Imaging of intracellular calcium stores in single permeabilized lens cells

Grant C. Churchill1 and Charles F. Louis1,2

Departments of 1 Biochemistry and 2 Veterinary PathoBiology, University of Minnesota, St. Paul, Minnesota 55108


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Intracellular Ca2+ stores in permeabilized sheep lens cells were imaged with mag-fura 2 to characterize their distribution and sensitivity to Ca2+-releasing agents. Inositol 1,4,5-trisphosphate (IP3) or cyclic ADP-ribose (cADPR) released Ca2+ from intracellular Ca2+ stores that were maintained by an ATP-dependent Ca2+ pump. The IP3 antagonist heparin inhibited IP3- but not cADPR-mediated Ca2+ release, whereas the cADPR antagonist 8-amino-cADPR inhibited cADPR- but not IP3-mediated Ca2+ release, indicating that IP3 and cADPR were operating through separate mechanisms. A Ca2+ store sensitive to IP3, cADPR, and thapsigargin appeared to be distributed throughout all intracellular regions. In some cells a Ca2+ store insensitive to IP3, cADPR, thapsigargin, and 2,4-dinitrophenol, but not ionomycin, was present in a juxtanuclear region. We conclude that lens cells contain intracellular Ca2+ stores that are sensitive to IP3, cADPR, and thapsigargin, as well as a Ca2+ store that appears insensitive to all these agents.

inositol 1,4,5-trisphosphate; cyclic ADP-ribose; calcium pools; mag-fura 2; epithelium


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

MANY EXTRACELLULAR SIGNALS increase cytosolic Ca2+ by activating the release of Ca2+ from intracellular stores (2). In the best-characterized mechanism for Ca2+ release, the second messenger inositol 1,4,5-trisphosphate (IP3) activates intracellular Ca2+ channels (2). Although its mechanism is not as well characterized as that of IP3, cyclic ADP-ribose (cADPR) also activates Ca2+ release from intracellular stores in sea urchin eggs (6, 20) and certain mammalian cells (5, 9, 11, 13, 14, 18).

Intracellular Ca2+ stores have commonly been characterized by their sensitivity to Ca2+-releasing agents, either physiological or pharmacological. Most cells contain Ca2+ pools that are sensitive to IP3, thapsigargin, and ionomycin (2), and some cells contain a Ca2+ pool that is sensitive to cADPR (5, 6, 9, 11, 13, 14, 18, 20). These Ca2+ pools overlap to varying degrees, suggesting that the intracellular Ca2+ stores are functionally organized into distinct compartments sensitive to only certain agents (2, 6, 9, 11, 13, 14, 18, 20). The location of intracellular Ca2+ stores sensitive to IP3, inhibitors of the intracellular Ca2+ pump, activators of ryanodine receptors, or ionomycin has been revealed by direct imaging of permeabilized cells, the organelles of which contain a Ca2+-sensitive dye (11, 12, 15-17, 26, 27, 29). In contrast, the location of cADPR-sensitive Ca2+ stores has not been defined, because the cells containing the Ca2+ stores that have been imaged were unresponsive to cADPR (17, 29). Nevertheless, it has been demonstrated that certain intracellular Ca2+ stores such as the envelope of isolated nuclei (10) and isolated zymogen granules (9) are sensitive to cADPR and IP3.

The ocular lens is a transparent tissue containing only two cell types: fiber cells, which lack organelles and make up the bulk of its mass, and epithelial cells, which contain organelles and form a single layer on its anterior surface (22). In the lens the loss of Ca2+ homeostasis is implicated in the loss of transparency and cataract formation (7), so it is important to better define the mechanisms by which Ca2+ is regulated in the lens. Duncan and co-workers (8) demonstrated that permeabilized human lens cells in suspension exhibit thapsigargin-sensitive, ATP-dependent Ca2+ uptake and IP3-mediated Ca2+ release. However, the location of the intracellular Ca2+ stores, their sensitivity to other Ca2+-releasing agents, and the overlap among the various Ca2+ pools are unknown for lens cells of any species.

The objective of this study was to characterize the intracellular Ca2+ stores of mammalian lens cells in terms of their distribution and sensitivity to Ca2+-releasing agents. Intracellular Ca2+ stores in permeabilized cells were imaged with mag-fura 2 and fluorescence microscopy (15). A sheep lens cell culture system (28) was used in which the cells exhibit agonist-mediated Ca2+ signaling (4) as well as cell-to-cell Ca2+ waves (3). We conclude that lens cells contain IP3-, cADPR-, and thapsigargin-sensitive intracellular Ca2+ stores that are distributed throughout the cell, as well as Ca2+ stores that are insensitive to all these agents that are localized in a juxtanuclear region.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Materials. cADPR and 8-amino-cADPR were generous gifts from Dr. Timothy Walseth (University of Minnesota). Sheep eyes were obtained from John Morrell (Sioux Falls, SD). Medium 199 and Hanks' balanced salt solution (HBSS) were obtained from GIBCO (Grand Island, NY). Fetal calf serum was obtained from Hyclone (Logan, UT). Mag-fura 2-AM was obtained from Molecular Probes (Eugene, OR). Ionomycin, thapsigargin, D-IP3, and L-IP3 were obtained from LC Laboratories (Woburn, MA). Saponin, digitonin, ADP-ribose, beta -NAD+, ATP, creatine, creatine kinase (porcine), heparin (6-kDa average fragment size), and all other chemicals were obtained from Sigma Chemical (St. Louis, MO).

Cell culture. Primary cultures of cells isolated from the equatorial region of fresh sheep lenses were prepared as described previously (28). The sheep lens epithelial cells used in this study were cultured for 4-34 days.

Monitoring intracellular store Ca2+ concentration. Cells were maintained in HBSS supplemented with 10 mM HEPES (HBSS-H, pH 7.2) before they were loaded with mag-fura 2 by incubation in 6 µM mag-fura 2-AM for 1 h at 37°C, which promotes compartmentalization of the dye into intracellular organelles (15). After they were loaded, cells were rinsed three times with HBSS-H and incubated for 20 min at 37°C to promote complete hydrolysis of the AM ester. The glass coverslip with attached cells formed the bottom of a microincubation culture chamber (model MS 200D, Medical Systems, Greenvale, NY). The chamber was mounted on the stage of an inverted epifluorescence microscope (model IM 35, Zeiss) supported on a vibration-isolated table (Technical Manufacturing, Peabody, MA). Cells were viewed through a ×40, 1.3-NA, oil-immersion objective lens (Fluor 40, Nikon, Melville, NY).

Mag-fura 2 was excited with light from a 50-W mercury lamp alternately filtered to 340 or 380 nm. Fluorescence emission was filtered to >510 nm, focused with a ×20 lens, and monitored with a silicon intensified target camera (model VE-1000, Dage-MTI, Michigan City, IN). The camera's gain and kilovolts were set to manual and initially adjusted for the fluorescence intensity of intact cells but were increased to monitor permeabilized cells to compensate for the smaller fluorescence due to the loss of cytosolic dye. Images were captured and processed with the software package Image-1/Fluorescence (Universal Imaging, West Chester, PA). Unprocessed images were recorded in digital form to an optical memory disk recorder (model TQ-3031F, Panasonic, Secaucus, NJ). Images were captured at 512 × 480 pixels at 256 intensity values. The software performed a background subtraction and a shading correction and then calculated the 340 nm-to-380 nm ratios for each matched pixel pair from the 340- and 380-nm intensity images and displayed the resulting image in pseudocolor. Thresholding was used to eliminate pixels that were too dim at either wavelength to calculate an accurate ratio.

Cells were permeabilized by exchanging the HBSS-H with an intracellular buffer lacking ATP and containing saponin (50 µg/ml) or digitonin (10-15 µg/ml). The intracellular buffer was composed of (in mM) 120 KCl, 20 NaCl, 10 HEPES, 3 MgSO4, 1 EGTA, 0.75 CaCl2, 3 (or 5) dithiothreitol, 3 Na2ATP, and 30 creatine phosphate and 10 U/ml creatine kinase. Free Ca2+ concentration was calculated to be 300 nM. The extent of permeabilization was monitored on-line. When ~75% of the cells showed a decrease in fluorescence intensity due to the loss of cytosolic mag-fura 2, the detergent was washed out by several exchanges of intracellular buffer supplemented with ATP (equivalent to 10-30 times the chamber volume). Unless otherwise noted, images were captured every 30 s to minimize photobleaching of the mag-fura 2.

Additions of chemicals and solution changes. The major problem encountered with solution additions or exchanges was that the fluid flow caused the permeabilized cells to detach from the coverslip. Therefore, to minimize fluid flows during solution additions, a 1 × 1-cm piece of Kimwipe was placed on the edge of the chamber in contact with the bathing solution. New solutions were transferred onto the edge of the Kimwipe with a pipette, which allowed gravity flow of the solution into the chamber at a relatively slow rate. Solutions were removed with an aspirator, the height of which could be adjusted to control the volume in the chamber (0.3-5 ml). Ca2+-releasing agents were added to the cells by dilution of the stock solution (never >1% of the final volume bathing the cells) into ~100 µl of the intracellular buffer and addition of this to the chamber. Then 100 µl of the intracellular buffer were removed and readded five times from different regions of the microincubator to ensure complete mixing of the added compound.

Data analysis and presentation. Data were analyzed only from regions that exhibited a stable mag-fura 2 fluorescence ratio for at least 5 min in the absence of a Ca2+-releasing agent. The results are from single experiments that are representative of the most frequently observed response for a given treatment. The number of independent experiments (n) is taken as the response of a field of 5-30 cells to a given treatment. All experiments were repeated a minimum of three but typically many more times.

Ca2+ concentrations are not presented as absolute values but, rather, as the ratio of mag-fura 2 fluorescence at 340-nm excitation to that at 380-nm excitation, because the amount of Mg2+ bound to mag-fura 2 is unknown, making the estimate of Ca2+ concentration potentially inaccurate (16, 27). Nevertheless, changes in Ca2+ concentration are reflected by the mag-fura 2 340 nm-to-380 nm fluorescence ratio, which is directly proportional to the concentration of free Ca2+ (dissociation constant = 53 µM) (15, 25).


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Monitoring Ca2+ concentration in intracellular stores with mag-fura 2. The first step in this study was to determine whether, as reported previously in other cell types (15), the Ca2+ concentration in intracellular stores in lens cells could be directly monitored with mag-fura 2. After mag-fura 2 loading but before permeabilization, most cells exhibited a similar fluorescence ratio throughout the cells (Fig. 1Ba, ratio, cell 2); however, certain cells exhibited a higher fluorescence ratio (Fig. 1Ba, ratio, cell 1), likely because of the presence of mag-fura 2 in intracellular compartments. After the addition of the intracellular buffer with 300 nM Ca2+, the cells began to round up and pull apart from one another (Fig. 1Bb), and the mag-fura 2 ratio decreased in the cells that had an elevated fluorescence ratio (Fig. 1, A and Bb, ratio). This decrease in the mag-fura 2 ratio was apparently due to the release of Ca2+ from intracellular stores and was likely due to the mechanical strain imposed on the cells during the change in morphology, since switching to low-Ca2+-concentration medium can trigger a transient increase in cytosolic Ca2+ concentration (data not shown). After incubation for ~9 min in an intracellular buffer containing digitonin (10 µg/ml) without ATP, most cells were permeabilized on the basis of a loss of fluorescence intensity that was most evident over nuclear regions (Fig. 1Bc, 340 nm). The overall decrease in fluorescence intensity with permeabilization is not evident in Fig. 1Bc, because the camera's amplification (gain and kV settings) was increased to compensate for the loss of dye from the cytosol. The remaining fluorescence was concentrated in certain regions that are likely organelles with trapped dye, which appeared as evenly fluorescent regions or punctate vesicular compartments (Fig. 1Bc, 340 nm). After the addition of 3 mM ATP, the mag-fura 2 fluorescence ratio increased rapidly for ~5 min and then more slowly over the next 20 min (Fig. 1A). These data demonstrate that mag-fura 2 can be used to monitor the Ca2+ concentration in the intracellular stores of sheep lens cells.


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Fig. 1.   Permeabilization and Ca2+ uptake into intracellular stores in mag-fura 2-loaded sheep lens epithelial cells. A: change in mag-fura 2 fluorescence ratio after lowering of extracellular Ca2+, addition of digitonin, and addition of ATP. B: field of cells presented as fluorescence at 340-nm excitation (340 nm) and pseudocolor images (ratio). Color corresponds to ratio as indicated by calibration scale in A. Camera's gain and kV were increased after permeabilization (b and c) to compensate for decrease in fluorescence resulting from loss of cytosolic mag-fura 2. Thus loss in mag-fura 2 fluorescence intensity on permeabilization is not evident in these images, except over nuclei. Two cells outlined in pseudocolor images in B were used to obtain graphs of Ca2+ concentration vs. time in A. Cells were loaded with mag-fura 2-AM at 37°C for 1 h, rinsed with Hanks' balanced salt solution-HEPES, and imaged (a). Hanks' balanced salt solution-HEPES was then exchanged with intracellular buffer containing 300 nM free Ca2+, no ATP, and 15 µg/ml digitonin (b). Cells were washed with digitonin-free intracellular buffer (c) and incubated in a buffer containing 3 mM ATP, 30 mM creatine phosphate, and 10 U/ml creatine kinase (d). Data are from a single experiment representative of response observed in 599 of 911 cells in 49 experiments in which 49 cultures were used.

IP3 releases Ca2+ from intracellular stores. That the Ca2+ concentration in intracellular stores could be monitored in permeabilized lens cells enabled the activity of Ca2+-releasing agents to be tested by directly adding them to the medium bathing the permeabilized cells. IP3 mobilizes Ca2+ in many cell types (2), and 0.1-5 µM has been shown to mobilize Ca2+ from permeabilized human lens cells in suspension (8). Figure 2 shows the effect of various concentrations of IP3 on the intracellular store Ca2+ concentration in a cytoplasmic region of a permeabilized sheep lens cell. The addition of submaximal concentrations (0.16-8 µM) of IP3 resulted in a rapid release of only a fraction of the Ca2+ that could be released with maximal concentrations (>= 16 µM) of IP3 (Fig. 2). This phenomenon is termed quantal Ca2+ release and is commonly reported during IP3-mediated Ca2+ release in other cell types (2, 21).


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Fig. 2.   Effect of inositol 1,4,5-trisphosphate (IP3) concentration ([D-IP3]) on intracellular store Ca2+ concentration in permeabilized lens cells. Increasing concentrations of IP3 were added at arrows. Mag-fura 2 ratio was obtained from a cytoplasmic region (~10 µm2). Data are from a single experiment representative of response observed in 73 of 134 cells in 11 similar experiments in which 11 cultures were used.

cADPR releases Ca2+ from intracellular stores. cADPR is known to mobilize Ca2+ from intracellular stores in sea urchin eggs (6, 20), as well as in certain mammalian cells (5, 9, 11, 13, 14, 18). To determine whether cADPR could release Ca2+ from permeabilized lens cells, cADPR was added to the buffer bathing permeabilized sheep lens cells. The effect of cADPR on the intracellular store Ca2+ concentration was variable; therefore, the results from three experiments are presented, which depict the different types of responses of permeabilized lens cells to added cADPR. In the experiment shown in Fig. 3A, 10 µM cADPR elicited a monophasic, nonquantal decrease in the intracellular store Ca2+ concentration that did not fully deplete these Ca2+ stores, even after 20 min. In the experiment shown in Fig. 3B, 0.5 µM cADPR elicited a monophasic, nonquantal decrease in the intracellular store Ca2+ concentration; the rate of Ca2+ release was not accelerated by the addition of higher concentrations of cADPR. In contrast, in the experiment shown in Fig. 3C, 0.5 µM cADPR elicited a rapid, quantal decrease in the Ca2+ concentration in intracellular stores. The subsequent addition of 5 µM cADPR led to no further Ca2+ release, whereas the addition of 50 µM cADPR resulted in further quantal Ca2+ release (Fig. 3C). A similar quantal Ca2+ release is shown in Fig. 3D; however, in this experiment the cells were monitored for 10 min before the first addition of the cADPR, demonstrating that the Ca2+ concentration in the intracellular stores was stable until the addition of cADPR. These results indicate that although cADPR can mobilize Ca2+ from intracellular stores in sheep lens cells, the response to cADPR addition was more variable than that obtained with IP3 in terms of the rate of Ca2+ release and whether the Ca2+ release was quantal.


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Fig. 3.   Effect of cyclic ADP-ribose (cADPR) concentration ([cADPR]) on intracellular store Ca2+ concentration in permeabilized lens cells. Indicated concentrations of cADPR were added at arrows. Different types of responses were observed after addition of cADPR: a relatively slow monophasic decrease in Ca2+ concentration (A), a slow decrease in Ca2+ concentration that did not accelerate with increasing concentrations of cADPR (B), and a relatively rapid quantal decrease in the Ca2+ concentration (C and D). Data are from single experiments representative of rates of release observed in 77 of 336 cells (A), 14 of 336 cells (B), and 112 of 336 cells (C and D) in 36 similar experiments in which 17 cultures were used.

Specificity of IP3- and cADPR-mediated Ca2+ release. To determine whether IP3 and cADPR released Ca2+ from intracellular stores through specific and separate mechanisms, the effect of specific antagonists and analogs of these Ca2+-releasing messengers was evaluated. In the first approach the responses to IP3 and cADPR were assessed in the presence of the IP3 antagonist heparin (2) or the cADPR antagonist 8-amino-cADPR (31). Heparin (100 µg/ml) blocked the Ca2+ release mediated by 16 µM IP3, but not that mediated by 10 µM cADPR (Fig. 4A). Conversely, 8-amino-cADPR (40 µM) blocked the Ca2+ release mediated by 10 µM cADPR, but not that mediated by 16 µM IP3 (Fig. 4B). These results indicate that IP3 and cADPR are acting through separate mechanisms.


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Fig. 4.   Specificity of IP3- and cADPR-dependent Ca2+ release from intracellular stores in permeabilized lens cells. A: effect of IP3 antagonist heparin (100 µg/ml) on Ca2+ release mediated by IP3 (16 µM) or cADPR (10 µM). B: effect of cADPR antagonist 8-amino-cADPR (8NH2-cADPR, 40 µM) on Ca2+ release mediated by cADPR (10 µM) or IP3 (16 µM). C: effect of compounds structurally similar to IP3 or cADPR on Ca2+ release: nonphysiological enantiomer L-IP3 (32 µM); hydrolytic product of cADPR, ADPR (50 µM); precursor to cADPR, beta -NAD+ (200 µM); and cADPR (50 µM). Data are from single experiments representative of response observed in 29 of 50 cells in 5 similar experiments (A) and 19 of 21 cells in 4 similar experiments (B) in which 8 cultures were used and 65 of 77 cells in 5 similar experiments in which 5 cultures were used (C).

In the second approach the Ca2+-releasing activity of IP3 or cADPR analogs was evaluated. IP3 is an enantiomer, and the naturally occurring D-form has a 100- to 1,000-fold greater affinity than the L-form for the IP3 receptor (24). In sea urchin eggs, cADPR is formed from beta -NAD+ and is metabolized to ADPR (20). Ca2+ was not released from intracellular Ca2+ stores in permeabilized lens cells after the addition of 32 µM L-IP3, 50 µM ADPR, or 200 µM beta -NAD+, but Ca2+ was released after the addition of 50 µM cADPR (Fig. 4C). Collectively, these results indicate that IP3 and cADPR release Ca2+ through specific mechanisms.

Spatial distribution and functional overlap of the intracellular Ca2+ stores sensitive to IP3, cADPR, and thapsigargin. The next set of experiments was designed to reveal whether lens cells contained a single intracellular Ca2+ store sensitive to all Ca2+-releasing agents or multiple intracellular Ca2+ stores sensitive to only certain agents. Also, to determine whether functionally distinct Ca2+ stores were also spatially distinct, as demonstrated recently in astrocytes and myocytes (12), three regions of interest were defined: one over the nucleus, a second over a juxtanuclear region, and a third over a cytoplasmic region. An experiment in which the IP3-sensitive Ca2+ stores were depleted before the addition of cADPR is shown in Fig. 5, A and C. The addition of 16 µM IP3 released a portion of the stored Ca2+ from all three regions monitored (Fig. 5, A and C), and the subsequent addition of 32 µM IP3 led to the release of additional Ca2+ (Fig. 5, A and C). The subsequent addition of 30 µM cADPR failed to release any additional Ca2+, indicating that IP3 had completely depleted the Ca2+ stores sensitive to cADPR. The addition of thapsigargin released a small amount of additional Ca2+ from all three intracellular regions. The addition of 10 µM ionomycin released the remaining Ca2+ from all the intracellular Ca2+ stores in all regions of the cell. The nuclear region appears to have ionomycin-insensitive Ca2+ stores, but this is an artifact arising from the low fluorescence intensity of mag-fura 2 in this region after permeabilization of the cells (Fig. 1B). The weaker the mag-fura 2 fluorescence, the more the ratio will be influenced by noise. The pixel intensities due to noise above the background will be approximately equal at 340- and 380-nm excitation, yielding a ratio of ~1. This "noise" ratio is averaged with the ratio reporting Ca2+, presumably 0.6, on the basis of the other regions of the cell that have sufficient dye to accurately report on Ca2+, resulting in an apparent ratio of 0.8. 


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Fig. 5.   Spatial distribution and functional overlap of Ca2+ stores sensitive to IP3, cADPR, thapsigargin, and ionomycin in permeabilized lens cells. A and D: changes in mag-fura 2 fluorescence ratio of 3 subcellular regions after addition of indicated compounds. B and E: fluorescence images of permeabilized cells showing intracellular distribution of Ca2+ stores based on mag-fura 2. C and F: images of monitored cells in which mag-fura 2 fluorescence ratio is presented in pseudocolor according to calibration scale. Three intracellular regions used to obtain traces in A and D are indicated and correspond to nuclear (circles), juxtanuclear (triangles), and cytoplasmic (squares) regions. Compounds were added as indicated by arrows at concentrations and sequence as follows: 16 and 32 µM IP3, 30 µM cADPR, 1 µM thapsigargin (Tg), and 10 µM ionomycin (iono) in A and 1 and 30 µM cADPR, 16 µM IP3, 1 µM thapsigargin, and 10 µM ionomycin in B. Data are from single experiments representative of responses observed in 55 of 96 cells in 8 similar experiments in which 8 cultures were used and 71 of 145 cells in 11 similar experiments in which 11 cultures were used. Ca2+ was not released by initial addition of IP3 in 75 of 88 cells or cADPR in 110 of 120 cells. Thapsigargin released a portion of stored Ca2+ in 204 of 208 cells, and ionomycin released all stored Ca2+ in 208 of 208 cells.

An experiment in which the cADPR-sensitive Ca2+ stores were depleted before the addition of IP3 is shown in Fig. 5, D and F. The addition of 1 µM cADPR resulted in a slow release of Ca2+ from all three intracellular regions (Fig. 5, D and F). The subsequent addition of 30 µM cADPR did not release appreciably more Ca2+, and all three regions reached a new steady state soon thereafter. The addition of IP3 released additional Ca2+ from all three regions, and this release was accelerated by the addition of thapsigargin. The addition of ionomycin rapidly released all the remaining Ca2+ from all three regions.

Thapsigargin-, IP3-, and cADPR-insensitive intracellular Ca2+ stores. In some cell types such as sea urchin eggs, the thapsigargin-sensitive Ca2+ pools completely overlap the IP3- and/or cADPR-sensitive Ca2+ pools (20); however, in other cell types, these Ca2+ pools are separable. For example, cADPR releases Ca2+ from a thapsigargin-insensitive Ca2+ pool in T lymphocytes (14), and IP3 and cADPR, but not thapsigargin, release Ca2+ from secretory granules isolated from pancreatic acinar cells (9). Therefore, it was of interest to determine whether cADPR or IP3 could release Ca2+ from a thapsigargin-insensitive store in lens cells. To address this issue, mag-fura 2-loaded permeabilized lens cells were treated sequentially with thapsigargin, IP3, and cADPR (Fig. 6). Thapsigargin almost fully depleted all the intracellular Ca2+ stores in the cytoplasmic regions but only partially depleted the intracellular Ca2+ stores in a juxtanuclear region (Fig. 6, A and Bb). Neither the thapsigargin-sensitive nor the thapsigargin-insensitive Ca2+ stores released any additional Ca2+ in response to the subsequent additions of 16 µM IP3 or 30 µM cADPR (Fig. 6A). These data demonstrate that the thapsigargin-sensitive Ca2+ stores appear to completely overlap the IP3- and cADPR-sensitive Ca2+ stores and that the thapsigargin-insensitive Ca2+ stores are also IP3 and cADPR insensitive. In some cell types, certain Ca2+ stores have been reported to contain a thapsigargin-resistant Ca2+ pump with an IC50 for thapsigargin of 5 µM compared with the more typical IC50 of ~0.2 µM (30); therefore, a higher concentration of thapsigargin was applied (10 µM) but was also without effect (Fig. 6A). In contrast, ionomycin rapidly depleted these Ca2+ stores (Fig. 6, A and Bc).


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Fig. 6.   Characterization of a thapsigargin-insensitive intracellular Ca2+ store in permeabilized lens cells. A: changes in mag-fura 2 fluorescence ratio of 2 intracellular regions after addition of indicated compounds. B: fluorescence images of permeabilized cell showing intracellular distribution of Ca2+ stores retaining mag-fura 2 (340 nm); mag-fura 2 fluorescence ratio is presented in pseudocolor according to calibration scale. Two intracellular regions used to obtain traces in A are indicated in B and correspond to a juxtanuclear region () and a cytoplasmic region (black-triangle). Compounds were added as indicated by arrows at concentrations and sequence as follows: 1 µM thapsigargin, 16 µM IP3, 30 µM cADPR, 10 µM thapsigargin, and 10 µM ionomycin. Data are from a single experiment representative of response observed in 6 similar experiments in which 6 cultures were used, and 31 of 68 cells showed a juxtanuclear thapsigargin-insensitive Ca2+ store that was sensitive to only ionomycin.

Mag-fura 2 fluorescence has been shown to respond to mitochondrial Ca2+ concentration (16); therefore, it was considered that the thapsigargin-, IP3-, and cADPR-insensitive Ca2+ stores identified in sheep lens cells might be mitochondria. Mitochondrial Ca2+ can be released by agents that dissipate their electrochemical gradient (17). After the addition of thapsigargin to deplete all but the thapsigargin-insensitive Ca2+ stores, the addition of the proton ionophore 2,4-dinitrophenol did not release Ca2+ from the thapsigargin-insensitive Ca2+ stores, whereas ionomycin rapidly released this Ca2+ (Fig. 7). These results indicate that the thapsigargin-, IP3-, and cADPR-insensitive Ca2+ stores were not mitochondria.


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Fig. 7.   Effect of 2,4-dinitrophenol (DNP) on thapsigargin-insensitive intracellular Ca2+ stores. Changes in mag-fura 2 fluorescence ratio of 2 intracellular regions after addition of 1 µM thapsigargin, 100 µM 2,4-dinitrophenol, and 10 µM ionomycin are indicated by arrows. Traces correspond to a cytoplasmic region () and a juxtanuclear region (black-triangle). Data are from a single experiment representative of responses observed in 7 similar experiments in which 7 cultures were used, and 35 of 88 cells showed a juxtanuclear thapsigargin-insensitive Ca2+ store that was insensitive to 2,4-dinitrophenol.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Intracellular Ca2+ stores in permeabilized sheep lens cells were imaged with mag-fura 2 to characterize their distribution and sensitivity to Ca2+-releasing agents. The intracellular Ca2+ stores exhibited ATP-dependent Ca2+ uptake and IP3- and cADPR-mediated Ca2+ release. These results are consistent with the previous report of IP3-mediated Ca2+ release in permeabilized human lens cells (8) and with the ability of an inhibitor of phospholipase C to prevent agonist-mediated cytosolic Ca2+ increases in sheep lens cells (4).

In permeabilized lens cells the response to cADPR was less consistent than the response to IP3 in regard to the cADPR concentration dependence and the rate of Ca2+ release. This may relate to the loss of a soluble factor(s) from the permeabilized cells such as calmodulin, which has been shown to be required for cADPR-mediated Ca2+ release in sea urchin egg homogenates (19, 20). The variable rate of Ca2+ release in response to even maximal concentrations of cADPR may relate to the number of cADPR-activated Ca2+ channels per intracellular compartment in different lens cells in these primary cultures. If only a few such channels are present, then the number of channels would be the rate-limiting step in Ca2+ store depletion, and the rate of release would likely be independent of cADPR concentration. In contrast, cells with a greater number of cADPR-dependent channels would likely exhibit a more pronounced concentration dependence. Although the conditions required to improve the consistency of the response to cADPR remain to be defined, as does the physiological significance of cADPR in lens cells, it is clear that cADPR can stimulate Ca2+ release from intracellular Ca2+ stores in sheep lens cells.

By directly imaging intracellular Ca2+ stores in permeabilized lens cells, we have demonstrated that IP3 and cADPR can release Ca2+ from stores that appear to be distributed throughout the entire cell. The IP3- and cADPR-sensitive Ca2+ stores in lens cells appeared to be functionally distinct in terms of the relative overlap of the these two Ca2+ pools. In some experiments the IP3- and cADPR-sensitive Ca2+ pools completely overlapped, whereas in other experiments cADPR released only a portion of the IP3-releasable Ca2+ pool. Similarly, the portion of the thapsigargin-sensitive Ca2+ pool released by IP3 was variable. Taken together, these results indicate heterogeneity in the Ca2+ stores in lens cells. This heterogeneity was not manifested as spatially distinct Ca2+ stores sensitive to IP3 or cADPR, however, indicating that the Ca2+ stores might be organized into distinct compartments on a scale that is below the spatial resolution of conventional microscopy or organized along the axis through which the cells are viewed. Inasmuch as conventional microscopy was used in this study, only the average Ca2+ in all stores through the thickness of the cell was detected.

Regardless of the intracellular organization of the Ca2+ stores, our data indicate the presence of at least three types of functionally distinct Ca2+ stores in lens cells. These Ca2+ pools may represent physically distinct Ca2+ stores with different complements of Ca2+ channels and pumps. In one type of Ca2+ store there would be channels activated by IP3 and cADPR as well as a thapsigargin-sensitive Ca2+ pump. In the second type of Ca2+ store there would be Ca2+ channels activated by IP3 but not cADPR as well as a thapsigargin-sensitive Ca2+ pump. In the third type of Ca2+ store there would be no Ca2+ channels activated by either IP3 or cADPR and no thapsigargin-sensitive Ca2+ pump. The relative abundance of each of these Ca2+ stores in any intracellular region would determine the degree of functional overlap among the various Ca2+ pools in that region.

This third type of Ca2+ store that was insensitive to IP3, cADPR, and thapsigargin was functionally and spatially distinct from the Ca2+ stores sensitive to these agents. This store's juxtanuclear location indicates that it may be the Golgi apparatus, although this could not be proven with organelle-selective dyes, because they failed to resolve the organelle distribution in lens cells (data not shown). IP3- and thapsigargin-insensitive Ca2+ pools have been reported previously in other cell types (16, 23, 30). Similar to lens cells, a Ca2+ store in a juxtanuclear region exhibited reduced responsiveness to IP3 in BHK-21 cells; however, unlike lens cells, this store was sensitive to thapsigargin (17). Hofer et al. (17) proposed that the partial Ca2+ release attained in the intracellular regions containing the Golgi apparatus arose from superimposition of the Golgi apparatus Ca2+ stores (IP3 insensitive) with the endoplasmic reticulum Ca2+ stores (IP3 sensitive). In conclusion, regardless of its identity, lens cells contain an intracellular compartment that stores Ca2+ but does not release Ca2+ in response to the Ca2+-releasing agents used in this study and thus would not serve as a Ca2+ store releasable by IP3 or cADPR.

The variability of the permeabilized lens cells to the Ca2+-releasing agents, together with the variable sizes of the Ca2+ pools in different lens cells and experiments, might relate to the differentiation of the lens cells from epithelial to fiber cells that occurs in this lens cell culture system (28). Lens cell differentiation involves large changes in the chemistry and structure of the cells (1, 22). Of particular note is the fragmentation and dispersion of the Golgi apparatus, followed by the loss of the endoplasmic reticulum and all organelles (1). During differentiation, remodeling of the intracellular Ca2+ stores could result in changes in their sensitivity to Ca2+-releasing agents and changes in the relative sizes of the Ca2+ pools sensitive to a given agent.

In conclusion, we have demonstrated that sheep lens cells contain Ca2+ stores that are sensitive to IP3, cADPR, and thapsigargin and that these stores are distributed throughout the cell. Additionally, Ca2+ stores are also present in some lens cells that are insensitive to IP3, cADPR, thapsigargin, and 2,4-dinitrophenol and are localized in a juxtanuclear region. It will be important to examine whether depletion of any of these Ca2+ stores is responsible for Ca2+ regulation defects that result in cataract formation.


    ACKNOWLEDGEMENTS

We thank Tim Walseth (University of Minnesota) for generously providing cyclic ADP-ribose and 8-amino-cyclic ADP-ribose and for helpful discussions.


    FOOTNOTES

G. C. Churchill was partially supported by a doctoral dissertation fellowship awarded by the Graduate School, University of Minnesota. This research was supported by National Eye Institute Grant EY-05684.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: C. F. Louis, Dept. of Veterinary PathoBiology, University of Minnesota, 1988 Fitch Ave., Rm. 295, St. Paul, MN 55108.

Received 15 April 1998; accepted in final form 12 October 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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