Acute hypoxia activates store-operated Ca²⁺ entry and increases intracellular Ca²⁺ concentration in rat distal pulmonary venous smooth muscle cells

Abstract

Rationale

Exposure to acute hypoxia causes vasoconstriction in both pulmonary arteries (PA) and pulmonary veins (PV). The mechanisms on the arterial side have been studied extensively. However, bare attention has been paid to the venous side.

Objectives

To investigate if acute hypoxia caused the increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i), and Ca²⁺ influx through store-operated calcium channels (SOCC) in pulmonary venous smooth muscle cells (PVSMCs).

Methods

Fluorescent microscopy and fura-2 were used to measure effects of 4% O₂ on [Ca²⁺]_i and store-operated Ca²⁺ entry (SOCE) in isolated rat distal PVSMCs.

Measurements and main results

In PVSMCs perfused with Ca²⁺-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca²⁺ stores in the sarcoplasmic reticulum (SR) and nifedipine to prevent Ca²⁺ entry through L-type voltage-depended Ca²⁺ channels (VDCC), hypoxia markedly enhanced both the increase in [Ca²⁺]_i caused by restoration of extracellular [Ca²⁺] and the rate at which extracellular Mn²⁺ quenched fura-2 fluorescence. Moreover, the increased [Ca²⁺]_i in PVSMCs perfused with normal salt solution was completely blocked by SOCC antagonists SKF-96365 and NiCl₂ at concentrations that SOCE >85% was inhibited but [Ca²⁺]_i responses to 60 mM KCl were not altered. On the contrary, L-type VDCC antagonist nifedipine inhibited increase in [Ca²⁺]_i to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca²⁺]_i caused by hypoxia was completely abolished by perfusion with Ca²⁺-free KRBS.

Conclusions

These results suggest that acute hypoxia enhances SOCE via activating SOCCs, leading to increased [Ca²⁺]_i in distal PVSMCs.

Introduction

In pulmonary circulation, pulmonary veins (PV) not only serve as channels through which oxygenated capillary blood flows into the left atrium, but also participates in regulation of the distention and recruitment of alveolar wall capillaries and ventilation-perfusion cooperated with pulmonary arteries (PA). Vasoconstriction of both PA and PV contributes to an increased pulmonary vascular resistance (1-5). Despite the physiological importance, little attention has been paid to the contractile response in PV. Previous studies indicate that a number of vasoconstrictor stimuli could induce PV constriction, such as hypoxia (2,5-12), endothelin (1,4,13-15), leukotrienes (16,17), thromboxane (3,18), and platelet-activating factor (1,4). However, the underlying mechanisms are largely unexplored.

In vascular smooth muscle, elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in smooth muscle cells is an essential signal for cell contraction and vasoconstriction (19). The rise in [Ca²⁺]_i could result from the followings (20,21): (I) influx of Ca²⁺ from extracellular fluid through L-type voltage-dependent Ca²⁺ channels (VDCC), receptor-operated Ca²⁺ channels (ROCC), or store-operated Ca²⁺ channels (SOCC); (II) release of Ca²⁺ from internal storage sites, such as the sarcoplasmic reticulum (SR); (III) reduced efflux of Ca²⁺ via plasmalemmal Ca²⁺-ATPases and Na⁺/ Ca²⁺ exchange. Ca²⁺ entry through SOCC, also known as store-operated Ca²⁺ entry (SOCE), is triggered by the depletion of SR Ca²⁺ stores and is essential to refill Ca²⁺ in the SR and maintain intracellular Ca²⁺ homeostasis (22-25). We recently demonstrated the presence of SOCE and expression of SOCC component proteins TRPC1, TRPC4, and TRPC6 in rat distal PA smooth muscle and PASMCs (22,26-34). SOCE was found to play a major role in hypoxia-induced increase of [Ca²⁺]_i in PASMCs, and hypoxic vasoconstriction in PA rings (30,31,35-38). Similarly, we found TRPC and SOCE are also present in PV and pulmonary venous smooth muscle cells (PVSMCs) (30); However, it is unknown if they contributes to hypoxic PV constriction or not. As the first step, this study was designed to examine the effect of hypoxia on [Ca²⁺]_i and the role of SOCE in hypoxic changes of [Ca²⁺]_i in PVSMCs.

Methods

Cell isolation and culture

Animal protocol was approved by the Animal Care and Use Committee of the Johns Hopkins University School of Medicine. The isolation and culture of PVSMCs have been described previously (30,39). Briefly, distal (>4th generation) intrapulmonary veins were dissected from lungs of pentobarbital sodium (65 mg/kg i.p.) anesthetized adult male Wistar rats (Harlan, Frederick, MD). After carefully removing adventitia and endothelium, PVSMCs were enzymatically harvested, plated on 25-mm coverslips in six-well dishes, cultured for 4-6 days until they reached 40-60% confluence in Smooth Muscle Growth Medium (Clonetics, Walkersville, MD) containing 5% serum in a humidified atmosphere of 5% CO₂-95% air at 37 °C. Cells were starved in Smooth Muscle Basal Medium (Clonetics, Walkersville, MD) with 0.3% serum for 24 h before experiments. Cellular purity was >98%, as assessed by the morphological appearance under phase-contrast microscopy and immunofluorescence staining for α-actin (39).

Measurement of [Ca²⁺]_i

As previously described (30,40,41), coverslips with PVSMCs were incubated with 5 µM fura-2 AM (Invitrogen, Carlsbad, CA), mounted in a closed polycarbonate chamber clamped in a heated aluminum platform (PH-2; Warner Instrument, Hamden, CT) on the stage of a Nikon TSE 100 Ellipse inverted microscope (Nikon, Melville, NY) and perfused at 1 mL/min with KRB solution. The perfusate was equilibrated in heated reservoirs with 5% CO₂ and either 16% O₂ (normoxia) or 4% O₂ (hypoxia). For depolarization of cells, the perfusate KCl concentration was increased to 60 mM, while NaCl was decreased to 62.7 mM. The chamber temperature was maintained at 37 °C with an in-line heat exchanger and dual-channel heater controller (models SF-28 and TC-344B; Warner Instrument).

After removal of extracellular dye by 10 min of normoxic perfusion, [Ca²⁺]_i was assessed from the ratio of fura-2 fluorescence emitted at 510 nm after excitation at 340 nm to that after excitation at 380 nm (F₃₄₀/F₃₈₀) measured in 20-30 cells using a xenon arc lamp, interference filters, electronic shutter, ×20 fluorescence objective, and a cooled charge-coupled device imaging camera. Data were collected online with InCyte software (Intracellular Imaging, Cincinnati, OH). [Ca²⁺] _i was estimated from F₃₄₀/F₃₈₀ measured in calibration solutions with [Ca²⁺] of 0-1,350 nM (Invitrogen, Carlsbad, CA).

Measurement of SOCE

SOCE was assessed in two ways, Ca²⁺ restoration and Mn²⁺ quenching, as previously described (30,40). Briefly, PVSMCs were perfused for at least 10 min with normoxic or hypoxic Ca²⁺-free KRB solution containing 5 µM nifedipine and 10 µM CPA. SOCE was evaluated from the increase in [Ca²⁺]_i caused by restoration of extracellular [Ca²⁺] in the continued presence of nifedipine and CPA. Second, we monitored the fura-2 fluorescence excited at 360 nm before and after the addition of MnCl₂ (200 µM) to the cell perfusate. SOCE was evaluated from the rate at which fura-2 fluorescence was quenched by Mn²⁺, which entered the cell as a Ca²⁺ surrogate and reduced fura-2 fluorescence upon binding to the dye.

Drugs and materials

Unless otherwise specified, all reagents were obtained from Sigma.

Statistical analysis

Data were expressed as means ± SE; n was the number of experiments, which is equal to animals providing PVSMCs. When fura-2 fluorescence was measured, the number of cells in each experiment was 20-30, as indicated in Results and the Figure legends 1-3. Statistical analyses were performed using Student’s t-test. Differences were considered significant when P<0.05.

Results

[Ca²⁺]_i responses to acute hypoxia in PVSMCs

As shown in Figure 1A and 1B, acute hypoxia (4% O₂) induced a marked increase in [Ca²⁺]_i that rose quickly to a peak of [Ca²⁺]_i =78±8 nM (n=8; P<0.0001), followed by a lower plateau before it returned to baseline by the perfusion of PVSMCs with normoxia (16% O₂). However, the L-type VDCC antagonist nifedipine administered at a 5 µM concentration blocked [Ca²⁺]_i response to acute hypoxia by only 50% (Figure 1C). Peak △[Ca²⁺]_i averaged 36±10 nM in PVSMCs treated with 4% O₂ and 5 µM nifedipine (n=5; P<0.01), compared with 72±7 nM (n=7) in PVSMCs treated with 4% O₂, but without nifedipine (Figure 1D). In contrast, 5 µM nifedipine completely blocked the response of [Ca²⁺]_i to 60 mM KCl (Figure 1E). Peak △[Ca²⁺]_i averaged 136±27 nM in control PVSMCs (n=5; P<0.01), whereas it only averaged 13±3 nM (n=5) in PVSMCs treated with 5 µM nifedipine (Figure 1F).

Figure 1.

(A) Time course of [Ca²⁺]_i before and after hypoxic perfusion (4% O₂) in rat distal PVSMCs (n=8 experiments in 185 cells) and in control cells perfused with sustained normoxia (16% O₂) (n=6 experiments in 138 cells); (B) Average peak change in [Ca²⁺]_i obtained from cells shown in (A). *P<0.0001 vs. 16% O₂; (C) Effect of 5 μM nifedipine on [Ca²⁺]_i response to 4% O₂ in rat distal PVSMCs (n=5 experiments in 128 cells); (D) Average peak change in [Ca²⁺]_i obtained from cells shown in (A). *P<0.01 vs. 4% O₂; (E) Effect of 5 μM nifedipine on [Ca²⁺]_i response to 60 mM KCl in rat distal PVSMCs (n=5 experiments in 147 cells); (F) Average peak change in [Ca²⁺]_i obtained from cells shown in (C). *P<0.001 vs. control.

SOCE in hypoxic and normoxic PVSMCs

SOCE in hypoxic and normoxic PVSMCs was assessed in two ways. First, we measured the peak increase in [Ca²⁺]_i resulting from restoration of extracellular [Ca²⁺] to 2.5 mM in PVSMCs perfused with Ca²⁺-free Krebs Ringer bicarbonate solution (KRBS) containing 10 µM CPA and 5 µM nifedipine. As shown in Figure 2A, [Ca²⁺]_i was greater in hypoxic cells than in normoxic ones, the peak △[Ca²⁺]_i caused by restoration averaged 500±22 nM (n=5; P<0.0001) in hypoxic PVSMCs, compared with 267±9 nM (n=5) in normoxic PVSMCs (Figure 2B). SOCC antagonists, i.e., SKF-96365 and Ni²⁺, have been demonstrated to block SOCE in various cell types including smooth muscle cells such as PASMCs (22,26,32,40,42) and PVSMCs (30). In addition, 50 µM SKF-96365 and 500 µM Ni²⁺ inhibited SOCE by >75% in rat distal PVSMCs during normoxia (30). Therefore, we evaluated their effects on enhancement of SOCE in acute hypoxic PVSMCs. As shown in Figure 2C,D, both 50 µM SKF-96365 and 500 µM NiCl₂ decreased Ca²⁺ entry in response to extracellular Ca²⁺ restoration, with the decrease of peak △[Ca²⁺]_i response happened from 500±22 nM (n=5) in untreated control cells to an average of 112±19 nM in cells perfused with 50 µM SKF-96365 (n=5; P<0.0001; Figure 2C,D) and 94±16 nM in cells perfused with 500 µM NiCl₂ (n=5; P<0.0001; Figure 2C,D).

Figure 2.

(A) Effect of restoration of extracellular [Ca²⁺] to 2.5 mM in distal PVSMCs perfused with Ca²⁺-free KRB solution containing 10 μM CPA and 5 μM nifedipine during normoxia (n=5 experiments in 133 cells) and hypoxia (n=5 experiments in 131 cells); (B) Maximum increase in [Ca²⁺]_i after (between 15 and 30 min, P<0.0001 vs. 16% O₂) restoration of extracellular [Ca²⁺] in cells exposed to normoxia and hypoxia; (C) Time course of effects of 50 μM SKF-96365 and 500 μM NiCl₂ on [Ca²⁺]_i change (△[Ca²⁺]_i) after the restoration of extracellular [Ca²⁺] to 2.5 mM in hypoxic PVSMCs perfused with Ca²⁺-free KRB solution containing 10 μM CPA and 5 μM nifedipine; (D) Average peak change in △[Ca²⁺]_i after (between 15 and 30 min) the restoration of extracellular [Ca²⁺] in hypoxic cells exposed to 50 μM SKF-96365 (n=5 experiments in 132 cells), 500 μM NiCl₂ (n=5 experiments in 135 cells), or control (n=5 experiments in 131 cells). * Significant difference from respective control (P<0.05).

Second, we measured the rate of Mn²⁺ quenched fura-2 fluorescence, which was thought to be a more specific index of Ca²⁺ influx. In PVSMCs perfused with Ca²⁺-free KRBS containing nifedipine but no CPA, Mn²⁺ quenching, expressed as the percentage decrease in fluorescence from time 0, after Mn²⁺ administration during normoxia. It was not different from the spontaneous decrease in fluorescence measured in normoxic cells that were not exposed to Mn²⁺ [(16±2)% vs. (14±1)%, n=5, P=0.4; Figure 3A,B]. However, acute hypoxia in the absence of CPA increased Mn²⁺ quenching approximately for 2-fold [(29±2)% vs. (16±2)%, n=5, P<0.002; Figure 3A,B]. As shown in Figure 3C,D, in normoxic PVSMCs perfused with Ca²⁺-free KRB solution containing both nifedipine and CPA, Mn²⁺ administration resulted in a (41±1)% decrease in fura-2 fluorescence, and acute hypoxia further enhanced the increased Mn²⁺ quenching by (58±2)% (n=5, P<0.0001). Consistently with the results of the peak increase in [Ca²⁺]_i, SOCC antagonists SKF-96365 and Ni²⁺ also reversed increases in Mn²⁺ quenching caused by hypoxia alone [(14±2)% vs. (29±2)% for 50 µM SKF-96365, n=5, P<0.001; (16±3)% vs. (29±2)% for 500 µM NiCl₂, n=5, P<0.01; Figure 3E,F] or hypoxia with CPA [(20±2)% vs. (58±2)% for 50 µM SKF-96365, n=5, P<0.0001; (19±2)% vs. (58±2)% for 500 µM NiCl₂, n=5, P<0.0001; Figure 3G,H].

Figure 3.

(A) and (B) Quenching of fura-2 fluorescence at 360 nm by 200 μM Mn²⁺ in distal PVSMCs perfused with Ca²⁺-free KRB solution containing nifedipine (5 μM), but no CPA. (A) Time course of fluorescence normalized to fluorescence at time 0 (F/F₀) during normoxia (n=5 experiments in 134 cells) and hypoxia (n=5 experiments in 136 cells); (B) Mn²⁺ quenching, expressed as the percentage decrease in fluorescence from time 0, after Mn²⁺ administration in normoxia there was no significant difference with the spontaneous decrease in fluorescence measured in normoxic cells not exposed to Mn²⁺ (n=5 experiments in 141 cells, P>0.4), but in acute hypoxia, this rate was increased by approximate 2-fold (P<0.002). Quenching of fura-2 fluorescence at 360 nm by 200 μM Mn²⁺ in distal PVSMCs perfused with Ca²⁺-free KRB solution containing both nifedipine (5 μM) and CPA (10 μM); (C) Time course of fluorescence normalized to fluorescence at time 0 (F/F₀) during normoxia (n=5 experiments in 132 cells) and hypoxia (n=5 experiments in 129 cells); (D) Mn²⁺ quenching, expressed as the percentage decrease in fluorescence from time 0, was greater in cells exposed to hypoxia than that in normoxia (P<0.0001); (E) Effects of 50 μM SKF-96365 and 500 μM NiCl₂ on time course of fura-2 fluorescence at 360 nm normalized to values at time 0 before and after administration of MnCl₂ (200 μM) to hypoxic PVSMCs perfused with Ca²⁺-free KRB solution containing nifedipine (5 μM), but no CPA; (F) Average change in fura-2 fluorescence at 360 nm in hypoxic PVSMCs exposed to SKF-96365 (n=5 experiments in 126 cells), NiCl₂ (n=5 experiments in 131 cells), or vehicle control (n=5 experiments in 136 cells) at 10 min after administration of MnCl₂. * Significant difference from control (P<0.01); (G0 Effects of 50 μM SKF-96365 and 500 μM NiCl₂ on time course of fura-2 fluorescence at 360 nm normalized to values at time 0 before and after administration of MnCl₂ (200 μM) to hypoxic PVSMCs perfused with Ca²⁺-free KRB solution containing both nifedipine (5 μM) and CPA (10 μM); (H) Average change in fura-2 fluorescence at 360 nm in hypoxic PVSMCs exposed to SKF-96365 (n=5 experiments in 133 cells), NiCl₂ (n=5 experiments in 132 cells), or control (n=5 experiments in 129 cells) at 10 min after administration of MnCl₂. * Significant difference from control (P<0.0001).

Discussion

The present study provided evidence indicating that acute hypoxia increased [Ca²⁺]_i and SOCE in rat distal PVSMCs. Acute hypoxia induced a rapid sustained increase in [Ca²⁺]_i in PVSMCs (Figure 1A,B). Application of L-type VDCC antagonist nifedipine (5 µM) partially prevented the effect of acute hypoxia on [Ca²⁺]_i, which indicates the participation of other pathways (Figure 1C,D). Similar results have been observed in PASMCs previously (43,44). For instance, Cornfield et al. reported that acute hypoxia increased [Ca²⁺]_i in fetal distal PASMCs and verapamil attenuated the hypoxia-induced increase in [Ca²⁺]_i (43). Salvaterra et al. also found that verapamil and nifedipine attenuated the hypoxia-induced increases in [Ca²⁺]_i in PASMCs by only 44% and 35%, respectively (44). Because nifedipine administered at a 5 µM concentration completely blocked the [Ca²⁺]_i response to 60 mM KCl (Figure 1E,F) but only partially attenuated the hypoxia-induced increases in [Ca²⁺]_i in PVSMCs, these results suggest that acute hypoxia causes influx of Ca²⁺ through L-type VDCC, as well as other signaling pathways in PVSMCs.

SOCC mediates SOCE and plays a critical role in maintaining Ca²⁺ homeostasis. In order to know whether [Ca²⁺]_i responding to hypoxia in PVSMCs requires SOCE initiated by SR Ca²⁺ release which is the same as in distal PASMCs reported in recent papers (31,45), we measured the effects of restoring extracellular Ca²⁺ in PVSMCs perfused with Ca²⁺-free KRBS. The perfusate contained CPA to deplete SR Ca²⁺ stores and activate SOCC and nifedipine to block Ca²⁺ entry through L-type VDCC. As shown in Figure 2A and 2B, acute hypoxia enhanced the increase in [Ca²⁺]_i elicited by Ca²⁺ restoration, indicating greater SOCE in hypoxic cells. This result is consistent with previous investigations in PASMCs (31).

Based on previous information, hypoxia is able to evoke the release of intracellular Ca²⁺ stores (23,45-47), which activates the SOCC via STIM1-dependent mechanism (27,48). The activated SOCC then induces the extracellular Ca²⁺ influx via SOCC as a form of SOCE (31). The increase in SOCE caused by acute hypoxia in the presence of CPA could be the result of enhanced activation of SOCC through the effects of acute hypoxia on the channels themselves (31). Further investigation is needed to clarify the underlying mechanisms.

To further confirm that acute hypoxia could cause greater SOCE, we also determined the rate of Mn²⁺ quenching of fura-2 fluorescence in PVSMCs. Mn²⁺ is a Ca²⁺ surrogate that reduces fura-2 fluorescence upon binding to the dye. Since the intensity of fluorescence excited at 360 nm is the same (isosbestic) for Ca²⁺-bound and Ca²⁺-free fura-2, changes in fluorescence induced by Mn²⁺ can be assumed to be due to Mn²⁺ alone. As shown in Figure 3A-D, a spontaneous decline was found in fura-2 fluorescence in normoxic PVSMCs not exposed to Mn²⁺. A similar phenomenon was observed in PASMCs and thought to be due to photo bleaching of the dye (31). During normoxia, Mn²⁺ quenching in PVSMCs treated with nifedipine did not differ from the spontaneous decline of fluorescence in cells not exposed to Mn²⁺ (Figure 3A-D). The lack of difference suggests that Ca²⁺ entry through pathways other than VDCC was negligible during normoxia. In contrast, the rate of Mn²⁺ quenching in the absence of CPA increased during hypoxia (Figure 3A,B), and this increase was blocked by SKF-96365 and NiCl₂ (Figure 3E,F). These results indicate that acute hypoxia alone is also able to induce SR Ca²⁺ release and thus facilitates SOCE in PVSMCs. These results are also consistent with previous reports concerning PASMCs (46-50). Meanwhile, the rate of Mn²⁺ quenching in the presence of CPA was greater in hypoxic than normoxic PVSMCs (Figure 3C,D), further indicating that acute hypoxia increased SOCE through SOCC.

SOCC antagonists SKF-96365 and NiCl₂ are commonly used as pharmacological tools to identify the presence and the role of SOCE in various smooth muscle cell preparations or vascular tissues from different species (22,36,40,51-53). SKF 96365, known as a specific inhibitor of nonselective cation channels and that without effect on the intracellular Ca²⁺ stores, is identified to be able to block PASMCs SOCC while does not affect VDCC in our previous study. However, besides its main inhibiting effects on the Ca²⁺ entry, previous publications also reported some tiny pharmacological side effects of SKF 96365, such as: (I) at higher concentrations, some inhibition of SKF 96365 on internal Ca²⁺ release was observed, and in some conditions in either intact or permeabilized cells, SKF 96365 appeared to cause some discharge of intracellular Ca²⁺ stores while the selective occurrence of such effects is only relative; (II) SKF 96365 had little effect on ATP-gated channels in arterial smooth muscle cells (52). Despite these slight pharmacological side effects, SKF 96365 was still proved as a valuable selective inhibitor of SOCC. In PVSMCs, we previously observed that SKF-96365 (50 µM) or NiCl₂ (500 µM) at the concentration that blocked >75% of SOCE did not affect VDCC, and the inhibitory effects of SKF 96365 and NiCl₂ on SOCE were not altered by hypoxia (30). Therefore, in this study, we also utilized them at the above mentioned concentrations in PVSMCs, and found 50 µM SKF-96365 or 500 µM NiCl₂ blocked hypoxia induced enhancement of both the [Ca²⁺]_i response to extracellular Ca²⁺ restoration (Figure 2C,D) and the rate of Mn²⁺ quenching (Figure 3E-H) in PVSMCs.

In summary, we observed that acute hypoxia caused an increase in [Ca²⁺]_i and enhancement of SOCE in rat distal PVSM. This increase in [Ca²⁺]_i was inhibited by SOCC inhibitors. These results suggest that hypoxia may enhance the SOCE induced by SR Ca²⁺ depletion in pulmonary vein. SOCC may be composed of TRPC and activated by STIM1. Further investigation is needed to evaluate these possibilities and to clarify the role of SOCC, SOCE and STIM1 in hypoxic pulmonary venous vasoconstriction.