Modulation of Pancreatic Acinar Cell to Cell Coupling during ACh-evoked Changes in Cytosolic Ca2+
1999; Elsevier BV; Volume: 274; Issue: 1 Linguagem: Inglês
10.1074/jbc.274.1.282
ISSN1083-351X
AutoresMarc Chanson, Patrice Mollard, Paolo Meda, Susanne Suter, Habo J. Jongsma,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoThe temporal changes in cytosolic free Ca2+ ([Ca2+]i), Ca2+-dependent membrane currents (Im), and gap junctional current (Ij) elicited by acetylcholine (ACh) were measured in rat pancreatic acinar cells using digital imaging and dual perforated patch-clamp recording. ACh (50 nm-5 μm) increased [Ca2+]i and evokedIm currents without alteringIj in 19 of 37 acinar cell pairs. Although [Ca2+]i rose asynchronously in cells comprising a cluster, the delay of the [Ca2+]i responses decreased with increasing ACh concentrations. Perfusion of inositol 1,4,5-trisphosphate (IP3) into one cell of a cluster resulted in [Ca2+]i responses in neighboring cells that were not necessarily in direct contact with the stimulated one. This suggests that extensive coupling between acinar cells provides a pathway for cell-to-cell diffusion of Ca2+-releasing signals. Strikingly, maximal (1–5 μm) ACh concentrations reduced Ijby 69 ± 15% (n = 9) in 25% of the cell pairs subjected to dual patch-clamping. This decrease occurred shortly after the Im peak and was prevented by incubating acinar cells in a Ca2+-free medium, suggesting that uncoupling was subsequent to the initiation of the Ca2+-mobilizing responses. Depletion of Ca2+-sequestering stores by thapsigargin resulted in a reduction of intercellular communication similar to that observed with ACh. In addition, ACh-induced uncoupling was prevented by blocking nitric oxide production with l-nitro-arginine and restored by exposing acinar cells to dibutyryl cGMP. The results suggest that ACh-induced uncoupling and capacitative Ca2+ entry are regulated concurrently. Closure of gap junction channels may occur to functionally isolate nearby cells differing in their intrinsic sensitivity to ACh and thereby to allow for sustained activity of groups of secreting cells. The temporal changes in cytosolic free Ca2+ ([Ca2+]i), Ca2+-dependent membrane currents (Im), and gap junctional current (Ij) elicited by acetylcholine (ACh) were measured in rat pancreatic acinar cells using digital imaging and dual perforated patch-clamp recording. ACh (50 nm-5 μm) increased [Ca2+]i and evokedIm currents without alteringIj in 19 of 37 acinar cell pairs. Although [Ca2+]i rose asynchronously in cells comprising a cluster, the delay of the [Ca2+]i responses decreased with increasing ACh concentrations. Perfusion of inositol 1,4,5-trisphosphate (IP3) into one cell of a cluster resulted in [Ca2+]i responses in neighboring cells that were not necessarily in direct contact with the stimulated one. This suggests that extensive coupling between acinar cells provides a pathway for cell-to-cell diffusion of Ca2+-releasing signals. Strikingly, maximal (1–5 μm) ACh concentrations reduced Ijby 69 ± 15% (n = 9) in 25% of the cell pairs subjected to dual patch-clamping. This decrease occurred shortly after the Im peak and was prevented by incubating acinar cells in a Ca2+-free medium, suggesting that uncoupling was subsequent to the initiation of the Ca2+-mobilizing responses. Depletion of Ca2+-sequestering stores by thapsigargin resulted in a reduction of intercellular communication similar to that observed with ACh. In addition, ACh-induced uncoupling was prevented by blocking nitric oxide production with l-nitro-arginine and restored by exposing acinar cells to dibutyryl cGMP. The results suggest that ACh-induced uncoupling and capacitative Ca2+ entry are regulated concurrently. Closure of gap junction channels may occur to functionally isolate nearby cells differing in their intrinsic sensitivity to ACh and thereby to allow for sustained activity of groups of secreting cells. connexin acetylcholine intracellular free calcium concentration Krebs-Ringer bicarbonate fluo-3-acetoxymethyl ester dimethyl sulfoxide inositol 1,4,5-trisphosphate nitric oxide ohm. Gap junctions are intercellular channels formed by twelve subunits of membrane proteins called connexins (Cx).1 Six subunits are contributed by each cell to form hemichannels, the docking of which provides a low resistance pathway for exchange of ions and small molecules between cells in contact. Gap junctional coupling was shown to be involved in the control of embryonic development, cell proliferation, electrical conduction, and metabolic cooperation (1Bennett M.V.L. Barrio L.C Bargiello T.A. Spray D.C. Hertzberg E. Sàez J.C. Neuron. 1991; 6: 305-320Abstract Full Text PDF PubMed Scopus (860) Google Scholar, 2Kumar N.M. Gilula N.B. Cell. 1996; 84: 381-388Abstract Full Text Full Text PDF PubMed Scopus (1630) Google Scholar, 3Goodenough D.A. Goliger J.A. Paul D.L. Annu. Rev. Biochem. 1996; 65: 475-502Crossref PubMed Scopus (1074) Google Scholar). Because gap junction channels allow for the potential passage of molecules of a molecular mass up to 1000 Da, it is conceivable that second messengers produced in one cell can diffuse between neighboring cells to coordinate their individual response. In support of this hypothesis, the passage of Ca2+ waves has been reported in epithelial cells, glial cells, and various cultured cells (4Sanderson M.J. News Physiol. Sci. 1996; 11: 262-269Google Scholar).The exocrine pancreas represents a valuable model to search for the role of gap junctional coupling in signal transduction of nonexcitable tissues. Acinar cells are extensively electrically and chemically coupled by Cx32- and Cx26-built gap junction channels (5Meda P. Bruzzone R. Chanson M. Bosco D. Hertzberg E.L. Johnson R.G. Modern Cell Biology. 7. Alan R. Liss, Inc., New York1988: 353-364Google Scholar). A major group of secretagogues in these cells are the Ca2+-mobilizing agonists, including cholecystokinin and acetylcholine (ACh). In the highly polarized acinar cells, cytosolic Ca2+ ([Ca2+]i) initially rises within the apical secretory region and, when stimulation is sufficient, subsequently spreads as a wave toward the basal pole of the cell (6Kasai H. Agustine G.J. Nature. 1990; 348: 735-738Crossref PubMed Scopus (313) Google Scholar, 7Nathanson M.H. Padfield P.J. O'Sullivan A.J. Burghstahler A.D. Jamieson J.D. J. Biol. Chem. 1992; 267: 18118-18121Abstract Full Text PDF PubMed Google Scholar, 8Toescu E.C. Lawrie A.M. Petersen O.H. Gallacher D.V. EMBO J. 1992; 11: 1623-1629Crossref PubMed Scopus (124) Google Scholar, 9Kasai H. Li Y.X. Miyashita Y. Cell. 1993; 74: 669-677Abstract Full Text PDF PubMed Scopus (316) Google Scholar). Intercellular propagation of Ca2+ oscillations and/or Ca2+ waves elicited by Ca2+-mobilizing secretagogues has been reported to correlate with gap junctional activity (7Nathanson M.H. Padfield P.J. O'Sullivan A.J. Burghstahler A.D. Jamieson J.D. J. Biol. Chem. 1992; 267: 18118-18121Abstract Full Text PDF PubMed Google Scholar, 10Petersen C.C.H. Petersen O.H. FEBS Lett. 1991; 284: 113-116Crossref PubMed Scopus (18) Google Scholar, 11Stauffer P.L. Zhao H. Luby-Phelps K. Moss R.L. Star R.A. Muallem S. J. Biol. Chem. 1993; 268: 19769-19775Abstract Full Text PDF PubMed Google Scholar, 12Ngezahayo A. Kolb H.-A. Pfluegers Arch. 1993; 422: 413-415Crossref PubMed Scopus (22) Google Scholar, 13Yule D.I. Stuenkel E. Williams J.A. Am. J. Physiol. 1996; 271: C1285-C1294Crossref PubMed Google Scholar). Increasing evidence indicates that open gap junctions coordinate the frequency of Ca2+ oscillations within individual cells of a same acinus which, in turn, regulates enzyme secretion. This hypothesis, however, is in apparent contradiction with the observation that these secretagogues also evoke acinar cell uncoupling, both in vitro and in vivo, at concentrations that maximally stimulate enzyme secretion (14Petersen O.H. Ueda N. J. Physiol. (Lond.). 1975; 247: 461-471Crossref Scopus (17) Google Scholar, 15Iwatsuki N. Petersen O.H. J. Physiol. (Lond.). 1978; 274: 81-96Crossref Scopus (46) Google Scholar, 16Meda P. Bruzzone R. Chanson M. Bosco D. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4901-4904Crossref PubMed Scopus (52) Google Scholar, 17Chanson M. Orci L. Meda P. Am. J. Physiol. 1991; 261: G28-G36PubMed Google Scholar). Thus, the role of gap junctional coupling during acute stimulation of pancreatic acinar cells remains tantalizing.One of the first questions to address is whether rises in [Ca2+]i and changes in junctional coupling evoked by acinar cell stimulation are parallel events. The application of the patch-clamp technique to dissociated acinar cells has revealed that the kinetic of Ca2+-dependent membrane current activation reflects that of the [Ca2+]i changes (18Osipchuk Y.V. Wakui M. Yule D.I. Gallacher D.V. Petersen O.H. EMBO J. 1990; 9: 697-704Crossref PubMed Scopus (218) Google Scholar, 19Maruyama Y. Inooka G. Li Y.X. Miyashita Y. Kasai H. EMBO J. 1993; 8: 3017-3022Crossref Scopus (96) Google Scholar, 20Thorn P. Lawrie A.M. Smith P. Gallacher D.V. Petersen O.H. Cell. 1993; 74: 661-668Abstract Full Text PDF PubMed Scopus (425) Google Scholar). However, accurate monitoring of junctional conductance under dual whole-cell recording conditions has been limited because spontaneous uncoupling occurs within seconds, presumably as a result of cytoplasm dialysis (21Neyton J. Trautmann A. Nature. 1985; 317: 331-335Crossref PubMed Scopus (242) Google Scholar, 22Chanson M. Bruzzone R. Spray D.C. Regazzi R. Meda P. Am. J. Physiol. 1988; 255: C699-C704Crossref PubMed Google Scholar, 23Somogyi R. Kolb H.-A. Pfluegers Arch. 1988; 412: 54-65Crossref PubMed Scopus (42) Google Scholar). To bypass this problem, we applied here a dual perforated patch-clamp approach, which preserves the integrity of the internal milieu (24Horn R. Marty A. J. Gen. Physiol. 1988; 92: 145-159Crossref PubMed Scopus (1454) Google Scholar, 25Takens-Kwak B.R. Jongsma H.J. Pfluegers Arch. 1992; 422: 198-200Crossref PubMed Scopus (87) Google Scholar), to monitor pairs of acinar cells stimulated with increasing ACh concentrations for both Ca2+-dependent membrane and gap junctional currents. In parallel experiments, changes in [Ca2+]i were monitored by digital imaging of fluo-3-loaded acinar cells. Under these conditions, we observed that ACh-induced closure of gap junction channels parallels the phase plateau of the [Ca2+]i response, but not the initial peak. We further show that acinar cell uncoupling requires the presence of extracellular Ca2+ and parallels capacitative Ca2+ entry.DISCUSSIONOur results describe the temporal relationship between changes in [Ca2+]i and gap junctional conductance within pairs of pancreatic acinar cells exposed to ACh. Uncoupling of pancreatic acinar cells by concentrations of Ca2+-mobilizing secretagogues that maximally stimulate exocrine secretion is well documented (14Petersen O.H. Ueda N. J. Physiol. (Lond.). 1975; 247: 461-471Crossref Scopus (17) Google Scholar, 15Iwatsuki N. Petersen O.H. J. Physiol. (Lond.). 1978; 274: 81-96Crossref Scopus (46) Google Scholar, 16Meda P. Bruzzone R. Chanson M. Bosco D. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4901-4904Crossref PubMed Scopus (52) Google Scholar, 17Chanson M. Orci L. Meda P. Am. J. Physiol. 1991; 261: G28-G36PubMed Google Scholar). However, the intracellular mechanism that mediates this effect is not known (38Chanson M. Meda P. Hall J.E. Zampighi G.A. Davis R.M. Progress in Cell Research. 3. Elsevier Science Publishers B.V., Amsterdam1993: 199-205Google Scholar). Using a dual perforated patch-clamp approach and confocal digital imaging, we report here that ACh-induced uncoupling and [Ca2+]i changes had distinct kinetics. Thus, decrease in junctional conductance of acinar cells consistently develops after the initial [Ca2+]i peak and is maximal during the [Ca2+]i plateau. Furthermore, ACh-induced uncoupling was no longer detected when reloading of internal Ca2+ stores was prevented by incubation of the cells in a Ca2+-free medium. These results suggest that depletion-activated Ca2+ entry was a key determinant for the ACh-induced uncoupling of acinar cells.In many nonexcitable cells, depletion of intracellular Ca2+stores by IP3 is the primary mechanism by which cell surface receptors activate Ca2+ influx. This phenomenon, which is termed capacitative Ca2+ entry (33Putney J.W. Bird G. St J. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (392) Google Scholar), has been involved in the control of Ca2+ oscillations (32Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar, 39Tsien R.W. Tsien R.Y. Annu. Rev. Cell Biol. 1990; 6: 715-760Crossref PubMed Scopus (1011) Google Scholar), secretion (40Parekh A.B. Penner R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7907-7911Crossref PubMed Scopus (153) Google Scholar), and enzymatic regulation (41Chiono M. Mahey R. Tate G. Cooper D.M. J. Biol. Chem. 1995; 270: 1149-1155Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The signal that couples store depletion to Ca2+ entry has not yet been identified (33Putney J.W. Bird G. St J. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (392) Google Scholar). In pancreatic acinar cells, however, there is definite evidence that NO produced by nitric-oxide synthase mediates the stimulation of cGMP formation by cholinergic agonists. cGMP, in turn, is known to modulate Ca2+ entry (29Xu X. Star R.A. Tortorici G. Muallem S. J. Biol. Chem. 1994; 269: 12645-12653Abstract Full Text PDF PubMed Google Scholar, 36Bahnson T.D. Pandol S.J. Dionne V.E. J. Biol. Chem. 1993; 268: 10808-10812Abstract Full Text PDF PubMed Google Scholar, 37Gukovskaya A. Pandol S. Am. J. Physiol. 1994; 266: G350-G356PubMed Google Scholar, 42Pandol S.J. Schoeffield-Payne M.S. J. Biol. Chem. 1990; 265: 12846-12853Abstract Full Text PDF PubMed Google Scholar, 43Xu X. Kitamura K. Lau K.S. Muallem S. Miller R.T. J. Biol. Chem. 1995; 270: 29169-29175Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Consistent with these data, we observed that activation of capacitative Ca2+ entry by either depletion of internal Ca2+stores with thapsigargin or by exposure of acinar cells to dibutyryl cGMP decreased intercellular communication to an extent similar to that observed with ACh alone. Side effects of these agents on intercellular communication appear unlikely because uncoupling was abolished in acini that were depleted for their internal Ca2+ and incubated in a Ca2+-free medium. In addition, cell uncoupling evoked by ACh was prevented in the presence of a nitric-oxide synthase inhibitor, supporting the view that gap junctional conductance and capacitative Ca2+ entry are concurrently regulated. These results, however, do not rule out the possibility that capacitative Ca2+ entry may activate another intracellular pathway leading to modulation of junctional conductance. Support for this idea is provided by the earlier observation that okadaic acid, a phosphatase inhibitor that modulates capacitative Ca2+ entry (44Berlin R.D. Preston S.F. Cell Calcium. 1993; 14: 379-386Crossref PubMed Scopus (34) Google Scholar, 45Parekh A.B. Terlau H. Stühmer W. Nature. 1993; 364: 814-818Crossref PubMed Scopus (319) Google Scholar), also prevents ACh-induced uncoupling (38Chanson M. Meda P. Hall J.E. Zampighi G.A. Davis R.M. Progress in Cell Research. 3. Elsevier Science Publishers B.V., Amsterdam1993: 199-205Google Scholar). Although several studies have shown that gap junction channels are blocked by cGMP (25Takens-Kwak B.R. Jongsma H.J. Pfluegers Arch. 1992; 422: 198-200Crossref PubMed Scopus (87) Google Scholar) and nitric oxide (46Lu C. McMahon D.G. J. Physiol. (Lond.). 1997; 499: 689-699Crossref Scopus (73) Google Scholar), our data provide the first observation that ACh-induced uncoupling is linked to capacitative Ca2+entry.Previous in vitro and in vivo studies have documented a relationship between intercellular communication and the secretory activity of pancreatic acinar cells (17Chanson M. Orci L. Meda P. Am. J. Physiol. 1991; 261: G28-G36PubMed Google Scholar, 30Meda P. Bruzzone R. Knodel S. Orci L. J. Cell Biol. 1986; 103: 475-483Crossref PubMed Scopus (61) Google Scholar, 47Chanson M. Fanjul M. Bosco D. Nelles E. Suter S. Willecke K. Meda P. J. Cell Biol. 1998; 141: 1267-1275Crossref PubMed Scopus (63) Google Scholar). In this context, gap junctional coupling is thought to coordinate the Ca2+ response of individual acinar cells within an acinus and thereby to regulate exocytosis (11Stauffer P.L. Zhao H. Luby-Phelps K. Moss R.L. Star R.A. Muallem S. J. Biol. Chem. 1993; 268: 19769-19775Abstract Full Text PDF PubMed Google Scholar, 48Bosco D. Soriano J.V. Chanson M. Meda P. J. Cell. Physiol. 1994; 160: 378-388Crossref PubMed Scopus (17) Google Scholar). This idea is supported by our present finding that the large and stable junctional conductance observed in most acinar cell pairs was not altered during changes in [Ca2+]i evoked by ACh. In agreement with a previous study using cholecystokinin (13Yule D.I. Stuenkel E. Williams J.A. Am. J. Physiol. 1996; 271: C1285-C1294Crossref PubMed Google Scholar), we observed that rises in [Ca2+]i were asynchronous in acinar cells stimulated with low concentrations of ACh. Increasing the agonist concentration was associated with shortening of the delay between the onset of the Ca2+ responses, suggesting that these cells were coupled in terms of Ca2+mobilization. Also, perfusion of IP3 into one cell evoked a rise in [Ca2+]i in neighboring cells even though not necessarily in those that directly contacted the stimulated one. This observation suggests that rat pancreatic acinar cells differ in their ability to mobilize Ca2+ from internal stores, as indicated by previous studies reporting similar heterogeneity in [Ca2+]imobilization (31Willems P.H.G.M. Van Emst-De Vries S.E. Van Os C.H. De Pont J.J.H.H.M. Cell Calcium. 1993; 14: 145-159Crossref PubMed Scopus (46) Google Scholar) and amylase secretion (48Bosco D. Soriano J.V. Chanson M. Meda P. J. Cell. Physiol. 1994; 160: 378-388Crossref PubMed Scopus (17) Google Scholar, 49Bosco D. Chanson M. Bruzzone R. Meda P. Am. J. Physiol. 1988; 254: G664-G670PubMed Google Scholar). This differential responsiveness may be essential to provide a properly modulable response to agonist-specific stimulation (11Stauffer P.L. Zhao H. Luby-Phelps K. Moss R.L. Star R.A. Muallem S. J. Biol. Chem. 1993; 268: 19769-19775Abstract Full Text PDF PubMed Google Scholar, 50Yule D.I. Lawrie A.M. Petersen O.H. Cell Calcium. 1991; 12: 145-151Crossref PubMed Scopus (97) Google Scholar).These results, however, are not immediately reconcilable with the observation that ACh decreases gap junctional coupling while maximally stimulating the secretory activity of acinar cells (30Meda P. Bruzzone R. Knodel S. Orci L. J. Cell Biol. 1986; 103: 475-483Crossref PubMed Scopus (61) Google Scholar, 47Chanson M. Fanjul M. Bosco D. Nelles E. Suter S. Willecke K. Meda P. J. Cell Biol. 1998; 141: 1267-1275Crossref PubMed Scopus (63) Google Scholar, 48Bosco D. Soriano J.V. Chanson M. Meda P. J. Cell. Physiol. 1994; 160: 378-388Crossref PubMed Scopus (17) Google Scholar). The blockade of acinar cell-to-cell communication is known to enhance the basal release of amylase in vitro and in vivo(16Meda P. Bruzzone R. Chanson M. Bosco D. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4901-4904Crossref PubMed Scopus (52) Google Scholar, 26Chanson M. Bruzzone R. Bosco D. Meda P. J. Cell. Physiol. 1989; 139: 147-156Crossref PubMed Scopus (52) Google Scholar, 30Meda P. Bruzzone R. Knodel S. Orci L. J. Cell Biol. 1986; 103: 475-483Crossref PubMed Scopus (61) Google Scholar, 47Chanson M. Fanjul M. Bosco D. Nelles E. Suter S. Willecke K. Meda P. J. Cell Biol. 1998; 141: 1267-1275Crossref PubMed Scopus (63) Google Scholar). Under conditions of gap junction blockade, the potency of several agonists in stimulating exocytosis has also been found to be reduced when the effect of acinar cell uncoupling on basal secretion was taken into account (11Stauffer P.L. Zhao H. Luby-Phelps K. Moss R.L. Star R.A. Muallem S. J. Biol. Chem. 1993; 268: 19769-19775Abstract Full Text PDF PubMed Google Scholar, 16Meda P. Bruzzone R. Chanson M. Bosco D. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4901-4904Crossref PubMed Scopus (52) Google Scholar, 30Meda P. Bruzzone R. Knodel S. Orci L. J. Cell Biol. 1986; 103: 475-483Crossref PubMed Scopus (61) Google Scholar). Therefore, uncoupling may provide acinar cells with a mechanism to sustain enzyme release during acute stimulation by increasing their rate of basal secretion. In this context, the delayed uncoupling evoked by ACh may compartmentalize cells that are highly sensitive to ACh from cells that are less sensitive to the secretagogue, therefore decreasing the effective volume of cytoplasm of interconnected cells. This regulation may be essential to ensure that the intracellular levels of critical factor(s) are maintained to allow for sustained activity of groups of actively secreting cells. Future studies should determine whether uncoupling induced by Ca2+ store depletion is a common mechanism to control junctional communication in other types of nonexcitable cells. Gap junctions are intercellular channels formed by twelve subunits of membrane proteins called connexins (Cx).1 Six subunits are contributed by each cell to form hemichannels, the docking of which provides a low resistance pathway for exchange of ions and small molecules between cells in contact. Gap junctional coupling was shown to be involved in the control of embryonic development, cell proliferation, electrical conduction, and metabolic cooperation (1Bennett M.V.L. Barrio L.C Bargiello T.A. Spray D.C. Hertzberg E. Sàez J.C. Neuron. 1991; 6: 305-320Abstract Full Text PDF PubMed Scopus (860) Google Scholar, 2Kumar N.M. Gilula N.B. Cell. 1996; 84: 381-388Abstract Full Text Full Text PDF PubMed Scopus (1630) Google Scholar, 3Goodenough D.A. Goliger J.A. Paul D.L. Annu. Rev. Biochem. 1996; 65: 475-502Crossref PubMed Scopus (1074) Google Scholar). Because gap junction channels allow for the potential passage of molecules of a molecular mass up to 1000 Da, it is conceivable that second messengers produced in one cell can diffuse between neighboring cells to coordinate their individual response. In support of this hypothesis, the passage of Ca2+ waves has been reported in epithelial cells, glial cells, and various cultured cells (4Sanderson M.J. News Physiol. Sci. 1996; 11: 262-269Google Scholar). The exocrine pancreas represents a valuable model to search for the role of gap junctional coupling in signal transduction of nonexcitable tissues. Acinar cells are extensively electrically and chemically coupled by Cx32- and Cx26-built gap junction channels (5Meda P. Bruzzone R. Chanson M. Bosco D. Hertzberg E.L. Johnson R.G. Modern Cell Biology. 7. Alan R. Liss, Inc., New York1988: 353-364Google Scholar). A major group of secretagogues in these cells are the Ca2+-mobilizing agonists, including cholecystokinin and acetylcholine (ACh). In the highly polarized acinar cells, cytosolic Ca2+ ([Ca2+]i) initially rises within the apical secretory region and, when stimulation is sufficient, subsequently spreads as a wave toward the basal pole of the cell (6Kasai H. Agustine G.J. Nature. 1990; 348: 735-738Crossref PubMed Scopus (313) Google Scholar, 7Nathanson M.H. Padfield P.J. O'Sullivan A.J. Burghstahler A.D. Jamieson J.D. J. Biol. Chem. 1992; 267: 18118-18121Abstract Full Text PDF PubMed Google Scholar, 8Toescu E.C. Lawrie A.M. Petersen O.H. Gallacher D.V. EMBO J. 1992; 11: 1623-1629Crossref PubMed Scopus (124) Google Scholar, 9Kasai H. Li Y.X. Miyashita Y. Cell. 1993; 74: 669-677Abstract Full Text PDF PubMed Scopus (316) Google Scholar). Intercellular propagation of Ca2+ oscillations and/or Ca2+ waves elicited by Ca2+-mobilizing secretagogues has been reported to correlate with gap junctional activity (7Nathanson M.H. Padfield P.J. O'Sullivan A.J. Burghstahler A.D. Jamieson J.D. J. Biol. Chem. 1992; 267: 18118-18121Abstract Full Text PDF PubMed Google Scholar, 10Petersen C.C.H. Petersen O.H. FEBS Lett. 1991; 284: 113-116Crossref PubMed Scopus (18) Google Scholar, 11Stauffer P.L. Zhao H. Luby-Phelps K. Moss R.L. Star R.A. Muallem S. J. Biol. Chem. 1993; 268: 19769-19775Abstract Full Text PDF PubMed Google Scholar, 12Ngezahayo A. Kolb H.-A. Pfluegers Arch. 1993; 422: 413-415Crossref PubMed Scopus (22) Google Scholar, 13Yule D.I. Stuenkel E. Williams J.A. Am. J. Physiol. 1996; 271: C1285-C1294Crossref PubMed Google Scholar). Increasing evidence indicates that open gap junctions coordinate the frequency of Ca2+ oscillations within individual cells of a same acinus which, in turn, regulates enzyme secretion. This hypothesis, however, is in apparent contradiction with the observation that these secretagogues also evoke acinar cell uncoupling, both in vitro and in vivo, at concentrations that maximally stimulate enzyme secretion (14Petersen O.H. Ueda N. J. Physiol. (Lond.). 1975; 247: 461-471Crossref Scopus (17) Google Scholar, 15Iwatsuki N. Petersen O.H. J. Physiol. (Lond.). 1978; 274: 81-96Crossref Scopus (46) Google Scholar, 16Meda P. Bruzzone R. Chanson M. Bosco D. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4901-4904Crossref PubMed Scopus (52) Google Scholar, 17Chanson M. Orci L. Meda P. Am. J. Physiol. 1991; 261: G28-G36PubMed Google Scholar). Thus, the role of gap junctional coupling during acute stimulation of pancreatic acinar cells remains tantalizing. One of the first questions to address is whether rises in [Ca2+]i and changes in junctional coupling evoked by acinar cell stimulation are parallel events. The application of the patch-clamp technique to dissociated acinar cells has revealed that the kinetic of Ca2+-dependent membrane current activation reflects that of the [Ca2+]i changes (18Osipchuk Y.V. Wakui M. Yule D.I. Gallacher D.V. Petersen O.H. EMBO J. 1990; 9: 697-704Crossref PubMed Scopus (218) Google Scholar, 19Maruyama Y. Inooka G. Li Y.X. Miyashita Y. Kasai H. EMBO J. 1993; 8: 3017-3022Crossref Scopus (96) Google Scholar, 20Thorn P. Lawrie A.M. Smith P. Gallacher D.V. Petersen O.H. Cell. 1993; 74: 661-668Abstract Full Text PDF PubMed Scopus (425) Google Scholar). However, accurate monitoring of junctional conductance under dual whole-cell recording conditions has been limited because spontaneous uncoupling occurs within seconds, presumably as a result of cytoplasm dialysis (21Neyton J. Trautmann A. Nature. 1985; 317: 331-335Crossref PubMed Scopus (242) Google Scholar, 22Chanson M. Bruzzone R. Spray D.C. Regazzi R. Meda P. Am. J. Physiol. 1988; 255: C699-C704Crossref PubMed Google Scholar, 23Somogyi R. Kolb H.-A. Pfluegers Arch. 1988; 412: 54-65Crossref PubMed Scopus (42) Google Scholar). To bypass this problem, we applied here a dual perforated patch-clamp approach, which preserves the integrity of the internal milieu (24Horn R. Marty A. J. Gen. Physiol. 1988; 92: 145-159Crossref PubMed Scopus (1454) Google Scholar, 25Takens-Kwak B.R. Jongsma H.J. Pfluegers Arch. 1992; 422: 198-200Crossref PubMed Scopus (87) Google Scholar), to monitor pairs of acinar cells stimulated with increasing ACh concentrations for both Ca2+-dependent membrane and gap junctional currents. In parallel experiments, changes in [Ca2+]i were monitored by digital imaging of fluo-3-loaded acinar cells. Under these conditions, we observed that ACh-induced closure of gap junction channels parallels the phase plateau of the [Ca2+]i response, but not the initial peak. We further show that acinar cell uncoupling requires the presence of extracellular Ca2+ and parallels capacitative Ca2+ entry. DISCUSSIONOur results describe the temporal relationship between changes in [Ca2+]i and gap junctional conductance within pairs of pancreatic acinar cells exposed to ACh. Uncoupling of pancreatic acinar cells by concentrations of Ca2+-mobilizing secretagogues that maximally stimulate exocrine secretion is well documented (14Petersen O.H. Ueda N. J. Physiol. (Lond.). 1975; 247: 461-471Crossref Scopus (17) Google Scholar, 15Iwatsuki N. Petersen O.H. J. Physiol. (Lond.). 1978; 274: 81-96Crossref Scopus (46) Google Scholar, 16Meda P. Bruzzone R. Chanson M. Bosco D. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4901-4904Crossref PubMed Scopus (52) Google Scholar, 17Chanson M. Orci L. Meda P. Am. J. Physiol. 1991; 261: G28-G36PubMed Google Scholar). However, the intracellular mechanism that mediates this effect is not known (38Chanson M. Meda P. Hall J.E. Zampighi G.A. Davis R.M. Progress in Cell Research. 3. Elsevier Science Publishers B.V., Amsterdam1993: 199-205Google Scholar). Using a dual perforated patch-clamp approach and confocal digital imaging, we report here that ACh-induced uncoupling and [Ca2+]i changes had distinct kinetics. Thus, decrease in junctional conductance of acinar cells consistently develops after the initial [Ca2+]i peak and is maximal during the [Ca2+]i plateau. Furthermore, ACh-induced uncoupling was no longer detected when reloading of internal Ca2+ stores was prevented by incubation of the cells in a Ca2+-free medium. These results suggest that depletion-activated Ca2+ entry was a key determinant for the ACh-induced uncoupling of acinar cells.In many nonexcitable cells, depletion of intracellular Ca2+stores by IP3 is the primary mechanism by which cell surface receptors activate Ca2+ influx. This phenomenon, which is termed capacitative Ca2+ entry (33Putney J.W. Bird G. St J. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (392) Google Scholar), has been involved in the control of Ca2+ oscillations (32Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar, 39Tsien R.W. Tsien R.Y. Annu. Rev. Cell Biol. 1990; 6: 715-760Crossref PubMed Scopus (1011) Google Scholar), secretion (40Parekh A.B. Penner R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7907-7911Crossref PubMed Scopus (153) Google Scholar), and enzymatic regulation (41Chiono M. Mahey R. Tate G. Cooper D.M. J. Biol. Chem. 1995; 270: 1149-1155Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The signal that couples store depletion to Ca2+ entry has not yet been identified (33Putney J.W. Bird G. St J. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (392) Google Scholar). In pancreatic acinar cells, however, there is definite evidence that NO produced by nitric-oxide synthase mediates the stimulation of cGMP formation by cholinergic agonists. cGMP, in turn, is known to modulate Ca2+ entry (29Xu X. Star R.A. Tortorici G. Muallem S. J. Biol. Chem. 1994; 269: 12645-12653Abstract Full Text PDF PubMed Google Scholar, 36Bahnson T.D. Pandol S.J. Dionne V.E. J. Biol. Chem. 1993; 268: 10808-10812Abstract Full Text PDF PubMed Google Scholar, 37Gukovskaya A. Pandol S. Am. J. Physiol. 1994; 266: G350-G356PubMed Google Scholar, 42Pandol S.J. Schoeffield-Payne M.S. J. Biol. Chem. 1990; 265: 12846-12853Abstract Full Text PDF PubMed Google Scholar, 43Xu X. Kitamura K. Lau K.S. Muallem S. Miller R.T. J. Biol. Chem. 1995; 270: 29169-29175Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Consistent with these data, we observed that activation of capacitative Ca2+ entry by either depletion of internal Ca2+stores with thapsigargin or by exposure of acinar cells to dibutyryl cGMP decreased intercellular communication to an extent similar to that observed with ACh alone. Side effects of these agents on intercellular communication appear unlikely because uncoupling was abolished in acini that were depleted for their internal Ca2+ and incubated in a Ca2+-free medium. In addition, cell uncoupling evoked by ACh was prevented in the presence of a nitric-oxide synthase inhibitor, supporting the view that gap junctional conductance and capacitative Ca2+ entry are concurrently regulated. These results, however, do not rule out the possibility that capacitative Ca2+ entry may activate another intracellular pathway leading to modulation of junctional conductance. Support for this idea is provided by the earlier observation that okadaic acid, a phosphatase inhibitor that modulates capacitative Ca2+ entry (44Berlin R.D. Preston S.F. Cell Calcium. 1993; 14: 379-386Crossref PubMed Scopus (34) Google Scholar, 45Parekh A.B. Terlau H. Stühmer W. Nature. 1993; 364: 814-818Crossref PubMed Scopus (319) Google Scholar), also prevents ACh-induced uncoupling (38Chanson M. Meda P. Hall J.E. Zampighi G.A. Davis R.M. Progress in Cell Research. 3. Elsevier Science Publishers B.V., Amsterdam1993: 199-205Google Scholar). Although several studies have shown that gap junction channels are blocked by cGMP (25Takens-Kwak B.R. Jongsma H.J. Pfluegers Arch. 1992; 422: 198-200Crossref PubMed Scopus (87) Google Scholar) and nitric oxide (46Lu C. McMahon D.G. J. Physiol. (Lond.). 1997; 499: 689-699Crossref Scopus (73) Google Scholar), our data provide the first observation that ACh-induced uncoupling is linked to capacitative Ca2+entry.Previous in vitro and in vivo studies have documented a relationship between intercellular communication and the secretory activity of pancreatic acinar cells (17Chanson M. Orci L. Meda P. Am. J. Physiol. 1991; 261: G28-G36PubMed Google Scholar, 30Meda P. Bruzzone R. Knodel S. Orci L. J. Cell Biol. 1986; 103: 475-483Crossref PubMed Scopus (61) Google Scholar, 47Chanson M. Fanjul M. Bosco D. Nelles E. Suter S. Willecke K. Meda P. J. Cell Biol. 1998; 141: 1267-1275Crossref PubMed Scopus (63) Google Scholar). In this context, gap junctional coupling is thought to coordinate the Ca2+ response of individual acinar cells within an acinus and thereby to regulate exocytosis (11Stauffer P.L. Zhao H. Luby-Phelps K. Moss R.L. Star R.A. Muallem S. J. Biol. Chem. 1993; 268: 19769-19775Abstract Full Text PDF PubMed Google Scholar, 48Bosco D. Soriano J.V. Chanson M. Meda P. J. Cell. Physiol. 1994; 160: 378-388Crossref PubMed Scopus (17) Google Scholar). This idea is supported by our present finding that the large and stable junctional conductance observed in most acinar cell pairs was not altered during changes in [Ca2+]i evoked by ACh. In agreement with a previous study using cholecystokinin (13Yule D.I. Stuenkel E. Williams J.A. Am. J. Physiol. 1996; 271: C1285-C1294Crossref PubMed Google Scholar), we observed that rises in [Ca2+]i were asynchronous in acinar cells stimulated with low concentrations of ACh. Increasing the agonist concentration was associated with shortening of the delay between the onset of the Ca2+ responses, suggesting that these cells were coupled in terms of Ca2+mobilization. Also, perfusion of IP3 into one cell evoked a rise in [Ca2+]i in neighboring cells even though not necessarily in those that directly contacted the stimulated one. This observation suggests that rat pancreatic acinar cells differ in their ability to mobilize Ca2+ from internal stores, as indicated by previous studies reporting similar heterogeneity in [Ca2+]imobilization (31Willems P.H.G.M. Van Emst-De Vries S.E. Van Os C.H. De Pont J.J.H.H.M. Cell Calcium. 1993; 14: 145-159Crossref PubMed Scopus (46) Google Scholar) and amylase secretion (48Bosco D. Soriano J.V. Chanson M. Meda P. J. Cell. Physiol. 1994; 160: 378-388Crossref PubMed Scopus (17) Google Scholar, 49Bosco D. Chanson M. Bruzzone R. Meda P. Am. J. Physiol. 1988; 254: G664-G670PubMed Google Scholar). This differential responsiveness may be essential to provide a properly modulable response to agonist-specific stimulation (11Stauffer P.L. Zhao H. Luby-Phelps K. Moss R.L. Star R.A. Muallem S. J. Biol. Chem. 1993; 268: 19769-19775Abstract Full Text PDF PubMed Google Scholar, 50Yule D.I. Lawrie A.M. Petersen O.H. Cell Calcium. 1991; 12: 145-151Crossref PubMed Scopus (97) Google Scholar).These results, however, are not immediately reconcilable with the observation that ACh decreases gap junctional coupling while maximally stimulating the secretory activity of acinar cells (30Meda P. Bruzzone R. Knodel S. Orci L. J. Cell Biol. 1986; 103: 475-483Crossref PubMed Scopus (61) Google Scholar, 47Chanson M. Fanjul M. Bosco D. Nelles E. Suter S. Willecke K. Meda P. J. Cell Biol. 1998; 141: 1267-1275Crossref PubMed Scopus (63) Google Scholar, 48Bosco D. Soriano J.V. Chanson M. Meda P. J. Cell. Physiol. 1994; 160: 378-388Crossref PubMed Scopus (17) Google Scholar). The blockade of acinar cell-to-cell communication is known to enhance the basal release of amylase in vitro and in vivo(16Meda P. Bruzzone R. Chanson M. Bosco D. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4901-4904Crossref PubMed Scopus (52) Google Scholar, 26Chanson M. Bruzzone R. Bosco D. Meda P. J. Cell. Physiol. 1989; 139: 147-156Crossref PubMed Scopus (52) Google Scholar, 30Meda P. Bruzzone R. Knodel S. Orci L. J. Cell Biol. 1986; 103: 475-483Crossref PubMed Scopus (61) Google Scholar, 47Chanson M. Fanjul M. Bosco D. Nelles E. Suter S. Willecke K. Meda P. J. Cell Biol. 1998; 141: 1267-1275Crossref PubMed Scopus (63) Google Scholar). Under conditions of gap junction blockade, the potency of several agonists in stimulating exocytosis has also been found to be reduced when the effect of acinar cell uncoupling on basal secretion was taken into account (11Stauffer P.L. Zhao H. Luby-Phelps K. Moss R.L. Star R.A. Muallem S. J. Biol. Chem. 1993; 268: 19769-19775Abstract Full Text PDF PubMed Google Scholar, 16Meda P. Bruzzone R. Chanson M. Bosco D. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4901-4904Crossref PubMed Scopus (52) Google Scholar, 30Meda P. Bruzzone R. Knodel S. Orci L. J. Cell Biol. 1986; 103: 475-483Crossref PubMed Scopus (61) Google Scholar). Therefore, uncoupling may provide acinar cells with a mechanism to sustain enzyme release during acute stimulation by increasing their rate of basal secretion. In this context, the delayed uncoupling evoked by ACh may compartmentalize cells that are highly sensitive to ACh from cells that are less sensitive to the secretagogue, therefore decreasing the effective volume of cytoplasm of interconnected cells. This regulation may be essential to ensure that the intracellular levels of critical factor(s) are maintained to allow for sustained activity of groups of actively secreting cells. Future studies should determine whether uncoupling induced by Ca2+ store depletion is a common mechanism to control junctional communication in other types of nonexcitable cells. Our results describe the temporal relationship between changes in [Ca2+]i and gap junctional conductance within pairs of pancreatic acinar cells exposed to ACh. Uncoupling of pancreatic acinar cells by concentrations of Ca2+-mobilizing secretagogues that maximally stimulate exocrine secretion is well documented (14Petersen O.H. Ueda N. J. Physiol. (Lond.). 1975; 247: 461-471Crossref Scopus (17) Google Scholar, 15Iwatsuki N. Petersen O.H. J. Physiol. (Lond.). 1978; 274: 81-96Crossref Scopus (46) Google Scholar, 16Meda P. Bruzzone R. Chanson M. Bosco D. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4901-4904Crossref PubMed Scopus (52) Google Scholar, 17Chanson M. Orci L. Meda P. Am. J. Physiol. 1991; 261: G28-G36PubMed Google Scholar). However, the intracellular mechanism that mediates this effect is not known (38Chanson M. Meda P. Hall J.E. Zampighi G.A. Davis R.M. Progress in Cell Research. 3. Elsevier Science Publishers B.V., Amsterdam1993: 199-205Google Scholar). Using a dual perforated patch-clamp approach and confocal digital imaging, we report here that ACh-induced uncoupling and [Ca2+]i changes had distinct kinetics. Thus, decrease in junctional conductance of acinar cells consistently develops after the initial [Ca2+]i peak and is maximal during the [Ca2+]i plateau. Furthermore, ACh-induced uncoupling was no longer detected when reloading of internal Ca2+ stores was prevented by incubation of the cells in a Ca2+-free medium. These results suggest that depletion-activated Ca2+ entry was a key determinant for the ACh-induced uncoupling of acinar cells. In many nonexcitable cells, depletion of intracellular Ca2+stores by IP3 is the primary mechanism by which cell surface receptors activate Ca2+ influx. This phenomenon, which is termed capacitative Ca2+ entry (33Putney J.W. Bird G. St J. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (392) Google Scholar), has been involved in the control of Ca2+ oscillations (32Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar, 39Tsien R.W. Tsien R.Y. Annu. Rev. Cell Biol. 1990; 6: 715-760Crossref PubMed Scopus (1011) Google Scholar), secretion (40Parekh A.B. Penner R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7907-7911Crossref PubMed Scopus (153) Google Scholar), and enzymatic regulation (41Chiono M. Mahey R. Tate G. Cooper D.M. J. Biol. Chem. 1995; 270: 1149-1155Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The signal that couples store depletion to Ca2+ entry has not yet been identified (33Putney J.W. Bird G. St J. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (392) Google Scholar). In pancreatic acinar cells, however, there is definite evidence that NO produced by nitric-oxide synthase mediates the stimulation of cGMP formation by cholinergic agonists. cGMP, in turn, is known to modulate Ca2+ entry (29Xu X. Star R.A. Tortorici G. Muallem S. J. Biol. Chem. 1994; 269: 12645-12653Abstract Full Text PDF PubMed Google Scholar, 36Bahnson T.D. Pandol S.J. Dionne V.E. J. Biol. Chem. 1993; 268: 10808-10812Abstract Full Text PDF PubMed Google Scholar, 37Gukovskaya A. Pandol S. Am. J. Physiol. 1994; 266: G350-G356PubMed Google Scholar, 42Pandol S.J. Schoeffield-Payne M.S. J. Biol. Chem. 1990; 265: 12846-12853Abstract Full Text PDF PubMed Google Scholar, 43Xu X. Kitamura K. Lau K.S. Muallem S. Miller R.T. J. Biol. Chem. 1995; 270: 29169-29175Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Consistent with these data, we observed that activation of capacitative Ca2+ entry by either depletion of internal Ca2+stores with thapsigargin or by exposure of acinar cells to dibutyryl cGMP decreased intercellular communication to an extent similar to that observed with ACh alone. Side effects of these agents on intercellular communication appear unlikely because uncoupling was abolished in acini that were depleted for their internal Ca2+ and incubated in a Ca2+-free medium. In addition, cell uncoupling evoked by ACh was prevented in the presence of a nitric-oxide synthase inhibitor, supporting the view that gap junctional conductance and capacitative Ca2+ entry are concurrently regulated. These results, however, do not rule out the possibility that capacitative Ca2+ entry may activate another intracellular pathway leading to modulation of junctional conductance. Support for this idea is provided by the earlier observation that okadaic acid, a phosphatase inhibitor that modulates capacitative Ca2+ entry (44Berlin R.D. Preston S.F. Cell Calcium. 1993; 14: 379-386Crossref PubMed Scopus (34) Google Scholar, 45Parekh A.B. Terlau H. Stühmer W. Nature. 1993; 364: 814-818Crossref PubMed Scopus (319) Google Scholar), also prevents ACh-induced uncoupling (38Chanson M. Meda P. Hall J.E. Zampighi G.A. Davis R.M. Progress in Cell Research. 3. Elsevier Science Publishers B.V., Amsterdam1993: 199-205Google Scholar). Although several studies have shown that gap junction channels are blocked by cGMP (25Takens-Kwak B.R. Jongsma H.J. Pfluegers Arch. 1992; 422: 198-200Crossref PubMed Scopus (87) Google Scholar) and nitric oxide (46Lu C. McMahon D.G. J. Physiol. (Lond.). 1997; 499: 689-699Crossref Scopus (73) Google Scholar), our data provide the first observation that ACh-induced uncoupling is linked to capacitative Ca2+entry. Previous in vitro and in vivo studies have documented a relationship between intercellular communication and the secretory activity of pancreatic acinar cells (17Chanson M. Orci L. Meda P. Am. J. Physiol. 1991; 261: G28-G36PubMed Google Scholar, 30Meda P. Bruzzone R. Knodel S. Orci L. J. Cell Biol. 1986; 103: 475-483Crossref PubMed Scopus (61) Google Scholar, 47Chanson M. Fanjul M. Bosco D. Nelles E. Suter S. Willecke K. Meda P. J. Cell Biol. 1998; 141: 1267-1275Crossref PubMed Scopus (63) Google Scholar). In this context, gap junctional coupling is thought to coordinate the Ca2+ response of individual acinar cells within an acinus and thereby to regulate exocytosis (11Stauffer P.L. Zhao H. Luby-Phelps K. Moss R.L. Star R.A. Muallem S. J. Biol. Chem. 1993; 268: 19769-19775Abstract Full Text PDF PubMed Google Scholar, 48Bosco D. Soriano J.V. Chanson M. Meda P. J. Cell. Physiol. 1994; 160: 378-388Crossref PubMed Scopus (17) Google Scholar). This idea is supported by our present finding that the large and stable junctional conductance observed in most acinar cell pairs was not altered during changes in [Ca2+]i evoked by ACh. In agreement with a previous study using cholecystokinin (13Yule D.I. Stuenkel E. Williams J.A. Am. J. Physiol. 1996; 271: C1285-C1294Crossref PubMed Google Scholar), we observed that rises in [Ca2+]i were asynchronous in acinar cells stimulated with low concentrations of ACh. Increasing the agonist concentration was associated with shortening of the delay between the onset of the Ca2+ responses, suggesting that these cells were coupled in terms of Ca2+mobilization. Also, perfusion of IP3 into one cell evoked a rise in [Ca2+]i in neighboring cells even though not necessarily in those that directly contacted the stimulated one. This observation suggests that rat pancreatic acinar cells differ in their ability to mobilize Ca2+ from internal stores, as indicated by previous studies reporting similar heterogeneity in [Ca2+]imobilization (31Willems P.H.G.M. Van Emst-De Vries S.E. Van Os C.H. De Pont J.J.H.H.M. Cell Calcium. 1993; 14: 145-159Crossref PubMed Scopus (46) Google Scholar) and amylase secretion (48Bosco D. Soriano J.V. Chanson M. Meda P. J. Cell. Physiol. 1994; 160: 378-388Crossref PubMed Scopus (17) Google Scholar, 49Bosco D. Chanson M. Bruzzone R. Meda P. Am. J. Physiol. 1988; 254: G664-G670PubMed Google Scholar). This differential responsiveness may be essential to provide a properly modulable response to agonist-specific stimulation (11Stauffer P.L. Zhao H. Luby-Phelps K. Moss R.L. Star R.A. Muallem S. J. Biol. Chem. 1993; 268: 19769-19775Abstract Full Text PDF PubMed Google Scholar, 50Yule D.I. Lawrie A.M. Petersen O.H. Cell Calcium. 1991; 12: 145-151Crossref PubMed Scopus (97) Google Scholar). These results, however, are not immediately reconcilable with the observation that ACh decreases gap junctional coupling while maximally stimulating the secretory activity of acinar cells (30Meda P. Bruzzone R. Knodel S. Orci L. J. Cell Biol. 1986; 103: 475-483Crossref PubMed Scopus (61) Google Scholar, 47Chanson M. Fanjul M. Bosco D. Nelles E. Suter S. Willecke K. Meda P. J. Cell Biol. 1998; 141: 1267-1275Crossref PubMed Scopus (63) Google Scholar, 48Bosco D. Soriano J.V. Chanson M. Meda P. J. Cell. Physiol. 1994; 160: 378-388Crossref PubMed Scopus (17) Google Scholar). The blockade of acinar cell-to-cell communication is known to enhance the basal release of amylase in vitro and in vivo(16Meda P. Bruzzone R. Chanson M. Bosco D. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4901-4904Crossref PubMed Scopus (52) Google Scholar, 26Chanson M. Bruzzone R. Bosco D. Meda P. J. Cell. Physiol. 1989; 139: 147-156Crossref PubMed Scopus (52) Google Scholar, 30Meda P. Bruzzone R. Knodel S. Orci L. J. Cell Biol. 1986; 103: 475-483Crossref PubMed Scopus (61) Google Scholar, 47Chanson M. Fanjul M. Bosco D. Nelles E. Suter S. Willecke K. Meda P. J. Cell Biol. 1998; 141: 1267-1275Crossref PubMed Scopus (63) Google Scholar). Under conditions of gap junction blockade, the potency of several agonists in stimulating exocytosis has also been found to be reduced when the effect of acinar cell uncoupling on basal secretion was taken into account (11Stauffer P.L. Zhao H. Luby-Phelps K. Moss R.L. Star R.A. Muallem S. J. Biol. Chem. 1993; 268: 19769-19775Abstract Full Text PDF PubMed Google Scholar, 16Meda P. Bruzzone R. Chanson M. Bosco D. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4901-4904Crossref PubMed Scopus (52) Google Scholar, 30Meda P. Bruzzone R. Knodel S. Orci L. J. Cell Biol. 1986; 103: 475-483Crossref PubMed Scopus (61) Google Scholar). Therefore, uncoupling may provide acinar cells with a mechanism to sustain enzyme release during acute stimulation by increasing their rate of basal secretion. In this context, the delayed uncoupling evoked by ACh may compartmentalize cells that are highly sensitive to ACh from cells that are less sensitive to the secretagogue, therefore decreasing the effective volume of cytoplasm of interconnected cells. This regulation may be essential to ensure that the intracellular levels of critical factor(s) are maintained to allow for sustained activity of groups of actively secreting cells. Future studies should determine whether uncoupling induced by Ca2+ store depletion is a common mechanism to control junctional communication in other types of nonexcitable cells. We thank D. Bosco, N. Guérineau, M. B. Rook, and S. Verheule for continuous support during the preparation of this work, and Luc Analbers and Isabelle Duperrut for excellent technical help.
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