Produção Nacional
Revisado por pares
The Spatial Distribution of Inositol 1,4,5-Trisphosphate Receptor Isoforms Shapes Ca2+ Waves
2007; Elsevier BV; Volume: 282; Issue: 13 Linguagem: Inglês
10.1074/jbc.m700746200
ISSN1083-351X
AutoresErick Hernández, M. Fátima Leite, Mateus T. Guerra, Emma A. Kruglov, Oscar Bruña‐Romero, Michele Ângela Rodrigues, Dawidson Assis Gomes, Frank J. Giordano, Jonathan A. Dranoff, Michael H. Nathanson,
Tópico(s)Cellular transport and secretion
ResumoCytosolic Ca2+ is a versatile second messenger that can regulate multiple cellular processes simultaneously. This is accomplished in part through Ca2+ waves and other spatial patterns of Ca2+ signals. To investigate the mechanism responsible for the formation of Ca2+ waves, we examined the role of inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms in Ca2+ wave formation. Ca2+ signals were examined in hepatocytes, which express the type I and II InsP3R in a polarized fashion, and in AR4-2J cells, a nonpolarized cell line that expresses type I and II InsP3R in a ratio similar to what is found in hepatocytes but homogeneously throughout the cell. Expression of type I or II InsP3R was selectively suppressed by isoform-specific DNA antisense in an adenoviral delivery system, which was delivered to AR4-2J cells in culture and to hepatocytes in vivo. Loss of either isoform inhibited Ca2+ signals to a similar extent in AR4-2J cells. In contrast, loss of the basolateral type I InsP3R decreased the sensitivity of hepatocytes to vasopressin but had little effect on the initiation or spread of Ca2+ waves across hepatocytes. Loss of the apical type II isoform caused an even greater decrease in the sensitivity of hepatocytes to vasopressin and resulted in Ca2+ waves that were much slower and delayed in onset. These findings provide evidence that the apical concentration of type II InsP3Rs is essential for the formation of Ca2+ waves in hepatocytes. The subcellular distribution of InsP3R isoforms may critically determine the repertoire of spatial patterns of Ca2+ signals. Cytosolic Ca2+ is a versatile second messenger that can regulate multiple cellular processes simultaneously. This is accomplished in part through Ca2+ waves and other spatial patterns of Ca2+ signals. To investigate the mechanism responsible for the formation of Ca2+ waves, we examined the role of inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms in Ca2+ wave formation. Ca2+ signals were examined in hepatocytes, which express the type I and II InsP3R in a polarized fashion, and in AR4-2J cells, a nonpolarized cell line that expresses type I and II InsP3R in a ratio similar to what is found in hepatocytes but homogeneously throughout the cell. Expression of type I or II InsP3R was selectively suppressed by isoform-specific DNA antisense in an adenoviral delivery system, which was delivered to AR4-2J cells in culture and to hepatocytes in vivo. Loss of either isoform inhibited Ca2+ signals to a similar extent in AR4-2J cells. In contrast, loss of the basolateral type I InsP3R decreased the sensitivity of hepatocytes to vasopressin but had little effect on the initiation or spread of Ca2+ waves across hepatocytes. Loss of the apical type II isoform caused an even greater decrease in the sensitivity of hepatocytes to vasopressin and resulted in Ca2+ waves that were much slower and delayed in onset. These findings provide evidence that the apical concentration of type II InsP3Rs is essential for the formation of Ca2+ waves in hepatocytes. The subcellular distribution of InsP3R isoforms may critically determine the repertoire of spatial patterns of Ca2+ signals. Cytosolic Ca2+ exerts wide ranging effects as a second messenger (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell Biol. 2000; 1: 11-21Crossref PubMed Scopus (4395) Google Scholar). In hepatocytes Ca2+ regulates such diverse functions as bile secretion, glucose release, cell metabolism, gene transcription, and apoptosis (2Leite M.F. Nathanson M.H. Arias I.M. The Liver: Biology and Pathobiology. Lippincott, Williams, and Wilkins, Philadelphia2001: 537-554Google Scholar). Spatial patterns of Ca2+ signals, such as Ca2+ waves, help encode the information that is responsible for such regulation (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell Biol. 2000; 1: 11-21Crossref PubMed Scopus (4395) Google Scholar, 2Leite M.F. Nathanson M.H. Arias I.M. The Liver: Biology and Pathobiology. Lippincott, Williams, and Wilkins, Philadelphia2001: 537-554Google Scholar). Ca2+ waves have specifically been implicated in the regulation of such processes as fluid and electrolyte secretion, exocytosis, cell-cell communication, and morphogenesis (3Gilland E. Miller A.L. Karplus E. Baker R. Webb S.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 157-161Crossref PubMed Scopus (110) Google Scholar, 4Kasai H. Augustine G.J. Nature. 1990; 348: 735-738Crossref PubMed Scopus (313) Google Scholar, 5Ito K. Miyashita Y. Kasai H. EMBO J. 1997; 16: 242-251Crossref PubMed Scopus (126) Google Scholar, 6Nathanson M.H. Burgstahler A.D. Mennone A. Fallon M.B. Gonzalez C.B. Saez J.C. Am. J. Physiol. 1995; 269: G167-G171Crossref PubMed Google Scholar, 7Robb-Gaspers L.D. Thomas A.P. J. Biol. Chem. 1995; 270: 8102-8107Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). In hepatocytes, Ca2+ wave formation is due entirely to the inositol 1,4,5-trisphosphate (InsP3) 2The abbreviations used are: InsP3, inositol 1,4,5-trisphosphate; InsP3R, inositol 1,4,5-trisphosphate receptor; ACh, acetylcholine; pfu, plaque-forming units; EGFP, enhanced green fluorescent protein; m.o.i., multiplicity of infection; ANOVA, analysis of variance. 2The abbreviations used are: InsP3, inositol 1,4,5-trisphosphate; InsP3R, inositol 1,4,5-trisphosphate receptor; ACh, acetylcholine; pfu, plaque-forming units; EGFP, enhanced green fluorescent protein; m.o.i., multiplicity of infection; ANOVA, analysis of variance. receptor (InsP3R), because that is the only intracellular Ca2+ release channel in this cell type (8Hirata K. Pusl T. O'Neill A.F. Dranoff J.A. Nathanson M.H. Gastroenterology. 2002; 122: 1088-1100Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and because Ca2+ waves in hepatocytes do not rely on influx of extracellular Ca2+ (9Nathanson M.H. Burgstahler A.D. Fallon M.B. Am. J. Physiol. 1994; 267: G338-G349Crossref PubMed Google Scholar). However, it is not entirely understood how InsP3Rs regulate the formation and spread of Ca2+ waves. There are three isoforms of the InsP3R, each with distinct biophysical properties (10Bezprozvanny I. Watras J. Ehrlich B.E. Nature. 1991; 351: 751-754Crossref PubMed Scopus (1430) Google Scholar, 11Hagar R.E. Burgstahler A.D. Nathanson M.H. Ehrlich B.E. Nature. 1998; 396: 81-84Crossref PubMed Scopus (228) Google Scholar, 12Ramos-Franco J. Fill M. Mignery G.A. Biophys. J. 1998; 75: 834-839Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). There is also variability in the expression and subcellular distribution of each isoform among different cell and tissue types (8Hirata K. Pusl T. O'Neill A.F. Dranoff J.A. Nathanson M.H. Gastroenterology. 2002; 122: 1088-1100Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 13Hirata K. Dufour J.-F. O'Neill A.F. Bode H.-P. Cassio D. St-Pierre M.V. LaRusso N.F. Leite M.F. Nathanson M.H. Hepatology. 2002; 36: 284-296Crossref PubMed Scopus (69) Google Scholar, 14Hirata K. Nathanson M.H. Burgstahler A.D. Okazaki K. Mattei E. Sears M.L. Investig. Ophthalmol. Vis. Sci. 1999; 40: 2046-2053PubMed Google Scholar, 15Sugiyama T. Yamamoto-Hino M. Wasano K. Mikoshiba K. Hasegawa M. J. Histochem. Cytochem. 1996; 44: 1237-1242Crossref PubMed Scopus (26) Google Scholar, 16Yule D.I. Ernst S.A. Ohnishi H. Wojcikiewicz R.J.H. J. Biol. Chem. 1997; 272: 9093-9098Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Hepatocytes express only the type I and the type II isoforms of the InsP3R (8Hirata K. Pusl T. O'Neill A.F. Dranoff J.A. Nathanson M.H. Gastroenterology. 2002; 122: 1088-1100Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In addition, the type I InsP3R is distributed relatively uniformly throughout the hepatocyte, whereas the type II isoform is concentrated in the apical region (8Hirata K. Pusl T. O'Neill A.F. Dranoff J.A. Nathanson M.H. Gastroenterology. 2002; 122: 1088-1100Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). It has been hypothesized that the expression and subcellular distribution of each isoform determines the spatial patterns of Ca2+ signals in cells. Because the type II InsP3R is the isoform with the highest affinity for InsP3 (17Newton C.L. Mignery G.A. Südhof T.C. J. Biol. Chem. 1994; 269: 28613-28619Abstract Full Text PDF PubMed Google Scholar), it has been suggested that Ca2+ waves begin in the apical region of hepatocytes because the type II isoform is concentrated there (8Hirata K. Pusl T. O'Neill A.F. Dranoff J.A. Nathanson M.H. Gastroenterology. 2002; 122: 1088-1100Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The purpose of this study was to determine whether the subcellular distribution of InsP3R isoforms is important for organizing the pattern of Ca2+ waves. Materials—Acetylcholine (ACh), arginine-vasopressin, bovine serum albumin, and penicillin-streptomycin were obtained from Sigma. Dulbecco's modified Eagle's medium and Liebowitz 15 (L-15) medium were from Invitrogen. Fluo-4/acetoxymethyl ester, TO-PRO-3, and rhodamine-conjugated phalloidin were from Molecular Probes (Eugene, OR). All other reagents were of the highest quality commercially available. Cloning of Rat cDNA Sequences for Type I, II, and III Isoforms of the InsP3R—The cDNA for the type I and type II isoforms of the InsP3R was synthesized by reverse transcription-PCR using template RNA isolated from the brain and the liver of rats, respectively. RNA from RIN cells was used as a template for the cDNA of the type III isoform. The RNA was isolated by the RNAqueous™ kit (Ambion Inc., Austin, TX). The sequences for each forward primer contained an XbaI site, and the sequence for each reverse primer contained a HindIII site. Sequences were designed to be ∼200 bp in length in order to maximize their stability upon production by adenovirus. Vector NTI software (Invitrogen) was used to identify regions of each isoform that had minimal (16-19%) homology with the other two isoforms (Fig. 1). This approach was taken so that each of the three antisense sequences would have little likelihood of inhibiting expression of InsP3R isoforms in a nonspecific fashion. The resulting primers for each of the three isoforms were as follows: (a) type I isoform 5′ (5′-aagctttACATCTGCAGGGCCTGTAACAA-3′, position 4699-4720) and type I isoform 3′ (5′-ctagaGCAGTGGTAAACTCGGAACACG-3′, position 4873-4895), which generates a 196-bp cDNA fragment; (b) type II isoform 5′ (5′-agctttTTCTCAGCCCTCCTTTGGGTAG-3′, position 6787-6809) and type II isoform 3′ (5′-tctagaTTCCCACAAAACTCACCAGGA-3′, position 6964-6986), which generates a 199-bp cDNA fragment; (c) type III isoform 5′ (5′-aagctttTGTGGGGTAGCATCTCCTTCAA-3′, position 6739-6761) and type III isoform 3′(5′-tctagaGAAGGACCAGTGCCACAATGAG-3′, position 6920-6942), which generates a 203-bp cDNA fragment. The obtained cDNA sequence for each isoform was then subcloned into the dual promoter (pCR II) vector (Invitrogen) for amplification, and then the identities were confirmed by direct sequencing. Antisense Oligonucleotides and Adenovirus Construction—Each cDNA was cloned into an adenovirus vector in an antisense orientation as described previously (18Lu C.Y. Giordano F.J. Rogers K.C. Rothman A. J. Mol. Cell. Cardiol. 1996; 28: 1703-1713Abstract Full Text PDF PubMed Scopus (7) Google Scholar). Briefly, the cDNA sequence for each InsP3R isoform was directionally sub-cloned into the adenoviral shuttle vector pCMVPLPASR, between the HindIII and XbaI polycloning sites in an antisense orientation, and each of the three antisense sequences was confirmed by sequencing. This construct was co-transfected with the plasmid pJM17 into 293 cells. Homologous recombination resulted in adenoviral particles expressing the antisense construct (19McGrory W.J. Bautista D.S. Graham F.L. Virology. 1988; 163: 614-617Crossref PubMed Scopus (549) Google Scholar). The resultant adenoviruses were tested by PCR to ensure that each expressed the correct sequence. Individual viral plaques were isolated and amplified, and then the recombinant adenoviruses were purified and concentrated using CsCl step gradients followed by dialysis against 10% glycerol in phosphate-buffered saline. Viral stocks consisting of 8 × 1010 plaque-forming units (pfu)/ml for adenoviral antisense for the type I InsP3R, 7 × 1010 pfu/ml for the type II receptor, and 5 × 1010 pfu/cell for the type III receptor were produced after four rounds of amplification for each construct. Viruses were then stored in aliquots at -80 °C in buffer with 10% glycerol. DsRed and enhanced green fluorescent protein (EGFP) (Clontech) were used to form adenoviral DsRed and EGFP constructs, respectively. These were amplified and purified as described above and then used to monitor the efficacy of infection. Infection Conditions—Both cell lines and animals were used for adenoviral studies. AR4-2J pancreatoma and RIN insulinoma cell lines (ATCC, Manassas, VA) were cultured in high glucose Dulbecco's modified Eagle's medium with 10% calf serum and antibiotics. Cells were seeded onto culture dishes at a density of 8 × 105 cells/dish. After an initial incubation period of 24 h, the cells were infected with adenovirus at a multiplicity of infection (m.o.i.) of 40 followed by a further 24-48 h of incubation. Male Sprague-Dawley rats (200-225 g; Charles River Breeding Laboratories) were used for all animal studies. For adenoviral studies, animals were anesthetized with 4% pentobarbital (0.4 ml intraperitoneally) and then adenoviral constructs (5 × 109 pfu/ml) were injected via the portal vein, which was exposed by laparotomy. After repair of the surgical wound, animals were allowed to recover and then were sacrificed 48 h later. At the time of sacrifice, livers were used for immunofluorescence or else used to isolate hepatocyte couplets and triplets as described below. All animal experiments were performed under the guidelines of the Yale University IACUC. Immunoblotting—Immunoblots were used to test the efficacy of adenoviral antisense constructs and were performed as described previously (8Hirata K. Pusl T. O'Neill A.F. Dranoff J.A. Nathanson M.H. Gastroenterology. 2002; 122: 1088-1100Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 13Hirata K. Dufour J.-F. O'Neill A.F. Bode H.-P. Cassio D. St-Pierre M.V. LaRusso N.F. Leite M.F. Nathanson M.H. Hepatology. 2002; 36: 284-296Crossref PubMed Scopus (69) Google Scholar). Briefly, cells were lysed at 4 °C with lysis buffer; the lysate underwent centrifugation, and the protein concentration of the supernatant was determined spectrophotometrically. Twenty five μg of total cellular protein was separated by SDS-PAGE using a 7.5% polyacrylamide gel. Membranes were blocked with nonfat milk and then incubated at room temperature with InsP3R isoform-specific antibodies (8Hirata K. Pusl T. O'Neill A.F. Dranoff J.A. Nathanson M.H. Gastroenterology. 2002; 122: 1088-1100Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 13Hirata K. Dufour J.-F. O'Neill A.F. Bode H.-P. Cassio D. St-Pierre M.V. LaRusso N.F. Leite M.F. Nathanson M.H. Hepatology. 2002; 36: 284-296Crossref PubMed Scopus (69) Google Scholar). An affinity-purified polyclonal antibody directed against the C terminus of the type I isoform of the InsP3R (11Hagar R.E. Burgstahler A.D. Nathanson M.H. Ehrlich B.E. Nature. 1998; 396: 81-84Crossref PubMed Scopus (228) Google Scholar) was used at a dilution of 1:1000, an affinity-purified polyclonal antibody against the C terminus of the type II isoform (20Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar) was used at 1:100, and a monoclonal antibody against the N terminus of the type III isoform (11Hagar R.E. Burgstahler A.D. Nathanson M.H. Ehrlich B.E. Nature. 1998; 396: 81-84Crossref PubMed Scopus (228) Google Scholar) was used at a dilution of 1:500. Membranes were washed and incubated with peroxidase-conjugated secondary antibodies, and then protein-antibody conjugates were detected by enhanced chemiluminescence (Amersham Biosciences). Immunofluorescence—Confocal immunofluorescence to detect the subcellular distribution of InsP3R isoforms was performed as described previously (8Hirata K. Pusl T. O'Neill A.F. Dranoff J.A. Nathanson M.H. Gastroenterology. 2002; 122: 1088-1100Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 13Hirata K. Dufour J.-F. O'Neill A.F. Bode H.-P. Cassio D. St-Pierre M.V. LaRusso N.F. Leite M.F. Nathanson M.H. Hepatology. 2002; 36: 284-296Crossref PubMed Scopus (69) Google Scholar). Briefly, frozen rat liver sections were fixed in 4% formaldehyde, followed by tissue permeabilization in 0.5% Triton X-100. After blocking steps, the liver sections were incubated with primary antibody against specific InsP3R isoforms and then rinsed with phosphate-buffered saline and 1% bovine serum albumin. The specimens were then incubated with Alexa 488 secondary antibody (Molecular Probes) and co-labeled with rhodamine-conjugated phalloidin (Molecular Probes) to facilitate the recognition of the apical and the basolateral pole of hepatocytes (8Hirata K. Pusl T. O'Neill A.F. Dranoff J.A. Nathanson M.H. Gastroenterology. 2002; 122: 1088-1100Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 9Nathanson M.H. Burgstahler A.D. Fallon M.B. Am. J. Physiol. 1994; 267: G338-G349Crossref PubMed Google Scholar). For AR4-2J cells, the isolated cells were seeded onto glass coverslips and incubated for 48 h at 37 °C, then fixed with 4% formaldehyde, and permeabilized with 0.5% Triton X-100. Primary and secondary antibodies were the same as used for liver immunofluorescence, and TO-PRO-3 (Molecular Probes) was used to label the nucleus of the cultured cells. An MRC-1024 confocal microscope (Bio-Rad) was used for all imaging studies. Images were obtained by excitation at 488 nm and observation at 505-550 nm to detect Alexa 488. Tissue specimens were excited at 543 nm and observed at >585 nm to detect rhodamine phalloidin, whereas cells in culture were excited at 647 nm and observed at >680 nm to detect TO-PRO-3. Isolation of Hepatocytes—Isolated rat hepatocyte couplets and triplets were used for single cell imaging, because these cells maintain structural and functional polarity in short term culture (8Hirata K. Pusl T. O'Neill A.F. Dranoff J.A. Nathanson M.H. Gastroenterology. 2002; 122: 1088-1100Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 9Nathanson M.H. Burgstahler A.D. Fallon M.B. Am. J. Physiol. 1994; 267: G338-G349Crossref PubMed Google Scholar, 21Graf J. Gautam A. Boyer J.L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6516-6520Crossref PubMed Scopus (129) Google Scholar). Cells were isolated in the Cell Isolation Core of the Yale Liver Center, as described previously (8Hirata K. Pusl T. O'Neill A.F. Dranoff J.A. Nathanson M.H. Gastroenterology. 2002; 122: 1088-1100Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 9Nathanson M.H. Burgstahler A.D. Fallon M.B. Am. J. Physiol. 1994; 267: G338-G349Crossref PubMed Google Scholar, 21Graf J. Gautam A. Boyer J.L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6516-6520Crossref PubMed Scopus (129) Google Scholar). Briefly, rat livers were perfused with Hanks' A and then Hanks' B medium containing 0.05% collagenase (Roche Applied Science) and 0.8 units of trypsin inhibitor (Sigma) per unit of tryptic activity. Livers were minced and passed through serial nylon mesh filters, and the resultant cells were washed. Isolated hepatocytes were resuspended in L-15 medium with 50 units of penicillin and 50 mg of streptomycin. The cells were then seeded onto glass coverslips and incubated at 37 °C for 2-4 h before used. Measurement of Cytosolic Ca2+—Cytosolic Ca2+ was monitored in individual cells and subcellular regions by time lapse confocal microscopy as described previously (8Hirata K. Pusl T. O'Neill A.F. Dranoff J.A. Nathanson M.H. Gastroenterology. 2002; 122: 1088-1100Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 11Hagar R.E. Burgstahler A.D. Nathanson M.H. Ehrlich B.E. Nature. 1998; 396: 81-84Crossref PubMed Scopus (228) Google Scholar, 13Hirata K. Dufour J.-F. O'Neill A.F. Bode H.-P. Cassio D. St-Pierre M.V. LaRusso N.F. Leite M.F. Nathanson M.H. Hepatology. 2002; 36: 284-296Crossref PubMed Scopus (69) Google Scholar, 22Hirata K. Nathanson M.H. Sears M.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8381-8386Crossref PubMed Scopus (32) Google Scholar). Briefly, AR4-2J cells or isolated rat hepatocytes were incubated with Fluo-4/acetoxymethyl ester (6 μm) for 30 min at 37 °C. Coverslips seeded with the cells were transferred to a custombuilt perfusion chamber on the stage of an MRC-1024 confocal microscope (Bio-Rad), and the cells were then perfused with HEPES-buffered solution. Fluo-4 was excited at 488 nm and observed at 505-550 nm. In most experiments a ×63 objective was used to observe the cells, but a ×20 objective was used in a limited series of studies to monitor population responses to agonists. Increases in Ca2+ were expressed as percent increase in Fluo-4 fluorescence intensity. Statistics—All results are expressed as mean ± S.D. Student's t test was used for comparisons between groups, whereas repeated measures ANOVA was used for comparisons among larger groups. A p value less than 0.05 was used to indicate a statistically significant difference. GraphPad Prism software (San Diego, CA) was used for all statistical tests. Adenoviral Antisense Can Selectively Decrease Expression of Specific InsP3R Isoforms—Antisense sequences were designed for regions of each InsP3R isoform with little homology to the other two isoforms (Fig. 1), and an adenoviral delivery system was used. AR4-2J cells were used as a tool to examine the efficacy of the adenoviral antisense constructs because these cells, like hepatocytes, are epithelia that almost exclusively express the types I and II InsP3R (20Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). In addition, hepatocytes and AR4-2J cells express these two isoforms in similar proportions (20Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar), and AR4-2J cells can be induced to differentiate into a hepatocyte phenotype (23Shen C.N. Slack J.M.W. Tosh D. Nat. Cell Biol. 2000; 2: 879-887Crossref PubMed Scopus (358) Google Scholar). Confocal immunofluorescence confirmed that AR4-2J cells express the type I and II isoforms of the InsP3R (Fig. 2). Both isoforms were distributed uniformly throughout the cytosol (Fig. 2, A and D). The cells were colabeled with the nuclear stain TO-PRO-3 (Fig. 2, B and E), which suggested the presence of the type I isoform of the InsP3R in the nucleus as well (Fig. 2C), although the nuclear labeling may have been nonspecific. There was minimal nuclear expression of the type II isoform (Fig. 2F). Thus, the cytosolic distribution of these two receptor isoforms was similar in AR4-2J cells. The efficacy of the isoform-specific adenoviral antisense constructs was determined in several steps. First, the efficiency of adenoviral infection was optimized by infecting cells with an adenovirus-DsRed construct. This allowed visual confirmation of successful infection. The efficiency of infection of AR4-2J cells was 100% using an m.o.i. of 40 (not shown), so this m.o.i. was used for infection with antisense constructs as well. Immunoblotting was then performed to analyze the effects of each adenoviral antisense construct. Treatment of AR4-2J cells with the adenoviral antisense construct for the type I InsP3R decreased the expression of the type I InsP3R (Fig. 3A) but not the type II InsP3R (Fig. 3B). Similarly, treatment of these cells with the construct for the type II InsP3R decreased the expression of the type II InsP3R (Fig. 3C), without affecting the expression of the type I InsP3R (Fig. 3D). In each case, expression of the respective isoform decreased markedly by 24 h post-infection and remained low after 48 h. Cells infected with an m.o.i. of less than 40 exhibited less pronounced decreases in the expression of the corresponding InsP3R isoform (not shown). Expression of type I and type II InsP3R was not inhibited by infection with adenovirus construct for DsRed (Fig. 3, A and C), demonstrating that the inhibition was not a nonspecific effect of adenoviral infection. Finally, the efficacy of the adenoviral antisense construct for the type III InsP3R was examined. RIN cells were used for these experiments because this cell type nearly exclusively expresses the type III isoform (11Hagar R.E. Burgstahler A.D. Nathanson M.H. Ehrlich B.E. Nature. 1998; 396: 81-84Crossref PubMed Scopus (228) Google Scholar, 20Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). The expression of this isoform in RIN cells was decreased only slightly 24 h after infection but decreased dramatically 48 h after infection (Fig. 3E). The basis for the longer time interval needed to decrease expression of this isoform is unclear. The type III InsP3R is degraded as quickly as the other two InsP3R isoforms following stimulation of phosphoinositide hydrolysis (20Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). However, InsP3R turnover is much slower in nonstimulated cells (24Joseph S.K. J. Biol. Chem. 1994; 269: 5673-5679Abstract Full Text PDF PubMed Google Scholar), so it is possible that the type III isoform has a longer half-life than the other two isoforms. These results demonstrate that each of the three adenoviral antisense constructs effectively inhibits expression of the corresponding InsP3R isoform.FIGURE 3Efficacy and specificity of adenoviral antisense constructs for each InsP3R isoform. A, effect of type I antisense (AS) on expression of the type I InsP3R in AR4-2J cells. The adenoviral construct markedly decreases expression of the type I isoform both 24 and 48 h after infection (left and right lanes, respectively), relative to uninfected control cells (ctrl). In contrast, expression of this isoform was not decreased 24 or 48 h after infection with the adenoviral DsRed construct (left and right lanes, respectively). B, expression of the type II isoform is not decreased 24 or 48 h after infection with the antisense construct for the type I InsP3R (left and right lanes, respectively), relative to uninfected control cells. C, effect of type II antisense on expression of the type II InsP3R in AR4-2J cells. The adenoviral construct markedly decreases expression of the type II isoform both 24 and 48 h after infection (left and right lanes, respectively), relative to uninfected control cells. In contrast, expression of this isoform was not decreased 24 or 48 h after infection with the adenoviral DsRed construct. D, expression of the type I isoform is not decreased 24 or 48 h after infection with the antisense construct for the type II InsP3R (left and right lanes, respectively), relative to uninfected control cells. E, effect of type III antisense on expression of the type III InsP3R in RIN cells. The adenoviral construct markedly decreases expression of the type III isoform 48 h (right) but not 24 h (left) after infection, relative to uninfected control cells. Each immunoblot was performed using 25 μg of protein/lane, and cells were infected with each adenovirus at a concentration of 5 × 1010pfu/ml (m.o.i. = 40).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Type I and II InsP3Rs Both Contribute to Ca2+ Signals in AR4-2J Cells—To investigate the relative contribution of the type I and II InsP3R isoforms to Ca2+ signaling in AR4-2J cells, the cells were infected with adenoviral constructs as described above. Cells were then stimulated with ACh (10 μm) to induce an InsP3-mediated increase in cytosolic Ca2+ (Fig. 4A). The amplitude of the ACh-induced Ca2+ signal was reduced by 75% in cells treated with adenoviral antisense for either the type I or type II InsP3R (p < 10-10 relative to uninfected controls, n = 20-22 cells in each group) (Fig. 4, A and B). Infection with the adenovirus DsRed construct did not affect the amplitude of the ACh-induced Ca2+ signal (p = 0.12), suggesting that the inhibition was not a nonspecific effect of adenoviral infection (n = 20 in both the noninfected and DsRed group) (Fig. 4B). The amplitude of the ACh-induced Ca2+ signal was not reduced significantly in cells treated with adenoviral antisense for either the type I or II InsP3R if cells were stimulated with lower concentrations of ACh (Fig. 4C). To characterize the effects of InsP3R isoforms on Ca2+ signals more completely, the relationship between ACh concentration and percent of responding cells (Fig. 4D) and time delay before a Ca2+ signal begins (Fig. 4E) were examined. The percent of cells responding to ACh was lower than controls in cells treated with adenoviral antisense for either the type I or II InsP3R, but only at minimal concentrations of ACh (Fig. 4D). Similarly, the time delay before the onset of ACh-induced Ca2+ signals was lower in controls than in cells treated with adenoviral antisense for either the type I or II InsP3R. The time delay was prolonged in cells expressing either type of antisense and stimulated with lower ACh concentrations, but the time delay persisted at higher ACh concentrations only in those cells expressing antisense for the type II InsP3R (Fig. 4E). These findings demonstrate that the loss of InsP3Rs induced by the adenoviral constr
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