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(Investigative Ophthalmology and Visual Science. 1999;40:2046-2053.)
© 1999 by The Association for Research in Vision and Ophthalmology, Inc.

Relationship between Inositol 1,4,5-Trisphosphate Receptor Isoforms and Subcellular Ca2+ Signaling Patterns in Nonpigmented Ciliary Epithelia

Keiji Hirata1, Michael H. Nathanson2, Angela D. Burgstahler2, Keisuke Okazaki1, Elisabetta Mattei1 and Marvin L. Sears1

From the Departments of 1 Ophthalmology and Visual Sciences, 2 Medicine and Cell Biology, Yale University School of Medicine, New Haven, Connecticut.

Abstract

PURPOSE. Subcellular Ca2+ signaling patterns, such as Ca2+ waves, gradients, and oscillations, are an important aspect of cell regulation, but the molecular basis for these signaling patterns is not understood. Because Ca2+ release patterns differ among isoforms of the inositol 1,4,5-trisphosphate (InsP3) receptor, the relationship between the distribution of these isoforms and subcellular Ca2+ signaling patterns in nonpigmented epithelial (NPE) cells was investigated.

METHODS. The distributions of the types I, II, and III InsP3 receptors were determined in NPE cells by immunofluorescence, and subcellular Ca2+ signaling patterns in these cells were examined by confocal line scanning microscopy.

RESULTS. The type I InsP3 receptor was concentrated at the basal pole of NPE cells, whereas the type III receptor was localized to the apical pole. The type II InsP3 receptor was not expressed in detectable amounts. Acetylcholine induced increases in Ca2+ that were mediated by InsP3, and these Ca2+ increases began as Ca2+ waves that were initiated at the apical pole, in the region of the type III InsP3 receptor. Acetylcholine occasionally induced sustained or repetitive Ca2+ increases that were prominent at the basal pole, in the region of the type I InsP3 receptor, but only subtle or absent apically.

CONCLUSIONS. Because the type I InsP3 receptor is thought to be responsible for repetitive Ca2+ release events, and the type III InsP3 receptor instead is suited to initiate Ca2+ signals, the subcellular distribution of these two isoforms corresponds to the Ca2+ signaling patterns observed in this cell type. Differential subcellular expression of InsP3 receptor isoforms may be an important molecular mechanism by which NPE cells organize their Ca2+ signals in space and time.

Spatial and temporal patterns of cytosolic Ca2+ (Cai2+) signals are highly organized in many cell types and play an important role in regulating cell function.1 2 For example, spatial patterns of Cai2+ signals, such as Cai2+ waves and gradients, direct functions such as secretion3 4 and cell migration,5 and temporal Cai2+ signaling patterns such as oscillations direct functions such as gene expression.6 7 The molecular basis for the subcellular organization of Cai2+ signals is not completely understood, though.

Cai2+ signaling in epithelia and other nonexcitable cells generally is mediated by Ca2+ release via the inositol 1,4,5-trisphosphate (InsP3) receptor.1 2 Three isoforms of this receptor have been identified, and many cell types express more than one of these isoforms.8 9 10 11 The function of the type I InsP3 receptor has been characterized in greatest detail12 ; the receptor functions as a Ca2+ channel in the presence of InsP3, but the open probability of that channel exhibits a bell-shaped dependence on the concentration of Cai2+.13 14 This Ca2+ dependence of the type I InsP3 receptor is thought to be important for the formation of certain types of Cai2+ signaling patterns, such as Cai2+ oscillations.15 16 The type III InsP3 receptor also is an InsP3-gated Ca2+ channel17 ; however, unlike the type I receptor, the type III receptor functions purely as a positive feedback Ca2+ channel.17 It has been proposed that this characteristic of the type III InsP3 receptor would enable this isoform to act preferentially as a trigger for Ca2+ release.17 This difference in dependence on Cai2+ thus suggests that the subcellular distribution of these two isoforms could provide a mechanism by which subcellular Cai2+ signals are organized. The goal of the present study was to examine the relationship between subcellular Cai2+ signaling patterns and the subcellular distribution of these two InsP3 receptor isoforms in one type of polarized epithelial cell, the nonpigmented epithelium (NPE) of the ciliary epithelial bilayer of the eye.

Materials and Methods

Animals and Materials
Male albino New Zealand rabbits weighing 2 to 3 kg obtained from Millbrook Farms (Amherst, MA) were used for all experiments. Acetylcholine (ACh), atropine, U73122, heparin (average molecular weight 6000), de-N-sulfated heparin, trypsin, and sulforhodamine 101 (Texas red) were obtained from Sigma Chemical (St. Louis, MO). Fluo-3 in acetoxy-methoxylated (AM) form and Pluronic F-127 were obtained from Molecular Probes (Eugene, OR). All other chemicals were of the highest quality commercially available.

Antibodies
The type I InsP3 receptor was labeled using affinity-purified rabbit polyclonal antibody T210, directed against the 19 C-terminal amino acids of the mouse type I InsP3 receptor.18 19 The type II InsP3 receptor was labeled using affinity-purified rabbit polyclonal antibody CT2 directed against the C-terminal portion of the rat type II InsP3 receptor.9 The type III InsP3 receptor was labeled using a commercially obtained mouse monoclonal antibody directed against the N-terminal portion of the human type III InsP3 receptor (mab InsP3R-3; Transduction Laboratories, Lexington, KY).17 20 The M3 muscarinic ACh receptor was labeled using a commercially obtained mouse monoclonal antibody21 raised against affinity-purified calf forebrain receptor (M35; Argene, North Massapequa, NY).

Preparation of Isolated Ciliary Epithelium
Isolated ciliary bilayer epithelium was prepared as described previously,22 23 with slight modification. Briefly, rabbits were anesthetized with an intramuscular injection of ketamine hydrochloride and xylazine, then euthanatized by intravenous injection of pentobarbital sodium and phenytoin sodium. The eyes were enucleated promptly, then the anterior segments were isolated after careful removal of the lens. From the isolated anterior segment of the eye, ciliary processes were separated from the iris and cut into 10 to 20 strips, each 2 to 3 mm in length. In selected experiments, the NPE layer was mechanically separated from the bilayer,24 then individual NPE cells were obtained by trypsin digestion of the monolayer in EDTA as described previously,25 maintained in HEPES-buffered M199 solution (Sigma) containing 10% fetal calf serum, and examined in short-term culture. All procedures conformed with NIH recommendations and the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research.

Confocal Immunofluorescence Histochemistry
Sections of rabbit ciliary epithelia were labeled with isoform-specific antibodies to determine the subcellular distributions of the types I, II, and III InsP3 receptors. Specimens were colabeled with rhodamine-phalloidin (Molecular Probes) because this stain facilitates identification of the apical and basolateral poles of epithelial cells.26 27 Additional sections were labeled with the M3 muscarinic ACh receptor antibody M35 plus rhodamine phalloidin, to determine the distribution of that receptor on NPE cells.

Immunochemistry was performed on 4-µm-thick frozen sections of rabbit ciliary epithelia. Tissue was fixed by perfusion with 4% paraformaldehyde in 0.12 M sodium phosphate buffer (pH 7.4), cryopreserved overnight in 15% sucrose, and frozen in isopentane/liquid nitrogen. After quenching with 50 mM NH4Cl and 16% goat serum in phosphate-buffered saline with Triton X-100, the sections were labeled overnight with a 1:10 dilution of antibody T210, a 1:250 dilution of antibody CT2, a 1:50 dilution of InsP3R-3 antibody, or a 1:200 dilution of antibody M35, then washed and incubated with either fluorescein isothiocyanate (FITC)–conjugated (Sigma) or Alexa 488–conjugated (Molecular Probes) anti–mouse or anti–rabbit secondary antibody, along with rhodamine–conjugated phalloidin.26 Negative controls were labeled with preimmune serum rather than with anti–InsP3 receptor antibodies but were otherwise processed as noted above. Specimens were examined with a Bio–Rad MRC-600 Confocal Microscope equipped with a krypton/argon mixed gas laser (Richmond, CA). To ensure specificity of InsP3 receptor staining, images were obtained using confocal machine settings (i.e., aperture, gain, and black level) at which no fluorescence was detectable in negative control samples labeled with preimmune serum. Double-labeled specimens were serially excited at 488 nm and observed at >515 nm to detect FITC, then excited at 568 nm and observed at >585 nm to detect rhodamine. This approach eliminated bleed-through of FITC fluorescence into the rhodamine channel.28

Confocal Microscopic Measurements of Cytosolic Ca2+
Isolated ciliary epithelial bilayers were prepared as described above, then loaded with fluo-3/AM (50 µM) and Pluronic F-127 for 1 hour at room temperature in Hanks’ balanced salt solution containing 10% fetal calf serum. Specimens were then placed between two glass coverslips in a gravity-driven perifusion chamber on the stage of a Zeiss Axiovert microscope (Thornwood, NY), and perifused at room temperature at a rate of 1 to 2 ml/min. Nonpigmented epithelial cells within the tissue were observed through a x63 1.40 NA objective using either a Bio–Rad MRC-600 or a Bio–Rad MRC-1024 laser scanning confocal imaging system. An argon laser was used to excite the dye at 488 nm, and emission signals above 515 nm were collected. Optical sections 1 to 2 µm in thickness were obtained. Neither autofluorescence nor other background signals were detectable at the machine settings used, and there was no change in size, shape, or location of cells during the experiments. In most experiments, two-dimensional images consisting of 768 x 512 pixels (0.26 µm/pixel) were recorded at a rate of 1 frame/s on an optical disc recorder and analyzed subsequently, using the mean pixel values of preselected areas to monitor intensity changes. Increases in Cai2+ were expressed as (F/F0) x 100%.22 29 In selected experiments, tissues instead were examined using the line scanning mode of the confocal microscope, to increase temporal resolution (to 10 or 200 msec). In this mode, fluorescence is determined at each point along a single line across the image, rather than at each point across the entire image.22 30 Line scans were displayed as images consisting of 768 x 512 pixels, with a spatial resolution of 0.26 µm/pixel (in the "x" direction) and a temporal resolution of 10 or 200 msec/pixel (in the "y" direction). Velocities of Cai2+ waves in individual cells were determined from the rate at which initial fluorescence increases moved along the scan line.30

Microinjection Studies
In selected studies, individual isolated NPE cells were stimulated with ACh to confirm their responsiveness to this agonist, then either heparin (1 mg/ml) or de-N-sulfated heparin (1 mg/ml) was delivered into the cells by microinjection, and the cells were restimulated with ACh. Cells were loaded with fluo-3/AM, then examined by confocal video microscopy as described above. Micropipettes with an internal diameter of <0.5 µm were made from glass capillary tubes using a Narishige PD-5 micropipette puller. A series 5171 Eppendorf micromanipulator was used for positioning, and an Eppendorf series 5242 microinjector was used for pressure-microinjections.31 Micropipettes were loaded with heparin or its de-N-sulfated analogue dissolved in an intracellular-like buffer (150 mM KCl plus 1 mM HEPES), and Texas red was coinjected as a marker of successful microinjection.31

InsP3 Measurement
Segments of ciliary epithelia were isolated as described above, then equilibrated for 15 minutes in Ringer’s solution consisting of NaCl (120 mM), KCl (2.8 mM), CaCl2 (2 mM), MgCl2 (2 mM), HEPES (10 mM), and glucose (10 mM). Either ACh (10 µM) or buffer was added for 2, 5, or 10 seconds, then stimulation was stopped by adding 20% perchloric acid and placing the samples on ice. The acid extracts were centrifuged, then the supernatants were removed and neutralized with KOH, HEPES, and EDTA, and InsP3 was measured in the neutralized extracts using a radiobinding assay (Amersham). The pellets were solubilized in NaOH for protein determination using a BCA-Protein Assay kit (Pierce). Results were expressed as picomoles of InsP3 per milligram of protein.

Results and Discussion

Localization of InsP3 Receptor Isoforms in NPE Cells
The subcellular distribution of the types I, II, and III InsP3 receptors was investigated by confocal immunofluorescence histochemistry. Ciliary epithelial bilayers were labeled with either antibody T210, CT2, or mab InsP3R-3 and colabeled with rhodamine–conjugated phalloidin to identify the apical and basolateral margins of the NPE cells (Fig. 1) . T210 labeling was limited to the basolateral pole of NPE cells and was found on the basolateral pole of pigmented epithelial cells as well (Figs. 1B 1C) . In contrast, the type III InsP3 receptor antibody labeled the apical pole of NPE cells (Figs. 1F 1G) . No such basal or apical labeling was seen in the NPE in tissue stained instead with preimmune serum (Figs. 1D 1H) . Unlike antibody T210 or mab InsP3R-3, antibody CT2 did not label NPE cells (Figs. 1J 1K) , even though this antibody has been used by others20 32 and by us (unpublished observation) to label the type II InsP3 receptor in other epithelia. Thus, like other cell types,8 9 11 including other epithelia,10 20 32 multiple InsP3 receptor isoforms are expressed in NPE cells. In particular, our findings suggest that NPE cells express types I and III but not the type II InsP3 receptor. Apical localization of the InsP3 receptor, especially the type III isoform, has also been shown in other epithelia, including pancreatic and salivary acinar cells.20 28 32 However, in those epithelia the type I and type II isoforms are predominantly expressed in the apical region as well.20 32 The finding that type I and type III InsP3 receptors are concentrated in different regions of the NPE cell, whereas the type II receptor is minimally expressed, thus suggests that this may provide a novel system in which to compare the function of InsP3R-I and InsP3R-III when the two are coexpressed in a single cell.



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  		Figure 1. Subcellular localization of the types I, II, and III InsP3 receptors in NPE cells, visualized by confocal immunofluorescence histochemistry. Ciliary epithelial bilayers in this figure are oriented so that the top layer of cells is the NPE and the bottom layer is the pigmented epithelium (PE). The apical membranes of the NPE and PE are in contact. (A) Rhodamine–conjugated phalloidin labeling. Scale bar, 10 µm. (B) Same tissue segment, with type I InsP3 receptor labeling. (C) Superimposure of (A) and (B) shows that the type I InsP3 receptor is concentrated at the basal pole of the NPE, as well as at the basal pole of the PE. (D) Negative control for the type I InsP3 receptor, labeled with preimmune serum and counterstained with FITC-conjugated anti-rabbit secondary antibody (green) plus rhodamine phalloidin (red). (E) A separate tissue section labeled with rhodamine-conjugated phalloidin. (F) Same tissue segment, with type III InsP3 receptor labeling. (G) Superimposure of (E) and (F) shows the type III InsP3 receptor is concentrated at the apical pole. (H) Negative control for the type III InsP3 receptor. (I) A separate tissue section labeled with rhodamine-conjugated phalloidin. (J) Same tissue segment, with type II InsP3 receptor labeling. No (green) labeling is seen. (K) Superimposure of (I) and (J). (L) Negative control for the type II InsP3 receptor.

 
ACh-Induced Ca2+ Signaling in NPE cells Is Mediated by InsP3
To determine the relationship between the distribution of InsP3 receptor isoforms and Cai2+ signaling patterns, we tried to identify an agonist that increases Cai2+ via InsP3 in NPE cells. Acetylcholine increases Cai2+ in NPE cells,22 33 34 35 so we examined whether this increase is mediated by InsP3. Acetylcholine (10 µM) increased fluo-3 fluorescence by 175% ± 25% (mean ± SEM) in these cells but by only 10% ± 1% when cells were stimulated in the presence of 10 µM atropine (n = 10 experiments; P < 0.0001 by paired t-test). In separate studies, ACh increased fluo-3 fluorescence by 126% ± 18% in the presence of 1.26 mM extracellular Ca2+ and by 110% ± 14% in Ca2+-free medium (n = 10 experiments; P > 0.05). These findings demonstrate that ACh increases Cai2+ in NPE cells via stimulation of muscarinic receptors, leading to release of Ca2+ from intracellular stores. It has previously been shown that carbachol stimulates production of inositol polyphosphates, including InsP3, in NPE cells,36 so we examined the time course of InsP3 production. Acetylcholine (10 µM) induced a net increase of 0.3, 6.8, and 9.9 pmol InsP3/mg protein after 2, 5, and 10 seconds of stimulation, respectively. These values correspond to increases of 1%, 20%, and 32% relative to InsP3 content of unstimulated controls. To demonstrate a causal link between ACh-induced InsP3 production and Cai2+ signaling in NPE cells, we examined the effects of the phospholipase C inhibitor U73122.37 Ciliary epithelial bilayers were sequentially stimulated, first with ACh (10 µM), then with ACh + U73122 (10 µM), and then with ACh again. Fluo-3 fluorescence was monitored in groups of at least 10 adjacent NPE cells, and we found that the ACh-induced increase in Cai2+ was reversibly inhibited by U73122 (Fig. 2) . To investigate whether this Ca2+ release is mediated by activation of the InsP3 receptor, cells were microinjected with either heparin (1 mg/ml), which is a high-affinity competitive antagonist for the InsP3 receptor,38 or de-N-sulfated heparin (1 mg/ml), which neither inhibits InsP3 binding to its receptor nor blocks InsP3-induced Ca2+ release from microsomes.39 As an extra control, only cells that responded to ACh were subsequently injected with heparin or its de-N-sulfated analogue, then each of those cells were restimulated with ACh after microinjection. Ten of 11 cells did not respond to ACh after injection with heparin (Figs. 3 A, 3B, 3C); fluorescence increased by 125% ± 30% in these cells when stimulated before heparin injection, but by only 9% ± 1% after injection (P < 0.005 by paired t-test). In contrast, 6 of 7 cells responded to ACh after injection with de-N-sulfated heparin (Fig. 3D) ; fluorescence increased by 108% ± 31% in these cells when stimulated before injection, and by 102% ± 31% after injection (P = 0.31). Taken together, these studies demonstrate that ACh increases Cai2+ in NPE cells by stimulation of muscarinic receptors, which then leads to phospholipase C–mediated mobilization of intracellular Ca2+ stores by activation of InsP3 receptors.



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  		Figure 2. The phospholipase C inhibitor U73122 (10 µM) inhibits ACh (10 µM)-induced Cai2+ signals in NPE cells. NPE cells within ciliary bilayers were monitored using time-lapse confocal microscopy as they were sequentially stimulated with ACh, then ACh + U73122, and then ACh again. Result is representative of that seen in four separate groups of NPE cells from three separate experimental preparations.

 


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  		Figure 3. Heparin but not de-N-sulfated heparin blocks ACh-induced Cai2+ signals in isolated NPE cells, as revealed by double-channel time-lapse confocal microscopy. (A) Confocal image of an isolated NPE cell microinjected with heparin (1 mg/ml), plus free Texas red as a marker of successful injection. (B) Simultaneous image of the same cell (arrow) and its neighbor obtained before stimulation with ACh, which shows loading of both cells with the Ca2+ dye fluo-3. (C) Subsequent to stimulation with ACh, an increase in fluo-3 fluorescence is seen in a neighboring cell but not in the cell microinjected with heparin. (D) Acetylcholine-induced increases in fluo-3 fluorescence are blocked in cells microinjected with heparin (n = 10) but not in cells microinjected with de-N-sulfated heparin (n = 6). Values are mean ± SEM (*P < 0.005).

 
To determine the polarity of muscarinic receptors on NPE cells, ciliary epithelial bilayers were labeled by confocal immunofluorescence histochemistry. Ciliary bilayers were labeled with monoclonal antibody M35 directed against the M3 subtype of the muscarinic receptor, because this subtype often links to InsP3-mediated Cai2+ signaling in epithelia,21 40 and because previous pharmacological studies suggest this subtype is present on NPE cells.36 Tissue specimens were colabeled with rhodamine–conjugated phalloidin to identify the apical and basolateral margins of the NPE cells (Fig. 4) . M35 labeling was limited to the basal pole of NPE cells. The M3 receptor directly couples to G proteins that activate phospholipase C–ß,1 41 which suggests that stimulation with ACh would preferentially generate InsP3 in the basolateral region rather than apically.



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  		Figure 4. Subcellular localization of the M3 muscarinic ACh receptor in NPE cells, visualized by confocal immunofluorescence histochemistry. (A) The ciliary epithelial bilayer of the eye, labeled with rhodamine-conjugated phalloidin. The NPE (top layer) and PE (bottom layer) are oriented so that their apical membranes are in contact. Scale bar, 10 µm. (B) Same tissue segment, labeled with antibody M35 directed against the M3 muscarinic receptor. (C) Superimposure of (A) and (B), revealing that the M3 receptor is concentrated at the basal pole of the NPE. (D) Negative control for the M3 receptor, stained only with Alexa 488–conjugated anti-rabbit secondary antibody (green) plus rhodamine phalloidin (red). No nonspecific antibody labeling is observed.

 
Subcellular Organization of Ca2+ Signals in NPE Cells
To observe the subcellular organization of ACh-induced Cai2+ signals, NPE cells within intact ciliary epithelial bilayers were examined using confocal line scanning microscopy.22 30 This approach permitted examination of NPE cells in a system in which their structural polarity was maintained,22 and in which spatial and temporal resolutions were maximized while photobleaching was minimized.42 To determine the site of initiation of ACh-induced Cai2+ signals, images were collected every 10 msec. Cai2+ signals always began in the apical region, then traveled as a wave from the apical to the basal pole in each of 6 NPE cells (Fig. 5) . The wave speed was no different in Ca2+-containing versus Ca2+-free medium (23.2 ± 1.6 versus 24.4 ± 1.9 µm/sec, respectively; P = 0.60). These findings demonstrate that Cai2+ waves are initiated in the apical region of NPE cells, then propagate to the basal region purely via release of Ca2+ from intracellular stores. This polarized apical-to-basal pattern of Cai2+ wave propagation is similar to the pattern observed in other epithelia, including pancreatic,30 43 44 45 lacrimal,46 and salivary20 acinar cells and hepatocytes.26 InsP3 receptor isoforms have been localized in both pancreatic and salivary acinar cells, and in each of these types of acinar cell, each type of isoform present is concentrated apically.20 32 Therefore, from these previous studies it has not been possible to determine whether one of these isoforms would preferentially behave as a trigger for Ca2+ release. Because type I and type III InsP3 receptors are spatially separated in NPE cells, these cells provide a novel system in which to investigate this question. Although the type I InsP3 receptor is in the same region as the M3 ACh receptor, where increases in InsP3 likely originate, Cai2+ signals nonetheless began in the region of the type III receptor instead. This finding suggests that the type III isoform may have a much lower threshold than the type I isoform for InsP3-mediated Ca2+ release. This similarly suggests that the type III InsP3 receptor serves to initiate Cai2+ signals in cells that coexpress the type I and type III isoforms. Furthermore, this finding supports the hypothesis that the role of the type III InsP3 receptor is to act as a trigger for cellular Ca2+ release.17



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  		Figure 5. Acetylcholine-induced increases in Cai2+ begin as apical-to-basal Cai2+ waves in NPE cells. (A) Confocal image of a segment of the isolated ciliary bilayer loaded with fluo-3. The confocal line scan in (B) was performed along the white horizontal line across this image, which runs along the apical-to-basal pole of an NPE cell. Pseudocolor scale is shown at bottom. (B) Line scan collected during stimulation with 10 µM ACh. Fluorescence intensity along the x axis reflects distance (across the scan line) and along the y axis reflects time (between serial scans). Line scans were obtained every 10 msec for a total of 5.12 seconds (from top to bottom). The increase in fluorescence begins apically, then spreads to the opposite (basal) pole. Results are representative of those seen in 6 preparations. (C) Graphical representation of the fluorescence intensity over time at an apical and a basal point in the NPE cell that was scanned. The increases in Cai2+ (arrows) within the NPE cell occur 300 msec apart, and the two points are separated by a distance of 6.11 µm, which corresponds to a wave speed of 20.4 µm/s.

 
To observe subcellular Cai2+ signaling patterns that occur after the initial Cai2+ wave, NPE cells were examined for 100 seconds rather than 5 seconds. Confocal line scanning microscopy was used here as well, but line scans were collected at a frequency of 200 msec rather than 10 msec. At the lowest ACh concentration perifused (0.1 µM), an increase in Cai2+ was detected in only 23% (n = 6 of 26) of NPE cells. In contrast, a Cai2+ increase was detected in 66% (n = 65 of 98) of cells stimulated with higher ACh concentrations (0.5, 1, 5, or 10 µM). Repetitive Cai2+ spikes, persistent Cai2+ gradients across the cytosol, or both were detected in 36 of the 71 cells that responded to ACh (Fig. 6) . Among these cells, repetitive Cai2+ increases were either of greater amplitude, more sustained, or only present in the basolateral region, whereas persistent Cai2+ gradients were manifested as prolonged increases in Cai2+ in the basolateral region relative to the apical region (Fig. 6) . Localized increases in Cai2+, including localized Cai2+ oscillations, also occur in pancreatic acinar cells,44 45 but those Cai2+ increases are restricted to a region in which both the type I and type III InsP3 receptors are expressed.28 45 47 Localized Cai2+ increases have been reported in the presynaptic region of neurons48 and in the subplasmalemma of neuroendocrine cells3 as well, but those increases are thought to occur by Ca2+ influx rather than localized release of intracellular Ca2+ stores. The current work provides evidence that localized persistent or repetitive increases in Cai2+ may be driven preferentially by Ca2+ released from the type I rather than the type III InsP3 receptor. This differential signaling pattern by distinct Ca2+ storage pools supports the hypothesis that the role of the type III InsP3 receptor is to initiate cellular Cai2+ signals,17 whereas the type I InsP3 receptor instead drives Cai2+ oscillations and other longer-term Cai2+ signaling patterns.



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  		Figure 6. Examples of distinct apical and basolateral Cai2+ signaling patterns in NPE cells. Cells were stimulated with ACh while examined by confocal line scanning microscopy, using a collection rate of 200 msec per line (note that apical and basal signals appear to begin simultaneously because of the expanded time scale). (A) Periodic Cai2+ spikes with a frequency of ~0.1 s-1 are seen in the basolateral but not the apical region. (B) An increase in Cai2+ persists for >1 minute in the basolateral region, but Cai2+ is elevated apically for <20 seconds. (C) There is a sustained ~45% increase in fluo-3 fluorescence, with superimposed Cai2+ spikes (frequency, ~0.1 second-1) in the basolateral region, whereas apically there is only a ~15% increase with no superimposed Cai2+ spikes.

 
Although these findings suggest specific and complementary roles for the types I and III InsP3 receptors, whether the type II InsP3 receptor also plays a distinctive role in Cai2+ signaling is not addressed here. In B cells that normally coexpress all three InsP3 receptor isoforms, B-cell receptor stimulation results in InsP3-mediated Cai2+ signaling when expression of one or even two of the isoforms is disrupted.11 This finding suggests that each isoform may provide a redundant Cai2+ signaling mechanism in this cell type.11 In B cells that have been genetically engineered to express only a single isoform of the InsP3 receptor, Cai2+ signaling patterns are different for each isoform.49 In addition, the function of the type II receptor has recently been described at the single channel level, and it differs from the function of the type I InsP3 receptor.50 Together, these findings suggest that each InsP3 receptor isoform contributes to cellular Cai2+ signaling but in a unique way. Although several types of epithelium express all three InsP3 receptor isoforms,10 20 32 colocalization of the various isoforms in those cell types had made it difficult to determine their relative contribution to Cai2+ signaling in epithelia until now.

What is the functional significance of the current findings? The ability to generate Cai2+ gradients, waves, and oscillations may be critical for secretion to occur in polarized epithelia. For example, apical increases in Cai2+ direct exocytosis,4 51 because localized intense increases in Cai2+ in the apical region induce targeting of vesicles to the apical membrane.4 Apical increases in Cai2+ also can direct the movement of subapical actin, which may mechanically facilitate secretion.26 52 53 54 Apical-to-basal Cai2+ waves direct vectorial movement of electrolytes such as Cl- and Na+.4 43 Finally, repetitive increases in Cai2+ (i.e., Cai2+ oscillations) direct repetitive membrane fusion and exocytic events.51 55 The current work provides evidence that in cells coexpressing the type I and type III InsP3 receptors, the type III receptor is responsible for initiating Cai2+ signals, whereas repetitive or sustained increases in Cai2+ may instead be driven by the type I receptor.

The NPE is unusual among epithelia because it transports fluid and electrolytes from the apical to the basal pole, and secretion occurs basolaterally rather than apically.24 25 56 Therefore, it may be preferable for NPE cells to generate sustained or repetitive Cai2+ signals basolaterally rather than apically. Although it can be speculated that the subcellular distribution of InsP3 receptor isoforms organizes subcellular Cai2+ signaling patterns in all cells, the novel functional requirements of NPE cells may provide a unique cell model in which to investigate this hypothesis.

Acknowledgements

The authors thank Barbara E. Ehrlich for useful discussions. We also thank Alden Mead for help with isolation of ciliary epithelial bilayers, Pietro DeCamilli and Kohji Takei for generously providing InsP3R-1 antibody T210, and Richard Wojcikiewicz for generously providing InsP3R-2 antibody CT2.

Footnotes

Reprint requests: Marvin L. Sears, Department of Ophthalmology and Visual Science, Yale University School of Medicine, 330 Cedar Street, New Haven, CT 06520-8061.

Supported by NIH Grants EY-08879, EY-00785, and DK-45710; the E. Matilda Ziegler Foundation; an Established Investigator Grant from the American Heart Association; a Pilot and Feasibility Grant from the Cystic Fibrosis Foundation; and the Morphology Core Facility of the Yale Liver Center (NIH DK-34989).

Submitted for publication January 21, 1999; revised April 9, 1999; accepted April 28, 1999.

Proprietary interest category: N.

References

  1. Berridge, MJ (1993) Inositol trisphosphate and calcium signalling Nature 361,315-325[Medline][Order article via Infotrieve]
  2. Clapham, DE (1995) Calcium signaling Cell 80,259-268[Medline][Order article via Infotrieve]
  3. Cheek, TR, Jackson, TR, O’Sullivan, AJ, Moreton, RB, Berridge, MJ, Burgoyne, RD (1989) Simultaneous measurements of cytosolic calcium and secretion in single bovine adrenal chromaffin cells by fluorescent imaging of fura-2 in cocultured cells J Cell Biol 109,1219-1227[Abstract/Free Full Text]
  4. Ito, K, Miyashita, Y, Kasai, H. (1997) Micromolar and submicromolar Ca2+ spikes regulating distinct cellular functions in pancreatic acinar cells EMBO J 16,242-251[Medline][Order article via Infotrieve]
  5. Hahn, K, DeBiasio, R, Taylor, DL (1992) Patterns of elevated free calcium and calmodulin activation in living cells Nature 359,736-738[Medline][Order article via Infotrieve]
  6. Dolmetsch, RE, Xu, K, Lewis, RS (1998) Calcium oscillations increase the efficiency and specificity of gene expression Nature 392,933-936[Medline][Order article via Infotrieve]
  7. Li, W, Llopis, J, Whitney, M, Zlokarnik, G, Tsien, RY (1998) Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression Nature 392,936-941[Medline][Order article via Infotrieve]
  8. Newton, CL, Mignery, GA, Südhof, TC (1994) Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP3) receptors with distinct affinities for InsP3 J Biol Chem 269,28613-28619[Abstract/Free Full Text]
  9. Wojcikiewicz, RJH (1995) Type I, II, and III inositol 1,4,5-trisphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types J Biol Chem 270,11678-11683[Abstract/Free Full Text]
  10. Sugiyama, T, Yamamoto–Hino, M, Wasano, K, Mikoshiba, K, Hasegawa, M. (1996) Subtype-specific expression patterns of inositol 1,4,5-trisphosphate receptors in rat airway epithelial cells J Histochem Cytochem 44,1237-1242[Abstract]
  11. Sugawara, H, Kurosaki, M, Takata, M, Kurosaki, T. (1997) Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor EMBO J 16,3078-3088[Medline][Order article via Infotrieve]
  12. Mignery, GA, Newton, CL, Archer, BT, Sudhof, TC (1990) Structure and expression of the rat inositol 1,4,5-trisphosphate receptor J Biol Chem 265,12679-12685[Abstract/Free Full Text]
  13. Bezprozvanny, I, Watras, J, Ehrlich, BE (1991) Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum Nature 351,751-754[Medline][Order article via Infotrieve]
  14. Finch, EA, Turner, TJ, Goldin, SM (1991) Calcium as a coagonist of inositol 1,4,5-trisphosphate-induced calcium release Science 252,443-446[Abstract/Free Full Text]
  15. Harootunian, AT, Kao, JPY, Paranjape, S, Tsien, RY (1991) Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3 Science 251,75-78[Abstract/Free Full Text]
  16. Dupont, G, Goldbeter, A. (1993) One-pool model for Ca2+ oscillations involving Ca2+ and inositol 1,4,5-trisphosphate as co-agonists for Ca2+ release Cell Calcium 14,311-322[Medline][Order article via Infotrieve]
  17. Hagar, RE, Burgstahler, AD, Nathanson, MH, Ehrlich, BE (1998) Type III InsP3 receptor channel stays open in the presence of increased calcium Nature 396,81-84[Medline][Order article via Infotrieve]
  18. Takei, K, Stukenbrok, H, Metcalf, A, et al (1992) Ca2+ stores in Purkinje neurons: endoplasmic reticulum subcompartments demonstrated by the heterogeneous distribution of the InsP3 receptor, Ca2+-ATPase, and calsequestrin J Neurosci 12,489-505[Abstract]
  19. Mignery, GA, Sudhof, TC, Takei, K, De Camilli, P. (1989) Putative receptor for inositol 1,4,5-trisphosphate similar to ryanodine receptor Nature 342,192-195[Medline][Order article via Infotrieve]
  20. Lee, MG, Xu, X, Zeng, WZ, et al (1997) Polarized expression of Ca2+ channels in pancreatic and salivary gland cells: correlation with initiation and propagation of [Ca2+]i waves J Biol Chem 272,15765-15770[Abstract/Free Full Text]
  21. Alvaro, D, Alpini, G, Jezequel, AM, et al (1997) Role and mechanisms of action of acetylcholine in the regulation of rat cholangiocyte secretory function J Clin Invest 100,1349-1362[Medline][Order article via Infotrieve]
  22. Hirata, K, Nathanson, MH, Sears, ML (1998) Novel paracrine signaling mechanism in the ocular ciliary epithelium Proc Natl Acad Sci USA 95,8381-8386[Abstract/Free Full Text]
  23. Sears, ML, Yamada, E, Cummins, D, Mori, N, Mead, A, Murakami, M. (1991) The isolated ciliary bilayer is useful for studies of aqueous humor formation Trans Am Ophthalmol Soc 89,131-152[Medline][Order article via Infotrieve]
  24. Chen, S, Sears, M. (1997) A low conductance chloride channel in the basolateral membranes of the non-pigmented ciliary epithelium of the rabbit eye Curr Eye Res 16,710-718[Medline][Order article via Infotrieve]
  25. Edelman, JL, Sachs, G, Adorante, JS (1994) Ion transport asymmetry and functional coupling in bovine pigmented and nonpigmented ciliary epithelial cells Am J Physiol Cell Physiol 266,C1210-C1221[Abstract/Free Full Text]
  26. Nathanson, MH, Burgstahler, AD, Fallon, MB (1994) Multi-step mechanism of polarized Ca2+ wave patterns in hepatocytes Am J Physiol Gastrointest Liver Physiol 267,G338-G349[Abstract/Free Full Text]
  27. Fallon, MB, Gorelick, FS, Anderson, JM, Mennone, A, Saluja, A, Steer, ML (1995) Effect of cerulein hyperstimulation on the paracellular barrier of rat exocrine pancreas Gastroenterology 108,1863-1872[Medline][Order article via Infotrieve]
  28. Nathanson, MH, Fallon, MB, Padfield, PJ, Maranto, AR (1994) Localization of the type 3 inositol 1,4,5-trisphosphate receptor in the Ca2+ wave trigger zone of pancreatic acinar cells J Biol Chem 269,4693-4696[Abstract/Free Full Text]
  29. Schlosser, SF, Burgstahler, AD, Nathanson, MH (1996) Isolated rat hepatocytes can signal to other hepatocytes and bile duct cells by release of nucleotides Proc Natl Acad Sci USA 93,9948-9953[Abstract/Free Full Text]
  30. Nathanson, MH, Padfield, PJ, O’Sullivan, AJ, Burgstahler, AD, Jamieson, JD (1992) Mechanism of Ca2+ wave propagation in pancreatic acinar cells J Biol Chem 267,18118-18121[Abstract/Free Full Text]
  31. Nathanson, MH, Burgstahler, AD, Mennone, A, Boyer, JL (1996) Characterization of cytosolic Ca2+ signaling in rat bile duct epithelia Am J Physiol Gastrointest Liver Physiol 271,G86-G96[Abstract/Free Full Text]
  32. Yule, DI, Ernst, SA, Ohnishi, H, Wojcikiewicz, RJH (1997) Evidence that zymogen granules are not a physiologically relevant calcium pool: defining the distribution of inositol 1,4,5-trisphosphate receptors in pancreatic acinar cells J Biol Chem 272,9093-9098[Abstract/Free Full Text]
  33. Farahbakhsh, NA, Cilluffo, MC (1994) Synergistic effect of adrenergic and muscarinic receptor activation on [Ca2+]i in rabbit ciliary body epithelium J Physiol (Lond) 477,215-221[Abstract/Free Full Text]
  34. Schutte, M, Diadori, A, Wang, C, Wolosin, JM (1996) Comparative adrenocholinergic control of intracellular Ca2+ in the layers of the ciliary body epithelium Invest Ophthalmol Vis Sci 37,212-220[Abstract/Free Full Text]
  35. Ohuchi, T, Yoshimura, N, Tanihara, H, Kuriyama, S, Ito, S, Honda, Y. (1992) Ca2+ mobilization in nontransformed ciliary nonpigmented epithelial cells Invest Ophthalmol Vis Sci 33,1696-1705[Abstract/Free Full Text]
  36. Wax, MB, Coca–Prados, M. (1989) Receptor-mediated phosphoinositide hydrolysis in human ocular ciliary epithelial cells Invest Ophthalmol Vis Sci 30,1675-1679[Abstract/Free Full Text]
  37. Yule, DI, Williams, JA (1992) U73122 inhibits Ca2+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells J Biol Chem 267,13830-13835[Abstract/Free Full Text]
  38. Ghosh, TK, Eis, PS, Mullaney, JM, Ebert, CL, Gill, DL (1988) Competitive, reversible, and potent antagonism of inositol 1,4,5-trisphosphate-activated calcium release by heparin J Biol Chem 263,11075-11079[Abstract/Free Full Text]
  39. Tones, MA, Bootman, MD, Higgins, BF, Lane, DA, Pay, GF, Lindahl, U. (1989) The effect of heparin on the inositol 1,4,5-trisphosphate receptor in rat liver microsomes FEBS Lett 252,105-108[Medline][Order article via Infotrieve]
  40. Caulfield, MP (1993) Muscarinic receptors: characterization, coupling and function Pharmacol Ther 58,319-379[Medline][Order article via Infotrieve]
  41. Wu, D, Jiang, H, Katz, A, Simon, MI (1993) Identification of critical regions on phospholipase C-ß1 required for activation by G-proteins J Biol Chem 268,3704-3709[Abstract/Free Full Text]
  42. Nathanson, MH, Burgstahler, AD (1992) Subcellular distribution of cytosolic Ca2+ in isolated rat hepatocyte couplets: evaluation using confocal microscopy Cell Calcium 13,89-98[Medline][Order article via Infotrieve]
  43. Kasai, H, Augustine, GJ (1990) Cytosolic Ca2+ gradients triggering unidirectional fluid secretion from exocrine pancreas Nature 348,735-738[Medline][Order article via Infotrieve]
  44. Thorn, P, Lawrie, AM, Smith, PM, Gallacher, DV, Petersen, OH (1993) Local and global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists and inositol trisphosphate Cell 74,661-668[Medline][Order article via Infotrieve]
  45. Kasai, H, Li, YX, Miyashita, Y. (1993) Subcellular distribution of Ca2+ release channels underlying Ca2+ waves and oscillations in exocrine pancreas Cell 74,669-677[Medline][Order article via Infotrieve]
  46. Toescu, EC, Lawrie, AM, Petersen, OH, Gallacher, DV (1992) Spatial and temporal distribution of agonist-evoked cytoplasmic Ca2+ signals in exocrine acinar cells analysed by digital image microscopy EMBO J 11,1623-1629[Medline][Order article via Infotrieve]
  47. Thorn, P, Moreton, R, Berridge, M. (1996) Multiple, coordinated Ca2+-release events underlie the inositol trisphosphate-induced local Ca2+ spikes in mouse pancreatic acinar cells EMBO J 15,999-1003[Medline][Order article via Infotrieve]
  48. Llinas, R, Sugimori, M, Silver, RB (1992) Microdomains of high calcium concentration in a presynaptic terminal Science 256,677-679[Abstract/Free Full Text]
  49. Miyakawa, T, Maeda, A, Yamazawa, T, Hirose, K, Kurosaki, T, Iino, M. (1999) Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes EMBO J 18,1303-1308[Medline][Order article via Infotrieve]
  50. Ramos–Franco, J, Fill, M, Mignery, GA (1998) Isoform-specific function of single inositol 1,4,5-trisphosphate receptor channels Biophys J 75,834-839[Medline][Order article via Infotrieve]
  51. Maruyama, Y, Inooka, G, Li, YX, Miyashita, Y, Kasai, H. (1993) Agonist-induced localized Ca2+ spikes directly triggering exocytotic secretion in exocrine pancreas EMBO J 12,3017-3022[Medline][Order article via Infotrieve]
  52. Watanabe, S, Smith, CR, Phillips, MJ (1985) Coordination of the contractile activity of bile canaliculi: evidence from calcium microinjection of triplet hepatocytes Lab Invest 53,275-279[Medline][Order article via Infotrieve]
  53. Watanabe, N, Tsukada, N, Smith, CR, Phillips, MJ (1991) Motility of bile canaliculi in the living animal: implications for bile flow J Cell Biol 113,1069-1080[Abstract/Free Full Text]
  54. Nathanson, MH, Burgstahler, AD, Mennone, A, Fallon, MB, Gonzalez, CB, Saez, JC (1995) Ca2+ waves are organized among hepatocytes in the intact organ Am J Physiol Gastrointest Liver Physiol 269,G167-G171[Abstract/Free Full Text]
  55. Tse, A, Tse, FW, Almers, W, Hille, B. (1993) Rhythmic exocytosis stimulated by GnRH-induced calcium oscillations in rat gonadotropes Science 260,82-84[Abstract/Free Full Text]
  56. Sears J, Sears M. Circadian rhythms in aqueous humor formation. In: M. Civan, ed. From Secretion to Glaucoma. Academic Press, Current Topics in Membranes vol. 45: p. 203–232, 1998.



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