Phosphorylation of Inositol 1,4,5-Trisphosphate Receptors in Parotid Acinar Cells
2002; Elsevier BV; Volume: 277; Issue: 2 Linguagem: Inglês
10.1074/jbc.m106609200
ISSN1083-351X
AutoresJason I.E. Bruce, Trevor J. Shuttleworth, David R. Giovannucci, David I. Yule,
Tópico(s)Pancreatic function and diabetes
ResumoAcetylcholine-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. 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. intracellular calcium concentration inositol 1,4,5-trisphosphate inositol 1,4,5-trisphosphate receptor acetylcholine carbamylcholine (carbachol) protein kinase A ryanodine receptor dibutyryl cAMP streptolysin-O, CICR, calcium-induced calcium release phospholipase C 4-choro-m-cresol endoplasmic reticulum physiological saline solution N-(2-hydroxyethyl)ethylenediaminetriacetic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Rp-adenosine-3:5′-cylic monophosphorothioate Calcium is a ubiquitous second messenger that is critically important in the regulation of a variety of cellular functions (1Clapham D.E. Cell. 1995; 80: 259-268Google Scholar, 2Penner R. Neher E. J. Exp. Biol. 1988; 139: 329-345Google Scholar, 3Hardingham G.E. Chawla S. Johnson C.M. Bading H. Nature. 1997; 385: 260-265Google Scholar). 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 (4Putney J.W.J. Science. 1998; 279: 191-192Google Scholar, 5Berridge M.J. Nature. 1997; 386: 759-760Google Scholar). 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 1interacting with those that elevate cAMP. Such cross-talk may alter the sensitivity of a variety of Ca2+ transport processes (6Putney J.W.J. Annu. Rev. Physiol. 1986; 48: 75-88Google Scholar,7Bugrim A.E. Cell Calcium. 1999; 25: 219-226Google Scholar). 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 (6Putney J.W.J. Annu. Rev. Physiol. 1986; 48: 75-88Google Scholar, 8Baum B.J. Sreenby L.M. The Salivary System. CRC Press, Inc., Boca Raton, FL1987: 123-134Google Scholar). 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 (9Petersen O.H. Gallacher D.V. Annu. Rev. Physiol. 1988; 50: 65-80Google Scholar), and, in addition, exert regulatory control over the exocytotic machinery required for protein secretion (6Putney J.W.J. Annu. Rev. Physiol. 1986; 48: 75-88Google Scholar). 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 β-adrenoreceptors (10Larsson O. Olgart L. Acta Physiol. Scand. 1989; 137: 231-236Google Scholar, 11Bobyock E. Chernick W.S. J. Dent. Res. 1989; 68: 1489-1494Google Scholar, 12Baldys-Waligorska A. Pour A. Moriarty C.M. Dowd F. Biochim. Biophys. Acta. 1987; 929: 190-196Google Scholar, 13Yoshimura K. Hiramatsu Y. Murakami M. Biochim. Biophys. Acta. 1998; 1402: 171-187Google Scholar). Although cAMP could have direct effects on ion channels (14Kraus-Friedmann N. Cell Calcium. 2000; 27: 127-138Google Scholar) and/or exocytotic proteins (15Fujita-Yoshigaki J. Cell Signal. 1998; 10: 371-375Google Scholar), an alternative hypothesis is that cAMP interacts directly with the Ca2+ signaling machinery to account for the synergistic effects (6Putney J.W.J. Annu. Rev. Physiol. 1986; 48: 75-88Google Scholar, 13Yoshimura K. Hiramatsu Y. Murakami M. Biochim. Biophys. Acta. 1998; 1402: 171-187Google Scholar, 16McKinney J.S. Desole M.S. Rubin R.P. Am. J. Physiol. 1989; 257: C651-C657Google Scholar). 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 (16McKinney J.S. Desole M.S. Rubin R.P. Am. J. Physiol. 1989; 257: C651-C657Google Scholar, 17Tanimura A. Nezu A. Tojyo Y. Matsumoto Y. Am. J. Physiol. 1999; 276: C1282-C1287Google Scholar, 18Zhang X. Wen J. Bidasee K.R. Besch Jr., H.R. Wojcikiewicz R.J.H. Lee B. Rubin R.P. Biochem. J. 1999; 340: 519-527Google Scholar, 19Takemura H. Yamashina S. Segawa A. Biochem. Biophys. Res. Commun. 1999; 259: 656-660Google Scholar, 20Tojyo Y. Tanimura A. Nezu A. Matsumoto Y. Eur. J. Pharmacol. 1998; 360: 73-79Google Scholar, 21Zhang X. Wen J. Bidasee K.R. Besch H.R.J. Rubin R.P. Am. J. Physiol. 1997; 273: C1306-C1314Google Scholar, 22Rubin R.P. Adolf M.A. J. Pharmacol. Exp. Ther. 1994; 268: 600-606Google Scholar, 23Ozawa T. Biochem. Biophys. Res. Commun. 1998; 246: 422-425Google Scholar). 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 situphosphorylation 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. 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 (24Evans R.L. Bell S.M. Schultheis P.J. Shull G.E. Melvin J.E. J. Biol. Chem. 1999; 274: 29025-29030Google Scholar). 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. 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. Isolated parotid acinar cells were incubated in the above attachment media containing 10 μm fura-2FF/AM (KD for Ca2+ ∼13 μm; see Ref. 25Hyrc K.L. Bownik J.M. Goldberg M.P. Cell Calcium. 2000; 27: 75-86Google Scholar) for 60 min at 37 °C. Permeabilization and subsequent Ca2+ uptake and Ca2+ release experiments were similar to those reported in pancreatic acinar cells (26van de Put F.H. Elliott A.C. J. Biol. Chem. 1996; 271: 4999-5006Google Scholar, 27van de Put F.H. Elliott A.C. J. Biol. Chem. 1997; 272: 27764-27770Google Scholar). 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 μmCa2+, 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. Total inositol phosphate production in response to forskolin and/or carbachol (CCh) was determined by a similar method to that described previously (28Shuttleworth T.J. Thompson J.L. J. Biol. Chem. 1998; 273: 32636-32643Google Scholar). Briefly, isolated parotid acinar cells were incubated with 5 μCi/mlmyo-[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. Totalmyo-[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. Parotid acinar cells were isolated from 4 mice as described above and metabolically labeled by incubating for 2 h with 14 μCi/ml 32PO 4− (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 4−-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 4−, 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 (29Montminy M. Annu. Rev. Biochem. 1997; 66: 807-822Google Scholar, 30Pearson R.B. Kemp B.E. Methods Enzymol. 1991; 200: 62-81Google Scholar). 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.). 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 samplet test. Occasionally, statistical significance was determined between groups of experiments where an unpaired ttest 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. 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. 31Yule D.I. Lawrie A.M. Gallacher D.V. Cell Calcium. 1991; 12: 145-151Google Scholar). 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. Using the adenylate cyclase activator forskolin, we investigated the effects of elevating intracellular cAMP levels on CCh-evoked [Ca2+]i signaling (33Seamon K.B. Padgett W. Daly J.W. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3363-3367Google Scholar). 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 (34Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Google Scholar) 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.Figure 2A 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).View Large Image Figure ViewerDownload (PPT) 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. 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 andB; 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. 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 InsP3production. 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. 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) (35Straub S.V. Giovannucci D.R. Yule D.I. J. Gen. Physiol. 2000; 116: 547-560Google Scholar). We pharmacologically separated these two Ca2+release pathways by inhibiting RyRs with 500 μm ryanodine (36Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51Google Scholar). 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 (32Lee M.-G. Xu X. Zeng W. Diaz J. Wojcikiewicz R.J.H. Kuo T.H. Wuytack R. Racymaekers L. Muallem M. J. Biol. Chem. 1997; 272: 15765-15770Google Scholar). 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. 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 μmforskolin 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. 36Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51Google Scholar, 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. 37Verma V. Carter C. Keable S. Bennett D. Thorn P. Biochem. J. 1996; 319: 449-454Google Scholar). 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 (38Zorzato F. Scutari E. Tegazzin V. Clementi E. Treves S. Mol. Pharm
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