Phorbol Esters and Norepinephrine Destabilize crlB-Adrenergic ...

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THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 269, No. 3, Issue of January’ 21, pp. 1705-1710,1994 Printed in U.S.A.

8 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Phorbol Esters and NorepinephrineDestabilize crlB-Adrenergic Receptor mRNA in Vascular Smooth Muscle Cells* (Received for publication, February 17, 1993, and in revised form, September 2, 1993)

Nicholas J. Izzo, Jr.$Q,Thomas N. TulenkoT, and Wilson S . ColucciSII From the $Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115 and the llDepartment of Physiology, MedicalCollege of Pennsylvania, Philadelphia, Pennsylvania 19129

The mechanism by which norepinephrine (NE) The intracellular second messengers that mediate a-AR(a-AR) mRNA stimulated contraction inVSMC have been studied in detail. down-regulates alB-adrenergic receptor was studied in rabbit aortic smooth musclecells. NE, Both calcium that is mobilized from intracellular stores and phorbol esters, and bradykinin each decreased a-AR extracellular calcium that enters the cell byway of membrane mRNA levels by 70-80%. The protein kinaseC inhib- channels contribute to thecontractile response to NE (4).In itor (+)-l-(5-isoquinolinesulfonyl)-2-methylpipera-addition, activation of PKC may play a role in sustainingthe zinedihydrochloride (H-7) abolishedthe effects of contractile response to NE (5). The mobilization of intracelphorbol esters andNEanddecreasedbasalmRNA lular calcium and theactivation of PKC aremediated by NElevels by 52 f 3%.Neither ryanodinenor EGTA inhib- stimulated increases in the cellular levels of IPS and DAG, ited down-regulation of a-AR mRNA by NE. Actinomycin D caused a-AR mRNA level to decrease with a respectively (6). The mechanism by which a-AR activation effect of H-7to causes the influx of extracellular calcium is less clear, but half-life of 3.2 f 0.4 h and blocked the decrease basala-AR mRNA level. Both NE and phorbol may involve PKC (7) or direct coupling of a-AR to calcium esters increased the rate ofa-AR mRNA degradation. channels (6). Chronic stimulation by catecholamines results in thedownIn NE-desensitized cells, phorbol esters and bradykiregulation of a-AR in several tissues, including vascular nin each caused the expected down-regulationa-AR of smooth muscle (8, 9). In VSMC cultured from the rabbit mRNA. The protein phosphatase inhibitor okadaic acid prolonged the normally transient effect of NE for at aorta, NEcauses down-regulation of a-AR (10,ll) associated least 24 h. We conclude that protein kinase C exerts with a rapid (peak effect, 4 h), concentration-dependent (EC, two opposing effects on a-AR mRNA levels, 1) a de- = 200 nM), decrease in the level of mRNA for the a-AR to crease in the stability of the mRNA that requires the less than 20% of control values ( l l ) , suggesting that NE may sustained phosphorylation of a protein kinase C sub- regulate the density of VSMC WAR, at least in part, through strate and 2) a permissive effect on a-AR gene tran- a reduction in receptor synthesis. Despite continued exposure scription. to NE, WAR mRNA levelsreturn to control levels in approximately 24 h (11). We addressed three issues related to the mechanism by which NE regulates a-AR mRNA level. First, we wished to The stimulation of a-AR’ on VSMC by NE plays a major determine the mechanism by whichNE actsto down-regulate role in the regulation of vascular tone. In addition, there is a-AR mRNA. Since this effect is blocked by the highly WARevidence that a-AR may play an important role in the cellular selective antagonist prazosin, but not by propranolol ( l l ) , it biology of VSMC. In vascular smooth muscle, stimulation of must involve second messenger pathways coupled to the aa-AR can cause cell proliferation (l), increased DNA synthe- AR. Therefore, we focused on the roles of calcium and PKC sis (2), the expression of platelet-derived growth factor (3), in modulating the level of a-AR mRNA. Second, since the and activation of early response genes such as c-fos.’ Thus, second messenger pathways stimulated by a-AR in VSMC the a-ARmay bean important locus for regulating the effects of the sympathetic nervous system and catecholamines on the are not unique to the a-AR, it was of interest to determine whether a-AR mRNA levelscan be regulated in aheterologous contractile state andcellular biology of VSMC. manner. To approach this issue, we examined the effect of *This workwas supported, in part, by National Institutes of bradykinin, a hormone that, like NE, activates PKC and Health grants HL425309 and HL30496. The costs of publication of causes calcium mobilization in VSMC (12). Third, we wished this article were defrayed in part by the payment of page charges. to elucidate the mechanism for the transient nature of NEThis article must therefore be hereby marked “advertisement” in mediated down-regulation of a-AR mRNA in VSMC.

accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. § Supported by National Research Service Award HL08324. )ISandoz Established Investigator of the American Heart Association. To whom correspondence should be addressed: Cardiovascular Div., Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02114. Tel.: 617-732-5894; Fax: 617-732-5132. The abbreviations used are: a-AR, alB-adrenergic receptor; VSMC, vascular smooth muscle cell; NE, norepinephrine; PKC, protein kinase C; DAG, diacylglycerol; PDBu, phorbol12,13-dibutyrate, PMA, phorbol 12-myristate 13-acetate; H-7, (+)-1-(5-isoquinolinesulfonyl)-2-methylpiperazinedihydrochloride; BSB, balanced salt buffer; IPZ, inositol bisphosphate; IPS, inositol trisphosphate. * N. Izzo and W. Colucci, unpublished observations.

MATERIALS AND METHODS

Cell Culture-VSMCwere cultured and passaged as previously described (131, except that experimental plates were grown in MI99 media (Sigma) supplemented with 5% fetal calf serum (Life Technologies, Inc.), subsequently referred to as “growth medium.” Inositol Phosphate Assay-VSMC were grown to confluence on 35mm dishes. Each dish of cells was prelabeled with 15 pCiof [3H] myoinositol (NEN Research Products) added in 1 ml of growth medium for 18-24 h prior to harvesting. Prior to assay, the dishes were washed three times with serum-free medium M199 and incubated with 1 ml of the same at 37 “C for 3 h.

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Protein Kinase C Regulates a--Adrenergic Receptor mRNA

Cells were equilibrated for 15 min at 37 "C with a balanced salt with an EC5,of approximately 100 nM (Fig. 1B). For both buffer containing glucose and lithium (BSB/Li, concentrations in hormones, DAG levels reached a plateau between 5 and 30 mM: NaCI, 100; KCl, 5; MgC12, 1; CaC12,1.5; HEPES, 20; glucose 10; LiCl, 50). Stimulation of cells with agonist was begun by aspirating min (Fig. 1C). NE and bradykinin stimulated DAG accumuvalues of approximately 300 and 80 nM, the BSB/Li and quickly adding 1 ml of fresh BSB/Li-containing lation with drugs or vehicle and incubating at 37 "C for the indicated times. respectively (Fig. 10). Inositol trisphosphates were extracted and isolated by ion exchange Thesedata 1) confirm that, in VSMC, NE causes the chromatography as described by Alexander et al. (14). accumulation of inositol phosphates and DAG, 2) indicate DAG Assay-VSMC were grown to confluence on 35-mm dishes. that calcium and/or PKC may be involved in the regulation DAG was assayed essentially as per Griendling et al. (15). Twentyfour h prior to assay, cells were prelabeled with 1 pCi/ml of [3H] of a-AR mRNA by NE, and 3) demonstrate that bradykinin, arachidonic acid (NEN Research Products) in growth medium. It was like NE, stimulates the accumulation of inositol phosphates found that serum-containing growth medium, per se,increased DAG and DAG and causes the mobilization of calcium. formation. Therefore, cells were washed three times with 1 ml of Effects of PKC Activators and Inhibitors-To evaluate the serum-free medium M199and incubated in the same medium at 37 "C ability of PKC to regulate a-AR mRNA in VSMC, cells were for 3 h prior to the addition of drugs. Under these conditions, DAG exposed to phorbol esters known to activate PKC (19). Exformation in control cells was stable over the 30-min time period used for agonist stimulation (-2 f 3% versus t = 0;p = not significant; posure of VSMC to PDBu (100 nM) for 4 h caused a 77 f 4% n = 8). To assess the effects of NE and bradykinin, cells were decrease (n = 6, p < 0.001) in the level of a-AR mRNA (Fig. equilibrated in BSB/glucose for 15 min (37 "C), the BSB/glucose was 2). Likewise, exposure to PMA (100 nM) for 4 h caused a 76 aspirated, and 1ml of the same containing drugs or vehicle wasadded. f 3% decrease ( n = 8; p < 0.002) in a-AR mRNA level (Fig. Cells were incubated (37 "C) for the indicated times, and the assay 2). Continued exposure to PDBu resulted in a sustained was stopped by aspirating the medium and quickly placing the dish decrease in the level of a-AR mRNA for at least 24 h (Fig. 2). on ice.Diacylglycerolwas extracted and measured by thin layer chromatography, as described by Griendling et nl. (15). All data reflect The pattern of a sustained decrease in a-AR mRNA level the drug-induced change in DAG normalized to matched control with PDBu differed from the effect of NE which caused only a transient decrease in mRNA level that was maximal at 4 h plates in thesame assay. Measurement of CytosolicCalciumConcentration-VSMCwere and resolved completely by 24 h (see Ref. 11and Fig. 2). dispersed from culture flasks by brief incubation with trypsin/EDTA It was previously found that PMA caused a concentration(3-5 min), followed by centrifugation (300 X g for 5 min). The cell dependent decrease in thedensity of a-AR inVSMC, with an pellet was immediately resuspended in a calcium-free Krebs/HEPES of approximately 3 nM (20). The exposure of VSMC to buffer with 0.1% bovine serum albumin and rinsed three times with this buffer. Fura-Z/AM (5 p ~ Molecular ; Probes, Inc., Eugene, OR) PMA for 24 h caused a concentration-dependent decrease in was added to the cell suspension and allowed to incubate for an a-AR mRNA, with an ECso in the range of 1-3 nM ( d a t a not additional 60 min at room temperature. Pluronic F-127 (0.03%, shown). The biologically inactive phorbol esters, 4-a-phorbol Molecular Probes, Inc., Eugene, OR) was used to solubilize the dye 12-myristate 13-acetate (100 nM), had no effect on a-AR to aid in loading. The cells were spun again in Krebs/HEPES buffer mRNA level (Fig. 2), thereby excluding a nonspecific effect and immediately resuspended in the same buffer. Cells were then incubated with or without ryanodine (10 p M ) for 30 min in Krebs/ of the phorbol moiety. These observations indicate that actiHEPES buffer. The suspension was again washed three times with vation of PKC, alone, can down-regulate a-AR mRNA levels. To examine the role of PKC in mediating the effect of NE Krebs/HEPES buffer and 1.0 mM calcium added and held on ice until assayed (within 30 min) using a Jasco (CAF-100) spectrofluorimeter. on a-AR mRNA level, VSMC werepretreated with the PKC Aliquots (1ml) of suspended cells containing 1X IO6cells were placed inhibitor, H-7 (21). In cells treated with H-7 (100 p ~ 4 ,h), in a temperature-regulated (37 "C) cuvette, and fluorescence intensity basal a-AR mRNA level decreased to 52 f 3% of control ( n ratio (340/380) measured at 505-nm emission with reciprocal excitation at 340 and 380 nm, respectively. Cytosolic calcium was measured = 8) (Figs. 3 and 4).3 In cells pretreated with H-7 (100 p ~ 1, as described by Grynkiewicz et al. (16). Norepinephrine-stimulated h), the addition of PMA (100 nM, 4 h) or NE (10 p ~ 4 ,h) calcium efflux was measured using %az+, as previously described caused no further change in a-AR mRNA relative to H-7 (Figs. 3 and 4). The addition of HA1004 (100 PM),an inhibitor (13). Preparation of RNA and Northern Analysis-For Northern anal- of cyclic AMP dependent protein kinase (21), to cell cultures yses, RNA from three confluent 100-mm plates was pooled for each for 24 h had no effect on basal a-AR mRNA levelsor the NEexperimental condition. Total RNA wasextracted by the guanidinium stimulated decrease (Fig. 3). isothiocyanate/cesium chloride method, and analyzed by Northern These observations indicate that PKC activation causes hybridization, as previously described (ll), with a full-length cDNA probe for the hamster WAR (17). Relative levels of WAR mRNA down-regulation of a-AR mRNA and contributesto theeffect were determined by laser densitometry of autoradiographs of the of NE, whereas basal PKC activity is required for full a-AR hybridized filters. mRNA expression. Following the analysis of WAR mRNA,all filters were probed with Effect ofNE and PMA on a-ARmRNA Stability-In VSMC a [32P]Pi-labeledoligonucleotide complementary to 18 S ribosomal treated with actinomycin D (5 pgfml), a-AR mRNA levels RNA, washed and autoradiographed, as described by Lee et al. (18). All autoradiographic values for WAR mRNA levels were normalized decreased with a half-life of 3.2 f 0.1 h (n = 24; Fig. 5). This relative to the level of 18 S ribosomal RNA to correct for potential concentration of actinomycin D inhibited [3H]uridineuptake by 299% (data not shown). By comparison, NE (10 PM) and differences in theamount of RNA loaded. Data Analysis-Data are presented as the mean f S.E. Statistical PMA (100 nM) caused more rapid decreases in a-AR mRNA analysis was performed by analysis of variance and two-tailed non- levels with half-lives of 0.9 f 0.2 h ( p < 0.01 uersus actinopaired or paired t tests, as appropriate. mycin D;n = 16) and 1.4 k 0.2 h ( p 0.02 uersw actinomycin RESULTS

SecondMessenger Activation by NE and Bradykinin in VSMC-Both NE and bradykinin caused rapid increases in IP2 and IPS that were evident by 15 s (Fig. lA).NE and bradykinin stimulated inositol phosphate accumulation with EC, values of approximately 600 and 100 nM, respectively (Fig. 1B). We previously found that NE stimulated calcium efflux with an EC,of approximately of 100 nM in these VSMC (13). Bradykinin likewise stimulated calcium efflux,

D; n = 18),respectively, after a lag period of approximately 1 h. These observations indicate that 1)under basal conditions, the half-life of WAR mRNA is relatively short and 2) both NE andphorbol esters decrease the stability of a-AR mRNA. As noted above, H-7 at 4 h caused a decrease in a-AR mRNAlevel comparable in magnitude to that caused by actinomycin D (49 k 5%of control, n = 10). The combination Staurosporine (20 nM) and sphingosine (150 nM) similarly decreased a-AR mRNA level.

C Regulates alB-AdrenergicReceptor mRNA

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FIG. 1. Stimulation of inositol phosphate accumulation and DAG formation by NE and bradykinin. Panel A , After addition of and IPz (A,A) were extracted and measured as described 100 p~ NE (0,A) or 100 p~ bradykinin (0,A)for the times indicated, IPB(0,O) under “Materials and Methods.” Panel B , the indicated concentration of NE (0)or bradykinin (0)was added to VSMC for 5 min prior to as described under “Materials and Methods.” Bradykinin-stimulated ‘%a2+ efflux ).( was assessed at theindicated measurement of IP3 (0,0), or 100 p~ bradykinin (0)for the concentrations, as described under “Materials and Methods.” Panel C, after addition of 100 pM NE (0) times indicated, DAG was extracted and measured as described under “Materials and Methods.” Panel D,the indicated concentration of NE (0) or bradykinin (0)was added to VSMC for 5 min prior to measurement of DAG. The maximal increases in DAG activity were 18 2% ( n = 6; p < 0.001) and 86 k 8% ( n = 6; p < 0.001) for 30 min stimulation with NE (100 p ~ and ) bradykinin (100 p ~ )respectively. , Data are normalized to vehicle-treated plates. The datadepicted are for duplicate determinations in one of three similar experiments.

of actinomycin D and H-7 caused no further decrease over that caused by H-7 alone (46 f 7%; n = 5). H-7 had no effect on the level of mRNA for a-subunit of Gi2 ( n = 2, data not shown), indicating that the decrease in WAR was not due to nonspecific degradation of mRNA. Takentogether,these findings suggest that H-7 caused a decrease in a-AR mRNA level by inhibiting transcription, ratherthan by a decrease in mRNA stability. Inhibition of Calcium Pathways-To determine the role of calcium mobilizing pathways in the NE-mediated down-regulation of WAR mRNA, two types of experiments were performed. First, to evaluate the role of intracellular calcium

mobilization, ryanodine wasused to block NE-stimulated intracellular calcium mobilization (22), asconfirmed by fluorescence spectroscopy of cells loaded with fura-Z/AM. In control cells, NE (10 NM) caused a rapid increase inpeak cytosolic calcium from 86 f 5 to 208 f 15 nM ( p < 0.05; n = 6). Pretreatment of VSMC with ryanodine (10 PM, 1 h) had no effect on basal cytosolic calcium (71 f 15 nM; p = not significant; n = 6), but abolished the NE-stimulated increase in cytosolic calcium. Although ryanodine pretreatment abolished the effect of NE on cytosolic calcium, it had no effect on the NE-induced down-regulation of WAR mRNA, which was of comparable magnitude in control (-73 f lo%,n = 3)

Protein Kinase C Regulates ala-Adrenergic Receptor mRNA

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FIG.2. VSMC were treated with vehicle (VEH), NE (10 PDBu (1 MM), PMA (100 p ~ ) 4, ~ p h o r b o l12-myristate 13-acetate (4a-PMA, 100 nM), or bradykinin ( B K , 1 pM) for 4 h (open bars)or 24 h (hatched bars).Total RNA was prepared

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and analyzed by northern hybridization with probes for a-AR cDNA and for 18 S rRNA, and quantified, as described under “Materials and Methods.” Data are expressed as a percentage of the level in vehicle-treated control plates. The datadepicted are the mean& S.E. from three to eight experiments. * = p < 0.05 uersus vehicle; * * = p < 0.05 uersus same agonist a t 4 h.

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FIG. 5. Disappearance rate ofa-AR mRNA under basal conditions (0)and in the presence of NE (10 PM, A) or PMA (10 nM, 0).In the presence of actinomycin D (5 pcglrnl) to block transcription of RNA, a-AR mRNA levels decreased with a half-life of 3.2 f 0.1 h ( n = 24). The rate of a-AR mRNA decrease was significantly more rapid in the presence of NE (half-life = 0.9 f 0.2 h; n = 16; p < 0.01 uersus actinomycin D) or PMA (half-life = 1.4 f 0.2 h; p < 0.02 uersus actinomycin D; n = 18). Total RNA was prepared and analyzed by Northern hybridization with probes for a-AR and 18 S RNA, as described under “Results.”

and ryanodine-pretreated(-82 f 6%, n = 3) cells. Sei et al. (23)found that a-AR-mediated stimulation of atrial natriuretic factor mRNAlevel in cardiac myocytes was substantially dependent on the influx of extracellular calcium. T o evaluate the possible role of extracellular calcium influx 28s in mediating the effect of NE on a-AR mRNA levels in a-AR-, VSMC, cells were incubated for 2 h in calcium-free medium containing 2 mM EGTA. This treatment had no effect on 18s basal a-AR mRNA level, and did not affect the magnitude of a-AR mRNA down-regulation by NE (-73%; n = 2) or PDBu (-54%; n = 2). Taken together, these data suggest that neither a rise in FIG. 3. Effect of protein kinase inhibitors H-7 and HA-1004 on NEandPMA-induced decreases in a-AR mRNA level. intracellular calcium nor theinflux of extracellular calcium is VSMC were treated with H-7 (100 p ~ )HA , 1004 (100 p ~ or) vehicle necessary for NE-induced down-regulation of a-AR mRNA for 1 hour prior to addition of NE (10 p ~ or) PMA (0.1 p M ) for an in VSMC. additional 4 h. Total RNA was prepared and hybridized with a-AR Heterologous Down-Regulation of a-AR mRNA-Since bracDNA, as described under“Materialsand Methods.” The results dykinin, like NE, increases cellular levels of DAG, and preshown are typical of five such experiments, summarized inFig. 4. sumably also activates PKC, we predicted that bradykinin would decrease a-AR mRNA levels in VSMC. Exposure of VSMC to bradykinin (100 nM, 4 h) caused a 71 f 8% ( n = 4; p < 0.005) decrease in a-AR mRNA level (Fig. 2). As with NE, this effect was transient, such that by 24 h the mRNA level had returned to controllevels (Fig. 2). Thus, bradykinin can cause heterologous down-regulation of the a-AR mRNA level in VSMC. Transient Nature of the NE Effect-It is known that exposure of VSMC to NE or phorbol esters results in loss of NE-stimulated cellularresponses in VSMC (20, 24, 25). Therefore, we hypothesized that the transient nature of the NE-induced a-AR mRNA down-regulation in VSMC is due to a-AR-specificdesensitization. Totestthis hypothesis, VSMC were pretreated with NE (10 PM) for 24 h, after which VEH NE PMA VEH NE PMA the a-AR mRNA level was similar to that in control cells CONTROL 100 p M H-7 FIG. 4. The effect of H-7 on NE- and PMA-induced down- pretreated withvehicle (Fig. 6). After 24-h pretreatment with NE, the subsequentexposure regulation of the a-AR mRNA. VSMC were exposed to NE (10 to NE for an additional 4 h had no effect on a-AR mRNA p ~or) PMA (10 nM) for 4 h in the absence(CONTROL)or presence of H-7 (100 PM, 1 h prior to NE or PMA). Total RNAwas prepared level (Fig. 6), whereas both PDBu and bradykinin caused and analyzed by Northern hybridization with probes for a-AR and down-regulation of a-AR mRNA level (Fig. 6). This observa18 S RNA, as described under “Materials and Methods.” Data are tion suggests that the transient nature of the NE-induced normalized for 18 S RNA and presented as a percentage of the level in vehicle-treated control plates. The results are the mean f S.E. of decrease in a-AR mRNA level reflects events at a receptorfive separate experiments. * = p < 0.01 uersus vehicle-treated control specific locus (e.g. the a-AR and/or itscoupling pathway). Since the effects of PKC are due to phosphorylation of cells.

+ +

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Protein Kinase

C Regulates culs-Adrewrgic Receptor mRNA

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is blocked by the a-AR-selective antagonist prazosin, but not the &adrenergic receptor antagonist propranolol ( l l ) , it can be concluded that this effect is mediated by one or more second messengers coupled to the WAR. In the rabbit aortic VSMC used forthese experiments, we have previously shown that NE stimulatescalcium mobilization via the a-adrenergic receptor (13). The present experiments further demonstrate that in these cells, NE increases inositol phosphate and DAG levels, and causes a rise in the intracellular calcium concentration. Since the activation of PK-C was not directly measured in thisstudy, we can only speculate that itwas activated by the small increase in DAG that was observed.Nevertheless, CON NE PDBu BK these data indicate that the alB-AR in these cells is coupled -4hrto thegeneration of IPS andDAG, and therefore that calcium mediators of the NE-induced decrease FIG. 6. Effect of 20-hpretreatmentwith NE (10 p ~ , and PK-C are potential hatched bars) or vehicle (open bars) on the down-regulation in a-AR mRNA level. The effects of phorbol esters indicate that activation of of a-AR mRNA level induced by a subsequent 4-h exposure to NE (10p ~ ) PDBu , (0.1p ~ ) or , bradykinin (10pM). Data are PKC can decrease WAR mRNA level.Both PDBu and PMA presented asa percentage of the value in control cells pretreated withcaused rapid decreases in a-AR mRNA to a level comparable vehicle (first open bar), In control cells (CON), a-AR mRNA level after 24-h pretreatment with norepinephrine was similar to that in to thatcaused by NE (approximately 80%).H-7, an inhibitor vehicle-treated control cells. The a-AR mRNA level determined by of PKC (21),abolished the phorbol-stimulated decrease in anorthern hybridization was normalized to the 18 S rRNA level as AR mRNA. In these experiments, a specific action of phorbol described under "Materials and Methods." The data depicted are the esters tostimulate PKC issupported by 1)the high sensitivity means f S.E. for three experiments. * = p < 0.05 versus vehicle- to phorbol esters consistent with the ECbofor PKC (19), 2) pretreated control;t = p < 0.05 versus NE-pretreated control. the lack of effect of a biologically inactive phorbol ester, and 3) inhibition of the effect of the phorbol ester by H-7. H-7 also inhibited the NE-stimulated decrease in a-AR mRNA. Whereas H-7 is an equipotent inhibitorof both PKC and cyclic AMP-dependent protein kinase, HA1004 is a relatively selective inhibitor of the latter (21). Since HA1004 had no effect on NE-stimulateda-AR mRNA regulation, the inhibitory effects of H-7 were most likely due to theinhibition of PKC. In vascular smooth muscle, NE causes the mobilization of intracellular calcium stores due to theinteraction of IPS with its receptor on the sarcoplasmic reticulum (4, 22). Ryanodine binds to theintracellular calcium release channel, locking the channel open and thereby resulting in depletion of intracelI lular calcium stores that are sensitive to NE (22). Pre-expo0 4 24 sure of rat aortic strips toryanodine prevents subsequent NEHours stimulated contraction and calcium transients (27). Pre-exFIG. 7. Effect of okadaic acid on NE-stimulated down-reg- posure of VSMC to ryanodine abolished the NE-stimulated ulation of a-AR mRNA. VSMC were treated with NE (10 PM, 01, okadaic acid (20 nM, 0 )or the combination of NE and okadaic acid increase in intracellular calcium concentration, as assessed by fura-2. Under these identical conditions, the NE-stimu(A) for theindicatedtimes. a-AR mRNA level was assessedas described in Methods. The data are the means f S.Efrom three lated decrease in a-AR mRNAlevelwas notattenuated, experiments. * = p < 0.05 versus NE or okadaic acid, alone. indicating that mobilization of intracellular calcium is not required for this action of NE. A necessary role for extracelsubstrate proteins, we predicted that inhibition of dephospho- lular calcium is also unlikely, since the effect of NE was not rylation would cause a-AR-mediated effects to be sustained, attenuated by removal of extracellular calcium in the presence despite attenuation of the receptor-stimulated production of of EGTA. Thus,neither the mobilization of intracellular DAG and/or PKC activation. Treatment of cells with okadaic calcium nor the influx of extracellular calcium is required for acid (20 nM), an inhibitor of phosphoprotein phosphatases NE-induced down-regulation of a-AR mRNA. (26), had no effect on basal a-AR mRNA level (Fig. 7). In Although the decrease in WAR mRNA caused by phorbol cells treated with NE and okadaic acid (20 nM) concurrently, ester is sustained for at least 24 h, the down-regulation caused a-AR mRNA level decreased at 4 h and, in contrast to the by NE is transient, despite replenishment of hormone (Ref. transient effect of NE alone, the decrease was sustained for 11 and Fig. 2). The observation that bradykinin and PDBu 24 h (Fig. 7). decreased a-AR mRNA level in NE-treated cells that were insensitive to NE, confirmed our hypothesis that the transient DISCUSSION nature of the decrease in a-AR mRNA in VSMC is due to These data indicate that activation of PKC is capable of desensitization of the a-AR pathway. The ability of okadaic causing a decrease in the level of a-AR mRNA, and further, acid, a phosphoprotein phosphatase inhibitor (26), to sustain that NE-induced down-regulation of a-AR mRNA requires NE-induced a-AR mRNA down-regulation for at least 24 h, the activity of PKC. In contrast, neither the mobilization of further indicates that theeffect of NE requires the continued intracellular calcium stores nor the influx of extracellular phosphorylation of a substrate for PK-C. The basal half-life of a-AR mRNA determined in the calcium is necessary for the down-regulation of a-AR mRNA by NE. Since the down-regulation of a-AR mRNA in VSMC presence of actinomycin D was approximately 3 h. Both NE

Protein Kinase C Regulates aIB-AdrenergicReceptor mRNA

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and PMA caused more rapid decreases in mRNA level than did actinomycin D, indicating that these agents act, at least in part, by destabilizing a-AR mRNA. In contrast, the PKC inhibitor H-7 caused a decrease in a-AR mRNA level at 4 h that was comparable in magnitude to that caused by actinomycinD, andthe effect of the combination of H-7and actinomycin D was comparable to that caused by each agent alone. This observation suggests that H-7 causes a decrease in basal a-AR mRNA level by inhibiting transcription. Of note, Hu et al. (28) found that in DDT, MF-2 cells derived from the hamster vas deferens, PMA caused an increase in aAR transcription rate. Thus, NEmay cause opposing changes in a-AR mRNAlevelvia its effects ontranscriptionand mRNA stability. However, the data presented here indicate that regardless of a potential effect of PKC to increase transcription of the a-AR gene, in rabbit VSMC this action is overshadowed by a PKC-mediated decrease in a-AR mRNA stability. The data we have presented here maybe integrated as depicted in Fig. 8. Stimulation of the a-AR by NE results in activation of phospholipase C and increased production of DAG. Other agonists for phospholipase C-coupled receptors, such as bradykinin, may also increase DAG. Activation of PK-C by phorbol esters, or possibly by a hormone-stimulated increase in DAG, results in the phosphorylation of one or more substrate that decrease AR mRNA stability and may increase basal gene transcription. In rabbit VSMC, the predominant effect of NEand phorbol esters is to decrease mRNA stability. This PKC-mediated down-regulation of aAR mRNA is terminated by a phosphatase sensitive to okadaic acid. It is of interest that our findings in rabbit VSMC differ strikingly from observations in DDT, mF-2 cells. In contrast NE +a16-AR

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