Plasma and intracellular membrane inositol 1,4,5-trisphosphate receptors mediate the Ca2+ increase associated with the ATP-induced increase in ciliary beat frequency

Nelson P. Barrera,1 Bernardo Morales,2 and Manuel Villalón1

1Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, and 2Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile

Submitted 7 August 2003 ; accepted in final form 1 June 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
An increase in intracellular free Ca2+ concentration ([Ca2+]i) has been shown to be involved in the increase in ciliary beat frequency (CBF) in response to ATP; however, the signaling pathways associated with inositol 1,4,5-trisphosphate (IP3) receptor-dependent Ca2+ mobilization remain unresolved. Using radioimmunoassay techniques, we have demonstrated the appearance of two IP3 peaks occurring 10 and 60 s after ATP addition, which was strongly correlated with a release of intracellular Ca2+ from internal stores and an influx of extracellular Ca2+, respectively. In addition, ATP-dependent Ca2+ mobilization required protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II activation. We found an increase in PKC activity in response to ATP, with a peak at 60 s after ATP addition. Xestospongin C, an IP3 receptor blocker, significantly diminished both the ATP-induced increase in CBF and the initial transient [Ca2+]i component. ATP addition in the presence of xestospongin C or thapsigargin revealed that the Ca2+ influx is also dependent on IP3 receptor activation. Immunofluorescence and confocal microscopic studies showed the presence of IP3 receptor types 1 and 3 in cultured ciliated cells. Immunogold electron microscopy localized IP3 receptor type 3 to the nucleus, the endoplasmic reticulum, and, interestingly, the plasma membrane. In contrast, IP3 receptor type 1 was found exclusively in the nucleus and the endoplasmic reticulum. Our study demonstrates for the first time the presence of IP3 receptor type 3 in the plasma membrane in ciliated cells and leads us to postulate that the IP3 receptor can directly trigger Ca2+ influx in response to ATP.

transduction mechanisms; P2Y receptor; calcium influx


CILIATED CELLS FROM OVIDUCTAL MUCOSA play an important role in the control of the mucociliary transport velocity of gametes and embryos (27). These cells modify their ciliary beat frequency (CBF) in response to a variety of chemical, electrical, and mechanical signals. For example, extracellular ATP is a powerful activator of CBF in cells derived from rabbit oviduct (46), rabbit trachea (19, 42), human trachea (21), and frog palate and esophagus (12, 39, 50), and an inhibitor of CBF in rat brain ependymal cells (31). Recently, we demonstrated that the increase in CBF induced by ATP in ciliated oviductal cells was mediated by P2Y2 receptor activation (28). Furthermore, we provided evidence that stimulation of ciliary activity by ATP requires phospholipase C (PLC) activation, Ca2+ mobilization by Ca2+ released from intracellular stores through inositol 1,4,5-trisphosphate (IP3) receptor activation and Ca2+ influx, and protein kinase C (PKC) activation. It is well known that in ciliated cells, a rise in intracellular free Ca2+ concentration ([Ca2+]i) through release from intracellular stores results in an increase in CBF (15, 19, 28, 42, 46). Furthermore, airway epithelial cells demonstrate Ca2+ wave propagation that is mediated by the activation of IP3 receptors (14, 37). However, the role and subcellular distribution of IP3 receptors involved in the ATP-dependent [Ca2+]i increase in ciliated cells remain unknown.

In mammals, at least three IP3 receptor types (types 1, 2, and 3) are encoded by three distinct genes (2, 38), and the expression of the IP3 receptors has been reported in ciliated cells from both rat olfactory cilia (8) and murine oviduct (29). IP3 receptors participate in Ca2+ signaling by mediating intracellular Ca2+ release (16) and regenerative Ca2+ signals (9). Interestingly, the type 3 receptor mediates regulation of Ca2+ flux across the plasma (34, 35, 44) and nuclear (40) membranes.

IP3 receptors can be regulated in a number of ways. They contain binding sites for Ca2+, and elevation in free [Ca2+] from nanomolar to micromolar concentrations modifies receptor properties (36). Differential Ca2+ sensitivity of Ca2+ activation sites between IP3 receptor types 1 and 3 (5, 25) and a hypothetical differential subcellular localization and expression of IP3 receptor types in the same ciliated cell could evoke complex Ca2+ responses to extracellular agonist stimulation. In addition, several cytosolic factors, including Ca2+/calmodulin (CaM) (1), and phosphorylation by protein kinase A (PKA) (51), PKC (10), and CaM-dependent protein kinase II (CaMKII) (53) can modulate the activity of IP3 receptors. However, whether these factors are associated with the ATP transduction pathways in oviductal ciliated cells is an unresolved issue.

Although the activating effect of ATP on CBF has been demonstrated in ciliated cells, the presence and mechanisms of activation of IP3 receptor associated with the ATP-dependent control of CBF are still not completely understood. In the present study, we show that the participation of the extracellular and intracellular Ca2+ reservoirs in the ATP response is dependent on IP3 receptor activation and that PKC and CaMKII are involved in Ca2+ mobilization. Furthermore, we have analyzed the quantitative and temporal relationships between ATP-induced CBF increase and changes in [IP3] and [Ca2+]i. In addition, we have demonstrated the differential subcellular localization of IP3 receptor types 1 and 3 in the same oviductal ciliated cell, showing that only the type 3 receptor is present in plasma membrane. Finally, we discuss the relationship between timing and subcellular localization of IP3 receptor activity in ciliated cells and suggest that both receptor types can play specific roles in the induction of the ATP-dependent [Ca2+]i increase.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and solutions. ATP, the PKC activator phorbol 12-myristate 13-acetate (PMA), EGTA, and Hanks' balanced salt solution were obtained from Sigma-Aldrich (St. Louis, MO). Fura 2-AM was obtained from Molecular Probes (Eugene, OR). The PLC inhibitor U-73122, the IP3 receptor inhibitor xestospongin C, the PKC blocker bisindolylmaleimide I (GF-109203X), the CaM blocker W-7, the CaMKII inhibitor peptide 281–309 (CBP), and the Ca2+ pump ATPase blocker thapsigargin were obtained from Calbiochem-Novabiochem International (San Diego, CA). Stock solutions of ATP, W-7, and CBP were prepared in water (pH 7.4) and diluted in Hanks' buffer just before use. In addition, the CBP solution was added to the Rose chamber in a hyposmotic medium (~35% reduction of normal osmolarity, ~290 mosM, in Hanks' buffer) during the period of incubation. After this incubation, this solution was changed to Hanks' buffer. All other reagents were dissolved in dry dimethyl sulfoxide (DMSO) at a final concentration of 0.1%. At this concentration, DMSO had no effect on CBF.

Culturing of ciliated cells from hamster oviductal epithelium. Experiments were performed in primary cultures of hamster oviductal epithelium obtained using the procedure previously described by Verdugo et al. (45). Adult (2–3 mo old) hamsters were anesthetized with 30% halothane and then decapitated. The oviducts were removed and placed in Hanks' solution (Sigma-Aldrich) at pH 7.4. The distal portion of the oviduct (i.e., the fimbria) was cut into small (4–8 mm2) pieces and placed in N-hydroxysuccinimide culture medium (in mM: 137 NaCl, 5.09 KCl, 1.14 Na2HPO4, 0.18 KH2PO4, 0.923 MgCl2, 0.91 CaCl2, 4.07 NaHCO3, 21.5 glucose, and 0.2 glutamine, supplemented with 1.0% vitamins, 1.0% essential amino acids, 1.0% nonessential amino acids, 1.0% pyruvate and antibiotics, 0.2 mg/ml neomycin, and 0.12 mg/ml penicillin, pH 7.2–7.4) for 30–40 min at 37°C. The tissue was then transferred to sterile Hanks' solution, and the ciliated epithelium (which has >95% ciliated cells) was mechanically removed from the rest of the tissue.

Explants of ciliated epithelium were grown on glass coverslips previously coated with a solution of 0.5% gelatin. A strip of dialysis membrane was used to cover the explants and secure them on the glass coverslip before assembly into Rose chambers filled with culture medium supplemented with 10% horse serum, 1 mg/ml neomycin, and 1.21 mg/ml penicillin. Explant-containing Rose chambers were incubated at 37°C for up to 7 days until the culture formed a monolayer of ciliated cells. When this cell monolayer showed spontaneous ciliary activity, the chambers were opened and the cultures were washed three times with fresh Hanks' solution before CBF measurements were performed. All procedures were approved by the Pontifical Catholic University of Chile Animal Care and Ethics Committee.

Measurement of CBF. CBF was monitored and recorded by performing microphotodensitometry according to a procedure described previously (47). Briefly, the spectral structure of light-scattering fluctuations produced by the moving cilia of a single cell was detected with a photodiode, and the signal was processed online using a digital spectrum computer card (model R360; Rapid System, Jersey City, NJ) installed on a personal computer. The CBF was derived from the power spectrum obtained by performing fast Fourier transform of the data. The instant averaged spectrum was recorded for further analysis. CBF in individual ciliated cells was recorded.

Measurement of [Ca2+]i. [Ca2+]i was determined using a spectrofluorometric technique described previously (13). Primary cell cultures exhibiting spontaneous ciliary activity at 37°C were loaded with fura 2-AM for 30 min. Cells were excited at 349 and 380 nm in a Nikon Diaphot microscope equipped with a Photon Technology International spectrofluorometer (Lawrenceville, NJ) for ratio analysis. After 10 min of stabilization, the intensity ratio was continuously recorded and cultures were superfused with solutions containing the drugs to be tested. Fluorescence emission intensity was detected with a photomutiplier and acquired and analyzed using a fluorescence analysis program (FELIX version 1.1). To study the possible toxicity of the continuous excitation of fura 2 to ciliated cells, we determined basal CBF in individual ciliated cells (n = 15) incubated with the fluorescent indicator and either unexcited or excited at 349 and 380 nm. We did not observe a difference in the basal CBF between the two conditions. In addition, the time course of the ATP effect on CBF was similar in both protocols (n = 8).

Extraction and quantification of IP3. Oviductal ciliated cells obtained by mechanical extraction of the epithelium of fimbria were placed in Hanks' medium at 37°C in an atmosphere of 5% CO2 and incubated with ATP. The reaction was stopped with 1 M cold trichloroacetic acid (TCA). The tissue was homogenized using the XL Microson ultrasonic cell disruptor (Heat Systems Ultrasonics, Farmingdale, NY). Briefly, each homogenate was centrifuged for 10 min at 2,000 g. The supernatant was collected and the TCA was extracted using 2 volumes of a 3:1 solution of 1,1,2-trichloro-1,2,2-trifluoroethane-trioctylamine. The resulting aqueous phase, which contains IP3, was removed using a transfer pipette. Samples were maintained at –70°C until analysis. The IP3 concentration ([IP3]) in 20 µg of protein from oviductal ciliated cells was determined using a commercial radioreceptor assay kit (NEN Life Sciences, Boston, MA). The [IP3] was expressed as picomoles of IP3 per microgram of protein. Epithelial protein content was determined using the Bradford method (3). A recovery of 98% was observed after the addition of a known amount (1 pmol) of IP3 to the culture medium. The sensitivity of the assay was 0.05 pmol IP3. In all cases, cross reactivity with inositol 1,4-bisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate was <2%.

Extraction of PKC and quantification of PKC activity. Oviductal ciliated cells obtained by mechanical extraction of the epithelium of fimbria were placed in Hanks' medium at 37°C in an atmosphere of 5% CO2 and incubated with ATP. Samples were homogenized and centrifuged at 100,000 g for 60 min at 4°C for PKC extraction. The supernatant was collected, and the protein concentration was determined as described previously (3). The PKC activity in 20 µg of protein was determined using a commercial ELISA kit (Calbiochem). The PKC activity was expressed as milliunits of PKC per microgram of protein.

Immunocytochemical localization of IP3 receptors in cultured ciliated cells. Immunofluorescence and confocal microscopy were used to determine the cellular localization of the IP3 receptors. Cultured ciliated cells were fixed for 30 min at room temperature in 3% paraformaldehyde in PBS containing 2 mM MgCl2 and 0.2 mM CaCl2 (PBSCaMg). Samples were washed three times in PBSCaMg. Next, ciliated cells were permeabilized with 0.2% Triton X-100 in PBSCaMg for 10 min. Samples were washed four times in PBS containing 0.2% gelatin (PBS-gel) and incubated overnight at 4°C with the purified primary antibodies: anti-IP3 receptor type 1 (polyclonal; Affinity BioReagents, Golden, CO) and/or anti-IP3 receptor type 3 (monoclonal; BD Transduction Laboratories, San Diego, CA), dilution 1:50. Samples were washed four times in PBS-gel and then incubated at 37°C for 60 min either with secondary antibody Cy2-conjugated, affinity-purified goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), dilution 1:50, or with Cy3-conjugated, affinity-purified goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), dilution 1:100, as appropriate. Samples were washed three times in PBS-gel and three times in PBS and then mounted in Fluoromount G (Electron Microscopy Sciences, Hatfield, PA). Fluorescence was detected using an LSM 510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). Controls for the specificity of IP3 receptor immunofluorescence included 1) omitting the primary antibody, 2) analyzing the cross-reaction between primary and secondary antibodies, and 3) incubating the antibodies either with an excess of neutralizing peptide (Affinity BioReagents) that reacts with anti-IP3 receptor type 1 antibody or with HeLa lysate (BD Transduction Laboratories) for anti-IP3 receptor type 3 antibody.

Immunocytochemical subcellular localization of IP3 receptors in oviductal ciliated cells. Immunogold electron microscopy was used to determine the subcellular localization of the IP3 receptors. Tissue samples from oviductal epithelium of hamster fimbria were fixed for 4 h at room temperature in 4% paraformaldehyde containing Zamboni's fixative in 0.1 M PBS at pH 7.3. Samples were rinsed in 0.1 M PBS for 12 h at 4°C and pH 7.3, dehydrated, and embedded in LR White resin (London Resin, Reading, UK) at 50°C. Sections were placed on nickel grids and rinsed for 30 min in PBS containing 1% BSA. The grids were floated with the section side down for 1 h at room temperature on drops of primary antiserum diluted in PBS containing 1% BSA (primary antibodies were the same as those used for immunofluorescence, with dilutions of 1:30 for primary antibody anti-IP3 receptor type 1 and 1:80 for primary antibody anti-IP3 receptor type 3). The sections were then rinsed dropwise with the same buffer. The grids were treated in the same manner with goat anti-rabbit or anti-mouse antibodies conjugated to 40-nm gold particles (Kirkegaard & Perry Laboratories, Gaithersburg, MD), diluted 1:30 in the above buffer. The sections were then postfixed with 0.5% glutaraldehyde for 5 min. After a water rinse, the grids were counterstained for 5 min with 1% uranyl acetate. Electron microscopy was performed using a Siemens 101 electron microscope (Siemens, Iselin, NJ) operated at 80 keV. Controls for the specificity of IP3 receptor immunogold labeling included 1) omitting the primary antibody, 2) analyzing the cross-reaction between primary and secondary antibodies, and 3) incubating the antibodies either with an excess of neutralizing peptide (Affinity BioReagents) that reacts with anti-IP3 receptor type 1 antibody or with HeLa lysate (BD Transduction Laboratories) for anti-IP3 receptor type 3 antibody.

Data analysis. Statistical comparisons between different experimental conditions were made using ANOVA with SPSS version 8.0 software (SPSS, Chicago, IL). The criterion for a significant difference was a final value of P < 0.05. Data are expressed as means ± SE; n refers to the number of cultures or samples analyzed. The curves were fitted to logistic equations using the Inplot computer program (GraphPad Software, San Diego, CA). Analysis of regression and correlation was performed using SPSS version 8.0 with stepwise methods, with significance set at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
[Ca2+]i increase induced by extracellular ATP in ciliated cells. We previously suggested that the increase in CBF induced by ATP in oviductal ciliated cells involves Ca2+ mobilization from intracellular reservoirs and extracellular medium (28). We therefore first focused on quantifying and characterizing the effect of ATP on [Ca2+]i in single ciliated cells from the oviduct. Figure 1A shows the time course of the effect of extracellular ATP on [Ca2+]i. After the addition of 100 µM ATP, we observed two components of the response. First, the transient [Ca2+]i increase reached the maximum average value of 387.8 ± 32.0 nM over basal [Ca2+]i (70.6 ± 5.4 nM; data not shown) within 10.8 ± 0.2 s, and then the response diminished to a plateau of 56.2 ± 7.0 nM, which was higher than basal levels. To analyze the effect of ATP on [Ca2+]i, we fitted the response decay to a simple exponential equation with the form [Ca2+]i = a + b·e(–t/{tau}), where a + b represents the peak value, a is the plateau value, and {tau} is the decay time constant. Figure 1B shows a distribution of the peak and plateau values (describing the [Ca2+]i increase) in relation to ATP concentration. Concentration-response curves of peak and plateau values between 1 nM and 100 µM ATP were fitted using the equation y = max{1 + (EC50·[A]–1)nH}–1, where max is the maximal effect, EC50 represents the agonist concentration necessary to obtain the half-maximal effect, [A] is the ATP concentration, and nH is the Hill coefficient. Analysis of the curves revealed EC50 values of 6.45 ± 0.68 µM for the peak and 8.31 ± 1.02 µM for the plateau. Student's t-test analysis demonstrated statistically significant differences between the two values (P < 0.05), suggesting a higher efficacy of ATP in the generation of the transient component. Taken together, these results demonstrate a concentration-dependent [Ca2+]i response for the two components.



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Fig. 1. Effect of ATP on intracellular free Ca2+ concentration ([Ca2+]i). A: time course of [Ca2+]i increase induced by extracellular 100 µM ATP. The application and removal of ATP are indicated by bar at top. Trace corresponds to average of 14 responses of individual cells. Continuous line represents fitted data using the exponential equation described in RESULTS. B: concentration-response curves of changes in [Ca2+]i for peak ({square}) and plateau ({triangleup}) values. Each point represents the average maximal value ± SE for 8–10 experiments. Values between the 2 curves at each point are significantly different (P < 0.05; ANOVA).

 
IP3 receptor activation during [Ca2+]i increase induced by ATP. Recently, we found that a reduction in the extracellular Ca2+ concentration ([Ca2+]e), from 1 mM to 10 nM, as well as inhibition of IP3 receptor activation, reduced the CBF increase induced by ATP (28). We therefore analyzed IP3 receptor activity during the ATP-induced [Ca2+]i increase. Figure 2A shows that a reduction of [Ca2+]e to 10 nM (using EGTA) resulted in a 94.4 ± 1.7% inhibition of the plateau value of [Ca2+]i (P < 0.05), without causing significant changes in the peak value. In addition, the CBF and basal values of [Ca2+]i were not modified in the Ca2+-free medium. The maximum value of the CBF increase induced by ATP was diminished by only 15.3 ± 2.4% in the Ca2+-free medium (28); however, there was a significant reduction in the plateau value, suggesting that the Ca2+ influx is not required to trigger the ATP-dependent ciliary response but may be needed to maintain or amplify it. On the other hand, as shown in Fig. 2B, the first component of the [Ca2+]i increase induced by ATP was significantly diminished in the presence of xestospongin C, an inhibitor of IP3 receptor activation (11). In the presence of 5 µM xestospongin C added 10 min before ATP application, the peak value was diminished by 81.9 ± 4.5% (P < 0.05), and there was also a reduction in the plateau value of 58.8 ± 3.6% (P < 0.05) compared with the control response (note also that 35% of the treated cells exhibited total inhibition). The use of Ca2+-free medium did not change the {tau} value ({tau} = 25.3 ± 1.7 s) compared with control ({tau} = 23.1 ± 1.3 s), suggesting that the origin of the first component of the response and its decay is accounted for by Ca2+ mobilization from intracellular Ca2+ stores induced by IP3 receptor activation. Because thapsigargin-dependent intracellular Ca2+ stores participate in the ATP-induced CBF response (28), we used this Ca2+ ATPase pump blocker to study the effect of IP3 receptor activation on the [Ca2+]i increase. When the ciliated cells were preincubated with 1 µM thapsigargin, the addition of 100 µM ATP caused a smaller increase in [Ca2+]i with respect to the control (Fig. 2C). However, the effect of 100 µM ATP on [Ca2+]i was almost completely abolished after thapsigargin addition in the presence of extracellular EGTA (~10 nM extracellular Ca2+; Fig. 2C). Interestingly, abolition of the ATP effect (P = 0.001) was also observed when ciliated cells were preincubated with thapsigargin followed by treatment with 5 µM xestospongin C for 10 min before ATP application (Fig. 2D). These results suggest an IP3 receptor activation-dependent Ca2+ influx. Taken together, these results indicate that the first component of the [Ca2+]i increase depends on IP3 receptor activation and that this component is required to initiate the ATP-induced increase in CBF. Moreover, it is possible that the Ca2+ influx is mediated directly by IP3 receptor activation. On the other hand, Morales et al. (28) obtained a complete inhibition of the CBF increase induced by ATP in the presence of a PLC inhibitor, U-73122 (6, 32). Because the PLC activation-dependent [IP3] increase should involve the participation of an IP3 receptor, we determined whether PLC inhibition blocked the [Ca2+]i increase. Figure 2E demonstrates complete inhibition of ATP-induced [Ca2+]i increase in the presence of 1 µM U-73122, suggesting that both components of the response require PLC activation. Interestingly, the delay in the response evoked by ATP in the presence of the different pharmacological agents used is similar to that in control ATP responses, suggesting the participation of the same ATP-activated transduction pathways in the different experimental approaches. Taken together, these results suggest that an initial component, which is dependent on IP3 receptor activation, initiates the CBF increase. Subsequently, Ca2+ influx probably induced by IP3 receptor activation is involved in the maintenance or amplification of the CBF response induced by ATP.



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Fig. 2. Participation of inositol 1,4,5-trisphosphate (IP3) receptor activation in the [Ca2+]i increase induced by ATP. A: incubation of ciliated cells with EGTA induced reduction in plateau value in response to 100 µM ATP. Trace corresponds to average of 10 responses of individual cells. B: xestospongin C (5 µM) reduced peak value and partially reduced plateau value in response to 100 µM ATP. Trace corresponds to average of 10 responses of individual cells. C: thapsigargin (1 µM) induced a [Ca2+]i increase, and subsequently 100 µM ATP triggered a lower increase in [Ca2+]i compared with control response (black trace), which is inhibited in the presence of extracellular EGTA (gray trace). Traces correspond to average of 6 responses of individual cells. D: xestospongin C (5 µM) blocked Ca2+ influx induced by 100 µM ATP. Trace corresponds to average of 6 responses of individual cells. E: application of 1 µM U-73122 totally blocked response of [Ca2+]i to 100 µM ATP. Trace corresponds to average of 8 responses of individual cells. Bars indicate incubation time in the presence of each agent.

 
Increased IP3 synthesis in response to ATP and the participation of PKC in the [Ca2+]i increase. Because the PLC pathway and IP3 receptor activation are responsible for the [Ca2+]i increase induced by ATP, we studied the effect of ATP on IP3 synthesis in oviductal ciliated cells. Figure 3A shows the time course of the [IP3] response induced by 100 µM ATP (n = 12). We observed a maximum increase in [IP3] at 10 s (66.95 pmol IP3·µg–1 protein) after ATP addition, followed by a mean response between 30 s and 4 min, which was 9.5 ± 1.5-fold lower than the maximum response (P < 0.05). It is important to emphasize that a significant [IP3] increase had already started 6 s after ATP addition. Consistent with the participation of PLC in the ATP response, a complete block in the [IP3] increase was observed when the ciliated cells were preincubated with 1 µM U-73122 (n = 5) in the presence of ATP. Figure 3B demonstrates a concentration-response curve for [IP3] in the presence of ATP. The same equation used in Fig. 1C fitted the data between 1 nM and 100 µM. EC50 was 0.29 ± 0.5 µM of the [IP3] increase, which is ~20-fold lower than the EC50 of the peak value of the [Ca2+]i increase. In addition, the EC50 for CBF increase induced by ATP was 10.2 µM (28). These results suggest that the sensitivity of the ATP transduction pathways tends to decrease along with an advance in the signaling cascade. However, knowledge of the generation of second messengers at each time point during the response would be required to determine how dynamic responses involved in the ATP transduction pathways are sequentially activated. In support of the idea of a strong relationship between IP3 synthesis and Ca2+ mobilization, the maximum change in IP3 synthesis had a high correlation (r = 0.9501) with the peak Ca2+ value (Fig. 3C). We also observed that the second, lower [IP3] increase at 60 s had a high correlation (r = 0.9615) with the plateau value (Fig. 3C), suggesting that both the intracellular Ca2+ mobilization from internal stores and the Ca2+ influx can be related to two IP3 peaks occurring at different times. To summarize the effect of ATP on ciliary activity, in Fig. 3D, we superimposed the time courses of CBF, [Ca2+]i, and [IP3] increases induced by 100 µM ATP. Interestingly, these three responses had similar time courses (P < 0.05; ANOVA), and, as previously postulated, we observed an order in the sequence of responses to ATP, beginning with an [IP3] increase that evoked a [Ca2+]i increase and a later cellular response, the CBF increase.



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Fig. 3. IP3 concentration ([IP3]) increase and participation of protein kinase C (PKC) in [Ca2+]i increase induced by ATP. A: time course of effect of 100 µM ATP on [IP3]. ATP induced [IP3] increase ({square}), which was completely blocked by coincubation with 1 µM U-73122 ({blacktriangleup}). Bar indicates application of ATP ± U-73122. Data are means ± SE (n = 10). B: concentration-response curve of maximum change in [IP3] induced by ATP. Data are means ± SE (n = 8). C: Pearson correlation between peak value and maximum change in [IP3] ({bullet}) and between plateau value and change in [IP3] at 60 s after ATP application ({circ}). Both correlations show statistical significance at P < 0.05. Data are means ± SE (n = 15). D: summary of time courses of [IP3] ({square}) (n = 10), [Ca2+]i (trace showing average of 10 individual responses), and ciliary beat frequency (CBF) ({circ}) (n = 20) increases induced by 100 µM ATP. Note that delay in initiation of responses was 6 and 10 s for [IP3] and [Ca2+]i increases, respectively, but the cellular response, CBF increase, was not observed for ~30 s. Data are means ± SE. E: small inhibition of [Ca2+]i increase induced by ATP in presence of 2 µM GF-109203X. Trace corresponds to average of 6 responses of individual cells. F: effect of phorbol 12-myristate 13-acetate (PMA) on [Ca2+]i. Application of 500 nM PMA induced no change in [Ca2+]i; however, [Ca2+]i increase induced by 100 µM ATP had a higher plateau value in presence of this PKC activator. Trace corresponds to average of 8 responses of individual cells. G: time course of effect of 100 µM ATP on PKC activity. ATP induced increase in PKC activity that reached maximum value at 60 s ({circ}); however, PKA activity was not changed in presence of ATP ({square}). Data are means ± SE (n = 12). H: concentration-response curve of maximum change in PKC activity induced by ATP. Data are means ± SE (n = 12).

 
Activation of the PLC pathway is known to evoke the production of diacylglycerol, the activator of PKC (30). Therefore, three experimental approaches were used to examine the participation of PKC in the [Ca2+]i increase. First, ciliated cells were preincubated with a PKC inhibitor, GF-109203X (41). Figure 3E shows that in the presence of 2 µM GF-109203X, there was a reduction of 54.0 ± 3.8% in the plateau value compared with the control response. However, the change in the peak value (14.5 ± 2.7%) was not statistically significant. Second, cells were stimulated by ATP in the presence of 500 nM PMA, a PKC activator (33). As shown in Fig. 3F, PMA did not evoke any change in basal [Ca2+]i. These results demonstrate that activation of PKC did not bring about an increase in [Ca2+]i but that activation of this pathway is necessary to produce the ATP response resulting in [Ca2+]i influx. Furthermore, PKC was not involved in the [IP3] increase induced by ATP because preincubation with GF-109203X or PMA evoked no change in IP3 production (data not shown). In addition, we observed a reduction of 78.5 ± 3.6% in the increase in CBF induced by ATP when ciliated cells were preincubated with 2 µM GF-109203X, and we observed a transient CBF increase (maximum of 61.2 ± 2.9%) after PMA treatment (28). Figure 3F shows that coincubation with 100 µM ATP and 500 nM PMA produced a plateau value 1.8-fold higher than the control response; however, this coapplication induced no change in the peak value. Third, we quantified PKC activity in oviductal ciliated cells stimulated with ATP. Figure 3G shows the time course of the effect of 100 µM ATP on PKC activity; there was a peak at 60 s that correlated with the appearance of the plateau component. The effect of ATP on PKC activity was dependent on ATP concentration (Fig. 3H), with an EC50 of 9.3 ± 0.2 µM. Because PKA has been implicated in the control of CBF through a release of Ca2+ from intracellular stores (4), we quantified PKA activity in oviductal ciliated cells. Figure 3G shows that PKA was not involved in the ATP transduction pathways. Taken together, these results suggest that PKC is a positive modulator of the Ca2+ influx and that PKC is probably a second messenger downstream of [Ca2+]i in the ATP effect.

Localization of IP3 receptors in oviductal ciliated cells. On the one hand, we report that IP3 receptors are present in ciliated cells and that they participate in the transduction pathways induced by ATP; on the other hand, it was previously suggested that multiple IP3 receptor intracellular locations could be related to the differing roles of these receptors in Ca2+ mobilization (9, 34). To determine the location of IP3 receptor types 1 and 3 in oviductal ciliated cells, immunofluorescence and confocal microscopy were performed with cultured ciliated cells using anti-IP3 receptor types 1 and 3 antibodies (Fig. 4). Figure 4A is a photomicrograph of red fluorescence indicating the localization of IP3 receptor type 3 in ciliated cells, and Fig. 4B shows the corresponding phase-contrast image. Figure 4C represents the localization of IP3 receptor type 1 in ciliated cells detected by green fluorescence, and Fig. 4D shows the corresponding phase-contrast image. In control experiments without primary antibodies, no fluorescence was detected in ciliated cells (data not shown). Interestingly, when the two receptor types were visualized in the same cell (Fig. 4, E–H), the type 3 receptor (red fluorescence in Fig. 4E) was concentrated at the apical pole of the cell, whereas the type 1 receptor (green fluorescence in Fig. 4F) was homogeneously distributed. To extend our observation, we performed immunogold electron microscopy to determine the subcellular location of IP3 receptor types 1 and 3 in oviductal ciliated cells (Fig. 5). IP3 receptor type 1-reacting gold particles were found in the nucleus, endoplasmic reticulum, and intracellular vesicles, whereas practically no gold particles were found in the nuclear and plasma membranes (see Fig. 5C, solid bar). IP3 receptor type 3-reacting gold particles were also found in the nucleus and the endoplasmic reticulum of oviductal ciliated cells; however, in contrast to IP3 receptor type 1, type 3-reacting gold particles were found in the nuclear and plasma membranes (see Fig. 5C, shaded bar). Representative photomicrographs of the subcellular localization of IP3 receptor type 3-reacting gold particles in plasma membrane (Fig. 5A) and nucleus (Fig. 5B) are shown. In parallel, control experiments without primary antibody demonstrated that no gold particles were found in identical ciliated tissues (data not shown). This evidence demonstrates a diverse intracellular distribution of the IP3 receptor types 1 and 3 in ciliated cells, which could be related to the [Ca2+]i increase induced by ATP-dependent IP3 receptor activation from several Ca2+ stores, including the nucleus, the endoplasmic reticulum, and the extracellular medium.



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Fig. 4. Localization of IP3 receptor types 1 and 3 in oviductal ciliated cells. A: immunofluorescence localization of IP3 receptor type 3 in cultured ciliated cells. B: ciliated cells shown in phase-contrast image. C: immunofluorescence localization of the IP3 receptor type 1 in cultured ciliated cells. D: ciliated cells shown in phase-contrast image. E and F: simultaneous detection by immunofluorescence of both IP3 receptor types 3 and 1 in the same ciliated cell. G and H: ciliated cell in phase-contrast image (G) and merged fluorescence image (H).

 


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Fig. 5. Subcellular localization of IP3 receptor types 1 and 3 in oviductal ciliated cells. A and B: representative photomicrographs of sections incubated with anti-IP3 receptor type 3 antibody followed by immunogold electron microscopy. Gold particle labeling is localized to the plasma membrane (PM) (A) and nucleus (N) (B). Bars and arrows indicate scale and gold particles, respectively. C: density of gold particles observed for IP3 receptor type 1 (black bar) and type 3 (gray bar) in N, endoplasmic reticulum (ER), PM, vesicles (V), and nuclear membrane (NM). Data are means ± SE (n = 10). Asterisk indicates significant statistical difference in density of gold particles between both types of IP3 receptors.

 
Participation of the CaM pathway in the ATP effect in ciliary activity. Because of the [Ca2+]i increase induced by ATP, which is responsible for the increment in CBF, we anticipated the participation of CaM, a known calcium-dependent protein, in the control of ciliary activity (52). We therefore asked whether this protein is capable of modifying the Ca2+ mobilization induced by ATP. Figure 6A shows that preincubation with 50 µM W-7, a CaM inhibitor, brought about 78.2 ± 5.2% inhibition of the maximum CBF induced by 100 µM ATP. In addition, Fig. 6C shows that preincubation with 50 µM W-7 caused only a small, transient increase in [Ca2+]i in response to ATP. To determine whether CaMKII, a CaM activity-dependent kinase (53), is involved in the transduction pathway activated by ATP, we preincubated the ciliated cells for 15 min (through hyposmotic shock) with 30 µM CBP, a synthetic peptide that inhibits CaMKII (49) by blocking CaM activation. After this preincubation, the solution was replaced by control Hanks' buffer for 10 min before application of 100 µM ATP (Fig. 6, B and D). Interestingly, the time course of the CBF response to ATP showed a 74.3 ± 3.7% inhibition of the maximum response in the presence of CBP (Fig. 6B), which was very similar to the extent of CaM inhibition (P < 0.05). Moreover, the effect of 30 µM CBP also resulted in a small, transient increase in [Ca2+]i in the presence of ATP (Fig. 6D). Therefore, a high degree of similarity was found when we compared the time courses of CBP and W-7 effects on [Ca2+]i and CBF increases induced by ATP, which suggests that the effect of CaMKII can explain the CaM response temporally and quantitatively because CaMKII is a second messenger downstream of CaM activation. Parallel experiments showed that hyposmotic shock without CBP did not change the response of CBF or [Ca2+]i to ATP. On the other hand, the [Ca2+]i increase induced by 100 µM ATP was almost completely blocked when ciliated cells were pretreated with 1 µM thapsigargin followed by 30 µM CBP. These results suggest that CaMKII is necessary to produce complete Ca2+ entry involved in the ATP effect on CBF.



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Fig. 6. Participation of Ca2+-calmodulin pathway in the effect of ATP on ciliated cells: effect of 50 µM W-7 and 30 µM CBP on CBF (A and B) and [Ca2+]i (C and D) increases induced by 100 µM ATP (n = 12). Preincubation with W-7 (A, {square}) or CBP (B, {circ}) for 10 min blocked CBF increase induced by ATP (A, {blacksquare}, or B, {bullet}). Data are means ± SE (n = 10). Moreover, preincubation with W-7 (C) or CBP (D) evoked only a small, transient increase in [Ca2+]i induced by ATP. Traces correspond to average of 8 responses of individual cells. Bars indicate incubation time with reagents.

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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This report demonstrates for the first time the presence and the subcellular localization of IP3 receptor types 1 and 3 in ciliated cells. We also experimentally explored the dynamic processes generating the IP3 and [Ca2+]i increases induced by ATP and their association with the cellular response, an increase in CBF. The complete [Ca2+]i response is described by two components: first, a transient peak component dependent on IP3 receptor activation, followed by a second component consisting of an elevated plateau dependent on Ca2+ influx, which is mediated by IP3 receptor activation. Judging by the similarity in the forms of the time courses of [IP3] response, [Ca2+]i response, PKC activity, and CBF increase, it is likely that the transduction mechanisms are highly coordinated to control the physiological response of ciliary activity.

Xestospongin C almost completely blocked the first component and reduced the second component of the response to ATP. Furthermore, after treatment with both thapsigargin and xestospongin C, the second component of the [Ca2+]i increase induced by ATP was also almost completely blocked. These results indicate an interaction between these components through IP3 receptor activation-dependent Ca2+ influx. Direct activation of Ca2+ entry by plasma membrane IP3 receptors has been characterized electrophysiologically in a variety of cells, including T-lymphocytes (20), olfactory neurons (22), vascular endothelial cells (43), and a carcinoma cell line (17). Moreover, a tight functional interaction between IP3 receptors and store-operated channels can regulate capacitative Ca2+ entry (18). This result indicates the direct participation of IP3 receptors in the induction of the Ca2+ influx, an idea that is supported by our observation that the IP3 receptor type 3 is localized in the plasma membrane of the oviductal ciliated cells. Because the half-life of IP3 is 9 ± 2 s in single cells (48), and because the generation of the maximum increase in [IP3] synthesis was transient, an additional increase in [IP3] synthesis would be necessary to generate the latter Ca2+ increase because of the Ca2+ influx. Significantly, there was a good correlation between the generation of the second [IP3] peak at 60 s and the [Ca2+]i change evoked by Ca2+ influx, an observation that supports the possibility that IP3 participates directly in triggering the ATP-dependent Ca2+ influx. Electrophysiological recordings of Ca2+ channels are needed to determine the function of IP3 receptors involved in the control of Ca2+ influx in oviductal ciliated cells.

Because contamination of commercial samples of adenosine nucleotides has been reported (23), the [Ca2+]i increase could in theory be induced by ADP rather than by ATP, acting via P2Y receptors coupled to Ca2+ mobilization. Regarding this concern, we have never observed any change in CBF in the presence of ADP (28). Moreover, ADP did not change [Ca2+]i in ciliated cells. Furthermore, the EC50 and maximal effect parameters that describe the concentration-response curve of the [Ca2+]i increase induced by ATP were not affected in the presence of ADP (data not shown). Consequently, potential traces of ADP contaminating commercially available ATP should not modify the interpretation of our results.

The IP3 receptor types 1 and 3 share a subcellular distribution, with both forms being localized to the nucleus and the endoplasmic reticulum. Although we anticipate that both IP3 receptors participate in the Ca2+ response induced by ATP, at this moment there is no evidence for any selectivity of xestospongin C between the IP3 receptor types 1 and 3, supporting the possibility that these receptors could induce the [Ca2+]i increase necessary to evoke a CBF increase in oviductal ciliated cells. The IP3 receptor type 3 (which possesses 5-fold greater affinity for IP3 than the type 1 receptor) can be activated before type 1 by a small change in [IP3], which can increase the [Ca2+]i to 100–200 nM (5). This hypothesis is supported by the single-channel differential Ca2+ activation properties that result in an apparent higher in vivo IP3 sensitivity of the type 3 receptor under resting levels of Ca2+ compared with type 1 (25). On the other hand, it has been reported that higher concentrations of [Ca2+]i (≤500 nM) cause a decrease in the IP3 binding to IP3 receptor type 1 while increasing binding to the type 3 receptor (5). In contrast to this observation, Mak et al. (25) found a similar inhibition of the activities of IP3 receptor types 1 and 3 by Ca2+ in this concentration range. Considering the subcellular distribution of the IP3 receptor types 1 and 3, it is possible that the first Ca2+ mobilization originates from the nucleus and the endoplasmic reticulum via activation of the type 3 receptor, after which IP3 receptor type 1, also localized in these organelles, participates in Ca2+ mobilization. Later, probably when [Ca2+]i has reached 400 nM, the IP3 receptors become inactivated by Ca2+ and induce the exponential decay in the response (7). It is important to emphasize that the inactivation of the IP3 receptor by IP3 can also contribute to the exponential decay in the response without changes in [Ca2+]i (24). This exponential decay was unaffected by Ca2+ influx, supporting the idea that IP3 receptor type 3 in the plasma membrane could be activated after intracellular IP3 receptor types 1 and 3 because of subsequent generation of IP3. Taken as a whole, this scenario would explain the increase in [Ca2+]i; however, experiments to control separately the activation of the two types of IP3 receptor associated with Ca2+ mobilization are required to completely understand the molecular mechanism of Ca2+ mobilization in ciliated cells.

We found that PKC activity is stimulated by ATP and is necessary to modulate Ca2+ influx induced by ATP. However, Ca2+ mobilization was not triggered in this process. Furthermore, PKC also has a role downstream of Ca2+ influx because this mediator can transiently increase CBF independently (28). On the other hand, a PKC effect upstream of the [IP3] increase can probably be excluded because the PKC activity did not modify the concentration of this mediator in any of the experiments performed in this study.

Our results suggest that the increase in CBF induced by ATP in oviductal ciliated cells requires Ca2+-dependent proteins such as CaM and CaMKII. The similarity in the time courses of the effects of CaM and CaMKII inhibitors on [Ca2+]i and CBF increases induced by ATP can be explained by the activation of CaM and the subsequent increase in CaMKII activity. CaMKII may target the IP3 receptor type 3, because these proteins have been found to be colocalized in the apical domain in gastrointestinal tissues and the IP3 receptor type 3 has potential phosphorylation sites for CaMKII (26). On the other hand, an effect upstream of the [IP3] increase induced by ATP is probably unlikely because changes in CaM and CaMKII activities did not affect the concentration of [IP3] in our experiments.

In summary, this study was focused on the elucidation of the relationship between the subcellular distribution and the functional effects of the IP3 receptors, which we postulate are associated with the ATP transduction pathway. Further experiments examining the ATP transduction pathway in oviductal ciliated cells are required to understand completely the role of IP3 receptors in the control of [Ca2+]i in these cells.


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 DISCUSSION
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This work was supported by Fondo Nacional de Investigación Cientifica y Tecnológica Grants 2010120 and 1040804 and Dirección General de Postgrado, Investigación, Centros y Programas de la P. Universidad Católica de Chile Grant 354/2002. N. P. Barrera was supported by a fellowship from the Comisión Nacional de Investigación Cientifica y Tecnológica.


    ACKNOWLEDGMENTS
 
We thank Drs. G. Owen, J. M. Edwardson, and C. Taylor for comments on the manuscript and Y. González and B. Kerr for technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Villalón, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Casilla 114-D, Santiago, Chile (E-mail: mvilla{at}bio.puc.cl)

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


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