Depletion of Ca2+ stores in the sarcoplasmic reticulum (SR) activates extracellular Ca2+ influx via capacitative Ca2+ entry (CCE). Here, CCE levels in proliferating and growth-arrested human pulmonary artery smooth muscle cells (PASMCs) were compared by digital imaging fluorescence microscopy. Resting cytosolic free Ca2+ concentration ([Ca2+]cyt) in proliferating PASMCs was twofold higher than that in growth-arrested cells. Cyclopiazonic acid (CPA; 10 μM), which inhibits SR Ca2+-ATPase and depletes inositol 1,4,5-trisphosphate-sensitive Ca2+ stores, transiently increased [Ca2+]cytin the absence of extracellular Ca2+. The addition of 1.8 mM Ca2+ to the extracellular solution in the presence of CPA induced large increases in [Ca2+]cyt, indicative of CCE. The CPA-induced SR Ca2+ release in proliferating PASMCs was twofold higher than that in growth-arrested cells, whereas the transient rise of [Ca2+]cytdue to CCE was fivefold greater in proliferating cells. CCE was insensitive to nifedipine but was significantly inhibited by 50 mM K+, which reduces the driving force for Ca2+ influx, and by 0.5 mM Ni2+, a putative blocker of store-operated Ca2+ channels. These data show that augmented CCE is associated with proliferation of human PASMCs and may be involved in stimulating and maintaining cell growth.
- vascular smooth muscle cells
- growth factors
- sarcoplasmic reticulum
- digital imaging
- fura 2
cytosolic calcium signals control numerous cell functions, including contraction (36, 37), gene expression (11, 32), and cell proliferation (4, 22, 25). By increasing the cytosolic free Ca2+ concentration ([Ca2+]cyt), a variety of vasoactive agonists cause smooth muscle cell (SMC) contraction and/or stimulate cell growth (4, 36, 37). The source of the agonist-mediated increase in [Ca2+]cytis predominantly Ca2+ stored in the sarcoplasmic reticulum (SR). In addition, depletion of SR activates an influx of extracellular Ca2+through store-operated Ca2+channels in the plasma membrane, termed capacitative Ca2+ entry (CCE) (30, 31). Other sarcolemmal Ca2+ channels, including voltage-gated (27) and receptor-operated channels (3), also likely participate in agonist-induced Ca2+ influx.
Maintenance of a sufficient level of Ca2+ within SR ([Ca2+]SR) is critical for normal cell function. Indeed, [Ca2+]SRexerts profound control over cell growth and progression through the cell cycle; depletion of SR Ca2+stores induces growth arrest (33) and may trigger apoptosis (16). CCE is a major pathway involved in refilling SR Ca2+ stores. CCE-regulated stores communicate with the plasma membrane to modulate Ca2+ entry as a function of [Ca2+]SR. CCE also appears to be important for prolonging agonist-induced Ca2+ signals; whereas agonist-induced mobilization of Ca2+ is a transient phenomenon, CCE is a persistent and sustained process. Because Ca2+ diffuses between the cytosol and nucleus (1, 32), CCE-induced elevation of [Ca2+]cytmay not only activate Ca2+-dependent cytosolic processes, but also activate nuclear processes such as gene expression and DNA fragmentation, which, among other things, are related to cell cycle regulation and apoptosis (8, 11).
The aim of the present study was to test the idea that the proliferation of human pulmonary artery SMCs (PASMCs) is associated not only with elevated [Ca2+]cytand [Ca2+]SRbut also with enhanced CCE. To that end, levels of resting [Ca2+]cyt, evoked SR Ca2+ release, and CCE in proliferating and growth-arrested human PASMCs were compared.
Cell preparation and culture.
Human lung specimens were obtained from patients undergoing lung-heart transplantation or lobectomy for bronchogenic carcinoma under a protocol approved by the Institutional Human Subjects Investigational Study Review Committee. The patients showed no evidence of pulmonary or systemic arterial hypertension by physical examination, electrocardiogram, echocardiogram, or pathological examination of the resected lung tissue. Lung segments were removed under sterile conditions and placed in cold (4°C), sterile Hanks’ balanced salt solution (HBSS) for transport to the laboratory.
Details of the method used for isolation and culture of PASMCs have been published (41). Briefly, intrapulmonary arterial branches were incubated for 20 min in HBSS containing 2 mg/ml collagenase (Worthington Biochemical, Freehold, NJ). After the incubation, the adventitia was carefully stripped, and the endothelium was removed. PASMCs in the remaining smooth muscle were dissociated by digestion for 45 min at 37°C with 2.0 mg/ml collagenase, 0.5 mg/ml elastase, and 1.0 mg/ml BSA (Sigma, St. Louis, MO). The dissociated cells were resuspended and plated on either 25-mm coverslips for use in fluorescent microscopy experiments or on 12-mm coverslips photoetched with a lettered grid for counting (Eppendorf, Hamburg, Germany). The plated cells were maintained in smooth muscle growth medium supplemented with 5 μg/ml insulin, 0.5 ng/ml human epidermal growth factor, 2 ng/ml human fibroblast growth factor, and 5% fetal bovine serum (SMGM2; Clonetics, San Diego, CA), under a humidified atmosphere of 5% CO2-95% air at 37°C. Onday 6, the cells were divided into two groups: one was continued in culture with SMGM2, and the other was placed in smooth muscle basal medium (SMBM; Clonetics) lacking serum and growth factors (GF). Fluorescence microscopy experiments were performed 2–3 days later; three to five coverslips were used for each condition in each experiment. SMC proliferation was assessed by testing the effect of serum plus GF on cell number. On day 6, PASMCs in each of five 175 × 175-μm squares on the 12-mm coverslips were counted. Then, on day 8, the same areas were relocated and the cells were recounted; cell numbers are expressed as percentages of the numbers onday 6.
The purity of cultures was verified immunocytochemically as previously described (41). Briefly, PASMCs were fixed in ice-cold 95% ethanol and labeled with the nucleic acid stain 4′,6′-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR) to identify all cells. SMCs were then identified with a monoclonal antibody against SMC α-actin (Boehringer Mannheim, Indianapolis, IN); FITC-conjugated goat anti-mouse antibody was used as the secondary stain (Jackson ImmunoResearch Laboratories, West Grove, PA). All DAPI-stained cells in the primary cultures cross-reacted with the SMC α-actin antibody, indicating the purity of the SMC cultures.
Measurement of [Ca2+]cyt.
Details of the digital imaging methods employed for measuring [Ca2+]cythave been published (13). Briefly, PASMCs grown on coverslips were loaded with fura 2 by incubating them in culture medium containing 3.3 μM fura 2-AM (TEFLABS, Austin, TX) for 30 min at 20–22°C, under an atmosphere of 5% CO2-95% air. The cells were then superfused for 20–30 min with physiological salt solution (PSS) at a rate of 2.0 ml/min (at 35°C) to remove extracellular fura 2-AM and to permit intracellular esterases to cleave intracellular fura 2-AM into active fura 2. The imaging system is designed around an Axiovert-100 microscope (Carl Zeiss, Thornwood, NY) optimized for ultraviolet transmission. The fluorochromes were excited with a Polychrome II illumination system (Applied Scientific Instruments, Eugene, OR), and fluorescent images were recorded with a Gen III ultrablue intensified charge-coupled device (CCD) camera (Stanford Photonics, Palo Alto, CA) with a 1,134 × 486-pixel CCD format. Image acquisition and analysis were performed with a MetaFluor/MetaMorph imaging system (Universal Imaging, West Chester, PA). Video frames containing images of cell fluorescence, as well as the corresponding background images (fluorescence after removing the cells from the field) were digitized at a resolution of 512 horizontal × 480 vertical pixels and at eight-bit gray scale resolution (256 gray levels). To improve the signal-to-noise ratio, 4–8 consecutive video frames were averaged at the video frame rate (30 frames/s). [Ca2+]cytwas calculated from the ratio of fura 2 fluorescence excited at 380 and 360 nm (15). In most experiments, four to eight cells in a single field were imaged, and one arbitrarily chosen, peripheral, cytosolic area (10–12 × 10–12 pixels) from each cell was spatially averaged.
It is virtually certain that some fura 2 was sequestered within mitochondria and SR, which are typically concentrated in the perinuclear region (12, 38). Because these organelles accumulate high concentrations of Ca2+, the fura 2 signal emanating from them could potentially introduce an error when estimating [Ca2+]cyt. Therefore, the fluorescent signal used to calculate [Ca2+]cytwas measured in peripheral cytoplasmic areas where few if any intracellular organelles are present (14).
Depletion of the intracellular Ca2+ stores was induced by a reversible inhibitor of the SR Ca2+-ATPase, CPA, in the absence of extracellular Ca2+. CPA selectively blocks Ca2+ uptake into the SR store (20), thereby promoting its depletion and triggering CCE. CCE was measured as the transient rise in [Ca2+]cytafter subsequent addition of Ca2+.
Solutions and reagents.
Standard PSS contained (in mM) 140 NaCl, 5.0 KCl, 1.2 NaH2PO4, 5.0 NaHCO3, 1.4 MgCl2, 1.8 CaCl2, 11.5 glucose, and 10 HEPES (titrated to pH 7.4 with NaOH). In Ca2+-free PSS, CaCl2 was omitted and 50 μM EGTA was added. For high-K+ solutions, the NaCl was isosmotically replaced by 50 mM KCl. Serotonin (5-HT), CPA, and nifedipine were purchased from Sigma. All other reagents were analytic grade or of the highest purity available.
The numerical data presented inresults are the means ± SE fromn single cells from a total of at least four independent experiments. Student’s tests for paired data were used to calculate the significance of the differences between means.
Comparison of resting [Ca2+]cyt, CPA-evoked SR Ca2+ release, and CCE in proliferating and growth-arrested PASMCs.
To characterize CCE in proliferating and growth-arrested PASMCs and to study the relationship between CCE and cell proliferation, the cells were initially cultured in SMGM2containing serum plus GF, which induce cell growth and division (4). Some cells were then growth-arrested by placing them in SMBM without serum and GF for 2 days. As shown in Table1, maintenance of PASMCs in the absence of serum and GF induced cells to enter a quiescent state: cell number remained essentially unchanged (104.6%) compared with control (100%). Despite their arrested growth, the cells retained normal morphology, viability, and responsiveness to vasoconstrictors such as 5-HT (Fig.1 A). In contrast, when PASMCs were cultured in medium containing serum plus GF for the same period of time, cell number increased by almost threefold (288.3%).
The resting [Ca2+]cytin PASMCs cultured with serum plus GF was twofold higher than that in cells cultured without them (170 ± 5 vs. 88 ± 4 nM; Fig.1 C). Depletion of SR Ca2+ stores with 10 μM CPA evoked biphasic changes in [Ca2+]cyt(Fig. 1, A andB): there were transient increases in [Ca2+]cyt, reflecting leakage of Ca2+ from SR, followed by decreases resulting from Ca2+ buffering, sequestration by CPA-insensitive organelles, and Ca2+ extrusion (13, 14). At a concentration of 10 μM, CPA apparently evoked maximal responses in both growth-arrested and proliferating cells, because doubling the concentration had no additional effect (not shown).
Depletion of the SR Ca2+ store is known to promote CCE (31). Consistently, in the continued presence of CPA and extracellular Ca2+, a secondary phase of the Ca2+signal, mediated by Ca2+ entry from the extracellular solution, was observed (Fig. 1,A andB). In proliferating PASMCs, the secondary phase of the Ca2+response (or CCE) was significantly larger than in growth-arrested cells (1,285 ± 126 vs. 287 ± 19 nM;P < 0.001; Fig.1 C). The stored Ca2+, evaluated by measuring the CPA-induced transient increases in [Ca2+]cyt, was also significantly larger in proliferating PASMCs (760 ± 95 vs. 396 ± 42 nM; P < 0.05; Fig.1 C). On the other hand, the CPA-induced transient increases in [Ca2+]cytin the presence of extracellular Ca2+ could be attributable to both Ca2+ release and influx. Therefore, to eliminate the contribution of extracellular Ca2+, the experiments were repeated with Ca2+-free medium.
The peak amplitudes of CPA-induced Ca2+ transients in Ca2+-free medium were virtually unchanged (Figs. 1 and 2), and [Ca2+]cytrapidly approached the basal level. Subsequent addition of 1.8 mM Ca2+ to the extracellular solution in the presence of CPA evoked large increases in [Ca2+]cytdue to CCE (Fig. 2, B andCc’). The time course of CCE was biphasic: there was an initial, early peak [Ca2+]cytfollowed by a decline to a sustained plateau (Fig.2 B). The plateau phase lasted for up to 20 min (the longest period examined; not shown), and the effects of CPA were completely reversible (Fig. 2,Cd andCd’).
The initial CPA-induced transient increase in [Ca2+]cyt, which reflects Ca2+ leakage from SR, was twofold greater in proliferating PASMCs than in quiescent cells (785 ± 31 vs. 390 ± 29 nM), whereas the transient rise of [Ca2+]cytdue to CCE was fivefold greater in proliferating cells (1,813 ± 73 vs. 370 ± 28 nM) (Fig. 2 D). Thus CCE is substantially augmented in proliferating PASMCs.
Comparison of CPA-induced Mn2+ quenching of fura 2 fluorescence in proliferating and growth-arrested PASMCs.
Augmentation of CCE in proliferating cells was further confirmed by using extracellular Mn2+ as a surrogate ion. The influx of Mn2+was evaluated from the rate at which Mn2+ quenched fura 2 fluorescence excited at 360 nm (the isosbestic point) after CPA-evoked depletion of SR Ca2+ stores. Because levels of resting Mn2+ entry (leakage) in untreated proliferating and growth-arrested cells were comparable, all measurements of Mn2+ quenching were expressed after subtraction of the resting leakage. The CCE channel proved permeable to Mn2+(28); further, the rate of Mn2+entry stimulated by depletion of SR Ca2+ stores was 5.07 ± 0.12-fold greater (n = 41;P < 0.05) in proliferating PASMCs than in growth-arrested cells (Fig. 3).
Inhibition of CPA-induced Ca2+ influx in PASMCs by Ni2+ and high K+ concentration.
CCE is largely determined by the Ca2+ driving force (Ca2+ electrochemical gradient) and the conductance of the store-operated Ca2+ channels. Membrane depolarization inhibits CCE in the A7r5 vascular SMC line by reducing the driving force for Ca2+ influx (35). Therefore, to further characterize CCE, the effect of 50 mM K+ on CPA-activated Ca2+ influx in proliferating PASMCs was studied. Figure 4 shows that application of high-K+ solution during the plateau phase of the response to CPA caused [Ca2+]cytto rapidly decline almost to the resting level. When the concentration of K+ in the superfusion solution was restored to normal, [Ca2+]cytrapidly increased again. The CPA-activated Ca2+ influx was also completely and reversibly blocked by 0.5 mM Ni2+, an inhibitor of the store-activated Ca2+ channel (Fig.4). Any contribution made by Ca2+influx through L-type voltage-gated Ca2+ channels was eliminated by the continuous presence of 1 μM nifedipine. Similar results were observed with growth-arrested PASMCs (not shown).
There is growing evidence that, by promoting the refilling of intracellular Ca2+ stores and by sustaining elevated [Ca2+]cyt, CCE plays an essential role in cell signaling in both excitable and unexcitable cells (5, 17, 31). The presence of CCE in SMCs is likely associated with their partial reliance on inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular stores, depletion of which is the major trigger for activation of CCE (17). The data presented here show that CCE is substantially augmented in proliferating PASMCs.
Cell proliferation is associated with increased [Ca2+]cyt; the elevated [Ca2+]cytis apparently required for the cascade of metabolic reactions leading to mitosis to proceed (22, 25). Different phases of the cell cycle have different Ca2+ requirements, however (4), and thus the mechanisms responsible for regulating intracellular Ca2+ may also participate in regulating the cell cycle. For instance, expression of sarco(endo)plasmic reticulum Ca2+-ATPase is closely associated with cell growth and proliferation (23, 39). Close correlation between SR Ca2+ content and the onset of DNA syntheses and proliferation of DDT1MF-2 hamster SMCs was also observed (33).
The results of the present study demonstrate that the proliferation of human PASMCs is associated not only with elevated [Ca2+]cytand [Ca2+]SRbut also with enhanced CCE. As already noted, CCE in proliferating PASMCs was characterized by an augmented transient rise of [Ca2+]cytthat decayed to a sustained plateau level (Fig.2 B). The sustained elevation in [Ca2+]cytdue to CCE persisted for 20 min (the longest time examined) and was also substantially augmented in proliferating PASMCs. Because CCE is Ca2+ sensitive (5, 21), its biphasic nature may be attributable to positive and negative Ca2+ feedback pathways sequentially activated by Ca2+entering through CCE channels (5); i.e., as [Ca2+]cytrises, the rate of Ca2+ entry would initially pass through an activation phase followed by an inactivation phase (5). The decline in [Ca2+]cytafter the transient increase would thus result, at least in part, from inactivation of Ca2+ influx. This is consistent with the notion that periodic, Ca2+-induced inactivation of the Ca2+ release-activated Ca2+ influx (10) may be responsible for the oscillatory time course of CPA-induced Ca2+ entry. Ultimately, however, [Ca2+]cytis determined by the balance between Ca2+ influx and extrusion. Most likely, plasmalemmal Ca2+- and Mg2+-ATPase, Na+/Ca2+exchange, and cytosolic buffering all affect the magnitudes and time courses of CCE-induced changes in [Ca2+]cyt.
In addition to causing Ca2+influx, the depletion of SR Ca2+stores also stimulated Mn2+ entry (Fig. 3), suggesting that in addition to Ca2+, store-operated channels are permeable to certain other divalent cations. Moreover, the magnitude of that permeability in proliferating PASMCs was significantly greater than that in growth-arrested cells. In contrast to observations on [Ca2+]cyt, the apparent elevation of [Mn2+]cytwas not transient. This may be in part a consequence of the fact that Mn2+ is a poor substrate for the plasmalemmal Ca2+-ATPase (9) and the Na+/Ca2+exchanger (2).
Voltage-gated Ca2+ channels are not the pathway through which PASMCs gain Ca2+ when Ca2+ stores are depleted. The inhibition of L-type Ca2+ channels by nifedipine blocked depolarization-induced increases in [Ca2+]cytbut did not affect CCE (Fig. 4). Although a specific blocker of the store-operated Ca2+ channels is lacking (for review, see Ref. 28), CCE can be inhibited by impermeant cations (e.g., Ni2+) and by membrane depolarization (reduced Ca2+ driving force). In proliferating PASMCs, both Ni2+and 50 mM K+ significantly inhibited CCE in the presence of nifedipine (Fig. 4), which is consistent with the findings of Skutella and Ruegg (35) showing that 55 mM KCl blocked thapsigargin-induced45Ca2+influx into the A7r5 vascular SMC line (35).
The importance of Ca2+ influx in the regulation of cell proliferation stimulated by GF has been extensively studied (18, 24, 26). Many of those studies attribute increases in Ca2+ entry to noncapacitative mechanisms. For example, platelet-derived growth factor (PDGF) activates Ca2+ entry in vascular SMCs in the absence of intracellular Ca2+ mobilization (18). The blockade of IP3-dependent intracellular Ca2+ release by microinjecting heparin did not prevent PDGF-evoked Ca2+ entry, whereas the microinjection of monoclonal antibodies to phosphatidylinositol 4,5-bisphosphate (PIP2) blocked the Ca2+ entry. These data suggest that PDGF-induced Ca2+ entry requires ongoing receptor occupancy and involves the phospholipase Cγ-mediated breakdown of PIP2 (18, 24).
It has been suggested that CCE and receptor-coupled Ca2+ influx have separate roles in cell function (7). In this regard, it cannot be ruled out that CCE may act as a safety device to prevent Ca2+ store depletion, while receptor-coupled influx may control cell growth. I would not exclude the possibility that receptor-coupled Ca2+ influx is involved in maintaining the elevated [Ca2+]cytand, correspondingly, [Ca2+]SR, in proliferating cells. Nonetheless, the data presented in this study indicate that proliferation of human PASMCs is associated with augmented CCE.
At the moment, the precise nature of the Ca2+-dependent mechanisms involved in the regulation of cell growth are not yet completely understood. It has been demonstrated that the protooncogenec-myb plays a critical role in regulating cell cycle progression and Ca2+ homeostasis in vascular SMCs (19, 34). Synchronized vascular SMCs exhibit a rise in [Ca2+]cytat the G1/S interface that is immediately preceded by a twofold increase in the expression ofc-myb (34). Recent studies showed that the functional level of c-mybregulates not only the G1/S transition but also the resting [Ca2+]cytand the sizes of the thapsigargin- and caffeine-sensitive Ca2+ stores in cultured vascular SMCs (19).
The molecular identity of CCE channels and the mechanisms by which SR depletion activates them remain unclear. It has been suggested that mammalian cells homologous for theDrosophila transient receptor potential (TRP) gene might form CCE channels; overexpression of TRP genes in mammalian cells increased CCE (6). It is, therefore, logical to propose that augmented CCE in proliferating PASMCs is associated with increased TRP gene expression. Indeed, Wang and colleagues (40) showed that the expression of TRP1 mRNA in proliferating human PASMCs was significantly increased compared with that in the growth-arrested cells.
CCE appears to play a central role in a diverse array of physiological processes including cell proliferation (5, 28, 29). It has been shown that CCE is absent in the T lymphocytes of a patient suffering from a primary immunodeficiency in which T cells do not proliferate (29). The data from this study indicate that CCE activity can change depending on the developmental state of cultured PASMCs and is augmented in the proliferation state. These findings suggest that the role of CCE can be substantially increased in such pathogenic phenomena as pulmonary hypertension when vascular SMC proliferation is the key event in the development of vascular remodeling.
I gratefully acknowledge Dr. J. Wang for his assistance with the technique of cell culture, Drs. J. V. Conte, J. X.-J. Yuan, and L. J. Rubin for their help in obtaining human tissue, and Drs. J. X.-J. Yuan, M. Juhaszova, and M. P. Blaustein for the critical review of the manuscript.
Address for reprint requests and other correspondence: V. A. Golovina, Dept. of Physiology, Univ. of Maryland School of Medicine, 655 West Baltimore St., Baltimore, MD 21201 (E-mail:).
This work was supported by a grant from the American Heart Association, Mid-Atlantic Affiliate (to V. A. Golovina), and National Heart, Lung, and Blood Institute Grants HL-32276 (to M. P. Blaustein) and HL-54043 (to J. X.-J. Yuan).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
- Copyright © 1999 the American Physiological Society