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Department of Pharmacology, Faculty of Medicine and Health Sciences, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, Quebec, Canada J1H 5N4

(Requests for offprints should be addressed to G Guillemette; Email: gaetan.guillemette{at}usherbrooke.ca)

	Abstract

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In non-excitable cells, the inositol 1,4,5-trisphosphate receptor channel, which plays a major (IP₃R) is an intracellular Ca²⁺ role in Ca²⁺ signalling. Three isoforms of IP₃R have been identified (IP₃R-1, IP₃R-2 and IP₃R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP₃R are poorly understood. AR4-2J cells, which express almost exclusively (~86%) the IP₃R-2, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) influences IP₃R-2-mediated Ca²⁺ release. Using an immunoprecipitation approach, we confirmed that AR4-2J cells express almost exclusively the IP₃R-2 isoform. Using an in vitro phosphorylation assay, we showed that the immunopurified IP₃R-2 was efficiently phosphorylated by exogenous PKC. In intact AR4-2J cells metabolically labelled with ³²Pi, we showed that phorbol-12-myristate-13-acetate (PMA) and Ca²⁺ mobilizing agonists cause the phosphorylation of IP₃R-2. In saponin-permeabilized AR4-2J cells, IP₃-induced Ca²⁺ release was reduced after a pre-treatment with PMA or with exogenous PKC. PMA also reduced the Ca²⁺ response of intact AR4-2J cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. These results demonstrate that PKC decreases the Ca²⁺mobilizing activity of IP₃R-2 and thus exerts a negative feedback on the agonists-induced Ca²⁺ response of AR4-2J cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are intracellular Ca²⁺channels that are activated by the second messenger IP₃, generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) by phospholipase C in response to Ca²⁺-mobilizing hormones (Mikoshiba 1997, Patel et al. 1999). IP₃Rs are located on the endoplasmic reticulum and their opening liberates stored Ca²⁺that diffuses in the cytosol and triggers Ca²⁺-dependent cellular responses. IP₃Rs play major roles in agonists-induced intracellular Ca²⁺release and also in store operated Ca²⁺ entry, a process whereby the depletion of intracellular Ca²⁺ store causes the opening of Ca²⁺ channels in the plasma membrane (for review see Smyth et al. 2006). The elevation of cytosolic free Ca²⁺ concentration is a dynamic signal regulating a variety of important cellular functions such as secretion, contraction, metabolism and gene transcription (Berridge et al. 2003).

In mammalian cells, three subtypes of IP₃Rs (IP₃R-1, IP₃R-2 and IP₃R-3), derived from three distinct genes, have been identified but the actual role of each subtype in physiological Ca²⁺ signalling remains to be established (Patterson et al. 2004, Bezprozvanny 2005, Futatsugi et al. 2005). IP₃Rs subtypes are highly homologous and have the same overall structure, including an N-terminal ligand-binding domain, a C-terminal channel domain and an intervening coupling domain or regulatory domain containing binding sites for Ca²⁺, nucleotides, calmodulin, diverse proteins and modulatory factors, as well as putative phosphorylation sites for several protein kinases (Patel et al. 1999, Patterson et al. 2004). IP₃Rs associate in tetrameric structures that form functional Ca²⁺channels. Individual cell types can express more than one IP₃R subtype, which may form different populations of homo- and hetero-tetrameric channels (Joseph et al. 1995, Monkawa et al. 1995, Wojcikiewicz & He 1995, Nucifora et al. 1996). The relatively poor homology between the regulatory domains of the three IP₃Rs subtypes is likely to confer specific pharmacological and functional properties to each subtype. The functional significance of subtype diversity has not been clearly established for homo-tetrameric channels and much less so for hetero-tetrameric channels. It is therefore important to characterize the specific pharmacological properties and the regulatory mechanisms of each IP₃R subtype.

The protein kinase C (PKC) activator diacylglycerol is co-generated with IP₃ from the cleavage of PIP₂ by phospholipase C. The regulatory domains of IP₃Rs contain several putative phosphorylation sites (S/TXR/K) for PKC. Previous studies showed that PKC increases IP₃-induced Ca²⁺ release in tissues expressing in large majority the subtype IP₃R-1 (Matter et al. 1993, Poirier et al. 2001), but little is known about the effect of PKC on IP₃R-2 and IP₃R-3. The rat pancreatoma cell line AR4-2J expresses IP₃R-2 almost exclusively and thus constitutes a good model for the study of this particular IP₃R subtype. The purpose of the present study was therefore to verify whether IP₃R-2 is a substrate for PKC and, if so, to determine the functional consequences of IP₃R-2 phosphorylation by PKC. Using in vitro and in cellulo phosphorylation assays, we demonstrated that IP₃R-2 is phosphorylated by PKC. Spectrofluorometric Ca²⁺ measurement assays revealed that PKC decreases IP₃-induced Ca²⁺ release. We conclude that in AR4-2J cells, that express predominantly the IP₃R-2 subtype, PKC is exerting a negative regulatory role on intracellular Ca²⁺ mobilization.

	Materials and Methods

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Materials

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and penicillin–streptomycin–glutamine were from Gibco Life Technologies. IP₃ was from Alexis Biochemicals (San Diego, CA, USA). Anti-IP₃R-1 antibody was obtained from Affinity Bioreagent (Golden, CO, USA). Anti-IP₃R-3 was obtained from BD Biosciences Transduction Laboratories (Mississauga, ON, USA). Anti-IP₃R-2 antibody was obtained by immunizing New Zealand rabbits with the C-terminus of IP₃R-2 coupled with keyhole limpet haemocyanin (Poitras et al. 2000). Protein AG-agarose was from Santa Cruz Technology (Santa Cruz, CA, USA), ATP, BSA, creatine phosphokinase, phosphocreatine, diolein, phosphatidylserine, poly-L-lysine hydrobromide, saponin and thapsigargin were from Sigma-Aldrich. Carbachol (CCh), free acid (fura2), fura2/AM, bisindolyl-maleimide I (GF 1), phorbol 12-myristate 13-acetate (PMA), okadaic acid, cyclosporine A (CSA), PKC isozyme sampler set and phosphatase inhibitor cocktail set II were from Calbiochem (San Diego, CA, USA). Proteases inhibitors cocktail (Complete) was from Roche Molecular Biochemicals (Laval, QC, Canada). PKC was obtained from Promega. [-³²P]-ATP and [³²P]Pi (orthophosphate) were from Perkin–Elmer (Boston, MA, USA).

Cell culture

The rat pancreatoma AR4-2J cell line was cultured in complete DMEM (supplemented with 15% heat-inactivated FBS, 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin) at 37 °C in a humidified atmosphere containing 5% CO₂. When 80% confluent, cells were subcultured (1:8 dilution) every 6–7 days using 0.25% trypsin, 1 mM EDTA and fed every other day with fresh DMEM.

In vitro phosphorylation of IP₃R-2

AR4-2J cells (2 x 10⁷ cells/ml) were solubilized for 1 h at 4 °C, under gentle agitation, in lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with the proteases inhibitors cocktail Complete (1X). The solubilized material was centrifuged at 30 000 x g for 30 min and the supernatant was immunoprecipitated for 16 h, at 4 °C, under gentle agitation, with anti-IP₃R-2 antibody and 50 µl protein AG-agarose beads. The immunoprecipitated IP₃Rs were phosphorylated for different time periods at 30 °C in phosphorylation buffer (25 mM Tris–HCl pH 7.4, 1.8 mM CaCl₂, 10 mM MgCl₂) containing 10 µCi [-³²P]ATP, 50 µM non-radioactive ATP, 500 µg/ml phosphatidylserine, 50 µg/ml diolein and 0.15 U of PKC in a final volume of 50 µl. Immune complexes were then washed twice with 1 ml ice-cold PBS (137 mM NaCl, 2.8 mM KCl, 1.5 mM KH₂PO₄ and 8 mM Na₂HPO₄) supplemented with 1 mM EGTA and resuspended in 45 µl of 1 x Laemmli buffer. Samples were heated for 5 min at 95 °C and loaded onto a 5% SDS-PAGE gel that was run at 20 mA for 120 min. IP₃R-2 protein was stained with Coomassie blue and gels were vacuum-dried for 1 h at 80 °C and exposed for 24 h on a BioMax MR film with an intensifying screen. Bands intensities were determined by densitometry using a Hewlett Packard Scan Jet 5100c (Hewlett-Packard Co.) and integrated peak areas were determined using the gel analysis Quantity One software (version 4.2; Bio-Rad Laboratories).

In cellulo phosprorylation of IP₃R-2

AR4-2J cells (5 x 10⁶ cells) were washed twice with phosphate-free DMEM and incubated in the same buffer for 5 h at 37 °C in the presence of 70 µCi/ml ³²P_i. Cells were stimulated for different time periods with 2 µM PMA or other agonists, washed and immediately solubilized with lysis buffer supplemented with 100 nM CSA, 100 nM okadaic acid, (1X) phosphatases inhibitors cocktail set II and (1X) proteases inhibitors cocktail complete. Insoluble material was precipitated by centrifugation at 13 000 g for 25 min at 4 °C. IP₃R-2 was immunoprecipitated for 1 h at 4 °C with anti-IP₃R-2 antibody and with 50 µl protein AG-agarose beads. Immune complexes were washed thrice with ice-cold lysis buffer and resuspended in 45 µl Laemmli buffer (1X). Samples were loaded onto a 4% SDS-PAGE gel that was subjected to a constant current of 15 mA for 150 min. The gel was vacuum dried for 1 h at 80 °C and subjected to autoradiography with Biomax MR film (Kodak).

Dynamic video imaging of cytosolic Ca²⁺

Fluorescence from fura2-loaded cells was monitored as previously described (Auger-Messier et al. 2004). Briefly, AR4-2J cells grown on glass coverslips were washed twice with 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid (HEPES)-buffered physiological saline solution (HBSS: 20 mM HEPES pH 7.4, 120 mM NaCl, 5.3 mM KCl, 0.8 mM MgSO₄, 1.8 mM CaCl₂ and 11.1 mM dextrose) and loaded with 0.1 µM fura2/AM for 20 min at room temperature in the dark. Cells were then washed and bathed in fresh HBSS for 20 min to ensure complete hydrolysis of the fura2/AM before inserting the coverslip into a circular open-bottom chamber that was mounted on the stage of a Zeiss Axiovert microscope fitted with an Attofluor Digital Imaging and Photometry System (Attofluor Inc., Rockville, MD, USA). The system allows simultaneous data acquisition from up to 99 user-defined variably sized regions of interest per field of view. Fluorescence from isolated fura2-loaded cells was monitored by videomicroscopy using alternative excitatory wavelengths of 334 and 380 nm and recording emitted fluorescence at 510 nm. All experiments were done at room temperature and the data expressed as intracellular free Ca²⁺ concentration (nM) calculated from 334/380 fluorescence ratio according to Grynkiewicz et al.(1985). Data acquisition was typically at 3 s intervals and lasted for 600 s.

IP₃-induced Ca²⁺ release

AR4-2J cells (50 x 10⁶ cells grown in 10-cm dishes) were resuspended in 2 ml cytosol-like buffer (20 mM Tris–HCl pH 7.4, 110 mM KCl, 10 mM NaCl, 5 mM KH₂PO₄ and 2 mM MgCl₂), supplemented with 0.5 µM fura2 acid, 20 units creatine kinase, 20 mM phosphocreatine, 1 mM ATP, 100 nM okadaic acid and 100 nM CSA. Cells were pre-treated for 10 min with vehicle or with 2 µM PMA and permeabilized with 100 µg/ml saponin. Ambient Ca²⁺ concentrations were monitored at 37 °C using a Hitachi F-2000 spectrofluorometer (Hitachi Scientific Instruments Inc.) with alternative excitation wavelengths of 340 and 380 nm and with emission wavelength of 510 nm. Each record was calibrated by the addition of a known amount of CaCl₂ to the mixture. At the end of each recording, maximal fluorescence ratio (R_max) and minimal fluorescence ratio (R_min) were determined by adding successively an excess of CaCl₂ (1.8 mM) and 30 mM EGTA. Free Ca²⁺ concentrations were calculated according to Grynkiewicz et al.(1985).

Statistical analysis

All experiments were performed at least thrice. Means ± S.D. were calculated based on three to five independent experiments. Data were analysed statistically using Student’s t-test. Results were considered statistically significant when P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
IP₃R-2 is abundantly expressed in AR4-2J cells

Using an immunoprecipitation and Coomassie blue staining approach, we evaluated the relative expression level of IP₃Rs subtypes in AR4-2J cells. Anti-IP₃R-1 antibody immunoprecipitated a small amount of IP₃R-1 (higher band migrating with an Mr of 260 kDa) together with a similar amount of IP₃R-2 (lower band migrating with an Mr of 230 kDa; Fig. 1A, lane R1). Anti-IP₃R-2 antibody immunoprecipitated a large amount of IP₃R-2 together with a small amount of IP₃R-1 (Fig. 1A, lane R2). Anti-IP₃R-3 antibody did not immunoprecipitate any significant amount of IP₃R (data not shown). The identities of IP₃R subtypes were confirmed by immunoblotting with selective anti-IP₃R antibodies and by partial sequencing using a Q-TOF mass spectrometry approach (data not shown). Fig. 1B shows the quantitative densitometric evaluation of data shown in Fig. 1A. Given the relative intensities of the different bands, these results suggest that the whole population of IP₃R-1 forms hetero-tetrameric Ca²⁺ channels with IP₃R-2 and that the major proportion of IP₃R-2 forms homo-tetrameric channels. As previously described (Wojcikiewicz 1995), these results confirm that AR4-2J cells express in large majority the IP₃R-2 subtype and thus constitute a good model to study the pharmacological and functional properties of IP₃R-2.

View larger version (26K): [in this window] [in a new window]   Figure 1 IP3Rs expressed in AR4-2J cells. (A) IP3Rs from 20 x 106 AR4-2J cells were solubilized with Triton X-100, immunoprecipitated (IP) with selective anti-IP3R-1 (R1) or anti-IP3R-2 (R2) antibodies, resolved by SDS-PAGE (4%) and stained with Coomassie blue. (B) Densitometric analysis of the bands shown in A. IP3R-1 (solid bar), IP3R-2 (open bar). These typical results are representative of three independent experiments.

After solubilization in lysis buffer, IP₃Rs from AR4-2J cells were immunoprecipitated with anti-IP₃R-2 antibody and phosphorylated in vitro as described in Materials and Methods. The material was resolved on a 5% SDS-PAGE. Fig. 2A (upper panel) shows that IP₃R-2 was a good substrate for PKC. Phosphorylation of IP₃R-2 occurred in a time-dependent manner reaching a maximal level within 15 min. As expected, when PKC was omitted, no significant phosphorylation of IP₃R-2 occurred (Fig. 2A, upper panel, lane 1). Coomassie blue staining of the same gel indicates that an equal amount of IP₃R-2 was loaded in each lane (Fig. 2A, lower panel). Commassie blue staining was routinely used in further experiments to ensure that equal amounts of IP₃Rs were loaded on each gel lane. To determine the stability of IP₃R-2 phosphorylation under our in vitro conditions, after maximal phosphorylation with PKC, IP₃R-2 was washed and re-incubated in phosphorylation buffer without PKC. Under these conditions, no significant dephosphorylation of IP₃R-2 was detected for as long as 60 min (Fig. 2B). The in vitro phosphorylation assay was done with different PKC isoforms. Interestingly, the conventional PKC isoforms , ßI and were the most efficient to phosphorylate IP₃R-2 (Fig. 2C). These isoforms are activated by Ca²⁺and diacylglycerol, two second messengers generated by activation of phospholipase C. Therefore, these PKC isoforms are the most susceptible to feedback on the IP₃Rs during cell stimulation with a Ca²⁺-mobilizing hormone. Together, these results clearly indicate that IP₃R-2 is a good substrate for conventional PKC isoforms.

View larger version (94K): [in this window] [in a new window]   Figure 2 In vitro phosphorylation of IP3R-2. IP3Rs from 20 x 106 AR4-2J cells were solubilized with Triton X-100, immunoprecipitated with anti-IP3R-2 antibody, incubated at 30 °C with PKC in the presence of [32P]-ATP, and resolved by SDS-PAGE (5%). (A) The phosphorylation of IP3R-2 for different time periods with a PKC preparation from rat brain (3 U/ml). In (B) IP3R-2 was phosphorylated in vitro for 30 min with PKC, washed with phosphorylation buffer to remove PKC and incubated at 30 °C for different time periods to verify the stability of the phosphorylation. (C) The in vitro phosphorylation of IP3R-2 for 30 min at 30 °C with different recombinant PKC isozymes (3 U/ml). In each panel, the upper shows the autoradiogram and the lower shows the Coomassie blue staining of the same bands. These typical results are representative of at least three independent experiments.

AR4-2J cells were metabolically labelled with [³²P]P_i and stimulated for different time periods with PMA, a direct activator of PKC. Under basal conditions, IP₃R-2 was already phosphorylated (Fig. 3A, time 0), however, its level of phosphorylation was increased after stimulation of AR4-2J cells with PMA (Fig. 3A). Densitometric analysis of band intensities revealed that treatments with PMA for 10 or 15 min significantly increased the phosphorylation of IP₃R-2 (Fig. 3B). AR4-2J cells endogenously express different Gq-coupled receptors for the Ca²⁺ mobilizing agonists CCh, cerulein (Cer) and ATP. These receptors can activate phospholipase C that produces the PKC activator diacylglycerol. Fig. 3C shows that the phosphorylation of IP₃R-2 was increased upon stimulation of AR4-2J cells with Ca²⁺ mobilizing agonists. Densitometric analysis of these results (Fig. 3D) revealed that CCh and Cer significantly increased the phosphorylation of IP₃R-2, whereas the minor effect of ATP was not statistically significant (likely due to a low level of purinergic receptor expression in our cell line). Together, these results suggest that endogenous PKC can phosphorylate IP₃R-2 in AR4-2J cells.

View larger version (19K): [in this window] [in a new window]   Figure 3 In cellulo phosphorylation of IP3R-2. AR4-2J cells were labelled for 5 h with [32P]Pi and then stimulated for different time periods with 2 µM PMA (A) and (B) or for 3 min with different agonists: 100 µM carbachol (CCh), 10 nM cerulein (Cer) or 100 µM ATP (C) and (D). After solubilization in lysis buffer, IP3R-2 (arrow) was immunoprecipitated, resolved SDS-PAGE (4%) and autoradiographed. (A) and (C) show the autoradiograms (B) and (D) the densitometric analysis (mean ± S.D. *P < 0.05 compared with control) from three independent experiments.

We used a direct IP₃-induced Ca ²⁺release assay that measures ambient Ca²⁺ concentrations by fura2 fluorescence to study the functional consequences of IP₃R phosphorylation by PKC. Fig. 4A (upper panel) shows a typical experiment where 0.1 µ M IP₃ released 4.0 nmol Ca²⁺ from permeabilized AR4-2J cells (the amount of Ca²⁺ released was calibrated by the exogenous addition of 4 nmol CaCl₂). In PKC-treated permeabilized cells (Fig. 4A, lower panel), 0.1 µM IP₃ released 2.5 nmol Ca²⁺, an amount which is smaller than in untreated cells. Histograms shown in Fig. 4B indicate that PKC-treated permeablized AR4-2J cells released significantly less Ca²⁺ than untreated cells in response to 0.1 µM IP₃. These results show that PKC decreases IP₃-induced Ca²⁺ release in AR4-2J cells.

View larger version (10K): [in this window] [in a new window]   Figure 4 PKC decreases IP3-induced Ca 2+ release. Permeabilized AR4-2J cells (5 x 107 cells/assay) were permeabilized in a cytosol-like medium containing 0.5 µM fura2 acid and 100 µg/ml saponin. Cells were treated for 5 min with vehicle or with PKC (0.5 IU/ml). IP3-induced Ca2+ release was measured as described in Materials and Methods. (A) Typical traces where a dose of 0.1 µ M IP3 partially releases stored Ca2+ from vehicle-treated (upper) or PKC-treated cells (lower). The amount of Ca2+released was calibrated by addition of 4 nmol exogenous Ca2+ (C). (B) Average Ca2+ released by 0.1 µM IP3 means ± S.D. of eight independent experiments; *P < 0.05), for cells pre-treated with vehicle (solid bar) or with PKC (open bar).

View larger version (22K): [in this window] [in a new window]   Figure 5 PKC decreases the apparent affinity of IP3Rs in permeabilized AR4-2J cells. Intact AR4-2J cells were pre-treated for 10 min with vehicle (A) or with PMA (B), permeabilized with saponin in a cytosol-like medium containing 0.5 µM fura2 acid and then stimulated with increasing doses of IP3. The amount of Ca2+ released was calibrated by adding a known amount (6 nmol) of exogenous Ca2+(C) and the total content of the Ca2+ pool was released with 2 µM Ionomycin (Io). (C) The dose–response curves for IP3-induced Ca 2+ released from permeabilized AR4-2J cells pre-treated with PMA (solid symbols) or with vehicle (open symbols). These typical results are representative of results obtained in at least three independent experiments.

To determine the functional consequence of PKC-mediated phosphorylation, we also evaluated the cytosolic Ca²⁺ concentration in intact AR4-2J cells loaded with fura2/AM. Fig. 6A shows that when cells were incubated in a medium prepared without Ca²⁺(nominally Ca²⁺-free), a low dose of the Ca²⁺-mobilizing agonist CCh (CCh, 3 µM) produced a transient Ca²⁺ increase with a peak amplitude of 135 ± 25 nM Ca²⁺. After a 5-min pre-treatment with 2 µM PMA, the effect of 3 µM CCh was very importantly reduced to a small transient with peak amplitude of 48 ± 19 nM Ca²⁺. The effect of PMA on CCh-induced intracellular Ca²⁺ mobilization was completely abolished after a pre-treatment with the PKC inhibitor GF1 (Fig. 6C). AR4-2J cells are sensitive to epidermal growth factor (EGF), which activates a receptor-tyrosine kinase that in turn activates phospholipase C and produces IP₃. Fig. 6B shows that, in a nominally Ca²⁺-free medium, a high dose of EGF (100 ng/ml) caused a relatively weak Ca²⁺ increase with a peak amplitude of 30 ± 8 nM Ca²⁺. After a 5-min pre-treatment with PMA, 100 ng/ ml EGF produced a barely detectable Ca²⁺ transient with a peak amplitude of 7 ± 5 nM Ca²⁺ (Fig. 6B). Because this series of experiments was done in the absence of extracellular Ca²⁺, it is likely that the Ca²⁺ transients produced by CCh and EGF were due to IP₃-induced Ca²⁺ release from intracellular stores.

View larger version (17K): [in this window] [in a new window]   Figure 6 PKC decreases carbachol-induced Ca2+ response in intact AR4-2J cells. AR4-2J cells were loaded with fura2/AM (0.15 µM) and bathed in a nominally Ca2+-free HBSS. Cells were imaged using a Zeiss Axiovert microscope (Prior Scientific Inc., Rockland, MA, USA; 40 x oil immersion objective) coupled to an Attofluor imaging system (Attofluor Inc.). (A) and (B) show representative recordings of intracellular Ca2+ levels before and after the addition of 3 µM CCh (A) or of 100 ng/ml EGF (B) to cells pre-treated for 5 min with vehicle (grey trace) or with 2 µM PMA (black trace). Each trace shows the average response of 60–80 cells per microscope field. (C) The average Ca2+ responses to 3 µM CCh (means ± S.D. *P < 0.05) obtained from three independent experiments, for cells pre-treated for 5 min with vehicle, or with 2 µM PMA, and/or for 15 min with 100 nM GF1.

View larger version (19K): [in this window] [in a new window]   Figure 7 PKC modifies carbachol-induced Ca2+ responses in single AR4-2J cells. AR4-2J cells were loaded with fura2/AM (0.15 µM), bathed in a nominally Ca2+-free HBSS and then stimulated with 3 µM CCh. (A) A typical example of CCh-induced Ca2+ oscillations. (B) A typical example of CCh-induced Ca2+ transient. (C) The proportion of cells that did not respond (NR) or that responded to 3 µM CCh with Ca2+ oscillations or with a Ca2+ transient, after a 5-min pre-treatment with vehicle or with PMA (2 µM), and/or after a 15-min pre-treatment with GF1 (100 nM; means ± S.D.*P < 0.05). These results were obtained with at least 200 single cells, in three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Using two different protocols, we directly evaluated the functional consequence of IP₃R-2 phosphorylation by PKC. In the first protocol, saponin-permeabilized AR4-2J cells were treated with exogenous PKC and their IP₃-induced Ca²⁺ release activity was measured. In the second protocol, intact AR4-2J cells were pre-treated with PMA and then permeabilized and their IP₃-induced Ca²⁺ release activity was measured. Both protocols revealed that PKC decreases IP₃-induced Ca²⁺ release. Because PKC did not affect the ionomycin-sensitive Ca²⁺ pool, the diminished Ca²⁺ release must be attributed to a diminution of IP₃R-2 activity. IP₃ dose–response curves suggested that PKC decreases the apparent affinity of IP₃R-2 in AR4-2J cells. Using intact AR4-2J cells incubated in a Ca²⁺-free medium, we showed that PKC reduces the global Ca²⁺ response induced by CCh and by EGF, two agonists that produce IP₃ through different pathways. A thorough review of the literature reveals conflicting results regarding the regulatory effect of PKC on intracellular Ca²⁺ release. On the one hand, a few studies concluded that PKC increases IP₃-induced Ca²⁺ release. In nuclei isolated from rat liver (unknown composition of IP₃R/channel), PKC enhanced IP₃-induced Ca²⁺ release (Matter et al. 1993). In bovine adrenal glomerulosa cells, which express all three subtypes of IP₃R (but a large majority of IP₃R-1), PKC enhanced IP₃-induced Ca²⁺ release (Poirier et al. 2001). In rat brain microsomes, a tissue containing exclusively the IP₃R-1 subtype, PKC also increased IP₃-induced Ca²⁺ release (Cameron et al. 1995). On the other hand, in permeabilized rabbit pancreatic acinar cells (unknown composition of IP₃R/channel), PKC decreased IP₃-induced Ca²⁺ release (Willems et al. 1989). In a recent study, we showed that PKC decreases IP₃-induced Ca²⁺ release in RINm5F cells that express almost exclusively the IP₃R-3 subtype (Caron et al. 2007). These results suggest that the regulatory effect of PKC is qualitatively different depending on the specific subtype of IP₃R phosphorylated. To our knowledge, we are the first to clearly show that PKC decreases IP₃-induced Ca²⁺ release in a tissue expressing almost exclusively the IP₃R-2 subtype.

Using intact AR4-2J cells bathed in a Ca²⁺-free medium, we consistently observed that the effect of PKC on CCh- and EGF-induced Ca²⁺ responses was more important than its effect on IP₃-induced Ca²⁺ release from permeabilized cells. These results suggest that PKC may down-regulate more than one component of the intra-cellular Ca²⁺ signalling cascade. Indeed, several studies have shown that PKC is responsible for the desensitization of some Gq-coupled receptors (Lynch et al. 1985, Biden et al. 1988), whereas other studies showed that PKC may disrupt the interaction between the G protein and phospholipase C (Orellana et al. 1987, Smith et al. 1987). In the same way, PKC has been shown to inhibit the tyrosine kinase activity of EGF receptor, thus down-regulating the IP₃/PLC pathway (Davis 1988, Lund et al. 1990). A recent study also showed that PKC negatively regulates the activity of TRPC3, TRPC4 and TRPC5, which are Ca²⁺ channels responsible for the capacitative Ca²⁺ entry into cells (Venkatachalam et al. 2003). Interestingly, PKC is exerting an inhibitory effect on all these components of the Ca²⁺ signalling cascade (Gq-coupled receptors, Gq coupling, tyrosine kinase receptors, Ca²⁺ entry channels). Therefore, by acting at different levels of the Ca²⁺ signalling cascade, PKC is providing diverse negative feedback loops that may produce additive inhibitory effects on intracellular Ca²⁺ increase. These effects of PKC are likely required to protect the cells against deleterious effects of sustained elevation of Ca²⁺.

In conclusion, we have shown that PKC phosphorylates IP₃R-2, thus diminishing its sensitivity to IP₃. This regulatory effect of PKC is a further negative feedback mechanism through which PKC controls intracellular Ca²⁺ elevation.

	Acknowledgements

 
This work was supported by a grant from the Canadian Institutes of Health Research. G A is the recipient of a studentship from the Natural Sciences and Engineering Council of Canada. A Z C is the recipient of a studentship from the Heart and Stroke Foundation of Canada. We declare that there is no conflict of interest that would prejudice the impartiality of the research reported.

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Received in final form 7 December 2006
Accepted 10 December 2006
Made available online as an Accepted Preprint 28 December 2006

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