Contribution of elevated intracellular calcium to pulmonary arterial myocyte alkalinization during chronic hypoxia

Abstract Abstract

In the lung, exposure to chronic hypoxia (CH) causes pulmonary hypertension, a debilitating disease. Development of this condition arises from increased muscularity and contraction of pulmonary vessels, associated with increases in pulmonary arterial smooth muscle cell (PASMC) intracellular pH (pH_i) and Ca²⁺ concentration ([Ca²⁺]_i). In this study, we explored the interaction between pH_i and [Ca²⁺]_i in PASMCs from rats exposed to normoxia or CH (3 weeks, 10% O₂). PASMC pH_i and [Ca²⁺]_i were measured with fluorescent microscopy and the dyes BCECF and Fura-2. Both pH_i and [Ca²⁺]_i levels were elevated in PASMCs from hypoxic rats. Exposure to KCl increased [Ca²⁺]_i and pH_i to a similar extent in normoxic and hypoxic PASMCs. Conversely, removal of extracellular Ca²⁺ or blockade of Ca²⁺ entry with NiCl₂ or SKF 96365 decreased [Ca²⁺]_i and pH_i only in hypoxic cells. Neither increasing pH_i with NH₄Cl nor decreasing pH_i by removal of bicarbonate impacted PASMC [Ca²⁺]_i. We also examined the roles of Na⁺/Ca²⁺ exchange (NCX) and Na⁺/H⁺ exchange (NHE) in mediating the elevated basal [Ca²⁺]_i and Ca²⁺-dependent changes in PASMC pH_i. Bepridil, dichlorobenzamil, and KB-R7943, which are NCX inhibitors, decreased resting [Ca²⁺]_i and pH_i only in hypoxic PASMCs and blocked the changes in pH_i induced by altering [Ca²⁺]_i. Exposure to ethyl isopropyl amiloride, an NHE inhibitor, decreased resting pH_i and prevented changes in pH_i due to changing [Ca²⁺]_i. Our findings indicate that, during CH, the elevation in basal [Ca²⁺]_i may contribute to the alkaline shift in pH_i in PASMCs, likely via mechanisms involving reverse-mode NCX and NHE.

Many chronic lung diseases are characterized by prolonged exposure to hypoxic conditions, with resulting development of pulmonary hypertension. The pathogenesis of hypoxic pulmonary hypertension is associated with changes in the pulmonary vasculature that include both contraction and growth of pulmonary arterial smooth muscle cells (PASMCs). The exact mechanisms underlying these changes are incompletely understood, although it is becoming apparent that these changes may be related to alterations in intracellular Ca²⁺ or pH homeostasis.

Our previous studies have revealed that exposure to chronic hypoxia (CH) increased the expression of Na⁺/H⁺ exchanger isoform 1 (NHE1), enhanced the activity of Na⁺/H⁺ exchange, and created an alkaline shift in intracellular pH (pH_i) in PASMCs.^1,2 Increased pH_i is associated with PASMC proliferation in response to growth factors^3,4 and, under some conditions, with enhanced smooth muscle contraction.^5-8 A role for Na⁺/H⁺ exchange in modulating PASMC growth during CH was initially suggested by studies indicating that inhibitors of Na⁺/H⁺ exchange attenuated pulmonary vascular remodeling in chronically hypoxic rats.⁹ Moreover, an absence of alkalinization correlated with reduced remodeling in a murine model of hypoxic pulmonary hypertension.² Later studies demonstrated that genetic loss of NHE1 prevented CH-induced pulmonary vascular remodeling¹⁰ and attenuated hypoxia-induced proliferation and migration in vitro.¹¹

Prolonged hypoxic conditions have also been shown to cause influx of Ca²⁺ into PASMCs through store-operated^12,13 or voltage-gated¹⁴ Ca²⁺ channels, resulting in increased intracellular calcium concentration ([Ca²⁺]_i). [Ca²⁺]_i is well known to contribute to a variety of cell functions, with increased [Ca²⁺]_i contributing to PASMC contraction^13,15-19 and playing a role in smooth muscle cell proliferation^20-23 and migration.²⁴

Interactions between pH_i and [Ca²⁺]_i have been demonstrated in smooth muscle under normoxic conditions.^25,26 For example, although increasing [Ca²⁺]_i alone was not sufficient to alter pH_i,^27,28 certain agonists that increased [Ca²⁺]_i, including thrombin and angiotensin II, were found to also cause acidification.^26,28,29 Others found that increasing or decreasing pH_i resulted in an increase in Ca²⁺ influx.^6,8,30-32 These studies suggest that the regulation of [Ca²⁺]_i and pH_i could be interdependent. Both [Ca²⁺]_i and pH_i increase in PASMCs during CH; however, it is not known whether these changes in pH and Ca²⁺ homeostasis are related or occur via completely independent mechanisms. Thus, the goals of this study were to determine (i) the relationship between pH_i and [Ca²⁺]_i in PASMCs during normoxia, (ii) whether this relationship is altered following exposure to CH, and, if so, (iii) the mechanisms involved. We used fluorescent microcopy with the Ca²⁺-senstive dye, Fura-2, and the pH-sensitive dye, BCECF, in transiently cultured PASMCs isolated from normoxic and chronically hypoxic rats to determine the effects of changing [Ca²⁺]_i on pH_i and vice versa.

Methods

Hypoxic exposure

All protocols were approved by the Johns Hopkins Animal Care and Use Committee. Adult male Wistar rats (250–350 g) were placed in a hypoxic chamber for 3 weeks as described elsewhere.¹³ The chamber was continuously gassed with a mixture of room air and 100% N₂ to maintain $10\%+0.5\%$ O₂ and low CO₂ concentrations (<0.5%). O₂ levels inside the chamber were continuously monitored (PRO-OX, RCI Hudson, Anaheim, CA), and this value was used by a servo controller to adjust N₂ inflow. Animals were allowed free access to food and water and were removed from the chamber for less than 5 minutes twice a week to replenish food and water supplies and to clean the cages. Normoxic animals were kept next to the chamber in room air and were thus exposed to similar temperatures and a similar light/dark cycle.

Isolation of pulmonary arterial smooth muscle cell

A total of 3,846 cells were analyzed for this study. The methods for obtaining primary cultures of rat PASMCs have been previously described.¹⁵ Briefly, animals were deeply anesthetized with sodium pentobarbital (130 mg/kg via intraperitoneal injection) and the heart and lungs were removed en bloc. Intrapulmonary arteries (200–600 μm outer diameter) were dissected and cleaned of connective tissue in ice-cold HEPES-buffered saline solution (HBSS) containing 130 mM NaCl, 5 mM KCl, 1.2 mM MgCl₂, 10 mM HEPES, and 10 mM glucose with pH adjusted to 7.2 with 5 M NaOH. The arteries were opened and the lumen gently scraped to remove endothelial cells. The arteries were allowed to recover for 30 minutes in cold (4°C) HBSS followed by 20 minutes in reduced-Ca²⁺ HBSS (20 μM CaCl₂) at room temperature. The tissue was enzymatically digested for 20–25 minutes at 37°C in reduced-Ca²⁺ HBSS containing collagenase (type I; 1,750 U/mL), papain (9.5 U/mL), bovine serum albumin (2 mg/mL), and dithiothreitol (1 mM). After digestion, single smooth muscle cells were dispersed by gentle trituration in Ca²⁺-free HBSS and were plated on 25-mm glass coverslips. PASMCs were cultured in Ham’s F-12 media containing 0.5% fetal calf serum and 1% penicillin/streptomycin for 24–48 hours.

Measurement of [Ca²⁺]_i

[Ca²⁺]_i was measured in rat PASMCs as previously described.¹³ Cells were incubated with 5 μM Fura-2 AM, a membrane permanent (acetoxymethyl ester) form of Fura-2, for 60 minutes at 37°C under an atmosphere of 16% O₂ and 5% CO₂. Following incubation, cells were placed in a laminar flow cell chamber perfused with modified Kreb’s solution (KRB) containing 118.3 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 25 mM NaHCO₃, 11 mM glucose, and 1.2 mM KH₂PO₄ and then gassed with 16% O₂ and 5% CO₂. Cells were washed for 15 minutes at 37°C to remove extracellular dye and allow complete de-esterification of cytosolic dye. Ratiometric measurement of Fura-2 fluorescence was performed in a workstation based on a Nikon TES 100 Ellipse inverted microscope with epi-fluoresence attachments. The collimated light beam from a xenon arc lamp was filtered by interference filters at 340 and 380 nm and focused onto PASMCs via a 20× fluorescence objective (Super Flour 20, Nikon). Light emitted from the cell was returned through the objective and detected by an imaging camera. An electronic shutter was used to minimize photobleaching of dye. Protocols were executed and data were collected online with InCyte software (Intracellular Imaging, Cincinnati, OH). [Ca²⁺]_i values were calculated from an in vitro calibration curve and thus should be considered an estimate of true [Ca²⁺]_i.

pH_i measurements

pH_i within PASMCs was monitored using the cell-permeant pH-sensitive dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyflourescein (BCECF AM) as described elsewhere.^1,2 Cells were placed in the perfusion chamber, perfused with KRB solution, and excited with light filtered at 490 and 440 nm. Light emitted from the cells was detected at 530 nm. The ratio of 490 to 440 nm emission was calculated and pH_i was estimated from in situ calibration after each experiment. For calibration, PASMCs were perfused with a high K⁺ solution containing 105 mM KCl, 1 mM MgCl₂, 1.5 mM CaCl₂, 10 mM glucose, 20 mM HEPES-Tris, and 0.01 mM nigericin to allow pH_i to equilibrate to external pH (42). A two-point calibration was created from fluorescence measured as pH_i was adjusted with KOH from 6.5 to 7.5. Intracellular H⁺ ion concentration ([H⁺]_i) was determined from pH_i using the formula $p{H}_i=-\log ({[H}^{+}{]}_i)$. For both [Ca²⁺]_i and pH_i measurements, cells were perfused using a multi-input, single-output system connected to reservoirs containing control solutions or solutions containing various antagonists. Switching between reservoirs allowed for rapid exchange of the chamber solution.

Drugs and solutions

Ethyl isopropyl amiloride (EIPA), SKF-96365 (SKF), and bepridil (BPD) were made up as 10⁻² M stock solutions in DMSO (for EIPA and BPD) or distilled water and refrigerated until use. KB-R7943 was made up as a 10⁻¹ M stock solution in DMSO, aliquoted, and frozen until use. Dichlorobenzamil (DCB) was made as a 10-mM stock in de-ionized water, aliquoted, and stored at 4°C until use. All other drugs were solubilized in de-ionized water on the day of the experiment. Drugs were diluted to final working concentrations in perfusate solutions. Vehicle (DMSO 1:1,000 dilution) had no effect on either [Ca²⁺]_i or pH_i. KB-R7943 (KBR) was obtained from Tocris (Ellisville, MO). Fura-2 AM and BCECF AM were obtained from Molecular Probes (Eugene, Oregon). All other chemicals were from Sigma-Aldrich.

Data analysis

All values are expressed as mean ± SEM; n refers to the number of cells. All experiments were performed a minimum of 3 times (10–27 cells/experiment) using cells obtained from a minimum of 3 different animals. pH values were converted to [H⁺] values for statistical analysis. For agents that induced a change in pH_i or [Ca²⁺]_i, statistical analysis was performed by comparison of values before and after treatment using paired Student t test. The effect of chronic hypoxia or inhibitors on the response to agents that caused a change in [Ca²⁺]_i or pH_i was evaluated by comparing the change in pH_i or [Ca²⁺]_i induced in the presence and absence of inhibitors or between normoxia and hypoxia, as a group using ANOVA. A P value <0.05 was accepted as statistically significant.

Results

Effect of CH on pH_i and [Ca²⁺]_i in PASMCs

Resting [Ca²⁺]_i in PASMCs isolated from rats exposed to CH was significantly higher compared to resting [Ca²⁺]_i measured in cells isolated from normoxic rats (Fig. 1A). Similarly, basal pH_i in PASMCs from rats exposed to CH was increased compared to PASMCs isolated from normoxic rats (Fig. 1B). The baseline values of both [Ca²⁺]_i and pH_i are qualitatively and quantitatively similar to those observed in our previous studies.^1,2,13,15

Figure 1.

Bar graphs showing mean ± SEM values for resting intracellular Ca²⁺ concentration ([Ca²⁺]_i; A) and basal intracellular pH (pH_i; B) in pulmonary arterial smooth muscle cells isolated from rats exposed to normoxia ($n=204$ for [Ca²⁺]_i and $n=550$ for pH_i) and chronic hypoxia ($n=455$ for [Ca²⁺]_i and $n=934$ for pH_i). Asterisk indicates a significant difference from the normoxic value ($P<0.05$).

Effect of depolarization or Ca²⁺ channel blockers on [Ca²⁺]_i

Exposure to KCl (80 mM), which causes depolarization and Ca²⁺ entry through voltage-dependent Ca²⁺ channels, increased [Ca²⁺]_i in PASMCs from both normoxic and chronically hypoxic rats (Fig. 2A). In normoxic PASMCs, the increase in [Ca²⁺]_i in response to KCl was slightly lower than that observed in hypoxic PASMCs. In normoxic cells, removal of extracellular Ca²⁺ or exposure to NiCl₂ (500 nM), a nonspecific inhibitor of Ca²⁺ entry through nonselective cation channels (NSCCs), which includes store-operated Ca²⁺ channels, had no significant effect on basal [Ca²⁺]_i. Somewhat surprisingly, application of SKF-96365 (10 μM), a more specific NSCC inhibitor, caused a slight, but statistically significant, increase in [Ca²⁺]_i in normoxic cells. In contrast, in hypoxic PASMCs, removal of extracellular Ca²⁺ or application of NiCl₂ or SKF-96365 decreased [Ca²⁺]_i. These decreases in basal [Ca²⁺]_i in PASMCs from chronically hypoxic rats in response to Ca²⁺-free conditions or inhibition of NSCCs are consistent with our previous observations.¹³

Figure 2.

A, Effect of exposure to KCl (80 mM; $n=97$ for normoxic and $n=138$ for hypoxic); removal of extracellular Ca²⁺ (Ca²⁺-free; $n=83$ for normoxic and $n=69$ for hypoxic); treatment with NiCl₂ (500 nM; $n=72$ for normoxic and $n=79$ for hypoxic) or treatment with SKF96365 (SKF; 10 μM; $n=79$ for normoxic and $n=47$ for hypoxic) on intracellular Ca²⁺ ([Ca²⁺]_i) in rat pulmonary arterial smooth muscle cells (PASMCs). B, Change in intracellular pH (pH_i) induced in PASMCs from normoxic and chronically hypoxic rats by exposure to KCl ($n=42$ for normoxic and $n=37$ for hypoxic); removal of extracellular Ca²⁺ ($n=92$ for normoxic and $n=34$ for hypoxic); treatment with NiCl₂ ($n=89$ for normoxic and $n=42$ for hypoxic) or treatment with SKF ($n=55$ for normoxic and $n=66$ for hypoxic). Data are expressed as mean ± SEM change (Δ) in [Ca²⁺]_i or pH_i. Asterisk indicates significant difference from baseline; two asterisks indicate significant difference between normoxic and hypoxic values.

Effect of changing [Ca²⁺]_i on pH_i

Increasing [Ca²⁺]_i by exposure to KCl (80 mM) increased pH_i in PASMCs from both normoxic and chronically hypoxic rats (Fig. 2B). Similar to the greater increase in [Ca²⁺]_i induced by KCl in chronically hypoxic PASMCs, the magnitude of the KCl-induced increase in pH_i was also greater in hypoxic PASMCs than in cells from normoxic rats. Although exposure to KCl increased pH_i, the removal of extracellular Ca²⁺ using a Ca²⁺-free solution caused a significant decrease in pH_i only in cells from chronically hypoxic rats. Qualitatively similar results were obtained with NiCl₂ and SKF, which had no significant effect on pH_i in normoxic cells but significantly decreased pH_i in PASMCs isolated from chronically hypoxic rats. Interestingly, Ca²⁺-free solution and NiCl₂ caused quantitatively similar reductions in both [Ca²⁺]_i and pH_i in PASMCs from hypoxic animals, whereas the effect of SKF was approximately 50% less in both cases.

Effect of removal of bicarbonate or exposure to NH₄Cl on pH_i

To alter pH_i, PASMCs were exposed to HEPES-buffered extracellular solution, which removes the contribution of Cl-/HCO₃-exchangers in pH_i homeostasis, or to 3 or 10 mM NH₄Cl, which causes alkalinization due to buffering of intracellular H⁺. As expected, based on our previous observations and studies in guinea pig pulmonary vascular smooth muscle,^1-3 removal of bicarbonate caused a brief increase in pH_i that subsided to a sustained reduction in pH_i in cells from both normoxic and chronically hypoxic rats (Fig. 3A). Conversely, exposure to either 3 or 10 mM NH₄Cl caused a significant increase in pH_i in PASMCs from both normoxic and chronically hypoxic rats. Although 3 mM NH₄Cl induced a change in pH_i that was quantitatively similar in normoxic and hypoxic cells, the increase in pH_i induced by 10 mM NH₄Cl was substantially larger in cells from rats exposed to CH compared with normoxia. In most cells, the response to NH₄Cl was a maintained increase in pH_i, although in some cells, there was a transient large increase in pH_i that then decreased to a sustained level that was below the peak but still above basal levels. In all experiments, pH_i was measured after 10 minutes of exposure to either bicarbonate-free solution or NH₄Cl, during the sustained phase of the response.

Figure 3.

A, Effect of removal of extracellular bicarbonate (HEPES; $n=28$ cells for normoxic and $n=32$ cells for hypoxic) and exposure to 3 mM ($n=88$ for normoxic and $n=77$ for hypoxic) or 10 mM ($n=25$ for normoxic and $n=32$ for hypoxic) ammonium chloride (NH₄Cl) on intracellular pH (pH_i) in pulmonary arterial smooth muscle cells (PASMCs) from normoxic and chronically hypoxic rats. B, Changes in intracellular Ca²⁺ ([Ca²⁺]_i) induced by exposure of PASMCs to HEPES-buffered extracellular solution ($n=85$ for normoxic and $n=48$ for hypoxic) or NH₄Cl (3 mM: $n=69$ for normoxic and $n=82$ for hypoxic; 10 mM: $n=100$ for normoxic and $n=48$ for hypoxic). Asterisk indicates significant difference from baseline; two asterisks indicate significant difference between normoxic and hypoxic values.

Increasing pH_i by exposure to 3 or 10 mM NH₄Cl had no significant effect on [Ca²⁺]_i in PASMCs isolated from normoxic or chronically hypoxic rats (Fig. 3B), although a small subset of cells exposed to 10 mM NH₄Cl (21 of 100 in normoxic; 7 of 48 in hypoxic) exhibited a transient increase in [Ca²⁺]_i that rapidly returned to basal levels. Because pH_i and [Ca²⁺]_i were not measured simultaneously in the same cells, it is unclear whether the cells that exhibited a transient increase in [Ca²⁺]_i also exhibited a transient overshoot in pH_i in response to NH₄Cl. Decreasing pH_i by perfusing the cells with a HEPES-buffered solution caused a very small but statistically significant decrease in [Ca²⁺]_i in PASMCs from normoxic rats, whereas no effect on [Ca²⁺]_i was observed in PASMCs from chronically hypoxic rats.

Role of Na⁺/H⁺ exchange in mediating changes in pH_i induced by changing [Ca²⁺]_i

We^1,2 and others^3,33 have previously reported that Na⁺/H⁺ exchange contributes to regulation of pH_i in PASMCs. To assess the contribution of Na⁺/H⁺ exchange in regulating pH_i in PASMCs from normoxic and chronically hypoxic rats, cells were exposed to EIPA, a Na⁺/H⁺ exchange inhibitor. Blockade of Na⁺/H⁺ exchange with EIPA (10 μM) caused a significant decrease in pH_i in PASMCs from normoxic animals (Fig. 4A). Consistent with our previously reported results, EIPA caused a decrease in pH_i in PASMCs isolated from chronically hypoxic rats that was greater than the decrease observed in normoxic PASMCs. EIPA caused a small but statistically significant increase in baseline [Ca²⁺]_i in PASMCs from normoxic animals (Fig. 4B) and a small but statistically significant decrease in [Ca²⁺]_i in chronically hypoxic PASMCs. When PASMCs were pretreated with EIPA, the changes in pH_i induced by KCl, removal of extracellular Ca²⁺, or exposure to NiCl were abolished (Fig. 4C).

Figure 4.

Role of Na⁺/H⁺ exchange in mediating Ca²⁺-dependent changes in intracellular pH (pH_i). Effect of ethyl isopropyl amiloride (EIPA; 10 μM) on basal pH_i (A) and intracellular Ca²⁺ ([Ca²⁺]_i; B) in pulmonary arterial smooth muscle cells (PASMCs) from normoxic ($n=40$ for pH_i and $n=42$ for [Ca²⁺]_i) or chronically hypoxic ($n=49$ for pH_i and $n=100$ for [Ca²⁺]_i) rats. Asterisk indicates significant difference from baseline; two asterisks indicate significant difference between normoxic and hypoxic values. C, EIPA prevented the change in pH_i induced by changing [Ca²⁺]_i via perfusion with KCl (80 mM; $n=42$ for untreated and $n=47$ for EIPA), Ca²⁺-free solution ($n=92$ for untreated and $n=35$ for EIPA) or NiCl₂ (500 nM; $n=89$ for untreated and $n=31$ for EIPA) in PASMCs from chronically hypoxic rats. Data are presented as mean ± SEM. Two asterisks indicate significant difference between values in the presence and absence of EIPA.

Is Na⁺/Ca²⁺ exchange involved in regulating pH_i and [Ca²⁺]_i?

A main mechanism regulating Ca²⁺ extrusion in PASMCs is the Na⁺/Ca²⁺ exchanger (NCX).^34-36 This exchanger generally transports one Ca²⁺ ion out of the cell in exchange for 3 Na⁺ ions into the cell; however, under certain conditions the exchanger can reverse, resulting in Ca²⁺ influx. Although acute hypoxia has been suggested to alter NCX activity,^35,36 it is not known whether either forward-mode (Ca²⁺ extrusion) or reverse-mode (Ca²⁺ entry) NCX contributes significantly to PASMC Ca²⁺ homeostasis during CH. The role of NCX in regulating resting [Ca²⁺]_i levels in PASMCs was tested by addition of 50 μM BPD or 15 μM DCB, general NCX inhibitors. In normoxic cells, both BPD and DCB increased [Ca²⁺]_i, consistent with blockade of Ca²⁺ extrusion (Fig. 5A). Addition of KB-R7943 (KBR; 10 μM), an inhibitor selective for reverse-mode (Ca²⁺ entry) NCX, to normoxic PASMCs had no significant effect on [Ca²⁺]_i. In contrast, when chronically hypoxic PASMCs were treated with BPD, DCB, or KBR, basal [Ca²⁺]_i decreased to a similar extent, presumably due to blockade of Ca²⁺ entry via reverse-mode Na⁺/Ca²⁺ exchange.

Figure 5.

Role of Na⁺/Ca²⁺ exchange in mediating Ca²⁺-dependent changes in intracellular pH (pH_i). All data are presented as mean ± SEM. A, Effect of bepridil (BPD; $n=119$ for normoxic and $n=72$ for hypoxic), dichlorobenzamil (DCB; $n=86$ for normoxic and $n=68$ for hypoxic), and KB-R7943 (KBR; $n=97$ for normoxic and $n=127$ for hypoxic) on basal intracellular Ca²⁺ ([Ca²⁺]_i) in pulmonary arterial smooth muscle cells (PASMCs) from normoxic and chronically hypoxic rats. B, Effect of BPD ($n=90$ for normoxic and $n=39$ for hypoxic), DCB ($n=70$ for normoxic and $n=70$ for hypoxic), and KBR ($n=89$ for normoxic and $n=83$ for hypoxic) on basal pH_i in PASMCs. A and B, asterisk indicates significant difference from baseline; two asterisks indicate significant difference between normoxic and hypoxic values. C, The effect of pretreating cells from chronically hypoxic rats with BPD, DCB, or KBR on the change in pH_i induced by changing [Ca²⁺]_i with 80 mM KCl ($n=37$, 63, 111, and 37 for control, BPD, DCB, and KBR, respectively), Ca²⁺-free extracellular solution ($n=34$, 74, 103, and 35 for control, BPD, DCB, and KBR, respectively), or 500 nM NiCl₂ ($n=42$, 89, 98, and 56 for control, BPD, DCB, and KBR, respectively). Asterisk indicates significant difference from baseline; two asterisks indicate significant difference between values in the absence (control hypoxic) and presence of Na⁺/Ca²⁺ exchange inhibitor.

Because reverse-mode NCX appeared to participate in regulation of [Ca²⁺]_i levels in PASMCs from chronically hypoxic, but not normoxic, animals, we tested whether reverse-mode NCX was contributing to either basal pH_i or changes in pH_i during stimulation in PASMCs following exposure to CH. We found that, while DCB and KBR had no significant effect on basal pH_i, BPD caused a slight but statistically significant decrease in basal pH_i in PASMCs from normoxic rats (Fig. 5B). In cells from hypoxic rats, all three NCX inhibitors decreased pH_i. When PASMCs from chronically hypoxic rats were pretreated with BPD, DCB, or KBR, changes in pH_i observed when [Ca²⁺]_i was increased by KCl or decreased by removal of extracellular Ca²⁺ or addition of NiCl₂ were significantly attenuated (Fig. 5C).

Discussion

During CH, PASMCs exhibit increases in both [Ca²⁺]_i and pH_i. Although our previous studies suggested that the elevation in [Ca²⁺]_i and alkaline shift in pH_i were associated with increased expression of NSCCs and Na⁺/H⁺ exchangers, respectively, it was unclear whether activation of these channels/transporters might also be altered and, if so, whether the changes were interdependent. Thus, the goal of the current study was to determine whether the elevation in basal [Ca²⁺]_i contributed to the alkaline shift in pH_i and vice versa. Our findings indicate that, during CH, changes in PASMC pH_i do not significantly impact [Ca²⁺]_i, whereas changes in basal [Ca²⁺]_i may contribute to the elevation in pH_i.

We found that KCl was able to increase [Ca²⁺]_i in PASMCs from both normoxic and chronically hypoxic animals and that the effect of KCl appears to be enhanced in cells from hypoxic animals. This would be consistent with recent reports showing upregulation of voltage-gated Ca²⁺ channel expression and increased KCl-induced Ca²⁺-influx in PASMCs derived from chronically hypoxic mice.³⁷ In contrast, measures designed to reduce [Ca²⁺]_i were only effective in PASMCs from chronically hypoxic animals, likely due to the low basal [Ca²⁺]_i levels observed in normoxic cells. The lack of effect of Ca²⁺ channel blockers on basal [Ca²⁺]_i in normoxic PASMCs is consistent with our previous results and those of other laboratories.^12,13,15,38 Similar to our previous results,³⁹ blockade of NSCCs with SKF caused a small but statistically significant increase in basal [Ca²⁺] under normoxic conditions. A similar SKF-induced increase in [Ca²⁺]_i has been noted in frog mesenteric endothelial cells⁴⁰ and a megakaryoblastic cell line,⁴¹ as well as in endothelial cells at higher concentrations.⁴² Although the exact mechanism underlying the increase in [Ca²⁺]_i is unknown, SKF has been shown to depolarize smooth muscle⁴³ and, in endothelial cells, SKF can block K⁺ currents with an estimated 50% inhibitory concentration near the concentrations used to block NSCC.⁴² Because of these uncertainties, we did not use SKF in the experiments designed to test the role of NCX and/or NHE in Ca²⁺-induced changes in pH_i.

Interestingly, in cells isolated from chronically hypoxic animals, removal of extracellular Ca²⁺ and application of NiCl₂ caused similar reductions in [Ca²⁺]_i, whereas the effect of SKF was significantly less. One explanation for these differences is that, while SKF is considered a relatively selective inhibitor of Ca²⁺ entry through NSCCs, removal of extracellular Ca²⁺ and application of NiCl₂ are less selective for specific Ca²⁺ channels/transporters. Indeed, both removal of Ca²⁺ and application of NiCl₂ also would prevent Ca²⁺ entry through voltage-gated Ca²⁺ channels or reverse-mode Na⁺/Ca²⁺ exchange.⁴⁴ The lack of effect of nifedipine or verapamil on baseline [Ca²⁺]_i in PASMCs from chronically hypoxic animals^13,15 indicates that these channels do not contribute significantly to maintenance of elevated basal [Ca²⁺]_i and thus would not be expected to contribute to the decrease in [Ca²⁺]_i observed with Ca²⁺ removal. In contrast, application of BPD, DCB, or KBR, all of which can block reverse-mode NCX, reduced [Ca²⁺]_i selectively in PASMCs isolated from chronically hypoxic animals. This finding is consistent with data demonstrating that reverse-mode NCX contributes to maintenance of elevated basal [Ca²⁺]_i in myocytes from hypertensive rats⁴⁵ and influences PASMC proliferation.⁴⁶ Although the magnitude of the decrease in [Ca²⁺]_i in response to NCX inhibitors was much less than that induced by extracellular Ca²⁺ removal, signifying a small role for Ca²⁺ entry through NCX in maintenance of basal [Ca²⁺]_i, it was qualitatively similar to the difference between the effect of SKF and those of NiCl₂ or removal of extracellular Ca²⁺, suggesting the contributions of NSCCs and NCX to maintenance of resting [Ca²⁺]_i might be additive.

Regardless of whether increased by KCl or decreased by blockade of Ca²⁺ entry, changing [Ca²⁺]_i induced similar directional changes in pH_i. These results support other reports indicating that changes in [Ca²⁺]_i can influence smooth muscle pH_i regulation^27,28,47-49 and indicate that the mechanisms involved in maintaining elevated basal [Ca²⁺]_i may contribute to alkalination of basal pH_i in PASMCs from chronically hypoxic animals. Interestingly, in rat aortic smooth muscle, changing Ca²⁺ was also found to change pH_i,²⁶ but in the opposite direction, with increased [Ca²⁺]_i causing a reduction in pH_i mediated by Ca²⁺-ATPase. Whether the contrast between our study and the previous report is due to differences in vascular bed, presence of bicarbonate, or other factors remains to be determined.

As expected, removal of bicarbonate or inhibition of Na⁺/H⁺ exchange reduced pH_i in PASMCs. In both cases, the magnitude of the reduction was greater in PASMCs from hypoxic rats, reflecting the role of Na⁺/H⁺ exchange in maintaining increased basal pH_i during CH. In contrast, application of NH₄Cl increased pH_i, due to buffering of intracellular H⁺ ions. At the lower concentration, the effect of NH₄Cl on pH_i was similar in cells from normoxic and hypoxic animals and equal in magnitude to the effect of CH on pH_i. At the higher concentration, the effect of NH₄Cl was greater in PASMCs from hypoxic rats; the exact reason for this difference is not clear. Nonetheless, none of the methods employed to change pH_i (removal of bicarbonate, application of NH₄Cl, or inhibition of Na⁺/H⁺ exchange) significantly altered [Ca²⁺]_i, suggesting that the alkaline shift in pH_i observed in PASMCs from chronically hypoxic rats does not contribute to the elevation in basal [Ca²⁺]_i. Although it is clear that none of the manipulations designed to change pH_i had a significant impact on [Ca²⁺]_i in our cells, our results stand in contrast to other reports in which changing pH_i elicited changes in [Ca²⁺]_i in ferret PASMCs,⁸ rat aortic smooth muscle,^6,25,30 canine kidney cells,⁵⁰ A7r5 cells,³¹ endothelial cells,⁵¹ and pancreatic acinar cells.⁵² On the other hand, studies involving platelets^53-55 also failed to show a change in [Ca²⁺]_i when pH_i was adjusted. The reason for the differences between studies remains to be determined, but variations in species, cell type, presence of bicarbonate, the methods used to alter pH_i, and/or the magnitude of the change in pH_i induced by interventions are likely contributing factors.^6,30,52

One aspect that should be taken into consideration when measuring the effect of changes in pH_i on [Ca²⁺]_i is that the dissociation constant (K_d) for Fura-2 is pH-sensitive. Over the range of pH_i from 7.0 to 7.5, K_d will vary little, but with acidic pH (<6.5), K_d can increase substantially.⁵⁶ For the range of basal pH values and changes in pH_i reported in this study (average range of 6.8–7.2 and changes of −0.04 to 0.96 units), K_d will vary slightly and does not significantly alter [Ca²⁺]_i values, with a calculated difference of less than 20 nM between corrected and uncorrected values at the highest change in pH_i that we observed (0.96 units). These modest changes in magnitude are consistent with those reported by others.^30,52 Thus, it is unlikely that pH-induced alterations in K_d obscured changes in [Ca²⁺]_i.

In PASMCs from chronically hypoxic animals, all of the changes in pH_i induced by changing [Ca²⁺]_i were blocked by pretreatment with EIPA, indicating that the changes were mediated entirely via alterations in Na⁺/H⁺ exchange activity. Ca²⁺-dependent regulation of Na⁺/H⁺ exchange has been described in other cell types,^{27,48,49,57,58} and NHE1 has been demonstrated to complex with, and be activated by, calcium-calmodulin, perhaps providing a link between [Ca²⁺]_i and Na⁺/H⁺ exchange activity.⁵⁹ Interestingly, an increase in [Ca²⁺]_i was not requisite for calcium-calmodulin activation, which also occurred following tyrosine phosphorylation,⁵⁹ and other studies found that simply increasing [Ca²⁺]_i was not sufficient to induce Na⁺/H⁺ exchange activation, but instead acted to modulate exchanger activity under certain conditions.²⁷ Moreover, Na⁺/H⁺ exchange activity is heavily influenced by the intracellular Na⁺ concentration ([Na⁺]_i); reductions in [Na⁺]_i enhance, whereas elevations in [Na⁺]_i reduce, exchanger activity. Thus, in response to our manipulations, it was not clear whether the changes in pH_i observed were directly related to the changes in [Ca²⁺]_i or were secondary to changes in [Na⁺]_i, as might occur with changes in NCX activation. The fact that the SKF-induced increase in [Ca²⁺]_i observed in PASMCs from normoxic animals had no significant effect on pH_i appeared to rule out a direct effect of increased [Ca²⁺]_i and instead supported the latter possibility. Under normal resting conditions, NCX operates in forward, or Ca²⁺-extrusion, mode; however, when the cell is depolarized, such as during CH^60-62 or exposure to high external [K⁺], the exchanger operates in reverse, or Ca²⁺-entry, mode. In this case, Na⁺ extrusion would be expected to enhance Na⁺/H⁺ exchange activity and increase pH_i. Thus, inhibiting reverse-mode NCX would be expected to not only decrease Ca²⁺ entry and [Ca²⁺]_i but also lead to accumulation of intracellular Na⁺, which would in turn serve to reduce Na⁺/H⁺ exchange activity and lower pH_i. Indeed, blockade of reverse-mode NCX prevented the changes in pH_i induced by KCl, Ca²⁺-free solution or NiCl₂. A role for [Na⁺]_i could also explain the effect of NSCC inhibition in chronically hypoxic PASMCs, as preliminary data suggest that these channels may contribute to depolarization of basal membrane potential in chronically hypoxic PASMCs (data not shown). Although alterations in NCX activity have been shown to modulate [Na⁺]_i,⁴⁴ further experiments will be required to determine whether this indeed occurs in PASMCs from chronically hypoxic rats and whether changes in [Na⁺]_i are responsible for mediating the NCX-dependent changes in Na⁺/H⁺ exchange activity.

One limitation in defining the role of NCX in mediating changes in [Ca²⁺]_i and pH_i in our study is the use of pharmacologic inhibitors, which could have off-target effects. We attempted to more selectively identify the role of NCX using small interfering RNA approaches; however, the long half-life of the protein, which has been reported at ∼33 hours in cardiomyocytes,⁶³ prevented us from achieving adequate knockdown in PASMCs with this method. Similar difficulties have been noted using antisense oligionucleotides in vascular smooth muscle.⁶⁴ However, we believe the use of 3 different inhibitors, with different off-target effects, helps to offset concerns about the specificity of the pharmacological approach.

In summary, our findings indicate that, in PASMCs from chronically hypoxic rats, Ca²⁺ entry via NSCCs and reverse-mode NCX contributed to the sustained increase in basal [Ca²⁺]_i observed in these cells (Fig. 6). Moreover, Na⁺ efflux through reverse-mode NCX may facilitate increased activation of Na⁺/H⁺ exchange activity, contributing to the alkaline shift in pH_i. In contrast, the alkaline shift in pH_i does not appear to contribute to the maintenance of elevated basal [Ca²⁺]_i. Future studies will be required to elucidate whether the changes in pH_i are due to the change in basal [Ca²⁺]_i induced by hypoxia, or are secondary to other effects of the mechanisms involved in Ca²⁺ homeostasis (i.e., Na⁺ influx and/or membrane potential).

Figure 6.

Schematic illustrating proposed mechanism by which changes in intracellular Ca²⁺ ([Ca²⁺]_i) influence intracellular pH (pH_i) in pulmonary arterial smooth muscle cells (PASMCs) during chronic hypoxia (CH). CH has been shown to upregulate the expression of canonical transient receptor potential (TRPC) proteins, leading to increased Ca²⁺ influx through nonselective cation channels (NSCC) and elevated intracellular Ca²⁺ concentrations ([Ca²⁺]_i). Because these channels can also conduct Na⁺, activation may also contribute to depolarization. Depolarization during CH leads to Ca²⁺ influx through reverse-mode Na⁺/Ca²⁺ exchange (NCX), which, along with upregulation of Na⁺/H⁺ exchanger isoform 1 (NHE1) expression, leads to increased activation of Na⁺/H⁺ exchange and elevated intracellular pH (pH_i). Both alkaline pH_i and elevated [Ca²⁺]_i have been associated with PASMC proliferation and contraction. Inhibitors are shown in red. Dashed gray line represents hypothesized pathway requiring additional confirmation. DCB: dichlorobenzamil; KBR: KB-R7943; SKF: SKF96365; BPD: bepridil; EIPA: ethylisoproplyamiloride.

Source of Support: This work was supported by National Institutes of Health grants HL 073859 and HL 084762.

Conflict of Interest: None declared.