Ca²⁺ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum

Abstract

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca²⁺ concentration ([Ca²⁺]_i) by inducing Ca²⁺ release from the sarcoplasmic reticulum (SR) and Ca²⁺ influx through store- and voltage-operated Ca²⁺ channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca²⁺ release, we measured [Ca²⁺]_i with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca²⁺-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP₃R), respectively, or 4% O₂ caused rapid transient increases in [Ca²⁺]_i, indicating intracellular Ca²⁺ release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca²⁺ release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca²⁺ release to caffeine and hypoxia but not norepinephrine. The IP₃R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca²⁺ release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca²⁺]_i that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca²⁺]_i induced by hypoxia, as well as the subsequent Ca²⁺ influx that sustained this increase, required release of Ca²⁺ from both RyR and IP₃R, and 2) the SR Ca²⁺ stores accessed by RyR, IP₃R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca²⁺-ATPase.

accumulating evidence indicates that hypoxic pulmonary vasoconstriction (HPV) is mediated by an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in pulmonary arterial smooth muscle cells (PASMC) caused by release of Ca²⁺ from sarcoplasmic reticulum (SR) and secondary Ca²⁺ entry through sarcolemmal store-operated Ca²⁺ channels (SOCC). For example, acute hypoxia increased store-operated Ca²⁺ entry (SOCE), as measured by Mn²⁺ quenching of fura-2 fluorescence and [Ca²⁺]_i responses to restoration of extracellular Ca²⁺ in PASMC perfused with Ca²⁺-free physiologic salt solutions (35, 48). Antagonists of SOCC, such as SKF-96365 and NiCl₂, blocked hypoxia-induced increases in SOCE and [Ca²⁺]_i in PASMC and HPV in isolated lungs but not the increases in PASMC [Ca²⁺]_i or pulmonary vasoconstriction induced by KCl (35, 48, 52). Preventing SOCE by removal of extracellular Ca²⁺ or knockdown of stromal interaction molelcule 1 (STIM1), a 90-kDa transmembrane Ca²⁺-binding protein found in SR and sarcolemma that activates SOCE upon depletion of SR Ca²⁺ (3), blocked the sustained but not the initial increase in [Ca²⁺]_i generated by hypoxia in PASMC (28, 34, 42).

In vascular smooth muscle, SR Ca²⁺ is released through channels in the SR membrane known as ryanodine and inositol 1,4,5-trisphosphate receptors (RyR, IP₃R). RyR and IP₃R each have three subtypes (RyR1–3; IP₃R1–3), all of which are expressed in PASMC (9, 54, 57, 59, 60). Pharmacologically, RyR can be activated by caffeine and inhibited by the polycyclic polyhydroxylic diterpene ryanodine, whereas IP₃R can be activated by agonists of sarcolemmal G-protein- or tyrosine kinase-linked receptors, which stimulate production of IP₃ by phospholipase C, and inhibited by xestospongin C (XeC), an alkaloid produced by the Australian marine sponge species Xestospongia (13, 20, 22).

The SR Ca²⁺ stores accessed by RyR and IP₃R may be the same or different. Store depletion by cyclopiazonic acid (CPA) or thapsigargin, which inhibit Ca²⁺-ATPases that pump Ca²⁺ into the SR, prevented contractile responses to both norepinephrine and caffeine in rat pulmonary arteries (15). In freshly isolated rat PASMC, caffeine prevented increases in [Ca²⁺]_i in response to norepinephrine and, conversely, norepinephrine prevented increases in [Ca²⁺]_i in response to caffeine (50). These results suggested that RyR and IP₃R accessed the same SR Ca²⁺ store in rat PASMC. In contrast, CPA or thapsigargin blocked contractions and increases in [Ca²⁺]_i induced by phenylephrine or angiotensin II but not caffeine in canine freshly isolated PASMC and pulmonary arteries (16, 17). Furthermore, depletion of SR Ca²⁺ stores with ryanodine and caffeine eliminated [Ca²⁺]_i responses to caffeine but not to angiotensin II (17). These results suggested that RyR and IP₃R accessed separate SR Ca²⁺ stores in canine PASMC and that the SR Ca²⁺-ATPase inhibited by CPA or thapsigargin in these cells maintained stores accessed by IP₃R but not RyR.

There is evidence that Ca²⁺ release from RyR plays an important role in HPV. Ryanodine or ryanodine plus caffeine strongly inhibited or abolished hypoxia-induced increases in [Ca²⁺]_i in rat PASMC (8, 46) and hypoxic constriction in pulmonary arteries isolated from rats (7–9, 14), pigs (27), and dogs (16). By itself, caffeine blocked the early transient increase and partially inhibited the steady-state increase in [Ca²⁺]_i caused by dithionite-induced hypoxia in rat PASMC (42). Hypoxic pulmonary arterial constriction and increases in PASMC [Ca²⁺]_i were reduced in mice deficient in the RyR1 or RyR3 isoforms of the receptor (25, 60) and enhanced in mice deficient in FK506 binding protein 12.6 (FKBP12.6), which stabilizes RyR in a closed state (58). However, other data suggest that RyR plays a minor role in HPV. Ryanodine or ryanodine plus caffeine had little or no effect on HPV in rat pulmonary arteries (19, 40). High concentrations of ryanodine (≥100 μM) caused incomplete inhibition of [Ca²⁺]_i and contractile responses to hypoxia (∼65 and 50%, respectively) in rat PASMC and pulmonary arterial strips (60) and reversed HPV by only 20–30% in nitric oxide synthase-inhibited isolated rat lungs (32).

It is even less clear whether Ca²⁺ release from IP₃R contributes to HPV. In rat pulmonary arteries, tissue IP₃ levels increased during norepinephrine-induced constriction but did not change during hypoxia-induced constriction (18). Thapsigargin or CPA, which were thought to deplete IP₃R- but not RyR-linked SR Ca²⁺ stores, potentiated HPV in canine pulmonary arteries (16) and isolated rat lungs (32) but inhibited HPV in rat pulmonary arteries (7, 14). In canine PASMC, SOCE induced by depletion of RyR- and IP₃R-linked Ca²⁺ stores was not blocked by high concentrations of ryanodine or further increased by hypoxia, and SOCE induced by hypoxia was not further increased by store depletion (35, 36), suggesting that store depletion and hypoxia might induce SOCE by a similar mechanism, such as activation of IP₃R. However, the IP₃R antagonist XeC inhibited SOCE induced by store depletion but did not alter SOCE induced by hypoxia, indicating that IP₃R did not contribute to the effects of hypoxia (36). These interpretations are complicated by the rapid and marked decrease in baseline [Ca²⁺]_i that occurred in canine PASMC upon exposure to Ca²⁺-free perfusate alone (33, 35, 36). This decrease, which did not occur in rat distal PASMC (28, 29, 47–49), indicates that a large influx of Ca²⁺ was required to maintain [Ca²⁺]_i at normal levels in canine PASMC under basal conditions. The nature of this influx and its influence on [Ca²⁺]_i responses to hypoxia and evaluation of SOCE in these cells remain unclear.

The present study was performed to clarify the role played by SR Ca²⁺ release from RyR and IP₃R in the [Ca²⁺]_i response of rat distal PASMC to acute hypoxia.

METHODS

Isolation and culture of PASMC.

Animal protocols were approved by the Johns Hopkins Animal Care and Use Committee. As previously described (47), distal (>4th generation) pulmonary arteries were dissected from lungs of male Wistar rats (300–500 g body wt) anesthetized with pentobarbital sodium (65 mg/kg ip). Endothelium was removed by rubbing with a cotton swab. PASMC were obtained by enzymatic digestion, plated on glass coverslips, and either maintained for 1–2 days in basal medium (Ham's F-12 medium with l-glutamine, Mediatech, Manassas, VA) containing 0.5% fetal calf serum, 1% streptomycin, and 1% penicillin (freshly isolated cells) or cultured for 3–4 days in Smooth Muscle Growth Medium 2 (Clonetics, Walkersville, MD) containing 5% serum, 1% streptomycin, and 1% penicillin (cultured cells). In cultured cells, serum concentration was decreased to 0.3% 24 h before an experiment to stop cell growth. Cellular purity was >98%, as assessed by morphological appearance under phase-contrast microscopy and immunofluorescence staining for α-actin (47).

Measurement of [Ca²⁺]_i.

PASMC on coverslips were loaded with 7.5 μM fura-2 (Molecular Probes, Eugene, OR) for 60 min at 37°C under an atmosphere of 5% CO₂-95% air and then mounted in a closed polycarbonate chamber clamped in a heated aluminum platform (PH-2; Warner Instruments, Hamden, CT) on the stage of a Nikon TSE 100 Ellipse inverted microscope (Melville, NY). The chamber was perfused at 1 ml/min with Krebs-Ringer bicarbonate solution (KRBS), which contained the following (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl₂, 0.57 MgSO₄, 1.18 KH₂PO₄, 25 NaHCO₃, and 10 glucose. The KRBS was equilbrated with a normoxic gas mixture (16% O₂-5% CO₂) at 38°C in heated reservoirs and led via stainless steel tubing and manifold through an in-line heat exchanger (SF-28; Warner Instruments) controlled by a dual channel heater controller (TC-344B, Warner Instruments) set to maintain chamber temperature at 37°C. To allow complete removal of extracellular dye and stabilization of chamber temperature, PASMC were perfused for 15 min before initiation of the experimental protocols described below. In each experiment, fura-2 fluorescence at 510 nm after excitation at 340 and 380 nm (F₃₄₀, F₃₈₀) was measured at 6-s intervals in 20–30 PASMC using a collimated light beam from a xenon arc lamp filtered by interference filters at 340 and 380 nm and focused onto cells visualized with a ×20 fluorescence objective (Super Fluor 20; Nikon) 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⁺ concentrations of 0–1,350 nM (Molecular Probes, Eugene, OR).

Experimental protocols.

To determine how the absence of the extracellular Ca²⁺ concentration ([Ca²⁺]_e) affected [Ca²⁺]_i responses of PASMC to hypoxia, cells were perfused with normal (n = 9) or Ca²⁺-free (n = 4) KRBS and sequentially exposed to normoxia for 5 min and then hypoxia for 10 min. Ca²⁺-free KRBS contained 0.5 mM EGTA and no CaCl₂, but otherwise its composition was the same as normal KRBS. During hypoxia, perfusates were equilibrated with 4% O₂-5% CO_2.. After hypoxia, all cells were perfused with normoxic normal KRBS.

To confirm that hypoxia caused Ca²⁺ release from RyR and IP₃R and to determine whether SR Ca²⁺ stores accessed by these receptors were the same or different, we measured the effects of preexposure to caffeine (30 mM), norepinephrine (300 μM), or CPA (10 μM) on subsequent responses to caffeine (n = 4, 5, and 5, respectively), norepinephrine (n = 4, 5, and 4, respectively), and hypoxia (n = 4, 4, and 4, respectively) in primary cultures of PASMC perfused with Ca²⁺-free KRBS. Preexposure to caffeine and norepinephrine occurred at 2–5 min of Ca²⁺-free perfusion, while preexposure to CPA occurred at 2–15 min. Subsequent exposures to caffeine, norepinephrine, and hypoxia occurred at 10–15 min. Responses to caffeine (n = 11), norepinephrine (n = 9), and hypoxia (n = 12) measured at 10–15 min in PASMC not subjected to preexposures provided control values. To assess the effect of cell culture on compartmentation of SR Ca²⁺ stores, we also determined the effects of preexposure to CPA on subsequent responses to caffeine (n = 3) and norepinephrine (n = 3) in freshly isolated PASMC. Similarly treated freshly isolated cells not subjected to preexposures served as controls (n = 3 and 3, respectively).

To determine the contribution of RyR and IP₃R to hypoxia-induced intracellular Ca²⁺ release in PASMC, we measured the effects of 5-min exposure to caffeine (30 mM), norepinephrine (300 μM), or hypoxia (4% O₂) on [Ca²⁺]_i after 2 min of perfusion with Ca²⁺-free KRBS followed by 8 min of perfusion with Ca²⁺-free KRBS containing either ryanodine (10 μM; n = 4, 3, and 5, respectively) or XeC (0.1 μM; n = 4, 4, and 4, respectively). PASMC not exposed to ryanodine or XeC served as controls (n = 11, 9, and 12, respectively). To determine whether Ca²⁺ release from RyR and/or IP₃R was required for the [Ca²⁺]_i response to hypoxia, PASMC perfused with normal KRBS containing ryanodine (10 μM; n = 3) or XeC (0.1 μM; n = 3) were sequentially exposed to normoxia for 10 min and hypoxia for 20 min. Again, cells not exposed to these antagonists served as controls (n = 6).

Statistical analysis.

Data are expressed as means ± SE, and n equals both the number of experiments and the number of animals that provided cells. Statistical analyses were performed using t-tests or ANOVA, as appropriate. When significant F-ratios were obtained with ANOVA, pair wise comparisons of means were performed using least significant differences. Comparisons with control values were made using Dunnett's two-tailed test. Differences were considered significant when P < 0.05.

Solutions and drugs.

Stock solutions of norepinephrine and caffeine were made up in water on the day of the experiment. Stock solutions of ryanodine and CPA were made up in DMSO and stored at −20°C until use. XeC was stored at −20°C and made up in DMSO on the day of the experiment. Ryanodine, XeC, caffeine, and norepinephrine were obtained from CalBiochem (La Jolla, CA). All other reagents were obtained from Sigma Chemical (St. Louis, MO).

RESULTS

Effect of Ca²⁺-free perfusion on the [Ca²⁺]_i response to hypoxia.

The time course of mean [Ca²⁺]_i in PASMC exposed to hypoxia is shown in Fig. 1A. Normoxic baseline [Ca²⁺]_i was not different in cells perfused with normal and Ca²⁺-free KRBS (140 ± 31 vs. 147 ± 77 nM; P > 0.9). In PASMC perfused with normal KRBS, exposure to hypoxia for 10 min caused a rapid increase in [Ca²⁺]_i to a maximum of 286 nM after 3.3 min of exposure. [Ca²⁺]_i then slowly declined to 231 nM at the end of exposure and fell rapidly to baseline upon restoration of normoxia. In cells perfused with Ca²⁺-free KRBS, hypoxia again rapidly increased [Ca²⁺]_i; however, the response (147 to 268 nM) peaked earlier (1.5 min of exposure) and was followed by a rapid fall to baseline (3.8 min of exposure). During the remainder of the exposure, [Ca²⁺]_i declined more slowly and then rose rapidly to baseline upon restoration of normoxia and normal [Ca²⁺]_e. The peak Δ[Ca²⁺]_i induced by hypoxia during the first 5 min of exposure was not different in PASMC perfused with normal and Ca²⁺-free KRBS (Fig. 1B; 192 ± 30 vs. 121 ± 59 nM, P > 0.1); however, the average Δ[Ca²⁺]_i during the last 5 min of hypoxia was greater in cells perfused with normal KRBS (106 ± 28 vs. −40 ± 6 nM; P < 0.007).

Fig. 1.

A: time course of mean change in intracellular Ca²⁺ concentration ([Ca²⁺]_i) during acute hypoxia (4% O₂) in rat distal pulmonary arterial smooth muscle cells (PASMC) exposed to normal (Control) or zero extracellular Ca²⁺ concentration ([Ca²⁺]_e = 0). B: mean values of peak and average [Ca²⁺]_i responses (Δ[Ca²⁺]_i) measured, respectively, during the first (5–10 min) and last (11–15 min) 5-min halves of the 10-min period of hypoxia (4% O₂) in rat distal PASMC exposed to normal (Control) or zero extracellular Ca²⁺ concentration ([Ca²⁺]_e = 0).

Compartmentation of SR Ca²⁺ stores in rat distal PASMC.

The effects of preexposure to caffeine (RyR agonist), norepinephrine (IP₃R agonist), or CPA (SR Ca²⁺-ATPase pump antagonist) on [Ca²⁺]_i responses to subsequent exposures to caffeine, norepinephrine, and hypoxia in PASMC perfused with Ca²⁺-free KRBS are shown in Fig. 2. These responses were compared with those obtained in control PASMC, which were not preexposed but were perfused with Ca²⁺-free KRBS for the same amount of time as preexposed cells. All preexposures caused transient increases in [Ca²⁺]_i (Fig. 2A) and abolished postexposure increases to caffeine (P < 0.001), norepinephrine (P < 0.001), and hypoxia (P < 0.002; Fig. 2B). Preexposure to CPA also abolished increases in [Ca²⁺]_i induced by caffeine and norepinephrine in freshly isolated rat distal PASMC (Table 1).

Fig. 2.

A: time course of mean change in [Ca²⁺]_i from baseline (Δ[Ca²⁺]_i) induced by 5-min exposures to 30 mM caffeine (Caff, first row), 300 μM norepinephrine (NE, second row), or hypoxia (4% O₂, third row) in rat distal PASMC perfused with Ca²⁺-free Krebs Ringer bicarbonate solution (KRBS) ([Ca²⁺]_e = 0) after preexposure to 30 mM caffeine, 300 μM norepinephrine, or 10 μM cyclopiazonic acid (CPA), as indicated by the top horizontal bars. Control PASMC were treated similarly but not subjected to preexposures. B: mean peak postexposure change in [Ca²⁺]_i (Δ[Ca²⁺]_i) induced by caffeine (left), norepinephrine (middle), and 4% O₂ (right) in rat distal PASMC perfused with Ca²⁺-free KRBS after no preexposure [none (Control)] or preexposure to caffeine, NE, or CPA.

Table 1.

Effects of CPA on SR Ca²⁺ release induced by caffeine and norepinephrine in freshly isolated rat distal PASMC

	Peak Δ[Ca2+]i, nM
Stimulus	Control	CPA (10 μM)	P Value
Caffeine (30 mM)	290 ± 94	−3 ± 2	<0.04
Norepinephrine (300 μM)	249 ± 14	3 ± 6	<0.001

Values are means ± SE. PASMC, pulmonary arterial smooth muscle cells; CPA, cyclopiazonic acid; SR, sarcoplasmic reticulum; Δ[Ca²⁺]_i, change in intracellular Ca²⁺ concentration.

Contribution of SR ryanodine and IP₃ receptors to hypoxia-induced Ca²⁺ release.

The effects of RyR and IP₃R antagonists on Ca²⁺ release induced by caffeine, norepinephrine, and hypoxia are shown in Fig. 3. In control cells perfused with Ca²⁺-free KRBS, baseline [Ca²⁺]_i remained stable (111 ± 7 nM during the 1st min vs. 107 ± 7 nM during the 10th min of perfusion; P > 0.2). At 10–15 min, caffeine, norepinephrine, and hypoxia caused rapid transient increases in [Ca²⁺]_i (Fig. 3A) that averaged 129 ± 19, 165 ± 23, and 125 ± 22 nM, respectively (Fig. 3B). Addition of the RyR antagonist ryanodine (10 μM) to the perfusate did not alter baseline [Ca²⁺]_i (83 ± 6 nM during min 1 vs. 81 ± 6 nM during min 10; P > 0.3) or the [Ca²⁺]_i response to norepinephrine (94 ± 14 nM, P = 0.09) but markedly reduced [Ca²⁺]_i responses to caffeine (30 ± 14 nM; P < 0.006) and hypoxia (8 ± 3 nM; P < 0.004). Perfusion with Ca²⁺-free KRBS containing the IP₃R antagonist XeC (0.1 μM) did not alter baseline [Ca²⁺]_i (84 ± 9 nM during minute 1 vs. 78 ± 8 nM during minute 10, P > 0.1) or the [Ca²⁺]_i response to caffeine (94 ± 18 nM; P = 0.28) but markedly reduced [Ca²⁺]_i responses to norepinephrine (36 ± 14 nM; P < 0.003) and hypoxia (28 ± 12 nM; P < 0.02).

Fig. 3.

A: time course of mean change in [Ca²⁺]_i from baseline (Δ[Ca²⁺]_i) caused by 30 mM caffeine (first row), 300 μM NE (second row), or hypoxia (4% O₂, third row) after treatment with ryanodine (10 μM) or xestospongin C (0.1 μM) beginning at 2 min in rat distal PASMC perfused with Ca²⁺-free KRBS ([Ca²⁺]_e = 0). Untreated cells served as controls. B: mean peak changes in [Ca²⁺]_i (Δ[Ca²⁺]_i) induced by caffeine (left), norepinephrine (middle), and 4% O₂ (right) in rat distal PASMC perfused with Ca²⁺-free KRBS after treatment with ryanodine (Ry; 10 μM) or xestospongin C (XeC; 0.1 μM). Untreated cells served as controls (Con).

Contribution of SR ryanodine and IP₃ receptors to the [Ca²⁺]_i response to hypoxia.

The effects of RyR and IP₃R antagonists on the [Ca²⁺]_i response to hypoxia in PASMC perfused with normal KRBS are shown in Fig. 4. Similar to control cells shown in Fig. 1, exposure of control PASMC to 4% O₂ for 10 min rapidly increased mean [Ca²⁺]_i from a baseline of 117 nM to a maximum of 243 nM at 3.5 min of exposure, followed by a slow decline to 212 nM at the end of exposure, and then a rapid fall to baseline upon restoration of normoxia (Fig. 4A). In PASMC treated with ryanodine (10 μM) or XeC (0.1 μM), this response was abolished except for an initial transient increase that was much smaller than the initial increase observed in control cells (Fig. 4B: 15 ± 5 and 32 ± 10 nM in cells treated with ryanodine and XeC, respectively, vs. 160 ± 33 nM in control cells; P < 0.02).

Fig. 4.

A: time course of the mean change in [Ca²⁺]_i from baseline (Δ[Ca²⁺]_i) induced by hypoxia (4% O₂) after treatment with ryanodine (10 μM) or xestospongin C (0.1 μM) beginning at 2 min in rat distal PASMC perfused with normal KRBS. Untreated cells served as controls. B: mean peak changes in [Ca²⁺]_i (Δ[Ca²⁺]_i) induced by hypoxia (4% O₂) in rat distal PASMC perfused with Ca²⁺-free KRBS after treatment with ryanodine (10 μM) or xestospongin C (0.1 μM). Untreated cells served as controls.

DISCUSSION

In rat distal PASMC, acute hypoxia caused a rapid increase in [Ca²⁺]_i that was transient when [Ca²⁺]_e was zero but sustained when [Ca²⁺]_e was normal (Fig. 1), consistent with previous observations in this preparation (48, 49) and rat main PASMC exposed to dithionite-generated hypoxia (42). These results confirm that acute hypoxia caused transient release of Ca²⁺ from intracellular stores followed by sustained influx of Ca²⁺ from extracellular fluid.

We previously found that sustained [Ca²⁺]_i responses of rat distal PASMC to acute hypoxia were completely blocked by knockdown of STIM1 (28), the protein that activates SOCC in response to decreased [Ca²⁺] in the SR lumen (3), or by SKF-96365 or NiCl₂, pharmacological antagonists of SOCC used at concentrations that did not alter [Ca²⁺]_i responses to KCl (48). In contrast, nifedipine, an antagonist of L-type voltage-operated Ca²⁺ channels (VOCC) used at concentrations that abolished [Ca²⁺]_i responses to KCl, only partially inhibited sustained [Ca²⁺]_i responses to hypoxia (48). These results suggested that acute hypoxia released Ca²⁺ from the SR, leading to activation of SOCC and SOCE, which in turn led to activation of VOCC and voltage-operated Ca²⁺ entry (VOCE). The mechanisms by which SOCE might activate VOCE are unknown, but possibilities include PASMC depolarization secondary to activation of Ca²⁺-dependent Cl⁻ channels (5, 12, 23), Ca²⁺-dependent inhibition of voltage-dependent K⁺ channels (38), and/or influx of Na⁺ (2) through SOCC, which are likely to be nonspecific cation channels with reversal potentials near 0 mV (37). The present study focuses on hypoxia-induced release of Ca²⁺ from SR, which initiates these downstream events.

To determine whether hypoxia released Ca²⁺ from SR stores accessed by RyR or IP₃R and whether these stores were the same or independent, we measured the effects of preexposure to the RyR agonist, caffeine, the IP₃R agonist norepinephrine and the SR Ca²⁺-ATPase inhibitor CPA on subsequent [Ca²⁺]_i responses to caffeine, norepinephrine, or hypoxia in rat distal PASMC perfused with Ca²⁺-free KRBS. As shown in Fig. 2, all preexposures prevented all postexposure responses. The simplest explanation for these results is that caffeine-induced activation of RyR and norepinephrine-induced activation of IP₃R caused Ca²⁺ release from, and depletion of, a common SR Ca²⁺ store, which was maintained by a CPA-inhibitable Ca²⁺-ATPase and provided the Ca²⁺ released by hypoxia. Alternatively, RyR and IP₃R could access separate stores that were in sufficient proximity to allow the activation of one receptor to cause heterologous desensitization of the other via cytoplasmic signaling pathways (31). For example, Ca²⁺ released by activation of IP₃R could stimulate Ca²⁺ release from RyR, or vice-versa, leading to depletion of the separate stores and subsequent desensitization to both caffeine and norepinephrine. In this case, anatomically separate stores would function as a common store. In theory, it is also possible that signaling pathways between separate stores in close proximity could be Ca²⁺ independent and/or cause heterologous receptor desensitization via mechanisms other than Ca²⁺ store depletion; however, the nature of such pathways and whether they exist in smooth muscle is unknown (31).

The results shown in Fig. 2 are consistent with previous observations in rat pulmonary arteries (15) and freshly isolated rat PASMC exposed to normal [Ca²⁺]_e, where caffeine prevented contractile or Ca²⁺ release responses to norepinephrine or IP₃ and norepinephrine or IP₃ prevented responses to caffeine (50, 60). They are not consistent, however, with results obtained in canine pulmonary arteries (16) and freshly isolated canine PASMC exposed to normal [Ca²⁺]_e (17), where CPA blocked contractile and [Ca²⁺]_i responses to phenylephrine or angiotensin II but not caffeine, suggesting that RyR- and IP₃R-linked stores were independent and maintained by different mechanisms. Subsequent experiments from the same laboratory in cultured canine PASMC (33) indicated that CPA blocked responses to both caffeine and 5-hydroxytryptamine, which was thought to activate IP₃R. Since CPA blocked responses to 5-hydroxytryptamine but not caffeine in freshly isolated canine PASMC (33), these results suggested that culture of canine PASMC caused reorganization of the SR into a common Ca²⁺ store accessed by both RyR and IP₃R.

To evaluate the influence of cell culture in our preparation, we determined the effects of CPA on Ca²⁺ release induced by caffeine and norepinephrine in freshly isolated rat distal PASMC. In these cells, CPA prevented Ca²⁺ release to both caffeine and norepinephrine (Table 1), as it did in primary cultures of rat distal PASMC (Fig. 2). Thus cell culture did not account for the discrepancies between the results that we (Fig. 2) and others (50, 60) obtained in rat distal PASMC and those obtained in canine PASMC (17, 33). These discrepancies could be due to differences in vessels providing the PASMC, species, or other factors; however, the effects of SOCC and VOCC antagonists on vasoconstrictor responses to hypoxia and KCl in isolated rat lungs (52) were similar to their effects on [Ca²⁺]_i responses to hypoxia and KCl in primary cultures of rat distal PASMC (48), supporting the physiological relevance of our cell preparation.

Although the results shown in Fig. 2 suggest that caffeine, norepinephrine, and hypoxia released Ca²⁺ from a common SR store, they do not indicate whether hypoxic Ca²⁺ release involved RyR, IP₃R, or some other Ca²⁺ release pathway. To evaluate these possibilities, we determined the effects of the RyR antagonist ryanodine and the IP₃R antagonist XeC on [Ca²⁺]_i responses to caffeine, norepinephrine, and hypoxia in rat distal PASMC perfused with Ca²⁺-free KRBS. As shown in Fig. 3, ryanodine inhibited Ca²⁺ release to caffeine but not norepinephrine, and XeC inhibited Ca²⁺ release to norepinephrine but not caffeine, confirming that ryanodine and XeC had their intended actions. In contrast, Ca²⁺ release to hypoxia was virtually abolished by either antagonist. These results suggest that hypoxic Ca²⁺ release required activation of both RyR and IP₃R. Furthermore, Ca²⁺ release pathways other than RyR and IP₃R were not required.

The validity of these conclusions depends on the absence of nonspecific actions of ryanodine and XeC that could prevent Ca²⁺ release to hypoxia. For example, low concentrations of ryanodine can lock RyR in an open low-conductance state (4, 22, 43) and high concentrations of XeC can inhibit SR Ca²⁺-ATPase (4, 6). Such actions might deplete a common SR Ca²⁺ store and thereby prevent Ca²⁺ release to hypoxia; however, this possibility is unlikely because, as noted above, high concentrations of ryanodine did not prevent Ca²⁺ release to norepinephrine, and low concentrations of XeC did not prevent Ca²⁺ release to caffeine (Fig. 3).

The mechanisms by which hypoxia may have activated RyR and IP₃R in PASMC remain unclear. With respect to RyR, one possibility is cyclic ADP ribose (cADPR), which causes activation by binding to the channel or FKBP12.6, an associated regulatory protein (24, 44). Hypoxia increased cADPR concentration in PASMC, possibly by increasing cADPR synthesis via activation of AMP kinase (10, 11, 41) or decreasing cADPR catabolism via inhibition of ADP-ribosyl hydrolase (7, 8, 53). Another possibility is reactive O₂ species, which increased in the cytosol of hypoxic PASMC (51) and caused dissociation of FKBP12.6 from RyR, thereby activating the channel (26). With respect to IP₃R, PASMC are able to generate their own agonists, such as endothelin (1, 30, 45, 55, 56). Although hypoxia did not increase endothelin levels in PASMC (30) as it did in endothelial cells (21), normoxic levels of this agonist might generate enough IP₃ to activate IP₃R. In this case, basal Ca²⁺ release from IP₃R could facilitate stimulated Ca²⁺ release from nearby RyR, which could then cause Ca²⁺-induced Ca²⁺ release from IP₃R, leading to continued Ca²⁺ release from both RyR and IP₃R and eventual store depletion. Consistent with this possibility, exogenous endothelin was found to cause cross-activation of RyR and IP₃R in rat PASMC, where the channels appeared to be colocalized (57). Cross-activation would also explain why hypoxic Ca²⁺ release required both RyR and IP₃R. More investigation is needed to evaluate these and other possible mechanisms of hypoxic Ca²⁺ release in PASMC.

As noted above, we previously found that knockdown of STIM1 prevented the late sustained but not the early transient [Ca²⁺]_i response to hypoxia in rat PASMC perfused with normal KRBS (28). Since STIM1 senses SR [Ca²⁺] and transduces SOCC activation (3), these results imply that the direct contribution of Ca²⁺ release to the sustained hypoxic [Ca²⁺]_i response was activation of SOCE, and not some other effect, such as Ca²⁺-dependent alteration of sarcolemmal K⁺ or Cl⁻ channel activity (39, 57). If this is correct, and both RyR and IP₃R are required for hypoxic Ca²⁺ release (Fig. 3), then blocking either RyR or IP₃R should prevent the sustained [Ca²⁺]_i response to hypoxia. We tested this possibility by measuring the effects of ryanodine and XeC on the [Ca²⁺]_i response to hypoxia in rat distal PASMC perfused with normal KRBS. As shown in Fig. 4, both antagonists abolished the sustained increase in [Ca²⁺]_i caused by hypoxia in these cells. These results provide further support for the conclusion that Ca²⁺ release from RyR and IP₃R, depletion of SR Ca²⁺ stores, and SOCE are essential components of the [Ca²⁺]_i response to acute hypoxia in PASMC.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-51912 and HL-75113 (to J. T. Sylvester), HL-67191 (to L. A. Shimoda), and HL-093020 (to J. Wang).

DISCLOSURES

No conflicts of interest, financial or otherwise are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: J.W., L.A.S., and J.T.S. conception and design of research; J.W. performed experiments; J.W., L.A.S., and J.T.S. analyzed data; J.W., L.A.S., and J.T.S. interpreted results of experiments; J.W. and J.T.S. drafted manuscript; J.W., L.A.S., and J.T.S. edited and revised manuscript; J.W., L.A.S., and J.T.S. approved final version of manuscript; J.T.S. prepared figures.