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(The FASEB Journal. 2000;14:1731-1740.)
© 2000 FASEB

Hypoxia inhibits the Na+/Ca2+ exchanger in pulmonary artery smooth muscle cells

YONG-XIAO WANG, PRASAD K. DHULIPALA and MICHAEL I. KOTLIKOFF1

Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6046, USA

1Correspondence: Department of Animal Biology, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104-6046, USA. E-mail: mik{at}vet.upenn.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cellular mechanisms underlying hypoxic pulmonary vasoconstriction are not fully understood. We examined the effect of hypoxia on Ca2+ efflux from the cytosol in single Fura-2-loaded pulmonary artery myocytes. During mild hypoxia (pO2=50–60 Torr), peak [Ca2+]i was increased and the rate of Ca2+ removal from the cytosol was markedly slowed after stimuli that elevated [Ca2+]i. Removal of extracellular Na+ potentiated the peak [Ca2+]i rise and slowed the Ca2+ decay rate in cells recorded under normoxic conditions; it did not further slow the Ca2+ decay rate or potentiate the [Ca2+]i increase in hypoxic cells. An Na+/Ca2+ exchange current was recorded in isolated pulmonary artery myocytes. Switching from Li+ to Na+ (130 mM) revealed an inward current with reversal potential consistent with the Na+/Ca2+ exchange current in cells in which [Ca2+]i was clamped at 1 µM; similar currents, although smaller, were observed with normal resting [Ca2+]i using the perforated patch clamp technique. The Na+/Ca2+ exchange current was markedly inhibited in myocytes exposed to mild hypoxia. RT-PCR revealed the expression of specific alternatively spliced RNAs of NCX1 in rat pulmonary arteries. These findings provide an enhanced understanding of the molecular mechanisms underlying hypoxic sensing in pulmonary arteries.—Wang, Y.-X., Dhulipala, P. K., Kotlikoff, M. I. Hypoxia inhibits the Na+/Ca2+ exchanger in pulmonary artery smooth muscle cells.


Key Words: smooth muscle • ion channel • cytosolic calcium • reverse transcriptase polymerase chain reaction • hypoxic pulmonary vasoconstriction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HYPOXIA RESULTS IN pulmonary vasoconstriction, which is thought to serve as an important regulatory process directing pulmonary blood flow to well-ventilated regions of the lung, thereby efficiently maintaining arterial oxygenation. However, sustained hypoxic pulmonary vasoconstriction is a key factor in the development of pulmonary hypertension. Although the cellular and molecular mechanisms underlying hypoxic pulmonary vasoconstriction are not certain, hypoxia is known to produce an increase in intracellular Ca2+ concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells, leading to vasoconstriction (1 , 2) . Substantial experimental data suggest that the hypoxic increase in [Ca2+]i is associated with Ca2+ influx through sarcolemmal voltage-dependent Ca2+ channels. The opening of voltage-dependent Ca2+ channels after hypoxic challenge most likely results from the inhibition of voltage-dependent K+ channels (3 4 5 6 7 8 9 10 11) and activation of Ca2+-activated Cl- channels (12) . However, increasing evidence indicates that Ca2+ release from the sarcoplasmic reticulum also makes an important contribution to the hypoxic increase in [Ca2+]i (7 , 12 13 14 15 16 17) .

Transport proteins that remove Ca2+ from the cytosol, such as plasmalemmal Ca2+ ATPase, sarcoplasmic reticulum Ca2+-ATPase, mitochondrial Ca2+ uptake, and the plasmalemmal Na+/Ca2+ exchanger also contribute to the steady state level of [Ca2+]i (18 , 19) , and hypoxic inhibition of one or more of these Ca2+ removal systems could contribute to hypoxic vasoconstriction. We have recently found that after hypoxic increases in [Ca2+]i in single pulmonary artery myocytes, the return to basal [Ca2+]i was much slower than that observed after elevations of [Ca2+]i by other stimuli (12) . Moreover, hypoxia and chemical hypoxia (cyanide) have been shown to result in a marked slowing of cytosolic Ca2+ removal in cardiac myocytes, and this effect has been shown to be associated with inhibition of the Na+/Ca2+ exchanger (20 , 21) . To determine whether hypoxia inhibits the Na+/Ca2+ exchanger and thereby slows Ca2+ efflux, we measured Fura-2 fluorescence and Na+/Ca2+ exchange currents under control and hypoxic conditions, and determined the expression of exchanger message in pulmonary artery.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Single cell preparation
Single smooth muscle cells were isolated from rat resistance (external diameter<300 mm) pulmonary arteries, since these regional artery cells show a stronger response to hypoxia (8 , 22) . The procedure for isolation of single cells was modified from that described previously (12 , 23) . Female or male Sprague-Dawley rats (body weight, 300–350 g) were killed by intramuscular injection of a mixture of xylazine (10 mg/kg) and ketamine (60 mg/kg). The heart and lungs were rapidly removed en bloc and placed in normal physiological saline (PSS). Resistance pulmonary arteries were carefully dissected using a dissecting microscope, the endothelium removed by gentle rubbing with a cotton-tipped applicator stick, and the vessels cut into small pieces (1x10 mm). The tissue was incubated in nominally Ca2+-free PSS (ncPSS, 1.5 ml) containing 2 mg papain (Worthington, Freehold, N.J.) and 0.2 mg dithioerythritol (Sigma, St. Louis, Mo.) for 20 min (37°C), then in ncPSS containing 0.5 mg type H collagenase (Sigma), 1.0 mg type F collagenase (Sigma), and 100 µM Ca2+ for 10–15 min (37°C), and finally in ice-cold ncPSS for 15 min. Single cells were harvested by gentle trituration using fire-polished siliconized Pasteur pipettes and stored on ice for use up to 8 h. Mouse pulmonary artery myocytes were isolated using the same procedure.

The composition of normal PSS was (mM): 130 NaCl, 5.4 KCl, 1 MgSO4, 1.8 CaCl2, 10 HEPES, and 10 glucose. The nominally Ca2+-free solution had the same composition as the standard PSS, except CaCl2 was omitted. The pH of all solutions was adjusted to 7.4 with NaOH.

Fura-2 fluorescence measurement
Measurements of intracellular Ca2+ concentration ([Ca2+]i) were performed using high band width, single-excitation wavelength fluorescence measurements as described previously (24) . Cells were loaded with 5 µM Fura-2/AM (Molecular Probes, Eugene, Oreg.) for 20 min at 35°C and then transferred to the recording chamber. After a brief period to allow adhesion, cells were continuously perfused with prewarmed (35°C) bath solution. Experiments were initiated after 15 min of perfusion to wash out extracellular Fura-2/AM and to allow the conversion of intracellular dye into its non-ester form. Fura-2 was initially excited at 340 and 380 nm wavelengths (Xenon 75 W arc lamp and bandpass filter at 2 Hz) to calculate the initial [Ca2+]i and subsequently excited only at 380 nm during the experiment. The emission fluorescence at 510 nm was detected by a photo multiplier tube (Thorn Emi Electron Tubes, Middlesex, U.K.). Photo bleaching was minimized by inserting neutral density filters into the optical pathway and blocking excitation light via a shutter between the sampling periods. The background fluorescence was determined by removing the cell from the field after the experiment. Values of Rmax (maximum 340/380), Rmin (minimum 340/380), and Sf380/Sb380 (ration of 380 fluorescence at calcium-free and saturating Ca2+ concentrations) were determined using 10 µM ionomycin and 10 mM calcium or 10 µM ionomycin and 10 mM EGTA for saturating and calcium-free conditions, respectively. For single wavelength determination, [Ca2+]i during experiments was calculated by the following equation:

where F(0) is the prestimulus 380 fluorescence, {Delta}F(t) the 380 fluorescence during experiments, [Ca2+]i,0 the initial calcium calculated from the dual wavelength measurement, [Ca2+]i,t the calcium during experiments, KD the dissociation constant for calcium binding to Fura-2, and K'D = KD (Rmax/Rmin). The fluorescence signal was recorded on VCR tape, and then re-digitized using an A-D converter (TL-125, Scientific Solutions) for analysis.

The composition of normal bath solution was as above. Na+-free bath solution contained LiCl substituted for NaCl. For 80 mM K+ bath solution, 74.6 mM NaCl was replaced by equimolar KCl.

Reverse transcriptase polymerase chain reaction (RT-PCR)
Total RNAs from rat pulmonary arterial smooth muscle (endothelium removed) and heart were isolated by the acid guanidinium thiocyanate-phenol-chloroform method (25) . Single strand cDNA was synthesized from 2 µg RNA using Superscript II reverse transcriptase (Life Technologies, Gaithersburg, Md.). The resultant cDNA template was amplified with the sense and antisense oligonucleotide primers listed in Table 1 . These primers were designed to span the previously identified putative cytoplasmic region of variability in NCX1 (26) , as well as to distinguish between the expression of exons A and B. Although three Na+/Ca2+ exchanger isoforms (NCX1, NCX2, and NCX3) have been identified (27 28 29 30) , we only examined NXC1 because this isoform has recently been functionally linked to the maintenance of intracellular [Ca2+]i homeostasis in vascular myocytes (31) . The amplified products were electrophoresed on an agarose gel and visualized by ethidium bromide staining.


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  		Table 1. Oligonucleotide primers for polymerase chain reaction

Patch clamp recording
Voltage clamp experiments were performed using the nystatin perforated and standard whole-cell patch clamp techniques, as described previously (24) . Patch pipettes were pulled from borosilicate capillary glass (TW 15°F-4, WPI) using a Flaming/Brown micropipette puller (P-87, Shutter Instrument, Novato, Calif.). For perforated patch clamp experiments, pipettes filled with intracellular solution had a resistance of 3–4 M{Omega}. Nystatin was included in the pipette solution at a final concentration of 200–300 mg/ml. Junction potentials between the pipette and bath solutions were compensated just before seal formation. When electrical access was detected, cells were clamped at a holding potential of -60 mV. Membrane capacitance and series resistance were continuously monitored and compensated; acceptable access resistance was considered to be less than 40 M{Omega}. In some experiments the standard whole-cell technique was used to clamp [Ca2+]i. In this case, the resistance of the patch pipettes was from 1 to 3 M{Omega}. Voltage-command protocols were generated by the EPC-9 system (Heka Electronics, Lambrecht, Germany). Data were recorded on a Macintosh computer and VHS tape for off-line analysis.

The composition of normal bath solution was (mM): 20 NaCl, 110 Na-acetate, 1.8 CaCl2, 1 MgSO4, 10 HEPES, and 10 glucose (pH 7.4). For recordings of exchange currents, ouabain (20 µM), nisoldipine (5 µM), and BaCl2 (1 mM) were added to the bath solution to block Na+-K+ ATPase, voltage-dependent Ca2+ channels, and K+ channels, respectively. For Na+-free bath solution, NaCl and Na-acetate were replaced by LiCl and Li-acetate. The intracellular solution contained (mM): 15 CsCl, 110 Cs-acetate, 5 NaCl, 1.2 MgCl2, 3 ATP-Mg, and 10 HEPES (pH 7.3) for standard whole-cell recordings. EGTA and CaCl2 were added to clamp [Ca2+]i at either 100 nM or 1 µM (32) . In some experiments, the intracellular solution contained 20 mM HEPES to clamp intracellular pH at 7.3. For the perforated patch experiments, the intracellular solution was (mM): 15 CsCl, 110 Cs-acetate, 5 NaCl, 1.2 MgCl2, 3 EGTA, 1 CaCl2, and 10 HEPES (pH 7.3). All external and internal solutions were filtered before use (0.2 µm Acrodisc, Gelman).

Hypoxia
To study the hypoxic response, the perfusing solution was switched from a bath solution equilibrated with 21% O2 and 79% N2 (control) to a solution equilibrated with 6% O2 and 94% N2 (hypoxic bath solution). The oxygen tension of the solution was monitored by means of an oxygen electrode (OXEL-1, WPI) inserted into the recording chamber. To avoid equilibration with atmospheric O2, mineral oil was placed on top of the bath solution in the recording chamber. A modified chamber was used to allow input and output of bath solution below the oil level. Under this condition, the pO2 in the control solution was >140 Torr, whereas in the hypoxic solution the values were 50–60 Torr. At this level of hypoxia, no significant changes in the resting level of [Ca2+]i were observed.

Data analysis
All values are expressed as means ± SE. The Student’s t test for paired data was used to determine the significance of differences between observations. A P value of less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hypoxia slows Ca2+ removal from the cytosol and augments peak [Ca2+]i
We have previously noted that under conditions of severe hypoxia (pO2<40 Torr) or chemical hypoxia (cyanide) the rate of Ca2+ decay after caffeine-induced Ca2+ release is markedly slower than occurs under nonhypoxic conditions, in pulmonary artery smooth muscle cells (12) . This suggested that hypoxia may inhibit one or more cellular Ca2+ transport processes, thereby resulting in a slower removal of Ca2+ from the cytosol. To test this hypothesis, we first examined the rate of Ca2+ removal before and during exposure of myocytes to mild hypoxia in cells isolated from pulmonary resistance artery myocytes. The elevation of [Ca2+]i was achieved by exposing myocytes to a caffeine/high K+ (10/80 mM) solution for 2.5 s. Mild hypoxic conditions (50–60 Torr) were chosen since this level of hypoxia did not cause a significant elevation of [Ca2+]i.

As shown in Fig. 1A , application of caffeine/high K+ solution to a myocyte in normal physiological solution produced a rapid [Ca2+]i rise and typical decay with a time constant of ~6 s. By contrast, after 5 min perfusion of the same myocyte with the hypoxic solution the [Ca2+]i decay was roughly fourfold slower. In both conditions, the Ca2+ decay could be well fitted by single exponential equation. A second effect seen in this experiment is the marked augmentation of the initial peak [Ca2+]i achieved by exposure to the caffeine/high K+ solution. In the experiment shown, the normoxic peak [Ca2+]i was 631 nM, and during mild hypoxia the peak was 1012 nM. Hypoxia produced a slowing of the [Ca2+]i decay and increase in the peak [Ca2+]i in 8 identical experiments. As summarized in Fig. 1B , the mean rate of Ca2+ decay was slowed from 5.5 ± 0.6 to 30.3 ± 3.5 s by hypoxia and the mean [Ca2+]i increase was augmented from 663 ± 23 to 1055 ± 36 nM (n=8, P<0.05).



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  		Figure 1. Mild hypoxia increases the peak and slows the decay of the [Ca2+]i transient in rat pulmonary resistance artery smooth muscle. A) Brief (2.5 s) application of caffeine/80 mM K+ solution results in a transient increase in [Ca2+]i before (left) and after (right) exposure to mild hypoxia (50–60 Torr). The peak [Ca2+]i achieved was markedly increased and the rate of decline of [Ca2+]i slowed during hypoxia, although hypoxia at this level did not significantly change the resting level of [Ca2+]i. Insets show the [Ca2+]i decays fit by single exponential (white line) and the associated time constants ({tau}). B) Summary of results from 8 cells showing the effects of hypoxia on the magnitude of [Ca2+]i and the decay constant (1/{tau}). Error bars show SE; *P < 0.05 compared with control.

We next examined whether hypoxia similarly inhibited the rate of [Ca2+]i decay after intracellular Ca2+ release produced by a 2.5 s application of caffeine alone. As expected, after exposure of rat pulmonary artery myocytes to mild hypoxia for 5 min, the rate of [Ca2+]i decay was also significantly slowed and the peak [Ca2+]i increase potentiated. In a total of 3 cells tested, the time constant of Ca2+ decay was slowed from 4.2 ± 0.9 to 18.9 ± 3.8 s and the peak [Ca2+]i increase augmented from 629 ± 35 to 961 ± 59 nM (P<0.05). Similar effects of hypoxia were seen in mouse pulmonary artery smooth muscle cells. Figure 2A shows an example of one such experiment, in which Ca2+ responses were induced by a 2.5 s application of caffeine under control and hypoxic conditions, and summarizes the results from 6 experiments in which there was an approximate fivefold increase in the time constant and 50% increase in the peak [Ca2+]i.



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  		Figure 2. Mild hypoxia potentiates Ca2+ release and slows the [Ca2+]i decay in mouse pulmonary artery myocytes. A) Protocol as in Fig. 1 , except caffeine alone (2.5 s puff) is used to release intracellular Ca2+. During hypoxia (right) the peak [Ca2+]i is increased and the rate of decline of [Ca2+]i is slowed ~fourfold. Below, the Ca2+ decay for both conditions is shown with superimposed single exponential fits and the associated time constants. B) Summary of the effects of hypoxia on the magnitude of [Ca2+]i and the decay constant obtained from 6 mouse cells. *P < 0.05 compared with control.

Removal of extracellular Na+ slows Ca2+ removal from the cytosol
Removal of Ca2+ from the cytosol of vascular myocytes is predominantly achieved by sarcolemmal and sarcoplasmic reticulum (SR) Ca2+-ATPases and by the sarcolemmal Na+/Ca2+ exchanger (18 , 19 , 31) . Since hypoxia potentiated peak [Ca2+]i, we reasoned that inhibition of SR Ca2+-ATPase, perhaps due to a decline in available ATP, was unlikely to be causal. Previous studies have suggested an important role for the Na+/Ca2+ exchanger in Ca2+ homeostasis of vascular smooth muscle (33 34 35 36 37 38 39 40) . To determine the role of the Na+/Ca2+ exchanger in this process, we first examined Ca2+ decay rates in the absence of extracellular Na+, using the same protocol for elevation of [Ca2+]i. As shown in Fig. 3 , the time constant for decay of [Ca2+]i was markedly increased and peak [Ca2+]i enhanced when cells were exposed to the [Ca2+]i elevation protocol in the absence of extracellular Na+. In a total of 7 cells tested, removing the extracellular Na+ reduced the rate of Ca2+ decay from 5.8 ± 0.7 to 22.2 ± 4.1 s, and augmented [Ca2+]i increase from 659 ± 35 to 998 ± 26 nM (P<0.05).



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  		Figure 3. Removal of extracellular Na+ increases the peak and slows the decay of the [Ca2+]i. A) [Ca2+]i transients produced by a 2.5 s of caffeine/high K+ before (left) and during (right) exposure to Na+-free bath solution (Na+ replaced with equimolar Li+). In the absence of Na+, the magnitude of the [Ca2+]i transient was augmented and the rate of decay slowed, similar to the effect of hypoxia. Inset shows single exponential fits to the [Ca2+]i decays and the time constants for the fits. B) Summary of the effects of Na+-free solution on the amplitude and decay of the [Ca2+]i transient from 7 similar experiments. *P < 0.05 compared with control.

To further explore the relationship between removal of extracellular Na+ and hypoxia, we sought to determine whether the hypoxic slowing of the rate of [Ca2+]i removal from the cytosol and augmentation of peak [Ca2+]i were affected by removal of extracellular Na+. Experiments were performed in which the [Ca2+]i decay was examined under hypoxic conditions in the presence and absence of extracellular Na+. Figure 4A shows a typical example of these experiments, in which [Ca2+]i was elevated by exposure to caffeine/high K+ solution first under conditions of hypoxia alone (left) and then under conditions of hypoxia combined with Na+-free bath solution (right). As seen from this example, removing extracellular Na+ did not further slow the rate of decay of [Ca2+]i or enhance the peak increase in [Ca2+]i. The amplitude and decay rate of the initial [Ca2+]i transient were altered by hypoxia in a manner consistent with experiments shown in Figs. 1 and 2 . Similar observations were obtained from 6 different cells (Fig. 4B ). Thus, hypoxia and removal of extracellular Na+ have similar and nonadditive effects on [Ca2+]i, suggesting that the mechanism underlying hypoxic effects on [Ca2+]i may be associated with an inhibitory effect on the plasmalemmal Na+/Ca2+ exchanger. Such a mechanism may explain previous findings that the slowed rate of relaxation of isolated pulmonary arteries by hypoxia no longer occurs in the absence of extracellular Na+ (34) .



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  		Figure 4. In the presence of hypoxia, removal of extracellular Na+ does not further augment the [Ca2+]i transient or slow the rate of decay. A) Caffeine/high K+ evoke a large [Ca2+]i transient with a slow decay rate in the presence of hypoxia (left). After substitution with Na+-free solution, an equivalent [Ca2+]i transient was observed (right). The inset shows the fits of the individual decays. Note that the time constants are similar to those observed with hypoxia (Fig. 1) or Na+-free solutions (Fig. 3) alone. B) Summary of data obtained from 6 equivalent experiments.

Na+/Ca2+ exchange currents in rat pulmonary artery cells
We next sought to measure Na+/Ca2+ exchange currents in pulmonary artery myocytes using patch clamp methods. Dialyzed myocytes from rat pulmonary resistance arteries were voltage-clamped at -60 mV with [Ca2+]i clamped at 1 µM to maximize the exchange current. Currents associated with voltage-dependent Ca2+ channels, K+ channels, and Na+-K+ ATPase were blocked by nisoldipine, Ba2+, and ouabain in the bath solution and by Cs+ in the intracellular solution. The current reversibly activated when cells were switched from Na+-free (Li+ substitution) to 130 mM Na+ bath solution was defined as the Na+/Ca2+ exchange current (41) . In myocytes voltage-clamped at -60 mV, brief exposure to extracellular Na+ rapidly activated an inward current, which decayed with relatively slow kinetics when Na+ was removed (Fig. 5 ). In a total of 9 cells tested, the mean current amplitude in 1 µM Ca2+ internal solution was 25.3 ± 3.6 pA. To confirm that the inward current resulted from the electrogenic Na+/Ca2+ exchanger, experiments were performed to determine the current reversal potential. Myocytes were dialyzed with intracellular solution containing 5 mM Na+ and [Ca2+]i clamped at 1 µM and exposed to Na+-containing solution to activate the inward current. Ramp voltage protocols were imposed (-60 to 100 mV for 320 ms) before and during activation of the Na+-sensitive current. The subtracted ramp currents indicated that the Na+-sensitive current reversed at -54 mV (Fig. 5C ), quite close to the calculated equilibrium potential of the Na+/Ca2+ exchange current (-60 mV), assuming an exchange stoichiometry of 3Na+/1Ca2+ as observed for the cardiac exchanger (31 , 41) . Similar results were observed from 5 other cells. Na+/Ca2+ exchange currents were also detected in nonstimulated cells under conditions in which [Ca2+]i was maintained at physiological levels using the perforated patch clamp method. Under these conditions, a smaller inward current was observed; in 8 cells tested, the mean current amplitude was 12.8 ± 1.9 pA (Fig. 5B ). A smaller exchange current would be predicted from the shift of the current reversal potential due to the lower [Ca2+]i (assuming [Ca2+]i=100 nM and Nai=5 mM, ECa/Na=0 mV). Thus, a significant Na+/Ca2+ exchange current is present in pulmonary resistance artery myocytes.



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  		Figure 5. Isolation of the Na+-Ca2+ exchange current in pulmonary artery myocytes. A) Above: currents were recorded in a dialyzed, voltage-clamped myocyte held at -60 mV. Currents were blocked with switching from Na+-containing bath solution induced inward currents to Na+-free. Cells were voltage clamped at -60 mV using the patch clamp method. Ouabain (20 µM), nisoldipine (5 µM), and BaCl2 (1 mM) were added to the extracellular solution; the internal solution contained Cs+ (substituted for K+) and the free Ca2+ concentration was clamped at 1 µM. When the Li+-containing solution was replace with an otherwise identical solution containing Na+, an inward current developed. Below: the Na+-sensitive current was also observed in nondialyzed cells (extracellular solution as above). B) Graphs show the mean amplitude of the Li+-sensitive currents in both conditions. Numbers in brackets indicate the number of cells tested. C) Left: recording of the Li+-sensitive current in a dialyzed cell as in panel A. Ramp pulses from -60 mV to 100 mV for 320 ms were applied to determine the current reversal potential. Right: difference current obtained by subtracting the ramp current in Li+ from that recorded during the inward current. The instantaneous current reversal potential was 54 mV, close to the theoretical equilibrium potential of Na+/Ca2+ exchanger (60 mV).

We also sought to confirm expression of Na+/Ca2+ exchanger mRNA in this preparation and determine the expression of specific alternatively spliced RNAs. Primers were selected that spanned the variable region in the carboxyl terminus of NCX1 (26) . As shown in Fig. 6 , primer pairs that spanned the entire region identified two prominent cDNAs of the predicted size in pulmonary artery, whereas one band was detected in rat heart, similar to previous results in the rat aorta (26) . Similar results were observed using primers spanning exons B–F, which yielded a larger band with the predicted size of RNAs including exon B (363) as well as a smaller band of ~285 bp. Given the finding of a major band using a forward primer within exon B and the mutual exclusivity of expression of exons A and B (30) , these results exclude the expression of exon A to a high degree in pulmonary arterial smooth muscle. Moreover, the difference in size between the two major RT-PCR products in both cases was ~80 bp, consistent with the major expression of a single deletion variant, most likely either NCX1.2 (81 bp smaller) or NCX1.9 (87 bp smaller) (30) . These results agree with previous studies that report expression of a single, larger RNA for NCX1 in rat heart, but a second deletion variant in other tissues (26 , 30 , 42) .



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  		Figure 6. Expression of Na+/Ca2+ exchanger splice variant in pulmonary arteries. Rat heart and pulmonary artery RNAs were amplified with oligonucleotide primers designed to span the variable region of NCX1. Lanes 1 and 5: template and 1F/1R primers; lanes 2 and 6: reverse transcriptase (rt) and 1F/1R primers; lanes 3 and 7: rt, template, and 1F/2R primers; lanes 4 and 8: rt, template, and 2F/1R primers. The 2F primer was complementary to sequence from exon B, indicating the expression of that exon in smooth muscle and heart. The size of the smaller deletion variant expressed in pulmonary arterial smooth muscle is consistent with expression of exons BCE or BDE (see text).

Hypoxia inhibits Na+/Ca2+ exchange currents
Since hypoxia slowed the decay of [Ca2+]i in a manner consistent with inhibition of the Na+/Ca2+ exchanger, we next sought to directly determine the effect of hypoxia on Na+/Ca2+ exchange currents. Myocytes were voltage-clamped at -60 mV using the perforated patch clamp method and the exchange current recorded before and during exposure to mild hypoxia. In control cells, the exchange current could be repeatedly activated with little or no decline in magnitude. By contrast, as shown in Fig. 7 , the Na+/Ca2+ exchange current was markedly inhibited by exposure of cells to mild hypoxia, compared to the current level on initial exposure to Na+. In the experiment shown, the current was inhibited by almost 70%, from 12.2 to 3.7 pA. Figure 7B summarizes data from 6 identical experiments; the exchange current was reduced during hypoxia from 11.7 ± 1.5 to 4.3 ± 0.8 pA (P<0.05). These results indicate that hypoxia inhibits the plasmalemmal Na+/Ca2+ exchange current and taken together with the effect of Na+ removal on Ca2+ efflux suggest that inhibition of the exchanger underlies the slowed rate of Ca2+ efflux from the cytosol observed during hypoxia.



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  		Figure 7. Hypoxia inhibits the Na+-Ca2+ exchange current. A) Isolated Na+/Ca2+ exchange currents recorded from single myocytes isolated from a resistance pulmonary artery. Above: an equivalent current is observed with repeated exposure of the cell to Na+-containing solution. Below: after exposure of a different cell to mild hypoxia for 5 min, the exchange current is markedly inhibited relative to its level under normoxic conditions. Both cells were voltage-clamped at -60 mV using the nystatin method. Hypoxia markedly inhibited the exchange current detected when Li+ was replaced with Na+. B) Summary of 6 equivalent experiments; the exchange current was 37% of the control value at a pO2 of 50–60 Torr. *P < 0.05 compared with control

In myocardial cells, exposure to anoxia results in an inhibition of Na+/Ca2+ exchange currents, and this inhibition occurs secondary to an acidification of the cytosol (20 , 21) . To examine whether hypoxic inhibition of the Na+/Ca2+ exchanger in pulmonary artery myocytes requires cytosolic acidification, we increased the intracellular concentration of HEPES to 20 mM to maintain a constant intracellular pH (7.3), as described previously (21) . As shown in Fig. 8 , clamping intracellular pH with 20 mM HEPES did not affect hypoxic inhibition of the Na+/Ca2+ exchange current. In four experiments recorded with [Ca2+]i buffered to physiological levels (100 nM), the current was inhibited by 60% (from 9.6±1.1 to 3.8±0.5 pA (P<0.05; Fig. 8B ), similar to results obtained in undialyzed cells (63%; Fig. 7B ). Thus, intracellular acidification is not required for hypoxic inhibition of Na+/Ca2+ exchange currents in pulmonary artery myocytes.



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  		Figure 8. Hypoxic inhibition of the Na+-Ca2+ exchange current does not require intracellular acidification. A) Na+/Ca2+ exchange currents recorded in a resistance pulmonary artery myocyte dialyzed with 20 mM HEPES to clamp intracellular pH; [Ca2+]i was clamped at 100 nM. Exposure to mild hypoxia for 5 min significantly inhibited the Na+/Ca2+ exchange current. B) Summary of 4 equivalent experiments; the exchange current was inhibited by 60% after exposure to pO2 of 50–60 Torr, similar to the level observed in perforated patch experiments. *P < 0.05 compared with control.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Substantial data indicate that hypoxic pulmonary vasoconstriction occurs after an increase in [Ca2+]i in pulmonary artery smooth muscle cells (1 , 2) . Two mechanisms have been advanced to explain the hypoxic increase in [Ca2+]i: hypoxia-mediated depolarization leading to Ca2+ influx through plasmalemmal voltage-dependent Ca2+ channels (3 4 5 6 7 8 9 10 11) , and Ca2+ release from intracellular stores (7 , 12 13 14 15 16 17) . Here we demonstrate that hypoxia inhibits Ca2+ removal from the cytoplasm, thereby resulting in an elevation of [Ca2+]i. Hypoxia markedly slows the decay in [Ca2+]i observed after elevation due to depolarization and/or intracellular Ca2+ release (Figs. 1 , 2) . This delay in the return of [Ca2+]i to basal levels likely explains the finding that hypoxia potentiates vasoconstriction and inhibits vasodilation in isolated pulmonary artery rings and lungs (34 , 43) . Thus, after an elevation of [Ca2+]i associated with hypoxia or other stimuli, delayed Ca2+ removal by hypoxia would serve to sustain this elevation and produce a maintained vasoconstriction.

In addition to inhibiting cytosolic Ca2+ efflux, hypoxia markedly potentiated Ca2+ release. This result suggested that the slowed Ca2+ removal was not due to inhibition of the sarcoplasmic reticulum Ca2+ pump, since this would likely result in decreased SR Ca2+ loading and hence decreased Ca2+ release. Our findings that the effect was mimicked by removal of extracellular Na+ and that the Na+/Ca2+ exchange current was inhibited during hypoxia suggested that inhibition of the Na+/Ca2+ exchanger likely underlies the delayed cytosolic Ca2+ efflux. Moreover, such an inhibitory effect would explain hypoxic potentiation of intracellular Ca2+ release, since inhibition of the plasmalemmal exchanger would serve to increase SR loading.

Na+/Ca2+ exchanger activity and gene expression has been reported in blood vessels (26 , 30 , 42 , 44) and functional studies indicate that this exchanger plays an important role in the regulation of cytosolic Ca2+ in vascular myocytes (33 34 35 36 37 38 39 40) . However, we are not aware of direct measurements of the isolated exchange current similar to the protocol used by Kimura et al. in heart cells (41) . Using this protocol, we directly measured the Na+/Ca2+ exchange current by brief applications of Na+ to voltage-clamped cells recorded in Na+-free conditions, resulting in an inward current with a predicted current reversal potential (Fig. 5) . The current recorded in pulmonary artery myocytes has a mean amplitude of 25 pA in conditions of 1 µM internal Ca2+, which is ~10 times smaller than the current reported in heart cells recorded under similar conditions (41) . Previous reports have suggested a substantially lower Na+/Ca2+ exchanger activity in smooth muscle compared to cardiac sarcolemmal vesicles (45) , and it should be noted that the total membrane capacitance (proportional to surface area) of pulmonary vascular smooth muscle cells is roughly 10 to 20% that of heart cells. Under more physiological conditions (permeabilized patch preparation), the exchange current was closer to 10 pA. A current of this magnitude would be expected to have a substantial effect on the resting membrane potential (assuming a 1 gig ohm input resistance, the current would depolarize the pulmonary vascular myocyte by roughly 10 mV).

We report that the Na+/Ca2+ exchange current was markedly inhibited in the presence of mild hypoxia (Fig. 7) . When the pO2 was decreased to 50–60 Torr, the exchange current was ~30% of the current recorded under normoxic conditions. The Na+/Ca2+ exchange current in heart cells is inhibited by cytosolic acidification (46 , 47) , a mechanism that underlies the suppression of the current observed during exposure of heart cells to anoxia (21) . Our data indicate that the effects of physiological hypoxia on pulmonary vascular myocytes are not explained by a mechanism requiring alterations in intracellular pH. Hypoxic inhibition of the Na+/Ca2+ current was equivalent in cells dialyzed with 20 mM HEPES or nondialyzed cells without exogenous buffer, whereas in heart cells the effect of anoxia on the exchanger was abolished when the intracellular pH was clamped in this manner (21) . These data, taken together with the finding that the rate of decay of the [Ca2+]i transient is slowed by exposure to hypoxia or removal of extracellular Na+, strongly suggest that the Na+/Ca2+ exchanger plays an important role in Ca2+ homeostasis of pulmonary artery smooth muscle cells and is modulated by physiological hypoxia. Additional experimental support for the involvement of the Na+/Ca2+ exchanger in hypoxic pulmonary vasoconstriction comes from functional studies demonstrating that hypoxic inhibition of pulmonary artery vasodilation is reduced by removing extracellular Na+ (34) .

Hypoxia and low Na+ solutions markedly potentiated the amplitude of the transient (Figs. 1 2 3 4) . Since a significant Na+/Ca2+ exchange current is measured at basal levels of [Ca2+]i (100 nM), this effect is likely due to enhanced Ca2+ uptake into the SR associated with loss Ca2+ transport across the plasmalemma. Augmented SR buffering has been proposed as the mechanism underlying the potentiation of the [Ca2+]i transient and arterial contraction observed after removal of extracellular Na+ (31 , 48) .

To provide further information about the expression of specific Na+/Ca2+ exchanger isoforms in resistance pulmonary arteries, we performed RT-PCR using specific primer pairs designed to identify RNAs in the variable region of NCX1. NCX1 gene products have been identified as contributors to the regulation of [Ca2+]i in smooth muscle (31) . Using the terminology of Quednau et al. (30) , our results indicate that NCX1.11 and either NCX1.2 or NCX1.9, corresponding to the expression of exons BCDEF and BCD or BDE, respectively, are the major expressed RNAs in smooth muscle. The former conclusion follows from the detection of two bands using forward primers either 5' to exon A or within exon B' and from the size of the larger PCR product in both conditions, whereas the latter conclusion derives from the size difference between the two PCR products using both sets of primers. Though NCX1.2 most closely matches the size difference between the two PCR products, we cannot exclude that the smaller band is the slightly larger NCX1.9.

In summary, we have demonstrated that the Na+/Ca2+ exchanger is functionally expressed in pulmonary artery smooth muscle cells, that the exchange current is active under physiological resting conditions and plays an important role in the decay of the [Ca2+]i transient evoked by calcium release and/or depolarization, and that the exchanger is inhibited during hypoxic stimuli resulting in a slowing of Ca2+ removal from the cytosol and enhanced Ca2+ release. This effect is observed during mild, physiological hypoxic stimuli and may be an important mechanism in amplifying effects of hypoxia on Ca2+ influx or release (1 , 2) .


   ACKNOWLEDGMENTS
 
We thank Mr. Mario Brenes for technical assistance. This work was supported by NIH HL45239 and HL41084 (M.I.K.), the American Heart Association, the Pennsylvania Thoracic Association, and NIH HL64043 (Y.-X.W.).

Received for publication September 24, 1999. Revision received February 14, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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