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studies of this type offerthe possibilityof establishingunequivocally which hormones and neurotransrnittersstimulate the duct cells,how these agents interact,and the true composition of ductal fluid. Acknowledgments Recent work has been supported by grants from the Medical Research Council (United Kingdom) and the Cystic Fibrosis Research Trust (United Kingdom).
[ 18] D i s p e r s e d P a n c r e a t i c A c i n a r C e l l s and Pancreatic Acini B y D E L I A MENOZZI, ROBERT T . JENSEN, a n d JERRY D . G A R D N E R
Introduction Initially the secretory function of the pancreas in vitro was studied using slices or fragments of the gland. An advance in the study of pancreatic function in vitro was the development of a technique for preparing dispersed pancreatic acinar cells? -3 This preparation consisted of a homogeneous suspension of dispersed single acinar cells. A few years later a modification of the technique for preparing dispersed cells led to a method for preparing dispersed pancreatic acini.4-~ Dispersed acini represent clumps of pancreatic acinar cells containing 10-20 cells/clump. Preparation of Pancreatic Acinar Cells Dispersed pancreatic acinar cells are prepared with a minor modification of the technique developed by Amsterdam and Jamieson. 1-3 This preparation contains at least 96% acinar cells and these cells retain their responsiveness to pancreatic secretagogues for up to 5 hr in vitro. Dispersed acinar cells have been prepared using pancreas from rats, mice, guinea pigs, or rabbits. 1. Prepare the following stock solution in advance and store at 4": NaC1 (100 mM) KC1 (6 mM) ' A. Amsterdam and J. D. Jamieson, Proc. Natl. Acad. Sci. U.S.A. 69, 3028 (1972). 2 A. Amsterdam and J. D. Jamieson, J. Cell Biol. 63, 1037 (1974). 3 A. Amsterdam and J. D. Jamieson, J. Cell Biol. 63, 1057 (1974).
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KH2PO4 (2.5 raM) HEPES (24.5 raM; pH 7.4) Sodium pymvate (5 raM) Sodium glutamate (5 raM) Sodium fumarate (5 raM) Glucose (14 mM) Glutamine (2 raM) Soybean trypsin inhibitor (0.1 mg/ml) Eagle's basal amino acid mixture ([100×; 1% (v/v) ] Essential vitamin mixture [ 100 ×; 1% (v/v) ] . Prepare two separate wash solutions: Wash solution 1: Stock solution plus CaC12 (0.1 raM), MgCI2
(1.2 m g ) Wash solution 2: Stock solution plus CaC12 (1 mM), MgC12 (1.2 raM), albumin [4% (w/v)] . Prepare three separate digestion solutions: Digestion solution 1: Stock solution plus CaC12 (0.1 raM), MgC12 (1.2 mM), type I collagenase (0.75 mg/ml; Sigma, St. Louis, MO); type I-S hyaluronidase (1.5 mg/ml; Sigma) Digestion solution 2: Stock solution plus EGTA (2 raM) Digestion solution 3: Same as digestion solution 1 except use coUagenase (1.25 mg/ml) and hyaluronidase (2 mg/ml) 4. Prepare incubation solution: Stock solution plus CaCI2 (0.5 raM), MgC12 (1.2 raM), and albumin [1% (w/v)] The animal is sacrificed by CO2 narcosis and the pancreas is removed, trimmed of fat and mesentery, and pinned to a wax tray. Five milliliters of digestion solution 1 is injected into the tissue using a syringe and a 25gauge needle. The pancreas and the digestion solution are transferred to a siliconized 25-ml Erlenmeyer flask, gassed for 30 sec with 100% 02, capped, and incubated in a Dubnoff metabolic shaking incubator (160 oscillations/min) at 37 ° for 15 rain. After 15 min, the digestion solution is decanted and replaced with 8 ml of digestion solution 2. The flask is gassed with 100% 02, capped, and returned to the incubator for 5 rain at 37 °. At the end of the 5-rain incubation the digestion solution is decanted and the pancreas is washed twice with 10 ml of wash solution 1. The washes are discarded. Five milliliters of digestion solution 3 is added to the flask. The flask is gassed with 100% 02, capped, and returned to the incubator at 37 ° for 20 rain. After 20 min the pancreas is dispersed by pipetting up and down five times through a Pasteur pipet and then passing it in and out of a
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TABLE I UsEs OF PANCREATIC ACINAR CELLS
Function measured
Reference
Amylase secretion Protein secretion Receptor binding Cellular cAMP Cellular cGMP Calcium transport Hydrolysis of polyphosphoinositides Na+ and K + content
7- 9 3 10 11 12,13 14 15 9
syringe bearing a 19-gauge needle five times. The resulting cell suspension is passed through nylon mesh (Spectramesh, 70/~m; Spectrum Medical Industries, Los Angeles, CA) to remove large clumps of nondispersed tissue. To separate the acinar cells from the digestion solution and cell debris, the cell suspension is gently layered into two 15-conical centrifuge tubes each containing 6 ml of wash solution 2. The tubes are centrifuged at 50 g for 5 min and the supernatant is discarded. The cell pellets are combined and washed twice with 6 ml of wash solution 2 by centrifugation at 50 g for 5 min and resuspension. After the second wash, the supernatant is removed and discarded and the acinar cells are suspended in an appropriate volume of incubation solution. Uses of Pancreatic Acinar Cells
Table I fists the published uses of pancreatic acinar cells.T M Acinar cells can be permeabilized by electrical or chemical treatment; however, the preparation and applications of permeabilized acinar cells is reviewed in Chapter [ 19] in this volume. 4 S. R. Peikin, A. J. Rottman, S. Batzri, and J. D. Gardner, Am. J. Physiol. 235, 743 (1978). 5 j. A. Williams, M. Korc, and R. L. Dormer, Am. J. Physiol. 235, E517 (1978). A. Amsterdam, T. E. Solomon, J. D. Jamieson, Methods CellBiol. 20, 361 (1978). J. D. Gardner and M. J. Jackson, J. Physiol. (London) 270, 439 (1977). s D. E. Chandler and J. A. Williams, J. Membr. Biol. 32, 201 (1977). 9 j. A. Williams, P. Cary, and B. Moffat, Am. J. Physiol. 231, 1562 (1976). to j. p. Christophe, T. P. Coulon, and J. D. Gardner, J. Biol. Chem. 251, 4629 (1976). H j. D. Gardner, T. P. Coulon, and T. D. Adams, Gastroenterology70, 29 (1976). t2 j. p. Christophe, E. K. Frandsen, T. P. Coition, G. Krishna, and J. D. Gardner, J. Biol. Chem. 251, 4640 (1976). ~3R. N. Lopatin and J. D. Gardner, Biochim. Biophys. Acta 543, 465 (1978). 14I. Schultz, Am. J. Physiol. 239, G335 (1980). ~5j. W. Putney, Jr., G. M. Burgess, S. P. Halenda, J. S. McKinney, and R. P. Rubin, Biochem. J. 212, 483 (1983).
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Preparation of Dispersed Pancreatic Acini The procedure for preparing dispersed pancreatic acini is a minor modification of the technique reported by Peikin et al. 4 Others~,6have also reported methods for preparing dispersed acini. These techniques4-6 when used with rat pancreas result in acini that show a greater than fivefold increase in enzyme secretion in response to a maximally effective concentration of cholecystoldnin. Recently a technique has been reported for preparing acini using a short digestion period, m6Unfortunately this rapid method for preparing acini gives rise to cells that show a greater than fivefold increase in enzyme secretion only in response to a maximally effective concentration of a cholecystokinin analog. Freshly prepared dispersed acini can be used for at least 5 hr in vitro and have been very useful to investigate the mechanism of action of various secretagogues. Dispersed acini have been prepared using pancreas from mice, rats, guinea pigs, or rabbits. 1. Prepare the following stock solution: NaC1 (100 mM) KC1 (6 mM) Glucose (14 mM) KH2PO4 (2.5 mM) HEPES (24.5 mM; pH 7.4) MgCl2 (1.2 mM) Sodium pyruvate (5 mM) Sodium fumarate (5 mM) Sodium glutamate (5 mM) Glutamine, (2 raM) Soybean trypsin inhibitor (0.1 mg/ml) Eagle's basal amino acid mixture [100X; 1% (v/v)] Essential vitamin mixture [ 100 X; 1% (v/v) ] 2. Prepare two separate wash solutions; Wash solution 1: Stock solution plus CaC12 (2 mM) and albumin
[4%(w/v)] Wash solution 2: Stock solution plus CaC12 (2 mM) 3. Prepare digestion solution: Stock solution plus collagenase (564 U/ rag, 0.04 mg/ml type CLSPA; Worthington Biochemical Corp., Freehold, NJ), albumin [0.2% (w/v)], and CaC12 (2 raM) 4. Prepare incubation solution: Stock solution plus CaCI2 (0.5 raM) and albumin [ 1% (w/v) ] 16R. Bruzzone, P. A. Halban, A. Gjinovci, and E. Trimble, Biochem. J. 226, 621 (1985).
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The animal is sacrificed by CO2 narcosis and the pancreas is removed, trimmed of fat and mesentery, and pinned to a way tray. Five milliliters of digestion solution is injected into the tissue using a syringe and a 25-gauge needle. The pancreas and the digestion solution are transferred to a siliconized 25-ml Edenmeyer flask, gassed for 30 see with 100% 02, capped, and incubated in a Dubnoff metabolic shaking incubator (160 oscillations/ min) at 37 ° for 10 min. After 10 min of incubation, the digestion solution is decanted and replaced with 5 ml of fresh digestion solution. The flask is gassed, capped, and placed in the incubator for 10 min at 37 °. After the second 10-min incubation, the digestion solution is again replaced with 5 ml of fresh digestion solution and the flask is gassed, capped, and placed in the incubator for 10 min at 37 °. After the third 10-min incubation, the digestion solution is decanted and replaced with 5 ml of fresh digestion solution. The flask is gassed and capped, and the tissue is disrupted by shaking the flask vigorously by hand for 15 see. After discarding the duct system and large tissue fragments, the suspension of acini is gently layered into two 15-ml conical centrifuge tubes, each containing 6 ml of wash solution. The tubes are centrifuged at 200 g for 5 see and the supernatant is discarded. The cell pellets are combined and any large clumps of tissue are removed with a Pasteur pipet. The acini are washed twice with wash solution by centrifugation (200 g for 5 see) and resuspended. After the last wash, the supernatant is discarded and the acini are suspended in an appropriate volume of incubation solution. Uses of D i s p e r s e d P a n c r e a t i c Acini Table II lists the published uses of dispersed pancreatic acini. 17-4° 17j. D. Gardner and R. T. Jensen, in "Physiologyof the Gastrointestinal Tract" (L. R. Johnson, ed.), p. 831. Raven, New York, 1981. n j. D. Gardner and R. T. Jensen, in "Physiologyof the Gastrointestinal Tract" (L. R. Johnson, ed.), 2nd Ed., p. 1109. Raven, New York, 1987. 19S. R. Hootman and J. A. Williams, in "Physiologyof the GastrointestinalTract" (L. R. Johnson, ed.), 2nd Ed. p. 1129. Raven, New York, 198q 20j. A. Williams,A. C. Bailey,and E. Roach, Am. J. Physiol. 254, G521 (1988). 21R. S. Izzo, C. Pelleeehia,and M. Praisman,Am. J. Physiol. 255, G738 (1988). 22S. A. Rosenzweig,L. J. Miller, and J. D. Jamieson,J. CellBiol. 96, 1288 (1983). 23C. D. Logsdonand J. A. Williams,Biochem. J. 223, 893 (1984). 24S. Muallem,Annu. Rev. Physiol. 51, 83 (1989). 25G. R. Gunther and J. D. Jamieson, Nature (London) 280, 318 (1979). 26j. D. Gardner and A. J. Rottman, Biochim. Biophys. Acta 627, 230 (1980). 27D. B, Burnham and J. A. Williams,Am. J. Physiol. 246, G500 (1984). 28M. Noguehi, H. Adaehi, J. D. Gardner, and R. T. Jensen, Am. J. Physiol. 248, G692 (1985). 29F. S. Goreliek, J. A. Cohn, S. D. Freedman, N. G. Delahunt, J. M. Gershouni, and J. D. Jamieson, J. CellBiol. 97, 1294 (1983).
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TABLE II USES OF DISPERSED PANCREATIC ACINI
Function measured Amylase secretion Receptor binding Ligand internalization Cytosolic calcium 45Ca2+ transport Cellular cAMP Cellular cGMP Hydrolysis of polyphosphoinositides Formation of diacylglycerol Protein kinase C activity Calmodulin-dependent protein kinase activity cAMP-Dependent protein kinase activity cGMP-Dependent protein kinase activity Phosphorylation of cellular protein Prostaglandins and leukotrienes Ion channels and membrane potential Cytosolic pH Glucose uptake Amino acid uptake [3H]Thymidine incorporation into DNA
Reference 17-19 17,18 20 - 23 24 24 18,19 25,26 19 19 27,28 27,29 27,30,31 30 32,33 19 34 35 36,37 37,38 39,40
3oR. T. Jensen and J. D. Gardner, Gastroenterology 75, 806 (1978). 31O. Holian, C. T. Bombeck, andL. M. Nyhus, Biochem. Biophys. Res. Commun. 95, 553
(1980). 32 D. B. Burnham, H.-D, S61ing, and J. A. Williams, Am. J. Physiol. 254, GI30 (1988). 33C. K. Sung and J. A. Williams, Diabetes 39, 544 (1989). 34O. H. Petersen and D. V. Gullacher, Annu. Rev. Physiol. 50, 65 (1988). 3s K. S. Carter, L. R. Rutledge, M. L. Steer, and W. Silen, Am. £ Physiol. 253, G690 (1987). 36M. Korc, J. A. Williams, and I. D. Goldfine, J. Biol. Chem. 254, 7624 (1979). 37H. Sankaran, I. D. Goldfme, A. Bailey, V. Licko, and J. A. Williams, Am. £ Physiol. 242, G250 (1982). 3s y . Iwamoto and J. A. Williams, Am. J. Physiol. 238, G440 (1980). ~9C. D. Logsdon and J. A. Williams, Am. J. Physiol. 244, G675 (1983). 40 C. D. Logsdon and J. A. grdliams, Am. J. Physiol. 250, G440 (1986).
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Dispersed Pancreatic A c i n i in Culture The procedure for preparing dispersed pancreatic acini in culture is an aseptic modification of the techniques reported previously for preparing fresh dispersed pancreatic acini. This method allows one to study longterm actions of various agents. Short-term primary cultures are prepared according to a modification41 of the procedure described by Lodgson and Williams.39 The following culture medium is prepared in advance and stored at 4 °. 1 : 1 mixture of Ham's F-12 and Dulbecco's modified minimal essen-
tial medium (MEM) HEPES (10 mM; pH 7.4) Albumin (1.0 mg/ml) Soybean trypsin inhibitor (0.1 mg/ml) Ascorbic acid (0.1 raM) Epidermal growth factor (EGF) (2 nM) ITS: Insulin (5 pg/ml), transferrin (5 #g/ml), selenium (5 ng/ml) (Collaborative Research, Lexington, MA) Penicillin (100 U/ml) Streptomycin (100 #g/ml) Amphotericin B (25/~g/ml) Acini from one pancreas are suspended in 80-120 ml of culture medium and 10-ml aliquots of the suspension are placed in disposable plastic tissue culture flasks and incubated for up to 48 hr at 37 ° in a humidified atmosphere of 5% CO2 in air. Agents to be tested are added at appropriate times during the culture period. Pancreatic acini can be maintained in a differentiated state in suspension culture for up to 48 hr. The acini retain their ability to secrete enzymes, although cultured acini are less sensitive and responsive to agonists than freshly prepared acini. Pancreatic acini in short-term culture can be used to measure the same functions that have been measured using freshly prepared acini but have the advantage of permitting one to examine actions that require 1 to 2 days to became maximal.4t, 42 Long-term primary cultures (up to 14 days) have been described for pancreatic acini from mouse.4° A monolayer culture of pancreatic acini has recently been described.4° In this condition pancreatic acini first Ondergo a period of adaptation during which differentiation occurs. Next, the acinar 4t S. R. Hootman, M. E. Brown, J. A. Williams, and C. D. Logsdon, Am. J. Physiol. 251, G75 (1986). 42C. K. Sung, S. R. Hootman, E. L. Stuenkel, C. Kuroiwa, and J. A. Williams, Am. J. Physiol. 254, G242 (1988).
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cells spread and divide to form a confluent monolayer on collagen gel. These cultured cells retain their ability to respond to cholecystokinin in a trophic manner, but lack the normal content of secretory enzymes and do not show stimulation of enzyme secretion with pancreatic secretagogues. Perifusion of Pancreatic Acini
Pancreatic acini, prepared as described earlier in this chapter, are placed in a small chamber and immobilized using falters43 or gel.~,45 Incubation solution is continuously passed over the acini and the effluent is collected using a fraction collector. This procedure provides the advantage that a population of pancreatic acini can be subjected to a continuous flow of material in vitro, thereby both supplying fresh modifier and removing the products of the response, as is likely to happen in vivo. Moreover the outflow is collected continuously into fractions that are then analyzed, thereby permitting constant monitoring of the dynamics of the system. Despite the potential of this system in elucidating the kinetics of pancreatic enzyme secretion, this procedure has not been characterized extensively and used to study pancreatic acinar cell functions. This method has been used to measure amylase secretion,4a-45 45Ca2+ efflux,44 and cellular cAMP? 4 Strengths and Drawbacks of Various Preparations During the last decade dispersed pancreatic acini have been used much more extensively than acinar cells, primarily because acini are easier to prepare and show a much greater increase in enzyme secretion in response to secretagogues than do acinar cells. Table III compares the strengths and drawbacks of the various preparations.
43 K. Imamura, H. Wakasugi, H. Shinozaki, and H. Ibayashi, Jpn. J. Physiol. 33, 687 (1983). 44 E. K. Frandsen, in "Biology of Normal and Cancerous Exoerine Pancreatic Cells" (A. Ribet, L. Pradayrol, and C. Susini, eds.), p. 27. Elsevier/North-Holland Biochemical, Amsterdam, 1980. 45 p. Singh, I. Asada, A. Owlia, T. J. Collins, J. C. Thompson, Am. J. Physiol. 254, G217 (1988).
0 C3
()
!.
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studies of this type offerthe possibilityof establishingunequivocally which hormones and neurotransrnittersstimulate the duct cells,how these agents interact,and the true composition of ductal fluid. Acknowledgments Recent work has been supported by grants from the Medical Research Council (United Kingdom) and the Cystic Fibrosis Research Trust (United Kingdom).
[ 18] D i s p e r s e d P a n c r e a t i c A c i n a r C e l l s and Pancreatic Acini B y D E L I A MENOZZI, ROBERT T . JENSEN, a n d JERRY D . G A R D N E R
Introduction Initially the secretory function of the pancreas in vitro was studied using slices or fragments of the gland. An advance in the study of pancreatic function in vitro was the development of a technique for preparing dispersed pancreatic acinar cells? -3 This preparation consisted of a homogeneous suspension of dispersed single acinar cells. A few years later a modification of the technique for preparing dispersed cells led to a method for preparing dispersed pancreatic acini.4-~ Dispersed acini represent clumps of pancreatic acinar cells containing 10-20 cells/clump. Preparation of Pancreatic Acinar Cells Dispersed pancreatic acinar cells are prepared with a minor modification of the technique developed by Amsterdam and Jamieson. 1-3 This preparation contains at least 96% acinar cells and these cells retain their responsiveness to pancreatic secretagogues for up to 5 hr in vitro. Dispersed acinar cells have been prepared using pancreas from rats, mice, guinea pigs, or rabbits. 1. Prepare the following stock solution in advance and store at 4": NaC1 (100 mM) KC1 (6 mM) ' A. Amsterdam and J. D. Jamieson, Proc. Natl. Acad. Sci. U.S.A. 69, 3028 (1972). 2 A. Amsterdam and J. D. Jamieson, J. Cell Biol. 63, 1037 (1974). 3 A. Amsterdam and J. D. Jamieson, J. Cell Biol. 63, 1057 (1974).
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KH2PO4 (2.5 raM) HEPES (24.5 raM; pH 7.4) Sodium pymvate (5 raM) Sodium glutamate (5 raM) Sodium fumarate (5 raM) Glucose (14 mM) Glutamine (2 raM) Soybean trypsin inhibitor (0.1 mg/ml) Eagle's basal amino acid mixture ([100×; 1% (v/v) ] Essential vitamin mixture [ 100 ×; 1% (v/v) ] . Prepare two separate wash solutions: Wash solution 1: Stock solution plus CaC12 (0.1 raM), MgCI2
(1.2 m g ) Wash solution 2: Stock solution plus CaC12 (1 mM), MgC12 (1.2 raM), albumin [4% (w/v)] . Prepare three separate digestion solutions: Digestion solution 1: Stock solution plus CaC12 (0.1 raM), MgC12 (1.2 mM), type I collagenase (0.75 mg/ml; Sigma, St. Louis, MO); type I-S hyaluronidase (1.5 mg/ml; Sigma) Digestion solution 2: Stock solution plus EGTA (2 raM) Digestion solution 3: Same as digestion solution 1 except use coUagenase (1.25 mg/ml) and hyaluronidase (2 mg/ml) 4. Prepare incubation solution: Stock solution plus CaCI2 (0.5 raM), MgC12 (1.2 raM), and albumin [1% (w/v)] The animal is sacrificed by CO2 narcosis and the pancreas is removed, trimmed of fat and mesentery, and pinned to a wax tray. Five milliliters of digestion solution 1 is injected into the tissue using a syringe and a 25gauge needle. The pancreas and the digestion solution are transferred to a siliconized 25-ml Erlenmeyer flask, gassed for 30 sec with 100% 02, capped, and incubated in a Dubnoff metabolic shaking incubator (160 oscillations/min) at 37 ° for 15 rain. After 15 min, the digestion solution is decanted and replaced with 8 ml of digestion solution 2. The flask is gassed with 100% 02, capped, and returned to the incubator for 5 rain at 37 °. At the end of the 5-rain incubation the digestion solution is decanted and the pancreas is washed twice with 10 ml of wash solution 1. The washes are discarded. Five milliliters of digestion solution 3 is added to the flask. The flask is gassed with 100% 02, capped, and returned to the incubator at 37 ° for 20 rain. After 20 min the pancreas is dispersed by pipetting up and down five times through a Pasteur pipet and then passing it in and out of a
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TABLE I UsEs OF PANCREATIC ACINAR CELLS
Function measured
Reference
Amylase secretion Protein secretion Receptor binding Cellular cAMP Cellular cGMP Calcium transport Hydrolysis of polyphosphoinositides Na+ and K + content
7- 9 3 10 11 12,13 14 15 9
syringe bearing a 19-gauge needle five times. The resulting cell suspension is passed through nylon mesh (Spectramesh, 70/~m; Spectrum Medical Industries, Los Angeles, CA) to remove large clumps of nondispersed tissue. To separate the acinar cells from the digestion solution and cell debris, the cell suspension is gently layered into two 15-conical centrifuge tubes each containing 6 ml of wash solution 2. The tubes are centrifuged at 50 g for 5 min and the supernatant is discarded. The cell pellets are combined and washed twice with 6 ml of wash solution 2 by centrifugation at 50 g for 5 min and resuspension. After the second wash, the supernatant is removed and discarded and the acinar cells are suspended in an appropriate volume of incubation solution. Uses of Pancreatic Acinar Cells
Table I fists the published uses of pancreatic acinar cells.T M Acinar cells can be permeabilized by electrical or chemical treatment; however, the preparation and applications of permeabilized acinar cells is reviewed in Chapter [ 19] in this volume. 4 S. R. Peikin, A. J. Rottman, S. Batzri, and J. D. Gardner, Am. J. Physiol. 235, 743 (1978). 5 j. A. Williams, M. Korc, and R. L. Dormer, Am. J. Physiol. 235, E517 (1978). A. Amsterdam, T. E. Solomon, J. D. Jamieson, Methods CellBiol. 20, 361 (1978). J. D. Gardner and M. J. Jackson, J. Physiol. (London) 270, 439 (1977). s D. E. Chandler and J. A. Williams, J. Membr. Biol. 32, 201 (1977). 9 j. A. Williams, P. Cary, and B. Moffat, Am. J. Physiol. 231, 1562 (1976). to j. p. Christophe, T. P. Coulon, and J. D. Gardner, J. Biol. Chem. 251, 4629 (1976). H j. D. Gardner, T. P. Coulon, and T. D. Adams, Gastroenterology70, 29 (1976). t2 j. p. Christophe, E. K. Frandsen, T. P. Coition, G. Krishna, and J. D. Gardner, J. Biol. Chem. 251, 4640 (1976). ~3R. N. Lopatin and J. D. Gardner, Biochim. Biophys. Acta 543, 465 (1978). 14I. Schultz, Am. J. Physiol. 239, G335 (1980). ~5j. W. Putney, Jr., G. M. Burgess, S. P. Halenda, J. S. McKinney, and R. P. Rubin, Biochem. J. 212, 483 (1983).
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Preparation of Dispersed Pancreatic Acini The procedure for preparing dispersed pancreatic acini is a minor modification of the technique reported by Peikin et al. 4 Others~,6have also reported methods for preparing dispersed acini. These techniques4-6 when used with rat pancreas result in acini that show a greater than fivefold increase in enzyme secretion in response to a maximally effective concentration of cholecystoldnin. Recently a technique has been reported for preparing acini using a short digestion period, m6Unfortunately this rapid method for preparing acini gives rise to cells that show a greater than fivefold increase in enzyme secretion only in response to a maximally effective concentration of a cholecystokinin analog. Freshly prepared dispersed acini can be used for at least 5 hr in vitro and have been very useful to investigate the mechanism of action of various secretagogues. Dispersed acini have been prepared using pancreas from mice, rats, guinea pigs, or rabbits. 1. Prepare the following stock solution: NaC1 (100 mM) KC1 (6 mM) Glucose (14 mM) KH2PO4 (2.5 mM) HEPES (24.5 mM; pH 7.4) MgCl2 (1.2 mM) Sodium pyruvate (5 mM) Sodium fumarate (5 mM) Sodium glutamate (5 mM) Glutamine, (2 raM) Soybean trypsin inhibitor (0.1 mg/ml) Eagle's basal amino acid mixture [100X; 1% (v/v)] Essential vitamin mixture [ 100 X; 1% (v/v) ] 2. Prepare two separate wash solutions; Wash solution 1: Stock solution plus CaC12 (2 mM) and albumin
[4%(w/v)] Wash solution 2: Stock solution plus CaC12 (2 mM) 3. Prepare digestion solution: Stock solution plus collagenase (564 U/ rag, 0.04 mg/ml type CLSPA; Worthington Biochemical Corp., Freehold, NJ), albumin [0.2% (w/v)], and CaC12 (2 raM) 4. Prepare incubation solution: Stock solution plus CaCI2 (0.5 raM) and albumin [ 1% (w/v) ] 16R. Bruzzone, P. A. Halban, A. Gjinovci, and E. Trimble, Biochem. J. 226, 621 (1985).
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The animal is sacrificed by CO2 narcosis and the pancreas is removed, trimmed of fat and mesentery, and pinned to a way tray. Five milliliters of digestion solution is injected into the tissue using a syringe and a 25-gauge needle. The pancreas and the digestion solution are transferred to a siliconized 25-ml Edenmeyer flask, gassed for 30 see with 100% 02, capped, and incubated in a Dubnoff metabolic shaking incubator (160 oscillations/ min) at 37 ° for 10 min. After 10 min of incubation, the digestion solution is decanted and replaced with 5 ml of fresh digestion solution. The flask is gassed, capped, and placed in the incubator for 10 min at 37 °. After the second 10-min incubation, the digestion solution is again replaced with 5 ml of fresh digestion solution and the flask is gassed, capped, and placed in the incubator for 10 min at 37 °. After the third 10-min incubation, the digestion solution is decanted and replaced with 5 ml of fresh digestion solution. The flask is gassed and capped, and the tissue is disrupted by shaking the flask vigorously by hand for 15 see. After discarding the duct system and large tissue fragments, the suspension of acini is gently layered into two 15-ml conical centrifuge tubes, each containing 6 ml of wash solution. The tubes are centrifuged at 200 g for 5 see and the supernatant is discarded. The cell pellets are combined and any large clumps of tissue are removed with a Pasteur pipet. The acini are washed twice with wash solution by centrifugation (200 g for 5 see) and resuspended. After the last wash, the supernatant is discarded and the acini are suspended in an appropriate volume of incubation solution. Uses of D i s p e r s e d P a n c r e a t i c Acini Table II lists the published uses of dispersed pancreatic acini. 17-4° 17j. D. Gardner and R. T. Jensen, in "Physiologyof the Gastrointestinal Tract" (L. R. Johnson, ed.), p. 831. Raven, New York, 1981. n j. D. Gardner and R. T. Jensen, in "Physiologyof the Gastrointestinal Tract" (L. R. Johnson, ed.), 2nd Ed., p. 1109. Raven, New York, 1987. 19S. R. Hootman and J. A. Williams, in "Physiologyof the GastrointestinalTract" (L. R. Johnson, ed.), 2nd Ed. p. 1129. Raven, New York, 198q 20j. A. Williams,A. C. Bailey,and E. Roach, Am. J. Physiol. 254, G521 (1988). 21R. S. Izzo, C. Pelleeehia,and M. Praisman,Am. J. Physiol. 255, G738 (1988). 22S. A. Rosenzweig,L. J. Miller, and J. D. Jamieson,J. CellBiol. 96, 1288 (1983). 23C. D. Logsdonand J. A. Williams,Biochem. J. 223, 893 (1984). 24S. Muallem,Annu. Rev. Physiol. 51, 83 (1989). 25G. R. Gunther and J. D. Jamieson, Nature (London) 280, 318 (1979). 26j. D. Gardner and A. J. Rottman, Biochim. Biophys. Acta 627, 230 (1980). 27D. B, Burnham and J. A. Williams,Am. J. Physiol. 246, G500 (1984). 28M. Noguehi, H. Adaehi, J. D. Gardner, and R. T. Jensen, Am. J. Physiol. 248, G692 (1985). 29F. S. Goreliek, J. A. Cohn, S. D. Freedman, N. G. Delahunt, J. M. Gershouni, and J. D. Jamieson, J. CellBiol. 97, 1294 (1983).
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TABLE II USES OF DISPERSED PANCREATIC ACINI
Function measured Amylase secretion Receptor binding Ligand internalization Cytosolic calcium 45Ca2+ transport Cellular cAMP Cellular cGMP Hydrolysis of polyphosphoinositides Formation of diacylglycerol Protein kinase C activity Calmodulin-dependent protein kinase activity cAMP-Dependent protein kinase activity cGMP-Dependent protein kinase activity Phosphorylation of cellular protein Prostaglandins and leukotrienes Ion channels and membrane potential Cytosolic pH Glucose uptake Amino acid uptake [3H]Thymidine incorporation into DNA
Reference 17-19 17,18 20 - 23 24 24 18,19 25,26 19 19 27,28 27,29 27,30,31 30 32,33 19 34 35 36,37 37,38 39,40
3oR. T. Jensen and J. D. Gardner, Gastroenterology 75, 806 (1978). 31O. Holian, C. T. Bombeck, andL. M. Nyhus, Biochem. Biophys. Res. Commun. 95, 553
(1980). 32 D. B. Burnham, H.-D, S61ing, and J. A. Williams, Am. J. Physiol. 254, GI30 (1988). 33C. K. Sung and J. A. Williams, Diabetes 39, 544 (1989). 34O. H. Petersen and D. V. Gullacher, Annu. Rev. Physiol. 50, 65 (1988). 3s K. S. Carter, L. R. Rutledge, M. L. Steer, and W. Silen, Am. £ Physiol. 253, G690 (1987). 36M. Korc, J. A. Williams, and I. D. Goldfine, J. Biol. Chem. 254, 7624 (1979). 37H. Sankaran, I. D. Goldfme, A. Bailey, V. Licko, and J. A. Williams, Am. £ Physiol. 242, G250 (1982). 3s y . Iwamoto and J. A. Williams, Am. J. Physiol. 238, G440 (1980). ~9C. D. Logsdon and J. A. Williams, Am. J. Physiol. 244, G675 (1983). 40 C. D. Logsdon and J. A. grdliams, Am. J. Physiol. 250, G440 (1986).
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DISPERSED PANCREATIC ACINAR CELLS AND ACINI
277
Dispersed Pancreatic A c i n i in Culture The procedure for preparing dispersed pancreatic acini in culture is an aseptic modification of the techniques reported previously for preparing fresh dispersed pancreatic acini. This method allows one to study longterm actions of various agents. Short-term primary cultures are prepared according to a modification41 of the procedure described by Lodgson and Williams.39 The following culture medium is prepared in advance and stored at 4 °. 1 : 1 mixture of Ham's F-12 and Dulbecco's modified minimal essen-
tial medium (MEM) HEPES (10 mM; pH 7.4) Albumin (1.0 mg/ml) Soybean trypsin inhibitor (0.1 mg/ml) Ascorbic acid (0.1 raM) Epidermal growth factor (EGF) (2 nM) ITS: Insulin (5 pg/ml), transferrin (5 #g/ml), selenium (5 ng/ml) (Collaborative Research, Lexington, MA) Penicillin (100 U/ml) Streptomycin (100 #g/ml) Amphotericin B (25/~g/ml) Acini from one pancreas are suspended in 80-120 ml of culture medium and 10-ml aliquots of the suspension are placed in disposable plastic tissue culture flasks and incubated for up to 48 hr at 37 ° in a humidified atmosphere of 5% CO2 in air. Agents to be tested are added at appropriate times during the culture period. Pancreatic acini can be maintained in a differentiated state in suspension culture for up to 48 hr. The acini retain their ability to secrete enzymes, although cultured acini are less sensitive and responsive to agonists than freshly prepared acini. Pancreatic acini in short-term culture can be used to measure the same functions that have been measured using freshly prepared acini but have the advantage of permitting one to examine actions that require 1 to 2 days to became maximal.4t, 42 Long-term primary cultures (up to 14 days) have been described for pancreatic acini from mouse.4° A monolayer culture of pancreatic acini has recently been described.4° In this condition pancreatic acini first Ondergo a period of adaptation during which differentiation occurs. Next, the acinar 4t S. R. Hootman, M. E. Brown, J. A. Williams, and C. D. Logsdon, Am. J. Physiol. 251, G75 (1986). 42C. K. Sung, S. R. Hootman, E. L. Stuenkel, C. Kuroiwa, and J. A. Williams, Am. J. Physiol. 254, G242 (1988).
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cells spread and divide to form a confluent monolayer on collagen gel. These cultured cells retain their ability to respond to cholecystokinin in a trophic manner, but lack the normal content of secretory enzymes and do not show stimulation of enzyme secretion with pancreatic secretagogues. Perifusion of Pancreatic Acini
Pancreatic acini, prepared as described earlier in this chapter, are placed in a small chamber and immobilized using falters43 or gel.~,45 Incubation solution is continuously passed over the acini and the effluent is collected using a fraction collector. This procedure provides the advantage that a population of pancreatic acini can be subjected to a continuous flow of material in vitro, thereby both supplying fresh modifier and removing the products of the response, as is likely to happen in vivo. Moreover the outflow is collected continuously into fractions that are then analyzed, thereby permitting constant monitoring of the dynamics of the system. Despite the potential of this system in elucidating the kinetics of pancreatic enzyme secretion, this procedure has not been characterized extensively and used to study pancreatic acinar cell functions. This method has been used to measure amylase secretion,4a-45 45Ca2+ efflux,44 and cellular cAMP? 4 Strengths and Drawbacks of Various Preparations During the last decade dispersed pancreatic acini have been used much more extensively than acinar cells, primarily because acini are easier to prepare and show a much greater increase in enzyme secretion in response to secretagogues than do acinar cells. Table III compares the strengths and drawbacks of the various preparations.
43 K. Imamura, H. Wakasugi, H. Shinozaki, and H. Ibayashi, Jpn. J. Physiol. 33, 687 (1983). 44 E. K. Frandsen, in "Biology of Normal and Cancerous Exoerine Pancreatic Cells" (A. Ribet, L. Pradayrol, and C. Susini, eds.), p. 27. Elsevier/North-Holland Biochemical, Amsterdam, 1980. 45 p. Singh, I. Asada, A. Owlia, T. J. Collins, J. C. Thompson, Am. J. Physiol. 254, G217 (1988).
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