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
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ABSTRACT |
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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
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INTRODUCTION |
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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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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.
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METHODS |
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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 × 10Gases
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
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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
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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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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
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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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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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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aalkjaer, C.
Regulation of intracellular pH and its role in vascular smooth muscle function.
J Hypertens
8:
197-206,
1990[ISI][Medline].
2.
Cutaia, MV,
Parks N,
Centracchio J,
Rounds S,
Yip KP,
and
Sun AM.
Effect of hypoxic exposure on Na+/H+ antiport activity, isoform expression, and localization in endothelial cells.
Am J Physiol Lung Cell Mol Physiol
275:
L442-L451,
1998
3.
Farrukh, IS,
Hoidal JR,
and
Barry WH.
Effect of intracellular pH on ferret pulmonary arterial smooth muscle cell calcium homeostasis and pressure.
J Appl Physiol
80:
496-505,
1996
4.
Leach, RM,
Sheehan DW,
Chacko VP,
and
Sylvester JT.
Effects of hypoxia on energy state and pH in resting pulmonary and femoral arterial smooth muscles.
Am J Physiol Lung Cell Mol Physiol
275:
L1051-L1060,
1998
5.
Little, PJ,
Neylon CB,
Farrely CA,
Weissberg PL,
Cragoe EJ, Jr,
and
Bobik A.
Intracellular pH in vascular smooth muscle: regulation by sodium-hydrogen exchange and multiple sodium dependent HCO3 mechanisms.
Cardiovasc Res
29:
239-246,
1995[ISI][Medline].
6.
Madden, JA,
Dawson CA,
and
Harder DR.
Hypoxia-induced activation in small isolated pulmonary arteries from the cat.
J Appl Physiol
59:
113-118,
1985
7.
Madden, JA,
Vadula MS,
and
Kurup VP.
Effects of hypoxia and other vasoactive agents on pulmonary and cerebral artery smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
263:
L384-L393,
1992
8.
O'Donnell, ME,
and
Owen NE.
Regulation of ion pumps and carriers in vascular smooth muscle.
Physiol Rev
74:
683-721,
1994
9.
Quinn, DA,
Honeyman TW,
Joseph PM,
Thompson BT,
Hales CA,
and
Scheid CR.
Contribution of Na+/H+ exchange to pH regulation in pulmonary artery smooth muscle cells.
Am J Respir Cell Mol Biol
5:
586-591,
1991[ISI][Medline].
10.
Thomas, JA,
Buchsbaum RN,
Zimniak A,
and
Racker E.
Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.
Biochemistry
81:
2210-2218,
1979.
11.
Vadula, MS,
Kleinman JG,
and
Madden JA.
Effect of hypoxia on cytoplasmic pH (pHc) of cat pulmonary and cerebral artery smooth muscle cells (Abstract).
FASEB J
6:
A1244,
1992[ISI].
12.
Vadula, MS,
Kleinman JG,
and
Madden JA.
Effect of hypoxia and norepinephrine on cytoplasmic free Ca2+ in pulmonary and cerebral arterial myocytes.
Am J Physiol Lung Cell Mol Physiol
265:
L591-L597,
1993
13.
Wray, S.
Smooth muscle intracellular pH: measurement, regulation, and function.
Am J Physiol Cell Physiol
254:
C213-C225,
1988