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Preferential role of intracellular Ca²⁺ stores in regulation of isometric force in NIH 3T3 fibroblast fibres

Koji Nobe, Hiromi Nobe, Kazuo Obara and Richard J. Paul

MS 1078 Received 8 May 2000; accepted after revision 24 July 2000.

	ABSTRACT

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1. Fibroblast contraction plays a major role in wound repair, but the regulatory mechanisms are not well known. We investigated the relations between isometric force and intracellular calcium concentration ([Ca²⁺]_i) in fibroblast fibres. These fibres were made with mouse NIH 3T3 fibroblasts cultured with native collagen in a three-dimensional matrix.

2. Calf serum (CS; 30 %) elicited a monotonic increase in force that attained a maximum within 15 min and could be sustained indefinitely. In contrast, [Ca²⁺]_i increased to a peak at 3 min after CS stimulation, then returned to baseline levels by 10 min. Pretreatment with Ca²⁺-free medium or the Ca²⁺-channel antagonist nicardipine (10 µM) blocked the CS-induced [Ca²⁺]_i increase, but force was not affected.

3. KCl (50 mM) stimulation on the other hand, elicited a prolonged increase in [Ca²⁺]_i but did not increase force.

4. Inhibition of the endoplasmic reticulum Ca²⁺ release with Ca²⁺-ATPase inhibitors cyclopiazonic acid (5 µM) or thapsigargin (5 µM) nearly abolished (< 20 % control) the increase in [Ca²⁺]_i and force response to CS. Treatment with ryanodine (10 µM) and caffeine (20 mM) had a similar effect. The phospholipase C inhibitor U73122 (3 µM) reduced the CS-induced increases in [Ca²⁺]_i and force by 70 and 40 %, respectively.

5. We conclude that fibroblast isometric force is not coupled to Ca²⁺ arising from transmembrane influx but is correlated with the transient [Ca²⁺]_i increase due to release from intracellular stores. Store-released Ca²⁺ may initiate activation pathways for fibroblast force development, but is not required for force maintenance.

	INTRODUCTION

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Wound repair involving fibroblast contraction is one of many important physiological processes dependent on non-muscle contractility. Our understanding of the regulation of contractility in non-muscle cells has evolved with that of muscle itself. Currently by analogy to the better characterized mechanism for activation of smooth muscle, a widely-held view postulates that actin-myosin interaction is initiated by Ca²⁺-calmodulin activation of myosin light chain kinase leading to phosphorylation of the 20 kDa regulatory light chain of myosin. For smooth muscle, the initial phase of force development has been attributed to Ca²⁺ release from intracellular stores, whereas maintenance of force is dependent on extracellular Ca²⁺ (Rasmussen et al. 1987; Karaki et al. 1997). Recent reports for smooth muscle suggest that the relationship between the source of Ca²⁺ and contraction may be even more complex. Some Ca²⁺ sources were capable of eliciting an increase in [Ca²⁺]_i as indicated by fura-2, but were not coupled to force production (Abe et al. 1996; Tosun et al. 1998). Currently, little is known about the source(s) of Ca²⁺ coupled to force production in non-muscle cells. This is partly due to the difficulty in precisely quantifying force production in non-muscle cells. The wrinkling of silicon substrata (Harris et al. 1980) or shrinkage of collagen gels (Bellows et al. 1982; Farsi & Aubin, 1984; Mochitate et al. 1991) by cultured fibroblasts have been used to measure contractility in non-muscle cells. These methods are at best semi-quantitative. Moreover they are difficult to interpret because the accompanying changes of shape may reflect changes in cell shape or morphology. As cell shape reflects a balance between cytoplasmic contraction and resisting forces from cell adhesion and cytoplasmic stiffness (Chicurel et al. 1998), these measures do not necessarily reflect contraction, or as used here, activation of actin- myosin interaction.

Recently a model system was developed whereby cells cultured in a three-dimensional collagen matrix could be directly attached to a force transducer (Kolodney & Wysolmerski, 1992; Obara et al. 1995). With this fibroblast-collagen fibre, quantitative mechanical studies including not only force, but also stiffness and velocity measurements can be made (Obara et al. 2000). We used this system to study the relationship between force and [Ca²⁺]_i in NIH 3T3 fibroblasts. Our results indicate that Ca²⁺ from intracellular sources is strongly coupled to force production, whereas [Ca²⁺]_i associated with influx is surprisingly ineffective.

	METHODS

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Cell culture and fibroblast fibre preparation

NIH 3T3 fibroblasts (mouse clonal cell line) were subcultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % calf serum, 100 u ml^-1 penicillin and 100 µg ml^-1 streptomycin. The cells were grown on 100 mm dishes in 5 % CO₂ and 95 % air with incubation at 37°C. The cells were propagated using 0·04 % trypsin, and 0·02 % EDTA in phosphate-buffered saline, at pH 7·2 in a split ratio 1:3.

Fibroblast fibres were prepared according to Obara et al. (1995). Rat tail collagen solution was neutralized with 0·1 M NaOH in an ice bath. Dispersed cells were suspended in a solution which contained 2 × 10⁶ cells ml^-1 and 0·5 mg ml^-1 collagen in DMEM. A cell suspension of 2 ml was poured into a specially designed mould with three wells (0·8 cm × 5 cm × 0·5 cm deep) which were cut into a layer of silicone rubber in a 100 mm dish and placed in a CO₂ incubator at 37°C. After 2 h, an additional 1-1·5 ml DMEM was added to each well. The fibre preparations were incubated for 2-4 days.

Measurement of isometric force development

NIH 3T3 fibroblast fibres prepared as described above were cut into 5 mm pieces. One piece was used for measurements of isometric force and another piece was used for parallel [Ca²⁺]_i measurements (see below). For mechanical studies one piece was mounted between glass posts with a cyanoacrylate glue. One post was fixed and the other connected to a silicon strain gauge force transducer (SensoNor, AME801, Horten, Norway). The fibres were bathed in a Mops-buffered physiological salt solution (Mops-PSS), which contained (mM): NaCl, 140; KCl, 4·7; NaH₂PO₄, 1·2; EDTA, 0·02; MgSO₄, 1·2; CaCl₂, 2·5; glucose, 11·1 and Mops, 20, pH 7·4 at 37°C. To help ensure similar loading conditions, fibres were returned to their original length observed before cutting them from the mould.

Measurement of intracellular calcium concentration

Measurements of [Ca²⁺]_i levels were obtained from cells in reconstituted fibres loaded with the calcium-sensitive fluorescent dye fura-2 based on the techniques of Grynkiewicz et al. (1985). Fura-2 AM was prepared as a stock solution of 1 mM dye in DMSO. Our fura-2 loading solution contained: 3 µM fura-2 AM, 0·015 % Pluronic F₁₂₇ and 0·5 % DMSO in Mops-PSS buffer, and was sonicated for at least 5 min to facilitate dispersion of the fura-2 AM. A segment (10 mm) of fibroblast fibres was incubated in this solution at room temperature for 3 h. Fibroblasts were rinsed in Mops-PSS buffer for 20 min to remove extracellular and non-hydrolysed fura-2 AM. In control experiments, fibres incubated for up to 6 h under unloaded conditions or for 3 h in the fura-2 loading solution produced forces in the normal range (n = 3).

A segment (2 mm) of the fura-2-loaded fibroblast fibre was placed in a glass-bottom culture dish and covered with nylon mesh, which kept the fibre isometric; fibre length on the mesh was set to its original length before cutting from the mould. The fibre was placed in a chamber with a total volume 500 µl and perfused (5 ml min^-1) with Mops-PSS maintained at 37°C. [Ca²⁺]_i was measured with an Intracellular Imaging (INCA, Inc., Cincinnati, OH, USA) microscope-based system. The chamber with the fibre was placed in a Nikon Diaphot inverted microscope with fluorphase objectives, permitting illumination at 340 nm. Fluorescence images of cells excited at 340 and 380 nm and emitting at 510 nm were obtained with a Dage silicon-intensified target camera. After subtraction of background fluorescence, the 340 and 380 nm images were ratioed on a pixel by pixel basis at a frequency of 1 Hz. The ratios (R_340/380) were converted to [Ca²⁺]_i using a previously generated standard curve as described below. Quantitative analysis of the mean intracellular calcium was done by defining the outline of the cell, summing the calcium in all the pixels within the defined area, and dividing by the number of pixels.

Standard curve for Ca²⁺ calibration

Solutions containing known concentrations of free Ca²⁺ for standard curves were obtained from Molecular Probes, Inc. (Eugene, OR, USA). Fluorescence intensity was measured in 150 µl of each standard solution (0, 0·065, 0·100, 0·225, 0·351, 0·602 µM free calcium concentration) containing 13·3 µg ml^-1 fura-2 pentapotassium salt. The R_340/380 values obtained were used with the INCA system software for Ca²⁺ calibration of experimental data.

Materials

NIH 3T3 fibroblast cells were purchased from American Type Culture Collection (Manassas, VA, USA). Dulbecco's modified Eagle's medium (DMEM) and calf serum (CS) were purchased from Life Technologies (Grand Island, NY, USA). Rat tail collagen type-1 was purchased from Upstate Biotechnology (Lake Placid, NY, USA). 1-(2-(5'-Carboxyoxazol-2'-yl)-6-amino-benzofuran-5-oxy)-2-(2'-amino-5'-methyl-phenoxy)-ethane-N,N,N',N'-tetra-acetic acid (fura-2) and fura-2 penta-acetoxymethyl ester (fura-2 AM) were obtained from Molecular Probes (Eugene, OR, USA). Caffeine, cyclopiazonic acid (CPA), nicardipine, penicillin and streptomycin mixture, thapsigargin (TG), ryanodine, and {1-[6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl) amino) hexyl]-1H-pyrrole-2,5-dione} (U73122) were purchased from Sigma (St Louis, MO, USA). All other chemicals and materials were of reagent grade.

Data analysis

Data are presented as means ± S.E.M. Groups of data were compared using a one-way analysis of variance (ANOVA); the Bonferroni method was used to determine the level of significance of differences between groups. A P value of < 0·05 was taken as indicative of statistical significance.

	RESULTS

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[Ca²⁺]_i measurements in NIH 3T3 fibroblast fibres

Figure 1 shows the fura-2 Ca²⁺ images of NIH 3T3 fibroblasts in the collagen fibre. Under unstimulated conditions (Fig. 1A), fibroblasts were not uniformly distributed as seen in a monolayer culture but found clustered in groups of cells. These groups appeared to contain several hundred cells arranged in parallel with the long axis of whole collagen fibres. These groups of cells occupied approximately 70 % of the fibre.

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Figure 1. CS-induced increase in [Ca2+]i in NIH 3T3 fibroblast fibres A, typical R340/380 images of fura-2-loaded fibroblasts under resting conditions and stimulated with 30 % CS. B, time courses of changes in [Ca2+]i images in the selected area (yellow square in A). C, average values for [Ca2+]i in 4 cells as indicated.

Exposure to 30 % CS, which induces a maximum contraction (Obara et al. 1995, 2000), decreased the fluorescence intensity of the F₃₈₀ image and increased that of the F₃₄₀ image (data not shown). Thus the increase in R_340/380 is expected to reflect an increase in [Ca²⁺]_i. The CS-induced increase in [Ca²⁺]_i was detected in over 75 % of cells in the field. Images showing the time course of the changes in [Ca²⁺]_i in pseudocolour are shown in Fig. 1B and the corresponding calibrated values of [Ca²⁺]_i in each cell in Fig. 1C. Resting levels of [Ca²⁺]_i were 55·5 ± 5·0 nM (n = 8). Significant increases in [Ca²⁺]_i were typically detected in less than 45 s after treatment with 30 % CS. Maximal levels of [Ca²⁺]_i were detected 60-90 s after the stimulation, averaging 150·8 ± 2·3 nM (n = 8).

After attaining its peak level, [Ca²⁺]_i gradually decreased to pre-stimulus levels, despite the continued presence of CS. Ninety per cent of the cells returned to resting levels within 5 min after CS stimulation. The effects of CS treatment on [Ca²⁺]_i were repeatable. After a 5 min rinse with PSS, similar transient increases in [Ca²⁺]_i could be induced by CS. The magnitude of the peak increase in [Ca²⁺]_i was a function of CS concentration. The EC₅₀ value was approximately 10 % CS and 97·7 ± 0·9 % (n = 4) was obtained at 30 % CS (data not shown).

Figure 2 shows the comparisons between the CS-induced increases in [Ca²⁺]_i and isometric force measured in paired samples from the same collagen-reconstituted fibre. To insure valid comparison of force and [Ca²⁺]_i, controls for the fura-2 loading conditions were performed. No effects on isometric force generation of either incubation under mechanically unloaded conditions (up to 6 h), or in the presence of fura-2 AM (3 h) were observed (data not shown, n = 3).

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Figure 2. CS- and KCl -induced increase in [Ca2+]i and isometric force in NIH 3T3 fibroblast fibres A, isometric force measured in NIH 3T3 fibroblast fibres; 30 % CS (left panel) and 50 mM KCl (right panel). B, [Ca2+]i measured in fura-2-loaded NIH 3T3 fibroblast fibres. Typical changes in 4 cells during the reagent treatments are shown. C, average [Ca2+]i for all cells in field (n = 7). D, maximal effects of 30 % CS and 50 mM KCl on isometric force (dark bars) and [Ca2+]i (light bars). Each value represents the mean ± S.E.M. for at least 7 independent determinations. *Significantly different from the respective resting level at P < 0·05.

Fibre length was set to the original length in the mould, which yielded a resting tension of approximately 50 µN. Transferring the fibre from PSS to a bath containing 30 % CS induced a significant increase in isometric force, as previously reported (Obara et al. 1995, 2000). An increase in force could be detected almost immediately upon transfer of the fibre to a CS-containing bath. The transfer is demarcated by artifacts due to surface tension effects (Fig. 2). A maximal force of 105·5 ± 16·7 µN (n = 8) was observed within 15 min. After the peak response was achieved, force was maintained at over 80 % of this value for at least 30 min. The responses of both force and [Ca²⁺]_i to CS were repeatable after relaxing the fibre with PSS. Near maximal values for the increase in [Ca²⁺]_i was observed at 3 min post stimulation in each cell. At this time, isometric force had increased to approximately 40 % of its maximal response. By 5 min, [Ca²⁺]_i decreased to resting levels while isometric force continued to increase. Fifteen minutes after CS stimulation, isometric force was near maximal but over 95 % of cells showed resting [Ca²⁺]_i levels. Thus the time courses of isometric force and [Ca²⁺]_i post CS treatment were sharply different.

To investigate the effects of [Ca²⁺]_i on force further, KCl (50 mM) was added to depolarize the fibroblasts. This initiates Ca²⁺ influx via voltage-gated Ca²⁺ channels. Upon addition of KCl to fura-2-loaded fibroblast fibres, [Ca²⁺]_i rapidly increased. As shown in Fig. 2, the increase in [Ca²⁺]_i induced by KCl was maintained for 2-3 min then decreased. [Ca²⁺]_i attained a peak value of 179·2 ± 8·9 nM (n = 8), which was greater than that induced by 30 % CS. Surprisingly, however, KCl had no effect on force. Similar results were obtained with the Ca²⁺ ionophore, ionomycin (10 µM, data not shown). The difference in responses between CS and KCl stimulation can be clearly seen when the increases in [Ca²⁺]_i and isometric force elicited by KCl are expressed in terms of those induced by 30 % CS. In these terms, the KCl increase in [Ca²⁺]_i was 132·3 ± 11·4 % while that of force was only -1·9 ± 6·8 % (n = 8).

Effects of Ca²⁺ influx on CS-induced changes in [Ca²⁺]_i and isometric force in NIH 3T3 fibroblast fibres

We studied the effects of Ca²⁺ influx on CS-induced increases in [Ca²⁺]_i and force. Preincubation of a fibre with Ca²⁺-free PSS (0 Ca²⁺ + 5 mM EGTA) for 10 min did not affect the resting level of isometric force, but [Ca²⁺]_i was slightly decreased (Fig. 3). Ca²⁺-free PSS also blunted the Ca²⁺ transient to CS. [Ca²⁺]_i increased to a maximum averaging 79·8 ± 3·9 nM (n = 9) and this raised [Ca²⁺]_i back to the resting level in normal PSS for these fibres. However, in Ca²⁺-free PSS, CS induced a sustained increase in isometric force. The maximal value and rate of the [Ca²⁺]_i increase were not different from those in normal PSS. Similar results were obtained when extracellular Ca²⁺ was reduced by adding 10 mM EGTA to the normal PSS; these data are summarized in Fig. 5. We also used a calcium channel blocker, nicardipine to reduce Ca²⁺ influx without altering the extracellular [Ca²⁺] (Fig. 4). A 10 min incubation period with nicardipine (10 µM) did not affect either the resting [Ca²⁺]_i or force. Importantly, while the CS-induced force development was not significantly affected, the increase in [Ca²⁺]_i in response to CS was strongly inhibited by nicardipine (> 60 %, Fig. 5). When the increase in [Ca²⁺]_i and isometric force induced by 30 % CS are taken as 100 %, pretreatment with 5 mM EGTA in Ca²⁺-free PSS, 10 mM EGTA or 10 µM nicardipine significantly decreased [Ca²⁺]_i to 16·8 ± 4·0, 4·3 ± 0·5 and 36·1 ± 9·0 %, of the control CS response, respectively. In contrast, the developed force remained at control levels, 91·6 ± 10·1, 87·2 ± 8·9, and 96·2 ± 11·8 %, respectively.

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Figure 3. Effects of Ca2+-free PSS treatment on CS-induced isometric force developments and [Ca2+]i increases A, isometric force was measured during a control CS (30 %)-induced contraction/relaxation cycle in NIH 3T3 fibroblast fibres. Then the sample was treated for 10 min with 5 mM EGTA in Ca2+-free PSS, followed by addition of 30 % CS (in Ca2+-free PSS). B, parallel measurements of [Ca2+]i in fura-2-loaded NIH 3T3 fibroblast fibres; typical changes in 4 cells. C, mean [Ca2+]i for all cells (n = 10) in field.

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Figure 4. Effects of nicardipine on CS-induced increases in isometric force and [Ca2+]i A, isometric force was measured during a control CS (30 %)-induced contraction/relaxation cycle in NIH 3T3 fibroblast fibres. After relaxation in PSS, 10 µM nicardipine was added 10 min prior to the CS test stimulation. B, parallel measurements of [Ca2+]i in fura-2-loaded NIH 3T3 fibroblast fibres; typical changes in 4 cells. C, average [Ca2+]i for all cells in field (n = 7).

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Figure 5. The effects of inhibition of Ca2+ influx on CS-induced increases in isometric force and [Ca2+]i Mean data from experiments shown in Figs 3 and 4 and data from similar experiments with 10 mM EGTA pretreatment (37 °C, 5 min) are presented as a percentage of the CS-induced maximal response. A, isometric force: stacked-bar graph giving the mean steady-state isometric force. Light bars represent the force after a 10 min pretreatment with the agents listed on the abscissa. Dark bars represent the magnitude of the increase in force after subsequent addition of CS. Final height of the stacked bar represents the total level of force attained above the initial unstimulated baseline. B, intracellular Ca2+: light bars show the steady-state [Ca2+]i after 10 min pretreatment with the agents listed on the abscissa. Dark bars represent the maximum value of the transient increase in [Ca2+]i after subsequent addition of calf-serum (CS). Each value represents the mean ± S.E.M. of at least 7 independent determinations. *Significantly different from each CS response at P < 0·05.

Effects of Ca²⁺ release from intracellular stores on CS-induced changes in [Ca²⁺]_i and isometric force in NIH 3T3 fibroblast fibres

Pretreatment of fura-2-loaded fibres with the endoplasmic reticulum Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA, 5 µM) caused a transient increase in [Ca²⁺]_i (Fig. 6). This increase was detected just after CPA addition and the peak level was observed 90-120 s after treatment. [Ca²⁺]_i returned to resting levels within 5 min. The peak value was 141·8 ± 1·7 nM or 86·0 ± 5·2 % of the CS control (n = 4). CPA treatment elicited a moderate increase in isometric force (25·0 ± 2·0 µN or 32·8 ± 3·0 % of CS response; n = 4) which was maintained, despite the return of [Ca²⁺]_i to baseline levels. After a 10 min incubation period with CPA, 30 % CS was added to the fibre. CS had no significant effect on [Ca²⁺]_i and elicited only a small increase in isometric force. We also utilized an alternative inhibitor of the SR-calcium pump, thapsigargin (TG, 5 µM). TG alone increased force, in a manner similar to CPA; however, it elicited a somewhat greater increase in [Ca²⁺]_i. Similar to the results with CPA, addition of CS after preincubation with TG caused only small increases in force and [Ca²⁺]_i. These data are summarized in Fig. 9.

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Figure 6. Effects of cyclopiazonic acid (CPA) on CS-induced increases in isometric force and [Ca2+]i. A, isometric force measured during a control CS (30 %)-induced contraction/relaxation cycle in NIH 3T3 fibroblast fibres. CPA (5 µM) was added to PSS 10 min before a subsequent CS test stimulation. B, typical changes in [Ca2+]i for 5 cells during the treatments as indicated. C, average [Ca2+]i for all cells (n = 5) in field.

We next used caffeine and ryanodine pretreatment as an alternative approach to alter the Ca²⁺ release from intracellular stores (Fig. 7). Ryanodine and caffeine open the caffeine-sensitive Ca²⁺ channel on the endoplasmic reticulum (ER). Treatment with ryanodine (10 µM) for 5 min did not affect the resting level of [Ca²⁺]_i. Subsequent addition of 20 mM caffeine tended to slightly decrease [Ca²⁺]_i. In the presence of both caffeine and ryanodine, the effects of 30 % CS were significantly blunted. The increase in [Ca²⁺]_i and force in response to CS was decreased to 23·8 ± 6·3 and 17·1 ± 3·8 % of control, respectively (n = 6; Fig. 9). Following a rinse in PSS, addition of 50 mM KCl as previously observed (Fig. 2), dramatically increased [Ca²⁺]_i, but without affecting isometric force.

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Figure 7. Effects of ryanodine and caffeine on CS-induced increases in isometric force and [Ca2+]i A, isometric force measured during a control CS (30 %)-induced contraction/relaxation cycle in NIH 3T3 fibroblast fibres. Ryanodine (10 µM) and 20 mM caffeine were then added to PSS 10 min and 5 min before CS stimulation, respectively. After rinsing the fibres with PSS, 50 mM KCl was added. B, typical changes in [Ca2+]i observed in 4 cells during the treatments as indicated. C, mean [Ca2+]i for all cells (n = 10) in field.

We also used a phospholipase C inhibitor, U73122, to probe Ca²⁺-store release mechanisms further at the level of the InsP₃ release channel. As shown in Fig. 8, treatment with U73122 (3 µM) slightly decreased the resting Ca²⁺ levels and elicited a small sustained increase in force. As summarized graphically in Fig. 9, U73122 pretreatment for 10 min sharply blunted the CS-induced increase in [Ca²⁺]_i, similar to that of caffeine and ryanodine; force development was inhibited, but to a lesser extent (56·2 ± 3·4 % of CS control; n = 3).

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Figure 8. Effects of U73122 on CS-induced increases in isometric force and [Ca2+]i A, isometric force measured during a control CS (30 %)-induced contraction/relaxation cycle in NIH 3T3 fibroblast fibres. U73122 (3 µM) was added to the PSS 10 min before CS stimulation. B, typical changes in [Ca2+]i for 4 cells. C, average [Ca2+]i for all cells (n = 10) in field.

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Figure 9. Effect of inhibition of Ca2+ release from intracellular stores on the CS-induced increases in isometric force and [Ca2+]i Mean data from experiments shown in Figs 6-9 and for thapsigargin pretreatment are presented as a percentage of the CS-induced maximal response. A, isometric force: stacked-bar graph giving the mean steady-state isometric force. Light bars represent the force after a 10 min pretreatment with the agents listed on the abscissa. Dark bars represent the magnitude of the increase in force after subsequent addition of CS. Final height of the stacked bar represents the total level of force attained above the initial unstimulated baseline. B, intracellular Ca2+: light bars show the steady-state [Ca2+]i after 10 min pretreatment with the agents listed on the abscissa. Dark bars represent the maximum value of the transient increase in [Ca2+]i after subsequent addition of calf-serum (CS). Each value represents the mean ± S.E.M. of at least 7 independent determinations. *Significantly different from each CS response at P < 0·05.

We attempted to block the CS-induced increase in [Ca²⁺]_i using the intracellular Ca²⁺ buffer, BAPTA. As shown in Fig. 10, incubation with 50 µM BAPTA significantly reduced the increase in [Ca²⁺]_i (33·8 ± 4·8 % of control) and force, though to a lesser degree (72·0 ± 7·5 % of control, n = 3).

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Figure 10. Effects of BAPTA-AM on CS-induced increases in isometric force and [Ca2+]i A, isometric force measured during a control CS (30 %)-induced contraction/relaxation cycle in NIH 3T3 fibroblast fibres. BAPTA AM (50 µM) was added to the PSS 30 min prior to a second CS stimulation. B, typical changes in [Ca2+]i for 3 cells. C, mean [Ca2+]i for all cells (n = 7) in field.

	DISCUSSION

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We investigated the relation between [Ca²⁺]_i and isometric force of fibroblasts in three-dimensional culture in a collagen matrix. We used serum as the activator, for it reproducibly increases force over many contraction-relaxation cycles and the steady-state level is a function of the serum concentration (Kolodney & Elson, 1993; Obara et al. 1995, 2000). Perhaps our most striking observation was that we did not observe a one-to-one correlation of force and [Ca²⁺]_i. For example, CS elicited a stable increase in force, but [Ca²⁺]_i increased only transiently before returning to baseline. A similar transient increase in [Ca²⁺]_i was also observed in fibroblasts cultured in Petri dishes, so was unlikely to be due to culture in the collagen matrix. The effects of serum on [Ca²⁺]_i in cultured fibroblasts appear to depend on the type of fibroblast. Sustained increases in [Ca²⁺]_i were reported for fibroblasts cultured from chicken embryo (Kolodney et al. 1999) and rat heart (Brilla et al. 1998), whereas transient effects were reported for Swiss 3T3 (Newcomb et al. 1993) and NIH 3T3 (this study). These differences may depend on whether or not the fibroblasts arise from transformed cell lines. Nonetheless, in NIH 3T3 fibroblasts, the sustained increases in isometric force to CS did not require a steady-state elevation of [Ca²⁺]_i from baseline.

Not only can one observe maintained force in the absence of an increased [Ca²⁺]_i, but the converse is also readily observed. That is, one can measure significant increases in [Ca²⁺]_i, as reported by fura-2 ratiometric fluorescence, without changes in force. Our evidence indicates that Ca²⁺ arising from influx is not coupled to force generation, whereas that from intracellular Ca²⁺ stores seems to be the major factor. KCl depolarization increases Ca²⁺ influx into fibroblasts (Lovisolo et al. 1992) and elicits a larger increase in [Ca²⁺]_i than that induced by serum (Fig. 2), yet addition of KCl had no effect on force. We also observed a similar increase in [Ca²⁺]_i without a force response with the Ca²⁺ ionophore, ionomycin. This is similar to findings recently reported for chicken embryo fibroblasts (Kolodney et al. 1999).

In addition, the absence of a tight coupling between force and [Ca²⁺]_i ascribable to Ca²⁺ influx was further demonstrated when Ca²⁺ influx was inhibited by Ca²⁺-free PSS, EGTA or nicardipine (Fig. 5). In all cases when Ca²⁺ influx was inhibited, the [Ca²⁺]_i response to addition of CS was strongly inhibited, but isometric force generation was not affected.

On the other hand, a specific coupling of force to an increase in [Ca²⁺]_i attributable to Ca²⁺ release from intracellular stores is supported by several lines of evidence (Fig. 9). Inhibition of the endoplasmic reticulum (ER) Ca²⁺-ATPase with CPA or TG would lead to depletion of Ca²⁺ from this store. Both CPA and TG increased [Ca²⁺]_i, which was sustained with TG, but not CPA. These treatments also increased force in the same proportion to [Ca²⁺]_i as that observed for the control CS stimulation. Importantly, after these treatments to deplete Ca²⁺ stores, the effects of CS on both [Ca²⁺]_i and force were blunted to less than 20 % of control. We also attempted to deplete Ca²⁺ stores via the ryanodine-release channel with caffeine and ryanodine. This protocol reduced the CS-induced increases in [Ca²⁺]_i and force to similar values obtained by pretreatment with inhibitors of the endoplasmic reticulum Ca²⁺-ATPase. Use of the putative phospholipase C inhibitor, U73122, to prevent the formation of InsP₃ and release of Ca²⁺ from the ER via InsP₃ channels also significantly reduced CS increases in [Ca²⁺]_i and force.

Recently, Kolodney et al. (1999) showed that BAPTA AM (10 µM), a chelator of [Ca²⁺]_i, could significantly reduce (to 30 %) the serum-induced increase in [Ca²⁺]_i in cultured chicken embryo fibroblast cells. However, in fibres made from these cells, BAPTA AM treatment did not reduce the force response to serum. In our NIH 3T3 fibroblast fibres, BAPTA AM (50 µM) was associated with a similar reduction of the peak increase in Ca²⁺ and a moderate reduction of force (30 %) in response to CS (Fig. 10). We did not investigate whether higher concentrations of BAPTA AM could further reduce these parameters. There are several reports of non-specific effects at this and higher concentrations (Tojyo & Matsumoto, 1990; Richardson & Taylor, 1993; Sun et al. 1998); in particular, a 50 % reduction in ATP concentrations in rat parotid cells has been reported (Tojyo & Matsumoto, 1990). Our clearest result is that multiple agents which are known to affect Ca²⁺ release from intracellular stores also significantly reduce the CS-induced increase in force.

Perhaps the simplest explanation for the tight coupling of force with Ca²⁺ arising from stores and the lack of coupling to Ca²⁺ influx is that the fura-2 signals are arising from different compartments. This has been previously suggested for smooth muscle (Abe et al. 1996; Tosun et al. 1998). An understanding of the mechanism(s) underlying the observed disconnection between [Ca²⁺]_i and force ultimately depends on the mechanism for activation of actin-myosin interaction in these fibroblasts. If one assumes that myosin phosphorylation/dephosphorylation plays a major role, then the search for a possible mechanism would probably involve differences between CS-mediated and Ca²⁺ influx pathways. CS-mediated pathways can lead to second messengers which include diacylglycerol (DAG), an activator of C-kinase, and InsP₃, an activator of store Ca²⁺-release channels. Force maintenance in smooth muscle without sustained increases in [Ca²⁺]_i have been reported by Morgan and colleagues (Jiang & Morgan, 1987), and was suggested to be related to activation of thin-filament regulatory proteins, such as calmodulin or calponin (Menice et al. 1997). CS-mediated stimulation may also be coupled to activation of the Rho-kinase. This might lead to force generation in the absence of maintained [Ca²⁺]_i via inhibition of myosin light chain phosphatase (Gong et al. 1996; Somlyo & Somlyo, 2000).

Perhaps the most surprising element of the activation of fibroblast force production, given these pathways, is that release of Ca²⁺ from intracellular stores appears to be sufficient and necessary for activation. Though not as large as the CS-induced increases in force, agents causing release of Ca²⁺ from stores elicited a significant increase in force. Moreover, they blocked further responses to CS. Thus the involvement of a CS-mediated pathway plus Ca²⁺ release from stores is required for full activation of force. The nature of the specific role of intracellular stores remains to be elucidated, but is a major difference between the activation mechanisms of this non-muscle and those in smooth muscle.

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Acknowledgements

This work was supported in part by NIH to R.J.P., grant no. HL54829.

Corresponding author

R. J. Paul: Department of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, Cincinnati, OH 45267-0576, USA.

Email: richard.paul{at}uc.edu

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