Ca2+-dependent Protein Kinase-A Modulation of the Plasma Membrane Ca2+-ATPase in Parotid Acinar Cells
2002; Elsevier BV; Volume: 277; Issue: 50 Linguagem: Inglês
10.1074/jbc.m208393200
ISSN1083-351X
AutoresJason I.E. Bruce, David I. Yule, Trevor J. Shuttleworth,
Tópico(s)Ion channel regulation and function
ResumoCross-talk between cAMP and [Ca2+]i signaling pathways represents a general feature that defines the specificity of stimulus-response coupling in a variety of cell types including parotid acinar cells. We have reported recently that cAMP potentiates Ca2+ release from intracellular stores, primarily because of a protein kinase A-mediated phosphorylation of type II inositol 1,4,5-trisphosphate receptors (Bruce, J. I. E., Shuttleworth, T. J. S., Giovannucci, D. R., and Yule, D. I. (2002) J. Biol. Chem. 277, 1340–1348). The aim of the present study was to evaluate the functional and molecular mechanism whereby cAMP regulates Ca2+ clearance pathways in parotid acinar cells. Following an agonist-induced increase in [Ca2+]i the rate of Ca2+clearance, after the removal of the stimulus, was potentiated substantially (∼2-fold) by treatment with forskolin. This effect was prevented completely by inhibition of the plasma membrane Ca2+-ATPase (PMCA) with La3+. PMCA activity, when isolated pharmacologically, was also potentiated (∼2-fold) by forskolin. Ca2+ uptake into the endoplasmic reticulum of streptolysin-O-permeabilized cells by sarco/endoplasmic reticulum Ca2+-ATPase was largely unaffected by treatment with dibutyryl cAMP. Finally, in situ phosphorylation assays demonstrated that PMCA was phosphorylated by treatment with forskolin but only in the presence of carbamylcholine (carbachol). This effect of forskolin was Ca2+-dependent, and protein kinase C-independent, as potentiation of PMCA activity and phosphorylation of PMCA by forskolin also occurred when [Ca2+]i was elevated by the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid and was attenuated by pre-incubation with the Ca2+ chelator, 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The present study demonstrates that elevated cAMP enhances the rate of Ca2+ clearance because of a complex modulation of PMCA activity that involves a Ca2+-dependent step. Tight regulation of both Ca2+ release and Ca2+ efflux may represent a general feature of the mechanism whereby cAMP improves the fidelity and specificity of Ca2+ signaling. Cross-talk between cAMP and [Ca2+]i signaling pathways represents a general feature that defines the specificity of stimulus-response coupling in a variety of cell types including parotid acinar cells. We have reported recently that cAMP potentiates Ca2+ release from intracellular stores, primarily because of a protein kinase A-mediated phosphorylation of type II inositol 1,4,5-trisphosphate receptors (Bruce, J. I. E., Shuttleworth, T. J. S., Giovannucci, D. R., and Yule, D. I. (2002) J. Biol. Chem. 277, 1340–1348). The aim of the present study was to evaluate the functional and molecular mechanism whereby cAMP regulates Ca2+ clearance pathways in parotid acinar cells. Following an agonist-induced increase in [Ca2+]i the rate of Ca2+clearance, after the removal of the stimulus, was potentiated substantially (∼2-fold) by treatment with forskolin. This effect was prevented completely by inhibition of the plasma membrane Ca2+-ATPase (PMCA) with La3+. PMCA activity, when isolated pharmacologically, was also potentiated (∼2-fold) by forskolin. Ca2+ uptake into the endoplasmic reticulum of streptolysin-O-permeabilized cells by sarco/endoplasmic reticulum Ca2+-ATPase was largely unaffected by treatment with dibutyryl cAMP. Finally, in situ phosphorylation assays demonstrated that PMCA was phosphorylated by treatment with forskolin but only in the presence of carbamylcholine (carbachol). This effect of forskolin was Ca2+-dependent, and protein kinase C-independent, as potentiation of PMCA activity and phosphorylation of PMCA by forskolin also occurred when [Ca2+]i was elevated by the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid and was attenuated by pre-incubation with the Ca2+ chelator, 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The present study demonstrates that elevated cAMP enhances the rate of Ca2+ clearance because of a complex modulation of PMCA activity that involves a Ca2+-dependent step. Tight regulation of both Ca2+ release and Ca2+ efflux may represent a general feature of the mechanism whereby cAMP improves the fidelity and specificity of Ca2+ signaling. Intracellular calcium is perhaps the most ubiquitous second messenger system in biology, yet the molecular mechanisms that confer specificity of Ca2+-dependent processes continue to intrigue investigators. Regulation of both the spatial and temporal properties of intracellular Ca2+ signals is believed to underlie the specificity of stimulus-response coupling in a variety of cell types (1Carafoli E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1115-1122Google Scholar, 2Brini M. Carafoli E. Cell. Mol. Life Sci. 2000; 57: 354-370Google Scholar, 3Bootman M.D. Collins T.J. Peppiatt C.M. Prothero L.S. MacKenzie L. De Smet P. Travers M. Tovey S.C. Seo J.T. Berridge M.J. Ciccolini F. Lipp P. Semin. Cell Dev. Biol. 2001; 12: 3-10Google Scholar, 4Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Google Scholar). Recently an accumulation of evidence suggests that specific regulatory control over a variety of Ca2+ signaling pathways can be achieved by the concomitant activation of additional signaling pathways, in particular those that elevate cyclic AMP (5Bruce J.I.E. Shuttleworth T.J.S. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Google Scholar, 6Giovannucci D.R. Groblewski G.E. Sneyd J. Yule D.I. J. Biol. Chem. 2000; 275: 33704-33711Google Scholar, 7Straub S.V. Giovannucci D.R. Bruce J.I. Yule D.I. J. Biol. Chem. 2002; 27: 31949-31956Google Scholar, 8Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Google Scholar). Parotid acinar cells represent an excellent model system not only for the study of Ca2+ signaling in general but also to investigate cross-talk between cAMP and Ca2+ signaling (2Brini M. Carafoli E. Cell. Mol. Life Sci. 2000; 57: 354-370Google Scholar). This is because there is an abundance of evidence showing that both acetylcholine-evoked fluid secretion and exocytosis are potentiated markedly by cAMP-raising pathways (9Baldys-Waligorska A. Pour A. Moriarty C.M. Dowd F. Biochim. Biophys. Acta. 1987; 929: 190-196Google Scholar, 10Bobyock E. Chernick W.S. J. Dent. Res. 1989; 68: 1489-1494Google Scholar, 11Larsson O. 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In addition, we have demonstrated recently in parotid acinar cells that raising cAMP potentiates Ca2+ release from intracellular stores, primarily because of a PKA 1The abbreviations used are: PKA, protein kinase A; CCh, carbamylcholine (carbachol); PMCA, plasma membrane Ca2+-ATPase; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; PMA, phorbol 12-myristate 13-acetate; Bt2cAMP, dibutyryl cAMP; SL-O, streptolysin-O; CPA, cyclopiazonic acid; CaM, calmodulin; HEDTA, N-(2-hydroxyethyl)ethylenediaminetriacetic acid; FCCP, carbonyl cyanide 4-trifluoro-methoxyphenylhydrazone; BAPTA-AM, 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid; ER, endoplasmic reticulum; CPA, cyclopiazonic acid; CaMK, CaM kinase. 1The abbreviations used are: PKA, protein kinase A; CCh, carbamylcholine (carbachol); PMCA, plasma membrane Ca2+-ATPase; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; PMA, phorbol 12-myristate 13-acetate; Bt2cAMP, dibutyryl cAMP; SL-O, streptolysin-O; CPA, cyclopiazonic acid; CaM, calmodulin; HEDTA, N-(2-hydroxyethyl)ethylenediaminetriacetic acid; FCCP, carbonyl cyanide 4-trifluoro-methoxyphenylhydrazone; BAPTA-AM, 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid; ER, endoplasmic reticulum; CPA, cyclopiazonic acid; CaMK, CaM kinase.-mediated phosphorylation of type II inositol 1,4,5-trisphosphate receptors (5Bruce J.I.E. Shuttleworth T.J.S. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Google Scholar). This was hypothesized to be the major mechanism for the potentiation of fluid secretion by cAMP-raising agonists in the parotid. In addition to potentiation of Ca2+ release, it was also observed that the rate of Ca2+ clearance upon removal of CCh was potentiated substantially in the presence of forskolin. This may represent an important additional mechanism by which cAMP tightly controls the spatial and temporal properties of Ca2+signaling in parotid acinar cells. The aim of the present study was to systematically determine the molecular mechanisms responsible for the potentiation of Ca2+ clearance by cAMP in mouse parotid acinar cells. The study revealed a novel, complex mechanism by which cAMP can modulate Ca2+ signaling by potentiating PMCA activity in a Ca2+-dependent manner. Because the PMCA has been suggested to respond to dynamic fluctuations in cytosolic [Ca2+] due to its CaM binding properties (14Foder B. Scharff O. Cell Calcium. 1992; 13: 581-591Google Scholar, 15Penniston J.T. Enyedi A. J. Membr. Biol. 1998; 165: 101-109Google Scholar), further regulation by cAMP may be important for the fine tuning of Ca2+ signaling. This regulatory control likely contributes to the general mechanism by which cAMP-elevating agonists shape Ca2+ signaling, a process that may have relevance to the regulation of a wide array of specific functions in various cell types. Clusters of parotid acinar cells were isolated by collagenase digestion as described previously (5Bruce J.I.E. Shuttleworth T.J.S. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Google Scholar). Following isolation, cells were re-suspended in a HEPES-buffered physiological saline solution containing the following (in mm): 5.5 glucose, 137 NaCl, 0.56 MgCl2, 4.7 KCl, 1 Na2HPO4, 10 HEPES (pH 7.4), 1.2 CaCl2. Aliquots of cell suspensions were loaded with 2 μm fura-2/AM for 30 min at room temperature after which they were re-suspended in HEPES-buffered physiological saline solution and kept at 4 °C until ready for use. During experiments, cells were allowed to adhere to a glass coverslip, which formed the base of a gravity-fed perfusion chamber. Cells were perfused continually with HEPES-buffered physiological saline solution, and automatic valves were used for switching solutions. [Ca2+] imaging experiments were performed using an inverted epifluorescence Nikon microscope with a ×40 oil immersion objective lens (numerical aperture, 1.3). Most experiments utilized an imaging system consisting of a DG-4 illumination system (Sutter), a 12-bit progressive interline charged coupled device camera (Sensicam), and Axon Imaging Workbench acquisition software as described previously in more detail (5Bruce J.I.E. Shuttleworth T.J.S. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Google Scholar). This imaging system was used for most standard experiments that required a relatively slow image acquisition rate of 0.1 to 1 Hz and 100- to 300-ms exposure times. For the rapid atropine-evoked Ca2+clearance experiments faster acquisition rates of 10 to 20 Hz and 10- to 20-ms exposure times were required; therefore, an alternative imaging system was used. This contained essentially the same optics and camera but used a TILL polychrome IV monochromator illumination system and TILL VisION acquisition and analysis software (see Ref. 6Giovannucci D.R. Groblewski G.E. Sneyd J. Yule D.I. J. Biol. Chem. 2000; 275: 33704-33711Google Scholar for detailed description). All experiments were performed at room temperature. Measurement of Ca2+ uptake into the ER was isolated by imaging streptolysin (SL-O)-permeabilized parotid acinar cells loaded with the low affinity Ca2+-sensing fluorescent dye, fura-2FF/AM (10 μm) for 60 min at 37 °C as described previously (5Bruce J.I.E. Shuttleworth T.J.S. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Google Scholar). Briefly, cells were perfused continually with a Chelex-100 "scrubbed" cytosol-like medium containing the following (in mm): 135 KCl, 1.2 KH2PO4, 0.5 EGTA, 0.5N-hydroxyethylethylenediaminetriacetic acid (HEDTA), 0.5 nitriloacetic acid, and 20 HEPES/KOH (pH 7.1). MgCl2, CaCl2, and MgATP were added accordingly (as calculated by MaxChelator program) to give constant free Mg2+, Ca2+, and ATP concentrations of 0.9 mm, 0.06–1.0 μm, and 1 mm, respectively. Permeabilization was achieved by perfusion with an ATP-containing (but Ca2+-free) cytosol-like medium containing 0.4 international unit of SL-O. Because fura-2FF accumulates in virtually every compartment of the cell, permeabilization could be verified by monitoring the decline in the 360-nm excitation signal (isosbestic point for fura-2FF) as the cytosolic dye leaked out of the cell leaving dye trapped in intracellular organelles (<20% of pre-permeabilized cells). Image acquisition rate was 0.1 Hz (100-ms exposure). Measurement of organelle [Ca2+] was achieved similarly to fura-2 (340- and 380-nm excitation and 510-nm emission) with an image acquisition rate of 0.1 Hz and 300-ms exposure, and fura-2FF 340/380 ratio images were calculated online. Following perfusion of cells with cytosol-like medium devoid of SL-O, Ca2+, or ATP for 5–10 min, rapid Ca2+ uptake was achieved upon addition of 1 mm Mg-ATP and [Ca2+] between 0.06 and 1.0 μm. The fura-2FF 340/380 ratio, representing organelle [Ca2+], reached a maximum within 3 min presumably because of an equilibrium being established between opposing Ca2+fluxes across the organelle membrane (steady state). Subsequent addition of 3 μm inositol 1,4,5-trisphosphate, 30 μm cyclopiazonic acid (CPA), and 1 μm FCCP evoked a rapid decrease, slow decrease, or no change in fura-2FF ratio, respectively, confirming that Ca2+ was taken up into the ER. The rate of Ca2+ uptake into the ER for each cell at each ambient [Ca2+] was fit to a single exponential decay to yield the time constant (τ). The mean time constant was compared in the absence and presence of 100 μm dibutyryl cyclic AMP (Bt2cAMP). Parotid acinar cells were isolated from 4–6 mice as described above, aliquotted appropriately, and treated for 10 min with or without the following test reagents: 1 μm CCh, 10 μm forskolin, 150 nmphorbol 12-myristate 13-acetate (PMA) or 30 μm CPA. In some experiments, cells were pre-incubated with 20 μmBAPTA/AM for 30 min at room temperature to buffer any change in [Ca2+]i. Cells were then pelleted rapidly by centrifugation and re-suspended in 400 μl of ice-cold lysis buffer containing the following (in mm): 50 Tris-HCl (pH 7.4), 250 NaCl, 5 EDTA, 100 NaF, 0.1% Triton X-100, and EDTA-free complete protease inhibitor mixture tablets (Roche Molecular Biochemicals). Cell lysates were then sonicated, left on ice for 30 min, and vortexed every 5 min. Each lysate was incubated with the monoclonal anti-PMCA antibody (∼1 μg/mg protein; clone 5F10, Affinity Bioreagents) for 1 h, followed by 80 μl of protein A-agarose beads (Pierce) for another hour at 4 °C, to immunoprecipitate PMCA protein. As a secondary control (blank) an aliquot of cell lysates from untreated cells was incubated with beads without any antibodies. As described previously (5Bruce J.I.E. Shuttleworth T.J.S. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Google Scholar) the beads-protein complex was washed five times in lysis buffer and denatured by boiling in SDS sample buffer (Laemmli) for 5 min. Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis (7.5%) and Western blotted using the phospho-(Ser/Thr) PKA substrate antibody (Cell Signaling Technology) to detect phosphorylated PMCA protein. To confirm that approximately equal amounts of protein were loaded into each lane of the gel or whether the above treatments altered PMCA levels, nitrocellulose membranes were incubated in stripping solution (62.5 Tris-HCl (pH 6.7), 2% SDS, and 100 mβ-mercaptoethanol) for 30 min at 50 °C to dissociate any bound antibodies. The membrane was then re-probed subsequently by Western blotting with the anti-PMCA antibody. In some experiments quantification of phosphorylation was achieved by densitometric analysis of visible bands detected by the phospho-(Ser/Thr) PKA substrate antibody. This was performed by imaging nitrocellulose membranes exposed to chemiluminescence reagents (Westpico/Westfemto Super Signal; Pierce) using a 12-bit charged coupled device camera (Sensicam) and LabWorks imaging and analysis software (UVP Bioimaging Systems). This measures the total pixel intensity above background of equal sized areas of interest for each visible band. Duplicates on each membrane were averaged, and to account for variability in band intensities between gels comparisons were made between treated and control conditions using a paired Student's t test. Because of the nature of most experiments an unpaired experimental design was applied (unless otherwise stated in the text), whereby statistical significance was determined between groups of experiments (control and treated) using an unpaired t test or Mann Whitney test. Occasionally, statistical significance was determined, where appropriate, using a paired t test, Wilcoxan test for pairs, or one samplet test. For Ca2+ clearance data the rate of Ca2+ clearance was fit to a single exponential decay using Microcal Origin 5.0 software and quantified by comparing time constants (τ). 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. We have shown previously that elevated cAMP potentiates CCh-evoked Ca2+ release (5Bruce J.I.E. Shuttleworth T.J.S. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Google Scholar). In the present study, under similar conditions, the rate of Ca2+ clearance was determined following removal of CCh by fitting the falling phase of the change in fura-2 340/380 ratio to a single exponential decay (Fig. 1B). Time constants (τ) were compared quantitatively in the absence and presence of 10 μm forskolin using a paired t test (Fig. 1C). These initial experiments revealed that forskolin caused a 5.3 ± 1.2-fold increase in Ca2+ clearance by reducing τ from 12.6 ± 2.0 to 2.9 ± 0.5 s (Fig. 1C; n = 7 experiments, 34 cells). In some cells the rate of decrease in fura-2 340/380 ratio following removal of CCh was initially slow, suggesting that the rate of solution exchange was contributing to the variability. Therefore to increase the accuracy and precision of the data, experiments were repeated by initiating clearance of Ca2+ from the cytosol by adding a 10-fold higher concentration of the muscarinic receptor antagonist, atropine (10 μm), in the continued presence of 1 μmCCh (Fig. 2). This reduces any error associated with solution exchange, because as soon as just 10% of the perfusate exchanges, the atropine will have essentially displaced all the CCh from the receptors. Because under these conditions atropine will remain bound for a long time, the effect of forskolin was compared with unpaired control experiments. Under these conditions forskolin increased Ca2+ clearance ∼2.5-fold by reducing τ from 8.27 ± 1.2 to 3.43 ± 0.52 s (n = 14 experiments, 57 cells; see Fig. 2). Collectively, these experiments demonstrate that elevating intracellular cAMP levels by activation of adenylyl cyclase, with forskolin, potentiates Ca2+clearance pathways.Figure 2Forskolin potentiates the clearance of [Ca2+]i following addition of atropine in the presence of CCh. A, clearance of [Ca2+]i was initiated by addition of 10 μm atropine in the presence of 1 μm CCh.B, to improve the precision and accuracy, data were acquired 10 times faster (10 Hz versus 1 Hz as in Fig. 1) during the period in the dashed box in A. C, similar to Fig. 1 clearance of [Ca2+]i was fit to a single exponential decay. The mean τ was compared between control and forskolin-treated cells using an unpaired t test (*, p < 0.05). Under these conditions forskolin caused an ∼2.5-fold increase in the rate of Ca2+ clearance (n = 14 experiments, 57 cells).View Large Image Figure ViewerDownload (PPT) The major routes for Ca2+ clearance in non-excitable cells, such as parotid acinar cells, are believed to be Ca2+uptake into the ER by the SERCA, Ca2+ efflux across the plasma membrane by the PMCA, and Ca2+ uptake into mitochondria (1Carafoli E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1115-1122Google Scholar, 3Bootman M.D. Collins T.J. Peppiatt C.M. Prothero L.S. MacKenzie L. De Smet P. Travers M. Tovey S.C. Seo J.T. Berridge M.J. Ciccolini F. Lipp P. Semin. Cell Dev. Biol. 2001; 12: 3-10Google Scholar, 4Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Google Scholar). Therefore, to determine the specific molecular loci for the effects of elevated cAMP, on Ca2+ clearance, the following experiments were designed to physically or pharmacologically isolate these different Ca2+ clearance pathways. The rate of Ca2+ clearance during each experimental paradigm varied markedly and thus may not accurately represent the actual rate of each isolated Ca2+ clearance pathway in vivo or under physiological Ca2+signaling conditions. Nevertheless, the relative rate of Ca2+ clearance following a particular treatment under each condition was always consistent, thereby verifying each experimental protocol. One of the major routes for Ca2+clearance following stimulation is believed to be the re-uptake of Ca2+ into the ER by SERCA (1Carafoli E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1115-1122Google Scholar, 3Bootman M.D. Collins T.J. Peppiatt C.M. Prothero L.S. MacKenzie L. De Smet P. Travers M. Tovey S.C. Seo J.T. Berridge M.J. Ciccolini F. Lipp P. Semin. Cell Dev. Biol. 2001; 12: 3-10Google Scholar, 4Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Google Scholar). In addition, SERCA activity is potentiated by elevated cAMP in cardiac cells because of PKA-mediated phosphorylation of the inhibitory accessory protein, phospholamban (16Tada M. Inui M. J. Mol. Cell. Cardiol. 1983; 15: 565-575Google Scholar, 17Tada M. Inui M. Yamada M. Kadoma M. Kuzuya T. Abe H. Kakiuchi S. J. Mol. Cell. Cardiol. 1983; 15: 335-346Google Scholar, 18James P. Inui M. Tada M. Chiesi M. Carafoli E. Nature. 1989; 342: 90-92Google Scholar, 19Brittsan A.G. Kranias E.G. J. Mol. Cell. Cardiol. 2000; 32: 2131-2139Google Scholar). Therefore, a likely locus for the effects of elevated cAMP on Ca2+ clearance in parotid acinar cells is Ca2+ uptake into the ER. To isolate Ca2+ uptake into the ER, parotid acinar cells were permeabilized with SL-O, and Ca2+ uptake was initiated by perfusion with a cytosol-like solution (see "Experimental Procedures") containing 1 mm ATP and varying concentrations of ambient Ca2+ (0.06–1.0 μm). Ca2+ uptake was indicated by an increase in the fura-2FF 340/380 ratio, which reached a steady state between 0.5 and 3.5 min depending on ambient [Ca2+] (Fig. 3A). Once Ca2+uptake reached a steady state, addition of more Ca2+ failed to increase the fura-2FF 340/380 ratio further (data not shown). Application of inositol 1,4,5-trisphosphate evoked a rapid Ca2+ release confirming that Ca2+ had been taken up into the ER by SERCA Ca2+ pumps (5Bruce J.I.E. Shuttleworth T.J.S. Giovannucci D.R. Yule D.I. J. Biol. Chem. 2002; 277: 1340-1348Google Scholar). Moreover, the SERCA inhibitor CPA and removal of ATP evoked a slow Ca2+leak, whereas the mitochondrial uncoupler FCCP failed to have any effect (data not shown). The rate of Ca2+ uptake was fit to a single exponential decay, and the mean time constant (τ) was determined for each ambient (loading) [Ca2+] in the absence or presence of Bt2-cAMP and compared using an unpaired t test (Fig. 3B). The rate of Ca2+ uptake into the ER increased by ∼4-fold as the ambient [Ca2+] increased from 60 to 200 nm (mean τ decreased from 28.7 ± 1.4 s for 60 nm, to 16.7 ± 2.0 s for 100 nm, and to 7.8 ± 0.8 s for 200 nm ambient [Ca2+]). The rate of Ca2+ uptake did not further increase significantly by increasing ambient [Ca2+] above 200 nm (mean τ = 7.3 ± 0.8 s for 600 nm and mean τ = 7.6 ± 1.5 s for 1 μm ambient [Ca2+];n = 3–6 experiments, 9–22 cells). This suggests that the rate of Ca2+ uptake into the ER increases as cytosolic [Ca2+] increases over the range of 60–200 nm[Ca2+] but is activated maximally above 200 nm [Ca2+]. In pancreatic acinar cells, under almost identical conditions the rate of Ca2+ uptake was shown to be much slower and increased over a much broader range of ambient [Ca2+] (20van de Put F.H. Elliott A.C. J. Biol. Chem. 1997; 272: 27764-27770Google Scholar). This suggests that SERCA is much more active and sensitive to small elevations of [Ca2+]i above resting levels in parotid than in pancreatic acinar cells. This is consistent with our previous data, which shows that the deactivation of Ca2+-dependent Cl− currents was greater in parotid compared with pancreatic acinar cells and that this was due, in part, to a greater SERCA activity (21Giovannucci D.R. Bruce J.I. Straub S.V. Arreola J. Sneyd J. Shuttleworth T.J. Yule D.I. J. Physiol. 2002; 540: 469-484Google Scholar). Whatever the specific mechanisms that control the Ca2+-dependent Ca2+ uptake into the ER, the most important observation from the present study was that at all the ambient [Ca2+] tested (100, 200, and 600 nm) the rate of Ca2+ uptake into the ER was not affected significantly by treatment with 100 μmBt2-cAMP (Fig. 3B). To further assess the effect of Bt2-cAMP on Ca2+ uptake into the ER of parotid acinar cells, a paired experimental design was utilized in the following way. Ca2+uptake was initiated in the absence of Bt2-cAMP by addition of ATP and 0.2 μm Ca2+. Upon reaching steady state, 100 μm Bt2-cAMP was added, and removal of Ca2+ and ATP caused a slow decline in fura-2FF 340/380 ratio, presumably because of depletion of the ER Ca2+ store (Fig. 3C). Upon reaching a new steady state (10 to 15 min) a second Ca2+ uptake was initiated in the continued presence of Bt2-cAMP (Fig. 3C). Using this paired experimental design the second Ca2+ uptake (mean τ = 9.4 ± 0.5 s) in the absence of Bt2-cAMP was ∼30% slower than the first Ca2+ uptake (mean τ = 7.2 ± 0.4 s) during time matched control experiments. Nevertheless, under the same conditions the change in rate of Ca2+ uptake following treatment with Bt2-cAMP (1.27 ± 0.03-fold increase in τ) was not significantly different from time-matched control experiments (1.32 ± 0.04-fold increase in τ; see Fig. 3D). These data therefore reinforce the initial observation that elevation of cAMP fails to enhance Ca2+ uptake into the ER of permeabilized parotid acinar cells. Despite rigorous attempts to isolate Ca2+uptake into the ER of SL-O-permeabilized parotid acinar cells it remains unlikely that the effects of elevated cAMP on Ca2+clearance are because of enhanced SERCA activity. However, a simple explanation for the lack of effect of Bt2-cAMP could be the permeabilization process itself, which may wash away critical cytosolic factors, such as calmodulin or phospholamban, important for SERCA pump activity (16Tada M. Inui M. J. Mol. Cell. Cardiol. 1983; 15: 565-575Google Scholar, 17Tada M. Inui M. Yamada M. Kadoma M. Kuzuya T. Abe H. Kakiuchi S. J. Mol. Cell. Cardiol. 1983; 15: 335-346Google Scholar, 18James P. Inui M. Tada M. Chiesi M. Carafoli E. Nature. 1989; 342: 90-92Google Scholar, 19Brittsan A.G. Kranias E.G. J. Mol. Cell. Cardiol. 2000; 32: 2131-2139Google Scholar). To address this further, the effects of elevated cAMP on Ca2+ clearance in intact cells was determined following inhibition of SERCA activity by CPA. Intact parotid acinar cells were stimulated with 30 μm CPA, which slowly raised [Ca2+]i primarily because of leak from the ER and activation of capacitative Ca2+ entry (22Mason M.J. Garcia-Rodriguez C. Grinstein S. J. Biol. Chem. 1991; 266: 20856-20862Google Scholar, 23Takemura H. Putney Jr., J.W. Biochem. J. 1989; 258: 409-412Google Scholar, 24Takemura H. Hughes A.R. Thastrup O. Putney Jr., J.W. J. Biol. Chem. 1989; 264: 12266-12271Google Scholar, 25Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Google Scholar). The increase in [Ca2+]i reached a maximum and then declined slowly to a new steady state, representing a balance of Ca2+ efflux and Ca2+ influx. Removal of external Ca2+, by chelation with 1 mm EGTA, evoked an immediate clearance of Ca2+ that was primarily because of the PMCA. Similar to previous experiments, the rate of Ca2+ clearance was fit to a single exponential decay (Fig. 4A), and the time constants (τ) were compared between control and forskolin-treated cells using an unpaired experimental design (Fig. 4B). Under these conditions forskolin caused an ∼2-fold increase in the rate of Ca2+ clearance, as indicated by a decrease in the mean τ from 28.2 ± 2.1 in control cells (n = 5 experiments; 27 cells) to 16.4 ± 1.9 (n = 5 experiments; 22 cells) in forskolin-treated cells (Fig. 4B). Although the major mechanism for Ca2+ clearance during dynamic Ca2+ signaling is believed to be because of SERCA, these data suggest that potentiation of Ca2+ clearance by elevated cAMP may be because of a pathway other than SERCA, most likely the PMCA. Because the potentiation of Ca2+ clearance by elevated cAMP appears independent of SERCA activity, another Ca2+ clearance pathway such as Ca2+ efflux across the plasma membrane (PMCA activity) may be the target for the effects of cAMP. To test this hypothesis, experiments similar to those described in Fig. 2 were carried out in the continued presence of 1 mm La3+, a known inhibitor of PMCA (26Furukawa K. Tawada Y. Shigekawa M. J. Biol. Chem. 1988;
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