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Visualization of elementary mechanosensitive Ca²⁺-influx events, Ca²⁺ spots, in bovine lens epithelial cells

Hisayuki Ohata, Ken-ichi Tanaka, Naoto Maeyama, Masayuki Yamamoto and Kazutaka Momose

	ABSTRACT

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1. Local increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in several regions within the bovine lens epithelial cell during application of mechanical stress were clearly visualized in the presence of lysophosphatidic acid (LPA), a bioactive lysophospholipid, using real-time confocal microscopy. We called the phenomenon 'Ca²⁺ spots'.

2. Ca²⁺ spots started in a circular area with a radius of about 1.5 m. These Ca²⁺ spots spread concentrically, resulting in a mean global increase in [Ca²⁺]_i. The local increase often occurred in a stepwise manner or repetitively at the same region. The spatiotemporal properties of the Ca²⁺ spots were completely different from those of the Ca²⁺ wave induced by ATP, a Ca²⁺-mobilizing agonist.

3. Ca²⁺ spots were inhibited by decreasing the extracellular Ca²⁺ concentration or by the presence of Gd³⁺, an inhibitor of mechanosensitive (MS) channels, but not by thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ pump, suggesting that Ca²⁺ spots arise from Ca²⁺ influx through Gd³⁺-sensitive MS channels.

4. On the assumption that, in lens epithelial cells, the open probability of the MS channel is 0.4, the membrane potential is 56 mV and the channel conductance is 50 pS, the estimated maximum flux of Ca²⁺ in a Ca²⁺ spot (0.4 10^-17 to 4.7 10^-17 mol s^-1) was comparable to currents of one or a few MS channels.

5. On real-time three-dimensional confocal imaging analysis, which permitted simultaneous imaging of basal and apical planes of cells at 37.6 ms intervals, Ca²⁺ spots on the apical plane were more clearly visualized than those on the basal plane.

6. From these results, we propose that the Ca²⁺ spot is an elementary Ca²⁺-influx event through MS channels directly coupled with the first step in mechanoreception In addition, our results strongly suggest that LPA functions as an endogenous factor affecting mechanotransduction systems.

	INTRODUCTION

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Mechanotransduction mechanisms have been found not only in sensory cells but also in a wide variety of cell types (Banes et al. 1995; Sachs & Morris, 1998). It is well known that shear stress associated with blood flow causes release of nitric oxide and prostacyclin from endothelial cells through activation of mechanotransduction systems, resulting in vasodilatation and cell remodelling, specific cellular functions (Davies, 1995; Takahashi et al. 1997). In addition to their physiological role, mechanotransduction mechanisms are considered to be involved in the development of pathophysiological changes. After cataract surgery, residual lens epithelial cells undergo fibrous pseudometaplasia at the capsulotomy site when they come into contact with the implanted intraocular lens (Nishi et al. 1997). This suggests that the mechanical stress due to contact with the intraocular lens induces secondary cataract formation. In addition, it has been suggested that accumulation of sugar alcohols in the lens results in cellular swelling and Ca²⁺ accumulation in sugar cataracts (Azuma et al. 1992).

We reported previously that lysophosphatidic acid (LPA), a bioactive phospholipid, sensitizes changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in response to mechanical stress in cultured lens epithelial cells (Ohata et al. 1997). LPA is a lipid mediator with diverse biological activities in various cell types (Moolenaar, 1995, 1999; Goetzl & An, 1998) and is present in serum at physiologically relevant concentrations (Tigyi & Miledi, 1992; Eichholtz et al. 1993). In addition, secretory phospholipase A₂ produced by inflammatory cells has been reported to generate LPA (Fourcade et al. 1995). LPA is also present in the aqueous humour and the lacrimal gland fluid of the rabbit eye, and corneal injury results in increased production of LPA (Liliom et al. 1998). Furthermore, breakdown of the blood-aqueous barrier in postoperative pseudophakic inflammation (Nishi & Nishi, 1992) should increase the concentration of LPA in the aqueous humour. These processes may be related to the observation that cataracts tend to develop in some inflammatory ocular diseases, and suggest that LPA and mechanical stress affect cataract formation. Furthermore, we also demonstrated that the mechanical stress-induced increase in [Ca²⁺]i in the presence of LPA was due to Ca²⁺ influx through mechanosensitive (MS) channels, which are considered to convert mechanical stress to Ca²⁺ influx (Ghazi et al. 1998), because Gd³⁺, a blocker of MS channels (Yang & Sachs, 1989), inhibited the [Ca²⁺]_i response. Such LPA-induced responses have also been observed in cultured smooth muscle cells from the ileum (Ohata et al. 1995) and cultured lung epithelial cells (Ohata et al. 1996), but among these cells the contribution of Ca²⁺ influx through MS channels to the LPA-induced response was greatest in lens epithelial cells. MS channel activities have been measured, mostly using the single-channel patch-clamp technique, in many cell types, suggesting that they may function as receptors for mechanotransduction mechanisms (Sackin, 1995; Hamill & McBride, 1996; Ghazi et al. 1998). A stretch-inhibitable non-selective cation channel was recently cloned for the first time (Suzuki et al. 1999), but the physiological significance of this channel is unclear and the stretch-activated channel, which is considered to regulate Ca²⁺ influx mainly in response to mechanical stress on the cell membrane, has still not been identified at the molecular level.

Since our experimental mechanotransduction system using lens epithelial cells was considered to be suitable to examine the Ca²⁺ response through MS channels, we attempted to visualize the mechanical stress-induced Ca²⁺ response using a real-time confocal imaging system. Our results showed for the first time that the mechanical stress-induced local Ca²⁺ increase, which is completely inhibited by Gd³⁺, could be clearly visualized in the presence of LPA in cultured lens epithelial cells. These results suggest that the local Ca²⁺ increase, 'Ca²⁺ spots', may represent an elementary Ca²⁺-influx event through MS channels underlying mechanical stress-induced cellular responses and that LPA may function as an endogenous factor affecting mechanotransduction systems.

	METHODS

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Materials

Fluo-4 acetoxymethylester (fluo-4 AM) and fura-2 AM were obtained from Molecular Probes, Inc. (Eugene, OR, USA). Cremophor EL was purchased from Nacalai Tesque (Kyoto, Japan). Ionomycin was obtained from Calbiochem-Novabiochem (La Jolla, CA, USA). Lysophosphatidic acid (LPA; oleoyl) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). ATP was obtained from Sigma Chemical Co. (St Louis, MO, USA). Thapsigargin was obtained from Wako Pure Chemicals (Osaka, Japan). All other chemicals were commercial products of the highest grade available.

Cell culture

Ocular lenses were isolated from bovine eyes, which were obtained from an abattoir (Tokyoshibaurazouki Co., Tokyo, Japan). After removal of the lens fibril material, the capsules were plated in 35 mm Petri dishes with 1 ml of minimum essential medium (MEM; Gibco, Grand Island, NY, USA) containing 10 % fetal calf serum, according to the method of Hughes et al. (1975), and maintained at 37 °C in a humidified atmosphere of 95 % air-5 % CO₂. After 24 h, the culture medium was replaced with MEM containing 10 % fetal calf serum, and the medium was changed every 3 days until the cells had reached confluency. For determination of [Ca²⁺]_i, the cells were subcultured on glass coverslips 25 mm in diameter inside 35 mm Petri dishes. Confluent cells from 3 to 10 passages were used for the experiments.

Loading of Ca²⁺ indicator

To determine the [Ca²⁺]_i, the cultured cells on coverslips were rinsed several times with physiological saline solution (PSS) consisting of (mM): NaCl, 137; KCl, 2.7; CaCl₂, 1.8; MgCl₂, 1.0; glucose, 5.6; Hepes, 8.4; pH 7.4, and incubated with 2.5 M fura-2 AM or 5 M fluo-4 AM with 0.03 % cremophor EL in PSS for 1 h at room temperature. After loading, the coverslips were rinsed several times with PSS without the fluorophores.

Measurement of [Ca²⁺]_i using fura-2

Fura-2 fluorescence images with excitation at 340 and 380 nm were acquired at > 480 nm with a high-speed cooled digital CCD camera fluorescence imaging system (ARGUS HiSCA, Hamamatsu Photonics, Hamamatsu, Japan) and ratio images were obtained by dividing the image with excitation at 340 nm by that with excitation at 380 nm. Since the time required for acquisition of each fluorescence image and to change the wavelength was 18.0 ms, the temporal resolution was 72.0 ms frame^-1. The X-Y resolution of the system was approximately 2 m pixel^-1 with a Fluar 40 oil (NA 1.3; Zeiss). [Ca²⁺]_i was estimated by in situ calibration as follows. Firstly, normal PSS in the bath was changed to Ca²⁺-free PSS containing 1 mM EGTA and 10 M ionomycin, and the ratio image of the cells loaded with fura-2 was recorded after the ratio reached the minimal value (R _min). Secondly, the Ca²⁺-free PSS was changed to normal PSS containing 10 M ionomycin, and the ratio image was recorded after the ratio reached the maximal value (R_max). [Ca²⁺]_i was calculated from the R_min, R_max and the K_d of fura-2 (224 nM), according to the reported equation (Grynkiewicz et al. 1985).

Confocal imaging of [Ca²⁺]_i using fluo-4

Fluorescence images of fluo-4 were collected using a real-time confocal imaging system composed of a multi-pinhole Nipkow disk type confocal scanner (CSU10Z; Yokogawa Electric Co., Tokyo, Japan) and the ARGUS HiSCA. Fluo-4 was excited by light at a wavelength of 488 nm with a 3-6 mW argon ion laser. Fluo-4 fluorescence was measured at a wavelength of > 510 nm. Using the imaging system with Uplan Apo 60 W (NA 1.2; Olympus), fluo-4 images with 60 pixels 60 pixels with X-Y resolution of 0.67-1.3 m pixel^-1 and Z-axis resolution (full-width at half-maximum, FWHM) of 1.3 m were acquired with 17.3-19.2 ms exposure at 12 bit dynamic range. The temperature was kept at 32 °C, since the intracellular fluorescence of fluo-4 decreased rapidly at 37 °C, probably due to dye leakage. The intensity of fluo-4 fluorescence for each region of interest was divided by the averaged resting fluorescence intensity of more than 20 frames before stimulation of the same region, after subtraction of background fluorescence. The resultant relative fluorescence intensity (F/F₀) was used as an indicator of [Ca²⁺]_i. Image processing and analysis were performed using NIH Image (version 1.62; NIH, Bethesda, MD, USA).

Real-time three-dimensional confocal imaging of [Ca²⁺]_i

Real-time three-dimensional confocal imaging of [Ca²⁺]_i was performed as described previously (Ohata et al. 1999) using a real-time confocal imaging system with synchronized high-speed Z-axis scanning. Z-axis scanning with a closed loop Microscope Objective NanoPositioner (P-721.10; PI Polytec, Tokyo, Japan), a piezoelectric actuator mounted under the objective lens, was controlled by an electrical square wave with 37.6 ms cycle^-1 and 0.3 V amplitude with 0.15 V offset, generated by a synthesized function generator (FG120; Yokogawa Electric Co.) triggered by TTL level signal output from the ARGUS HiSCA system at the start of each exposure for synchronization with the exposure interval of the CCD camera. Fluo-4 fluorescence images with 40 pixels 40 pixels were acquired with 9.4 ms exposure continuously. The time required for positioning to a steady level on the Z-axis was about 8 ms. Therefore, although the image acquired during the positioning period was not available, fluo-4 fluorescence images at both the apical and basal planes at a distance of 3 m could be acquired reciprocally at a 37.6 ms interval, respectively, without decreasing spatial resolution.

Application of mechanical stress

Bath solution was perpendicularly spritzed onto the cells from a pipette at an appropriate constant flow rate (0.15 or 2.25 ml min^-1) for 3-4 s or 60 s using a Perista pump (Model SJ-1220; Atto Corp., Tokyo, Japan). The tip of the pipette was placed 0.2 mm over the cells of interest. The inside diameter of the tip was 0.5 mm. Exact placement of the tip of the pipette was achieved in each experiment using a microscope with a focus control unit. All of the cells observed (3-10 cells per microscopic field) could be stimulated simultaneously by this method under the same conditions. This mechanical stress did not affect acquisition of the fluorescence images and induced a reproducible response, although it might have involved a mix of both flow and pressure stress. In our method, the shear stress to the cells is considered to be maximum when the fluid stream passes through the periphery of the pipette tip. Maximal shear stress (, in N cm^-2) in the presence of a flow rate of 2.25 ml min^-1 was estimated using the following equation (assumptions: depth is 0.02 cm, width is 0.05 cm 3.14 cm in the flow stream):

= 6 Q (a²b)^-1 = 3.22 10^-4,

where represents the fluid viscosity (0.0009 Pa s), Q the flow rate (0.0375 ml s^-1), and a the depth and b the width of the flow stream. The pressure exerted by the fluid stream may be maximum in the centre of the pipette tip. The maximal pressure exerted by a flow rate of 2.25 ml min^-1 was measured in the same setting using a pressure transducer (LPU-0.1-350-0-II; Nihon Koden, Tokyo, Japan). The pressure was found to be less than 50 cmH₂O. The intensities of the shear and the pressure stresses occurred within the physiological range.

	RESULTS

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Mechanical stress-induced local increase in [Ca²⁺]_i in the presence of LPA

Using a real-time confocal imaging system, we monitored changes in [Ca²⁺]_i in cultured bovine lens epithelial cells loaded with fluo-4 during application of mechanical stress by spritzing a bath solution onto the cells in the presence of LPA (Fig. 1). [Ca²⁺]_i in the resting state was stable over the range 50-100 nM and was almost homogeneous within the fura-2-loaded cells (data not shown). Neither addition of 3 M LPA nor application of mechanical stress (2.25 ml min^-1) alone affected [Ca²⁺]_i, as shown in Fig. 1A. In the presence of LPA, mechanical stress caused local increases in [Ca²⁺]_i in several regions within the cell (Fig. 1B). Each local increase in [Ca²⁺]_i occurred independently and preceded the averaged increase over the whole area of the cell after a lag time during mechanical stress (Fig. 1B and C). We called the mechanical stress-induced local increase in [Ca²⁺]_i a 'Ca²⁺ spot'. The peak [Ca²⁺]_i in the starting region of Ca²⁺ spots was estimated as being of the order of several hundred nanomolar by fura-2 ratio imaging. The density of Ca²⁺ spots was about 1 (10³ m²)^-1. [Ca²⁺]_i returned to the resting level within several minutes after application of mechanical stress. This phenomenon was reproducible in the same cells for at least a few hours. Since similar phenomena were also observed using fura-2 ratiometric imaging (data not shown), although the spatiotemporal resolution was less than that with confocal imaging, it was clear that Ca²⁺ spots were not artifacts due to localization of fluo-4.

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Figure 1. Visualization of Ca2+ spots in lens epithelial cells during application of mechanical stress in the presence of LPA A, time course of averaged F/F0 of 30 cells loaded with fluo-4 that were exposed to 3 M LPA for 30 s and mechanically stimulated by spritzing bath solution onto the cells from a pipette at a constant flow rate of 2.25 ml min-1 (downward arrows). B, confocal fluorescence images (F/F0) of Ca2+ spots. Values under the images indicate the time after onset of mechanical stress. The colour scale represents F/F0. Arrows mark the first indications of each Ca2+ spot. Scale bar, 20 m. C, time course of changes in F/F0 in the starting region (1.3 m 1.3 m) of the Ca2+ spots. Each black line represents the time course of individual Ca2+ spots indicated by the corresponding number in B. Cells a, b and c correspond to the cells shown in the fluorescence image in B. The red lines indicate the time course of changes in averaged F/F0 in each cell.

Spatiotemporal properties of Ca²⁺ spots

To determine the area of the starting region in Ca²⁺ spots, we examined the spatial properties of the Ca²⁺ spot more closely at higher X-Y spatial resolution (0.67 m pixel^-1). Spatiotemporal changes of two different Ca²⁺ spots induced by mechanical stress of 2.25 ml min^-1 in the presence of 3 M LPA are shown as surface plots prepared using NIH Image in Fig. 2. In both Ca²⁺ spots (Fig. 2A and B), the [Ca²⁺]_i increase was confirmed as starting in a restricted circular area with a radius of about 1.5 m. [Ca²⁺]_i in the starting region of the Ca²⁺ spots was always higher than that in the surrounding area during the response to mechanical stress. The Ca²⁺ spot shown in Fig. 2A affected [Ca²⁺]_i over a large area of the cell and persisted for more than 1 s. On the other hand, the Ca²⁺ spot shown in Fig. 2B disappeared completely within 70 ms after onset without affecting the surrounding [Ca²⁺]_i and was the shortest lived and smallest of the Ca²⁺ spots observed under these conditions.

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Figure 2. Spatiotemporal changes in F/F0 in the region surrounding Ca2+ spots Cells loaded with fluo-4 were mechanically stimulated (2.25 ml min-1) in the presence of 3 M LPA. Spatiotemporal changes in F/F0 in each square area centred around the starting region of Ca2+ spots shown in the fluorescence image on the left are presented as surface plots prepared using NIH Image. Values under the images indicate the time after onset of the Ca2+ spot. Scale bars, 10 m. The resolution of the images is approximately 0.67 m 0.67 m (X and Y) by 1.3 m (FWHM). The Ca2+ spot shown in A is typical, while that in B is the shortest lived and smallest example.

We then compared the spatiotemporal properties of Ca²⁺ spots with those of Ca²⁺ waves induced by ATP, a Ca²⁺-mobilizing agonist, in X-Y ratio images, Y-T ratio images and the time course of changes in [Ca²⁺]_i in the starting region and the surroundings in cultured lens epithelial cells (Fig. 3). In the Ca²⁺ spot, the rate of rise and maximum level of [Ca²⁺]_i were greatest in the starting region and decreased in inverse relation to distance (4-16 m) from the starting region, as shown in Fig. 3A. On the other hand, the rate of rise and the maximum level of [Ca²⁺]_i in the Ca²⁺ wave induced by 10 M ATP were almost constant regardless of the distance (8-32 m) from the starting region (Fig. 3B). In addition, the maximum rate of rise of the Ca²⁺ spot was more than fivefold higher than that of the ATP-induced Ca²⁺ wave.

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Figure 3. Comparison of spatiotemporal properties of Ca2+ spot and ATP-induced Ca2+ wave A, a Ca2+ spot induced by mechanical stress (2.25 ml min-1) in the presence of 3 M LPA is shown in sequential X-Y ratio images (F/F0) and a Y-T ratio image (F/F0). The area indicated by the yellow line in the fluorescence image represents the region of the X-Y ratio images centred around the starting region of the Ca2+ spot. Values above the X-Y ratio images indicate the time after onset of mechanical stress. Scale bar, 10 m. The Y-T ratio image was reconstructed as the Y-axis shown by the vertical blue line in the fluorescence image versus time image prepared using NIH Image. The time scale of the Y-T ratio image corresponds with that of the lower graph, which represents the time course of changes in [Ca2+]i in the starting region (1.3 m 1.3 m) of the Ca2+ spot and in each region (1.3 m 1.3 m) at intervals of 1.3 m as shown by the corresponding coloured lines in the Y-T ratio images. B, Ca2+ wave induced by 10 M ATP in the lens epithelial cells, shown in the same manner as in A. In A and B, the dashed lines in the graphs represent the maximum rate of rise in each region shown by the parallel coloured lines.

Classification of Ca²⁺ spots and dependency on strength of mechanical stress and concentration of LPA

The time course of changes in [Ca²⁺]_i in the starting region of Ca²⁺ spots varied even between those observed within the same cell, as shown in Fig. 1C. Ca²⁺ spots could be classified into three types (Fig. 4A). Ca²⁺ spots which were affected by surrounding Ca²⁺ spots were excluded from the analysis. Type 1 was classified as a transient increase in [Ca²⁺]_i, where [Ca²⁺]_i returned to less than the midpoint between the resting level and the maximum level of the transient increase within 1 s after onset of the Ca²⁺ spot. Most of these Ca²⁺ spots disappeared within 1 s without contributing to the mean global increase in [Ca²⁺]_i. The Ca²⁺ spot shown in Fig. 2B is a typical example of this type. Type 2 was classified as a stepwise increase in [Ca²⁺]_i to the maximum level, which spread concentrically and finally contributed to the mean global increase in [Ca²⁺]_i. Type 3 Ca²⁺ spots showed a marked increase in [Ca²⁺]_i to the maximum level within 0.1 s and suddenly spread concentrically. In the presence of 3 M LPA, the rank order of the types of Ca²⁺ spots caused by mechanical stress of 2.25 ml min^-1 was type 2 > type 3 > type 1 (8:4:1, n = 132 Ca²⁺ spots; Fig. 4B). The time course changed depending on the strength of mechanical stress and the concentration of LPA, as shown in Fig. 4B. When the mechanical stress was 0.15 ml min^-1 in the presence of 3 M LPA, the percentage of type 1 Ca²⁺ spots increased, and the ratio of type 1, 2 and 3 Ca²⁺ spots was 3:6:2 (n = 27 Ca²⁺ spots), respectively. With mechanical stress of 0.15 ml min^-1, a decrease in the concentration of LPA markedly increased the percentage of type 1 and decreased that of types 2 and 3. In the presence of 0.3 M LPA, the rank order of the types of Ca²⁺ spots induced by mechanical stress of 0.15 ml min^-1 became type 1 > type 2 > type 3 (8:1.3:1, n = 31 Ca²⁺ spots). The density of the Ca²⁺ spots tended to decrease with decreasing strength of mechanical stress and concentration of LPA.

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Figure 4. Classification of Ca2+ spots and the dependency on strength of mechanical stress and concentration of LPA A, Ca2+ spots were classified into three types as shown depending on the differences in the time course of changes in [Ca2+]i in the starting region. Type 1, transient increase in [Ca2+]i; type 2, stepwise increase in [Ca2+]i; type 3, marked increase in [Ca2+]i. B, effects of strength of mechanical stress and concentration of LPA on the percentage of each type of Ca2+ spot. MS, mechanical stress; n, number of Ca2+ spots analysed.

Repetitive transient Ca²⁺ spots induced by a low level of mechanical stress

As shown in Fig. 4B, decreases in the concentration of LPA and level of mechanical stress induced transient Ca²⁺ spots (type 1) with less effect on the global increase in [Ca²⁺]_i. Furthermore, we observed that continuous application of a low level of mechanical stress (0.15 ml min^-1 for 60 s) caused repetitive transient Ca²⁺ spots (type 1) in the presence of a low concentration of LPA (0.3 M), as shown in Fig. 5. At least eight Ca²⁺ spots were repetitively observed within a single cell, and they disappeared within 0.2 s (Fig. 5C) without diffusing over a circular area with a radius of about 5 m (Fig. 5B). The frequency of the repetitive Ca²⁺ spots was 0.1-0.5 spikes s^-1 and tended to increase with time after the onset of mechanical stress (Fig. 5C). They did not contribute to the global increase in [Ca²⁺]_i, at least until 30 s after the onset of mechanical stress, but thereafter the mean [Ca²⁺]_i in the cells gradually increased with increasing frequency of Ca²⁺ spots.

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Figure 5. Visualization of repetitive transient Ca2+ spots induced by a low level of mechanical stress Cells were exposed to 0.3 M LPA for 30 s and then mechanically stimulated by spritzing bath solution onto the cells from a pipette at a constant flow rate of 0.15 ml min-1 for 60 s in the presence of LPA. A, confocal fluorescence images of Ca2+ spots in a cell. Arrows mark the first indications of each Ca2+ spot. Values under the images indicate the time after onset of mechanical stress. Scale bar, 20 m. B, Y-T ratio images of the Ca2+ spots indicated by red and blue arrows in A were reconstructed as the Y-axis versus time image using NIH Image. The time scale corresponds to that of the upper graph in C. C, time course of changes in F/F0 in the starting region (1.3 m 1.3 m) of the Ca2+ spots indicated by corresponding coloured arrows in A. The black line indicates the time course of changes in averaged F/F0 in the whole area of the cell.

Source of increased [Ca²⁺]_i in Ca²⁺ spots

We then examined whether Ca²⁺ spots arise from Ca²⁺ influx through plasmalemmal channels or from Ca²⁺ release from intracellular stores. Pretreatment with 3 M thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase pump (Thastrup et al. 1990), did not affect the spatiotemporal properties (Fig. 6A) or the density (Fig. 6B) of Ca²⁺ spots induced by mechanical stress of 2.25 ml min^-1 in the presence of 3 M LPA. We confirmed that the thapsigargin treatment increased [Ca²⁺]_i (data not shown). Furthermore, the [Ca²⁺]_i response to subsequent applications of 10 M ATP was abolished, indicating that the function of the intracellular Ca²⁺ stores was completely inhibited by the thapsigargin treatment. On the other hand, the density of Ca²⁺ spots was markedly decreased in cells exposed to bathing medium in which the concentration of CaCl₂ was decreased to 0.18 mM or to medium supplemented with 3 M Gd³⁺, an inhibitor of MS channels (Yang & Sachs, 1989; Hamill & McBride, 1996) (Fig. 6B). These results clearly indicate that Ca²⁺ spots arise from Ca²⁺ influx through Gd³⁺-sensitive MS channels, and not from Ca²⁺ release from intracellular stores.

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Figure 6. Effects of thapsigargin, a reduced concentration of CaCl2 and the presence of Gd3+ on the density of Ca2+ spots A, confocal fluorescence image and ratio images (F/F0) of Ca2+ spots in the presence of 3 M LPA in cells pretreated with 3 M thapsigargin for 3 min. Values under the images indicate the time after onset of mechanical stress (2.25 ml min-1). The colour scale represents F/F0. Arrows mark the first indications of each Ca2+ spot. Scale bar, 20 m. B, the cells were incubated in normal medium (Cont), in the presence of 3 M thapsigargin (TG), in bathing medium containing 0.18 mM CaCl2 (low Ca) or in the presence of 3 M GdCl3 (Gd3+) for 3 min before application of mechanical stress in the presence of 3 M LPA. Fluorescence images were acquired. The density of Ca2+ spots was obtained by dividing the number of Ca2+ spots in the image by the total area of cells in the image. Data are expressed as means ± S.E.M. (n = 3-15 experiments).

Estimation of the flux of Ca²⁺ in Ca²⁺ spots

Since Ca²⁺ spots are considered to be due to Ca²⁺ influx through MS channels, the flux of Ca²⁺ (J) associated with a Ca²⁺ spot can be estimated by the following equation:

J = B [Ca²⁺]_iVT^-1,

where B is the buffering power of the cell for Ca²⁺, [Ca²⁺]_i is the concentration change in the Ca²⁺ spot estimated by fura-2 ratio imaging, V is the volume occupied by the spot, and T is the time required for the rise in [Ca²⁺]_i. The flux of Ca²⁺ (J) within the circular area, the centre of which is the starting region for the spot and the [Ca²⁺]_i is not affected by other surrounding Ca²⁺ spots, was estimated on the assumption that the thickness of the cell was 4 m and B was 100 (Neher & Augustine, 1992). When J in a Ca²⁺ spot caused by mechanical stress of 2.25 ml min^-1 in the presence of 3 M LPA was calculated at 72 ms time intervals, the maximum value in each Ca²⁺ spot varied in the range 0.4 10^-17 to 4.7 10^-17 mol s^-1 and the mean value was 1.59 10^-17 ± 0.31 10^-17 mol s^-1 (± S.E.M., n = 13 Ca²⁺ spots; Fig. 7). This value was not dependent on the strength of mechanical stress or the concentration of LPA.

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Figure 7. Maximal Ca2+ flux for each Ca2+ spot induced by different conditions of mechanical stress and concentration of LPA The flux of Ca2+ associated with each Ca2+ spot was estimated in the circular area centred around the starting point of the Ca2+ spot and a radius of 10 m in the fura-2 ratio image as described in the text. Each symbol represents the maximum flux of Ca2+ associated with each Ca2+ spot induced by the indicated strengths of mechanical stress and concentrations of LPA. MS, mechanical stress. Horizontal bars represent averaged values of each flux of Ca2+.

Real-time three-dimensional confocal imaging of Ca²⁺ spots

To clarify whether Ca²⁺ spots occur on the basal membrane or the apical membrane of cells stimulated by mechanical stress, simultaneous imaging of a Ca²⁺ spot in the apical and basal planes of the cells was considered to be required, because the spatiotemporal properties of Ca²⁺ spots varied as shown in Fig. 1 and 2. We therefore performed simultaneous imaging of Ca²⁺ spots in the apical and basal planes at a distance of 3 m using our recently developed real-time three-dimensional confocal imaging system (Ohata et al. 1999) (Fig. 8). As shown in the sequential fluo-4 ratio images in Fig. 8A, the Ca²⁺ spot in the apical plane was more clearly visualized than that in the basal plane. In addition, the rate of rise of [Ca²⁺]_i in the starting region (1.3 m 1.3 m) was greatest in the apical plane (Fig. 8B). However, no significant differences were observed between the rate of rise of [Ca²⁺]_i in the starting region and that of the surrounding area in the basal plane.

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Figure 8. Simultaneous confocal imaging of Ca2+ spots at apical and basal planes of lens epithelial cells using the high-speed three-dimensional confocal imaging system Cells were exposed to 3 M LPA for 30 s and then mechanically stimulated (2.25 ml min-1). A, confocal images of Ca2+ spots in apical (top) and basal (bottom) planes. B, time course of changes in F/F0 in the starting region (arrowhead in A, 1.3 m 1.3 m; , apical plane; , basal plane) of the Ca2+ spot and in each region as shown in the scheme in A (1.3 m 1.3 m; filled symbols, apical plane; open symbols, basal plane). C, comparison between rates of rise of F/F0 in the starting region of the Ca2+ spot in apical and basal planes. Each symbol represents the rate of rise in the apical plane and relative rate of rise obtained by dividing the rate of rise in the apical plane by that in the basal plane of individual Ca2+ spots.

Since the ratio of the rate of rise of [Ca²⁺]_i in the starting region in the apical plane to that in the basal plane within a single Ca²⁺ spot was considered to be a reasonable parameter for estimating whether Ca²⁺ spots occur on the basal membrane or the apical membrane, we compared these values for 33 Ca²⁺ spots. As shown in Fig. 8C, in 20 of 33 Ca²⁺ spots (63.6 %), the rate of rise in the apical plane was more than 130 % of that in the basal plane. On the other hand, the values in the apical and the basal planes were almost the same in 12 Ca²⁺ spots (36.4 %), but there were no Ca²⁺ spots in which the rate of rise in the basal plane was more than 130 % of that in the apical plane. These results suggest that Ca²⁺ spots reflect Ca²⁺ influx on the apical membrane, which is exposed directly to mechanical stress.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

We visualized the localized increase in [Ca²⁺]_i, Ca²⁺ spots, induced by mechanical stress in the presence of LPA in cultured lens epithelial cells using real-time confocal microscopy. The following observations indicated that the Ca²⁺ spot is an elementary Ca²⁺ influx event through MS channels in response to mechanical stress. Firstly, Ca²⁺ spots were specifically induced by mechanical stress. Secondly, Ca²⁺ spots were inhibited by reducing the concentration of Ca²⁺ in the bathing medium to 0.18 mM or by addition of 3 M Gd³⁺, an inhibitor of MS channels (Yang & Sachs, 1989; Hamill & McBride, 1996), but not by thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase pump (Thastrup et al. 1990) (Fig. 6). Lastly, the spatiotemporal properties of Ca²⁺ spots indicated that they reflected Ca²⁺ supplied only from the restricted starting region diffusing passively into the surrounding area (Fig. 2 and 3A), while ATP-induced Ca²⁺ waves propagated actively by a mechanism involving inositol 1,4,5-trisphosphate-induced intracellular Ca²⁺ release (O'Connor et al. 1991) (Fig. 3B). Sigurdson et al. (1992) reported that gentle prodding of cultured heart cells with a pipette produced Ca²⁺ influx through stretch-activated ion channels. Furthermore, this work opined that treatment of the channels as point sources resulted in the diffusion of ions away from the channel with hemispherical contours of equal concentration according to Fick's law. Our findings prove this estimation to be true experimentally. Local increases in [Ca²⁺]_i have also been reported as Ca²⁺ sparks, elementary ryanodine-sensitive Ca²⁺-release events, in cardiac muscle (Cheng et al. 1993) and arterial smooth muscle (Nelson et al. 1995), but this mechanism is quite different from that of Ca²⁺ spots. Therefore, the Ca²⁺ spot is a specific phenomenon found as a localized increase in [Ca²⁺]_i due to mechanical stress-induced elementary Ca²⁺ influx through MS channels. Such local increases in [Ca²⁺]_i are considered to be physiologically suitable properties for detecting the strength and direction of mechanical stress, a stimulant with vector, unlike chemical stimulants.

Although the activity of MS channels has been measured using the single-channel patch-clamp technique in many cell types, MS channels have yet to be characterized at the molecular level (Sackin, 1995; Hamill & McBride, 1996; Ghazi et al. 1998). In lens epithelial cells, cation-selective MS channels with a conductance of 50 pS have also been detected (Cooper et al. 1986). We therefore estimated the flux of Ca²⁺ (J) in a Ca²⁺ spot to examine the relationship between the Ca²⁺ spots and the MS channels with a conductance of 50 pS detected by patch-clamp recording. On the assumption that the open probability of the MS channel is 0.4, the membrane potential is 56 mV and the channel conductance is 50 pS in lens epithelial cells (Cooper et al. 1986), the estimated maximum flux of Ca²⁺ in a Ca²⁺ spot (0.4 10^-17 to 4.7 10^-17 mol s^-1; Fig. 7) was comparable to currents of one or a few MS channels. The heterogeneity of the flux in each Ca²⁺ spot may be due to differences in the open probability of each channel, which is dependent on the focus of mechanical stress on the membrane around the MS channels. In addition, since it has been reported that more than five kinds of MS channel coexist within a single cell (Bowman et al. 1992; Ruknudin et al. 1993), the heterogeneity of the flux in Ca²⁺ spots may reflect the existence in the cell of several kinds of MS channels with different conductance. The differences between the three types of time course of Ca²⁺ spots were considered to be due to differences in the open probability and the duration of the period during which the open probability is high. The stepwise increase (type 2) and marked increase (type 3) in [Ca²⁺]_i in the starting region of Ca²⁺ spots (Fig. 4B) caused by a high level of mechanical stress (2.25 ml min^-1) in the presence of a high concentration (3 M) of LPA may reflect repetitive and continuous appearance of the period during which the open probability is high, respectively. These types of Ca²⁺ spots could contribute not only to the local increase in [Ca²⁺]_i, but also to the global increase in [Ca²⁺]_i in the cell. On the other hand, repetitive transient Ca²⁺ spots induced by a low strength of mechanical stress (0.15 ml min^-1) in the presence of a low concentration of LPA (0.3 M; Fig. 5) are believed to correspond to the changes in Ca²⁺ flux through MS channels observed using the single-channel patch-clamp technique in many cell types. Additionally, repetitive transient Ca²⁺ spots are presumably dependent on changes in open probability of MS channels, although the mechanism is unclear. This type of Ca²⁺ spot could stimulate cellular responses only within the restricted local region, but not the global response. These results suggested that the duration of the period during which the open probability is high is dependent on the strength of mechanical stress and concentration of LPA, in addition to supporting the suggestion that Ca²⁺ spots reflect Ca²⁺ influx through one or a few MS channels.

MS channels tend to be ubiquitous, occurring at uniform density of the order of 1 m^-2 as determined by patch-clamp recording (Moody & Bosma, 1989). However, the density of Ca²⁺ spots was much lower than that of MS channels. Although this discrepancy is considered to be due to differences in both the methods of mechanical stress, it may also be partially explained by underestimation of the number of Ca²⁺ spots, for the following reasons. Firstly, a large Ca²⁺ spot may cover a number of neighbouring small Ca²⁺ spots. Secondly, a small Ca²⁺ spot, which may correspond to Ca²⁺ flux arising from a single MS channel with a conductance of 50 pS and an open probability of less than 0.4, may not be detected by the method used here. Lastly, it is possible that not all MS channels respond to mechanical stress, but rather only those MS channels in the region where the mechanical force is focused may be activated. In other words, our results suggested that the starting regions of Ca²⁺ spots are the sites at which functional elements of mechanotransduction systems coexist with MS channels and are also the sites at which mechanical stress is focused, although the sites could not be distinguished anatomically from other sites in the cell. Therefore, Ca²⁺ spots are also considered to be useful for identification of sites at which mechanical stress is focused, whereas patch-clamp recording can detect MS channel activities only within a restricted area of a few square micrometres.

We then examined whether Ca²⁺ spots occur on the basal membrane or the apical membrane of cells stimulated by mechanical stress using our recently developed real-time three-dimensional confocal imaging technique (Ohata et al. 1999). The three-dimensional imaging of Ca²⁺ spots directly indicated that Ca²⁺ spots occur on the apical membrane (Fig. 8). These results corresponded with the data reported by Cooper et al. (1986) indicating that MS cation channel current exists on the apical membrane of frog lens epithelial cells. Our results also suggested that Ca²⁺ spots reflect Ca²⁺ influx through MS channels at restricted sites on the apical membrane.

Ca²⁺ spots are thought to occur physiologically or pathophysiologically, although they required the presence of LPA in the present study. LPA is present in normal human plasma at a concentration of 0.29 M and in the plasma of multiple myeloma patients at a concentration of 3.21 M (Sasagawa et al. 1999), concentrations corresponding with that required for induction of Ca²⁺ spots in the present study. In addition, LPA is also present in the normal aqueous humour of the rabbit eye at a concentration of 0.2 M, and corneal injury (Liliom et al. 1998) or breakdown of the blood-aqueous barrier occurring in postoperative pseudophakic inflammation (Nishi & Nishi, 1992) would increase the concentration of LPA in the aqueous humour. Therefore, Ca²⁺ spots observed in the presence of 0.3 M LPA may be involved in the regulation of physiological function or integrity of lens epithelial cells, and LPA at the micromolar level may cause cellular injury through activation of Ca²⁺ spots. These processes may be related to the observation that cataracts tend to develop in some inflammatory ocular diseases. Furthermore, our preliminary investigation showed that similar Ca²⁺ spots occurred in bovine aortic endothelial cells in the presence of LPA (Ikeuchi et al. 2000). Therefore, the occurrence of Ca²⁺ spots must be an elementary event underlying mechanical stress-induced cellular responses, and LPA may be an important endogenous factor affecting mechanotransduction. We have previously suggested that the sensitizing effect of LPA is an action specific to LPA via its membrane receptor. The sensitizing effect was not due to the amphipathic action of LPA (Ohata et al. 1997). However, further studies are required in order to clarify the LPA receptor subtypes and the signal transduction pathway associated with the sensitizing effect. These elucidations may also contribute to the understanding of the mechanisms which regulate MS channels in mechanotransduction systems.

In conclusion, we propose that the Ca²⁺ spot is an elementary Ca²⁺-influx event through MS channels underlying mechanical stress-induced cellular responses. This hypothesis should contribute to the elucidation of the molecular mechanisms of mechanotransduction systems as a phenomenon directly coupled with the first step in mechanoreception. In addition, our results strongly suggest that LPA functions as an endogenous factor affecting mechanotransduction systems.

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Acknowledgements

We are grateful to Dr Toru Kawanishi (National Institute of Health Sciences) for insightful comments. This study was supported in part by a grant-in-aid for a drug innovation science project (to T. Kawanishi and K. Momose) from the Japan Health Science Foundation and a grant-in-aid (to H. Ohata) for general scientific research from the Ministry of Education, Science, Sports and Culture of Japan.

Corresponding author

H. Ohata: Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.

Email: ohata{at}pharm.showa-u.ac.jp

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