Visualization of elementary mechanosensitive Ca2+-influx events, Ca2+ spots, in bovine lens epithelial cells.
Journal: 2001/August - Journal of Physiology
ISSN: 0022-3751
PUBMED: 11283223
Abstract:
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'. 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. 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. 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 x 10-17 to 4.7 x 10-17 mol x s(-1)) was comparable to currents of one or a few MS channels. 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. 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.
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J Physiol 532(Pt 1): 31-42

Visualization of elementary mechanosensitive Ca<sup>2+</sup>-influx events, Ca<sup>2+</sup> spots, in bovine lens epithelial cells

Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, 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: pj.ca.u-awohs.mrahp@ataho
Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Received 2000 Aug 15; Accepted 2000 Nov 29.

Abstract

  1. Local increases in the intracellular Ca concentration ([Ca]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 ‘Ca spots’.

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

  3. Ca spots were inhibited by decreasing the extracellular Ca concentration or by the presence of Gd, an inhibitor of mechanosensitive (MS) channels, but not by thapsigargin, an inhibitor of the endoplasmic reticulum Ca pump, suggesting that Ca spots arise from Ca influx through Gd-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 Ca in a Ca spot (0.4 × 10 to 4.7 × 10 mol s) 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, Ca spots on the apical plane were more clearly visualized than those on the basal plane.

  6. From these results, we propose that the Ca spot is an elementary Ca-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.

Abstract

Mechanotransduction mechanisms have been found not only in sensory cells but also in a wide variety of cell types (Banes et al. 1995; Sachs &amp; 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 Ca accumulation in sugar cataracts (Azuma et al. 1992).

We reported previously that lysophosphatidic acid (LPA), a bioactive phospholipid, sensitizes changes in intracellular free Ca concentration ([Ca]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 &amp; An, 1998) and is present in serum at physiologically relevant concentrations (Tigyi &amp; 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 &amp; 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 [Ca]i in the presence of LPA was due to Ca influx through mechanosensitive (MS) channels, which are considered to convert mechanical stress to Ca influx (Ghazi et al. 1998), because Gd, a blocker of MS channels (Yang &amp; Sachs, 1989), inhibited the [Ca]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 Ca 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 &amp; 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 Ca 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 Ca response through MS channels, we attempted to visualize the mechanical stress-induced Ca response using a real-time confocal imaging system. Our results showed for the first time that the mechanical stress-induced local Ca increase, which is completely inhibited by Gd, could be clearly visualized in the presence of LPA in cultured lens epithelial cells. These results suggest that the local Ca increase, ‘Ca spots’, may represent an elementary Ca-influx event through MS channels underlying mechanical stress-induced cellular responses and that LPA may function as an endogenous factor affecting mechanotransduction systems.

Acknowledgments

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.

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