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• W1984941270 abstract "Rat aorta smooth muscle cells which express all three α1-adrenoreceptors (α1A, α1B and α1D) were used to determine the effect of stimulation of α1-adrenergic receptor subtypes on cell growth. “Combined”α1-adrenoreceptor subtype stimulation with norepinephrine alone caused a concentration-dependent, prazosin-sensitive increase in protein content and synthesis: 48 h of stimulation at 1 μM increased cell protein to 216 ± 40% of time-matched controls (p = 0.008) and RNA to 140 ± 13% (p = 0.03); protein synthesis increased to 167 ± 13% (p < 0.01) after 24 h. Stimulation with norepinephrine plus the selective α1A/α1D antagonist 5-methylurapidil produced greater increases in α-actin mRNA (270 ± 40% at 8 h; p = 0.007), total cell protein (220 ± 45% at 24 h; p = 0.004), and RNA (135 ± 8% at 24 h; p = 0.01). These effects were prevented by pretreatment with the selective α1B antagonist chloroethylclonidine. Comparable results were obtained for intact aortae. Stimulation with norepinephrine plus 5-methylurapidil increased (p < 0.05) tissue protein, RNA, dry weight, and α-actin mRNA; and as in cultured cells, combined stimulation with norepinephrine alone attenuated these responses. By comparison, adventitia (fibroblasts) was unaffected. Removal of endothelial cells had no effect. α1B mRNA decreased by 42 ± 12% (p = 0.01) in cultured cells during combined α1-adrenoreceptor stimulation and by 23 ± 8% (p = 0.03) for intact aorta. α1D and β-actin mRNA were unchanged in cultured cells, aorta media, and adventitia. These findings suggest that prolonged stimulation of chloroethylclonidine-sensitive, possibly α1B-adrenoreceptors induces hypertrophy of arterial smooth muscle cells and that stimulation of 5-methylurapidil-sensitive, non-α1B-adrenoreceptors attenuates this growth response. Rat aorta smooth muscle cells which express all three α1-adrenoreceptors (α1A, α1B and α1D) were used to determine the effect of stimulation of α1-adrenergic receptor subtypes on cell growth. “Combined”α1-adrenoreceptor subtype stimulation with norepinephrine alone caused a concentration-dependent, prazosin-sensitive increase in protein content and synthesis: 48 h of stimulation at 1 μM increased cell protein to 216 ± 40% of time-matched controls (p = 0.008) and RNA to 140 ± 13% (p = 0.03); protein synthesis increased to 167 ± 13% (p < 0.01) after 24 h. Stimulation with norepinephrine plus the selective α1A/α1D antagonist 5-methylurapidil produced greater increases in α-actin mRNA (270 ± 40% at 8 h; p = 0.007), total cell protein (220 ± 45% at 24 h; p = 0.004), and RNA (135 ± 8% at 24 h; p = 0.01). These effects were prevented by pretreatment with the selective α1B antagonist chloroethylclonidine. Comparable results were obtained for intact aortae. Stimulation with norepinephrine plus 5-methylurapidil increased (p < 0.05) tissue protein, RNA, dry weight, and α-actin mRNA; and as in cultured cells, combined stimulation with norepinephrine alone attenuated these responses. By comparison, adventitia (fibroblasts) was unaffected. Removal of endothelial cells had no effect. α1B mRNA decreased by 42 ± 12% (p = 0.01) in cultured cells during combined α1-adrenoreceptor stimulation and by 23 ± 8% (p = 0.03) for intact aorta. α1D and β-actin mRNA were unchanged in cultured cells, aorta media, and adventitia. These findings suggest that prolonged stimulation of chloroethylclonidine-sensitive, possibly α1B-adrenoreceptors induces hypertrophy of arterial smooth muscle cells and that stimulation of 5-methylurapidil-sensitive, non-α1B-adrenoreceptors attenuates this growth response. INTRODUCTIONEvidence suggests that the sympathetic nervous system and α1-adrenoreceptors (AR) ( (1)The abbreviations used are: ARadrenergic receptorSMCsmooth muscle cellNEnorepinephrine5-MU5-methylurapidilCECchloroethylclonidineMOPS3-(N-morpholino)propanesulfonic acidbpbase pair(s)RPARNase protection assayPIphosphoinositide.) may exert trophic influences over SMCs during normal development and also contribute to the pathogenesis of vascular hypertrophy and atherosclerosis(1.Bauch H.J. Grunwald J. Visher P. Gerach U. Hauss W.H. Circulation. 1984; 69: 441-450Crossref PubMed Scopus (98) Google Scholar, 2.Vashisht R. Siam M. Franks P.J. O'Malley M.K. Br. J. Surg. 1992; 79: 1285-1288Crossref PubMed Scopus (37) Google Scholar). Hyperinnervation of blood vessels by catecholaminergic fibers in the genetic spontaneously hypertensive rat has been correlated with SMC hypertrophy and hyperplasia in these vessels(3.Head R.J. Blood Vessels. 1991; 28: 173-178PubMed Google Scholar). Also, sympathectomy attenuates normal growth, as well as hypertrophy of the vascular wall in hypertensive animals(4.Bevan R.D. Tsuru H. Blood Vessels. 1979; 16: 109-112PubMed Google Scholar, 5.Nyborg N.C.B. Mulvany M.J. Cardiovasc. Res. 1985; 19: 528-536Crossref PubMed Scopus (61) Google Scholar, 6.Hart M.N. Heistad D.D. Brody M.J. Hypertension. 1980; 2: 419-423Crossref PubMed Scopus (206) Google Scholar, 7.Lee R.M.K.W. Smeda J. Can. J. Physiol. Pharmacol. 1985; 63: 392-401Crossref PubMed Scopus (66) Google Scholar). There is considerable evidence that smoking, stress, and hypertension, which are key risk factors for atherosclerosis and hypertrophic vascular disease, are associated with elevated plasma catecholamines(8.Houston M.C. Dzau V.J. Loscalzo J. Creager M.A. Dzau V.J. Vascular Medicine. Little Brown, Boston1992: 621-657Google Scholar, 9.Kones R.J. Angiology. 1979; 30: 327-336Crossref PubMed Scopus (11) Google Scholar).Catecholamines have been shown to initiate not only immediate SMC responses such as contraction of blood vessels, but may also influence proliferation and growth of cultured vascular SMCs(10.Blaes N. Boissel J.P. J. Cell. Physiol. 1983; 116: 167-172Crossref PubMed Scopus (148) Google Scholar, 11.Geisterfer A.A.T. Peach M.J. Owens G.K. Circ. Res. 1988; 62: 749-756Crossref PubMed Scopus (1008) Google Scholar, 12.Holycross B.J. Peach M.J. Owens G.K. J. Vasc. Res. 1993; 30: 80-86Crossref PubMed Scopus (40) Google Scholar, 13.Jackson C.L. Schwartz S.M. Hypertension. 1992; 20: 713-736Crossref PubMed Scopus (236) Google Scholar). In nonconfluent, cultured rat and rabbit aortic SMCs, AR stimulation promotes cell proliferation(10.Blaes N. Boissel J.P. J. Cell. Physiol. 1983; 116: 167-172Crossref PubMed Scopus (148) Google Scholar, 13.Jackson C.L. Schwartz S.M. Hypertension. 1992; 20: 713-736Crossref PubMed Scopus (236) Google Scholar). Furthermore, α1 blockade reduces vascular collagen synthesis in the spontaneous hypertensive rat(14.Chickester C.O. Rodgers R.L. J. Cardiovasc. Pharmacol. 1987; 10: S21-S26Crossref PubMed Scopus (27) Google Scholar), and inhibits SMC proliferation induced by endothelial denudation (13.Jackson C.L. Schwartz S.M. Hypertension. 1992; 20: 713-736Crossref PubMed Scopus (236) Google Scholar, 15.O'Malley M.K. McDermott E.W.M. Mehigan D. O'Higgins N.J. Br. J. Surg. 1989; 76: 936-938Crossref PubMed Google Scholar) and angiotensin infusion (16.van Kleef E.M. Smits J.F.M. DeMey J.G.R. Cleutjens J.P.M. Lombardi D.M. Schwartz S.M. Daemen M.J.A.P Circ. Res. 1992; 70: 1122-1127Crossref PubMed Scopus (74) Google Scholar) in normal rats. In cholesterol-fed monkeys, elevated plasma norepinephrine (NE) greatly increased atherosclerotic lesion growth(17.Kukrega R.S. Datta B.N. Chakravarti R.N. Atherosclerosis. 1981; 40: 292-298Google Scholar). Infusion of NE over a 2-week period, at a level which did not cause sustained elevation of blood pressure, induced formation of atherosclerotic vascular lesions in rabbit aorta(18.Helin P. Lorenzen I. Garbarsch C. Matthiessen E. Atherosclerosis. 1970; 12: 125-132Abstract Full Text PDF PubMed Scopus (37) Google Scholar). There is also evidence that α1ARs mediate growth of myocardial cells. In cultured neonatal rat cardiac myocytes that possess both β1 and α1ARs, stimulation of α1ARs with NE increased cell protein, RNA, myocyte surface area, and contractile protein expression(19.Simpson P. Circ. Res. 1985; 56: 884-889Crossref PubMed Scopus (403) Google Scholar). However, no studies have examined whether α1ARs influence proliferation-independent growth of SMCs.Both molecular cloning and pharmacologic studies have shown that α1ARs are comprised of three closely related subtypes(20.Bylund D.B. Eikenberg D.C. Hieble J.P. Langer S.Z. Lefkowitz R.J. Minneman K.P. Molinoff P.B. Ruffolo Jr., R.R. Trendelenburg U. Pharmacol. Rev. 1994; 46: 121-136PubMed Google Scholar, 21.Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1130) Google Scholar). We have recently used polymerase chain reaction (22.Ping P. Faber J.E. Am. J. Physiol. 1993; 265: H1501-H1509Crossref PubMed Google Scholar) and RNase protection assays ( (2)A. E. Eckhart and J. E. Faber, submitted for publication.) to determine expression of αAR subtype mRNA by rat vascular SMCs. Freshly isolated and early passage cultured aortic and vena cava SMCs express both α1B and α1D mRNA(20.Bylund D.B. Eikenberg D.C. Hieble J.P. Langer S.Z. Lefkowitz R.J. Minneman K.P. Molinoff P.B. Ruffolo Jr., R.R. Trendelenburg U. Pharmacol. Rev. 1994; 46: 121-136PubMed Google Scholar), and it appears that both receptors are present on SMCs of rat aorta (24, 25, and (22.Ping P. Faber J.E. Am. J. Physiol. 1993; 265: H1501-H1509Crossref PubMed Google Scholar) and references therein). Rat aorta has been shown recently to also express α1A (formerly denoted “α1C”) mRNA(26.Perez D.M. Piascik M.T. Malik N. Gaivin R. Graham R.M. Mol. Pharmacol. 1994; 46: 823-831PubMed Google Scholar, 27.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, 28.Price D.T. Chari R.S. Berkowitz D.E. Meyers W. Schwinn D.A. Mol. Pharmacol. 1994; 46: 221-226PubMed Google Scholar, 29.Ford P.D.W. Williams T.J. Blue D.R. Clark D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar). Given this multiplicity of α1AR expression, the purpose of the present study was first to examine both in vitro and in situ, the effects of combined stimulation of the α1AR subtypes with NE alone on proliferation-independent growth, and expression of sarcomeric α-SMC-actin and cytoskeletal β-actin mRNAs by arterial and venous SMCs. Second, effects of combined stimulation were compared with those during treatment with NE plus antagonists, 5-methylurapidil (5-MU, selectivity = α1A > α1D > α1B) to favor α1B stimulation and after pretreatment with chloroethylclonidine (CEC, selectivity = α1B > α1D > α1A) to select for stimulation of non-α1BARs. The results suggest, in both cultured aorta SMCs and intact aorta media, that stimulation of CEC-sensitive α1ARs (possibly α1B) induces hypertrophy of aorta SMCs and that stimulation of 5-MU-sensitive, non-α1BARs antagonizes this response. Thus, alterations in the relative activity of different α1AR subtype signaling pathways may participate in both normal vascular growth and remodeling and also in vascular hypertrophic diseases.MATERIALS AND METHODSCell CulturePrimary cultures of vascular SMCs were each obtained from pooled thoracic aortae or vena cavae from eight adult Sprague-Dawley rats (200 g, Sasko, Omaha, NE) by a modification (22.Ping P. Faber J.E. Am. J. Physiol. 1993; 265: H1501-H1509Crossref PubMed Google Scholar) of the method of Turla et al.(30.Turla M.B. Thompson M.M. Corjay M.H. Owens G.K. Circ. Res. 1991; 68: 288-299Crossref PubMed Scopus (84) Google Scholar) and maintained in Medium 199 (M199) supplemented with 10% fetal bovine serum, 200 mg/ml L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were passaged at 90-95% confluence with 0.05% trypsin/EDTA every 5-7 days and seeded at a density of 5000 cells/cm2 on plastic plates. Viable cell number was determined by hematocytometry with trypan blue exclusion in duplicate. To favor the differentiated phenotype and quiescence, in all experiments cells were exposed to experimental conditions beginning at 4 days after reaching confluence in passage 4. A separate cell line was used in each experimental replicate. These cultures lacked contamination by other cell types as revealed by immunohistochemistry with monoclonal antibodies (Dako A/S, Glostrup, Denmark) against SMC-specific α-actin and von Willebrand factor.ProtocolsTo examine the effect of combined α1AR stimulation on SMC growth, as reflected by changes in total protein and RNA per cell(19.Simpson P. Circ. Res. 1985; 56: 884-889Crossref PubMed Scopus (403) Google Scholar), in the first experiment aorta or vena cava cells were exposed for 8, 24, and 48 h to 1 μM NE. In this and all subsequent experiments, 100 μM ascorbate was present to prevent NE degradation(19.Simpson P. Circ. Res. 1985; 56: 884-889Crossref PubMed Scopus (403) Google Scholar), plus 0.5 μM rauwolscine and 1 μM propranolol to minimize activation of α2- and βARs. Control cells in all experiments were time-matched and exposed to these agents, but not to NE (vehicle groups). In a second experiment cells were exposed to 1 μM NE and also to the α1 antagonist prazosin (1 μM). mRNAs for SMC-specific α-actin, β-actin, α1DAR, and α1BARs were measured (below). Effects of combined α1AR stimulation were also examined in aorta SMCs maintained in serum-free media after the second day of post-confluence (48 h before start of experiment). Serum-free media consisted of 50% DMEM, 50% F-12 media supplemented with 2.85 mg/ml insulin, 5 mg/liter transferrin, 35.2 mg/liter ascorbic acid, 6 μg/ml selenium, 100 units/ml penicillin, and 100 μg/ml streptomycin. Since responses were similar regardless of presence or absence of serum (see “Results”) subsequent experiments (below) were conducted in serum-free medium. In a third experiment, aorta SMCs were exposed for 24 h to NE (10-9 to 10-5M) for determination of concentration-response effects. In a fourth experiment aortic SMCs were exposed for 8 and 24 h to 1) vehicle, 2) 10 μM NE, 3) NE plus the α1A/D antagonist 5-MU (0.3 μM), or 4) NE after pretreatment with the selective α1B antagonist CEC (3 μM)(20.Bylund D.B. Eikenberg D.C. Hieble J.P. Langer S.Z. Lefkowitz R.J. Minneman K.P. Molinoff P.B. Ruffolo Jr., R.R. Trendelenburg U. Pharmacol. Rev. 1994; 46: 121-136PubMed Google Scholar, 24.Gurdal H. Johnson M.D. FASEB J. 1993; 7: A46Google Scholar, 25.Han C.D. Esbenshade T.A. Minneman K.P. Eur. J. Pharmacol. 1992; 226: 141-148Crossref PubMed Scopus (33) Google Scholar, 29.Ford P.D.W. Williams T.J. Blue D.R. Clark D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 31.Leech, C. J., Faber, J. E. (1995) Am. J. Physiol., in pressGoogle Scholar). Cells were incubated with CEC for 30 min at 37°C to alkylate and irreversibly inactivate α1BARs, followed by several media changes. During this incubation rauwolscine and propranolol were present to minimize alkylation of other ARs. CEC was not present during the subsequent 24-h exposure to NE. Concentrations of all antagonists were selected based on our previous determinations of their efficacy in in vitro vascular contractile studies (31.Leech, C. J., Faber, J. E. (1995) Am. J. Physiol., in pressGoogle Scholar, 32.Ikeoka K. Faber J.E. Am. J. Physiol. 1993; 265: H1988-H1995PubMed Google Scholar) and are in agreement with concentrations used by others (cf. Refs. 20, 25, 29, 31 and references therein).Organ CultureTo examine the effects of α1AR stimulation on SMC growth and mRNA expression in cells maintained “in situ” in a state more closely resembling the normal arterial wall environment than cultured SMC monolayers, an organ culture system was devised for the thoracic aorta with intima, media, and adventitia intact. In rat thoracic aorta the media is composed entirely of SMCs, while the adventitia is ≈ 95% fibroblasts(22.Ping P. Faber J.E. Am. J. Physiol. 1993; 265: H1501-H1509Crossref PubMed Google Scholar, 33.Ives H.E. Schultz G.S. Galardy R.E. Jamieson J.D. J. Exp. Med. 1978; 148: 1400-1413Crossref PubMed Scopus (74) Google Scholar). For each experiment, 25-mm lengths of thoracic aortae (1 per 200 g rat) were dissected under sterile conditions from 16 rats after CO2 asphyxiation and decapitation. Vessels were transferred to 4°C M199, and loose connective and fatty tissue were carefully removed. Eight aortae were randomly assigned to either the control or treated group in each experiment. Individual aortae were suspended horizontally from a stainless-steel wire in serum-free medium. Circumferential wall tension was provided by a second wire connected to an adjustable weight (0.4 g/mm vessel length). This tension was selected to achieve optimal preload based on contractile studies of isolated rat aortic rings. To examine the effect of combined α1AR subtype stimulation, in the first experiment vessels were exposed for 24 h to 10 μM NE, 10 μM ascorbic acid, 0.5 μM rauwolscine, and 1 μM propranolol in a 95% air, 5% CO2 incubator. Control (vehicle) vessels were exposed to these agents but not NE. In a second experiment to examine the effect of endothelial cell presence and of selective α1BAR stimulation on SMC and adventitial growth, both endothelial cell denuded and intact vessels (four groups; n = 3 vessels per group) were compared in an experimental design identical to the preceding one, but in the presence of 0.1 μM 5-MU. In these experiments a smaller number of vessels were studied, because only total RNA, protein RNA, α-actin, and vessel dry weight were measured. Endothelial cells were removed from vessels in 4°C M199 using a nylon wire technique which avoids mechanical damage and proliferative stimulation of the underlying smooth muscle media(34.Reidy M.A. Jackson C. Lindner V. Vasc. Med. Rev. 1992; 3: 156-167Crossref Google Scholar, 35.Fingerle J. Au Y.P. Clowes A.W. Reidy M.A. Arteriosclerosis. 1990; 10: 1082-1087Crossref PubMed Google Scholar). We have demonstrated that this technique results in removal of greater than 90% of the endothelial cells.2 In all experiments after 24 h, aortae were incubated in an enzyme solution (Hanks' buffered saline solution (Life Technologies, Inc.), penicillin (100 U/ml), streptomycin (10 μg/ml), 16 mM sodium bicarbonate, 1 mM calcium chloride dihydrate, pH 7.2, 2 mg/ml collagenase, 2 mg/ml soybean trypsin inhibitor, and 13.5 units/ml of elastase (Worthington)) at 37°C with 5% CO2, 95% air− Endothelial cells were then removed or sham-removed in the already denuded group by gently rubbing with a cotton tipped applicator, and the media and adventitia were separated with fine forceps using a dissection microscope and a 4°C tissue bath containing M199. Media and adventitia were washed several times, frozen in liquid nitrogen, powdered, and immediately placed in guanidinium thiocyanate (see below) and stored at −70°C until RNA extraction. Protein was determined for fresh media and adventitia as described below. Dry weights were determined for 5-mm lengths of intact vessels from each group after baking at 60°C for 4 h.RNA and Protein DeterminationTotal cellular RNA was determined (from duplicate plates or vessel extracts) using standard techniques(36.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62983) Google Scholar). RNA integrity was assessed, and variations in aliquoted amounts in each RNase protection assay were corrected (see below), according to film densitometry (UMAX UC630 film scanner and the Image Program (National Institutes of Health)) of 28 and 18 S rRNA bands resolved on ethidium bromide-stained, 1 × MOPS/formaldehyde, 1% agarose gels. RNA quantity and purity were determined spectrophotometrically. Total soluble protein was determined in duplicate using a modified (37.Goldschmidt R.C. Kimelberg H.K. Anal. Biochem. 1989; 177: 41-45Crossref PubMed Scopus (40) Google Scholar, 38.Akins R.E. Tuan R.S. BioTechniques. 1992; 12: 496-499PubMed Google Scholar) BCA assay (Pierce).Protein SynthesisFour-day, post-confluent SMCs maintained in serum-free media received a change of media containing low methionine (2 mg/liter), 100 μM ascorbate, 1 μM propranolol, and 10-9 to 10-5M NE (duplicate plates for each NE concentration). Eighteen hours later [35S]methionine (5 μCi/ml, 1000 Ci/mmol, Amersham) was added. After 6 h, cells were washed twice with 4°C phosphate-PBS and lifted with 0.05% trypsin-EDTA, which was then stopped with serum-containing M199. Pelleted cells were lysed with Nonidet P-40 at 4°C. The supernatant was treated with trichloroacetic acid at a final concentration of 10% in the presence of 100 μg/ml bovine serum albumen, and incubated for 30 min at 4°C. Trichloroacetic acid-precipitable counts were collected on Whatman GF/C filters and counted in Ecoscint H (National Diagnostics) after overnight shaking at 25°C.mRNA Quantificationα-Actin and β-actin plasmids contained, respectively, 191 and 526 base pairs (bp) of the 3′-untranslated region of the cDNA sequences. The 117-bp NcoI/BamHI fragment that encodes the third intracellular loop of the α1DAR was subcloned into pGEM-4Z (Promega). The 306-bp BamHI/PstI fragment that encodes the fourth and fifth transmembrane-spanning regions of the α1BAR was subcloned into pGEM-3Z. Identity and orientation of inserts was assessed by restriction enzyme analysis and sequencing. RNase protection assays (RPAs) were used to quantitate mRNA levels. In vitro transcribed [35P]rCTP-labeled cRNAs (riboprobes) and cold sense RNAs were made according to standard procedures. Each assay consisted of addition of a constant amount of a single labeled probe and total cell RNA corresponding to an equal number of cells or vessel length for control versus treatment groups (instead of constant amounts of RNA) to account for any treatment effects on cell size and number. Because total RNA per cell varied according to treatment groups (i.e. from hypertrophy), it was not appropriate to load a constant amount of cell RNA. Companion samples run beforehand on ethidium bromide gels for quantitation of 28 and 18 S rRNA (see above) were used to correct for variations in acutal versus expected amounts of cell RNA loaded. tRNA was added to bring each assay up to a constant amount of total RNA. Known amounts of sense RNA were similarly treated in each RPA to ensure molar excess of probe. Hybridization was performed at 57°C for 16 h and RNase digestions at 37°C for 30 min (α1D, α1B, and β-actin: RNase A (20 mg/ml, Boehringer Mannheim) + RNaseT1 (250 units/ml, Boehringer Mannheim); α-actin: RNase ONE (35 units/ml, Promega)). RNA hybrids were resolved on an 8 M urea, 6% polyacrylamide, 1 × TBE (Tris-borate-EDTA) gel. Dried gels were exposed to film (X-Omat, Kodak) with intensifying screens at −70°C for 3-96 h. Densitometry was performed as described above.Specificity of RPAsRiboprobes were tested for specificity using total RNA from liver, kidney, fresh aorta medial and adventitial layers, and cultured medial SMCs and also with homologous and heterologous sense RNAs for α1AR subtypes and actins. In agreement with polymerase chain reaction data (22.Ping P. Faber J.E. Am. J. Physiol. 1993; 265: H1501-H1509Crossref PubMed Google Scholar) and using up to 100 μg of RNA, the α1D riboprobe only detected mRNA in the aortic medial layer, adventitia, cultured SMCs, and kidney; the α1B probe only detected mRNA in the medial layer, cultured SMCs, liver, and kidney; the α-actin probe detected mRNA only in the medial layer of the aorta and cultured SMCs, and the β-actin probe detected mRNA in all of these tissues and cells. No cross-reactivity was found among riboprobes and heterologous sense RNAs for expected protected fragments.Analysis and StatisticsTotal RNA, protein, and mRNA species were determined on a per cell (SMC cultures) or per millimeter of vessel length (organ culture) basis for NE-treated and time-matched control (vehicle-treated) groups. Data are given as mean ± S.E.; means for groups with n sizes ≥ 3 were analyzed using t tests and analysis of variance, followed by Dunnett's test or the Bonferroni correction for multiple comparisons. A value of p < 0.05 was considered significant. n sizes represent the number of experimental replicates, each obtained from a separate cell culture line or group of pooled aortae maintained in organ culture.RESULTSEffect of Combined α1AR Subtype Stimulation on Aorta SMCsCombined stimulation of aorta SMC α1ARs with 1 μM NE in the presence of α2AR and βAR blockade did not induce cell proliferation or change cell viability (trypan blue exclusion) (Fig. 1). In both control (vehicle-treated), NE and NE + prazosin groups, viability averaged 97 ± 0.8% (data not shown). Total cell protein and RNA at 0 h in the control group were 713 ± 49 pg/cell and 24 ± 5 pg/cell, respectively, and were unchanged in the NE and NE + prazosin groups. Combined α1AR stimulation significantly increased cell RNA to 140 ± 13% of control (p = 0.03) at 48 h and increased cell protein to 167 ± 11% (p = 0.004) at 24 h and to 216 ± 40% (p = 0.008) at 48 h (Fig. 1). These increases were prevented by the α1 antagonist prazosin (Fig. 1). Because cells in defined, serum-free medium (n = 3) exhibited responses for this same protocol that did not differ significantly from cells in the presence of serum (n = 3), the data were combined (Fig. 1).This growth effect of combined α1AR subtype stimulation was further characterized by examining α-actin and β-actin mRNA. α1AR stimulation had no effect on levels of α- or β-actin mRNA, although α-actin tended to increase at the 24-h point (240% of control, p = 0.08) (Fig. 2). In contrast, in the presence of prazosin at the 8-, 24-, and 48-h points, mRNA (in percent of time 0) for α-actin was 85 ± 25, 70 ± 5, and 110 ± 22, respectively, and for β-actin was 92 ± 4, 95 ± 5, and 95 ± 18, respectively. α1BAR mRNA transiently decreased by 42 ± 12% of control (p = 0.01) at 8 h in serum-containing and by 38 ± 14% (p = 0.02) in serum-free medium and returned to control by 24 h in both groups (Fig. 2, data combined). In contrast, α1D mRNA evidenced no significant change. In the presence of prazosin, α1D and α1B mRNAs were not significantly different from those for control cells whose levels themselves did not change significantly over the 48-h interval. In the presence of NE plus prazosin at the 8-, 24-, and 48-h points, mRNA (in percent of time 0) for α1D was 107 ± 6, 100 ± 1, and 117 ± 20, respectively, and for α1B was 88 ± 30, 86 ± 10, and 119 ± 33, respectively. The observation that β-actin mRNA remained constant while total per cell RNA increased provides an internal standard that validates as specific the changes in α1B and α-actin mRNA.Figure 2:Combined stimulation of α1-adrenoreceptor subtypes decreased α1B and increased α-actin mRNA without altering α1D-adrenoreceptor and β-actin mRNA in aortic SMCs. Left panels, total RNA from 3 × 106 cells exposed to 8 and 24 h of NE ± vehicle (V; ascorbate, rauwolscine, and propranolol) was assayed for α1D and α1B mRNA using ribonuclease protection assay (RPA). Lane 1, probes alone (P); lane 2, probes + RNases A and T1 (PX); lane 3, zero hour (control); lane 4, 8-h NE; lane 5, 8-h vehicle; lane 6, 24-h NE; lane 7, 24-h Vehicle; lanes 8-10, three amounts of α1D or α1B sense RNA. Total RNA (70-80 μg) was loaded in lanes 3-7; lanes 2, 8, 9, and 10 balanced to 70-80 mg of RNA with yeast tRNA. Right panels, effect of combined stimulation of α1ARs with 1 μM NE on α1AR, α-actin, and β-actin mRNA per cell as assessed by RPA. n size refers to number of replicate experiments. ∗, p < 0.05 versus time-matched control cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Concentration-response experiments for per cell protein content and protein synthesis were conducted over 24 h of combined α1AR stimulation with NE (as in above protocol) in serum-free, defined medium. Combined stimulation caused dose-dependent increases in both protein content and synthesis relative to time-matched, vehicle-treated controls (Fig. 3). As in the preceding experiments, cell number was unchanged.Figure 3:Concentration-dependent stimulation of α1-adrenoreceptor for 24 h increased protein content (A) and protein synthesis ([35S]methionine incorporation) (B). C, time-matched control cells exposed to vehicle only. ∗ (∗∗), p < 0.05 (<0.01) versus control (analysis of variance plus Dunnett's test). NE concentrations given in -log molar. n sizes (replicate experiments) for all NE concentrations except 10-9 molar (n = 3) are n = 4 for A and n = 5 for B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Vena Cava Smooth MuscleThe effect of combined α1AR stimulation with 1 μM NE was also examined for passage 4, 4-day post-confluent thoracic vena cava SMCs maintained in serum-containing medium (n = 3 each for vehicle, NE, and NE + prazosin groups (data not shown)). There were no significant changes in viable cell number (0 h = 1.7 × 106± 0.2 cells/plate), total protein (0 h = 1028 ± 141 pg/cell), total RNA (0 h = 14.1 ± 2.8 pg/cell), or in α1B, α1D, α-actin, or β-actin mRNA levels at 8 and 24 h for any group.Selective Stimulation of Aorta SMC α1-Adrenoreceptor SubtypesTo determine the influence of stimulation of α1AR subtypes on aorta SMC growth, cells maintained in serum-free medium were treated for 8 and 24 h with 10 μM NE alone (combined α1AR stimulation), NE plus the competitive α1A/D antagonist 5-MU (0.3 μM), or NE after pretreatment with the irreversible α1B antagonist CEC (30 μM). All groups received rauwolscine, propranolol, and ascorbat" @default.
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