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(Investigative Ophthalmology and Visual Science. 2002;43:2341-2350.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Lysosomal Ca2+ Stores in Bovine Corneal Endothelium

Sangly P. Srinivas, Angeline Ong, Leanne Goon, Levina Goon and Joseph A. Bonanno

From School of Optometry, Indiana University, Bloomington, Indiana.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Acidic organelles, including Golgi bodies and lysosomes, are known to operate as Ca2+ storage sites in many cell types. This study demonstrates the presence of Ca2+ stores in lysosomes of bovine corneal endothelial cells (BCECs) and examines their interaction with Ins(1,4,5)P3-sensitive Ca2+ stores.

METHODS. Glycyl-L-phenylalanine-ß-naphthylamide (GPN) was used to release Ca2+ from lysosomes by inducing their selective osmotic swelling. Ca2+ released into the cytoplasm was measured with fura-2 or fura-PE3 fluorescent dyes. Fluorescence of acridine orange (AO), which selectively sequesters into acidic organelles, was used to establish swelling of lysosomes in response to GPN.

RESULTS. Exposure to GPN (100–200 µM) in cultured BCECs produced an increase in free cytosolic Ca2+ ([Ca2+]i) equivalent to approximately 79% of the peak response to uridine triphosphate (UTP), a P2Y agonist (n = 19). The endothelium of the freshly isolated cornea also produced [Ca2+]i transients similar to those in cultured BCECs; however, the peak [Ca2+]i increase was smaller (~43% of the peak response to UTP; n = 13). In cultured BCECs, the response to UTP was unaffected by pretreatment with GPN with extracellular calcium ([Ca2+]o) at 0 and 1.2 mM (n = 10). Neither pretreatment with thapsigargin (5 µM) nor with U73122 (a phospholipase C inhibitor; 10 µM) blocked the peak GPN response (n = 6). Exposure to 20 µM monensin produced a [Ca2+]i increase with [Ca2+]o at 0 and 1.2 mM and also reduced the subsequent peak response to GPN (n = 6).

CONCLUSIONS. GPN-sensitive lysosomal Ca2+ stores, distinct from Ins(1,4,5)P3-sensitive Ca2+ stores, are found in both cultured cells and fresh tissue. These stores are susceptible to depletion by the loss of the pH gradient across lysosomes and P2 agonists. The latter occurs through mechanisms independent of phospholipase C (PLC) activation or Ins(1,4,5)P3. The GPN stores also induce [Ca2+]o influx in response to their depletion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The homeostatic control of free cytosolic Ca2+ concentration ([Ca2+]i) is influenced by Ca2+ stores1 2 and Ca2+ transport mechanisms at the plasma membrane, consisting of pumps (i.e., Ca2+-adenosine triphosphatases [ATPases]), channels (receptor-operated Ca2+ channels, including capacitative Ca2+ entry [CCE]) through store-operated Ca2+ channels3 ), and translocators (i.e., Na+/Ca2+ exchange).1 The differences in the expression of these mechanisms contribute to the diversity in Ca2+ homeostasis among different cell types. For example, Ins(1,4,5)P3-sensitive Ca2+ stores, usually linked to a variety of G-protein–coupled cell surface receptors, are ubiquitous in mammalian cells,1 2 4 but ryanodine-sensitive stores are not widely expressed among nonmuscle cells4 5 (for exceptions, see Wu et al.6 ). In addition to Ins(1,4,5)P3-sensitive and ryanodine-sensitive stores,6 mitochondria1 7 and acidic organelles8 9 have also been reported to operate as Ca2+ storage organelles.

The specificity of the acidic organelles and their physiological significance as Ca2+ stores are not well understood in mammalian cells, although this has been relatively better examined in lower organisms.9 10 11 12 13 14 15 Consistent with the distribution of Ins(1,4,5)P3-sensitive and ryanodine-sensitive Ca2+ stores in the endoplasmic reticulum (ER), recent studies have pointed out that lysosomes16 17 and Golgi bodies,18 19 20 21 22 23 which form part of the extended ER network, may also operate as Ca2+ storage organelles. A study with yeast13 has demonstrated a P-type Ca2+-ATPase that is selectively targeted to Golgi bodies, suggesting a potential mechanism for Ca2+ accumulation. A Ca2+ binding protein (CALNUC or nucleobindin) with a single high-affinity, low-capacity Ca2+-binding site is expressed selectively in Golgi bodies.21 22 Haller et al.16 17 and Yagodin et al.,15 have found that lysosomes in Madin-Darby canine kidney (MDCK) cells and the S2 cell lines of Drosophila melanogaster operate as Ca2+ stores that are thapsigargin insensitive, functionally coupled to Ins(1,4,5)P3-sensitive Ca2+ stores, and incapable of activating CCE pathways. The coupling with Ins(1,4,5)P3-sensitive Ca2+ stores has been shown to be unidirectional, so that the agents that release Ins(1,4,5)P3-sensitive Ca2+ stores (e.g., thapsigargin or P2Y receptor agonists adenosine triphosphate [ATP] and uridine triphosphate [UTP]) also deplete the lysosomal Ca2+ stores.16 17 However, the depletion of lysosomal Ca2+ stores did not affect the Ins(1,4,5)P3-sensitive Ca2+ stores. These findings are somewhat similar to recent observations with Ca2+ stores associated with Golgi bodies,19 but are in contrast with mitochondrial Ca2+ stores, which buffer large intracellular Ca2+ overloads7 24 and are implicated in the modulation of CCE.7 25

In this study, the characteristics of lysosomal Ca2+ stores have been further explored in corneal endothelial cells and thereby demonstrate for the first time that acidic organelles function as Ca2+ storage sites in mammalian cells derived from primary cultures and native tissue. Corneal endothelial cells express a number of G-protein–coupled receptors capable of Ca2+ mobilization26 27 and therefore permit an investigation of the interrelationship between Ins(1,4,5)P3-sensitive and lysosomal Ca2+ stores. Specific questions investigated in this study included whether lysosomes are sites of Ca2+ storage in cultured and native bovine corneal endothelial cells (BCECs), whether there is a functional coupling between Ins(1,4,5)P3-sensitive and lysosomal Ca2+ stores, and whether the depletion of the lysosomal Ca2+ stores also induces extracellular Ca2+ influx.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Endothelial Cells
Primary cultures of BCECs) were established from bovine eyes, as described previously.27 Second- or third-passage cells were grown to confluence on 25-mm glass coverslips. Experiments were also conducted with endothelial cells associated with intact corneal buttons, as described previously.27 These were cut with an 18-mm trephine from fresh corneas dissected from eyes obtained from a local abattoir within 3 to 6 hours after death.

Chemicals
Lysosomal staining agent (LysoTracker Green DND-26; L-7526; Molecular Probes, Eugene, OR), acridine orange (AO; A-1301), and fura-2 were obtained from Molecular Probes (Eugene, OR). The green dye was supplied as a 50-mM stock solution in dimethylsulfoxide (DMSO). Stock solutions (100 mM) of AO were prepared in fresh Ringer’s solution every third day and kept at -20°C until use. Glycyl-L-phenylalanine-ß-naphthylamide (GPN) was obtained from Sigma (St. Louis, MO). A stock solution of GPN (100 mM) in ethyl alcohol was stored at -20°C. Thapsigargin, U73122, and U73343 were obtained from RBI (Natick, MA). Fura-PE3 was obtained from TefLabs (Austin, TX). Cell culture supplies were obtained from Gibco BRL (Grand Island, NY). All other chemicals were obtained from Sigma. Stock solutions of fura-PE3 (10 mM), fura-2 (10 mM), thapsigargin (10 mM), U73122 (10 mM), and U73343 (10 mM) were prepared in DMSO and stored at -20°C.

Solutions
All experiments were conducted with HCO3--free Ringer’s solution containing (in mM) 145 Na+, 4 K+, 0.6 Mg2+, 1.4 Ca2+, 120 Cl-, 1 HPO42-, 10 HEPES-, 28.5 gluconate, and 5 glucose. Ca2+-free Ringer’s solution was obtained by removing Ca2+ gluconate. Trace Ca2+ was chelated by 500 µM EGTA. Osmolarity of the Ringer’s solution, measured using a vapor pressure osmometer (model 5500; Wescor, Logan, UT), was adjusted to 300 ± 5 mOsM by adding sucrose. pH was adjusted to 7.5 at 37°C and equilibrated with air during perfusion.

Perfusion
A coverslip with a monolayer of cultured cells was mounted in a perfusion chamber (volume, 80 µL), maintained at 37°C, and placed on the stage of an inverted microscope equipped with an epifluorescence attachment.27 The flow of the perfusate, usually 320 to 480 µL/min, was achieved by gravity. A similar perfusion chamber, of which the bottom was formed by a glass coverslip, was used for trephined corneal buttons.27 The corneal button (18 mm in diameter) was placed on the top surface of the chamber coated with a thin layer of vacuum grease. The cornea was then held pressed down at the periphery on the top surface of the chamber by a clamp. On mounting, the endothelial cells faced the objective and were approximately 1 mm away from the bottom coverslip.

AO Fluorescence
Coverslips with a confluent monolayer of cells were placed in Ringers’ solution containing 200 µM AO at 25°C for approximately 6 minutes. AO was excited at 495 ± 10 nm. The emission (>530 nm) was passed through a dichroic centered at 510 nm and a 520-nm long-pass filter. The cells were viewed with a 63x objective (1.25 numeric aperture [NA]; Plan-Neofluar; Carl Zeiss, Thornwood, NY), and the field of view contained a minimum of six cells. The emission was detected by an intensified 8-bit charge-coupled device (CCD) camera. The fluorescence images were acquired every 8 seconds and analyzed in real time for average pixel intensities encompassing a set of predefined regions of interest (ROIs). The ROIs, which were manually selected, framed a concentric region outside the nucleus.

Spatial distribution of the acidic organelles was examined by confocal imaging (LSM 510; Carl Zeiss). The organelles were stained with AO and green fluorescent dye (LysoTracker Green; Molecular Probes). Cells were stained with the dye by incubation for approximately 20 minutes in Ringer’s solution containing 50 nM dye at 25°C.16 Both fluorescent probes were excited by an Ar laser (i.e., 488 nm). For the green fluorescence, emission was collected through a long-pass filter with a cutoff at 510 nm. For AO, dual-channel detection was used with an interference filter centered at 525 ± 10 nm for green fluorescence and long-pass filter for red fluorescence (>580 nm).

Measurement of [Ca2+]i in Cultured Cells
Cells were incubated in Ringer’s solution containing 10 µM fura-PE3-AM at 25°C for approximately 100 minutes in the presence of 0.025% nonionic detergent (Pluronic F-127; Biotium, Hayward, CA). Coverslips were then placed in dye-free Ringer’s solution for 40 minutes before use. Experiments (Figs. 9 10 11 12) were conducted with fura-2, in which the cells were loaded with a 10-µM solution at 25°C for approximately 40 minutes and placed in dye-free Ringer’s solution for 40 minutes before use. Fluorescence measurements were performed with an excitation monochromator (DeltaRam; Photon Technology International, Lawrenceville, NJ) adapted to an inverted microscope (Nikon, Tokyo, Japan), as described earlier. Cells were viewed with a 40x objective (1.3 NA, Fluor; Nikon). The excitation slits of the monochromator were adjusted to an approximately 5-nm bandwidth at 340 and 380 nm excitation wavelengths. The corresponding emissions, denoted by F340 and F380, were collected through a long-pass filter with a cutoff at 480 nm and directed to a photomultiplier operating in the photon-counting mode. In some experiments, cells were alternately exposed to 340 ± 11 and 380 ± 13 nm, by using interference filters, and the fluorescence emission was detected by the intensified CCD camera. The dichroic and the barrier filters were the same as those used with the imaging system described earlier. The images were acquired and analyzed in real time to determine average pixel intensities in a set of predefined ROIs framing boundaries of approximately six cells, similar to detection of AO fluorescence.



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  		Figure 9. GPN-induced [Ca2+]i mobilization in the presence of thapsigargin. Top: Where indicated, cells were first exposed to GPN (200 µM) and then to thapsigargin (5 µM). At the peak of response to thapsigargin, cells were exposed to GPN (200 µM) in the continued presence of thapsigargin (5 µM). All perfusates contained 1.2 mM Ca2+. The results are typical of those in six independent trials (i.e., six coverslips). Bottom: Summary of experiments similar to that shown at top. Leftmost two bars: experiments in which GPN was added after an initial exposure to thapsigargin. Data in the last three bars were obtained from experiments in which cells were exposed to GPN alone, followed by thapsigargin, and then thapsigargin (5 µM) + GPN (200 µM).

 


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  		Figure 10. UTP-induced [Ca2+]i mobilization in the presence of the PLC inhibitor U73122. (A) Where indicated, cultured cells were first exposed to UTP (50 µM) until a peak ratio was observed. Next, the cells were exposed to U73122 (10 µM), followed by UTP (50 µM) + U73122 (10 µM). The perfusate contained 1.2 mM Ca2+. The results are typical of those in four independent trials (i.e., four coverslips). (B) Similar to experiment shown in (A), but cells were exposed to U73122 for 4 minutes before the second exposure to UTP. (C) Similar to experiment shown in (A), but cells were exposed to U73122 for 7 minutes before the second exposure to UTP. Note that there was no response to UTP after 7 minutes of exposure to U73122. (D) Summary of experiments similar to that shown in (A). Leftmost two bars: experiments in which cells were exposed to U73122 for shorter periods before exposure to UTP.

 


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  		Figure 11. GPN-induced [Ca2+]i mobilization in the presence of the PLC inhibitor U73122. (A) Where indicated, cultured cells were first exposed to U73122 (10 µM) for approximately 9 minutes. Next, the cells were exposed to GPN (200 µM) + U73122 (10 µM). The perfusate contained 1.2 mM Ca2+. The results are typical of those in 20 independent trials (i.e., 20 coverslips). (B) Summary of experiments similar to those shown in (A). Left bar: control GPN response (from Fig. 3 ).

 


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  		Figure 12. GPN-induced [Ca2+]i mobilization in the presence of U73343, the inactive analogue of the PLC inhibitor U73122. All experiments were conducted with perfusate containing 1.2 mM Ca2+. (A) Where indicated, cultured cells were first exposed to UTP (50 µM) followed by U73343 (10 µM) for more than 7 minutes. Next, the cells were exposed to UTP in the continued presence of U73343. The results are typical of those in nine independent trials (i.e., nine coverslips). (B) Where indicated, cultured cells were first exposed to U73343 (10 µM) for more than 7 minutes. Next, the cells were exposed to GPN (200 µM) in the continued presence of U73343. The results are typical of those in 16 independent trials (i.e., 16 coverslips). (C) Summary of experiments similar to those shown in (A) and (B) and trials (n = 4) in which cells were exposed to GPN once before exposure to U73343. 1 and 2: exposure to UTP or GPN before and after treatment with U73343, respectively. Rightmost two bars: summary of experiments in Figure 3 shown for comparison with trials similar to those shown in (B).

 
Measurement of [Ca2+]i in Native Endothelial Cells
Endothelial cells associated with fresh corneas were loaded with fura-PE3 in a manner identical with that of cultured cells. The dye-loaded cornea was then trephined, and the resultant corneal button was mounted on the perfusion chamber described earlier. The cells were imaged using a long-working-distance, 40x objective (0.75 NA, water immersion, 1.2 mm working distance; Zeiss). Fluorescence was detected by the monochromator (DeltaRam; Photon Technology International) system as explained for the cultured cells. To improve sensitivity, however, the excitation light at 340 ± 5 nm was changed to 345 ± 5 nm, which enhanced the light throughput of the objective.27

Measurement of Mn2+ Influx
Relative rates of Ca2+ influx were measured using Mn2+ as a surrogate for Ca2+. In these experiments, 100 µM Ca2+ was replaced by 100 µM Mn2+. Fura-PE3–loaded cells were excited at 340, 360, and 380 nm, each with a bandwidth of 2.5 nm. Because fluorescence emission resulting from excitation at 360 nm (F360) is insensitive to changes in [Ca2+]i, it is a direct measure of Mn2+ influx and thus that of Ca2+.27 The ratio of F340/F380 was used to evaluate changes in [Ca 2+]i until Mn2+ influx became apparent by decline in both F340 and F380 intensities.27


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lysosomes in Cultured BCECs: Spatial Distribution
Figure 1A shows a typical staining pattern of AO observed by confocal microscopy. Consistent with the spectral characteristics of AO, the nuclear region appeared green (shown as light gray in the figure), because of the intercalation of AO to DNA. When AO is present at high concentrations, its fluorescence emission (at {lambda} >520 nm; denoted by F>520) is quenched by the formation of oligomers,28 29 30 31 resulting in orange fluorescence. Because AO preferentially accumulates in acidic organelles, orange spots (shown as bright spots in the figure) in the extranuclear region denote the spatial distribution of lysosomes around the nucleus in addition to other acidic organelles (e.g., endosomes). This pattern was further confirmed by the green fluorescent dye, which has a higher specificity for labeling acidic compartments.17 Unlike AO, the green dye showed no nonspecific labeling in the cytoplasm (Fig. 1B) .



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  		Figure 1. Labeling of acidic organelles in cultured corneal endothelial cells. Images were obtained by confocal microscopy immediately after labeling. (A) AO was excited at 488 nm. Images were collected through dual-channel detection. For green fluorescence, an interference filter with a peak at 520 ± 10 nm and for red fluorescence, a long-pass filter with a cutoff at 580 nm. The images were subsequently superimposed. The green fluorescence in the nucleus is due to AO that binds to DNA. (B) Green fluorescent dye was also excited at 488 nm. Emission was collected with a long-pass filter with a cutoff at 510 nm.

 
AO’s Response to GPN
When cultured cells were exposed to GPN, AO fluorescence increased (Fig. 2 , GPN at 100–200 µM; n = 6). GPN, a basic amine, accumulates in acidic organelles and is broken down by cathepsin C, leading to osmotic swelling.16 17 32 33 Furthermore, because cathepsin C is localized only in a subpopulation of lysosomes,32 33 the response to GPN shown in Figure 2 can be attributed to a decrease in AO concentration in the swelling lysosomes.



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  		Figure 2. GPN induced an increase in AO fluorescence. AO-labeled cells were excited at 475 nm, and emission more than 530 nm was detected by a CCD camera. The y-axis represents average pixel intensity of AO fluorescence (0–256 gray levels) from selected ROIs. Where indicated, the cells were exposed to GPN (100 µM). Each profile corresponds to a response in one ROI—10 total per field of view. Each ROI was contained in one cell and defined a region, so that the nucleus was excluded (inset). The results shown are typical of those in six independent experiments (i.e., six coverslips).

 
GPN-Induced [Ca2+]i Mobilization
In the presence of external Ca2+, exposure of cultured cells to GPN (100–200 µM) led to a transient increase in [Ca2+]i after an initial delay (Fig. 3 ; peak change in the F340/F380 ratio: {Delta}[ratio]peak = 0.69 ± 0.35; n = 19). This is equivalent to approximately 79% of the peak response to the P2 agonist, UTP.27 The increase in the ratio resulted from both an increase in F340 and a concomitant decrease in F380 (Fig. 3) , indicating that the GPN response underlies the increase in [Ca2+]i. When exposed to GPN for a second time after approximately 3 minutes, [Ca2+]i mobilization was less ({Delta}[ratio]peak = 0.40 ± 0.18, n = 12) than that in the first exposure.



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  		Figure 3. GPN exposure led to [Ca2+]i mobilization. Cultured cells loaded with fura-PE3 were excited at 340 ± 5 and 380 ± 5 nm sequentially. Where indicated, cells were exposed to GPN (100 µM) or UTP (100 µM). F340 and F380 are fluorescence emissions to excitation at 340 and 380 nm, respectively. F340/F380 is the ratio of emission intensities in response to excitation at 340 nm to that at 380 nm.

 
Figure 3 also shows that cells previously exposed to GPN responded to UTP. The extent of [Ca2+]i mobilization ({Delta}[ratio]peak = 0.88 ± 0.34; n = 10) was the same as that when cells had not been exposed to GPN.27 In addition, it is notable that the kinetics of the GPN response was relatively slower than the UTP response.

Figure 4 shows that endothelial cells associated with freshly isolated corneas also produced an increase in [Ca2+]i due to GPN. Although the temporal characteristics of the response were similar to that of cultured cells, the magnitude of the peak [Ca2+]i increase was less ({Delta}[ratio]peak = 0.17 ± 0.14; n = 13 in the first exposure and {Delta}[ratio]peak = 0.082 ± 0.045; n = 7 in the second exposure; P < 0.01). This response to GPN was equivalent to approximately 43% of the peak response to UTP,27 measured under identical conditions with freshly isolated tissue.



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  		Figure 4. GPN-induced [Ca2+]i mobilization in native endothelial cells. Cells loaded with fura-PE3 were excited at 345 ± 5 and 380 ± 5 nm sequentially. Where indicated, the endothelial surface was exposed to GPN (200 µM). F340/F380 is the ratio of emission intensities in response to excitation at 340 nm to that at 380 nm.

 
Exposure to GPN in the absence of extracellular Ca2+ also produced an increase in [Ca2+]i (Fig. 5 ; {Delta}[ratio]peak = 0.55 ± 0.39; n = 11), but a second exposure produced a smaller increase ({Delta}[ratio]peak = 0.09 ± 0.05; n = 5; P < 0.01). In six of eight experiments, a small transient increase in [Ca2+]i was noted, merely after removal of external Ca2+ (i.e., by substitution with Ca2+-free Ringer’s solution containing 500 µM EGTA; Fig. 5 , dotted rectangle 1), but this phenomenon was not explored. Unlike the response obtained in the presence of external Ca2+, a small increase in the ratio was noted immediately on the first exposure to GPN (in 5 of 11 experiments; Fig. 5 , dotted rectangle 2). This increase in the ratio, however, was concomitant with an increase in both F340 and F380 emission intensities. These increases were therefore not indicative of a increase in [Ca2+]i and were probably an artifact due to the formation of naphthylamine, a fluorescent byproduct of GPN hydrolysis16 17 (see the Discussion section). A similar artifact was evident on a second exposure to GPN (Fig. 3 , dotted rectangle 3), followed by the expected delayed, but reduced, [Ca2+]i increase. Figures 6A 6B and 6C summarize the data obtained from experiments similar to those shown in Figures 3 4 and 5 .



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  		Figure 5. GPN mobilized [Ca2+]i from intracellular stores. Where indicated, cultured cells were exposed to GPN (100 µM) in the absence of extracellular Ca2+. The Ringer’s solution also contained 500 µM EGTA. Rectangle 1: release of [Ca2+]i merely by removal of extracellular Ca2+; rectangles 2, 3: apparent increases in [Ca2+]i due to formation of certain fluorescent byproduct after hydrolysis of GPN.

 


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  		Figure 6. Summary of GPN-induced [Ca2+]i mobilization. Responses from the cells obtained by two consecutive exposures are shown connected. Some experiments contained only one exposure to GPN. (A) [Ca2+]o = 1.2 mM with cultured cells. (B) [Ca2+]o = 0 mM with cultured cells. The Ringer’s solution also contained 500 µM EGTA. (C) [Ca2+]o = 1.2 mM with freshly isolated tissue.

 
Effect of pH Gradient on GPN-Induced Ca2+ Mobilization
Monensin is well known to collapse pH gradients across acidic organelles by forming an Na+/H+ exchanger.11 To determine whether the GPN-sensitive Ca2+ stores are dependent on the pH gradient across lysosomal membranes, [Ca2+]i mobilization in response to GPN was examined after pretreatment with monensin. Exposure to monensin (20 µM) itself produced a significant increase in [Ca2+]i (Fig. 7A ; relative to resting Ca2+ levels), but exposure to GPN at the peak of the monensin response produced a further transient increase in [Ca2+]i (relative to resting Ca2+ levels; Fig. 7A ). These increases in Ca2+ effected by monensin and GPN, respectively, were also produced in the absence of external Ca2+ (Figs. 7B 7C) . These results, summarized in Figure 7D , indicate that monensin induced Ca2+ release from intracellular stores, and that its exposure partially affected the response to GPN.



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  		Figure 7. Monensin-induced Ca2+ mobilization partially affected GPN response. (A) Where indicated, cultured cells were exposed to monensin (20 µM). At the peak of the monensin response, cells were exposed to GPN (200 µM). (B) Where indicated, cultured cells were exposed to monensin (20 µM) and GPN (200 µM) sequentially in the absence of external Ca2+. After the resting Ca2+ level was reached, on completion of the GPN response, cells were exposed to Ca2+-rich Ringer’s solution ([Ca2+]o = 1.2 mM). (C) Where indicated, cultured cells were exposed to monensin (20 µM) in the absence of external Ca2+. After reaching the resting Ca2+ level on completion of the monensin response, cells were exposed to GPN (200 µM) and ATP (100 µM) sequentially. (D) Summary of data obtained from experiments similar to those shown in (A–C).

 
Figure 7C also shows that reexposure to Ca2+-rich Ringer’s solution (i.e., [Ca2+]o = 1.4 mM) after releasing the GPN-sensitive stores led to a rapid increase in [Ca2+]i, indicating stimulated influx of extracellular Ca2+.

GPN-Induced Extracellular Mn2+ Influx
To determine whether part of the [Ca2+]i increase in response to GPN was contributed by extracellular Ca2+, Mn2+ was used as a surrogate for Ca2+ and its influx was measured at the isosbestic point of fura-2 (i.e., 360 nm).27 Exposure to GPN in the presence of 100 µM Mn2+ and 1.2 mM Ca2+ led to a decline in F360 consistent with Mn2+ influx (Fig. 8A ; n = 11). The initial rapid decreases in F360, however, were artifacts caused presumably by either a spectral overlap of F360 with F380 emission intensities or the fact that the isosbestic point for fura-PE3 in BCECs is not at 360 nm. This was tested by increasing [Ca2+]i through exposure to ATP in the absence of Mn2+ and by measuring the fluorescence in response to excitation at all three wavelengths (i.e., F340, F360, and F380). Figure 8B shows that exposure to ATP also caused a rapid decrease in F360 concomitant with a decrease in F380. This is consistent with the observation that Mn2+ influx did not occur during the initial decrease in F360 in the presence of Mn2+ (Fig. 8A) . It may also be noted that both F360 and F380 recovered partially in the absence of external Mn2+ (Fig. 8B) compared with a continuous decline in the presence of Mn2+ (Fig. 8A) .



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  		Figure 8. GPN exposure led to an influx of extracellular Ca2+. Cells loaded with fura-PE3 were excited at 340, 360, and 380 nm sequentially. The left y-axis shows the emission intensities at 360 (F360), 340 (F340), and 380 nm (F380). The right y-axis shows the ratio (F340/F380). (A) Response to GPN. Where indicated, the cells were exposed to Ringer’s solution containing Mn2+ (100 µM) and subsequently to GPN (100 µM). The results are typical of those in 11 independent trials (i.e., 11 coverslips). (B) Response to ATP. Where indicated, the cells were exposed to Ringer’s solution without Mn2+ but containing ATP (100 µM) as a positive control. The results are typical of those in four independent trials (i.e., four coverslips).

 
Relationship to Ins(1,4,5)P3-Sensitive Ca2+ Stores
To determine the relationship between GPN-sensitive and Ins(1,4,5)P3-sensitive stores, several experiments were initiated. In the first series, an attempt was made to examine the extent of the GPN response before and after emptying Ins(1,4,5)P3-sensitive stores. In previous studies, in the absence of extracellular Ca2+, UTP has been shown to deplete Ins(1,4,5)P3-sensitive Ca2+ stores such that the [Ca2+]i increase was diminished in response to subsequent exposure to cyclopiazonic acid (10 µM; ER Ca2+-ATPase inhibitor) or to a second exposure to UTP itself.27 In the absence of extracellular Ca2+, initial exposure to UTP (100 µM) mobilized [Ca2+]i ({Delta}[ratio]Peak = 0.62 ± 0.15; n = 8), but subsequent exposure to GPN (200 µM) did not result in a detectable increase in [Ca2+]i (data not shown). By contrast, prior exposure to GPN did not prevent UTP-induced [Ca2+]i mobilization (n = 10; data not shown). In the presence of extracellular Ca2+, however, responses to GPN after exposure to UTP were significant ({Delta}[ratio]peak = 0.27 ± 0.12; n = 4), but were smaller compared with the response obtained when cells were previously exposed to GPN (P < 0.01). These data show that GPN-sensitive stores are sensitive to UTP. To determine whether this sensitivity is associated with Ins(1,4,5)P3-sensitive stores, the latter were depleted by thapsigargin, an irreversible ER Ca2+-ATPase inhibitor. As shown in Figure 9 (top), GPN induced a [Ca2+]i increase in the continued presence of thapsigargin. The extent of Ca2+ increase was close to that found in the absence of thapsigargin (summarized in Fig. 9 , bottom), indicating that the GPN response was independent of Ins(1,4,5)P3. To determine whether GPN caused an increase of Ins(1,4,5)P3, its potential release through the breakdown of membrane phospholipids was blocked by the inhibition of phospholipase C (PLC) with U73122. The time of exposure required to completely inhibit PLC-mediated Ca2+ release with U73122 was first determined using UTP to stimulate release from Ins(1,4,5)P3-sensitive stores. An exposure time of 7 minutes or more completely blocked the PLC-mediated increase in Ca2+ by UTP (100 µM; Fig. 10C ). Figure 10D summarizes similar experiments conducted for different durations. Based on these findings, cells were pretreated with U73122 (10 µM) for 7 to 9 minutes and then exposed to GPN in the continued presence of the inhibitor. A typical response to GPN in the presence of U73122 is shown in Figure 11A , and the results of all similar experiments are summarized in Figure 11B . Although the extent of the GPN response was reduced significantly (P < 0.01) compared with the control (Fig. 11B) , the GPN response was still much larger than that to UTP, which at 100 µM did not produce any detectable increase in Ca2+ after pretreatment with U73122 (Figs. 10C 10D) . A significant decrease in the GPN response compared with the control appeared to be nonspecific, because pretreatment with U73343 (10 µM), an inactive analogue of U73122, also caused similar decreases in response to UTP and GPN (Figs. 12) .


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GPN-Induced Lysosomal Swelling
AO, a weakly basic amine, has been used to stain acidic organelles such as trans-Golgi vesicles, endosomes, and lysosomes.28 29 30 31 34 35 The excitation spectrum of AO fluorescence is pH sensitive with a broad peak at approximately 475 nm. At low concentrations, the emission spectrum has a peak at approximately 530 nm. At high concentrations, however, AO molecules form oligomers and are fluorescent, with an emission more than 650 nm, whereas the emission at 530 nm is severely quenched.30 Thus, acidic compartments that accumulated AO appeared orange (Fig. 1A) . Fluorescent green staining was consistent with the specific labeling of acidic organelles, as demonstrated by punctate staining in the cytoplasm and absence of orange fluorescence in the nuclear region (Fig. 1B) .16 17

GPN is a well-known substrate for cathepsin C (dipeptidyl peptidase I),36 37 usually localized in a subpopulation of lysosomes.16 17 32 33 Breakdown of GPN by cathepsin C induces osmotic swelling of the lysosome, leading to loss of its membrane barrier and concomitant release of its contents into the cytoplasm.16 17 32 33 Figure 2 confirms that GPN caused lysosomal swelling in cultured BCECs by the dequenching of AO fluorescence as a result of a swelling-induced decrease in the intralysosomal concentration of AO.

GPN-Induced [Ca2+]i Mobilization
Exposure to GPN, at the same concentration and for the same period as in the previous experiment (Fig. 2) , caused an increase in the F340/F380 ratio (Fig. 3) . Both an increase in F340 and a decrease in F380 contributed to the observed increase in the ratio (see F340 and F380 profiles in Fig. 3 ). This indicates that naphthylamine, a weakly fluorescent byproduct of GPN hydrolysis16 17 (with excitation and emission peaks at 335 and 410 nm, respectively), is not a confounding factor in the observed increase in the F340/F380 ratio. Thus, results from experiments similar to that shown in Figure 3 are suggestive of GPN-induced [Ca2+]i mobilization. That GPN-induced [Ca2+]i mobilization is not an anomaly of cell culture was evident by the response to GPN obtained from endothelial cells of the freshly isolated cornea (Fig. 4) .

Effect of Intracellular Stores on GPN-Induced Ca2+ Mobilization
In several cell types, histones and certain other basic polypeptides activate Ca2+-cation influx.38 Therefore, to ascertain whether GPN, a small peptide, also contributes to [Ca2+]i influx by merely forming Ca2+ channels or by behaving in a manner similar to an ionophore, the increase in [Ca2+]i was measured in response to GPN in the absence of extracellular Ca2+. Trace Ca2+ in the Ringer’s solution was also chelated by EGTA (500 µM). Exposure to GPN produced a transient [Ca2+]i increase followed by a sharp decline (Fig. 5) . This clearly indicates that the GPN-induced [Ca2+]i increase is derived from intracellular stores.

GPN-Induced Extracellular Ca2+ Influx
The significant increase in [Ca2+]i (Fig. 7C) after exposure to Ca2+-rich Ringer’s solution after depletion of the GPN-sensitive stores suggests activation of Ca2+ influx pathways reminiscent of CCE observed on depletion of Ins(1,4,5)P3-sensistive stores.3 27 To ascertain the nature of Ca2+ influx on exposure to GPN, Mn2+ was used as a surrogate for Ca2+. In the absence of GPN, exposure to Mn2+ led to no significant changes in F340, F360, and F380 (Fig.8A) , indicating negligible Mn2+ influx through leakage pathways under resting conditions.27 After exposure to GPN, however, there was a decrease in F380 and a concurrent increase in F340, which are the emission intensities at Ca2+-sensitive wavelengths. A noticeable reduction in F360 concomitant with an increase in F340/F380 ratio was also apparent. This was found to be an artifact (noted earlier) caused presumably by either a spectral overlap of the emissions to excitations at 360 and 380 nm or by an isosbestic point different from 360 nm. In fact, P2 agonists exhibited similar profiles (Fig. 8B) , even in the absence of external Mn2+.24 These observations suggest that the GPN-induced increase in [Ca2+]i begins with Ca2+ release from GPN-sensitive stores and not by extracellular Ca2+ influx. In addition, the results can be taken to show that GPN itself does not form a Ca2+ channel or ionophore. Furthermore, we noted a continued decrease in F360 after the short decline (an artifact), but coinciding with the peak in F340/F380 (Fig 8A) . This indicates that the depletion of lysosomal stores leads to Ca2+ influx and is consistent with a significant increase in [Ca2+]i, resulting from the second exposure to GPN (Fig. 3) . The influx, however, must be small, because the response to GPN was without the distinct biphasic characteristics of the influx (Fig. 3) induced by P2 agonists, which are known to produce significant CCEs.27 The conclusion that the depletion of the GPN-sensitive store induces CCE is at variance with the observations in MDCK cells, in which depletion of the GPN-sensitive stores did not elicit extracellular Ca2+ influx.16 17 In the amoeba, Dictyostelium discoideum, however, Ca2+ stores associated with acidic organelles have been found to induce CCE on their depletion by fatty acids.9

Relationship to Ins(1,4,5)P3-Sensitive Stores
In the presence of thapsigargin, GPN caused a significant increase in [Ca2+]i (Fig. 9) . In fact, the responses were approximately the same as those obtained from the first exposure to GPN in the absence of thapsigargin (Fig. 9 , bottom). When the cells were exposed to thapsigargin, it can be assumed that the luminal concentration of Ca2+ in Ins(1,4,5)P3-sensitive stores reached that of cytosolic Ca2+ concentration (i.e., [Ca2+]i). Therefore, exposure to GPN could not have led to further increases in [Ca2+]i as a result of the formation of Ins(1,4,5)P3 itself or as a result of perturbations in components leading to release from Ins(1,4,5)P3-sensitive stores. Furthermore, the effects of GPN were not due to changes in Ins(1,4,5)P3, because U73122, at concentrations completely blocking UTP responses, did not inhibit the GPN responses completely (Fig. 11A) . The limited inhibition, however, could be attributed to nonspecific effects of the putative PLC inhibitor U73122.39 40 41 The lack of selectivity is also apparent in the decreased response to its inactive analogue, U73343 (Fig. 12) . From these considerations, it can be concluded that the GPN-induced [Ca2+]i increase is not derived from Ins(1,4,5)P3-sensitive stores, which is similar to findings in MDCK cells.16 17 This is also consistent with the observation that release of the GPN-sensitive stores does not affect Ca2+ mobilization in response to P2 agonists. The fact that the GPN-sensitive Ca2+ stores were concomitantly depleted after release of Ins(1,4,5)P3-sensitive Ca2+ stores (also after exposure to P2 agonists in MDCK cells16 17 ), however, implies that release from lysosomal Ca2+ stores may be modulated by Ins(1,4,5)P3, but that it is not linked to thapsigargin-sensitive stores. This is similar to Ca2+ stores associated with Golgi bodies, which are depleted in response to agents that stimulate release from Ins(1,4,5)P3-sensitive Ca2+ stores.19 In contrast, the depletion of lysosomal Ca2+ stores on activation of P2 receptors could also be attributed partly to release in the process of lysosomal fusion, which is induced in response to large increases in [Ca2+]i.42 43

Effect of Monensin on GPN-Inducible Stores
The results shown in Figure 7 are consistent with the notion that active transport of Ca2+ into the lumen of lysosomes is dependent on the pH gradient across the lysosomal membrane. Similar conclusions have been reached in PC12 cells where Ca2+ pools associated with acidic organelles have been shown to be susceptible for release by agents known to collapse the intracellular pH gradients (e.g., monensin, NH4Cl or chloroquine8 ). At the molecular level, the specific mechanisms that lead to active accumulation of Ca2+ in acidic organelles are relatively well demonstrated in parasites (e.g., Trypanosoma brucei,11 12 44 Toxoplasma gondii tachyzoites10 45 ) and in the amoeba (e.g., D. discoideum9 ). For example, it has been shown11 46 47 48 that Ca2+ transport into acidic organelles, referred to as acidocalcisomes, is dependent on a vanadate-sensitive Ca2+-ATPase and that Ca2+ release occurs through a Ca2+/nH+ exchanger when the pH gradient collapses. Accordingly, acidocalcisomes release Ca2+ in response to monensin, which mimics the Na+/H+ antiporter and hence collapses the lysosomal pH gradient.11

In conclusion, in the current study lysosomes formed an Ins(1,4,5)P3-independent Ca2+ store in cultured and native BCECs. The release of lysosomal Ca2+ led to Ca2+ influx, although the mechanism of activation of CCE pathways needs further investigation. This is the first study to demonstrate the presence of lysosomal Ca2+ stores in cells from primary culture and native tissue. In addition, this study demonstrates the functional activity of cathepsin C in corneal endothelial cells that had been previously shown only through histochemical methods.49


   Acknowledgements
 
The authors thank Amelia Loo for assistance during experiments shown in Figures 9 10 11 12 .


   Footnotes
 
Supported by National Eye Institute Grant EY11107 (SPS).

Submitted for publication October 16, 2001; revised February 4, 2002; accepted February 15, 2002.

Commercial relationships policy: N.

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

Corresponding author: Sangly P. Srinivas, 800 East Atwater Avenue, Indiana University, School of Optometry, Bloomington, IN 47405; srinivas{at}indiana.edu.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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