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J. Biol. Chem., Vol. 275, Issue 43, 33704-33711, October 27, 2000

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Targeted Phosphorylation of Inositol 1,4,5-Trisphosphate Receptors Selectively Inhibits Localized Ca²⁺ Release and Shapes Oscillatory Ca²⁺ Signals^*

David R. Giovannucci^§, Guy E. Groblewski^¶, James Sneyd, and David I. Yule

From the  Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, the ^¶ Department of Nutritional Science, University of Wisconsin, Madison, Wisconsin 53706, and the  Institute of Informational and Mathematical Sciences, Massey University, Auckland PB102-904, New Zealand

Received for publication, May 18, 2000, and in revised form, June 30, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The current study provides biochemical and functional evidence that the targeting of protein kinase A (PKA) to sites of localized Ca²⁺ release confers rapid, specific phosphoregulation of Ca²⁺ signaling in pancreatic acinar cells. Regulatory control of Ca²⁺ release by PKA-dependent phosphorylation of inositol 1,4,5-trisphosphate (InsP₃) receptors was investigated by monitoring Ca²⁺ dynamics in pancreatic acinar cells evoked by the flash photolysis of caged InsP₃ prior to and following PKA activation. Ca²⁺ dynamics were imaged with high temporal resolution by digital imaging and electrophysiological methods. The whole cell patch clamp technique was used to introduce caged compounds and to record the activity of a Ca²⁺-activated Cl current. Photolysis of low concentrations of caged InsP₃ evoked Cl currents that were inhibited by treatment with dibutryl-cAMP or forskolin. In contrast, PKA activators had no significant inhibitory effect on the activation of Cl current evoked by uncaging Ca²⁺ or by the photolytic release of higher concentrations of InsP₃. Treatment with Rp-adenosine-3',5'-cyclic monophoshorothioate, a selective inhibitor of PKA, or with Ht31, a peptide known to disrupt the targeting of PKA, largely abolished forskolin-induced inhibition of Ca²⁺ release. Further evidence for the targeting of PKA to the sites of Ca²⁺ mobilization was revealed using immunocytochemical methods demonstrating that the R_II subunit of PKA was localized to the apical regions of acinar cells and co-immunoprecipitated with the type III but not the type I or type II InsP₃ receptors. Finally, we demonstrate that the pattern of signaling evoked by acetylcholine can be converted to one that is more "CCK-like" by raising cAMP levels. Our data provide a simple mechanism by which distinct oscillatory Ca²⁺ patterns can be shaped.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Hormone-, neurotransmitter-, or growth factor-evoked increases in intracellular calcium exert control over a vast array of cellular functions, including secretion and gene expression (1-3). The regulatory control governing the fidelity and specificity of these processes can be encoded by the amplitude, frequency, or localization of cytosolic calcium signals ([Ca²⁺]_c) (4-6).¹ It is well established that [Ca²⁺]_c in nonexcitable cells is generally induced by InsP₃ production, following activation of G_q proteins coupled to phospholipase C (7, 8). Many cell types express multiple distinct receptors coupled to this general transduction pathway. It is, however, not understood how the selective activation of distinct receptors that utilize the same general intracellular signaling system, such as the G_q-coupled formation of InsP₃, can generate agonist-specific [Ca²⁺]_c (9-12). Pancreatic acinar cells represent an ideal cell model for investigating this phenomenon because despite the activation of a common Ca²⁺ release pathway by agonists, very different patterns of [Ca²⁺]_c emerge. Stimulation of cells with acetylcholine (ACh) or cholecystokinin (CCK) results in [Ca²⁺]_c that differ in spike frequency, level of base-line spiking, and local sites of initiation (11-14).

In a previous study, we showed that the acinar cell type III InsP₃R is rapidly and selectively phosphorylated by cAMP-dependent kinase A (PKA) following stimulation with physiological levels of CCK but not by ACh (15). This observation is consistent with data in the literature suggesting that CCK but not muscarinic stimulation results in coupling to G_s in addition to G_q (16). Thus, in this study we have investigated the possibility that selective phosphoregulation of the InsP₃R by PKA contributes to the shaping of cytosolic Ca²⁺ signals.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Isolation of Mouse Pancreatic Acinar Cells-- Single acinar cells or small two- to three-cell clusters were prepared by a standard collagenase digestion of pancreata from wild-type C57BL/6 mice. Briefly, 25-g mice were sacrificed in accordance with National Institutes of Health policy and established protocol with the Division of Laboratory Animal Medicine, University of Rochester following CO₂ gas asphyxiation and then cervical dislocation. The pancreas was removed, and the capsule was injected with Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 30 µg/ml collagenase P (Sigma), 1000 units Purified collagenase (Worthington), and 1 mg/ml soybean trypsin inhibitor. Following 20-30 min of digestion at 37 °C, the pancreas was triturated through a 10-ml Falcon pipette tip, passed through a 100-µm nylon mesh, and washed with Dulbecco's modified Eagle's medium containing 1% bovine serum albumen (Sigma), and isolated cells and small acini were collected by centrfugation at 100 × g for 3 min. Cell were plated onto poly-L-lysine-coated glass coverslips and allowed to adhere for 5 min prior to application of recording solution perifusate.

Electrophysiology-- Ionic currents were recorded at a sampling rate of 1 KHz using an Axopatch 200A patch clamp amplifier, Instrutech digital interface, and IGOR PRO/Pulse Control XOP software. The standard intracellular recording solution contained 140 mM KCl, 10 mM HEPES-KOH, 1.13 mM MgCl₂, 2 mM sodium ATP, 1 mM n-hydroxyethylethylenediaminetriacetic acid, and 0.001-0.1 mM D-myo-inositol 1,4,5-trisphosphate, P⁴⁽⁵⁾-1-(2-nitrophenyl)-ethyl ester (NPE-caged InsP₃), pH 7.3. The intracellular recording solution for the photolytic release of caged Ca²⁺ contained 130 mM KCl, 10 mM HEPES-KOH, 10 mM o-nitrophenyl EGTA, 5 mM CaCl₂, 2 mM magnesium ATP, 1.2 mM MgCl₂, pH 7.2. Under whole cell conditions, resting [Ca²⁺]_c and free [Mg²⁺] were estimated at 175 nM and 1 mM, respectively. Intervals of 4 min were maintained following patch rupture prior to and between stimuli to allow for sufficient equilibration with the patch pipette solution. The extracellular solution contained 140 mM NaCl, 10 mM HEPES-NaOH, 10 mM D-glucose, 4.7 mM KCl, 1.13 mM MgCl₂, 1 mM CaCl₂, pH 7.3.

Flash Photolysis and Digital Imaging-- Photolytic release was performed using a pulsed Xenon arc lamp (T.I.L.L. Photonics) and fiber optic guide fed to a dual port epifluorescence condenser attached to a Nikon TE-200 fluorescence microscope. A high intensity 0.5-ms flash of UV light (360 ± 7.5 nm) was reflected onto the plane of focus with a DM400 dichroic mirror and Nikon 40× oil immersion objective, 1.3 NA. At the high intensity setting about 80 J were discharged. In other experiments, a low intensity (0.1 J) continuous strobe discharge was used to uncage threshold levels of InsP₃. For simultaneous current recording and direct measurement of [Ca²⁺]_c, 75 µM Oregon Green 488 Bapta-2 was added to the patch pipette solution. [Ca²⁺]_c imaging was performed using a monochrometer-based illumination system and high speed CCD camera (T.I.L.L. Photonics). Cells were illuminated at 488 ± 15 nm, and fluorescence was collected through a 525 ± 25-nm band pass filter (Chroma). Images were acquired at 79-ms intervals and displayed as F/F_o = 100[(F  F_o)/F_o], where F is the recorded fluorescence and F_o was obtained from the mean of 15 sequential frames following equilibration with the patch dialysis solution and prior to stimulation. In some experiments, the acetoxymethylester of fura-2 was loaded into acinar cells and used to measure agonist-induced [Ca²⁺]_c. In this case, loaded cells were alternately excited at 340 and 380 ± 15 nm, and emitted light was collected through a 525 ± 25-nm bandpass filter. The ratio of emitted light resultant from excitation at these two wavelengths was utilized as a measure of [Ca²⁺]_c.

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting-- Immunoprecipitations were performed as follows. Samples were sonicated in 0.5 ml of ice-cold lysis buffer (100 mM NaF, 50 mM Tris, 150 mM NaCl, 10 mM EDTA, 1 mM benzamidine, 1% Triton X-100, 1% 2-mercaptoethanol, 10 mg/ml leupeptin, and 10 mg/ml pepstatin, at pH 7.4) and then sonicated. After 30 min on ice, the samples were centrifuged for 30 min at 10,000 × g. The supernatant was assayed for protein concentration. Samples of equal protein concentration were then incubated with antiserum overnight at 4 °C. Immunoprecipitation of individual InsP₃R subtypes was performed with excess antiserum (17). (No InsP₃R was detectable in the lysate after addition of protein A.) Immobilized protein A beads (Pierce) were added to each sample. As a control, samples were processed with no cellular lysate or with no immunoprecipitating antiserum to ascertain specific bands on the immunoblot. After rotating the samples for 2 h, the samples were microcentrifuged, and the supernatant was discarded. The beads were washed four times with lysis buffer, suspended in SDS-polyacrylamide gel electrophoresis sample buffer, and boiled. The proteins were then separated by SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose prior to immunoblotting (Schleicher & Schuell). Immunoreactivity was visualized using peroxidase-conjugated secondary antibodies followed by detection using the ECL detection system exposed on ECL Hyperfilm (Amersham Pharmacia Biotech).

Immunocytochemistry-- Subcellular localization was performed using standard immunohistochemical methods in isolated acini fixed with 2% paraformaldehyde as described previously (18). A Noran/Oz laser scanning confocal microscope was used to visualize the distribution of immunofluorescence.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Threshold level InsP₃-evoked Ca²⁺ Release Is Inhibited by PKA Activation-- To date, no consensus exists in the literature as to the physiological consequence of phosphorylation of individual InsP₃R types (19-23). Thus, we investigated the PKA-dependent regulation of Ca²⁺ release in isolated mouse pancreatic acinar cells using the controlled release of photoactivatable (caged) InsP₃ and selective pharmacological activators and inhibitors of PKA function. Whole cell patch clamp methods were used to measure Ca²⁺-activated ionic current evoked by the photolysis of caged compounds. This current has been previously established to faithfully report [Ca²⁺]_c, especially below the apical plasma membrane (6, 11, 24, 25).

As shown in Fig. 1 (A and C), high intensity UV light flash photolysis of a reproducible portion of 1-100 µM NPE-caged InsP₃, introduced via diffusional equilibration with the patch pipette solution, could repetitively evoke a Ca²⁺-activated current in a concentration-dependent manner. This indicated that prior exposure to Ca²⁺ had little effect on amplitude or kinetics of the whole cell current. A constant interval was maintained between flash discharge (3-5 min) to allow for equilibration of the cytosol with the cage-containing patch pipette. Application of the cell-permeable, hydrolysis-resistant cAMP analogue, dibutyryl cyclic adenosine monophosphate (dbcAMP) (60 µM), prior to flash photolysis of caged InsP₃, rapidly and reversibly reduced or abolished the amplitude of the Ca²⁺-activated current. Currents represent the maximum inhibition observed after a 3-5-min exposure to dbcAMP. Only cells that showed recovery to near control current levels within 15 min were included in the analysis. Surprisingly, it was only at the lowest concentrations of NPE-caged InsP₃ tested that this inhibitory effect was revealed as significant (Fig. 1B). Inhibition compared with control was 82.6 ± 9.0% (1 µM, n = 7), 36.8 ± 11.1% (3 µM, n = 9), 23.4 ± 11.2% (10 µM, n = 7), 20.2 ± 6.3% (30 µM, n = 8), and 5.8 ± 2.9% (100 µM, n = 5). The percentage of inhibition was determined by measuring the peak current in response to a high energy flash discharge prior to and after 3-5-min exposure and following removal of drug. The observation that only the smallest, threshold responses to InsP₃ were inhibited by PKA activation is consistent with a previous study demonstrating that PKA had little effect on Ca²⁺ release evoked by application of high doses of agonist (26-28).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   PKA activation inhibits Ca2+ release. A, application of 60 µM dbcAMP reversibly inhibited the Ca2+-activated ionic current evoked by low doses of InsP3 in whole cell patch clamped mouse acinar cells. The traces represent recordings from single or two- or three-cell clusters that were voltage clamped at a holding potential (Hp) of 30 mV at 25 °C. No significant changes in current were activated by flash photolysis when Hp was 0 mV (the reversal potential of both Ca2+-activated currents) or when caged compounds were omitted from the internal solution. B, the averaged data comparing the percentage of inhibition by dbcAMP of the ionic currents evoked by flash photolysis in cells loaded with different amounts of NPE-caged InsP3. The percentage of inhibition was determined by comparing peak current amplitude prior to and following dbcAMP treatment. The number of cells for each data set and statistical significance (p  0.01) is indicated (asterisk). C, repetitive photolytic release of InsP3 had no effect on peak amplitude or kinetics of current transients induced prior to and following treatment with dbcAMP.

It is unlikely that this reduction was mediated by direct inhibitory phosphoregulation of the Ca²⁺-activated Cl channel because, as shown in Fig. 2, no inhibition of peak current amplitude was observed over a range of currents (32-527 pA, 5 mM NP-EGTA; or 320-686 pA, 10 mM NP-EGTA) activated by the release of caged Ca²⁺. Average peak currents prior to and following treatment with 100 µM dbcAMP for 4 min were 217 ± 51 versus 243 ± 50 pA (n = 11, 5 mM NP-EGTA) and 519 ± 73 pA versus 571 ± 84 pA; (n = 5; 10 mM NP-EGTA). On average, flash photolysis of caged Ca²⁺ (10 mM NP-EGTA) transiently raised [Ca²⁺]_c to about 7 µM, as measured with the ratiometric, low affinity Ca²⁺ indicator, benzothiazola coumarin. These levels of Ca²⁺ are generally within a physiological range evoked apically by InsP₃ (1-10 µM) (29) and at which the Cl current is not likely to be saturated (29-31). It is generally believed that, unlike CFTR or voltage-activated Cl channels, cAMP does not directly modulate the Ca²⁺-activated Cl channel (32-34). Furthermore, we observed that PKA activation was found to directly inhibit InsP₃-evoked [Ca²⁺]_c in a permeablized mouse pancreatic acinar cell preparation, supporting our contention that the reduction in the Ca²⁺ signal largely resulted from a decreased Ca²⁺ release rather than an effect on the Ca²⁺-activated Cl channel or on Ca²⁺ clearance (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Lack of direct effect of PKA activation on Ca2+-activated Cl currents. A range of Ca2+-dependent ionic currents were directly activated in patch-clamped mouse acinar cell by the instantaneous, uniform elevation of [Ca2+]c by the photolytic release of caged Ca2+ (5 mM (A and B) or 10 mM NP-EGTA (C), 50% bound with Ca2+) prior to and following treatment for 4 min with 100 µM dbcAMP.

A reduction of the current activated by low levels of stimulation indicated that a subset of InsP₃R that exhibit the highest sensitivity for InsP₃ is preferentially modulated by dbcAMP. Because the pancreatic acinar cell expresses all isoforms of receptor together with heterotetrameric forms (35), it is difficult to categorically identify this high sensitivity receptor. The relative contribution of an InsP₃R type to the initial signal is presumably determined by several factors including the relative abundance of the particular receptor, the affinity of the receptor for InsP₃ and susceptibility to phosphoregulation by PKA. Because the type II receptor is a poor substrate for PKA phosphorylation (23), it would seem unlikely that this receptor contributes markedly to threshold level Ca²⁺ release and its modulation by PKA. In contrast, the type I receptor is a good substrate for phosphorylation by PKA and has been reported to have the highest affinity for InsP₃ but is expressed at very low levels in pancreatic acinar cells (17). The type III receptor has been shown to be an efficient substrate for PKA-mediated phosphorylation (15). In addition, the only quantitative study of receptor number in the exocrine pancreas has shown that the type III receptor is the most abundant receptor form (17) and, thus, by mass action would presumably contribute significantly to the initial Ca²⁺ release. In support of this contention, the type III receptor has also been proposed to serve as an initial trigger for Ca²⁺ release (36). Furthermore, the open probability of the receptor is modulated very steeply by near resting [Ca²⁺]_c (37). It should, however, be noted that the type III receptor has been reported, when studied in isolation, to have the lowest affinity for InsP₃ (23). These data together with the body of work in the literature indicate that the type III is a good candidate for the initial, PKA-modulated Ca²⁺ signal. A contributing role for the type I receptor alone or in heterologous association with the type III receptor, however, cannot be excluded.

Because Ca²⁺ spikes induced by low levels of ACh have been shown to initiate at specialized, InsP₃R-rich sites (trigger zones) tightly associated with the luminal borders and are often confined to the luminal pole (18, 29, 38, 39), we postulated that the effect of dbcAMP on Ca²⁺ release would also be manifested in this region. To test this hypothesis, we sought to selectively activate Ca²⁺ release at the acinar cell trigger zone. Thus, rather than using a high intensity flash discharge, we applied a continuous, low level photolytic strobe stimulus (indicated as I in the Fig. 3) to cells loaded with caged InsP₃ to achieve threshold activation levels of InsP₃. In most cases it was necessary to first determine empirically the concentration of caged InsP₃ that was needed to evoke threshold activation. As shown in Fig. 3, fluorescence digital imaging methods confirmed that this stimulation paradigm preferentially induced Ca²⁺ rises that were initiated and largely maintained in the luminal pole, characteristic of threshold Ca²⁺ release. Simultaneous patch clamp measurements revealed the activation of irregular current spikes that mirrored the evoked apical [Ca²⁺]_c. As expected, a subsequent, high intensity UV flash discharge (indicate as II in the Fig. 3) evoked a global Ca²⁺ rise that initiated in the luminal pole and activated a robust current.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Low intensity photolysis of NPE-caged InsP3 preferentially induces Ca2+ signals that initiate in and remain confined to the luminal pole. A, simultaneous measurements of the Ca2+-activated current spikes (lower trace) and the luminal (black) or basal (red) [Ca2+]c in a patch clamped acinar cell loaded with 75 µM OGB-2. Ca2+ release was activated by low intensity, strobe-induced photolysis of threshold amounts of InsP3 (dashed line) or by high intensity flash discharge (arrow). B, a corresponding series of fluorescence images from the same cell as in A show the spatial distribution of the Ca2+ signal at various time points. Images were obtained at 79-ms intervals and are displayed sequentially as every second (first row), fifth (second row), or 25th image frame (third row) during low intensity photolysis (I). The last row shows the [Ca2+]c evoked by high intensity flash photolysis (II) at sequential 79-ms intervals. The inset shows the transmitted light image of the cell doublet and placement of the regions of interest for measurement of luminal (black box) or basal (red box) [Ca2+]c.

To investigate the inhibitory effect of cAMP on the high sensitivity InsP₃R subset, cAMP levels were increased by treatment with 10 µM forskolin, and the Ca²⁺-activated current spike activity was evoked by 15-30 s of a continuous UV strobe. To accurately quantify the complex nature of the Ca²⁺ release events evoked by continuous strobe prior to and following forskolin application for 3-5 min, the time integral of the current produced was determined. As shown in Fig. 4A, forskolin treatment reversibly inhibited Ca²⁺ release at the trigger zone. Following wash-off of forskolin, current responses returned to control values within 5-20 min. Forskolin treatment induced nearly 83% inhibition of threshold Ca²⁺ release (Fig. 4B). Current integrals were, on average, reduced to 16.4 ± 9% of control values by forskolin treatment (p  0.0003, n = 10). Removal of forskolin restored the current integrals over 5-20 min to a level not significantly different from control values (150.7 ± 23%). Next, we tested whether PKA activation was required for the inhibitory effect of cAMP. As shown in Fig. 4 (C and D), inclusion of a specific, competitive PKA inhibitor, Rp-adenosine-3',5'-cyclic monophoshorothioate (Rp-cAMPS) (334 µM), in the patch pipette solution largely abrogated the forskolin-induced inhibition of spike activity, and the current integrals following forskolin treatment were not significantly different from control values (107 ± 7 and 124 ± 27%, respectively; n = 5). Taken together, these data strongly indicate that intracellular rises in cAMP negatively modulate InsP₃ induced Ca²⁺ release via PKA-dependent phosphoregulation of a specialized subset of InsP₃R localized at the acinar cell apical pole.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Ca2+-activated current spikes are inhibited by PKA activation. A, application of 10 µM forskolin for 3-5 min (solid line) induces the reversible inhibition of current spikes evoked by continuous low intensity UV strobe discharge (arrow and dashed line) in a single acinar cell. Breaks in the record indicate 3-5-min intervals between stimuli. Because current profiles evoked by 15-30 s of continuous strobe discharge exhibited complex kinetics, current integrals were used to quantify the changes in whole cell current. B, a comparison of the of the time-integrated current spikes induced prior to and following treatment with forskolin in cells loaded 1-10 µM NPE-caged InsP3. C, inclusion of 334 µM Rp-cAMPS in the patch pipette solution effectively blocked the forskolin-induced inhibition of current spikes evoked by low level photolytic release of InsP3. D, average current integrals measured during treatment with and following removal of 10 µM forskolin in the presence of Rp-cAMPS were not significantly different from control values. Number of cells for each data set and significance (asterisk) are indicated.

PKA Is Functionally Targeted to the Type III InsP₃ Receptor-- Because forskolin treatment likely induces a spatially uniform rise in cAMP, we suggest that signaling specificity is achieved not in the production of cAMP but in the compartmentalization of its effector, PKA. This co-localization has been shown by others to lead to rapid and efficient phosphorylation of a specific substrate, even on a uniform background of cAMP levels (40-47). Given the high degree of functional and structural organization exhibited by these polarized cells, we hypothesized that PKA might be targeted to the trigger zone to provide spatiotemporal control over the phosphoregulation of Ca²⁺ release. PKA targeting is often achieved by the association of the regulatory R subunit dimer with an protein kinase A-anchoring protein bearing a specific subcellular localization signal (46, 48). Immunohistochemical methods were performed to visualize the subcellular distribution of PKA in acinar cells using R_II and R_II subunit-specific monoclonal anitbodies. The R_II subunit exhibited diffuse cytosolic distribution (not shown). In contrast, the R_II subunit displayed a pattern of labeling that was confined to the apical and granular region of the cells (Fig. 5). This pattern of staining overlaps and is consistent with localization of PKA to the sites of InsP₃-induced Ca²⁺ release. It is within this region that the majority of InsP₃R co-localize with a highly structured sublumenal actin web (18, 39, 49).

View larger version (121K): [in this window] [in a new window]   Fig. 5.   The type III InsP3R and PKA co-localize. A confocal micrograph of small acinar cell clusters showing that the RII subunit of PKA is localized to the luminal secretory poles of the cells (arrowheads) but not to the basal or perinuclear regions (large arrows). No staining was evident when primary antibody was omitted.

To determine whether R_II subunits form molecular associations with type III InsP₃R, we immunoprecipitated proteins from acinar cell extracts using a monoclonal antibody to the type III InsP₃R. The enriched proteins were separated, and the Western blots were probed with actin, R_II, and R_II subunit-specific monoclonal antibodies. As shown in Fig. 6A, actin and both R_II subunits immunoprecipitated with the type III InsP₃R, suggesting that PKA is targeted to its potential kinase substrate, either directly or by a common tethering mechanism, possibly through actin or actin-binding proteins. To demonstrate specificity for the immunoprecipitation of PKA, lysates immunoprecipitated with type III InsP₃R and containing R_II (Fig. 6B, lane 1) were probed with a polyclonal antiserum recognizing all heterotrimeric G protein  subunits. Although G subunits were identified in whole pancreatic acinar cell lysates (Fig. 6B, lane 4), no G could be demonstrated to co-immunoprecipitate with type III InsP₃R antiserum (Fig. 6B, lanes 2 and 3). To investigate whether PKA was associated selectively with any particular InsP₃R type, individual receptors were immunoprecipitated from pancreatic lysates using subtype-specific antiserum (17). Probing of separated proteins with the antiserum used to immunoprecipitate confirmed that individual InsP₃R types had been immunoprecipitated (Fig. 6C, upper panel). In lane A, specific polyclonal antiserum raised against the C terminus of type I receptor were used (17, 18). In lane B, a polyclonal antiserum versus the type II receptor (17, 18), and in lane C, a monoclonal antibodies specific for type III receptor were utilized. Each antiserum was utilized in excess and is known to effectively and selectively immunoprecipitate its respective receptor (17, 18). In the lower panel, the same immune complexes were probed with antiserum raised against PKA-R_II. Only samples immunoprecipitated with type III InsP₃R contained PKA-R_II. These data strongly suggest that PKA is targeted specifically to type III receptors and, thus, is ideally placed to mediate rapid, specific phosphorylation of this receptor.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Physical interaction of type III InsP3R with PKA. A, Western blot analysis of proteins enriched from acinar cell lysates by immunoprecipitation (IP) with a monoclonal antibody to the type III InsP3R (-IP3RIII). The -IP3RIII pulls down the type III receptor (first lane), actin (second lane), RII subunit (third lane), and RII subunit (fourth lane) identified using -IP3RIII (1:1000) or polyclonal -actin (Zymed Laboratories Inc.) (1:1000) or subtype-specific monoclonal antibodies to the PKA regulatory subunits (1:500) (Transduction Labs) and visualized by chemiluminescence (mouse IgG 1:2000). Arrows indicate the standard molecular mass marker positions (in kDa). B, Western blot of lysates immunoprecipitated with type III InsP3R antiserum and probed with RII (lane 1); in lane 2 an equal amount of lysate was probed with G antiserum (Santa Cruz 1:1000); and in lane 3, twice the amount of lysate used in lanes 1 and 2 was probed with G, demonstrating no detectable protein. Lane 4 shows that G is present in whole pancreatic acinar cell lysates. C, PKA-RII subunits preferentially immunoprecipitate with type III InsP3R. InsP3R from lysates containing equal cell protein were immunoprecipitated with antiserum to individual InsP3R types.

To test the functional significance of type III InsP₃R-PKA co-localization, we disrupted PKA targeting in patch clamped acinar cells using a synthetic peptide containing the functional R_II binding motif of the thyroid protein kinase A-anchoring protein, Ht31. The Ht31 peptide has been shown to be an efficient and specific inhibitor of R_II protein kinase A-anchoring protein interactions (46, 48). Fig. 7A shows that inclusion of 30 µM Ht31 in the pipette solution could block the forskolin-induced inhibition of the apical Ca²⁺ release. As shown in Fig. 7B, no statistically significant inhibition was observed following continuous treatment with forskolin for 5 or 10 min or following removal of forskolin, compared with control responses (76.8 ± 15, 49.5 ± 27, and 139 ± 30% of control, respectively; n = 9).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Functional effect of inhibiting the targeting of PKA. A, inclusion of the Ht31 synthetic peptide (30 µM) in the patch pipette solution blocked forskolin-induced inhibition of current spikes evoked by low intensity photolysis. Intervals of 5 min were maintained following patch rupture prior to and between stimuli to allow for sufficient equilibration with the patch pipette solution. B, average current integrals measured during treatment with 10 µM forskolin (5 and 10 min) and following removal of forskolin, in the continuous presence of Ht31.

Functional Relevance of PKA Activation to the Shaping of Oscillatory Ca²⁺ Signatures-- Although our experiments provide evidence that the targeted phosphorylation of a subset of InsP₃R exerts negative regulatory control over local Ca²⁺ release events, we wondered whether this modulation could account for the distinctive patterns of Ca²⁺ oscillations induced by CCK or carbachol (CCh). Application of physiological doses of ACh or CCh to pancreatic acinar cells generates sinusoidal [Ca²⁺]_c oscillations with frequencies of 4-6/min that are superimposed on an elevated base line (11, 12). In contrast, CCK application induces oscillations consisting of base-line spikes of much longer period (1-2/min) (12, 50). To address this relationship, we tested whether PKA-dependent phosphorylation could convert CCh-induced oscillations into a pattern that resembled CCK-induced oscillations. Intact acinar cells were treated with low doses of either CCh or CCK to induce [Ca²⁺]_c oscillations and the [Ca²⁺]_c monitored with the Ca²⁺-sensitive dye, fura-2, prior to and following application of 60 µM dbcAMP. As shown in the representative trace in Fig. 8A, introduction of dbcAMP reduced both spike amplitude and the plateau component (defined as the average intra-spike level) and slowed the frequency of [Ca²⁺]_c oscillations, suggesting that activation of PKA was sufficient to shift the CCh-induced pattern of oscillations to one that was more "CCK-like". As expected, dbcAMP had little effect on CCK-induced [Ca²⁺]_c oscillations (Fig. 8B), where presumably the InsP₃R is already in a phosphorylated state. Analysis of the oscillation frequencies and plateau levels of [Ca²⁺]_c induced by these agonists are shown in Fig. 8 (C and D).

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Treatment with 60 µM dbcAMP alters carbachol- but not CCK-induced [Ca2+]c oscillations in intact acinar cells. Cells were loaded with 2 µM fura-2 AM and monitored by digital imaging. Representative fluorescence ratio changes evoked by bath application of low doses of carbachol (A) or CCK (B) prior to and following application of dbcAMP. C, the frequency of carbachol-induced [Ca2+]c oscillations was significantly reduced from 4.33 ± 0.36 oscillation/min to 2.47 ± 0.48 oscillation/min (p  0.003, n = 6) by dbcAMP, whereas the frequency of CCK-induced oscillations remained unchanged (0.99 ± 0.18 oscillation/min versus 1.0 ± 0.05 oscillation/min, n = 3). D, similarly, the elevated Ca2+ plateau observed during carbachol treatment was diminished by application dbcAMP (0.836 ± 0.064 ratio units versus 0.652 ± 0.037 ratio units; p  0.003, n = 6), whereas there was no significant effect on CCK-induced responses (0. 566 ± 0.017 ratio units versus 0.577 ± 0.035 ratio units, n = 3). The dashed line indicates averaged basal (resting) ratio values (0.517 ± 0.085) determined betwixt [Ca2+]c oscillations during the treatment periods.

Numerous factors have been proposed to provide a mechanism underlying agonist-specific [Ca²⁺]_c oscillations observed in pancreatic acinar cells (51, 52). These include the utilization by CCK receptor signaling of alternative second messengers such as cyclic adenosine diphosphate ribose and nicotinic acid adenine dinucleotide phosphate. Although no data are available regarding the ability of agonists to stimulate levels of these putative messengers, significant modulation of Ca²⁺ signaling has been reported (25, 53). It is clear that if the levels of these molecules are modulated by agonist stimulation, it must be through G_q activation, because both CCK and muscarinic signaling are abolished by maneuvers antagonizing G_q function. (54, 55). Recent reports have also suggested that pancreatic secretagogue receptors interact with differing RGS proteins to play a role in modulating signaling events (56, 57). Modulating the kinetics of InsP₃ production at the level of the cell surface receptors would be expected to have significant effects on the generation of [Ca²⁺]_c signals. Although the data presented in this study do not preclude any of the aforementioned proposals, selective modulation of the initial [Ca²⁺]_c by PKA appears to contribute significantly to shaping the Ca²⁺ signal.

We conclude that targeted, PKA-mediated phosphorylation specifically controls the functioning of a discrete subset of intracellular Ca²⁺ release channels that act as the initial trigger for InsP₃-induced Ca²⁺ release. Our data suggest that the type III InsP₃R, abundant in the apical trigger zone, fulfills this role. We contend that the negative allosteric modulation of the type III InsP₃R contributes significantly to the characteristic Ca²⁺ oscillations induced by CCK. Given that most cells have surface receptors that can couple to multiple G protein families (58, 59), cross-talk between divergent signaling pathways to modulate Ca²⁺ signaling at the level of the Ca²⁺ release channel is likely to be a widespread and important mechanism by which information can be encoded for the selective activation of distinct physiological end points.

	ACKNOWLEDGEMENTS

We thank Brain Giordano, John Puskas, and Dr. Patricia Hinkle for help with some of the immunocytochemical experiments; Dr. John Scott for kindly supplying the Ht31 peptide; Dr. R. J. H. Wojcikiewicz for providing InsP₃R-specific antisera; and Drs. Patricia Hinkle, Robert Dirksen, and Trevor Shuttleworth for comments on the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant DK 54568 (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. E-mail: david_giovannucci@urmc.rochester.edu.

Published, JBC Papers in Press, July 7, 2000, DOI 10.1074/jbc.M004278200

	ABBREVIATIONS

The abbreviations used are: [Ca²⁺]_c, cytosolic calcium concentration; NPE-caged InsP₃, D-myo-inositol 1,4,5-trisphosphate, P⁴⁽⁵⁾-1-(2-nitrophenyl)-ethyl ester; CCh, carbamylcholine, carbachol; CCK, cholecystokinin; NP-EGTA, o-nitrophenyl ethylenediaminetriacetic; dbcAMP, dibutyryl cyclic adenosine monophosphate; InsP₃, inositol 1,4,5-trisphosphate; InsP₃R, InsP₃ receptor(s); PKA, protein kinase A; Rp-cAMPS, Rp-adenosine-3',5'-cyclic monophoshorothioate; ACh, acetylcholine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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