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• W2004603227 abstract "Activation of phospholipase C (PLC) in neonatal rat cardiomyocytes (NCM) generates primarily inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in response to rises in intracellular Ca2+, or inositol 1,4-bisphosphate (Ins(1,4)P2) in response to norepinephrine (NE) (Matkovich, S. J. and Woodcock, E. A. (2000)J. Biol. Chem. 275, 10845–10850). To examine the PLC subtype mediating the α1-adrenergic receptor response, PLC-β1 and PLC-β3 were overexpressed in NCM using adenoviral infection (Ad-PLC-β1 NCM and Ad-PLC-β3 NCM, respectively) and PLC responses assessed from [3H]inositol phosphate (InsP) generation in the presence of 10 mm LiCl. The [3H]InsP response to NE (100 μm) was enhanced in Ad-PLC-β1NCM relative to cells infected with blank virus (Ad-MX NCM), but was reduced in Ad-PLC-β3 NCM. In contrast, the [3H]InsP response to ATP (100 μm) was not elevated in Ad-PLC-β1 NCM, and was enhanced rather than diminished in Ad-PLC-β3 NCM, showing that effects of the two PLC-β isoforms were specific for particular receptor types. PLC-δ1 overexpression selectively reduced NE-induced [3H]InsP responses, without affecting the ATP stimulation. The reduced NE response was associated with a selective loss of PLC-β1 expression in Ad-PLC-δ1 NCM. α1-Adrenergic receptor activation caused phosphorylation of PLC-β1 but not PLC-β3, whereas stimulation by ATP induced phosphorylation of PLC-β3 but not PLC-β1. Taken together, these studies provide evidence that NE-stimulated InsP generation in NCM is primarily mediated by PLC-β1, despite the presence of both PLC-β1 and PLC-β3 isoforms. Activation of phospholipase C (PLC) in neonatal rat cardiomyocytes (NCM) generates primarily inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in response to rises in intracellular Ca2+, or inositol 1,4-bisphosphate (Ins(1,4)P2) in response to norepinephrine (NE) (Matkovich, S. J. and Woodcock, E. A. (2000)J. Biol. Chem. 275, 10845–10850). To examine the PLC subtype mediating the α1-adrenergic receptor response, PLC-β1 and PLC-β3 were overexpressed in NCM using adenoviral infection (Ad-PLC-β1 NCM and Ad-PLC-β3 NCM, respectively) and PLC responses assessed from [3H]inositol phosphate (InsP) generation in the presence of 10 mm LiCl. The [3H]InsP response to NE (100 μm) was enhanced in Ad-PLC-β1NCM relative to cells infected with blank virus (Ad-MX NCM), but was reduced in Ad-PLC-β3 NCM. In contrast, the [3H]InsP response to ATP (100 μm) was not elevated in Ad-PLC-β1 NCM, and was enhanced rather than diminished in Ad-PLC-β3 NCM, showing that effects of the two PLC-β isoforms were specific for particular receptor types. PLC-δ1 overexpression selectively reduced NE-induced [3H]InsP responses, without affecting the ATP stimulation. The reduced NE response was associated with a selective loss of PLC-β1 expression in Ad-PLC-δ1 NCM. α1-Adrenergic receptor activation caused phosphorylation of PLC-β1 but not PLC-β3, whereas stimulation by ATP induced phosphorylation of PLC-β3 but not PLC-β1. Taken together, these studies provide evidence that NE-stimulated InsP generation in NCM is primarily mediated by PLC-β1, despite the presence of both PLC-β1 and PLC-β3 isoforms. phospholipase C inositol phosphate 4,5)P3, inositol 1,4,5-trisphosphate 4)P2, inositol 1,4-bisphosphate phosphatidylinositol phosphate 5)P2, phosphatidylinositol 4,5-bisphosphate phosphatidylinositol 4-phosphate phenylephrine polyacrylamide gel electrophoresis adrenergic receptor guanosine 5′-3-O-(thio)triphosphate pertussis toxin norepinephrine neonatal rat cardiomyocyte Dulbecco's modified Eagle's medium Receptor stimulation of phospholipase C (PLC)1 causes generation ofsn-1,2-diacylglycerol, which is able to activate protein kinase C isoforms (1Newton A.C. Trends Biochem. Sci. 1995; 20: 2-3Google Scholar), and Ins(1,4,5)P3, which releases Ca2+ from intracellular stores (2Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6157) Google Scholar). However, studies from our laboratory using intact heart and isolated cardiomyocytes have shown that, unlike responses in other cell types, α1-adrenergic receptor (α1-AR) stimulation of cardiomyocytes generates principally Ins(1,4)P2, which has no Ca2+-mobilizing function (3Woodcock E. Suss M. Anderson K. Circ. Res. 1995; 76: 252-260Crossref PubMed Google Scholar, 4Matkovich S.J. Woodcock E.A. J. Biol. Chem. 2000; 275: 10845-10850Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In marked contrast, increased intracellular Ca2+ itself stimulates robust Ins(1,4,5)P3 responses (4Matkovich S.J. Woodcock E.A. J. Biol. Chem. 2000; 275: 10845-10850Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Introduction of Ins(1,4,5)P3 into patch-clamped myocytes causes inward, depolarizing current via Na+/Ca2+ exchange (5Gilbert J.C. Shirayama T. Pappano A.J. Circ. Res. 1991; 69: 1632-1639Crossref PubMed Scopus (55) Google Scholar) and is associated with prolongation of the action potential and the consequent development of early and delayed afterdepolarizations (6Felzen B. Shilkrut M. Less H. Saparov I. Coleman R. Robinson R. Berke G. Binah O. Circ. Res. 1998; 82: 438-450Crossref PubMed Scopus (70) Google Scholar). Thus, Ins(1,4,5)P3 is potentially arrhythmogenic (7Lipp P. Laine M. Tovey S.C. Burrell K.M. Berridge M.J. Li W. Bootman M.D. Curr. Biol. 2000; 10: 939-942Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), and mechanisms that by-pass its generation may be cardioprotective. For these reasons, we sought to clarify the mechanisms responsible for primary generation of Ins(1,4)P2, rather than Ins(1,4,5)P3 in response to α1-AR activation. As an initial step in this process, we attempted to identify the PLC subtype mediating the response. The three major classes of mammalian PtdIns-specific PLC enzymes are β, γ, and δ (8Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar), and a new class, PLC-ε, has recently been identified (9Song C. Hu C.D. Masago M. Kariya K. Yamawaki-Kataoka Y. Shibatohge M. Wu D.M. Satoh T. Kataoka T. J. Biol. Chem. 2001; 276: 2752-2757Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). PLC-β isoforms can be activated by heterotrimeric G proteins of the Gq class via its activated α subunit (10Smrcka A.V. Hepler J.R. Brown K.O. Sternweis P.C. Science. 1991; 251: 804-807Crossref PubMed Scopus (695) Google Scholar, 11Wu D. Katz A. Lee C.-H. Simon M. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar, 12Taylor S.J. Chae H.Z. Rhee S.G. Exton J.H. Nature. 1991; 350: 516-518Crossref PubMed Scopus (610) Google Scholar) and are thus likely mediators of α1-AR responses. GTPγS-bound Gαq has been shown to stimulate both PLC-β1 and PLC-β3 to a similar extentin vitro (13Hepler J.R. Kozasa T. Smrcka A.V. Simon M.I. Rhee S.G. Sternweis P.C. Gilman A.G. J. Biol. Chem. 1993; 268: 14367-14375Abstract Full Text PDF PubMed Google Scholar, 14Jhon D.-Y. Lee H.-H. Park D. Lee C.-W. Lee K.-H. Yoo O.J. Rhee S.G. J. Biol. Chem. 1993; 268: 6654-6661Abstract Full Text PDF PubMed Google Scholar). The βγ subunits of heterotrimeric G proteins, especially those of the pertussis toxin (PTX)-sensitive Gi class, are able to activate PLC-β2 and PLC-β3 (15Camps M. Carozzi A. Schnabel P. Scheer A. Parker P. Gierschik P. Nature. 1992; 360: 684-686Crossref PubMed Scopus (513) Google Scholar, 16Park D. Jhon D.-Y. Lee C.-W. Lee K.-H. Rhee S.G. J. Biol. Chem. 1993; 268: 4573-4576Abstract Full Text PDF PubMed Google Scholar) but are poor stimulators of PLC-β1 (13Hepler J.R. Kozasa T. Smrcka A.V. Simon M.I. Rhee S.G. Sternweis P.C. Gilman A.G. J. Biol. Chem. 1993; 268: 14367-14375Abstract Full Text PDF PubMed Google Scholar). Of the four identified PLC-β isoforms, only PLC-β1 and PLC-β3 are expressed in heart (8Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar, 14Jhon D.-Y. Lee H.-H. Park D. Lee C.-W. Lee K.-H. Yoo O.J. Rhee S.G. J. Biol. Chem. 1993; 268: 6654-6661Abstract Full Text PDF PubMed Google Scholar). PLC-γ isoforms comprise a part of receptor and non-receptor tyrosine kinase cascades and can also be stimulated by phosphatidylinositol 3,4,5-trisphosphate generated in response to phosphoinositide 3-kinase activation (17Bae Y.S. Cantley L.G. Chen C.S. Kim S.R. Kwon K.S. Rhee S.G. J. Biol. Chem. 1998; 273: 4465-4469Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 18Falasca M. Logan S.K. Lehto V.P. Baccante G. Lemmon M.A. Schlessinger J. EMBO J. 1998; 17: 414-422Crossref PubMed Scopus (481) Google Scholar, 19Rameh L.E. Rhee S.G. Spokes K. Kazlauskas A. Cantley L.C. Cantley L.G. J. Biol. Chem. 1998; 273: 23750-23757Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Regulation of PLC-δ is less well understood. It is the most Ca2+-sensitive of the PLC subtypes (20Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (820) Google Scholar), and there have been reports of its activation by the GTP-binding protein transglutaminase II in response to α1-AR stimulation (21Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.J. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (528) Google Scholar, 22Feng J.-F. Rhee S. Im M.-J. J. Biol. Chem. 1996; 271: 16451-16454Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). The current experiments were undertaken to establish which PLC isoform mediates the distinctive InsP response to α1-AR activation in rat neonatal cardiomyocytes. NCM were prepared from 1–3-day-old Sprague-Dawley rat pups, essentially as described previously (23Simpson P. McGrath A. Savion S. Circ. Res. 1982; 51: 787-801Crossref PubMed Scopus (373) Google Scholar). NCM were isolated by repeated trypsin digestion with gentle mechanical dispersion, pre-plated twice for 30 min each to remove non-myocytes, and left to attach for 18 h in DMEM, 10% fetal calf serum, 0.1 mm bromodeoxyuridine, 50 units/ml penicillin G, and 50 μg/ml streptomycin sulfate onto uncoated dishes, at a typical seeding density of 700 cells/mm2. Medium was then replaced with a defined serum-free medium consisting of DMEM, 10 μg/ml human insulin, 10 μg/ml bovine apo-transferrin, 0.1 mm bromodeoxyuridine, 50 units/ml penicillin G, and 50 μg/ml streptomycin sulfate. Bromodeoxyuridine was omitted after 3 days. NCM were treated with adenovirus and labeled with [3H]inositol in defined inositol-free, serum-free medium for 48 h prior to experiments. Adenoviruses expressing PLC-β1, PLC-β3, and Gα11 were prepared as described previously (24Gasa R. Trinh K.Y., Yu, K. Wilkie T.M. Newgard C.B. Diabetes. 1999; 48: 1035-1044Crossref PubMed Scopus (24) Google Scholar). Rat PLC-δ1 cloned into theNotI site of pZipNeo (25Suh P.G. Ryu S.H. Moon K.H. Suh H.W. Rhee S.G. Cell. 1988; 54: 161-169Abstract Full Text PDF PubMed Scopus (234) Google Scholar) was a gift from Dr. S. G. Rhee. The NotI restriction fragment corresponding to PLC-δ1 was subcloned into pXCMV, which is an adenoviral shuttle vector formed by subcloning a pRcCMV expression cassette (Invitrogen) into pXCX3, which in turn is derived from pXCX2 (26Spessot R. Inchley K. Hupel T.M. Bacchetti S. Virology. 1989; 168: 378-387Crossref PubMed Scopus (41) Google Scholar). This construct was cotransfected into HEK293 cells with pJM17 (modified adenovirus backbone), and cultures were overlaid with agarose. Following recombination, individual plaques were selected and purified through sequential rounds of infection and agarose overlay. High titer viral stocks were produced by amplification of the virus through successive rounds of infection in HEK293 cells, with purification on CsCl gradients. Blank virus (Ad-MX) contained the adenoviral backbone but expressed no additional gene product. Rat PLCγ1 was subcloned into pShuttle-CMV and recombined with pAdEasy-1 (27Mitchell C.J. Kelly M.M. Blewitt M. Wilson J.R. Biden T.J. J. Biol. Chem. 2001; 276: 19072-19077Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Adenoviruses were used at a multiplicity of infection of 10–50 plaque-forming units/cell, corresponding to 0.5–2.5 × 107 plaque-forming units/35-mm well. Infection with adenovirus expressing an S65T-modified green fluorescent protein (kindly supplied by Dr. Ross Hannan, Molecular Physiology Laboratory, Baker Medical Research Institute) confirmed infection of >90% of cardiomyocytes present on each well. NCM were washed three times in ice-cold phosphate-buffered saline, and proteins extracted in ice-cold lysis buffer at pH 7.7 containing 20 mm Tris-HCl, 250 mm NaCl, 2 mm EDTA, 2 mm EGTA, 20 mm β-glycerophosphate, 1 mm sodium orthovanadate, 10% glycerol, 0.5% Nonidet P-40 with 1 mmphenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 1 μm pepstatin A, 5 μg/ml aprotinin, and 10 μg/ml leupeptin. Protein content of the supernatant was determined by the Bradford method with bovine serum albumin as standard using a dye reagent from Bio-Rad. Proteins from whole-cell lysates were separated on 7.5% denaturing polyacrylamide gels (28Cannon-Carlson S. Tang J. Anal. Biochem. 1997; 246: 146-148Crossref PubMed Scopus (62) Google Scholar) and electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell). Membranes were stained with Ponceau S (Sigma) to confirm equal protein loading and transfer efficiency, blocked in 5% nonfat dry milk for 1 h, and then probed with antibodies. Enhanced chemiluminescence detection was carried out according to the manufacturer's instructions (Amersham Pharmacia Biotech). Rabbit polyclonal antibodies against Gαq/11, PLC-β1, and PLC-β3 were obtained from Santa Cruz Biotechnology and used at a final concentration of 1 μg/ml for Gαq/11 and PLC-β3 and 2 μg/ml for PLC-β1. Mouse monoclonal anti-PLC-δ1antibody was from Upstate Biotechnology and used at a final concentration of 0.4 μg/ml. Mouse monoclonal anti-PLC-γ1 antibody was from Transduction Laboratories and used at a final concentration of 1 μg/ml. Secondary antibodies (Amersham Pharmacia Biotech ECL kit) were used at a dilution of 1:5000 for PLC-β1 and 1:10,000 for all other PLCs and Gαq/11. [3H]Inositol-labeled (48 h, 10 μCi/ml) NCM were washed with unlabeled medium and pretreated with 10 mm LiCl in DMEM for 10 min prior to the addition of agonist. For experiments involving NE, propranolol (1 μm) and β-mercaptoethanol (5 mm) were included. For experiments using the synthetic α1-AR agonist phenylephrine (PE), propranolol (1 μm) was included. After stimulation, [3H]InsPs were extracted with ice-cold 5% trichloroacetic acid, 2.5 mm EDTA, 5 mmsodium phytate and the supernatants subsequently treated with a 1:1 mixture of 1,1,2-trichlorotrifluoroethane:tri-n-octylamine to remove remaining trichloroacetic acid. The aqueous phase containing [3H]InsPs was prepared for high performance liquid chromatography, which was performed as described previously (29Woodcock E. Mol. Cell. Biochem. 1997; 172: 121-127Crossref PubMed Scopus (20) Google Scholar). Neonatal cardiomyocytes were incubated at 37 °C for 2 h in phosphate-free DMEM containing 80 μCi/ml [32P]orthophosphoric acid. Labeled cells were challenged with agonists for the specified times and subsequently lysed for 1 h at 4 °C in lysis buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 2 mm EDTA, 50 mm NaF, 0.1% SDS, 0.5% deoxycholic acid, 1% Triton X-100) containing protease inhibitors (1 μg/ml aprotinin, 5 μg/ml leupeptin, 0.7 μg/ml pepstatin A). The lysate was centrifuged at 16,000 ×g (4 °C, 15 min), and the resultant supernatant was pre-cleared with protein A-Sepharose for 1 h at 4 °C. After centrifugation (16,000 × g for 30 s, 4 °C), the supernatants were incubated at 4 °C overnight with PLC-β1 (4 μg) or PLC-β3 (2 μg) antibodies and protein A-Sepharose with gentle rocking. Precipitates were washed five times at 0 °C, extracted with sample buffer, and proteins separated on a 7.5% denaturing polyacrylamide gel.32P labeling was quantified using a Fujix BAS 1000 phosphorimager. Fetal calf serum specially selected for low endotoxin was obtained from the Commonwealth Serum Laboratories (Parkville, Australia). DMEM, Hepes, and other materials for the preparation of cell culture solutions and media were cell culture-grade obtained from Sigma and dissolved in MilliQ H2O. Norepinephrine bitartrate and ATP (disodium salt) were purchased from Sigma. [3H]Inositol (18.00 Ci/mmol) was obtained from Amersham Pharmacia Biotech (Bucks., United Kingdom). [32P]Orthophosphoric acid (3000 Ci/mmol) was from PerkinElmer Life Sciences or Bresatec, Pty. Ltd. Other reagents were obtained from Sigma or BDH/AnalaR and were of analytical reagent grade. Differences between treatment groups were assessed by one-way analysis of variance with Tukey's test for multiple comparisons, and accepted as statistically significant at a family error rate ofp < 0.05 (individual pairwise comparisons were significant at p < 0.02). Unless otherwise noted, results shown are from representative experiments performed in triplicate, which were repeated in independent NCM preparations at least three times. InsP responses to α1-adrenergic receptor activation are mediated by the Gq class of heterotrimeric G proteins that stimulate the β isoforms of PLC. PLC-β1 and PLC-β3isoforms are expressed in NCM, and both of these are potential targets for activated Gαq (13Hepler J.R. Kozasa T. Smrcka A.V. Simon M.I. Rhee S.G. Sternweis P.C. Gilman A.G. J. Biol. Chem. 1993; 268: 14367-14375Abstract Full Text PDF PubMed Google Scholar). To identify the PLC-β isoform principally mediating α1-AR-stimulated InsP responses in NCM, we overexpressed either PLC-β1 or PLC-β3 and measured [3H]InsP responses to NE. NCM were labeled with [3H]inositol, treated for 48 h with adenoviruses expressing PLC-β1 or PLC-β3, and then washed and pretreated with 1 μm propranolol and 10 mm LiCl prior to stimulation with 100 μm NE for 20 min. In Ad-MX NCM, NE increased [3H]InsP content (Fig.1). In comparison with Ad-MX NCM, Ad-PLC-β1 NCM showed an enhanced NE response, but NE responses in Ad-PLC-β3 NCM were significantly decreased (Fig. 1). These data demonstrate that overexpression of PLC-β1 has different effects on NE responses from overexpressed PLC-β3, and suggest that PLC-β1 is more likely to be a mediator of α1-AR responses. Neither PLC-β1 nor PLC-β3 overexpression caused any alteration in [3H]InsP content in unstimulated cells. Overexpressed PLC-β1 and PLC-β3 had opposite effects on [3H]InsP responses to NE, and it was necessary to determine if this finding was specific to α1-AR responses. Similar experiments were performed using ATP to stimulate [3H]InsP responses via P2Y2-purinergic receptors in NCM. [3H]InsP responses to 100 μm ATP in Ad-MX NCM, Ad-PLC-β1 NCM, or Ad-PLC-β3 NCM are shown in Fig. 1. In contrast to findings with NE, ATP-stimulated [3H]InsP responses were not altered by overexpression of PLC-β1 and furthermore were enhanced, rather than decreased, in Ad-PLC-β3 NCM. Thus, overexpressing each of the two PLC-β isoforms causes different effects on NE and ATP responses. The data suggest that PLC-β1 is the primary PLC isoform responsible for [3H]InsP responses to α1-adrenergic receptor stimulation, but PLC-β3 is a more likely mediator of responses to ATP. Experiments were performed to examine effects of overexpressed PLC-β1 or PLC-β3 on the contents of other intermediates potentially involved in receptor stimulation of InsP generation. Extracts of Ad-MX NCM, Ad-PLC-β1 NCM, and Ad-PLC-β3 NCM were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with subtype-selective PLC and Gαq/11 antibodies. As expected, overexpression of PLC-β1 increased PLC-β1 content, but surprisingly also increased the expression of PLC-δ1 (Fig.2). There was no change in the content of PLC-β3, PLC-γ1, or Gαq/11. Overexpression of PLC-β3 increased the amount of PLC-β3 with no change in PLC-β1, PLC-γ1, or Gαq/11 content, and there was no change in PLC-δ1 content. The finding that Ad-PLC-β1 NCM contained heightened levels of PLC-δ1 as well as PLC-β1 raised the possibility that PLC-δ1 contributed to the increased α1-AR response. Accordingly, experiments were performed with Ad-PLC-δ1 NCM. Whereas PLC-β1 or PLC-β3 overexpression did not increase basal activity, there was an elevated [3H]InsP content in Ad-PLC-δ1 NCM (Fig.3). Despite the increased basal activity, Ad-PLC-δ1 NCM had a reduced [3H]InsP response to NE (Fig. 3A). Thus, increased PLC-δ1 content does not account for the enhanced NE response seen in Ad-PLC-β1 NCM, and this instead most likely reflects the increased PLC-β1 availability. In contrast to findings with NE, overexpression of PLC-δ1did not reduce the InsP response to ATP (Fig. 3B). Thus, α1-AR stimulation of [3H]InsP generation was specifically disrupted by overexpression of PLC-δ1. To identify possible mechanisms by which PLC-δ1 overexpression might lead to a selective reduction in α1-AR-stimulated PLC activity, we examined the cellular content of PLC isoforms and Gαq/11. Cell lysates from Ad-MX NCM and Ad-PLC-δ1 NCM were separated by SDS-PAGE and blotted with antibodies to PLC-δ1, PLC-β1, PLC-β3, PLC-γ1, and Gαq/11. Treatment with virus encoding PLC-δ1 resulted in a marked elevation of PLC-δ1 content (Fig. 4). Levels of PLC-β3, PLC-γ1, and Gαq/11 were not altered by PLC-δ1overexpression, but PLC-β1 content was markedly reduced (Fig. 4). Thus, the selectively reduced NE-stimulated [3H]InsP response in Ad-PLC-δ1 NCM is associated with decreased PLC-β1 expression, and this may explain the reduced response to α1-AR stimulation. Overexpression studies provided evidence that PLC-β1 is the primary mediator of InsP responses to α1-AR activation, despite the presence of both PLC-β1 and PLC-β3 isoforms. However, such high levels of expression could potentially lead to unphysiological interactions. For this reason, it was necessary to find evidence for selective interaction between the receptors and the PLC-β subtypes at physiological expression levels. To do this we measured PLC-β isoform phosphorylation in response to stimulation with the synthetic α1-AR agonist PE or with ATP. PE was used in these experiments rather then NE to minimize any possible PLC-β3 phosphorylation mediated by protein kinase A (30Strassheim D. Williams C.L. J. Biol. Chem. 2000; 275: 39767-39772Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) following β-AR stimulation. NCM were labeled with [32P]orthophosphate for 2 h then stimulated with either PE (50 μm, plus 1 μm propranolol to block β-ARs) or ATP (100 μm) for 30 s or 1 min. Extracts were prepared, immunoprecipitated with PLC-β1 antibody, and subjected to SDS-PAGE as described under “Experimental Procedures.” No32P-labeled material with the expected molecular mass values of PLC-β1 (140 and 150 kDa for the two splice variants; Ref. 31Bahk Y.Y. Lee Y.H. Lee T.G. Seo J.K. Ryu S.H. Suh P.G. J. Biol. Chem. 1994; 269: 8240-8245Abstract Full Text PDF PubMed Google Scholar) was observed in immunoprecipitated extracts from unstimulated NCM or from NCM treated with ATP. However, stimulation with PE (50 μm) for 30 s caused the appearance of a phosphorylated band within this molecular mass range. Stimulation for 1 min was less effective. Subsequent ECL-Western blotting of the membrane confirmed the immunoprecipitation of PLC-β1 (Fig.5A). In similar experiments, 32P-labeled NCM were treated with 50 μm PE or 100 μm ATP for 30 s or 1 min, and extracts were immunoprecipitated with PLC-β3antibody. No 32P-labeled band corresponding to the predicted molecular mass of PLC-β3 (143 kDa), was detected in extracts immunoprecipitated with PLC-β3antibody from unstimulated cells or from cells stimulated with PE. However, stimulation with ATP for 1 min (30 s was less effective) caused phosphorylation of PLC-β3 (Fig. 5B). Thus, α1-AR and P2Y2R specifically phosphorylate PLC-β1 and PLC-β3, respectively. Results of these studies at physiological levels of PLC expression support the conclusions from the overexpression experiments that α1-ARs couple to PLC-β1 and P2Y2Rs couple to PLC-β3. The apparently selective interaction of P2Y2Rs to PLC-β3 might be explained if these coupled primarily to Gi rather than Gq/11 in NCM. To examine this possibility, we treated NCM with PTX (1 μg/ml, for 24 h) to inactivate G proteins of the Giclass. PTX treatment did not significantly reduce the stimulation by either NE (100 μm) or ATP (100 μm), providing evidence that neither receptor type was Gi-coupled (Fig.6A). In another series of experiments, Gα11 was overexpressed in NCM by treating with adenovirus encoding Gα11(Ad-Gα11) for 48 h (50 plaque-forming units/cell). Overexpression of Gα11 increased responses to both NE and ATP (Fig. 6B), supporting the notion that both responses are mediated by the Gq/11 class of heterotrimeric G protein. Thus, the data provide evidence that α1-ARs couple to PLC-β1 via Gq/11 while P2Y2Rs also couple via Gq/11 but in this case the PLC activated is PLC-β3. We have recently reported two distinct pathways of InsP generation in NCM, one involving Ins(1,4,5)P3 generation in response to elevated Ca2+ and the other involving primarily the hydrolysis of PtdIns(4)P to Ins(1,4)P2 following α1-AR activation (4Matkovich S.J. Woodcock E.A. J. Biol. Chem. 2000; 275: 10845-10850Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Generation of Ins(1,4,5)P3 is associated with the onset of arrhythmias (7Lipp P. Laine M. Tovey S.C. Burrell K.M. Berridge M.J. Li W. Bootman M.D. Curr. Biol. 2000; 10: 939-942Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 32Du X.-J. Anderson K. Jacobsen A. Woodcock E. Dart A. Circulation. 1995; 91: 2712-2716Crossref PubMed Scopus (84) Google Scholar, 33Jacobsen A.N. Du X.J. Lambert K.A. Dart A.M. Woodcock E.A. Circulation. 1996; 93: 23-26Crossref PubMed Scopus (65) Google Scholar); thus, hydrolysis of PtdIns(4)P to Ins(1,4)P2, by limiting changes in Ins(1,4,5)P3 generation, may be an important cardioprotective mechanism. It is essential, therefore, to identify the mechanisms responsible for the preferential hydrolysis of PtdIns(4)P to Ins(1,4)P2, and, as a first step, this report is concerned with identifying the PLC subtype mediating the response to α1-AR activation. α1-ARs couple via the Gq/11 class of heterotrimeric G proteins (34Rybin V. Han H.M. Steinberg S.F. G Proteins. 1996; 29: 344-361Crossref Scopus (6) Google Scholar) to PLC-β isoforms (11Wu D. Katz A. Lee C.-H. Simon M. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar), and of these the β1 and β3 subtypes are expressed in cardiomyocytes (8Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar, 14Jhon D.-Y. Lee H.-H. Park D. Lee C.-W. Lee K.-H. Yoo O.J. Rhee S.G. J. Biol. Chem. 1993; 268: 6654-6661Abstract Full Text PDF PubMed Google Scholar). We overexpressed the two candidate PLC-β subtypes in NCM using adenoviral infection and examined their effects on NE responses. Overexpression of PLC-β1 enhanced NE-stimulated [3H]InsP responses, in keeping with an activation mediated by the α subunit of Gq. However, even though Gαq has been shown to interact with PLC-β3, overexpression of this latter PLC isoform actually decreased rather than increased NE responses. Importantly, overexpression of the two PLC-β subtypes affected P2Y2R signaling differently, with overexpressed PLC-β3 actually increasing, rather than decreasing [3H]InsP responses (Fig. 1). Thus, taken together, the data imply that PLC-β1 is instrumental in mediating responses to NE, but not those to ATP. The mechanisms underlying the inhibitory effects of PLC-β3 overexpression on α1-AR-mediated responses have not been further elucidated. It is possible that the large excess of PLC-β3 relative to PLC-β1in Ad-PLC-β3 NCM competes for access to endogenous Gαq (13Hepler J.R. Kozasa T. Smrcka A.V. Simon M.I. Rhee S.G. Sternweis P.C. Gilman A.G. J. Biol. Chem. 1993; 268: 14367-14375Abstract Full Text PDF PubMed Google Scholar). Overexpression of PLC-γ1 (27Mitchell C.J. Kelly M.M. Blewitt M. Wilson J.R. Biden T.J. J. Biol. Chem. 2001; 276: 19072-19077Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) did not alter [3H]InsP responses to either NE or ATP, and the tyrosine kinase inhibitor genistein (50 μm) was not inhibitory (data not shown). Thus, neither receptor appears to be coupled to PLC-γ under the conditions of our experiments (35Puceat M. Vassort G. Biochem. J. 1996; 318: 723-728Crossref PubMed Scopus (51) Google Scholar). An unexpected finding of these studies was the apparently selective effect of PLC-β1 and PLC-δ1 on the expression of each other (Figs. 2 and 4). Overexpression of PLC-β1 caused increased expression of PLC-δ1, whereas overexpressed PLC-δ1reduced PLC-β1 content. The mechanism and the physiological relevance of these changes are unknown. However, in the context of the current studies, this cross-talk provided further support for PLC-β1 as the mediator of α1-AR signaling in NCM. Overexpression of PLC-δ1 decreased the α1-AR response, as expected if this were mediated by PLC-β1 (Fig. 4). The demonstration that overexpressed PLCs can have apparently “specific” effects on other isoforms made it imperative to show that α1-ARs and P2Y2Rs interact with PLC-β1 and PLC-β3 selectively, at physiological levels of the PLC isoforms. In non-transfected NCM, addition of ATP but not the α1-AR agonist phenylephrine caused rapid phosphorylation of PLC-β3. This again pointed to P2Y2R but not α1-AR coupling to PLC-β3, in agreement with the findings of the overexpression studies. Selective phosphorylation of PLC-β3 has been reported previously in ATP-stimulated Chinese hamster ovary cells (30Strassheim D. Williams C.L. J. Biol. Chem. 2000; 275: 39767-39772Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), even though PLC-β1 was present. Phosphorylation of PLC-β3 by protein kinase C, protein kinase A, or protein kinase G family members has been reported and is associated with decreased activity (36Yue C.P. Dodge K.L. Weber G. Sanborn B.M. J. Biol. Chem. 1998; 273: 18023-18027Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 37Yue C.P. Kus C.Y. Liu M.Y. Simon M.I. Sanborn B.M. J. Biol. Chem. 2000; 275: 30220-30225Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 38Xia C. Bao Z. Yue C. Sanborn B.M. Liu M. J. Biol. Chem. 2001; 276: 19770-19777Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Phosphorylation of PLC-β1 has previously been reported only in response to receptor tyrosine kinases, and in these studies was shown not to occur in response to G protein coupled receptors (39Faenza I. Matteucci A. Manzoli L. Billi A.M. Aluigi M. Peruzzi D. Vitale M. Castorina S. Suh P.G. Cocco L. J. Biol. Chem. 2000; 275: 30520-30524Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 40Martelli A.M. Billi A.M. Manzoli L. Faenza I. Aluigi M. Falconi M. DePol A. Gilmour R.S. Cocco L. FEBS Lett. 2000; 486: 230-236Crossref PubMed Scopus (46) Google Scholar). Such phosphorylation of PLC-β1 downstream of tyrosine kinases and growth signaling pathways appears to be a feature of undifferentiated cells, as this response decreases with differentiation (41Cocco L. Rhee S.G. Gilmour R.S. Manzoli F.A. Eur. J. Histochem. 2000; 44: 45-50PubMed Google Scholar, 42Cocco L. Martelli A.M. Gilmour R.S. Rhee S.G. Manzoli F.A. Biochim. Biophys. Acta. 2001; 1530: 1-14Crossref PubMed Scopus (92) Google Scholar). The observed selective phosphorylation of PLC-β1 following α1-AR activation in cardiomyocytes, where more than 90% of the cells have lost replicative ability, is thus a novel finding and most likely involves different mechanisms from those described previously. This is currently under investigation. Despite the finding that ATP and NE activate different PLC-β subtypes, both appear to couple to the Gq/11 class of heterotrimeric G proteins. Thus, downstream signaling specificity is conferred by the receptor rather than the particular G protein involved. The most obvious explanation for this is that the receptors, coupling proteins, and PLCs, possibly together with other factors such as RGS proteins, are held in close association by a scaffolding system. In Drosophila photoreceptors, PLC and other signaling components form multiprotein structures through a PDZ scaffold protein (INAD) (43Cook B. BarYaacov M. BenAmi H.C. Goldstein R.E. Paroush Z. Selinger Z. Minke B. Nat. Cell Biol. 2000; 2: 296-301Crossref PubMed Scopus (93) Google Scholar). Such scaffolding proteins have not been identified in mammalian cells, but mammalian PLCs have domains that recognize PDZ domains, making a similar scenario a possibility (20Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (820) Google Scholar, 44Hwang J.I. Heo K. Shin K.J. Kim E. Yun C.H.C. Ryu S.H. Shin H.S. Suh P.G. J. Biol. Chem. 2000; 275: 16632-16637Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In further support of a scaffolding principle, a recent study has identified multiple Gα-binding domains in the sequences of “activators of G protein signaling” (AGS molecules), suggesting a possible scaffolding function (45Bernard M.L. Peterson Y.K. Chung P. Jourdan J. Lanier S.M. J. Biol. Chem. 2001; 276: 1585-1593Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In addition, experiments using green fluorescent protein-tagged Gαq, showed a non-homogeneous distribution of Gαq in the plasma membrane, resembling clusters of receptor populations seen in previous studies (46Hughes T.E. Zhang H.L. Logothetis D.E. Berlot C.H. J. Biol. Chem. 2001; 276: 4227-4235Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Thus, it is possible that there are signaling complexes involving receptors, G proteins, and PLC enzymes in NCM and that these account for the observed PLC subtype-selective coupling to particular receptor classes. Discrete areas of plasma membrane enriched in PtdIns(4,5)P2have been demonstrated (47Tall E.G. Spector I. Pentyala S.N. Bitter I. Rebecchi M.J. Curr. Biol. 2000; 10: 743-746Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), and it is therefore possible that PLC can act on this substrate to generate Ins(1,4,5)P3 only when the PLC-containing complex and the PtdIns(4,5)P2 are co-localized. Such a mechanism could provide an explanation for the preferential hydrolysis of PtdIns(4)P following α1-AR activation. However, such a proposal requires that PtdIns(4)P is localized differently from PtdIns(4,5)P2, and this is currently unknown. Another possibility is that the specific phosphorylation of PLC-β1 in NCM, in some way alters the activity to cause a change in preferred or available substrate. Further studies are required to distinguish these possibilities. We thank Dr. Hongwei Qian for expert help with the phosphorylation studies and Bronwyn Kenney for technical assistance. We gratefully acknowledge Dr. Rosa Gasa and Dr. Chris Newgard for the PLC-β1 and PLC-β3adenoviruses." @default.
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