Localization of calcium and microfilament changes in mechanically stressed cells

We combined fluorescence labeling, digital image processing, and micromanipulation to investigate the intracellular events induced by inflicting a mechanical stress on rat basophilic leukemia cells. Our findings were as follows:

1. Most cells displayed a localized calcium rise in response to micropipet aspiration. This represented an average threefold increase as compared to resting level, and it was observed during the first 10 s following aspiration. A slow return to initial level occurred within about 3 min. Further, this calcium rise involved a mobilization of intracellular stores, since it was not prevented by adding a calcium chelator into the extracellular medium.
2. All micropipet-aspirated cells displayed a local accumulation of microfilaments, with a preferential localization in the cell protrusions or near the pipet tips.
3. No absolute correlation was found between the localization of calcium rise and cytoskeletal accumulation.
4. Cell deformability was decreased when intracellular calcium was maintained at a constant (high or low) level with ionomycin and/or EGTA.

It is concluded that cells have a general ability to respond to mechanical stimulation by a coordinated set of events. More parameters must be studied before the mechanisms of cell shape regulation are fully understood.     

Hepoxilin-Evoked Intracellular Reorganization of Calcium in Human Neutrophils: A Confocal Microscopy Study

Hepoxilin A₃has previously been shown to cause a rapid dose-dependent rise in intracellular calcium in intact human neutrophils in suspension. Two components have been observed, an initial rapid phase of intracellular calcium rise, followed by a slow decline to plateau levels that remain above the original baseline calcium levels. These changes have been suggested to involve the release of calcium from intracellular stores in the ER (initial rapid phase), while the slower rate of decline (plateau phase) was presumed to be due to calcium influx as it was abolished in zero calcium extracellular medium. The present study used confocal microscopy to examine the response to hepoxilin A₃at the subcellular level. Our results show that calcium dynamics in response to hepoxilin A₃varies in different subcellular compartments within the cell and that hepoxilin A₃evoked a persistent accumulation of calcium in organelles. The hepoxilin-evoked calcium sequestration was eliminated by prior exposure to CCCP, a mitochondrial uncoupler. CCCP also eliminated the plateau phase of the calcium response in cell suspension, suggesting that this phase was due to mitochondrial accumulation of calcium rather than calcium influx. Experiments with DiI-loaded cells, a membrane marker, showed that the nuclear calcium was not elevated by hepoxilin addition to the cells. These results demonstrate that hepoxilins evoke the release of calcium from the ER which is taken up by the mitochondria where it is tightly sequestered. These results offer an explanation of observations previously made with cell suspensions in which hepoxilin A₃was shown to inhibit the calcium mobilizing effects of chemotactic agents.     

UV-treated lipoproteins as a model system for the study of the biological effects of lipid peroxides on cultured cells. 4. Calcium is involved in the cytotoxicity of UV-treated LDL on lymphoid cell lines

In lymphoid cells pulsed with ‘cytotoxic’ concentrations of UV-treated LDL, the study of the variations of free cytosolic calcium concentration, of the influence of extracellular calcium and of the protective effect of calcium chelators suggests that both intra- and extracellular calcium could play a major role in the genesis of cell injury leading to cell death. (1) A dramatic sustained rise of cytosolic free calcium (the level of free cytosolic calcium was higher than 500 nmol /1 for 6 h or more) occurred several hours after the beginning of the pulse with UV-treated LDL (lag period between 6 and 12 h). (2) The rise of the free cytosolic calcium and the ‘cytotoxicity’ induced by UV-treated LDL were largely dependent on the concentration of extracellular calcium which has an effect on the uptake of UV-treated LDL and on the expression of the ‘cytotoxicity’ at the cellular level. (3) The study of the sequence of intracellular events showed that the cellular oxidative stress generated by oxidized LDL was followed by the rise of free cytosolic calcium and later by the rise of ‘cytotoxicity’ indexes. (4) The intracellular calcium chelators, BAPTA/AM and EGTA/AM, were able to partially protect lymphoid cells against the ‘cytotoxicity’ of oxidized LDL. The supposed mechanisms of the free cytosolic calcium rise and the respective role of calcium or/and other factors (for instance direct lesions of the plasma membrane by the oxidative stress due to oxidized LDL) in the genesis of cellular lesions leading to cell death are discussed.     

Control of cell division: a unifying hypothesis.

A constant feature of the initiation of cell division in a number of different cells is a rise in the intracellular level of calcium. The importance of cyclic nucleotides may depend on the way they interact with calcium. Cyclic AMP is apparently not an essential regulator of cell division but through its ability to modulate the intracellular level of calcium this cyclic nucleotide can exert profound effects on cell growth. In some systems (liver and salivary glands) cyclic AMP seems to augment the calcium signal whereas in others (lymphocytes and fibroblasts) it opposes calcium and can thus inhibit cell division. A rise in the level of calcium may be responsible for the parallel increase in cyclic GMP level which is usually associated with the stimulus to divide. An appealing feature of this calcium hypothesis is that it can account for the growth characteristics revealed by fibroblasts in tissue culture or embryonic cells during development. In both cases there is an initial phase of exponential growth during which I have proposed that the high level of calcium at mitosis persists into early G1 to provide the signal for the next division. In order to account for the sudden cessation of cell division at confluency, or at a specific stage during development, it is necessary to postulate that there is something different about the final mitosis which sets it apart from earlier mitoses. It is proposed that as the cells leave the last mitosis the level of calcium falls much more rapidly than it did during preceeding mitoses perhaps as a result of a more rapid rise in the level of cyclic AMP. This rapid rise in cyclic AMP level may have a dual function. Not only will it lower the level of calcium thus preventing further division, but it may also stimulate differentiation. Many of the embryonic cells which differentiate into specialized cells (lymphocytes, liver, salivary gland) retain the ability to divide if provided with appropriate stimuli. Although the nature of these stimuli vary considerably, they all seem to act by elevating the intracellular level of calcium.     

Intracellular calcium dynamics during photolysis.

The objective of this investigation was to gain a deeper understanding of the intracellular events that precede photolysis of cells. A model system, consisting of malignant melanoma cells pretreated with the calcium sensitive fluorescent dye, Fluo-3, was used to examine the intracellular calcium dynamics in single-cell photolysis experiments. Exposure of the cells to 632 nm laser light in the presence of photosensitizer, tin chlorin e6, resulted in a rise in intracellular calcium. The increase in intracellular calcium was blocked using a variety of calcium channel blocking agents, including verapamil, nifedipine, and nickel. Treatment with the channel blockers was also effective in either decreasing or eliminating cell death despite the presence of lethal doses of photosensitizer and irradiation. These results show that intracellular calcium rises prior to plasma membrane lysis, and that this early rise in intracellular calcium is necessary for membrane rupture.     

A selective requirement for elevated calcium in DNA degradation, but not early events in anti-Fas-induced apoptosis

Jurkat cells undergo apoptosis in response to anti-Fas antibody through a caspase-dependent death cascade in which calcium signaling has been implicated. We have now evaluated the role of calcium during this death cascade at the single cell level in real time utilizing flow cytometric analysis and confocal microscopy. Fluo-3 and propidium iodide were employed to evaluate calcium fluxes and to discriminate between viable and non-viable cells, respectively. Anti-Fas treatment of Jurkat cells resulted in a sustained increase in intracellular calcium commencing between 1 and 2 h after treatment and persisting until subsequent loss of cell membrane integrity. The significance of this rise in calcium was evaluated by buffering intracellular calcium with BAPTA and/or removing calcium from the extracellular medium and monitoring the effects of these manipulations on calcium signaling and components of the apoptotic process. Complete inhibition of the anti-Fas induced rise in intracellular calcium required both chelation of [Ca(2+)](i) and removal of extracellular calcium. Interestingly, this condition did not abrogate several events in Fas-induced apoptosis including cell shrinkage, mitochondrial depolarization, annexin binding, caspase activation, and nuclear poly(A)DP-ribose polymerase cleavage. Furthermore, calcium-free conditions in the absence of anti-Fas antibody weakly induced these apoptotic components. In marked contrast, calcium depletion did not induce DNA degradation in control cells, and inhibited apoptotic DNA degradation in response to anti-Fas. These data support the concept that the rise in intracellular calcium is not a necessary component for the early signal transduction pathways in anti-Fas-induced apoptosis in Jurkat cells, but rather is necessary for the final degradation of chromatin via nuclease activation.     

Intracellular mechanics and mechanotransduction associated with chondrocyte deformation during pipette aspiration

The present study utilised pipette aspiration and simultaneous confocal microscopy to test the hypothesis that chondrocyte deformation is associated with distortion of intracellular organelles and activation of calcium signalling. Aspiration pressure was applied to isolated articular chondrocytes in increments of 2 cm of water every 60 seconds up to a maximum of 10 cm of water. At each pressure increment, confocal microscopy was used to visualise the mitochondria and nucleus labelled with JC-1 and Syto-16, respectively. To investigate intracellular calcium signalling, separate cells were labelled with Fluo 4, rapidly aspirated to 5 cm of water and then imaged for 5 minutes at a tare pressure of 0.1 cm of water. Partial cell aspiration was associated with distortion of the mitochondrial network, elongation of the nucleus and movement towards the pipette mouth. Treatment with cytochalasin D or nocodazole produced an increase in cell aspiration indicating that both the actin microfilaments and microtubules provide mechanical integrity to the cell. When the data was normalised to account for the increased cell deformation, both actin microfilaments and microtubules were shown to be necessary for strain transfer to the intracellular organelles. Mitochondria and nucleus deformation may both be involved in chondrocyte mechanotransduction as well as cellular and intracellular mechanics. In addition, pipette aspiration induced intracellular calcium signalling which may also form part of a mechanotransduction pathway. Alternatively calcium mobilisation may serve to modify actin polymerisation, thereby changing cell mechanics and membrane rigidity in order to facilitate localised cell deformation. These findings have important implications for our understanding of cell mechanics and mechanotransduction as well as interpretation and modelling of pipette aspiration data.     

Calcium Independence of Phosphoinositide Hydrolysis-Induced Increase in Cyclic AMP Accumulation in SK-N-SH Human Neuroblastoma Cells

Previous work has shown that stimulation of muscarinic receptors in various cell lines increases intracellular cyclic AMP (cAMP) levels. This unusual response has been hypothesized to be mediated by stimulation of calcium/calmodulin-sensitive adenylate cyclase, secondary to inositol trisphosphate (IP3)-mediated calcium mobilization. To test this hypothesis, we stimulated muscarinic receptors in SK-N-SH human neuroblastoma cells while blocking the IP3-mediated rise in intracellular calcium concentration using two different methods. Loading cells with the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the carbachol-mediated intracellular calcium release without abolishing the carbachol-mediated increase in cAMP level. Similarly, in cells preexposed to carbachol, the agonist-induced change in intracellular calcium level was blocked, but the cAMP response was not. Thus, both of these methods failed to block the muscarinic receptor-mediated increase in cAMP level, thereby demonstrating that this cAMP level increase is not mediated by a detectable rise in intracellular calcium concentration.     

Tissue-Tissue Interaction-Triggered Calcium Elevation Is Required for Cell Polarization during Xenopus Gastrulation

The establishment of cell polarity is crucial for embryonic cells to acquire their proper morphologies and functions, because cell alignment and intracellular events are coordinated in tissues during embryogenesis according to the cell polarity. Although much is known about the molecules involved in cell polarization, the direct trigger of the process remains largely obscure. We previously demonstrated that the tissue boundary between the chordamesoderm and lateral mesoderm of Xenopus laevis is important for chordamesodermal cell polarity. Here, we examined the intracellular calcium dynamics during boundary formation between two different tissues. In a combination culture of nodal-induced chordamesodermal explants and a heterogeneous tissue, such as ectoderm or lateral mesoderm, the chordamesodermal cells near the boundary frequently displayed intracellular calcium elevation; this frequency was significantly less when homogeneous explants were used. Inhibition of the intracellular calcium elevation blocked cell polarization in the chordamesodermal explants. We also observed frequent calcium waves near the boundary of the dorsal marginal zone (DMZ) dissected from an early gastrula-stage embryo. Optical sectioning revealed that where heterogeneous explants touched, the chordamesodermal surface formed a wedge with the narrow end tucked under the heterogeneous explant. No such configuration was seen between homogeneous explants. When physical force was exerted against a chordamesodermal explant with a glass needle at an angle similar to that created in the explant, or migrating chordamesodermal cells crawled beneath a silicone block, intracellular calcium elevation was frequent and cell polarization was induced. Finally, we demonstrated that a purinergic receptor, which is implicated in mechano-sensing, is required for such frequent calcium elevation in chordamesoderm and for cell polarization. This study raises the possibility that tissue-tissue interaction generates mechanical forces through cell-cell contact that initiates coordinated cell polarization through a transient increase in intracellular calcium.     

Local Calcium Elevation and Cell Elongation Initiate Guided Motility in Electrically Stimulated Osteoblast-Like Cells

Background

Investigation of the mechanisms of guided cell migration can contribute to our understanding of many crucial biological processes, such as development and regeneration. Endogenous and exogenous direct current electric fields (dcEF) are known to induce directional cell migration, however the initial cellular responses to electrical stimulation are poorly understood. Ion fluxes, besides regulating intracellular homeostasis, have been implicated in many biological events, including regeneration. Therefore understanding intracellular ion kinetics during EF-directed cell migration can provide useful information for development and regeneration.

Methodology/Principal Findings

We analyzed the initial events during migration of two osteogenic cell types, rat calvarial and human SaOS-2 cells, exposed to strong (10–15 V/cm) and weak (≤5 V/cm) dcEFs. Cell elongation and perpendicular orientation to the EF vector occurred in a time- and voltage-dependent manner. Calvarial osteoblasts migrated to the cathode as they formed new filopodia or lamellipodia and reorganized their cytoskeleton on the cathodal side. SaOS-2 cells showed similar responses except towards the anode. Strong dcEFs triggered a rapid increase in intracellular calcium levels, whereas a steady state level of intracellular calcium was observed in weaker fields. Interestingly, we found that dcEF-induced intracellular calcium elevation was initiated with a local rise on opposite sides in calvarial and SaOS-2 cells, which may explain their preferred directionality. In calcium-free conditions, dcEFs induced neither intracellular calcium elevation nor directed migration, indicating an important role for calcium ions. Blocking studies using cadmium chloride revealed that voltage-gated calcium channels (VGCCs) are involved in dcEF-induced intracellular calcium elevation.

Conclusion/Significance

Taken together, these data form a time scale of the morphological and physiological rearrangements underlying EF-guided migration of osteoblast-like cell types and reveal a requirement for calcium in these reactions. We show for the first time here that dcEFs trigger different patterns of intracellular calcium elevation and positional shifting in osteogenic cell types that migrate in opposite directions.     

Dynamic study of cell mechanical and structural responses to rapid changes of calcium level

Cell shape control is complex since it may involve multiple cytoskeletal components and metabolic pathways. Here we present a kinetic study of the mechanical and structural responses of cells from the monocytic THP-1 line to a rapid increase of cytosolic calcium level. Cells were exposed to ionomycin in a medium of varying calcium concentration and they were probed at regular intervals for (1) cortical rigidity as determined with micropipette aspiration, and (2) content and distribution of polymerized actin, myosin or ABP-280, as determined with flow cytometry and/or confocal microscopy. An increase of free intracellular calcium level induced: (1) a biphasic deformability change with marked stiffening within a second, and significant softening a minute later; (2) a biphasic change of actin polymerization with initial decrease (within less than a second) and rapid recovery (within a few seconds); (3) a topographical redistribution of microfilaments with an oscillatory behavior of the cortical fraction, while no substantial redistribution of myosin or ABP-280 was detected. It is suggested that a regulation of cell rigidity might be achieved without any structural change by suitable modulation of the lifetime of bridges formed between microfilaments by actin binding proteins.     

Regulation of ciliary activity in the mammalian respiratory tract

A computer-assisted transillumination, photoelectronic technique has been used to measure the beat frequency of cilia of rabbit tracheal cells grown in culture. When ciliated cells are mechanically stimulated with a microprobe the cells respond rapidly by increasing the beat frequency of their cilia. This mechanosensitive response is not limited to the stimulated cell, but is communicated in all directions to neighboring cells. To characterize the progression of this communicated response we used an automated computer-assisted imaging system to examine high-speed films of responding cells. The time it takes for the response to be transmitted between cells is slow (1-3 sec) with each cell responding after a lag-time that is proportional to the distance of the cell from the stimulated cell. We have confirmed that gap junctions are present between cells and that adjacent or non-adjacent ciliated, as well as non-ciliated, cells are electrically coupled. To correlate the mechanosensitive response with intracellular calcium fluxes we have used fura-2, a calcium-specific fluorescent dye, and digital video microscopy. Mechanical stimulation of the cultured ciliated cells, in the presence of extracellular calcium, resulted in an initial increase in intracellular calcium, which was communicated to neighboring cells. Without extracellular calcium, mechanosensitivity of cultured cells was lost and a small decrease in intracellular calcium was observed in the stimulated cell. However, neighboring cells still displayed an increase in intracellular calcium. The time course and general pattern of calcium increase in adjacent cells was similar to the responses in ciliary activity produced by mechanical stimulation. Ciliary beat frequency is also elevated by beta-adrenergic drugs independently of mechanosensitivity. These responses are important because they could provide a dual regulatory mechanism for the control of mucus transport. Adrenergic agonists could provide non-specific control by increasing ciliary activity throughout the airways while mechanosensitivity could provide local control by increasing activity in those regions of heavy mucus load.     

Intracellular Ca(2+) accumulation is strain-dependent and correlates with apoptosis in aortic valve fibroblasts

Aortic valve (AV) disease is often characterized by the formation of calcific nodules within AV leaflets that alter functional biomechanics. In vitro, formation of these nodules is associated with osteogenic differentiation and/or increased contraction and apoptosis of AV interstitial cells (AVICs), leading to growth of calcium phosphate crystal structures. In several other cell types, increased intracellular Ca(2+) has been shown to be an important part in activation of osteogenic differentiability. However, elevated intracellular Ca(2+) is known to mediate cell contraction, and has also been shown to lead to apoptosis in many cell types. Therefore, a rise in intracellular Ca(2+) may precede cellular changes that lead to calcification, and fibroblasts similar to AVICs have been shown to exhibit increases in intracellular Ca(2+) in response to mechanical strain. In this study, we hypothesized that strain induces intracellular Ca(2+) accumulation through stretch-activated calcium channels. We were also interested in assessing possible correlations between intracellular Ca(2+) increases and apoptosis in AVICs. To test our hypothesis, cultured porcine AVICs were used to assess correlates between strain, intracellular Ca(2+), and apoptosis. Ca(2+) sensitive fluorescent dyes were utilized to measure real-time intracellular Ca(2+) changes in strained AVICs. Ca(2+) changes were then correlated with AVIC apoptosis using flow cytometric Annexin V apoptosis assays. These data indicate that strain-dependent accumulation of intracellular Ca(2+) is correlated with apoptosis in AVICs. We believe that these findings indicate early mechanotransductive events that may initiate AV calcification pathways.     

Transient inositol (1,4,5) trisphosphate accumulation under vasopressin stimulation in WRK1 cells: correlation with intracellular calcium mobilization

In the rat mammary tumoral cell line (WRK1 cells), vasopressin was previously described to stimulate a phospholipase C. In this study, we have analysed the effect of vasopressin both on intracellular calcium mobilization and on the accumulation of inositol phosphates. Maximal concentration of vasopressin simultaneously induces an accumulation of Ins(1,4,5)P3 and a rise of intracellular calcium concentration. Both these two phenomena are transient and exhibit similar kinetics. A sustained accumulation of InsP2, Ins(1,3,4)P3 and InsP are observed later. Yet no stimulation of InsP4 can be objectified. These results indicate that Ins(1,4,5)P3 is the major inositol phosphate involved in intracellular calcium mobilization.     

Measurement of local strain on cell membrane at initiation point of calcium signaling response to applied mechanical stimulus in osteoblastic cells

In adaptive bone remodeling, it is believed that bone cells such as osteoblasts, osteocytes and osteoclasts can sense mechanical stimuli and modulate their remodeling activities. However, the mechanosensing mechanism by which these cells sense mechanical stimuli and transduce mechanical signals into intracellular biochemical signals is still not clearly understood. From the viewpoint of cell biomechanics, it is important to clarify the mechanical conditions under which the cellular mechanosensing mechanism is activated. The aims of this study were to evaluate a mechanical condition, that is, the local strain on the cell membrane, at the initiation point of the intracellular calcium signaling response to the applied mechanical stimulus in osteoblast-like MC3T3-E1 cells, and to investigate the effect of deformation velocity on the characteristics of the cellular response. To apply a local deformation to a single cell, a glass microneedle was directly indented to the cell and moved horizontally on the cell membrane. To observe the cellular response and the deformation of the cell membrane, intracellular calcium ions and the cell membrane were labeled using fluorescent dyes and simultaneously observed by confocal laser scanning microscopy. The strain distribution on the cell membrane attributable to the applied local deformation and the strain magnitude at the initiation point of the calcium signaling responses were analyzed using obtained fluorescence images. From two-dimensionally projected images, it was found that there is a local compressive strain at the initiation point of calcium signaling. Moreover, the cellular response revealed velocity dependence, that is, the cells seemed to respond with a higher sensitivity to a higher deformation velocity. From the viewpoint of cell biomechanics, these results provide us a fundamental understanding of the mechanosensing mechanism of osteoblast-like cells.     

Subsecond and second changes in inositol polyphosphates in GH4C1 cells induced by thyrotropin-releasing hormone.

It has been demonstrated previously that thyrotropin-releasing hormone (TRH) induces changes in inositol polyphosphates in the GH3 and GH4C1 strains of rat pituitary cells within 2.5-5.0 s. TRH also causes a rapid rise in cytosolic free calcium concentration ([Ca2+]i) in these cells which is due largely to redistribution of cellular calcium stores. Therefore, it has been concluded that TRH acts to release sequestered calcium in these cells via enhanced generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. If this conclusion were correct, TRH-enhanced accumulation of Ins(1,4,5)P3 should occur at least as rapidly as the increase in [Ca2+]i. We have shown previously that the rise in [Ca2+]i induced by TRH occurs within about 400 ms; thus, it was important to investigate the subsecond time-course of changes in inositol phosphates caused by TRH. Using a rapid mixing device, we have measured changes in inositol polyphosphates on a subsecond time scale in GH4C1 cells prelabelled with myo-[2-3H]inositol. Although TRH did alter inositol polyphosphate metabolism within 500 ms, the changes observed did not reveal a statistically significant increase in Ins(1,4,5)P3 within time intervals of less than 1000 ms. Thus, we have been unable to demonstrate that a TRH-induced rise in Ins(1,4,5)P3 precedes or occurs concomitantly with the rise in [Ca2+]i in GH4C1 cells. Although these results do not disprove the current view that Ins(1,4,5)P3 mediates the action of TRH on intracellular calcium redistribution, we conclude that caution should be exercised in this, and possibly other cell systems, in accepting the dogma that all of the rapid, agonist-induced redistributions of intracellular calcium are mediated by Ins(1,4,5)P3.     

Changes of erythrocyte deformability induced by calcium accumulation and calmodulin inhibitors

The deformability of human erythrocytes was investigated with a rheoscope to study the role of intracellular calcium in the dynamic cytoskeletal structure. Calcium was loaded to or depleted from erythrocytes with a calcium ionophore (A 23187) in a Na- or a K-HEPES buffer. (1) After calcium loading in the Na-HEPES buffer, the cell volume of erythrocytes was greatly reduced due to dehydration. On the contrary, upon calcium-loading or -depletion in the K-HEPES buffer, the intracellular calcium content could be varied in the range of 1/4 to 3 times as much as that of control cells without the reduction of mean cell volume. Further incubation without A 23187 and calcium in the K-HEPES buffer enabled the calcium-loaded erythrocytes to restore the cell shape and the ATP concentration. (2) When intracellular calcium content was increased to above 1.5 times of the normal value, the deformability was distinctly decreased. On the other hand, the deformability was unchanged when the intracellular calcium content was reduced below the normal level. (3) The deformability, once decreased due to the calcium accumulation, was recovered by the treatment with a calmodulin inhibitor, W-7 or trifluoperazine, while these drugs were not effective on the deformability of control or calcium-depleted erythrocytes. We conclude that the membrane stiffness which influence the deformability of erythrocytes, is modulated by the intracellular calcium content through the interaction between the calcium-calmodulin complex and the cytoskeletal proteins.     

Cytochalasin induces an increase in cytosolic free calcium in murine B lymphocytes

Cytochalasin promotes the progression of anti-immunoglobulin-treated B lymphocytes to S phase. However, the intracellular events induced by cytochalasin which may mediate signaling for progression have not been elucidated. In this study, the effect of cytochalasin on the level of intracellular free calcium in murine splenic B lymphocytes was assessed by using the fluorescent calcium indicator Indo-1. Cytochalasins A, B, D, and E induced a rapid and sustained elevation of intracellular free calcium. The calcium response to cytochalasin derived largely from the influx of extracellular calcium, although a small, transient elevation in intracellular calcium persisted when the suspension medium was made calcium-free with EGTA, implicating an intracellular source for a portion of the calcium response. Single cell fluorescence studies revealed that cytochalasin elicited a calcium response in most splenic B cells in suspension, indicating that this phenomenon is not restricted to a subpopulation of responding B cells. Phorbol esters inhibited the B cell calcium response to cytochalasin, and an established response to cytochalasin was rapidly and completely reversed by subsequently administered phorbol ester. T cells that lack the cytochalasin pathway showed a markedly diminished calcium response that was only apparent at higher cytochalasin concentration. However, B cells from xid-defective [CBA/N X DBA/2]F1 males, which fail to respond to anti-immunoglobulin plus cytochalasin, showed a calcium response to cytochalasin similar to that of phenotypically normal F1 females. These data, along with the finding that the rise in intracellular calcium occurred in naive B cells as well as B cells previously treated with anti-immunoglobulin, suggest that there is no clear association between the calcium response induced by cytochalasin and the ability of cytochalasin to stimulate progression to S phase. However, this effect of cytochalasin may suggest a connection between actin filaments and calcium influx in B cells.     

Effect of PDGF-AB heterodimer on a corneal epithelial cell line.

Exposure of cells of a rabbit corneal epithelial cell line (SIRC) to platelet-derived growth factor-AB heterodimer (PDGF-AB) resulted in a rapid and transient elevation of cytosolic free calcium concentration with a maximum at 2 to 3 min after stimulation. The kinetics of the calcium response were dose-dependent, e.g., higher concentrations of PDGF-AB caused a faster rise in cytosolic free calcium concentration. Maximum response was achieved with 10 ng/ml PDGF; higher concentrations up to 100 ng/ml did not further enhance cytosolic free calcium concentration. The ED50 was calculated to be 5 ng/ml PDGF-AB. After complexing extracellular calcium, PDGF-AB still caused a significant rise in cytosolic free calcium concentration indicating a mobilization of calcium from intracellular stores. This rise, however, was less pronounced than in the presence of extracellular calcium. The elevation in cytosolic free calcium concentration was not accompanied by an increased mitotic or proliferative activity of the cells as checked by [3H]thymidine incorporation and counting of cell numbers after 3 days of continuous incubation with various concentrations of PDGF-AB or by alterations in cell size and cell volume. In contrast, alterations in cell shape with a remarkable amount of rounded and partially detached SIRC cells after addition of PDGF-AB were observed within 24 h. Moreover, PDGF-AB caused a reversible distortion of cytoskeletal components such as actin-containing microfilament bundles, microtubules, and vimentin filaments. The results suggest that PDGF-AB may act only as a competence factor for the stimulation of SIRC cells via modification of the intracellular calcium homeostasis.