Localization of the type 3 inositol 1, 4, 5-trisphosphate receptor in the ...

THEJOURNAL OF B m m l c a CHEMISTRY Vol. 269, No. 7,Issue of February 18, pp. 4693-4696, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Communication Localization of the Type 3 Inositol 1,4,5-Trisphosphate Receptor in the Ca2’ Wave Trigger Zone of Pancreatic Acinar Cells* (Received for publication, November 30, 1993, and in revised form, December 21, 1993)

not been established. Since agonist-induced Ca2+L waves are initiated by inositol 1,4,5-trisphosphate (InsP&mediated Ca2+ release in pancreatic acinar cells( 6 , 7), here we used immunocytochemistry to localize the InsPs receptor(InsP3R)in thiscell for secretion in epithelia, type. Ca2+isignaling also is important Ca2+isignaling patterns but the relationship between polarized and apical secretion is unclear. Since amylase is released in a Ca2+-dependent fashion fromthe apical pole of pancreatic acin a r cells (8, 9), we also measured amylase release in permeabilized acini to examinethe effect of InsPBon apical secretion.

Michael H. NathansonSg, Michael B. FallonS, Philip J. Padfield$ and AnthonyR. Marantoll

MATERIALSANDMETHODS

Animals and Materials-MaleSprague-Dawley rats (80-100g; Camm Research Lab Animals, Wayne, NJ) were maintained on Purina From the $Liver Center, Yale University School of rodent chow under a constant light cycle and used for all experiments. Medicine, New Haven, Connecticut 06510, the InsP, was from Boehringer Mannheim, streptolysin 0 (SLO)was from Wepartment of Internal Medicine, St. Louis University Wellcome Diagnostics, and low molecular weight heparin wasfrom School of Medicine, St. Louis, Missouri 63104,a n d the Sigma. All other chemicals were of the highest quality commercially IDepartments of Medicine a n d Biomedical Research, St. available. Elizabeth’s Medical Center, n f l s University School of Antibodies-Thetype 3 InsPsR waslabeledwithanti-GST-H3CT Medicine, Boston, Massachusetts 02135 (aH3CT), an affinity-purified polyclonal rabbit antiserum raised Agonist-induced cytosolic Ca2+ (Ca2+i) signals begin as against a GST fusion protein containing the COOH-terminal 27 amino apical-to-basal Ca2+i waves in pancreatic acinar cells acids of the human type 3 InsP3R (10). For negative controls of InsP,R and in other polarized epithelia. However, the basis of labeling, tissue sections were stained with the fraction of anti-GSTthis polarized Ca2f signaling pattern is unknown. Here H3CT antiserum that bound to an affinity matrix of only the GST we use immunocytochemistry to demonstrate that the camer protein. To screen for the presence of the type 1 InsP,Rby is localized to the Western blotting, another affinity-purified antiserum, anti-GST-H1CT type 3 inositol trisphosphate receptor (aHlCT), was prepared by raising and purifying antibodies against a extremeapex of pancreaticacinarcells,theregion GST fusion protein containing the COOH-terminal 30 amino acids of which corresponds to the trigger zone from which Ca2+i the human type 1InsP,R (10).Although the fusion proteins GST-H3CT signals originate in this cell type (Kasai, H., Li, Y. X., and and GST-H1CT contain 12 identical amino acids at the NH2 ends of the Miyashita, Y. (1993)Cell 74, 669877).We also show that segments derived from their respective InsP,Rs, as shown below, the inositoltrisphosphate-mediatedCa2+releaseinduces antisera aH3CT and aHlCT are highly specific for the full-length rat (and human, data not shown) type 3 and type 1 receptors, respectively amylase release frompermeabilizedpancreaticacini. Since Ca2+i signals begin by inositol trisphosphate-me- (Fig. 1).An antiserum specific for the type 2 InsPsR was not available. The apical region of acinar cells wasidentified using antibody SG7C12, diated Ca2+ release, these findings suggest that localization of the type 3 inositol trisphosphate receptor to the a mouse monoclonal antibody raised against a secretory carrier memprotein (SCAMP) found onthe cytoplasmic side of parotid apical trigger zone is responsible for the generation of apical- brane secretory vesicles, and also on pancreatic exocrine granules (11). The to-basal Ca2+i waves, and that this organization may be apical membrane of centroacinar cells and small intralobular duct cells important for regulating apical exocytosis in pancreatic was labeled with an affinity-purifiedpolyclonal chickenantibody raised acinar cells. against a 13-amino acid peptide in the COOH-terminal region of the mouse and rat cystic fibrosis transmembrane regulator (CFTR). This CFTR antibody colocalizes in rat pancreas with antibody 1468, an afAgonist-induced cytosolic Ca2+(Ca2+i)1 signals begin as Ca2+i finity-purified polyclonal rabbit antibody directed against a COOHwaves in a wide range of cell types (1,2).In polarized epithelia, terminal peptide of human CFTR that recognizes CFTR in both human and rodent pancreas (12, 13h2 including pancreatic and lacrimal acinar cells, agonist-induced Western Analysis of InsP, ReceptorTypes in Pancreas-Immuthe apical to the basal pole (3-5). noblotting of rat pancreatic lysates was performed to identify the preCa2+i waves travel from However, the basis of this polarizedCa2+isignaling patternhas dominant type of InsP,R present in the pancreas. Pieces of tissue were homogenized on ice for 10 min in lysis buffer containing 50 m Tris* This work was supported by a Clinician-Scientist Award from the HCl, 1 mM EDTA, 1 m phenylmethylsulfonyl fluoride, and 1%Triton American Heart Association and a grantfrom the Whitaker Foundation X-100, pH 7.5. Following centrifugation at 15,000 x g for 5 min at 4 “C, (both to M.H. N.),United States Public Health Service Grants lysates were either subjected directly to SDS-polyacrylamide gel elecDK02030 (toM. B. F.) and DK44475 (to A. R. M.), the Morphology Core trophoresis (14) or first immunoprecipitated with affmity-purified Facility of the Yale Liver Center (National Institutes of Health (NIH) aH3CT as described previously 110). Nitrocellulose blots were probed Grant P30 DK349891, and the Tufts Digestive Disease Center (NIH with aHlCT or aH3CT to detect type 1or type 3 InsP,Rs, respectively. Grant P30 DK34928). The costs of publication of this article were de- Immunoreactive bands were detected by incubation with protein Afrayed in part by the payment of page charges. This article must there- horseradish peroxidase conjugate (Bio-Rad) followed by visualization fore be hereby marked “aduertisement”in accordance with 18 U.S.C. with enhanced chemiluminescence reagents (Amersham Corp.). Section 1734 solely to indicate this fact. Immune Localization of the ripe 3 InsP, Receptor in Pancreas5 To whom correspondence should be addressed: Liver Study Unit, Immunohistochemistrywas performedon 5-pm-thick frozen sections of 1080 LMP,Yale University School of Medicine, 333 Cedar St., New rat pancreas. Tissue sections were fixed and permeabilized in ice-cold Haven, CT 06510. Tel.: 203-785-4138; Fax: 203-785-7273. The abbreviations used are: Ca2+,,cytosolic Ca2+;InsP3, D-myo-ino- acetone for 10 min and then blocked with phosphate-buffered saline sitol 1,4,5-trisphosphate; InsP3R, InsP, receptor; CFTR, cystic fibrosis solution containing 1%bovine serum albumin. To determine the subtransmembrane regulator; SLO, streptolysin 0; SCAMP, secretory car- cellular distribution of the type 3 InsP,R, specimens were double-larier membrane protein; GST, glutathione S-transferase; PIPES, 1,4- beled with aH3CT and antibody SG7C12. Labeled sections were then piperazinediethanesulfonicacid; CCK, cholecystokinin;ACh, acetylcholine. * C. R. Marino, unpublished observations.

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washed and incubated with rhodamine-conjugated goat anti-rabbit antibodies (Pierce Chemical Co.) and fluorescein-conjugated goat antimouse antibodies (Cappel). Negative controls for InsP,R labeling were stained with aH3CT antiserum purified against GST alone (as described above), and then with rhodamine-conjugated goat anti-rabbit antibodies. To distinguish acinar cells from adjacent centroacinar and duct cells, selected specimens were triple-labeled with aH3CT, SG7C12, and the chicken anti-CFTR antibody. Secondary antibodies for triplelabeling were fluorescein-conjugated goat anti-mouse antibodies, rhodamine-conjugated rabbit anti-chicken antibodies, and indodicar- 250 kDa bocyanine-conjugated donkey anti-rabbit antibodies (Jackson ImmuuH3CT noresearch Laboratories). Labeled specimens were examined with a n W MRC-600 confocal microscope equipped with akryptodargon mixed gas m laser. Using a filter wheel and different emission filters on multiple 8 a detectionchannels,specimenswereseriallyexcited at 488 nm and - 260 kDa observed a t >515 nm to detect fluorescein, excited at 568 nm and obUHlCT served at >585 nm to detect rhodamine, and excited at 647 nm and observed a t >680 nm to detect cyanine. This approach eliminated bleedFIG.1. Western blot analysisof rat cerebellar and pancreatic through of fluorescein fluorescence into the rhodamine and cyanine lysates for type 1 and type3 InsP, receptors. Two companion gels channels, and of rhodamine into the cyanine channel. Double-labeled were each loaded with lysates or immunoprecipitates from lysates (iniimages were reconstructed using VoxelMath software (Vital Images) tial amount of lysate protein is indicated in parentheses), blotted onto and triple-labeled images were reconstructed using Winmerge (Bio- nitrocellulose,and probed witheitheraffinity-purifiedanti-type 3 Rad),eachwith8-bitresolution. All imagesweredisplayed on a n InsP3R antiserum (aH3CT)or affinity-purified anti-type 1 InsPaR anIRIS-4D workstation (Silicon Graphics) and photographed using a n tiserum (aH1CT). Antiserum aH3CT does not recognize the type 1 InsP,R abundant in the cerebellum, but itdoes detect and immunopreAgfa 6564 film recorder. cipitate a 250 kDa band from pancreatic lysates. Antiserum aH1CT Preparation of Pancreatic Acini-Pancreatic aciniwereprepared from male Sprague-Dawley rats (80-100 g) asdescribed previously (3, failed to detect type 1 InsP3R in whole pancreatic lysates and did not 8). Briefly, the pancreas was removed following a n overnight fast and label the 250-kDa band immunoprecipitated by aH3CT. placed in a Ca2+- and Mg2+-freebuffer (pH 7.4) containing NaCl(97 m), KC1 (5 m), glucose (20 m),HEPES (20m),soybean trypsin inhibitor type 1-specific antibody, aH1CT. In contrast, thetype 1-specific (0.1 mg/ml), and bovine serum albumin(0.1%). The pancreas was finely antibody, aHlCT, did not detect type 1 InsP3R in pancreatic diced, transferred to a siliconized flask in buffer containing CaCI, (2 lysates (Fig. 1). Theseresults,together with the fact that mM), MgCl, (1.2 mM), and collagenase (400 unitsf5 ml), and gently aH3CT does not cross-react with the260-kDa cerebellar type 1 placed in aCorex agitated for5 min at 37 "C. This pancreatic tissue was 3 InsP3R is present inrat tube and shakenby hand for -5-10 min, then filtered through 200-pm InsP3R (Fig. 11, suggest that the type nylon mesh and rinsed in collagenase-free buffer. After preparation the pancreas, and at levels much greater than those of the type 1 isolated acini were washed into a potassium glutamate (139 mM)/PIPES InsP,R. (20 m ) buffer (pH 6.6) by repeated sedimentation and resuspension. Subcellular Localization of Q p e 3 ZnsP, Receptor in PanFor each secretion experiment 50 pl of acinar suspension was added to creas by Confocal Microscopy-To localize the InsP3R within 150 pl of permeabilization buffer containing 139m potassium gluta- pancreatic acinar cells, slices of rat pancreas were double-lamate, 20 mM PIPES, 2 m MgATP, 2 m free Mg'+, and 0.5 IU/mlSLO. The concentrations of the last threecomponents are given as the final beled with antibodies against the type 3 InsP3R (H3CT) and SCAMP (SG7C12), and then with fluorescently labeled anticoncentrations after addition of the acini. The acini were then incubated bodies as described above (Fig. 2). The apical region of acinar a t 37 "C and amylase release determined after 25 min. Measurement ofAmylase Secretion-Amylase release from permeabi- cells was identified by intense SG7C12 labeling of apical secrelized acini was determined under five conditions: (a)control, no addi- tory granules (Fig. 2 4 1 , as previously reported (11).Type 3 tions to incubation, ( b ) 100 PM Inspa, ( c ) 4 m EGTA, ( d ) 100 InsP, + 4 m EGTA, and (e) 10m free Ca2++ 4 mM EGTA. The concentration InsP3R labeling was detected as narrow apical staining surof CaCl, required to give 10 PM free Ca2+ in the presence of 4 mM EGTA rounding lumenal structures (Fig. 2B ). Double-label images was calculated as described previously (8).In addition, the effect of revealed that InsP3R staining was confined to the extreme + 10 m free Ca2+on amylase release was examined apical region of acinar cells (Fig. 2C). In contrast, no specific heparin (200 pg/ml) in selected preparations. In these studies, 50 pl aliquots of acini were staining wasobserved in specimens stained with negative conpreincubated with 50 pl of permeabilization buffer containing SLO 2 trol aH3CT anti-serum (Fig. 2 0 ) . These results demonstrate heparin for 5 min. This preincubation is required toallow the heparin that pancreatic acinar cells contain the type3 InsP3R, and that to diffuse into the permeabilized acini before stimulation of enzyme the receptor is localized to the apical pole in such cells. discharge. Amylase release was then stimulated by addition of 100 pl of Pancreatic acini clusteraboutthepancreaticductsinto buffer containing 10m free Ca2+.All steps were carried outat 37 "C. Cells were then centrifuged for 2 min at 2,000 x g, and the Bemfeld which they drain, so that centroacinar cells and the terminal lysed assay (15)was usedto measure amylase in each supernatant and portions of small intralobular ducts areimmediately adjacent cell pellet a s described previously (8). Amylase release was calculated to the exocrine cells comprising each acinus(13).To determine as a fraction of the total amfrom these measurements and expressed whether InsP3R labeling was present in centroacinar and duct ylase present. Amylase release was measured in acinarcells prepared from four rats. Measurements were made in quadruplicate for each of cells as well as acinar cells, pancreas slices were triple-labeled with antibodies directed against SCAMP, the type 3 InsP3R, these preparations. Statistical Analysis-Comparisons between groups were made using and CFTR as described above (Fig. 3). The apical region of repeated-measuresanalysis of variance (ANOVA). Resultsare ex- individual acinar cells was againidentified by SG7C12 labeling pressed a s mean * S.E. of apical secretory granules (Fig. 3 A ) , while CFTR labeling

identified centroacinar and small intralobular duct cells in close proximity (Fig. 3B). InsP3R labeling (Fig. 3C) was conDetection of o p e 3 ZnsP.? Receptor in Pancreas by Western tinuous with but not identical to CFTR labeling. Triple-label Blotting-Immunoblot analysis was used to test for the pres- images demonstrated that the InsP3R was concentrated in the ence of type 1 and type 3 InsP3R in rat pancreatic lysates. On apical region of acinar cells, CFTR was concentrated in duct blots probed with thetype 3-specific antibody, aH3CT, a single cells, and weak CFTR labeling co-localized with some InsP3R band of immunoreactivity with an apparentmolecular mass of labeling, which probably represents centroacinarcells (16)(Fig. 250 kDa was identified (Fig. 1).This protein also was immu- 30). These observations further suggest that the type 3 InsPsR noprecipitated with aH3CT and did not cross-react with the in pancreas is found predominantly in the apical region of RESULTS

Qpe 3 InsP3 Receptor in Exocrine Pancreas

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concentration of Ca2+ (10mM, in the presence of 4 m~ EGTA) induced a 263 2 35% increase in amylase release relative to controls (n = 4 experiments for all groups; p < 0.0001 by repeated measuresANOVA). The effects of heparin (200 pg/ml) on Ca2+-induced amylase release were also examined, since heparin has been used to block binding of InsP3 to InsP3Rs (6, 7) and heparinalso blocks InsP3 binding t o the type 3r e ~ e p t o r . ~ I n each of two experiments, heparin caused a25-30% reduction in amylase release from acini incubated with 10 mM Ca2+.These findings suggest that InsP3induces amylase releasefrom pancreatic acini, and that this effect is mediated almost entirelyby InsP3-induced Ca2+ release. Our results also suggest that the effects of heparin on InsP3-mediated Ca2+ signals must be interpreted withcaution,since heparin inhibitedInsP3-independent, Ca2+-mediated amylase release. DISCUSSION

FIG.2. Immune co-localizationof the type3 InsP,R and apical Ca2+isignals begin as Ca2+[ wavesin epithelia. It has been secretory granulesin rat pancreas, examined by confocal fluorescence microscopy. A, distribution of apical secretory vesicles in proposed that Ca2+; waves are initiated by InsP3-mediated Ca2+ release, thenpropagate by Ca2+-induced Ca2+ release(1, acinar cells, labeled by antibody SG7C12. E , distribution of the InsP,R, labeled with antibodyHBCT. C, superimposed imagesA and B, demon- 3, 17). Such Ca2+i waves usually are initiated apically, in panstrating theapical distributionof the InsPSR in pancreatic acinar cells. creatic acinar cells and in other polarized epithelia, even in D, negative control for InsP3R staining using aH3CT anti-serum affiCa2+-free medium (3-5), although up to 25% of acetylcholine ity-purified against GST alone. Scale bar (lower right) is 25 pm. (ACh)- and cholecystokinin (CCK)-induced Ca2+i waves may begin at the basolateralpole (3). Stimulationwith low concentrations of either CCK (6) or ACh (7) induces non-propagating Ca2+j increases that are localized to the apex. Similar patterns of polarized Ca2+isignaling are seen in pancreatic acinar cells microinjected in the basolateralregion with InsP3 (6,7);apical increases inCa2+iare observed regardless of the InsP, concentration (6, 7). In addition, little (7) or no (6) basolateral Ca2+j increase is induced by 5-20 p~ InsP3, while 100 PM InsP3 induces a large basolateralCa2+;increase that coincides with the apical increase (7). The highly localized apical region from which InsP3-mediated Ca2+; signals originate in isolated pancreatic acinar cells has been called the trigger zone (7). Since = ', the InsP3R forms the Ca2+ channel complex which gates the release of Ca2+ from InsP3-sensitive Ca2+ stores (17), InsP3mediated Ca2+isignals would be expected to begin at the same site as the receptor. Therefore, the finding in the current work FIG.3. Co-localizationof apical secretorygranules, thecystic that thetype 3 InsP3R is ina highly localized region in the apex fibrosis transmembrane regulator, and thetype 3 InsP,R in rat pancreas, examined by confocal fluorescence microscopy. A, dis- of pancreatic acinar cells strongly suggests that Ca2+isignals tribution of apical secretoryvesicles in acinarcells, labeled by antibody begin in the trigger zone in these cells because the InsP3-senSG7C12 with a fluorescein-conjugated secondary antibody. Scale bar sitive Ca2+stores associated with the type 3 receptor are local(lower right) is 10 p m . B,distribution of CFTR along the apical mem- ized there. Immunohistochemical evidence in rat intestinal brane of centroacinar and duct cells, which are adjacent to the acinar cells. Intense staining identifies intralobular duct cells, while weaker mucosa (10) and preliminary evidence in rat liver (18)demonstaining probably corresponds t o centroacinar cells. C, distribution of strates that theInsP,R are located in theapex of these epithethe InsP,R in the apical region of acinar cells, labeled with antibody lial cell types as well. Thus, currentevidence suggests that the H3CT and an indodicarbocyanine-conjugated secondary antibody. D, apical localization of type 3 InsP3Rs, and therefore polarized superimposed images A,E , and C , demonstrating that the InsP,R is concentrated at the extreme apex of the SCAMP-labeled acinar cells Ca2+zsignaling patterns, may be a general featureof polarized epithelia. Since high concentrationsofACh, CCK, or InsP3 may and not in adjacent ductcells labeled with CFTR. induce Ca2+i increases that begin basolaterally (3, 7), InsP3acinar cells and excluded from adjacent ducts which drain the sensitive Ca2+ stores that display a lower sensitivity to InsP, acinus. may be localized to that region (7). The current work suggests InsP, Mediates Amylase Release from Permeabilized Pancre- that type 1 InsP3Rs are not in pancreas, but the presence and atic Acini-Increases in Ca2+i can induce amylase releasefrom subcellular distribution of other receptor subtypes in pancreas has not been established (19, 20). Of note, most hormone repancreaticacini (8, 9), and stimuli that increase InsP3 are associated with increased release of amylase. To examine ceptors are located on the basolateralcell surface, so that phoswhether InsP3-mediated Ca2+ release is potentially responsible pholipase C-mediated InsP3 production would be expected to Apical localization for amylase secretion, we examined amylase release from per- occur at or near the basolateral membrane. meabilized acini, an assaysystem in which the effects of mem- of the InsP3R thus implies that InsP3 must diffuse from the basolateral to theapical pole in order to initiateCa2+i signalbrane-impermeant cytosolic messengers on amylaserelease can be observed directly (8, 9). InsP, (100 p ~ increased ) amy- ing. The diffusional range of InsP3 within the cytosol is up to25 lase release by 71 2 16% relative t o unstimulated acini, while p m (21),which is consistentwith this proposed model of InsP3this increase was reduced to only 8 2 6% above control values mediated Ca2+i signaling mechanisms. in acini incubated with InsP3 in thepresence of the Ca2+chelator EGTA (4 mM). For comparison, a maximally stimulatory A. R. Maranto, unpublished observations.

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What is the functional significance of the polarized distribu- fluid and electrolyte secretion by subcellular Ca2+i signals in tion of InsPsRs in pancreatic acinar cells? It is known that Ca2+j pancreatic acinar cells and other epithelia. is involved in the regulation of secretion in manycell types. The Acknowledgments-We thank J. David Castle for providing antibody apical location of the type3 InsP3R in acinarcells suggests that SG7C12 and Christopher R. Marino for uCFTR antibodies. We also local increases in Caz+j may be important in regulating the po- thank Philippe Male for expert photographic assistance. larized pattern of exocytosis and fluid secretionin thiscell type. REFERENCES Localized subplasmalemmal increases in Ca2+j can induce exo1 . Bemdge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205 cytosis in other cell types, as demonstrated by the release of 2. Jaffe, L. F. (1991)Proc. Natl. Acad. Sci. [I. S.A. 88,9883-9887 synaptic vesicles from neurons (22)and catecholamine secretion 3. Nathanson, M. H., Padfield, P. J., OSullivan, A. J., Burgstahler, A.D., and Jamieson, J. D. (1992) J. Eiol. Chem. 267,18118-18121 by adrenal chromaEncells (23,24).Increases in Ca2+i also can 4. Kasai, H., and Augustine, G. J. (1990)Nature 348, 73L738 induce release of amylase from pancreatic acini ( 8 ) . Further5. lbescu, E. C., Lawrie, A. M., Petersen, 0. H., and Gallacher, D. V.(1992) EMEO J. 11, 1623-1629 p ~ or) CCK (