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Physiologic electrical stimulation provokes intracellular calcium increase mediated by phospholipase C activation in human osteoblasts

LAYLA KHATIB^*, DAVID E. GOLAN and MICHAEL CHO^*,1

^* Department of Bioengineering, University of Illinois, Chicago, Illinois, USA; and
Departments of Biological Chemistry, Molecular Pharmacology, and Medicine, Harvard Medical School, Boston, Massachusetts, USA

¹Correspondence: Department of Bioengineering, University of Illinois at Chicago, 851 S. Morgan St., (M/C 063), Chicago, IL 60607, USA. Email: mcho{at}uic.edu

SPECIFIC AIMS

1) Electromechanically induced [Ca²⁺]_i increase in human osteoblasts: molecular mechanisms mediating cellular and tissue responses induced by non-invasive electrical stimulation (ES) remain to be elucidated, and the effort to identify the electrocoupling mechanisms is complicated by the fact that the electrical properties of cell depend on the mode, strength, and duration of ES exposure, suggesting that differential molecular mechanisms may be involved. However, one consistent cellular response is an increase in [Ca²⁺]_i that is likely to involve both Ca²⁺ influx across the plasma membrane via electrically and/or mechanically operated ion channels, and mobilization of intracellular Ca²⁺. 2) Role of extracellular Ca²⁺: the role of extracellular Ca²⁺ mediating [Ca²⁺]_i increase in response to physiological ES is determined by depleting Ca²⁺ from extracellular buffer, and the involvement of voltage-gated Ca²⁺ channels and stretch-activated cation channels mediating Ca²⁺ influx across the plasma membrane is tested. 3) Role of intracellular Ca²⁺: one potential electrocoupling mechanism may involve phospholipase (PLC)-coupled cell surface receptors. Because PLC activation is known to produce secondary messengers such as IP₃ and diacylglycerol (DAG) that binds to IP₃ receptors and alters cytoskeleton, respectively, a specific PLC antagonist is used to test release of intracellular Ca²⁺ from internal stores and inhibition of mechanically operated ion channels responsible for Ca²⁺ influx.

PRINCIPAL FINDINGS

1. Time dependence of [Ca²⁺]_i increase induced by electrical stimulation
Application of a small but physiologically relevant strength (e.g., 2 V/cm measured at the wound site) of ES was found to induce [Ca²⁺]_i increases in human osteoblasts. While control cells (i.e., cells not exposed to an ES) showed no significant changes in [Ca²⁺]_i over a 16 h time period of observation, cells exposed to a 2 V/cm ES showed no [Ca²⁺]_i increase for the first 20 min but reached the maximum 45-fold rise within the next 5 min from the [Ca²⁺]_i baseline 40 nM to 1.8 µM (Fig. 1 ). Subsequent monitoring of the fluorescence intensity showed a rapid decrease in the [Ca²⁺]_i level that may be attributed to either osmotic shrinkage or compromised cell membrane.

View larger version (11K): [in this window] [in a new window]   Figure 1. Time-dependent [Ca2+]i increase induced by ES. Human fetal osteoblasts were loaded with Fluo-3 and exposed to 2 V/cm ES for 30 min at room temperature. Control cells were treated identically, except that these cells were not exposed to ES. No changes in [Ca2+]i were recorded in control cells over a 16 h observation period (data not shown). Each point represents the mean ± SE of 4 independent experiments (n=32 cells).

2. Role of extracellular Ca²⁺
[Ca²⁺]_i increase can be mediated by Ca²⁺ influx across the plasma membrane and/or by activation of intracellular Ca²⁺ store. To test the hypothesis that [Ca²⁺]_i increase is induced by Ca²⁺ influx, cells were exposed to a 2 V/cm ES in Ca²⁺-depleted buffer. Although [Ca²⁺]_i increase was not completely inhibited by this treatment, the maximum [Ca²⁺]_i level attained was significantly reduced to 230 nM. Hence, a substantial portion of the ES-induced [Ca²⁺]_i increase is likely mediated by Ca²⁺ influx. At least two Ca²⁺ influx pathways may be involved. First, Ca²⁺ influx can be mediated by activation of voltage-gated Ca²⁺ channels (VGCCs). Using verapamil to block VGCCs did not inhibit the ES-induced [Ca²⁺]_i increase. Because application of a 2 V/cm ES is likely to change the membrane potential by no more than 10 mV, this finding is consistent with the notion that the physiological ES is unlikely to activate VGCCs directly. Second, osteoblastic cells are known to express stretch-activated cation channels (SACCs) which upon activation, act as nonselective cation channels. Blocking SACCs by Gd³⁺, a potent SACC inhibitor, did not completely inhibit the ES-induced [Ca²⁺]_i increase but again reduced the maximum [Ca²⁺]_i level to 240 nM. Based on these findings, SACCs are likely responsible for mediating [Ca²⁺]_i increases across the plasma membrane in response to physiologic ES.

3. Role of internal Ca2+ store
Release of internal Ca²⁺ in response to a 2 V/cm ES was tested. First, cells were treated with BAPTA-AM. Such treatment to chelate intracellular Ca²⁺ caused the maximum [Ca²⁺]_i level to rise to only 130 nM, indicating that in addition to Ca²⁺ influx via SACCs, the release of internal Ca²⁺ represents another major mechanism responsible for the ES-induced [Ca²⁺]_i increase. Concomitant treatment of cells with BAPTA and depletion of extracellular Ca²⁺ completely inhibited the ES-induced [Ca²⁺]_i increase. Second, release of Ca²⁺ from internal Ca²⁺ store can be initiated by activation of phospholipase C (PLC) at the cell surface. A specific PLC inhibitor (U73122, 25 µM final concentration) was used to test this hypothesis. Cells treated with U73122 before exposure to a 2 V/cm ES showed no increases in [Ca²⁺]_i (Fig. 2 ), while the treatment of cells with U73433 (a partially inactive analog of U73122) did not prevent such ES-induced [Ca²⁺]_i, indicating that PLC activation initiates the signaling cascades that lead to the release of intracellular Ca²⁺ and induce [Ca²⁺]_i increase.

View larger version (12K): [in this window] [in a new window]   Figure 2. Role of phospholipase C activation in mediating ES-induced [Ca2+]i increase. Cells were incubated with 25 µM U73122 (circles) or U73433 (squares) and exposed to 2 V/cm ES. While U73433 treatment did not affect the ES-induced [Ca2+]i increase (see control curve in Fig. 1 ), U73122 treatment completely inhibited the ES-induced [Ca2+]i increase. Points represent the mean ± SE of 2–4 independent experiments from 24 to 32 individual cells.

CONCLUSIONS AND SIGNIFICANCE

Results from the present study show that physiologic ES is capable of inducing increases in [Ca²⁺]_i. Application of 2 V/cm ES does not induce an immediate increase in [Ca²⁺]_i, however. Rather, there appears to be a latency period, the duration of which depends on ES strength, followed by a significant [Ca²⁺]_i increase from 40 nM to 1.8 µM. Depletion of extracellular Ca²⁺ or treatment of cells with Gd³⁺ partially inhibits the ES-induced [Ca²⁺]_i increase, as does treatment with the intracellular Ca²⁺ chelator BAPTA. Concomitant treatment of cells with BAPTA and depletion of extracellular Ca²⁺ completely inhibits the [Ca²⁺]_i increase. Treatment with the PLC inhibitor U73122 also completely prevents the ES-induced [Ca²⁺]_i increase. Together, these data suggest that physiologic (2 V/cm) ES activates PLC-coupled receptors at the cell surface, leading to Ca²⁺ influx across the plasma membrane via SACCs and to the release of Ca²⁺ from internal stores. In combination, these two mechanisms induce a robust increase in [Ca²⁺]_i.

While the latency period can be shortened by applying larger ES, the maximum [Ca²⁺]_i attained does not depend on ES strength. This behavior is analogous to activating a binary system characterized by a threshold level for activation. Thus, once the threshold (see below) required to activate Ca²⁺ pathways has been reached, stronger stimulation does not have an additive effect. The kinetics of reaching the threshold, however, is expected to depend on the strength of stimulation.

The mechanism of membrane protein electro-osmosis can be invoked to explain the observed threshold effect. Because most cell surface receptors are capable of lateral mobility and bear a net electrical charge, membrane proteins such as transferrin receptors, epidermal growth factor receptors, low density lipoprotein receptors, and integrins redistribute in the plane of the membrane and cluster in response to ES. In turn, receptor redistribution and clustering initiate signal transduction cascades. Our results are consistent with this mechanism. The kinetics of PLC redistribution, clustering, and activation may vary depending on the strength of ES. Once activated (i.e., once the threshold for this binary system has been reached), PLC could initiate signaling cascades (including production of the second messengers IP₃ and DAG) that cause Ca²⁺ release from internal stores and that stimulate other signaling molecules such as protein kinase C (PKC). The ability of U73122 but not U73433 to prevent the ES-induced [Ca²⁺]_i increase provides strong evidence for PLC involvement.

Because ES induces proteins to undergo structural changes that could alter functional properties of the proteins, conformational transitions induced by ES could also be a possible mechanism for PLC activation. However, ES strengths in the 10 KV/cm range have been required to produce this effect, and electroconformational changes are therefore unlikely to be involved in the present study. [Ca²⁺]_i increases can be induced by application of ES in modes other than dc (e.g., oscillatory). Application of oscillatory ES induces [Ca²⁺]_i increases in hepatocytes without PLC activation, suggesting that the electrocoupling mechanism in each particular case may depend on the mode, strength, and duration of ES and on the cell type. Selective, optimal application of ES would therefore have to be determined for each cell type. Recent results from our laboratory suggest that altered calcium spiking activity rather than a sustained [Ca²⁺]_i increase is induced in human neuroblastoma cells by 2 V/cm ES, underscoring the importance of cell type-dependent effects.

Activation of SACCs is likely to represent the main pathway for Ca²⁺ influx induced by 2 V/cm ES. The observation that Gd³⁺, the most potent SACC blocker, partially inhibits the ES-induced [Ca²⁺]_i increase suggests that ES is capable of coupling to SACCs. At least two mechanisms could be responsible for SACC activation. First, SACC activation could be mediated by ES-induced SACC redistribution and clustering (i.e., electro-osmosis). Second, because SACCs are mechanically operated, changes in cellular mechanics could cause SACC activation. We have previously demonstrated that treatment of cells with hypotonic solution (60% HBSS and 40% H₂O) induces both a morphologic change and an increase in [Ca²⁺]_i in the cells, and that the hypotonically induced [Ca²⁺]_i increase is blocked by Gd³⁺. These results suggest that SACC activation is associated with changes in cellular morphology and mechanics.

Based on our collective findings, an integrated model for electrocoupling mechanisms is proposed (Fig. 3 ). This model assumes that the critical first step required for transduction of ES into cellular responses involves PLC activation by redistributing and clustering cell surface receptors. PLC activation results in the generation not only of IP₃, which mobilizes internal Ca²⁺ stores and induces [Ca²⁺]_i increases, but also of DAG, which stimulates PKC. In turn, PKC, a known effector of cytoskeletal reorganization, leads to activation of mechanically operated ion channels. According to this model, SACC activation would be a consequence but not a cause of PLC-dependent changes in cytoskeletal organization.

View larger version (15K): [in this window] [in a new window]   Figure 3. Schematic diagram.

Finally, changes in both cell morphology and cell size are observed after the maximum [Ca²⁺]_i has been reached. These phenomena are accompanied by the loss of Fluo-3 fluorescence (dashed line in Fig. 1 ), suggesting either that Fluo-3 has exited from the cell because the integrity of the plasma membrane has been compromised or that Fluo-3 fluorescence has been self-quenched as a result of osmotic cell shrinkage. Importantly, ES-induced cell shrinkage and loss of Fluo-3 fluorescence depend critically on [Ca²⁺]_i. Treatments that completely prevent the [Ca²⁺]_i increase induced by 2 V/cm ES (i.e., chelation of intracellular Ca²⁺ and depletion of extracellular Ca²⁺or inhibition of PLC) are also effective in preventing cell shrinkage. This correlation suggests that influx of extracellular Ca²⁺ via SACCs and release of Ca²⁺ from internal stores could be mechanistically related to cell shrinkage. At least two mechanisms are possible: cell shrinkage could be induced either osmotically by activation of mitogen-activated protein kinase, or directly by opening of influx and efflux (e.g., Cl^–) pathways. We favor the latter mechanism for two reasons. First, treatment of cells with a mitogen-activated protein kinase inhibitor (SB202190, 50 µM final concentration) fails to prevent ES-induced cell shrinkage (unpublished observations). Second, incubation of cells with high (sub-mM) concentrations of Gd³⁺ not only prevents changes in cell morphology and size, and blocks ES-induced [Ca²⁺]_i increases, but also inhibits influx and efflux pathways. These observations suggest that ES-induced mechanical deformation of cells is prevented by blocking SACCs and other Gd³⁺-sensitive pathways that remain to be identified. Finally, we note that the use of electrical stimulation to activate mechanically operated channels and other signaling molecules may represent a novel model for cell activation, and that the proper description of cellular behavior in response to external electrical and mechanical stimuli may be cast more appropriately as electromechanical responses than as either electrical or mechanical responses.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1814fje;

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