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

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⁺ (Ca_i ²⁺) signals are highly organized in many cell types and play an important role in regulating cell function. ^{1 2} For example, spatial patterns of Ca_i ²⁺ signals, such as Ca_i ²⁺ waves and gradients, direct functions such as secretion ^{3 4} and cell migration, ⁵ and temporal Ca_i ²⁺ signaling patterns such as oscillations direct functions such as gene expression. ^{6 7} The molecular basis for the subcellular organization of Ca_i ²⁺ signals is not completely understood, though. 

Ca_i ²⁺ 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 detail ¹² ; 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 Ca_i ²⁺. ^{13 14} This Ca²⁺ dependence of the type I InsP3 receptor is thought to be important for the formation of certain types of Ca_i ²⁺ signaling patterns, such as Ca_i ²⁺ oscillations. ^{15 16} The type III InsP3 receptor also is an InsP3-gated Ca²⁺ channel ¹⁷ ; 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 Ca_i ²⁺ thus suggests that the subcellular distribution of these two isoforms could provide a mechanism by which subcellular Ca_i ²⁺ signals are organized. The goal of the present study was to examine the relationship between subcellular Ca_i ²⁺ 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 antibody ²¹ 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 ×63 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 × 512 pixels (0.26μ m/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 Ca_i ²⁺ were expressed as (F/F₀) × 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 × 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 Ca_i ²⁺ 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 others ^{20 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. 

ACh-Induced Ca²⁺ Signaling in NPE cells Is Mediated by InsP3

To determine the relationship between the distribution of InsP3 receptor isoforms and Ca_i ²⁺ signaling patterns, we tried to identify an agonist that increases Ca_i ²⁺ via InsP3 in NPE cells. Acetylcholine increases Ca_i ²⁺ 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 Ca_i ²⁺ 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 Ca_i ²⁺ 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 Ca_i ²⁺ 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 Ca_i ²⁺ 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. 

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 Ca_i ²⁺ 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–β, ^{1 41} which suggests that stimulation with ACh would preferentially generate InsP3 in the basolateral region rather than apically. 

Subcellular Organization of Ca²⁺ Signals in NPE Cells

To observe the subcellular organization of ACh-induced Ca_i ²⁺ 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 Ca_i ²⁺ signals, images were collected every 10 msec. Ca_i ²⁺ 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 Ca_i ²⁺ 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 Ca_i ²⁺ wave propagation is similar to the pattern observed in other epithelia, including pancreatic, ^{30 43 44 45} lacrimal, ⁴⁶ and salivary ²⁰ 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, Ca_i ²⁺ 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 Ca_i ²⁺ 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  

To observe subcellular Ca_i ²⁺ signaling patterns that occur after the initial Ca_i ²⁺ 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 Ca_i ²⁺ was detected in only 23% (n = 6 of 26) of NPE cells. In contrast, a Ca_i ²⁺ increase was detected in 66% (n = 65 of 98) of cells stimulated with higher ACh concentrations (0.5, 1, 5, or 10 μM). Repetitive Ca_i ²⁺ spikes, persistent Ca_i ²⁺ gradients across the cytosol, or both were detected in 36 of the 71 cells that responded to ACh (Fig. 6) . Among these cells, repetitive Ca_i ²⁺ increases were either of greater amplitude, more sustained, or only present in the basolateral region, whereas persistent Ca_i ²⁺ gradients were manifested as prolonged increases in Ca_i ²⁺ in the basolateral region relative to the apical region (Fig. 6) . Localized increases in Ca_i ²⁺, including localized Ca_i ²⁺ oscillations, also occur in pancreatic acinar cells, ^{44 45} but those Ca_i ²⁺ increases are restricted to a region in which both the type I and type III InsP3 receptors are expressed. ^{28 45 47} Localized Ca_i ²⁺ increases have been reported in the presynaptic region of neurons ⁴⁸ and in the subplasmalemma of neuroendocrine cells ³ 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 Ca_i ²⁺ 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 Ca_i ²⁺ signals, ¹⁷ whereas the type I InsP3 receptor instead drives Ca_i ²⁺ oscillations and other longer-term Ca_i ²⁺ signaling patterns. 

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 Ca_i ²⁺ signaling is not addressed here. In B cells that normally coexpress all three InsP3 receptor isoforms, B-cell receptor stimulation results in InsP3-mediated Ca_i ²⁺ signaling when expression of one or even two of the isoforms is disrupted. ¹¹ This finding suggests that each isoform may provide a redundant Ca_i ²⁺ 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, Ca_i ²⁺ 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 Ca_i ²⁺ 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 Ca_i ²⁺ signaling in epithelia until now. 

What is the functional significance of the current findings? The ability to generate Ca_i ²⁺ gradients, waves, and oscillations may be critical for secretion to occur in polarized epithelia. For example, apical increases in Ca_i ²⁺ direct exocytosis, ^{4 51} because localized intense increases in Ca_i ²⁺ in the apical region induce targeting of vesicles to the apical membrane. ⁴ Apical increases in Ca_i ²⁺ also can direct the movement of subapical actin, which may mechanically facilitate secretion. ^{26 52 53 54} Apical-to-basal Ca_i ²⁺ waves direct vectorial movement of electrolytes such as Cl⁻ and Na⁺. ^{4 43} Finally, repetitive increases in Ca_i ²⁺ (i.e., Ca_i ²⁺ 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 Ca_i ²⁺ signals, whereas repetitive or sustained increases in Ca_i ²⁺ 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 Ca_i ²⁺ signals basolaterally rather than apically. Although it can be speculated that the subcellular distribution of InsP3 receptor isoforms organizes subcellular Ca_i ²⁺ signaling patterns in all cells, the novel functional requirements of NPE cells may provide a unique cell model in which to investigate this hypothesis. 

 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.

 

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, 10μ m. (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.

Figure 2.

 

The phospholipase C inhibitor U73122 (10 μM) inhibits ACh (10μ M)-induced Ca_i ²⁺ 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.

Figure 3.

 

Heparin but not de-N-sulfated heparin blocks ACh-induced Ca_i ²⁺ 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).

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.

Figure 5.

 

Acetylcholine-induced increases in Ca_i ²⁺ begin as apical-to-basal Ca_i ²⁺ 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 Ca_i ²⁺ (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 20.4μ m/s.

Figure 6.

 

Examples of distinct apical and basolateral Ca_i ²⁺ 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 Ca_i ²⁺ spikes with a frequency of ∼0.1 s⁻¹ are seen in the basolateral but not the apical region. (B) An increase in Ca_i ²⁺ persists for >1 minute in the basolateral region, but Ca_i ²⁺ is elevated apically for <20 seconds. (C) There is a sustained ∼45% increase in fluo-3 fluorescence, with superimposed Ca_i ²⁺ spikes (frequency, ∼0.1 second^-1) in the basolateral region, whereas apically there is only a ∼15% increase with no superimposed Ca_i ²⁺ spikes.

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.