Contribution of Elevated Intracellular Calcium to Pulmonary Arterial Myocyte Alkalinization during Chronic Hypoxia

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 (200600 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)wasdetermined from pH_i using the formula pH = –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.

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

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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

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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.

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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).

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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.

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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).