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J. Biol. Chem., Vol. 277, Issue 2, 1340-1348, January 11, 2002

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Phosphorylation of Inositol 1,4,5-Trisphosphate Receptors in Parotid Acinar Cells

A MECHANISM FOR THE SYNERGISTIC EFFECTS OF cAMP ON Ca²⁺ SIGNALING^*

Jason I. E. Bruce, Trevor J. Shuttleworth, David R. Giovannucci, and David I. Yule

From the Department of Pharmacology & Physiology, School of Medicine and Dentistry, University of Rochester Medical Center, Rochester, New York 14642

Received for publication, July 13, 2001, and in revised form, October 29, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Acetylcholine-evoked secretion from the parotid gland is substantially potentiated by cAMP-raising agonists. A potential locus for the action of cAMP is the intracellular signaling pathway resulting in elevated cytosolic calcium levels ([Ca²⁺]_i). This hypothesis was tested in mouse parotid acinar cells. Forskolin dramatically potentiated the carbachol-evoked increase in [Ca²⁺]_i, converted oscillatory [Ca²⁺]_i changes into a sustained [Ca²⁺]_i increase, and caused subthreshold concentrations of carbachol to increase [Ca²⁺]_i measurably. This potentiation was found to be independent of Ca²⁺ entry and inositol 1,4,5-trisphosphate (InsP₃) production, suggesting that cAMP-mediated effects on Ca²⁺ release was the major underlying mechanism. Consistent with this hypothesis, dibutyryl cAMP dramatically potentiated InsP₃-evoked Ca²⁺ release from streptolysin-O-permeabilized cells. Furthermore, type II InsP₃ receptors (InsP₃R) were shown to be directly phosphorylated by a protein kinase A (PKA)-mediated mechanism after treatment with forskolin. In contrast, no evidence was obtained to support direct PKA-mediated activation of ryanodine receptors (RyRs). However, inhibition of RyRs in intact cells, demonstrated a role for RyRs in propagating Ca²⁺ oscillations and amplifying potentiated Ca²⁺ release from InsP₃Rs. These data indicate that potentiation of Ca²⁺ release is primarily the result of PKA-mediated phosphorylation of InsP₃Rs, and may largely explain the synergistic relationship between cAMP-raising agonists and acetylcholine-evoked secretion in the parotid. In addition, this report supports the emerging consensus that phosphorylation at the level of the Ca²⁺ release machinery is a broadly important mechanism by which cells can regulate Ca²⁺-mediated processes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Calcium is a ubiquitous second messenger that is critically important in the regulation of a variety of cellular functions (1-3). The spatio-temporal "shaping" of Ca²⁺ signals is thought to play an important role in defining the specificity of stimulus-response coupling both between cell types and within the same cell (4, 5). However, despite intensive investigation, the molecular mechanisms that control frequency- and/or amplitude-encoded Ca²⁺ oscillations, Ca²⁺ wave propagation, or localized Ca²⁺ release events remain poorly understood. An emerging body of evidence indicates that, in various systems, specific control over Ca²⁺ signals may be achieved by cross-talk between second messenger systems that raise [Ca²⁺]_i ¹ interacting with those that elevate cAMP. Such cross-talk may alter the sensitivity of a variety of Ca²⁺ transport processes (6, 7).

An example of this cross-talk occurs in the salivary gland, where both fluid and exocytotic secretion are controlled by separate neuronal and/or humoral inputs (6, 8). Specifically, neuronally released acetylcholine (ACh) activates acinar cell muscarinic receptors, leading to increased [Ca²⁺]_i via the phosphoinositide pathway. Elevations in [Ca²⁺]_i activate ion channels essential for unidirectional fluid secretion (9), and, in addition, exert regulatory control over the exocytotic machinery required for protein secretion (6). Muscarinic activation of both fluid secretion, and to a lesser extent exocytosis, has been shown to be dramatically potentiated by the concomitant activation of cAMP-raising pathways, such as by co-released vasoactive intestinal peptide, or by sympathetic stimulation of -adrenoreceptors (10-13). Although cAMP could have direct effects on ion channels (14) and/or exocytotic proteins (15), an alternative hypothesis is that cAMP interacts directly with the Ca²⁺ signaling machinery to account for the synergistic effects (6, 13, 16). Because parotid acinar cells have been used extensively to study Ca²⁺ signaling, this model system is ideally suited to investigate cross-talk between cAMP and Ca²⁺ signaling.

InsP₃ production, Ca²⁺ influx, and Ca²⁺ release from either InsP₃Rs or RyRs, are all potential targets for cAMP in modulating Ca²⁺signaling, however, the literature is equivocal as to the site of any interaction (16-23). No single study has been successful in unambiguously identifying a specific molecular target that accounts for the synergistic relationship between cAMP and Ca²⁺ signaling in parotid acinar cells. Therefore, the aim of the present study was to investigate the molecular target(s) for the interaction between cAMP and Ca²⁺ signaling in mouse parotid acinar cells. This was achieved using a combination of imaging (intact and SL-O-permeabilized cells), inositol phosphate assays and in situ phosphorylation experiments. These experimental paradigms revealed that cAMP dramatically potentiated Ca²⁺ release through PKA-mediated phosphorylation of InsP₃ receptors, likely the type II isoform. This regulatory control likely underlies the synergistic relationship between ACh and cAMP-elevating agonists in parotid acinar cells. These findings have broad implications and may represent a general feature for the regulation of Ca²⁺ release events that are linked to a vast array of specific functions in all cell types.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of Single Parotid Acinar Cells-- Single and small groups of parotid acinar cells were isolated by collagenase digestion of freshly dissected parotid glands from wild type Swiss Black mice using a technique similar to that described previously for rat parotid (24). Briefly, 25-g mice were killed by cardiac puncture immediately following CO₂ gas asphyxiation. Parotid glands were dissected, minced, and incubated for 60 min at 37 °C in Earle's minimum essential medium (Biofluids, Inc., Rockville, MD) containing 2 mM glutamine, 1% bovine serum albumin, and 0.04 mg/ml collagenase P (Roche Molecular Biochemicals, Mannheim, Germany). Minced tissue was dispersed by multiple trituration every 20 min. Cells were resuspended in bovine serum albumin-free Eagle's basal medium (Invitrogen) supplemented with 2 mM glutamine and penicillin/streptomycin and left on ice until ready for use.

Digital Imaging of [Ca²⁺]_i-- Isolated parotid acinar cells were resuspended in a HEPES-buffered physiological saline solution (HEPES-PSS) containing (in mM) 5.5 glucose, 137 NaCl, 0.56 MgCl₂, 4.7 KCl, 1 Na₂HPO₄, 10 HEPES (pH 7.4), 1.2 CaCl₂. The cells were then incubated in the above HEPES-PSS containing 2 µM fura-2/AM for 30 min at room temperature, after which they were washed once and resuspended in the above HEPES-PSS and kept on ice. Cells were allowed to adhere to a glass coverslip that formed the base of a gravity-fed perfusion chamber and continually perfused with HEPES-PSS. [Ca²⁺] imaging was performed using an inverted epifluorescence Nikon microscope with a 40× oil immersion objective lens (numerical aperture, 1.3). A field of 3-15 fura-2-loaded cells was excited alternately with light at 340 and 380 nm (± 10 nm bandpass filters, Chroma) using an illumination system (Sutter, DG-4). Fluorescence images (500 ± 45 nm) were captured and digitized at 12-bit resolution using an interline progressive scan CCD camera (Sensicam). Axon Imaging Workbench was used to drive the DG-4 and image acquisition by the camera. Images were acquired every second with an exposure of 200 ms. Background-subtracted and 340/380 ratio images were calculated on-line and stored immediately to hard disk. Images (480 × 640 pixels) were collected with no binning, thereby giving a spatial resolution of 0.225 µm/pixel. All experiments were performed at room temperature.

Measurement of Intra-ER Ca²⁺([Ca²⁺]_ER)-- Isolated parotid acinar cells were incubated in the above attachment media containing 10 µM fura-2FF/AM (K_D for Ca²⁺ ~13 µM; see Ref. 25) for 60 min at 37 °C. Permeabilization and subsequent Ca²⁺ uptake and Ca²⁺ release experiments were similar to those reported in pancreatic acinar cells (26, 27). Briefly, cells were constantly perfused with a Chelex-100 "scrubbed" cytosol-like medium containing (in mM) 135 KCl, 1.2 KH₂PO₄, 0.5 EGTA, 0.5 HEDTA, 0.5 nitriloacetic acid, and 20 HEPES/KOH (pH 7.1). MgCl₂, CaCl₂, and MgATP were added accordingly to give a constant free Mg²⁺, Ca²⁺, and ATP concentration of 0.9 mM, 200 nM, and 1 mM, respectively (as calculated by WEBMAXC version 2.1). Permeabilization was achieved by perfusion with an ATP-containing (but Ca²⁺-free) cytosol-like medium containing 0.4 IU of streptolysin-O (SL-O). Permeabilization was monitored and verified using the imaging system by exciting loaded cells with light at 360 nm, which is the isosbestic point for fura-2FF (360 ± 25 nm bandpass filter, Chroma). Images were acquired every 10 s (100-ms exposure) and the fluorescence in each cell monitored over time. The emitted fluorescence declined rapidly within 10 min as the plasma membrane permeabilized and all the cytosolic dye leaked out of the cell. The resultant fluorescence (<20% of pre-permeabilized cells) was caused by dye trapped in intracellular organelles. The cells were subsequently perfused with the cytosol-like medium devoid of SL-O, Ca²⁺, or ATP for 10 min. Measurement of [Ca²⁺]_ER was achieved by exciting permeabilized cells with light at 340 and 380 nm as with fura-2, except images were acquired every 10 s with an exposure of 300 ms. This was done to prevent photobleaching of the dye because emitted fluorescence was significantly lower than when the dye was trapped in the cytosol. Rapid Ca²⁺ uptake was achieved upon addition of 1 mM Mg-ATP and 0.2 µM Ca²⁺, reaching a steady state within 3 min. Upon loading the stores with Ca²⁺, permeabilized cells were stimulated with various concentrations of InsP₃ in the absence or presence of 100 µM dibutyryl cyclic AMP (Bt₂cAMP), 10 µM Rp-cAMPS, and/or 500 µM ryanodine. Ca²⁺ release was measured as a decrease in the fura-2FF 340/380 ratio.

Measurement of Inositol Phosphate Production-- Total inositol phosphate production in response to forskolin and/or carbachol (CCh) was determined by a similar method to that described previously (28). Briefly, isolated parotid acinar cells were incubated with 5 µCi/ml myo-[³H]inositol for 2 h, followed by three washes in HEPES-PSS. Cells were then incubated with 1 µM CCh and/or 10 µM forskolin for 5 min in the presence of 10 mM lithium. Total myo-[³H]inositol phosphates from each sample were extracted by solubilization with 0.5 M trichloroacetic acid, eluted on Dowex columns and detected by liquid scintillation. Total inositol phosphate generation was expressed as a percentage of total phosphoinositides, determined by counting the trichloroacetic acid-insoluble fraction by liquid scintillation.

Phosphorylation of Ca²⁺ Release Channels-- Parotid acinar cells were isolated from 4 mice as described above and metabolically labeled by incubating for 2 h with 14 µCi/ml ³²PO (PerkinElmer Life Sciences) in a phosphate-free saline solution containing (in mM) 109.7 NaCl, 4.5 KCl, 1.2 MgCl₂, 5.95 HEPES (free acid), 7.05 NaHEPES (pH 7.4) 1.13 CaCl₂ and 6 glucose. Following incubation, cells were washed three times in the above ³²PO-free media and aliquots treated with or without 10 µM forskolin for 10 min at 37 °C. Cells were then rapidly pelleted by centrifugation and resuspended in ice-cold lysis buffer containing (in mM) 50 Tris-HCl (pH 7.4), 250 NaCl, 5 EDTA, 100 NaF, 1 benzamidine, 1 dithiothreitol, 1% CHAPS, 10 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 0.7 µg/ml pepstatin A, 1 µg/ml aprotinin, 1 µg/ml antipain. Cell lysates were then sonicated, left on ice for 30 min, and vortexed every 5 min. InsP₃R or RyR protein was immunoprecipitated from the samples by incubating lysates with a mixture of antibodies (~1 µg of antibody/mg of protein) raised against all three InsP₃R types or all three RyR types for 1 h at 4 °C, followed by incubation with 80 µl of protein A-agarose beads (Pierce) for an additional 1 h at 4 °C. Antibodies directed against InsP₃R were CT₁ and CT₂ (kind gifts from Richard Wojcikiewicz, State University of New York, Syracuse, NY) and type III antibody (Transduction Laboratories). Antibodies directed against RyR were 34C directed against RyR₁ (Developmental Studies Hybridoma Bank, Iowa City, IA), C3-33 directed against RyR₂ (Affinity Bioreagents, Inc.), and anti-rabbit skeletal muscle RyR antibody directed against RyR₃ (Upstate Biotechnology, Lake Placid, NY). As a secondary control, aliquots of cell lysate from cells treated with forskolin were incubated with beads without any antibodies. Following incubation, the beads-protein complexes were washed five times in lysis buffer by repeated centrifugation and resuspension and then boiled in SDS-sample buffer (Laemmli). Immunoprecipitated proteins were separated by SDS-polyacrylamide gel (5%) electrophoresis (SDS-PAGE), after which the gels were vacuum-dried (Bio-Rad) and exposed to a PhosphorImager intensifier screen. Bands of the appropriate molecular weight, which corresponded to phosphorylated proteins that had incorporated ³²PO, were detected using a Molecular Dynamics PhosphorImager.

To determine whether phosphorylation of InsP₃Rs was mediated by PKA, H-89 and Rp-cAMPS were used to inhibit PKA in combination with an additional and complimentary approach using a phospho-PKA substrate antibody (Cell Signaling Technology). This antibody specifically recognizes proteins containing phosphorylated serine or threonine, with an arginine residue at position 3, but not the corresponding nonphosphorylated motif (29, 30). Although this antibody would not discriminate between substrates of PKA, protein kinase C, or cyclic GMP-dependent protein kinase, the combined use of forskolin and appropriate PKA inhibitors was used to implicate a specific role of PKA. Aliquots of isolated parotid acinar cells were treated with or without 10 µM forskolin, 50 nM okadaic acid, and/or the PKA inhibitors, H-89 (2 µM) and Rp-cAMPS (30 µM) for 10 min at 37 °C. Cells were then solubilized in lysis buffer similar to the method above. Lysates were then incubated with phospho-PKA substrate antibody (1:100 dilution) to immunoprecipitate phosphorylated proteins. Specific detection of phosphorylated type II InsP₃Rs was achieved by Western blotting with the CT₂ antibody. Whole cell lysates or immunoprecipitated proteins were denatured in SDS-sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose (Schleicher & Schuell) prior to immunoblotting with the CT₂ antibody. Immunoreactivity was visualized using peroxidase-conjugated secondary antibodies (Bio-Rad), followed by detection by Super Signal detection system (Pierce) exposed on XAR film (Eastman Kodak Co.).

Data Analysis and Experimental Design-- In all experiments (unless otherwise stated in the text), a paired experimental design was applied, whereby a particular experimental paradigm was repeated in the absence or presence of a test reagent(s) on the same cell. Therefore, statistical significance was determined, where appropriate, using a paired t test, Wilcoxon test for pairs, or one sample t test. Occasionally, statistical significance was determined between groups of experiments where an unpaired t test or Mann-Whitney test was used. For any parameter analyzed from several cells in a particular experiment, an average value was determined. These values were in turn averaged to give the values expressed in the text as means ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Carbachol-evoked [Ca²⁺]_i Changes-- To investigate the effects of elevated cAMP levels on [Ca²⁺]_i signaling in parotid acinar cells, we first characterized the types of [Ca²⁺]_i responses evoked by the muscarinic receptor agonist, CCh. Low concentrations of CCh (10-300 nM) caused an oscillatory increase in [Ca²⁺]_i in 86% of cells tested, which was characterized by a large initial spike followed by rapid sinusoidal oscillations superimposed over an elevated base line. These oscillations were complex in nature, and their frequency and amplitude varied markedly between cells. Higher concentrations of CCh (300 nM to 10 µM) induced a biphasic increase in [Ca²⁺]_i, which was characterized by a large initial spike followed by a sustained elevation. These patterns of [Ca²⁺]_i changes are typical of a variety of exocrine cells; however, the oscillation frequency was significantly higher than reported in pancreatic acinar cells (7-11/min in parotid compared with 4-6/min; see Ref. 31). Despite the complex nature of these CCh-evoked [Ca²⁺]_i changes, there was a consistent concentration-dependent increase in the magnitude of the initial spike-like increase in [Ca²⁺]_i. This initial [Ca²⁺]_i spike was interpreted to reflect Ca²⁺ release from intracellular stores and was quantitatively compared in the absence and presence of 10 µM forskolin using a paired experimental design.

Forskolin Potentiates Carbachol-evoked [Ca²⁺]_i Changes-- Using the adenylate cyclase activator forskolin, we investigated the effects of elevating intracellular cAMP levels on CCh-evoked [Ca²⁺]_i signaling (33). Repeated stimulations with CCh evoked [Ca²⁺]_i changes of equal magnitude (Fig. 1). Forskolin (10 µM) induced a dramatic and time-dependent potentiation of this CCh-evoked initial increase in [Ca²⁺]_i. Upon removal of forskolin, there was an equivalent time-dependent recovery (Fig. 1). On average, forskolin increased the CCh-evoked [Ca²⁺]_i response by 148.9 ± 8.5% after 3-5 min, which increased further to 177.1 ± 17.4% after 8-10 min of incubation with forskolin (see Fig. 1). To test whether the potentiation was the result of specific activation of PKA, cells were also pretreated with 2 µM H-89, an inhibitor of serine/threonine kinases such as PKA (34) prior to specific activation of PKA by treatment with forskolin. This completely prevented the potentiation of the CCh-evoked initial increase in [Ca²⁺]_i (Fig. 2), suggesting that the potentiation was caused by activation of PKA.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Forskolin causes potentiation of CCh-evoked [Ca2+]i changes in mouse parotid acinar cells. A, stimulation of cells with a low (0.3 µM) and higher (1 µM) concentration of CCh demonstrated a steep concentration-response effect of CCh on [Ca2+]i. Repeated stimulations with CCh in the absence (CCh-1 and CCh-2 in B) and presence of 10 µM forskolin (forsk-1 and forsk-2 in B) illustrate the dramatic potentiation of the initial peak CCh evoked [Ca2+]i response and subsequent recovery (wash-1 and wash-2 in B) after removal of forskolin. B, quantification of mean data (13 experiments, 87 cells) was achieved by expressing each CCh-evoked initial [Ca2+]i increase as a % of the first control CCh (CCh-1) response in the same cell. Statistical significance was determined using a paired one sample t test (*p < 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   A PKA inhibitor prevents the potentiation of the CCh-evoked [Ca2+]i response by forskolin. Using a similar experimental paradigm as Fig. 1, cells were pretreated with 2 µM H-89 for 5 min prior to treatment with 10 µM forskolin in combination with 2 µM H-89 for at least 5 min. A, representative trace from 6 separate experiments (24 cells). B, quantification of mean data revealed that the PKA inhibitor, H-89 completely prevented the potentiation by forskolin. Statistical significance was determined using a paired one sample t test (*p < 0.05).

In addition to the potentiation of the CCh-evoked initial increase in [Ca²⁺]_i, forskolin also converted oscillatory [Ca²⁺]_i changes into a sustained increase, suggesting a leftward shift in the CCh concentration-response curve compared with control cells (Fig. 3A). Consistent with this hypothesis, forskolin treatment enabled normally subthreshold concentrations of CCh (3-30 nM) to evoke oscillatory [Ca²⁺]_i responses (49 of 75 cells). Interestingly, in six of these cells, [Ca²⁺]_i oscillations were confined to the apical region of the cells (Fig. 3B), revealing conversion of a subthreshold response into a measurable threshold response. To identify the molecular site at which this potentiation was manifested, we systematically investigated the effects of forskolin on Ca²⁺ entry, InsP₃ production, and Ca²⁺ release channels in parotid acinar cells using a variety of biochemical and functional assays.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Forskolin sensitizes the CCh-evoked [Ca2+]i response. A, forskolin converted an oscillatory [Ca2+]i response into a sustained [Ca2+]i response in all cells tested (7 experiments, 58 cells). The dotted line represents a gap of 7 to 9 min during which no images were acquired. B, treatment with forskolin also resulted in a response to sub-threshold concentrations of CCh (10-30 µM) being converted from nonresponding to an oscillatory [Ca2+]i response (49 of 75 cells, 11 experiments). The trace is representative of 6 of these cells (2 experiments) where the oscillations were confined to the apical region of the cell. The red and blue trace represent the changes in [Ca2+]i defined by the corresponding red and blue boxes in the above brightfield image. The red and blue boxes represent the apical and basal region respectively of a single cell that is part of a small clump or acini. The above pseudo-color enhanced fluorescence ratio images were the corresponding images at a, b and c on the trace.

Effects of Forskolin on Ca²⁺ Entry-- A potential locus for the effects of cAMP on Ca²⁺ signals is the Ca²⁺ entry pathways. Stimulation of parotid acinar cells with low concentrations of CCh (100 nM) in Ca²⁺-free solution (nominal Ca²⁺) evoked [Ca²⁺]_i oscillations. These oscillations progressively decreased in amplitude (Fig. 4A), presumably in response to depletion of intracellular stores. Re-introduction of external Ca²⁺ in the continued presence of CCh produced a large increase in base-line [Ca²⁺]_i with oscillations of increasing amplitude superimposed (Fig. 4A). The elevated base-line [Ca²⁺]_i was interpreted to reflect capacitative Ca²⁺ entry. Repeating this paradigm in the presence of 10 µM forskolin caused a 169.3 ± 6.5% potentiation of the initial CCh-evoked [Ca²⁺]_i increase, which was not significantly different from that observed in the presence of external Ca²⁺ (177.1 ± 17.4%; Fig. 4B) This suggests that enhanced Ca²⁺ release, rather than Ca²⁺ entry, was responsible for potentiation of the initial CCh-evoked [Ca²⁺]_i increase. Re-introduction of external Ca²⁺ in the presence of forskolin produced a potentiation of the elevated base-line [Ca²⁺]_i (Fig. 4, A and B; 122.1 ± 11.3% higher than control), suggesting an effect on capacitative Ca²⁺ entry. This most likely reflected an indirect effect, resulting from the enhanced Ca²⁺ release and store depletion, on Ca²⁺ entry. However, additional direct effects of cAMP on Ca²⁺ entry cannot be completely excluded.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Ca2+ entry is not directly affected by forskolin treatment. A, representative experiment showing the effect of removal and subsequent re-introduction of external [Ca2+] on the CCh-evoked [Ca2+]i response in the absence and presence of forskolin. The dotted line represents a gap of 7 to 9 min during which no images were acquired. B, quantification of mean data revealed that the initial CCh-evoked [Ca2+]i increase was dramatically potentiated by forskolin even in the absence of external [Ca2+] (171 ± 13% increase, 4 experiments, 14 cells; * p < 0.05 determined by paired one sample t test). However this was not significantly different from the potentiation of the initial CCh-evoked [Ca2+]i increase by forskolin in the presence of external [Ca2+] (177 ± 17% increase from Fig. 1, statistical significance determined by unpaired Mann Whitney test).

Effects of Forskolin on InsP₃ Production-- Activation of muscarinic receptors leads to the generation of InsP₃ through G-protein-coupled activation of phospholipase C (PLC). Thus, the effects of forskolin could conceivably be caused by an interaction of cAMP with this receptor-signaling complex leading to an enhancement of InsP₃ generation. To test this idea we examined the effects of forskolin on inositol phosphate production by measuring [³H]inositol incorporation to assess PLC activity and thus InsP₃ production. CCh (1 µM) significantly increased inositol phosphates from 5.9 ± 0.2 to 9.8 ± 0.1% of total phosphoinositides (Fig. 5). In contrast, forskolin (10 µM) had no effect on either basal (6.1 ± 0.1%) or CCh-evoked (8.9 ± 0.3%) inositol phosphate turnover (Fig. 5). This indicated that increased cAMP does not directly stimulate the production of inositol phosphates and suggests a likely site of action is the Ca²⁺ release process itself.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   InsP3 generation is not directly affected by forskolin treatment. [3H]Inositol phosphate production, measured by incorporation of [3H]inositol into phospholipid, was assessed as an indirect measure of InsP3 generation. Inositol phosphate production was expressed as % of total phosphoinositides. 1 µM CCh significantly increased inositol phosphates from 5.9 ± 0.2% to 9.8 ± 0.1% of total phosphoinositides. Forskolin alone (6.1 ± 0.1%) or in combination with 1 µM CCh (8.9 ± 0.3%) did not significantly affect inositol phosphate production. Statistical significance was determined by unpaired students t test (* p < 0.05, n = 3).

Effects of Ryanodine on Carbachol-evoked [Ca²⁺]_i Changes-- In nonexcitable cells, InsP₃Rs are generally thought to be the trigger for Ca²⁺ release, whereas RyRs may have a role in propagating further release by Ca²⁺-induced Ca²⁺ release (CICR) (35). We pharmacologically separated these two Ca²⁺ release pathways by inhibiting RyRs with 500 µM ryanodine (36). Ryanodine alone failed to significantly affect the initial increase in [Ca²⁺]_i evoked by CCh (101.5 ± 7.3%; Fig. 6, A and C), suggesting that this event does not involve RyRs. However, when applied during a train of Ca²⁺ oscillations, ryanodine dramatically dampened the oscillatory [Ca²⁺]_i response (Fig. 6, A and B). These data therefore imply that RyRs may be important for propagating and maintaining Ca²⁺ oscillations in parotid acinar cells as is the case in pancreatic acinar cells (32).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effects of inhibition of RyRs on CCh-evoked [Ca2+]i responses and the potentiation by forskolin. A, 500 µM ryanodine dampened the oscillatory [Ca2+]i response but failed to significantly affect the initial CCh-evoked [Ca2+]i increase. B, using a similar experimental paradigm and in combination with 10 µM forskolin, ryanodine reduced the potentiation of the initial CCh-evoked [Ca2+]i increase by forskolin. The dotted line represents a gap of 7 to 9 min during which no images were acquired. C, quantification of mean data confirmed that ryanodine failed to significantly affect the initial CCh-evoked [Ca2+]i increase (101.5 ± 7.3% increase), but significantly reduced the potentiation by forskolin (125.2 ± 2.8% increase compared with 177.1 ± 17.4% in the absence of ryanodine; p < 0.05 as determined by Mann Whitney test). However the residual potentiation in the presence of both ryanodine and forskolin remained significant (*p < 0.05 as determined by one sampled t test).

When applied in combination with 10 µM forskolin, ryanodine significantly reduced the potentiation of the CCh-evoked [Ca²⁺]_i response from 177.1 ± 17.4% to 125.2 ± 2.8% of that observed in the absence of forskolin and ryanodine (Fig. 6, B and C). Nevertheless, the residual potentiation (125.2 ± 2.8%) remained significantly different from control. One possible interpretation of these data is that the potentiation of Ca²⁺ release by forskolin is caused by a direct effect of PKA on both RyRs and InsP₃Rs.

Direct Activation of RyR-- To address whether the potentiation by forskolin was caused by a direct effect of PKA on RyRs, we attempted to selectively activate RyRs. Caffeine (20 mM, n = 5 experiments, 22 cells) or low concentrations of ryanodine (0.01-10 µM, n = 8 experiments, 49 cells) consistently failed to affect resting [Ca²⁺]_i either in the absence or presence of forskolin (Fig. 7, A and B). In addition, 10 µM forskolin failed to affect resting [Ca²⁺]_i in any cell tested either in the presence or absence of ryanodine (data not shown). Because the effects of ryanodine are use-dependent (see Ref. 36, and references therein), two separate approaches were utilized to facilitate activation of RyRs. First, 10 µM ryanodine was added during a train of CCh-evoked [Ca²⁺]_i oscillations (see Fig. 7C) and continually applied after CCh was removed (similarly to Ref. 37). The second approach was to increase external [Ca²⁺] to 10 mM in an attempt to progressively elevate resting [Ca²⁺]_i, thereby increasing the sensitivity of RyRs to ryanodine (see Fig. 7D). However, ryanodine, either alone or in combination with forskolin, failed to affect resting [Ca²⁺]_i in any cell tested (see Fig. 7, C (three separate experiments, 15 cells) and D (four separate experiments, 29 cells). In contrast to conventional activators, such as caffeine or ryanodine, 4-chloro-m-cresol (CmC), a compound well documented to specifically activate RyRs (38-41), caused slow, concentration-dependent and readily reversible increases in resting [Ca²⁺]_i (Fig. 7E, three experiments, 14 cells). However, in contrast to CCh-evoked [Ca²⁺]_i increases, 10 µM forskolin failed to significantly affect the CmC-evoked [Ca²⁺]_i increases (Fig. 7F, five experiments, 33 cells). This indicates that RyR-mediated Ca²⁺ release in mouse parotid acinar cells is not directly affected by raising cAMP with forskolin, and suggests that PKA has a direct effect on InsP₃Rs. The role of RyRs may simply be to amplify the enhanced Ca²⁺ release from InsP₃Rs by CICR.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Attempts to directly activate RyRs fail to show any potentiation by forskolin. Representative experiments using several different paradigms to directly activate RyRs in the absence of InsP3R activation failed to evoke any change in [Ca2+]i either in the absence or presence of 10 µM forskolin; using A, 20 mM caffeine (5 experiments, 22 cells), B, low concentrations of ryanodine (0.01-10 µM; 8 experiments, 49 cells), C, application of ryanodine during a train of CCh-evoked [Ca2+]i oscillations (3 experiments, 15 cells), D, application of ryanodine when external [Ca2+] was elevated to 10 mM (4 experiments, 29 cells). E, concentration-dependent increase in resting [Ca2+]i evoked by CmC; (three experiments, 14 cells). F, 10 µM forskolin failed to affect 300 µM CmC-evoked increase in resting [Ca2+]i (5 experiments, 33 cells).

InsP₃-evoked Ca²⁺ Release from SL-O-permeabilized Cells-- To directly assess the effects of cAMP on InsP₃-evoked Ca²⁺ release, SL-O-permeabilized cells were used. Despite permeabilization, cells retained their polarity as indicated by apically located secretory granules. Following permeabilization, fluorescence became both dramatically reduced (<20%) and highly punctate, indicative of dye trapped within organelles (26, 42). Perfusion of permeabilized cells with a "cytosolic-like" medium devoid of Ca²⁺ and ATP caused a slow decline in the fura-2FF-340/380 ratio, which stabilized in 5-10 min, indicating Ca²⁺ store depletion. The subsequent addition of 0.2 µM Ca²⁺ and 1 mM Mg-ATP evoked a rapid increase in fura-2FF-340/380 ratio that reached a steady state within 3 min (Fig. 8, A-E), reflecting rapid uptake of Ca²⁺ into the ER. The observed rate was significantly faster than that reported in pancreatic acinar cells (~10 min to reach steady state; see Refs. 26 and 27).

View larger version (26K): [in this window] [in a new window]   Fig. 8.   InsP3-evoked Ca2+ release from SL-O-permeabilized cells is dramatically potentiated by Bt2cAMP, but unaffected by ryanodine. Perfusion of SL-O-permeabilized parotid acinar cells with 0.2 µM Ca2+ and 1 mM MgATP evoked rapid uptake of Ca2+ (indicated by arrow, A-E). Subsequent application of InsP3 evoked a rapid decrease in fura-2FF 340/380 ratio, indicative of Ca2+ release. A paired experimental design was utilized whereby a low concentration (0.3 µM), followed by a high concentration (3 µM) of InsP3 was applied first in the absence and then in the presence of treatment on the same cell. A to E are representative experiments showing time-matched controls (A, 4 experiments, 21 cells), effects of 100 µM Bt2cAMP (B, 5 experiments, 5 cells), the combined effect of Bt2cAMP and Rp-cAMPS (C, 4 experiments, 17 cells), 500 µM ryanodine (D, 4 experiments, 20 cells) and the combined effect of Bt2cAMP and ryanodine (E, 4 experiments, 17 cells) on InsP3-evoked Ca2+ release. F, quantification of mean data was achieved by expressing InsP3-evoked Ca2+ release in the presence of treatment (or 2nd control) relative to that in the absence of treatment (or 1st control) in the same cell. Statistical significance was determined using a paired one sample t test (*p < 0.05). This revealed that Ca2+ release was unaltered by repeated stimulation with InsP3 either over time (0.3 µM, 0.94 ± 0.06-fold change; 3.0 µM, 0.96 ± 0.03-fold change), or following treatment with 500 µM ryanodine (0.3 µM, 1.02 ± 0.05-fold change; 3.0 µM, 0.93 ± 0.05-fold change). However, 100 µM Bt2cAMP dramatically potentiated Ca2+ release evoked by 0.3 µM InsP3 (3.0 ± 0.5-fold change) but not by 3 µM InsP3 (0.95 ± 0.06-fold change). The potentiation by Bt2cAMP (3.0 ± 0.5-fold change) was prevented by Rp-cAMPS (1.1 ± 0.2-fold change) but not affected by co-treatment with ryanodine (2.37 ± 0.16-fold change) as compared using an unpaired Mann Whitney test. Inset shows mean InsP3 concentration-response curve, where Ca2+ release was expressed as % of the maximum evoked by 10 µM InsP3, which gave an EC50 of 0.64 ± 0.11 µM InsP3.

Following Ca²⁺ uptake, InsP₃ (0.1-10 µM) evoked a concentration-dependent stepwise decrease in [Ca²⁺]_ER. For each experiment, mean data were fit to a sigmoidal dose-response curve from which a mean half-maximum concentration (EC₅₀) was determined (Fig. 8F, inset; five separate experiments, 41 cells). This revealed a very steep InsP₃ concentration-response relationship for Ca²⁺ release with an EC₅₀ of 0.64 ± 0.11 µM and maximal release at 10 µM InsP₃, similar to previous reports (22, 43, 44). InsP₃-evoked Ca²⁺ release was also readily reversible, because removal of InsP₃ in the continued presence of Ca²⁺ and ATP caused a rapid return of [Ca²⁺]_ER to near pre-stimulatory levels, suggesting Ca²⁺ was rapidly taken back up into the ER (Fig. 8, A-F). This allowed the application of a general experimental paradigm consisting of stimulating with a low dose of InsP₃ (0.3 µM), which evoked 24.2 ± 0.1% maximal release, followed by a high dose (3 µM), which evoked nearly maximal release (93.1 ± 0.2%; Fig. 8F, inset). This paradigm was then repeated in the presence of 100 µM Bt₂cAMP (Fig. 8B) with or without 10 µM Rp-cAMPS or 500 µM ryanodine (Fig. 8, C-E). Time-matched controls were performed to account for loss of dye, photobleaching, or possible desensitization (Fig. 8A). Fig. 8F shows the mean data expressed as -fold increase in Ca²⁺ released by 0.3 and 3.0 µM InsP₃. Time-matched controls revealed that repeated stimulations with InsP₃ were not significantly different (Fig. 8, A and F). In contrast 100 µM Bt₂cAMP dramatically potentiated Ca²⁺ release evoked by 0.3 µM InsP₃ (3.0 ± 0.5-fold increase; Fig. 8, B and F) but did not significantly effect Ca²⁺ release evoked by 3 µM InsP₃ (0.95 ± 0.06-fold; Fig. 8F). This suggested that cAMP increased the sensitivity of InsP₃-evoked Ca²⁺ release without affecting the capacity of the stores to release Ca²⁺. Consistent with the effects of forskolin in intact cells, Bt₂cAMP failed to evoke Ca²⁺ release when applied in the absence of InsP₃ (Fig. 8B). In addition, 10 µM Rp-cAMPS, an inhibitor of PKA that competes for the cAMP-binding site (45), completely prevented the potentiation of InsP₃-evoked Ca²⁺ release by Bt₂cAMP (Fig. 8, C and F). This is further supportive of data from intact cells suggesting that the potentiation is PKA-mediated.

Similar to the effects of ryanodine on CCh-evoked Ca²⁺ release in intact cells, ryanodine also failed to significantly affect Ca²⁺ release from permeabilized cells evoked by either 0.3 µM (1.02 ± 0.05-fold change) or 3 µM InsP₃ (0.93 ± 0.05-fold change; Fig. 8, D and F (four separate experiments, 20 cells)). However, in contrast to intact cells, ryanodine failed to affect the potentiation of InsP₃-evoked Ca²⁺ release by Bt₂cAMP; release stimulated by 0.3 µM InsP₃ increased 2.37 ± 0.16-fold, whereas release stimulated by 3 µM InsP₃ was unaffected (1.04 ± 0.01-fold; Fig. 8, E and F (four separate experiments, 17 cells). Therefore, the lack of effect of ryanodine in SL-O-permeabilized cells suggests that, under these conditions, RyRs are functionally uncoupled from InsP₃Rs. Collectively, these data add credence to the notion that InsP₃Rs, and not RyRs, are directly modulated by PKA.

In Situ Phosphorylation of InsP₃R and RyRs-- To examine whether the potentiation of Ca²⁺ release correlated with PKA-mediated phosphorylation of InsP₃Rs and/or RyRs, two complimentary approaches were adopted using receptor specific antibodies to immunoprecipitate potentially phosphorylated protein. First, cells were metabolically labeled with ³²PO and protein that had incorporated ³²PO upon phosphorylation was detected by autoradiography. Following treatment with forskolin and subsequent immunoprecipitation with InsP₃R-specific antibodies, there was enhanced labeling of bands at the expected molecular weight for InsP₃Rs compared with control (Fig. 9A). This demonstrates that InsP₃Rs are directly phosphorylated, presumably by a PKA-mediated process. In contrast, under similar conditions using RyR-specific antibodies, no detectable signal was observed at the appropriate molecular weight for RyRs. Immunoprecipitation of RyR protein from skeletal muscle, cardiac muscle, and brain tissue lysates confirmed the avidity of RyR-specific antibody (data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 9.   PKA-mediated phosphorylation of InsP3Rs by treatment with forskolin. A, parotid acini, metabolically labeled with 32PO, were incubated with (+) or without () 10 µM forskolin for 10 min and samples were prepared as detailed under "Experimental Procedures." Treatment with forskolin dramatically increased the intensity of a band representative of a ~250 kDa protein, indicative of phosphorylated InsP3Rs. B, verification of Phospho-PKA substrate antibody in detecting phosphorylated proteins upon treatment of cells with (+) or without () 10 µM forskolin, 2 µM H-89, 30 µM Rp-cAMPS and/or 50 nM okadaic acid. Cell lysates were prepared as detailed under "Experimental Procedures," and protein samples run on a 7.5% SDS-polyacrylamide gel. Phosphorylated proteins were detected by Western blotting with the Phospho-PKA substrate antibody. C, detection of phosphorylated type II InsP3Rs upon treatment of cells with (+) or without () 10 µM forskolin, 2 µM H-89 or 30 µM Rp-cAMPS. Phosphorylated proteins were immunoprecipitated with the Phospho-PKA substrate antibody, run on a 5% SDS-polyacrylamide gel and phosphorylated type II InsP3Rs were detected by Western blotting with the CT2 antibody. * in A and C represent a secondary control whereby samples were treated identically but immunoprecipitating antibodies were omitted and represent nonspecific binding to protein-A beads.

Second, an alternative and complimentary approach used the phospho-PKA substrate antibody to immunoprecipitate phosphorylated protein, followed by specific detection of phosphorylated type II InsP₃R by immunoblot with the CT₂ antibody. The specificity of the phospho-PKA substrate antibody for phosphorylated protein was verified in Fig. 9B. A variety of proteins of different molecular weight were detected upon treatment with forskolin compared with untreated cells even though equal amounts of total protein were added into each lane (Fig. 9B, lane 3 versus lane 1). The phospho-PKA substrate antibody does not distinguish between substrates of PKA, protein kinase C, and cyclic GMP-dependent protein kinase (29, 30). To distinguish between activation of these kinases, cells were treated with the PKA inhibitors H-89 (2 µM) and Rp-cAMPS (30 µM) prior to and in the continued presence of forskolin to specifically activate PKA. This completely prevented the detection of visible bands (Fig. 9B, lane 4), which strongly suggests that forskolin evokes PKA-mediated phosphorylation of proteins. Furthermore, treatment of cells with the protein phosphatase inhibitor okadaic acid (50 nM) did not dramatically increase protein phosphorylation, suggesting that basal protein phosphatase activity was low and not affected by cell lysis (Fig. 9B, lanes 5 and 6). Finally, immunoprecipitation with the phospho-PKA substrate antibody and subsequent immunoblot with CT₂ antibody showed that forskolin dramatically increased the detection of phosphorylated type II InsP₃R (Fig. 9C, lanes 4 and 5), which was prevented by pretreatment with H-89 and Rp-cAMPS (Fig. 9C, lanes 6 and 7). Taken together, these data provide convincing evidence that forskolin causes PKA-mediated phosphorylation of type II InsP₃R.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present study provides functional and molecular evidence defining a specific site at which cAMP-raising agonists exert synergistic regulatory control over fluid secretion and exocytosis in mouse parotid acinar cells stimulated by muscarinic activation of [Ca²⁺]_i signaling (10-13, 16). The signal transduction pathway following muscarinic receptor stimulation and leading to an increase in intracellular Ca²⁺ is a rich source of sites for possible PKA regulation, which could account for the dramatic PKA-mediated potentiation of the CCh-evoked initial [Ca²⁺]_i increase. The effects of raising cAMP on various aspects of this signaling pathway were therefore systematically investigated. Specifically, Ca²⁺ entry has been suggested to be important not only in replenishing the stores with Ca²⁺ but also in maintaining agonist-evoked sustained [Ca²⁺]_i signals (4, 46), and in modulating the frequency of [Ca²⁺]_i oscillations by sensitizing Ca²⁺ release (47). In addition, in hepatocytes (48) and submandibular acinar cells (49), cAMP has been shown to potentiate InsP₃ production. However, in the present study, forskolin treatment was shown to have no appreciable effect on Ca²⁺ entry or CCh-evoked PLC activity and InsP₃ production. These data led us to conclude that the primary site of action of PKA is on the Ca²⁺ release process itself.

InsP₃-evoked Ca²⁺ release from InsP₃Rs underlies agonist-induced increases in [Ca²⁺]_i in a variety of nonexcitable cells including secretory epithelia (4, 32). We observed in a proportion of cells that forskolin altered the sensitivity of CCh-evoked [Ca²⁺]_i signals in such a way that apically confined [Ca²⁺]_i signals were initiated from subthreshold CCh concentrations. In a variety of exocrine cells, the apical region is regarded as the "trigger zone" from which Ca²⁺ waves are initiated (32, 50, 51). Conceptually, such a region could be established by the relative abundance of Ca²⁺ release channels and/or expression of the most sensitive channels. Both of these criteria are satisfied in rat parotid acinar cells; immunocytochemical studies have reported that the extreme apical region is highly enriched in all InsP₃R types (18, 19). Furthermore, of the three InsP₃Rs expressed, the type II InsP₃R, which has been reported to be the most sensitive to InsP₃ (52, 53), is the most abundant InsP₃R type in parotid acinar cells (18). Collectively, this supports the idea that cAMP potentiates Ca²⁺ release from the most sensitive InsP₃Rs located in the apical region of the cell.

Ca²⁺-induced Ca²⁺ release is important for the generation of both localized "spikes" and global increases in [Ca²⁺]_i (4, 32), and it is conceivable that modulation of CICR could contribute to the observed cAMP-mediated potentiation. Although InsP₃Rs themselves possess all the properties to support CICR (54), there is an emerging body of evidence that RyRs are important for CICR in nonexcitable cells (5, 35, 55). Both InsP₃Rs and RyRs have been shown to contain putative consensus sequences for phosphorylation by PKA (56, 57) and in some cases phosphorylation results in enhanced release (58-60). However, in mouse parotid acinar cells, we found that inhibition of RyRs with high concentrations of ryanodine failed to significantly affect the CCh-evoked initial increase in [Ca²⁺]_i, consistent with the notion that the mechanism underlying this release primarily involves InsP₃Rs. When applied during a train of Ca²⁺ oscillations, however, ryanodine dramatically inhibited oscillations, similarly to previous studies (35, 61). Of particular interest, ryanodine significantly reduced the potentiation of the CCh-evoked [Ca²⁺]_i response by forskolin (Fig. 6). An obvious explanation for this result is the direct phosphorylation of RyRs by PKA. However, we were unable to obtain any functional or biochemical evidence for this phosphorylation in parotid acinar cells. For example, various maneuvers designed to isolate Ca²⁺ release through RyR were not augmented by raising cAMP levels, and attempts to demonstrate direct phosphorylation of RyRs were unsuccessful. How can the effects of ryanodine on the initial PKA-potentiated release be reconciled with these data? One possibility is that the apparent contribution of RyRs after PKA treatment is simply the result of the enhanced release of Ca²⁺ by InsP₃Rs, resulting in an increased [Ca²⁺] in the vicinity of RyR, thereby increasing the possibility of CICR. This implies that RyRs in parotid acinar cells act to amplify Ca²⁺ signals that originate from the primary trigger source, which is InsP₃Rs, but only do so when a threshold [Ca²⁺]_i (or microdomain of Ca²⁺) is established. This is consistent with studies showing that RyR₃ is the predominant RyR type expressed in mouse parotid acinar cells, albeit in relatively low abundance compared with skeletal muscle (62). Single-channel data indicate that RyR₃ exhibit very low activity at [Ca²⁺] lower than 1 µM, but are dramatically activated by [Ca²⁺] above 1 µM (63). As such, RyRs would be functionally uncoupled at rest and only become activated by microdomains of Ca²⁺ created by Ca²⁺ released from neighboring InsP₃Rs.

Activation of PKA with Bt₂cAMP failed to evoke Ca²⁺ release from SL-O-permeabilized cells (Fig. 8B), a result consistent with the lack of effect of forskolin in intact cells and in agreement with similar studies using imaging of calcium green C18-labeled permeabilized parotid acinar cells (20). In contrast, cAMP has been demonstrated to evoke Ca²⁺ release from static suspensions of permeabilized cells and microsomal vesicles of rat parotid acinar cells (18, 21-23), a result that was attributed to activation of RyRs. Of interest in these studies is the fact that cAMP elevation evoked a relatively small Ca²⁺ release (~35 nM) compared with InsP₃ (~150 nM) (18, 21) and that this release occurred under conditions where basal [Ca²⁺] was estimated to be as high as 431 nM (22). At these [Ca²⁺], any RyRs would be expected to be in a sensitized state. The discrepancy between the present study and the aforementioned report underscores the difficulty in extrapolating averaged Ca²⁺ release from permeabilized cell suspensions to Ca²⁺ release in intact cells where resting [Ca²⁺]_i is likely to be closer to ~100 nM. Alternatively, the presence of such a ryanodine-sensitive cAMP-dependent Ca²⁺ release pathway, not observed in this study, may simply be the result of species differences. As stated previously, mouse parotid acinar cells express predominantly RyR₃ (62), whereas rat parotid acinar cells (revealed by reverse transcription-PCR) express predominantly RyR₁ (21). Because RyR₁ exhibit a lower threshold for activation by Ca²⁺ (63), any potentiation of Ca²⁺ release by cAMP would be amplified under conditions of elevated resting [Ca²⁺]_i (18, 21, 22). Such species differences could also explain the lack of effect of caffeine in mouse acinar cells, as RyR₃ are thought to be caffeine-insensitive (64, 65).

The lack of effect of ryanodine on InsP₃-evoked Ca²⁺ release in SL-O-permeabilized parotid acinar cells suggests that InsP₃Rs and RyRs are functionally uncoupled under these conditions, presumably as a result of high perfusion rates resulting in an effective infinite volume mimicking the cytoplasm. As such, microdomains of Ca²⁺ will likely fail to establish at sufficiently high levels to evoke CICR. In addition, many cytosolic components such as mobile buffers, which may be important for CICR, will be lost in permeabilized cells. Thus, this system allowed us to study the functioning of InsP₃Rs in isolation. The data obtained indicate that the potentiation of CCh-evoked [Ca²⁺]_i signals is mediated through InsP₃Rs, because activation of PKA still revealed potentiation under conditions where RyRs were functionally uncoupled. In support of this, treatment with forskolin caused phosphorylation of type II InsP₃Rs; the weight of evidence presented suggests a PKA-mediated mechanism. There is an emerging consensus that PKA may regulate Ca²⁺ release events directly at the level of the Ca²⁺ release channel. Previous studies have shown that InsP₃Rs contain several consensus sequences for PKA-mediated phosphorylation (56) and can be phosphorylated both in vitro (66, 67) and in vivo (68, 69). However, there exists no clear consensus as to the physiological consequence of InsP₃R-type-specific phosphorylation in terms of Ca²⁺ release. For example, phosphorylation of type I InsP₃R from cerebellum caused a decrease in Ca²⁺ release (66, 70), whereas others have shown an increase in Ca²⁺release (71). In addition, phosphorylation of type III InsP₃R in pancreatic acinar cells results in reduced Ca²⁺ release at low doses of InsP₃ (72), whereas phosphorylation of type II InsP₃R in liver enhances Ca²⁺ release (58, 60). This latter observation is of particular interest to the present study, as the type II InsP₃Rs are the dominant form expressed in parotid acinar cells (18). Despite that in vitro phosphorylation studies revealed that type II InsP₃Rs were relatively poor substrates for PKA phosphorylation (69), the present study convincingly demonstrated that forskolin treatment causes PKA-mediated phosphorylation of type II InsP₃Rs in mouse parotid acinar cells.

In summary, PKA-mediated phosphorylation of type II InsP₃Rs is a predominant mechanism for the potentiation of Ca²⁺ signaling in mouse parotid acinar cells. This largely explains the synergistic relationship between cAMP-raising agonists and ACh-evoked secretion in the parotid. These findings support the emerging consensus that phosphoregulation of InsP₃Rs is an important mechanism underlying the "shaping" of Ca²⁺ signals, and thus have broad implications for the fidelity of Ca²⁺-mediated processes.

	ACKNOWLEDGEMENTS

The RyR antibody (clone 34C), developed by J. Airey and J. Sutko, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD, National Institutes of Health (Bethesda, MD) and maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA). We thank Dr. R. J. H. Wojcikiewicz for providing InsP₃R antibodies (CT₁ and CT₂), Jodi Pilato for preparation of single parotid acinar cells, Jill Thompson for help with inositol phosphate and in situ phosphorylation experiments, Pauline Leakey and Sarah Wolbert for excellent technical assistance, and Dr. Patricia Hinkle and Steve Straub for helpful comments on the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DEO 13539 (to T. J. S. and D. I. Y.), GM 40457 (to T. J. S.), and DK54568 (to D. I. Y.).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.

To whom correspondence should be addressed: Dept. of Pharmacology & Physiology, School of Medicine and Dentistry, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-6128; Fax: 716-273-2652; E-mail: jason_bruce@urmc.rochester.edu.

Published, JBC Papers in Press, November 1, 2001, DOI 10.1074/jbc.M106609200

	ABBREVIATIONS

The abbreviations used are: [Ca²⁺]_i, intracellular calcium concentration; InsP₃, inositol 1,4,5-trisphosphate; InsP₃R, inositol 1,4,5-trisphosphate receptor; ACh, acetylcholine; CCh, carbamylcholine (carbachol); PKA, protein kinase A; RyR, ryanodine receptor; Bt₂cAMP, dibutyryl cAMP; SL-O, streptolysin-O, CICR, calcium-induced calcium release; PLC, phospholipase C; CmC, 4-choro-m-cresol; ER, endoplasmic reticulum; PSS, physiological saline solution; HEDTA, N-(2-hydroxyethyl)ethylenediaminetriacetic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Rp-cAMPS, Rp-adenosine-3:5'-cylic monophosphorothioate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES