α1-Adrenergic Receptor Subtype mRNAs Are Differentially Regulated by α1-Adrenergic and Other Hypertrophic Stimuli in Cardiac Myocytes in Culture and In Vivo
1996; Elsevier BV; Volume: 271; Issue: 10 Linguagem: Inglês
10.1074/jbc.271.10.5839
ISSN1083-351X
AutoresGregg Rokosh, Alexandre F.R. Stewart, Kevin Chang, Beth A. Bailey, Joel S. Karliner, Santiago Camacho, Carlin S. Long, Paul Simpson,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoThe three cloned α1-adrenergic receptor (AR) subtypes, α1B, α1C, and α1D, can all couple to the same effector, phospholipase C, and the reason(s) for conservation of multiple subtypes remain uncertain. All three α1-ARs are expressed natively in cultured neonatal rat cardiac myocytes, where chronic exposure to the agonist catecholamine norepinephrine (NE) induces hypertrophic growth and gene transcription. We show here, using RNase protection, that the α1-AR subtype mRNAs respond in distinctly different ways during prolonged NE exposure (12-72 h). α1B and α1D mRNA levels were repressed by NE, whereas α1C mRNA was induced. Changes in mRNA levels were mediated by an α1-AR, were not explained by altered mRNA stability, and were reflected in receptor proteins by [3H]prazosin binding. α1-AR-stimulated phosphoinositide hydrolysis and myocyte growth were not desensitized. Three other hypertrophic agonists in culture, endothelin-1, PGF2α, and phorbol 12-myristate 13-acetate, also induced α1C mRNA and repressed α1B mRNA. In myocytes from hearts with pressure overload hypertrophy, α1 mRNA changes were identical to those produced by NE in culture. These results provide the first example of a difference in regulation among α1-AR subtypes expressed natively in the same cell. Transcriptional induction of the α1C-AR could be a mechanism for sustained growth signaling through this receptor and is a common feature of a hypertrophic phenotype in cardiac myocytes. The three cloned α1-adrenergic receptor (AR) subtypes, α1B, α1C, and α1D, can all couple to the same effector, phospholipase C, and the reason(s) for conservation of multiple subtypes remain uncertain. All three α1-ARs are expressed natively in cultured neonatal rat cardiac myocytes, where chronic exposure to the agonist catecholamine norepinephrine (NE) induces hypertrophic growth and gene transcription. We show here, using RNase protection, that the α1-AR subtype mRNAs respond in distinctly different ways during prolonged NE exposure (12-72 h). α1B and α1D mRNA levels were repressed by NE, whereas α1C mRNA was induced. Changes in mRNA levels were mediated by an α1-AR, were not explained by altered mRNA stability, and were reflected in receptor proteins by [3H]prazosin binding. α1-AR-stimulated phosphoinositide hydrolysis and myocyte growth were not desensitized. Three other hypertrophic agonists in culture, endothelin-1, PGF2α, and phorbol 12-myristate 13-acetate, also induced α1C mRNA and repressed α1B mRNA. In myocytes from hearts with pressure overload hypertrophy, α1 mRNA changes were identical to those produced by NE in culture. These results provide the first example of a difference in regulation among α1-AR subtypes expressed natively in the same cell. Transcriptional induction of the α1C-AR could be a mechanism for sustained growth signaling through this receptor and is a common feature of a hypertrophic phenotype in cardiac myocytes. INTRODUCTIONThe natural catecholamines norepinephrine (NE) ( 1The abbreviations used are: NEnorepinephrineARadrenergic receptorET-1endothelin-1GAPDHglyceraldehyde-phosphate dehydrogenaseIPinositol phosphatePLCphospholipase CPMAphorbol myristate acetate5MU5-methylurapidil.) and epinephrine activate adrenergic receptors (ARs) in three families, α1, α2, and β. Multiple subtypes have been cloned within each family: three α1-ARs (B, C, and D), ( 2It has been suggested that the α1C be renamed the α1A(49.Hieble J.P. Bylund D.B. Clarke D.E. Eikenburg D.C. Langer S.Z. Lefkowitz R.J. Minneman K.P. Ruffolo Jr., R.R. Pharmacol. Rev. 1995; 47: 267-270PubMed Google Scholar). For clarity, we continue to use the name α1C here, since the designation α1A (or α1A/D) has also been applied to the α1D in many reports(49.Hieble J.P. Bylund D.B. Clarke D.E. Eikenburg D.C. Langer S.Z. Lefkowitz R.J. Minneman K.P. Ruffolo Jr., R.R. Pharmacol. Rev. 1995; 47: 267-270PubMed Google Scholar).) three α2-ARs (A, B, and C; also called C10, C2, and C4, respectively), and three β-ARs (1, 2, and 3)(1.Lomasney J.W. Allen L.F. Lefkowitz R.J. Weintraub B.D. Molecular Endocrinology: Basic Concepts and Clinical Correlations. Raven Press, Ltd., New York1995: 115-131Google Scholar). The reason(s) for conservation of multiple subtypes remain uncertain, since all subtypes in each family couple preferentially to the same effector when overexpressed, α1-ARs to activation of phospholipase C (PLC), α2-ARs to inhibition of adenylyl cyclase, and β-ARs to activation of adenylyl cyclase(1.Lomasney J.W. Allen L.F. Lefkowitz R.J. Weintraub B.D. Molecular Endocrinology: Basic Concepts and Clinical Correlations. Raven Press, Ltd., New York1995: 115-131Google Scholar).An intriguing difference among β-AR and α2-AR subtypes has been suggested recently, in the regulation of receptor levels during prolonged agonist exposure. Levels of the β3-AR (2.Thomas R. Holt B.D. Schwinn D.A. Liggett S.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4490-4494Crossref PubMed Scopus (106) Google Scholar, 3.Liggett S.B. Freedman N.J. Schwinn D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3665-3669Crossref PubMed Scopus (207) Google Scholar, 4.Nantel F. Marullo S. Krief S. Strosberg A.D. Bouvier M. J. Biol. Chem. 1994; 269: 13148-13155Abstract Full Text PDF PubMed Google Scholar) and the α2C-AR (5.Eason M.G. Liggett S.B. J. Biol. Chem. 1992; 267: 25473-25479Abstract Full Text PDF PubMed Google Scholar, 6.Eason M.G. Jacinto M.T. Theiss C.T. Liggett S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11178-11182Crossref PubMed Scopus (96) Google Scholar) are not down-regulated during long term agonist exposure, at least in some cells, in contrast with down-regulation of β1- and β2-ARs, and α2A- and α2B-ARs. Down-regulation of receptor expression is thought to be a major determinant of desensitization when catecholamine exposure is prolonged(7.Liggett S.B. Lefkowitz R.J. Sibley D.D. Houslay M. Regulation of Cellular Signal Transduction Pathways by Desensitization and Amplification. 7. John Wiley & Sons, Inc., New York1993: 71-97Google Scholar). Conversely, a receptor that is induced by agonist might be adapted to mediate catecholamine responses when sympathetic activity is increased chronically, a condition that occurs frequently in the intact organism.We have been studying a physiological response that develops over long periods of catecholamine exposure, α1-adrenergic induction of hypertrophic growth and gene transcription in primary cultures of neonatal rat cardiac myocytes(8.Simpson P. J. Clin. Invest. 1983; 72: 732-738Crossref PubMed Scopus (546) Google Scholar, 9.Simpson P. Circ. Res. 1985; 56: 884-894Crossref PubMed Scopus (403) Google Scholar, 10.Waspe L.E. Ordahl C.P. Simpson P.C. J. Clin. Invest. 1990; 85: 1206-1214Crossref PubMed Scopus (155) Google Scholar, 11.Kariya K. Farrance I.K.G. Simpson P.C. J. Biol. Chem. 1993; 268: 26658-26662Abstract Full Text PDF PubMed Google Scholar, 12.Kariya K. Karns L.R. Simpson P.C. J. Biol. Chem. 1994; 269: 3775-3782Abstract Full Text PDF PubMed Google Scholar, 13.Karns L.R. Kariya K. Simpson P.C. J. Biol. Chem. 1995; 270: 410-417Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). We have found recently that the cardiac myocytes express the mRNAs for all three cloned α1-AR subtypes, the α1B, the α1C, and the α1D, whereas cardiac fibroblasts do not express any α1-AR(14.Stewart A.F.R. Rokosh D.G. Bailey B.A. Karns L.R. Chang K.C. Long C.S. Kariya K. Simpson P.C. Circ. Res. 1994; 75: 796-802Crossref PubMed Scopus (93) Google Scholar). This system thus provides the opportunity to study regulation of all three α1-AR subtypes expressed natively in the same cell. Most prior studies of chronic agonist regulation of the β3- and α2C-ARs have been in transfected cells overexpressing these subtypes(3.Liggett S.B. Freedman N.J. Schwinn D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3665-3669Crossref PubMed Scopus (207) Google Scholar, 4.Nantel F. Marullo S. Krief S. Strosberg A.D. Bouvier M. J. Biol. Chem. 1994; 269: 13148-13155Abstract Full Text PDF PubMed Google Scholar, 5.Eason M.G. Liggett S.B. J. Biol. Chem. 1992; 267: 25473-25479Abstract Full Text PDF PubMed Google Scholar, 6.Eason M.G. Jacinto M.T. Theiss C.T. Liggett S.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11178-11182Crossref PubMed Scopus (96) Google Scholar), where transcriptional regulation of receptor expression would not occur.In the present study, we report that the α1-AR subtype mRNAs respond in distinctly different ways during chronic exposure to NE. The mRNAs encoding the α1B and the α1D were repressed, whereas α1C mRNA was induced. These mRNA changes were mediated through an α1-AR, were likely transcriptional in origin, and were followed by changes in receptor protein by radioligand binding. α1-AR-stimulated phosphoinositide hydrolysis and myocyte growth were not desensitized. Three other hypertrophic agonists, endothelin-1 (ET-1)(15.Shubeita H.E. McDonough P.M. Harris A.N. Knowlton K.U. Glembotski C.C. Brown J.H. Chien K.R. J. Biol. Chem. 1990; 265: 20555-20562Abstract Full Text PDF PubMed Google Scholar), PGF2α(16.Tsoporis J.N. Zeirhut W. Karns L.R. Simpson P.C. J. Mol. Cell. Cardiol. 1994; 26 (CLXXVIII)Google Scholar), and phorbol 12-myristate 13-acetate (PMA)(17.Henrich C.J. Simpson P.C. J. Mol. Cell. Cardiol. 1988; 20: 1081-1085Abstract Full Text PDF PubMed Scopus (141) Google Scholar), also induced α1C mRNA and repressed α1B mRNA. Changes identical to those produced by NE in culture were seen in myocytes isolated from hearts with hypertrophy produced by abdominal aortic banding. These results provide the first example of a difference in regulation among α1-AR subtypes expressed natively in the same cell. Transcriptional induction of the α1C-AR could be a mechanism for sustained growth signaling through this receptor and is a common feature of a hypertrophic phenotype in cardiac myocytes.EXPERIMENTAL PROCEDURESCell CultureMyocytes were isolated from the day-old rat heart using trypsin and were seeded at single-cell density in minimal essential medium with 5% calf serum (Hyclone) and 0.1 mM bromodeoxyuridine to inhibit fibroblast proliferation(18.Simpson P. Savion S. Circ. Res. 1982; 50: 101-116Crossref PubMed Scopus (444) Google Scholar, 19.Long C.S. Henrich C.J. Simpson P.C. Cell Regul. 1991; 2: 1081-1095Crossref PubMed Scopus (141) Google Scholar). After 26-28 h, cells were washed and incubated in serum-free minimal essential medium containing 10 μg/ml bovine transferrin (Hyclone), 10 μg/ml porcine insulin (Lily), 1.0 mg/ml bovine serum albumin (Armour), 0.1 mM bromodeoxyuridine, and 100 μM ascorbic acid as an antioxidant. Drugs or their vehicle were then added, and the cultures were incubated for increasing times at 37°C.RNase Protection AssayTotal RNA (20.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62983) Google Scholar) was used in RNase protection assay with probes specific for each of the three rat α1-AR mRNAs, α1B, α1C, and α1D, and with probes for glyceraldehyde-phosphate dehydrogenase (GAPDH) and β-actin, as described and validated previously(14.Stewart A.F.R. Rokosh D.G. Bailey B.A. Karns L.R. Chang K.C. Long C.S. Kariya K. Simpson P.C. Circ. Res. 1994; 75: 796-802Crossref PubMed Scopus (93) Google Scholar, 21.Rokosh D.G. Bailey B.A. Stewart A.F.R. Karns L.R. Long C.S. Simpson P.C. Biochem. Biophys. Res. Commun. 1994; 200: 1177-1184Crossref PubMed Scopus (130) Google Scholar). The rat β-actin probe (188 bases; Ambion) protected a fragment of 126 bases (coding sequence nucleotides 771-896)(22.Nudel U. Zakut R. Shani M. Neuman S. Levy Z. Yaffe D. Nucleic Acids Res. 1983; 11: 1759-1771Crossref PubMed Scopus (1017) Google Scholar). In most experiments, the autoradiographic bands for each mRNA were scanned at 600 dpi and quantified as arbitrary density units/μg of total RNA (30-50 μg of RNA in each assay) (Scan Analysis, Biosoft). In the pressure overload experiments, bands were quantified by phosphor imaging (Bio-Rad GS-363 Molecular Imager). In all cases, signals were corrected for background and were within the linear range of the assay.[3H]Prazosin BindingA total membrane fraction (100,000 × g pellet of a myocyte homogenate) was used for [3H]prazosin binding, with 10 μM phentolamine to define nonspecific binding, as described previously (14.Stewart A.F.R. Rokosh D.G. Bailey B.A. Karns L.R. Chang K.C. Long C.S. Kariya K. Simpson P.C. Circ. Res. 1994; 75: 796-802Crossref PubMed Scopus (93) Google Scholar). In competition binding, membranes in assay buffer (1.0 ml final volume) were incubated at 30°C in a shaking water bath with 22 concentrations of the α1-adrenergic antagonist 5-methylurapidil (5MU) (0.01-100 nM) for 5 min before the addition of 0.25 nM [7-methoxy-3H]prazosin (72 Ci/mmol, DuPont NEN). A [3H]prazosin KD of 0.103 ± 0.013 nM (n = 5) was used for competition binding analyses with the iterative curve-fitting program EBDA/LIGAND (23.Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7760) Google Scholar); NE treatment for 48 h had no effect on the [3H]prazosin KD by saturation binding (data not shown). Differences between competition binding curves were determined by two-factor repeated measures analysis of variance.Phosphatidylinositol HydrolysisMyocytes in triplicate 35-mm dishes were radiolabeled with 5 μCi/ml myo-[2-3H]inositol (4 Ci/mmol, Amersham Corp.) for 48 h, rinsed, and treated with vehicle or six concentrations of NE (1 nM to 100 μM) in the presence of 10 mM LiCl. After 10 min at 37°C, cellular [3H]inositol phosphates (IPs) were extracted with trichloroacetic acid and separated by anion-exchange chromatography, as described previously(24.Karliner J.S. Kagiya T. Simpson P.C. Experientia. 1990; 46: 81-84Crossref PubMed Scopus (30) Google Scholar).Cultured Myocyte GrowthMyocytes in triplicate 35-mm dishes were incubated with vehicle or four concentrations of NE (20 nM to 20 μM) for 72 h in the presence of 0.1 μCi/ml L-[U-14C]phenylalanine (498 mCi/mmol, DuPont NEN). Myocyte size was quantified from the content of radiolabeled protein per dish. Cell numbers are unchanged over time in these cultures, and >90% of the cells are myocytes(8.Simpson P. J. Clin. Invest. 1983; 72: 732-738Crossref PubMed Scopus (546) Google Scholar, 9.Simpson P. Circ. Res. 1985; 56: 884-894Crossref PubMed Scopus (403) Google Scholar).Aortic Banding and Isolation of Adult Cardiac MyocytesAdult male Sprague-Dawley rats (200-220 gm) were anesthetized with pentobarbital, and a 0.31-mm internal diameter Weck hemoclip was placed around the suprarenal abdominal aorta through a left flank incision. For sham controls, the clip was not closed. After 10-12 weeks, cardiac myocytes were isolated by retrograde perfusion of the heart with collagenase, as described previously(14.Stewart A.F.R. Rokosh D.G. Bailey B.A. Karns L.R. Chang K.C. Long C.S. Kariya K. Simpson P.C. Circ. Res. 1994; 75: 796-802Crossref PubMed Scopus (93) Google Scholar). Myocyte yield was 10.9 ± 0.2 × 106/heart for banded rats (n = 4) and 10.1 ± 0.6 × 106/heart for shams (n = 5), and >90% of cells were rod-shaped. An aliquot of the isolated myocytes was used to measure cell volume on a Coulter Multisizer, and the rest were used to prepare RNA(20.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62983) Google Scholar).Materials(-)-NE HCl, human/porcine ET-1, PMA, and (-)-timolol maleate were from Sigma; prazosin HCl and 5MU, from Research Biochemicals Inc. PGF2α was from Caymen Chemicals, [5,6-3H]uridine (37 Ci/mmol) was from DuPont NEN, and actinomycin D was from Calbiochem. Stock solutions were in 1 mM HCl, or in Me2SO for prazosin, actinomycin D, PGF2α, and PMA (final Me2SO ≤ 0.01 volume %).Data AnalysisResults are presented as the mean ± SE from the number of experiments indicated. Treated/control ratios were tested for deviation from unity by calculation of confidence limits. Mean values were compared by the paired, two-sided Student's t test.RESULTSIn this neonatal rat cardiac myocyte culture model, α1-AR-induced growth and gene expression are half-maximum at ~12-18 h and maximum at ~24-48 h of catecholamine exposure(9.Simpson P. Circ. Res. 1985; 56: 884-894Crossref PubMed Scopus (403) Google Scholar, 10.Waspe L.E. Ordahl C.P. Simpson P.C. J. Clin. Invest. 1990; 85: 1206-1214Crossref PubMed Scopus (155) Google Scholar); and these responses require continuous receptor occupancy by agonist(25.Simpson P. Circulation. 1984; 70 (II-198)Google Scholar, 26.Lee H.R. Henderson S.A. Reynolds R. Dunnmon P. Yuan D. Chien K.R. J. Biol. Chem. 1988; 263: 7352-7358Abstract Full Text PDF PubMed Google Scholar, 27.Sei C.A. Irons C.E. Sprenkle A.B. McDonough P.M. Brown J.H. Glembotski C.C. J. Biol. Chem. 1991; 266: 15910-15916Abstract Full Text PDF PubMed Google Scholar). Thus, continuous α1-AR signaling appears to occur over long times, seemingly counter to the well established concept of AR desensitization by agonist(7.Liggett S.B. Lefkowitz R.J. Sibley D.D. Houslay M. Regulation of Cellular Signal Transduction Pathways by Desensitization and Amplification. 7. John Wiley & Sons, Inc., New York1993: 71-97Google Scholar).To determine if prolonged catecholamine exposure produced atypical regulation of one or more of the three α1-AR subtypes in the myocytes, the α1-AR mRNAs were quantified by RNase protection after treatment with NE. A dose of NE was used (2 μM) that is maximum for myocyte growth and gene transcription(9.Simpson P. Circ. Res. 1985; 56: 884-894Crossref PubMed Scopus (403) Google Scholar, 10.Waspe L.E. Ordahl C.P. Simpson P.C. J. Clin. Invest. 1990; 85: 1206-1214Crossref PubMed Scopus (155) Google Scholar). After 24 h of NE exposure, α1B and α1D mRNA levels were reduced to about 26 and 42%, respectively, of those in control myocytes treated concurrently with vehicle (Fig. 1). Repression of α1B and α1D mRNAs was also evident when normalized to the levels at time 0, when NE was added (19 ± 1% and 14 ± 1% for α1B and α1D, respectively, n = 5, p < 0.05). ( 3As shown in Fig. 2(lane 2 versus lane 1), the level of α1D mRNA was markedly lower in the absence of serum; thus down-regulation of α1D mRNA by NE was greater when normalized to time zero than to the vehicle control.) In direct contrast to the decrease of α1B and α1D mRNAs, NE increased the abundance of α1C mRNA, by over 3-fold (Fig. 1). Induction of α1C mRNA was also observed when normalized to the level at time zero (4.4 ± 1.2-fold, n = 5, p < 0.05). As a control, NE had no significant effect on the mRNA for the "housekeeping" gene GAPDH (at 24 h, 1.16 ± 0.10-fold versus vehicle, 1.10 ± 0.08-fold versus time 0, n = 3, p = not significant). The nonselective α1-AR antagonist prazosin inhibited regulation of the α1-AR mRNAs by NE, whereas the β-AR antagonist timolol did not (Fig. 1). Thus activation of an α1-AR induced α1C mRNA and repressed α1B and α1D mRNAs.As shown in Fig. 2, the effects of NE on each mRNA were sustained for up to 72 h of continuous NE exposure. The effects were detectable at the earliest time studied (2 h; α1C 1.4-fold × control, α1B 76% of control, and α1D 37% of control; means from two experiments), were submaximum at 12 h (two experiments as in Fig. 3), and were maximum at ~24 h (four experiments as in Fig. 2).Figure 2:NE regulation of α1-AR mRNAs is sustained over 72 h. Cultured myocyte RNA was harvested for RNase protection assay at the time of 2 μM NE addition (0 h), and at 24, 48, and 72 h after the addition of NE (+) or vehicle(-). Protected fragments of the following sizes (bases) are shown: α1B, 432; α1C, 315; α1D, 217; and GAPDH, 316(21.Rokosh D.G. Bailey B.A. Stewart A.F.R. Karns L.R. Long C.S. Simpson P.C. Biochem. Biophys. Res. Commun. 1994; 200: 1177-1184Crossref PubMed Scopus (130) Google Scholar). Yeast transfer RNA (tRNA) was a control for nonspecific hybridization, and GAPDH was a control for input RNA. The same results were observed in three additional experiments, and the numbers were similar to those quantified in Fig. 1. At 72 h, the level of α1B mRNA in cells treated with NE alone was 25% of the vehicle control, whereas it was higher, 70% of control, with NE in the presence of timolol (2 μM), suggesting a β-AR contribution to persistent α1B mRNA down-regulation (mean from 3-4 experiments).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3:NE has no effect on α1-AR mRNA degradation. Cultured cardiac myocytes were treated with actinomycin D (+Act D) (0.05 μg/ml) or with its vehicle (-) in minimal essential medium with 1% calf serum for 3 h, and then 2 μM NE (+NE) or its vehicle(-) were added at time 0 (0 h). RNA was harvested at the times indicated (-3 to 12 h), and the α1-AR mRNAs and GAPDH mRNA were assayed by RNase protection. Protected fragments are as in Fig. 2, except a longer α1D riboprobe, 474 bases, was used to protect 446 bases of α1D mRNA (nucleotides 1752-2197(52.Lomasney J.W. Cotecchia S. Lorenz W. Leung W.-Y. Schwinn D.A. Yang-Feng T.L. Brownstein M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1991; 266: 6365-6369Abstract Full Text PDF PubMed Google Scholar)). The same results were obtained in an additional experiment, and receptor mRNA degradation half-lives (see "Results") were estimated from semilogarithmic plots of mRNA levels versus time in the presence of actinomycin D.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To test if the NE-induced changes in mRNA levels were transcriptional in origin, the myocytes were incubated with actinomycin D, at a concentration (0.05 μg/ml) that inhibited transcription by >95% within 3 h, as assayed by [3H]uridine incorporation into total RNA, but had no effect on cell viability over 12 h (data not shown). As shown in Fig. 3, all three α1-AR mRNAs disappeared rapidly in the presence of actinomycin D, with apparent degradation half-lives of ~2.5, 3, and 1 h for α1B, α1C, and α1D mRNAs, respectively (Fig. 3, lanes 1-3 and 5). Treatment with NE when transcription was inhibited by actinomycin D had no effect on the abundance of any α1-AR mRNA (Fig. 3, lanes 3-6). In the absence of actinomycin D, in contrast, the characteristic effects of NE on all three mRNAs were observed over the same time interval (12 h; Fig. 3, lanes 7 and 8). These results indicated that NE did not alter α1-AR mRNA stability and suggested that α1-AR stimulation regulated transcription of the α1-AR genes.Radioligand binding was used to test whether the changes in receptor subtype mRNAs were accompanied by parallel changes in receptor proteins. In competition binding assays with [3H]prazosin, the cloned rat α1C-AR has much higher affinity for the antagonist 5MU (4 nM) (28.Laz T.M. Forray C. Smith K.E. Bard J.A. Vaysse P.J.-J. Branchek T.A. Weinshank R.L. Mol. Pharmacol. 1994; 46: 414-422PubMed Google Scholar) than does the rat α1B-AR (122 ± 4 nM) (28.Laz T.M. Forray C. Smith K.E. Bard J.A. Vaysse P.J.-J. Branchek T.A. Weinshank R.L. Mol. 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In the cultured cardiac myocytes, competition binding with [3H]prazosin distinguished an α1-AR population with high affinity for 5MU (4.4-7.6 nM) and a population with low affinity (261-277 nM) (Table 1). The high affinity sites were assumed to reflect the α1C-AR, and the low affinity sites were assumed to reflect the α1B-AR and/or the α1D-AR. ( 4Receptor inactivation with chloroethylclonidine (28.Laz T.M. Forray C. Smith K.E. Bard J.A. Vaysse P.J.-J. Branchek T.A. Weinshank R.L. Mol. Pharmacol. 1994; 46: 414-422PubMed Google Scholar, 50.Perez D.M. Piascik M.T. Malik N. Gaivin R. Graham R.M. Mol. Pharmacol. 1994; 46: 823-831PubMed Google Scholar, 51.Hiramatsu Y. Muraok A.R. Kigoshi S. Muramatsu I. Br. J. Pharmacol. 1992; 105: 6-7Crossref PubMed Scopus (11) Google Scholar) did not resolve a more sensitive α1B-AR and a less sensitive α1D-AR in the population of α1-ARs with low affinity for 5MU (data not shown).) Exposure to NE for 72 h decreased the number of low affinity sites to 60% of control, consistent with down-regulation of the α1B and/or the α1D (Table 1). In contrast, NE exposure for 72 h doubled the number of high affinity sites, consistent with up-regulation of the α1C-AR (Table 1) and in agreement with the 3-fold increase in α1C mRNA (Fig. 1). Thus in the cardiac myocytes exposed chronically to NE, the α1C-AR became the predominant receptor, increasing from 26 to 55% of total α1-ARs (Table 1). It was noteworthy that total receptor number did not change, despite the significant shift in subtype proportions (Table 1).Tabled 1 Open table in a new tab PLC activation was used to test for desensitization of α1-AR signaling. α1-AR coupling to PLC in cultured neonatal rat cardiac myocytes is through an α1-AR with high affinity for 5MU (2 nM)(33.Knowlton K.U. Michel M.C. Itani M. Shubeita H.E. Ishihara K. Brown J.H. Chien K.R. J. Biol. Chem. 1993; 268: 15374-15380Abstract Full Text PDF PubMed Google Scholar), probably the α1C (see above for 5MU affinities of cloned α1-ARs). In myocytes exposed to 2 μM NE for 48 h, the EC50 for NE-stimulated total [3H]IP production was unchanged (0.7 μM for NE-treated cells versus 0.9 μM for control cells, mean of four experiments). Fractions corresponding to IP1, IP2, and IP3 were increased equally in NE-treated and control cells (data not shown), and maximum total [3H]IP responses were not different (3.6-fold for NE-treated cells versus 3.3-fold for controls). Thus there was no desensitization of α1-AR-mediated PLC activation with 72 h of NE exposure, consistent with prior studies in cultured myocytes (34.Abdellatif M.M. Neubauer C.F. Lederer W.J. Rogers T.B. Circ. Res. 1991; 69: 800-809Crossref PubMed Scopus (67) Google Scholar, 35.McDonough P.M. Brown J.H. Glembotski C.C. Am. J. Physiol. 1993; 264: H625-H630Crossref PubMed Google Scholar) and with unimpaired α1C-AR signaling. It was not possible to test for desensitization of α1B and/or α1D signaling, since biochemical responses coupled to these α1-ARs in myocytes have not been identified conclusively.There was also no desensitization of a physiological response to chronic α1-AR activation, NE-induced myocyte growth. In myocytes pretreated with 2 μM NE for 72 h, the EC50 for NE-stimulated protein accumulation over the subsequent 72 h was the same as in control myocytes (0.8 ± 0.3 μM for NE-treated cells versus 0.7 ± 0.3 μM for controls, n = 5, p = not significant).To test if induction and repression of the α1-AR subtype mRNAs required α1-AR occupancy, mRNA levels were measured after treatment of the myocytes with three other hypertrophic growth factors, ET-1(15.Shubeita H.E. McDonough P.M. Harris A.N. Knowlton K.U. Glembotski C.C. Brown J.H. Chien K.R. J. Biol. Chem. 1990; 265: 20555-20562Abstract Full Text PDF PubMed Google Scholar), PGF2α(16.Tsoporis J.N. Zeirhut W. Karns L.R. Simpson P.C. J. Mol. Cell. Cardiol. 1994; 26 (CLXXVIII)Google Scholar), and PMA(17.Henrich C.J. Simpson P.C. J. Mol. Cell. Cardiol. 1988; 20: 1081-1085Abstract Full Text PDF PubMed Scopus (141) Google Scholar). As shown in Fig. 4, ET-1, PGF2α, and PMA were all similar to NE, inducing α1C mRNA and repressing α1B mRNA. Interestingly, unlike NE, PGF2α tended to induce α1D mRNA, and PMA had no effect on α1D (Fig. 4).Figure 4:Hypertrophic stimuli induce α1C mRNA and repress α1B mRNA in culture and in vivo. Cultured neonatal rat cardiac myocytes were treated for 24 h with 2 μM NE, 100 nM ET-1, 10 μM PGF2α, 100 nM PMA, or their vehicle controls. Adult cardiac myocytes were isolated from the intact rat heart after 10-12 weeks of aortic banding. Total RNA was prepared and the α1-AR subtype mRNAs were quantified by RNase protection. For the growth factors in culture, values are the mean ± S.E. treated/control ratios for three separate experiments (five for PMA). For aortic banding, values are the mean banded/sham ratios for four banded rats and five shams; statistical analyses were done on the absolute phosphor imaging values. ∗, p < 0.05 versus vehicle control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The pattern of α1-AR mRNA regulation produced by NE in culture was also observed with a pressure overload stimulus for hypertrophy in the intact animal. Myocytes were isolated from the adult rat heart after 10-12 weeks of abdominal aortic banding. Banding stimulated myocyte hypertrophy, increasing the mean volume of isolated myocytes by ~20%, from 36,000 ± 300 μm3/myocyte with sham operation to 43,000 ± 900 μm3 with b
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