Ion exchange activity in pulmonary artery smooth muscle cells: the response to hypoxia

Jane A. Madden1,2, Daniel E. Ray3, Peter A. Keller2, and Jack G. Kleinman2,3

Departments of 1 Neurology and 3 Medicine, The Medical College of Wisconsin, Milwaukee 53226; and 2 Research Service, Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin 53295


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purposes of this study were to determine 1) the presence of the major ion transport activities that regulate cytoplasmic pH (pHc) in cat pulmonary artery smooth muscle cells, i.e., Na+/H+ and the Na+-dependent and -independent Cl-/HCO3- exchange, 2) whether pHc changes in cells from small (SPAs) and large (LPAs) pulmonary arteries during hypoxia, and 3) whether changes in pHc are due to changes in the balance of exchange activities. Exchange activities as defined by physiological maneuvers rather than molecular identity were ascertained with fluorescence microscopy to document changes in the ratio of the pHc indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Steady-state pHc was higher in LPA than in SPA normoxic smooth muscle cells. SPAs and LPAs possessed all three transport activities; in HCO3--containing normoxic solutions, Cl-/HCO3- exchange rather than Na+/H+ exchange set the level of pHc; in HCO3--containing hypoxic solutions, pHc increased in SPA and decreased in LPA cells; altering the baseline pHc of a cell type to that of the other did not change the direction of the pHc response during hypoxia. The absence of Na+ prevented hypoxia-induced alkalinization in SPA cells; in both cell types, inhibiting the Cl-/HCO3- exchange activities reversed the normal direction of pHc changes during hypoxia.

cat; sodium/hydrogen exchange; sodium-dependent chloride/bicarbonate exchange; sodium-independent chloride/bicarbonate exchange


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DESPITE A CONSIDERABLE research effort to elucidate the mechanism(s) of hypoxic pulmonary vasoconstriction, the sequence of cellular events leading to the contraction of the pulmonary vascular smooth muscle are not yet fully understood. In a study of cat pulmonary arteries, our laboratory (6) found that hypoxic pulmonary vasoconstriction is a function of the small (200- to 600-µm-diameter) arteries. Smooth muscle cells isolated from these small arteries contract when they are exposed to hypoxia (7), and the free cytoplasmic calcium concentration ([Ca2+]c) also increases (12). In contrast, arteries > 800 µm in diameter, as well as their smooth muscle cells, do not contract to hypoxia, and the [Ca2+]c decreases. In a preliminary report, our laboratory (11) found that during hypoxia the cytoplasmic pH (pHc) of small pulmonary artery (SPA) smooth muscle cells increased concomitantly with the increase in [Ca2+]c. In the large pulmonary artery (LPA) cells, pHc decreased in parallel with the decrease in [Ca2+]c. Changes in pHc can alter Ca2+/calmodulin binding to modify the magnitude of myosin phosphorylation and the contractile response for a given [Ca2+]c (1). Thus changes in pHc as well as in [Ca2+]c may play a role in determining whether a pulmonary artery does or does not constrict in response to hypoxia.

pHc regulation in smooth muscle cells is accomplished through the activities of several ion transport systems (Fig. 1). Some of these transport activities are poised to acidify the cell, i.e., Na+-independent Cl-/HCO3- exchange. Others work to alkalinize it, principally Na+/H+ and Na+-dependent Cl-/HCO3- exchange. Vasoactive agonists and cellular energy status can affect the activity and possibly the direction of ion transport activities in vascular smooth muscle (1, 5, 8, 13). For the smooth muscle cell to respond appropriately to agonists, the transport activities must be coordinated. For example, agonist stimulation of transport activity may increase the cycling of an ion between the cell and its environment, and this, in turn, may stimulate other transport activities to restore ionic homeostasis (13).


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Fig. 1.   Major ion transport mechanisms that regulate smooth muscle cytoplasmic pH (pHc) by acidification (Na+-independent Cl-/HCO3- exchange) or alkalinization (Na+/H+ and Na+-dependent Cl-/HCO3- exchange)

How pulmonary artery smooth muscle cells regulate pHc has not been extensively studied nor has it been determined whether any of the transport activities that regulate pHc are affected by hypoxia. Na+/H+ exchange has been shown indirectly in ferret pulmonary artery (3) and directly in guinea pig main pulmonary artery smooth muscle cells (9). In the guinea pig cells, Na+/H+ exchange played an active role in pHc regulation even in HCO3--containing buffers (9). Other than the aforementioned work by Quinn et al. (9), the presence and role of Na+-dependent and -independent Cl-/HCO3- exchange in pulmonary artery smooth muscle cells has not been studied.

The purposes of this study were to determine 1) the presence of the major ion transport activities that regulate pHc in cat pulmonary artery smooth muscle cells, i.e., Na+/H+ and Na+-dependent and -independent Cl-/HCO3- exchange; 2) whether pHc changes in cells from SPAs and LPAs during hypoxia; and 3) whether changes in pHc are due, at least in part, to changes in the balance of transport activities that regulate pHc. The presence and activity of the ion exchange activities as defined by conventional physiological maneuvers, not by determination of a specific molecular identity, was accomplished with fluorescence microscopy to document changes in the ratio of the fluorescence indicator, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), which reflects changes in pHc.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Isolation and Culture

This study was approved by the Animal Care and Use Committee of the Zablocki VA Medical Center (Milwaukee, WI). Adult mongrel cats (2.5-4.0 kg) of either sex were anesthetized with ketamine (15 mg/kg) and pentobarbital sodium (30 mg/kg) and decapitated. The animals were exsanguinated by severance of the carotid artery, and the lungs were removed under aseptic techniques and immediately placed in chilled sterile physiological saline solution (PSS). SPAs (200- to 600-µm diameter) and LPAs (>800-µm diameter) were dissected from portions of both lung lobes under sterile conditions and placed in sterile MEM plus penicillin and streptomycin (Sigma) until the dissection process was complete.

The dissected arteries were cut into 2-mm segments and placed in sterile centrifuge tubes containing 1 ml of enzyme solution. The tubes were then placed in a sterile incubator at 37°C for ~1 h. Enzyme digestion was stopped by the addition of an equal volume of smooth muscle basal medium and smooth muscle medium supplement plus penicillin and streptomycin (all from Sigma). The crude digestate was filtered through three staccups in series as previously described (7) to remove connective tissue and cell debris, and the resulting suspension was centrifuged for 10 min at 1,200 rpm at 4°C. The supernatant was removed and discarded, and the pellet was suspended in 1 ml of the medium. Approximately 0.1 ml of the suspension was plated onto coverslips coated with 0.1 mg/ml of poly-D-lysine (Sigma). The coverslips were put into culture dishes and left in a darkened hood for 30 min. Afterward, 2.5 ml of smooth muscle medium were added to the culture dishes, and they were put in a water-jacketed incubator at 37°C for 48-72 h before use.

Fluorescence Imaging

For fluorescence imaging studies of individual smooth muscle cell pHc, the cells were loaded with 5 µl of 5 µM BCECF (Molecular Probes) for 45 min at 37°C in a humidified atmosphere of 95% air-5% CO2. The coverslip containing the cells was placed in a specially constructed 2-ml volume chamber designed to sit on the stage of a Leitz upright microscope. Warm, gassed PSS from the reservoir was slowly perfused over the cells.

The cells were imaged at the selected wavelengths for BCECF (see pHc Calibration) with a SenSys air-cooled CCD camera (Photometrics, Tucson, AZ) and epifluorescence illumination. The epifluorescence illumination light path had an eight-position, computer-controlled filter wheel and shutter attached to the microscope. The delay between the two excitation wavelengths was on the order of 50 ms. The computer-controlled shutter was used to eliminate unnecessary specimen illumination. The microscope was linked to a Silicon Graphics SGI Indy computer equipped with RatioTool software (Inovision, Raleigh, NC).

Fluorescence measurements rely on the ratio principle, which involves selecting ion-sensitive excitation or emission wavelengths that increase or decrease on cation binding. Cells loaded with BCECF were sequentially excited at 450 and 500 nm and light emitted at 535 nm (10-nm bandwidth for all filters). The ratio of 500- to 450-nm excitation was directly proportional to pHc. Individual pulmonary artery muscle cells were selected in a field of view, and changes in their BCECF ratios were acquired every 5 s during an experiment with RatioTool software. The ratios were converted to pHc based on calibration curves (see pHc Calibration).

pHc Calibration

The pHc was determined from calibration curves for each cell type. The cells in the chamber were perfused sequentially with solutions (see Solutions) of pH 6.5, 7.0, and 7.5 that contained the K+ ionophore nigericin (10). This ionophore exchanges intracellular K+ for extracellular H+ and in the presence of a high concentration of K+, allows equalization of intracellular and extracellular pH. For each of the three solutions, the BCECF ratios were recorded every 5 s until the ratios were stable. The BCECF ratios obtained for each solution were plotted, and a straight line demonstrating the relationship between pH and fluorescence was fit to the curve. These curves were used to calculate pHc for the pulmonary artery smooth muscle cells used in the experiments.

Solutions

Enzyme solution. The enzyme solution contained 500 U/ml of collagenase (type II), 50 U/ml of elastase, 0.75% (wt/vol) bovine serum albumin, 4 mM ATP, and 0.25% (wt/vol) soybean trypsin inhibitor (Worthington Biochemical, Freehold, NJ).

Pucks saline solution. Pucks saline solution contained (in mM) 0.1 CaCl2 · 2H2O, 4.7 KCl, 1.18 KH2PO4, 1.19 MgSO4 · 7H2O, 120 NaCl, 0.116 Na2HPO4 · 7H2O, 5.5 D-glucose, and 0.013 phenol red (pH 7.34).

PSS. PSS contained (in mM) 141 Na+, 4.7 K+, 2.5 Ca2+, 0.72 Mg2+, 124 Cl-, 1.7 H2PO43-, 22.5 HCO3-, and 11 glucose. Solutions used for the ammonia pulse technique were prepared by replacing NaCl with 15 mM NH4Cl. HCO3--free solution was prepared by substituting 20 mM HEPES buffer.

Cl--free solution. This solution contained (in mM) 128 sodium gluconate, 3.3 potassium gluconate, 6 calcium gluconate (hemi Ca2+ salt), 1.2 KH2PO4, 0.72 MgSO4, 2.25 Na2SO4, 11 glucose, and 22.5 NaHCO3-.

Na+-free solution. This solution contained (in mM) 140 N-methyl-D-glucamine, 3.3 KCl, 1.2 KH2PO4, 0.72 MgSO4, 2.5 CaCl2, 11 glucose, and 118 HCl, adjusted to pH 7.37 with 2 M Tris buffer.

pH calibration solution. pH calibration solution contained (in mM) 140 K+, 2.5 CaCl2, 1 MgSO4, 50 HEPES, 10 µM nigericin, and 1 µM valinomycin. The solutions were titrated with 2 M Tris buffer to adjust pH to 6.5, 7.0, and 7.5.

Exchanger Inhibitors

Exchanger inhibitors were 3 × 10-5 M 5-(N,N-dimethyl)amiloride hydrochloride (DMA; Calbiochem, San Diego, CA) and 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; Sigma).

Gases

Calibrated gas mixtures were pumped from weather balloons through dispersion stones into the PSS reservoir. The gas mixtures used were normoxic control (PO2 140 Torr; PCO2 37 Torr), normoxic acidosis (PCO2 increased to 51 Torr), normoxic alkalosis (PCO2 decreased to 21 Torr), hypoxic control (PO2 50 Torr; PCO2 37 Torr), hypoxic acidosis and hypoxic alkalosis (PCO2 changed as above). The PSS from the reservoir and the cell chamber described in Fluorescence Imaging were sampled at regular intervals, and the PO2, PCO2, and pH were determined with a Corning model 278 blood gas analyzer. When the gas mixtures bubbling into the reservoir were changed during an experiment, the gas tension in the cell chamber changed within 2 min. The new values for the PO2, PCO2, and pH in the cell chamber were determined 5 min after the gas change, the point at which the BCECF ratios were stable.

Data and Statistical Analyses

pHc values for the cells are expressed as means ± SE for n cells. To determine differences, Student's paired and unpaired t-tests or ANOVA and Fisher's least significant difference (LDS) test were used as appropriate. A value of P < 0.05 was considered significant.


    RESULTS
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Steady-State and Hypoxic pHc

In HCO3--containing solution under normoxic conditions, the pHc of the smooth muscle cells from SPAs and LPAs averaged 6.92 ± 0.02 and 7.02 ± 0.01, respectively (P < 0.05 by unpaired Student's t-test). When the cells were then exposed to hypoxia, the BCECF ratios stabilized within 5 min. At this point, the pHc of the SPA cells had increased to 7.02 ± 0.01, whereas in LPA cells, it had decreased to 6.96 ± 0.01 (both at P < 0.05 by paired Student's t-test). Thus with hypoxia, SPA cells become more alkaline, whereas LPA cells become more acidic (Fig. 2A).


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Fig. 2.   Steady-state pHc of pulmonary artery smooth muscle cells. A: under control normoxic conditions, pHc was lower in small pulmonary artery (SPA; n = 25 cells) than in large pulmonary artery (LPA; n = 21 cells) smooth muscle cells (**P < 0.05, SPA vs. LPA cells). After 5 min of hypoxia, pHc was stable and had increased in SPA cells and decreased in LPA cells (*P < 0.05, normoxia vs. hypoxia). B: baseline pHc and the directional response to hypoxia. In LPA cells (n = 13), pHc was reduced to a level close to that of SPA cells (n = 20), but during hypoxia, the pHc still decreased in the LPA cells (*P < 0.05, hypoxic acidosis vs. normoxic acidosis for LPA cells). C: pHc of SPA cells (n = 7) was raised to a level close to that of LPA cells (n = 8), but during hypoxia, the pH still increased in the SPA cells (*P < 0.05 hypoxic alkalosis vs. normoxic alkalosis for SPA cells.

Because the baseline normoxic pHc differed between the two cell types, we wondered if baseline pHc determined the direction of the pHc change during hypoxia and if changing the baseline pHc would alter the direction of the hypoxic change. In LPA cells, the PCO2 of the bath was increased to 51 Torr to reduce the pHc closer to that of the SPA cells (6.88 ± 0.03 vs. 6.92 ± 0.01). However, when the LPA cells were then exposed to hypoxia, the pHc still decreased to 6.81 ± 0.04 (P < 0.05; Fig. 2B). In SPA cells, the PCO2 of the bath was decreased to 21 Torr to raise the pHc to a level approximately that of the LPA cells (7.07 ± 0.02 vs. 7.05 ± 0.01). Again, the direction of the pHc response to hypoxia was not changed; the pHc increased to 7.14 ± 0.02 (P < 0.05; Fig. 2C).

Na+/H+ Exchange Activity

The activity of Na+/H+ exchange in pHc recovery from an acid load was demonstrated with the NH4Cl pulse maneuver. Brief exposure to 15 mM NH4Cl resulted in alkalinization due to NH3 diffusion into the cells. The NH4Cl was then washed out, and pHc became acidotic due to NH3 diffusion out of the cells together with NH4+ dissociation. pHc thus decreased to a value below the normal baseline. Recovery from this acidosis can occur by a number of different exchange processes. Figure 3 shows the typical pattern of changes in SPA pHc during an NH4Cl experiment performed in the absence of CO2 and HCO3- to eliminate any contribution from Cl-/HCO3- exchange. This same pattern was also seen in the LPA cells.


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Fig. 3.   Representative curve of pHc changes in SPA smooth muscle cells during NH4Cl pulse and washout [HCO3--free physiological saline solution (PSS) in the absence and presence of dimethylamiloride (DMA)]. Under control conditions, the pHc, which becomes acidotic as a result of NH4Cl removal, returns to baseline as H+ is extruded in exchange for Na+. When DMA is present, this exchange is inhibited, and pHc remains acidotic. The LPA cells exhibited the same pattern.

Table 1 presents the pHc values obtained for SPA and LPA cells during this experiment. In the absence of the inhibitor DMA, pHc in SPAs and LPAs returned to baseline after NH4Cl washout in 8.36 ± 1.08 and 8.50 ± 1.42 min, respectively. With DMA, pHc recovery was minimal. These results are consistent with activation of Na+/H+ exchange being responsible for the recovery of pHc after intracellular acidification with an NH4Cl pulse.

                              
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Table 1.   pHc of SPA and LPA smooth muscle cells during NH4Cl experiments

When the same experiments were conducted in HCO3--containing PSS, pHc also returned to baseline after NH4Cl washout (Table 1) in 3.62 ± 0.45 and 4.57 ± 0.44 min, respectively. DMA added to the washout solution did not affect pHc recovery, but if Na+ was also removed from the washout fluid, pHc did not recover in either cell type (Table 1).

Although the above experiments demonstrated the presence and activity of Na+/H+ exchange in the SPA and LPA smooth muscle cells, it did not appear that this was either solely or predominantly responsible for maintaining pHc when the cells were bathed in solutions containing CO2 and HCO3-. In addition to the lack of effect of DMA on pHc recovery from an acid load (Table 1), adding DMA to SPA cells in HCO3--containing PSS at steady state resulted in only a slight and insignificant acidification from 6.92 ± 0.04 to 6.87 ± 0.04. Within 2.65 ± 0.26 min, the pHc had returned to baseline (n = 8 cells). When DMA was added to LPA cells, pHc remained constant at 7.01 ± 0.02 (n = 10 cells).

Under hypoxic conditions, it did not appear that the alkalinization observed in SPA cells was due to activation of the Na+/H+ exchanger to pump H+ out of the cells. Even in the presence of DMA, exposure to hypoxia still resulted in an increase in pHc from 6.88 ± 0.03 to 7.05 ± 0.04 (data not shown; P < 0.05 by paired Student's t-test; n = 8 cells).

Na+-Dependent and -Independent Cl-/HCO3- Exchange Activity

When Cl- was removed from the medium bathing the cells, the pHc in both SPA and LPA cells increased in the manner shown in Fig. 4 and to the values shown in Table 2. This change indicated intracellular alkalinization, presumably due to Cl- efflux and HCO3- influx or to the absence of extracellular Cl- to exchange for intracellular HCO3-. When pHc reached a new, higher steady state, extracellular Cl- was restored, and pHc decreased to baseline levels (Table 2). When DIDS, a blocker of anion transport, was present during the recovery phase, pHc remained significantly above baseline in both cell types (P < 0.05; Table 2). If Cl- was restored but Na+ was removed from the PSS, the pHc continued to decline to a level significantly below baseline (P < 0.05; Table 2). This suggests that the recovery from the cellular alkalosis is mediated by Na+-independent Cl-/HCO3- exchange


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Fig. 4.   Typical pHc changes in SPA smooth muscle cells in response to Cl--free HCO3--containing PSS and after restoring Cl-, Cl- plus 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), or 0-Na+ PSS. LPA cells responded similarly.


                              
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Table 2.   pHc of SPA and LPA smooth muscle cells during Cl- removal and restoration experiments

Na+-dependent Cl-/HCO3- exchange helps to maintain steady-state pHc in pulmonary artery smooth muscle cells by exchanging cellular Cl- for extracellular Na+ and HCO3- (Fig. 1). When Na+ was removed from the HCO3--containing PSS bathing SPA cells, the pHc decreased from 6.97 ± 0.02 to 6.45 ± 0.03 (P < 0.05; n = 7 cells), reflecting intracellular acidification. Returning Na+ to the PSS restored the pHc to baseline, even in the presence of DMA. LPA smooth muscle cells showed a similar response to Na+ removal, decreasing from 7.00 ± 0.02 to 6.59 ± 0.07 (P < 0.05; n = 6 cells). However, when Na+ was restored to LPA cells treated with DMA, the pHc increased above baseline to 7.84 ± 0.17 (P < 0.05). This result might be indicative of less intrinsic activity of Na+-independent Cl-/HCO3- exchange in LPA than in SPA cells.

When SPA cells were exposed to hypoxia in the absence of Na+, the pHc did not increase; rather it decreased from 6.86 ± 0.02 to 6.68 ± 0.02 (P < 0.05; n = 6 cells). In LPA cells, the pHc still decreased during hypoxia from 6.66 ± 0.08 to 6.49 ± 0.09 (P < 0.05; n = 7 cells). That the activity of both Cl-/HCO3- exchangers determines the pHc response to hypoxia is further suggested by the data shown in Fig. 5. When these exchangers were inhibited in SPA smooth muscle cells either with HEPES-buffered PSS or by exposing the cells to DIDS in the presence of CO2 and HCO3-, the pHc decreased during hypoxia (P < 0.05 by ANOVA and Fisher's LSD test; Fig. 5A). The pHc of similarly treated LPA cells increased in response to hypoxia (P < 0.05 by ANOVA and Fisher's LSD test; Fig. 5B). Under normoxic conditions, exposing SPA cells to DIDS resulted in a decline of 0.06 ± 0.01 pH units (P < 0.05) but no significant change in LPA cells (data not shown).


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Fig. 5.   Effect of DIDS on the pHc response to hypoxia. During hypoxia, the pHc of SPA cells decreased (A) and of LPA cells increased (B) in either HEPES-buffered PSS or HCO3--buffered PSS plus DIDS. Nos. in bars, no. of cells. *P < 0.05 by ANOVA and Fisher's least significant difference test.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The major findings of this study were that 1) in the presence of CO2 and HCO3-, pHc was higher in the smooth muscle cells of LPAs than of SPAs; 2) activation of Na+/H+ exchange as well as both Na+-dependent and -independent Cl-/HCO3- exchange could be demonstrated in SPA and LPA smooth muscle cells; 3) in the presence of CO2 and HCO3-, Cl-/HCO3- exchange rather than Na+/H+ exchange appeared to be more active in regulating pHc under normoxic conditions; 4) under hypoxic conditions, pHc increased in SPA cells and decreased in LPA cells; 5) altering the baseline pHc of either cell type to a level characteristic of the other did not change the usual direction of the pHc response during hypoxia; 6) the absence of Na+ but not the pharmacological inhibition of Na+/H+ exchange prevented the hypoxia-induced alkalinization in SPA cells; and 7) inhibiting Cl-/HCO3- exchange activity in the two cell types reversed the normal direction of pHc changes during hypoxia.

The finding in this study that under normoxic conditions pHc was higher in LPA cells than in SPA cells agrees with a preliminary report from our laboratory (11). The decreased pHc in hypoxic LPA cells is also consistent with that report and agrees with the recent study by Leach et al. (4) where pHc decreased in large-diameter pig pulmonary arteries during hypoxia. The increased pHc in hypoxic SPA smooth muscle cells seen in this work and in the previous study from our laboratory (11) are, to our knowledge, the only reports of this kind. The pHc changes in both cell types during hypoxia appear to be a consistent response to hypoxia rather than a function of baseline pHc. This was suggested by the finding that altering the baseline pHc of each type of cell to a level characteristic of the other, i.e., increasing the pHc of the SPA cells or decreasing it in LPA cells, did not change the usual direction of the pHc response during hypoxia.

The role of ion transport mechanisms in regulating pulmonary artery smooth muscle cell pHc has not been extensively studied. However, studies done in HCO3--free medium have provided evidence for Na+/H+ exchange activity in the pulmonary vasculature. For example, in ferret main pulmonary artery smooth muscle cells, the resting pHc and its recovery from acidosis depended on the external Na+ concentration and was most likely mediated by the Na+/H+ exchanger (3). Likewise, in guinea pig main pulmonary artery smooth muscle cells, Quinn et al. (9) demonstrated that the Na+/H+ exchanger played a significant role in regulating intracellular pH under steady-state conditions. Even in the presence of HCO3-, the Na+/H+ exchanger appeared to be very active in these cells. In rat aorta smooth muscle cells, Little et al. (5) found that the Na+/H+ exchanger regulated pHc under control conditions, but when cellular ATP was depleted, the activity of this exchanger was attenuated and the Na+- and HCO3- -dependent mechanisms were more active.

In the present study of cat pulmonary artery smooth muscle cells, activity of the three major ion transport mechanisms, Na+/H+ and both Na+-dependent and -independent forms of Cl-/HCO3- exchange, could be demonstrated. In CO2- and HCO3--containing solutions, Na+/H+ exchange appears to be less active than Na+-dependent Cl-/HCO3- exchange both at steady-state pHc and during recovery from an acid load. This was evidenced by the fact that the Na+/H+ exchange inhibitor DMA had no effect on baseline pHc in either cell type, nor did DMA inhibit recovery from an acid load.

Putative activity of a cell-acidifying Na+-independent Cl-/HCO3- exchange was demonstrated in both cell types by withdrawing and then restoring extracellular Cl-. pHc recovery after Cl- restoration did not occur in the presence of DIDS. Additionally, if Na+ was not present when Cl- was restored, the pHc continued to decline. This likely reflects continued activity of this exchange mechanism and inactivity of the cell alkalinizing exchange mechanisms, i.e., Na+-dependent Cl-/HCO3- exchange and Na+/H+ exchange. To our knowledge, this is the first evidence of a role for Na+-independent Cl-/HCO3- exchange in pulmonary artery smooth muscle cells.

We investigated whether the two Cl-/HCO3- exchange mechanisms observed under normoxic conditions might be involved in the pHc changes noted during hypoxia. Our results indicate that they were. In SPA cells treated with DMA, the pHc still increased during hypoxia, thereby ruling out a hypoxia-induced activation of the Na+/H+ exchanger. Na+ removal abrogated the alkalinization observed with hypoxia in SPA cells. In fact, the pHc decreased rather than increased in response to hypoxia. Additionally, in HCO3--free (HEPES-buffered) PSS and in DIDS plus HCO3--containing PSS, the pHc decreased during hypoxia in the SPA cells. These results suggest that activation of a Na+-dependent Cl-/HCO3- exchanger is responsible for the increase in pHc observed with hypoxia in SPA cells. In LPA cells bathed in HCO3--free (HEPES-buffered) PSS and in DIDS plus HCO3--containing PSS, the pHc increased during hypoxia. However, in Na+-free, HCO3--containing PSS, pHc decreased as usual in the hypoxic LPA cells, suggesting that the decrease was mediated, at least in part, by Na+-independent Cl-/HCO3-exchange activity.

The results of the present studies have been integrated into an overall model describing the balance of the acid-base exchange activities examined in these studies (Fig. 6). Assuming a hypothetical smooth muscle cell that contains equal numbers of functional acidifying and alkalinizing transport mechanisms, the steady-state pHc would be set at a point where the activity versus pHc curves of the two exchangers cross each other. Increasing the amount of acidifying exchange (e.g., Na+-independent Cl-/HCO3- exchange in SPA cells) would act to lower pHc. This decline in pHc would continue until an allosteric stimulation of activity of the countervailing transporter (in this case, Na+-dependent Cl-/HCO3- exchange) occurs because of the decreased pHc or cell HCO3-. At this point, the steady state would be restored, albeit at a lower pHc than in the hypothetical cell. Increasing the amount of alkalinizing exchange, Na+-dependent Cl-/HCO3- exchange in LPA cells would have the opposite effect, leading to a higher pHc than the hypothetical cell and, more to the point, higher than the pHc observed in the SPA cells. This describes the situation that pertains to normoxic conditions.


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Fig. 6.   Proposed balance of acidifying and alkalinizing transport activities in SPA and LPA smooth muscle cells during normoxia and hypoxia.

If an increase in the amount of Na+-dependent Cl-/HCO3- exchange in SPA occurs during hypoxia, pHc would increase as it did in these studies. The increase in pHc would continue until stimulation of the countervailing ion transport activity, Na+-independent Cl-/HCO3- exchange, occurs. Stimulation of Na+-independent Cl-/HCO3- exchange would serve to restore the steady state but at a higher pHc than during normoxia. Similarly, an increase in the amount of Na+-independent Cl-/HCO3- exchange in LPA cells during hypoxia would have the opposite effect, leading to a decline in pHc, again consistent with the results observed in these studies.

Why the pHc changes during hypoxia were reversed in SPA and LPA smooth muscle cells bathed either in HEPES-buffered or in HCO3--buffered PSS plus DIDS is not immediately evident. However, if the scenario outlined above pertains, the data could be interpreted as follows. In SPA cells in the presence of DIDS or in the absence of CO2 and HCO3-, Na+/H+ exchange, although present, is not sufficiently active either to raise pHc under normoxic conditions or to prevent acidification due to increasing metabolic acids during hypoxia. In LPA cells in the presence of DIDS or in the absence of CO2 and HCO3-, Na+/H+ exchange may have an enhanced role in regulating pHc. During hypoxia, this transport mechanism may, in a way analogous to our argument above for Cl-/HCO3- exchange, increase in activity, thereby increasing pHc.

Differences between cell types, preparations, and animal species as they relate to the proposed mechanisms that regulate hypoxic vasoconstriction have complicated the understanding of the response. The postulated roles for Cl-/HCO3- exchange in pHc control of the cat pulmonary artery smooth muscle cells may be specific to this species. Quinn et al. (9) found that Na+/H+ exchange appeared to play a more prominent role in pH control of guinea pig pulmonary artery smooth muscle cells. Therefore, without additional evidence from studies in other species and organs, it is difficult to generalize our findings. To determine the relative contributions of the acidifying and alkalinizing exchange mechanisms in different species and among various cell types, it will be necessary to study cells under physiological conditions, e.g., in the presence of HCO3- and CO2, and to know whether or not the data are derived from primary or passaged cells. Complete understanding of pHc regulation in the pulmonary vasculature will also require studying the roles of the ion exchange mechanisms in pulmonary artery endothelial cells. Evidence from Cutaia et al. (2) has shown that prolonged exposure to hypoxia decreases activity of Na+/H+ exchange isoform 1 of the Na+/H+ exchanger in human pulmonary artery endothelial cells, but whether or not this result is species and/or cell specific is difficult to know. Finally, investigations into the interaction between pHc and [Ca2+]c to determine whether changes in pHc and [Ca2+]c are linked to one another and whether this linkage occurs through changes in the activities of the known acid-base and Ca2+ transport mechanisms should also be performed.


    ACKNOWLEDGEMENTS

This study was supported by Veterans Affairs Medical Research funds awarded to J. A. Madden and J. G. Kleinman, by National Institute of Diabetes and Digestive and Kidney Diseases funds awarded to J. G. Kleinman, and by funds from the Division of Pulmonary Medicine of the Medical College of Wisconsin.


    FOOTNOTES

Present address of D. E. Ray: Pulmonary Associates, 1210 S. Cedarcrest Blvd., Suite 3200, Allentown, PA 18103.

Address for reprint requests and other correspondence: J. A. Madden, Neurology Research 151, VAMC, Milwaukee, WI 53295 (E-mail: jmadden{at}mcw.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 24 January 2000; accepted in final form 25 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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