing the agent or combination of agents under study. I-V curves obtained before and after addition of the test agent were compared to determine the influence of the agent. Each measurement was repeated five times using five cells in five different dishes.

We showed earlier that HIG-82 cells exhibit a normal Vm only when aggregated with each other (14). Therefore, the measurements reported here were carried out on a cell aggregated with other cells. The aggregates contained different numbers of cells (5-20 cells) because it was not convenient to restrict the study to aggregates with the same number of cells. Because the cells were connected electrically to each other by gap junctions (14), however, the I-V curve depended on the number and configuration of the cells in the aggregate. The current was higher for aggregates containing a larger number of cells. Consequently, a procedure was needed to standardize the curves so that the results from cells in aggregates of different sizes recorded under the same experimental conditions could be combined. It turned out that, by following the procedure described below, we could fit I-V curves from different aggregates to each other simply by changing the scale of the current (Fig. 1, A and B). Our ability to do so indicated that the varying number of cells in the aggregate only changed the magnitude of the current and did not change the shape of the I-V curves. We used the property of constancy of shape to compare the results obtained on cells in different aggregates.


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Fig. 1.   Effect of IL-1beta (1 ng/ml) on current-voltage (I-V) characteristics of 5 different synovial fibroblasts measured in different aggregates. A: curves obtained in bath solution. B: results of fitting each curve to mean by multiplying current for each point on curve by a constant coefficient determined using method of least squares. C: means ± SE of fitted curves. D: curves for same cells after 15 min of voltage clamp at -30 mV followed by 5 min of exposure to interleukin-1beta (IL-1beta ). E: results of multiplying current for each point on curve in D by previously determined constant coefficient for each cell. F: means ± SE of curves in E. Effect of IL-1beta was evaluated by comparing curves in C and F. Arrows indicate average curves.

For each experiment, I-V curves for five cells in different aggregates in bath solution were averaged (Fig. 1A). We used the average I-V curve to fit I-V curves for different cells to each other. The I-V curve for each cell was fitted to the average I-V curve by multiplying the current by a certain coefficient. The value of the coefficient for each cell was found by fitting its I-V curve to the average I-V curve, employing the method of least squares. The fitted I-V curves (Fig. 1B) were found to coincide well with each other. It is seen in Fig. 1, A and B, that even an I-V curve for a cell in a relatively large aggregate (with large current) coincides well with other I-V curves after fitting. Thus I-V curves for all cells were normalized to the I-V curve of the cell aggregate of average size. The means ± SE for the fitted I-V curves were used to display the behavior of the cells in bath solution (Fig. 1C).

The effect of IL-1beta (or of the other substances studied) on the I-V curve was obtained as follows. The I-V curve was measured for each cell after addition of IL-1beta (Fig. 1D; or other substances). Then, the current in the I-V curve was multiplied by the same coefficient found for the cell in bath solution (Fig. 1E). The mean ± SE of these curves (Fig. 1F) was then compared statistically with the results obtained in bath solution (Fig. 1C) to ascertain the effect of IL-1beta (or other substances). The procedure followed permitted comparison of results obtained from cells in aggregates of different sizes subjected to the same conditions.

The reversal potential (voltage corresponding to zero current) was found for each I-V curve, and reversal potentials of the cells in bath solution and those exposed to the substance under study were compared using the Wilcoxon signed rank test at a significance level of P < 0.05. The reversal potentials for the original and fitted (multiplied by a coefficient) I-V curves coincided with each other.

The systematic error of the measurement of Vm due to the electrode potential did not exceed 2 mV.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of IL-1beta . The effect of IL-1beta on the I-V curve of cells having a high Vm is shown in Fig. 1. After the initial (control) measurements in bath solution (Fig. 1A), the cells were held at -30 mV for 15 min before the addition of IL-1beta (1 ng/ml of bath solution), whereupon the I-V curves were measured again after 5 min (Fig. 1D). The results of normalizing each control curve to the mean and the means ± SE of these curves are shown in Fig. 1, B and C, respectively. The corresponding results following addition of IL-1beta are shown in Fig. 1, E and F. IL-1beta caused an increase in inward current and a shift in the reversal potential (Table 1), confirming our previous results (14).

                              
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Table 1.   Influence of different substances on reversal potential of synovial fibroblasts

Effect of IL-1beta in presence of PMA. PMA, an activator of PKC, mimicked the effect of IL-1beta on induction of metalloproteinase synthesis by HIG-82 cells at 0.15 µM (1), possibly indicating that protein phosphorylation by PKC was responsible for the membrane depolarization caused by IL-1beta . To test this hypothesis, we studied the effect of PMA on Vm using the same protocol as for IL-1beta . Control measurements showed that 1.5 µM PMA did not change the I-V curve or the Vm (Table 1). Thus activation of PKC by PMA did not produce membrane depolarization.

After the control measurements (Fig. 2A), 1.5 µM PMA was added to the bath solution and the cell was held at -30 mV for 15 min. IL-1beta was then added, and the I-V curve changed significantly within 5 min (Fig. 2B, Table 1). Thus activation of PKC by PMA did not eliminate the effect of IL-1beta on membrane depolarization.


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Fig. 2.   Effect of IL-1beta (1 ng/ml) on I-V characteristics (means ± SE of normalized curves) of 5 different synovial fibroblasts measured in different aggregates in presence of phorbol 12-myristate 13-acetate (PMA; 1.5 µM). A: I-V curves in bath solution. B: I-V curves after 15 min of voltage clamp at -30 mV and PMA treatment followed by exposure to IL-1beta for 5 min.

Effect of IL-1beta in presence of bisindolylmaleimide I. Although PKC alone was not responsible for changing the I-V curves following addition of IL-1beta , PKC could have functioned with other cell systems to produce membrane depolarization. To test this hypothesis, we studied the effect of IL-1beta in the presence of bisindolylmaleimide I, a protein kinase inhibitor that is specific for PKC and that inhibits the activity of all four PKC subtypes with similar potency (21). Addition of 3 µM bisindolylmaleimide I alone to the bath solution did not change the I-V curve or the Vm of the cells (Table 1).

After the control measurements (Fig. 3A), 3 µM bisindolylmaleimide I was added to the bath solution and the cells were held at -30 mV for 15 min before the addition of 1 ng/ml IL-1beta . The I-V curves did not change (Fig. 3B, Table 1), indicating that inhibition of PKC eliminated the effect of IL-1beta on the I-V curves. Because depolarization did not occur after addition of IL-1beta in the presence of an inhibitor of PKC, it can be concluded that activation of PKC by IL-1beta is necessary to produce membrane depolarization. Because activation of PKC alone (by PMA) did not cause membrane depolarization, it follows that IL-1beta activates not only PKC but other systems as well.


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Fig. 3.   Effect of IL-1beta (1 ng/ml) on I-V characteristics (means ± SE of normalized curves) of 5 different synovial fibroblasts measured in different aggregates in presence of bisindolylmaleimide I (3 µM). A: I-V curves in bath solution. B: I-V curves after 15 min of voltage clamp at -30 mV and bisindolylmaleimide I treatment followed by exposure to IL-1beta for 5 min.

Effect of PMA in presence of Ca2+ ionophore. Previous evidence suggested that the system responsible for intracellular Ca2+ homeostasis was the system that worked with PKC to mediate the effect of IL-1beta (7, 18). If so, then addition of PMA and Ca2+ ionophore together should produce membrane depolarization. Addition of 10 µM Ca2+ ionophore did not alter the Vm (Table 1).

After the control measurements (Fig. 4A), 1.5 µM PMA and 5 µM Ca2+ ionophore were added. After 15 min, an increase in inward current was observed (Fig. 4B), accompanied by a shift in reversal potential (Table 1). Thus activation of PKC and increased Ca2+ concen-tration resulted in a membrane depolarization similar to that produced by IL-1beta .


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Fig. 4.   Effect of PMA (1.5 µM) and Ca2+ ionophore (5 µM) on I-V characteristics (means ± SE of normalized curves) of 5 different synovial fibroblasts measured in different aggregates. A: I-V curves in bath solution. B: I-V curves after exposure to PMA and Ca2+ ionophore for 15 min.

Effect of IL-1beta in presence of nifedipine and verapamil. To clarify the role of Ca2+ in the effect of IL-1beta on Vm, we used the Ca2+ channel blockers nifedipine and verapamil. Neither 50 µM nifedipine nor 10 µM verapamil altered the Vm.

After the control measurements (Figs. 5A and 6A), 50 µM nifedipine or 10 µM verapamil was added to the bath solution and the cells were held at -30 mV for 15 min before the addition of 1 ng/ml IL-1beta . The I-V curves did not change significantly within 5 min (Figs. 5B and 6B, respectively), indicating that blocking of Ca2+ channels in the cytoplasmic membrane eliminated the effect of IL-1beta . Thus opening of membrane Ca2+ channels was essential for membrane depolarization caused by IL-1beta .


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Fig. 5.   Effect of IL-1beta (1 ng/ml) on I-V characteristics (means ± SE of normalized curves) of 5 different synovial fibroblasts measured in different aggregates in presence of nifedipine (50 µM). A: I-V curves in bath solution. B: I-V curves after 15 min of voltage clamp at -30 mV and nifedipine treatment followed by exposure to IL-1beta for 5 min.


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Fig. 6.   Effect of IL-1beta (1 ng/ml) on I-V characteristics (means ± SE of normalized curves) of 5 different synovial fibroblasts measured in different aggregates in presence of verapamil (10 µM). A: I-V curves in bath solution. B: I-V curves after 15 min of voltage clamp at -30 mV and verapamil treatment followed by exposure to IL-1beta for 5 min.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Protein expression by synovial cells in culture in response to IL-1beta typically occurs after exposure for 1-2 days (5, 8, 19), and there is some evidence suggesting that PKC and Ca2+ are involved somewhere in the signaling pathway (1). Before our work (14), the initial steps of IL-1beta transduction in synoviocytes were unstudied. We found that IL-1beta induced relatively fast (~5 min) switching of electrophysiological states of synovial fibroblasts, leading to transitions to a state of low Vm.

IL-1beta activates different signaling pathways in different cell types, and only some of the pathways involve PKC and Ca2+ (7, 16). The hypothesis of this study was that one particular pathway of the large number of theoretically possible pathways was responsible for the early electrophysiological response of synovial fibroblasts to IL-1beta . As hypothesized, we found that activation of PKC and Ca2+ influx were necessary and sufficient processes in the transduction of IL-1beta by synovial cells, under the conditions studied, to cause membrane depolarization.

Membrane depolarization occurred soon after ligation of IL-1beta by its receptor; within 5 min after addition of IL-1beta , the mean reversal potential of the cells decreased from -69 to -29 mV (Fig. 1, Table 1). Bisindolylmaleimide I alone had no effect on Vm, indicating that activation of PKC was not necessary to maintain a high resting potential. However, the IL-1beta -induced decrease in Vm was blocked by bisindolylmaleimide I (Fig. 3), thereby implicating participation of PKC in the effect of IL-1beta .

The Ca2+ channel blockers nifedipine and verapamil had no effect on Vm, indicating that passage of Ca2+ from the bath solution into the cell through voltage-gated ion channels was not necessary for maintenance of the cell resting potential. However, nifedipine (Fig. 5) and verapamil (Fig. 6) blocked the action of IL-1beta , indicating that Ca2+ influx was an essential step in the signal pathway of IL-1beta leading to membrane depolarization.

The question of whether PKC activation and Ca2+ influx were sufficient to produce depolarization was studied by exposing the cells to PMA and Ca2+ ionophore in the absence of IL-1beta . Neither agent alone affected the Vm or I-V characteristics of the cells. Thus neither activation of PKC alone nor Ca2+ influx alone was sufficient to mimic the effect of IL-1beta on Vm. Jointly, however, the agents produced a membrane depolarization that was indistinguishable from the effect of IL-1beta (Fig. 4).

It could be argued that PKC in the synovial cells was self-activating and that the basic role of IL-1beta was to increase Ca2+ influx, which then permitted the already activated PKC to catalyze processes that led to depolarization. However, this view is inconsistent with the observation that Ca2+ ionophore alone did not mimic the effect of IL-1beta (Table 1). A similar argument in favor of an effect of IL-1beta on PKC activation with spontaneously open Ca2+ channels is inconsistent with the evidence that PMA alone did not mimic the effect of IL-1beta (Table 1). It can be concluded, therefore, that the PKC was not self-activating and that Ca2+ channels were not spontaneously open but rather that both processes were activated by IL-beta .

Changes in Ca2+ influx and in intracellular Ca2+ have previously been implicated in the regulation of protease production by synovial cells (10, 22), but a clear picture of the role of PKC in the IL-1 signaling pathway in synovial cells has not emerged from previous PKC inhibitor studies. IL-1-induced expression of proteases (3), protease inhibitor (6), IL-1 (24), and arachidonic acid (4) was suppressed by PKC inhibitors. In other studies, however, different effects were seen. The inhibitors were associated with increased expression of PGE2 (20), a biphasic effect on IL-8 production (12), and no effect on proteases (11). One possible explanation for the range of observed responses is the occurrence of nonspecific reactions induced by the various PKC inhibitors. For example, IL-1 can induce expression of PGE2 (5), which is an agonist of cAMP (15); cAMP can downregulate stromelysin transcription (13). Thus the observation that staurosporine antagonized stromelysin production (3) could possibly have been due to a PKC-independent mechanism (upregulation by staurosporine of IL-1-induced PGE2, leading to increased levels of cAMP that inhibited stromelysin). The point is that inhibitor analysis is best in relatively simple cases in which the system has few different intermediate states in its signaling pathway. It is logically difficult to identify pathways when observations are made many hours after addition of the inhibitor because alternative and indirect signaling pathways potentially could increase the probability of an unspecified interaction with the inhibitor. This consideration underscores the importance of electrophysiological measurements in the study of IL-1 transduction because the short time scale of the measurements tends to exclude long-term processes, thereby making inhibitor analysis a more probative instrument.

IL-1 is a potent inducer of metalloproteinases by synovial cells, and this process probably plays an important role in the pathophysiology of joints (5, 8, 17, 19). Attempts at therapy based on reducing the levels of metalloproteinases in diseased joints do not appear to be successful (17), suggesting that blocking the disease earlier in its development might be a more effective strategy. This consideration provided the impetus for our study of the early electrical events associated with binding of IL-1beta by synovial cells.

We conclude that the depolarization that occurs as an early consequence of transduction of IL-1beta by voltage-clamped HIG-82 cells is caused by activation of PKC and Ca2+ influx. The role of the IL-1beta -induced depolarization in regulating protein expression by the cells remains to be determined, but the results reported here, taken together with observations that activation of PKC and Ca2+ influx also mediate the effect of IL-1beta on protease production (1, 2), suggest that electrophysiological changes are actually part of the pathway for expression of proteases in response to IL-1beta .

    ACKNOWLEDGEMENTS

This work was supported in part by the Center for Excellence in Arthritis and Rheumatology of the Louisiana State Medical Center and by the Hamilton Foundation.

    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: A. A. Marino, Dept. of Orthopaedic Surgery, Louisiana State University Medical Center, PO Box 33932, Shreveport, LA 71130-3932.

Received 2 July 1998; accepted in final form 13 September 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 276(1):C9-C15
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