Regulation of α1b-adrenergic receptor gene expression in rat liver cell lines

et Biq

Biochimica et Biophysica Acta 1219 (199z

adrenergic receptor g lines

ression in rat lfi

Chun-ling Deng, Lawreno L

~tt *

~rtmentof Physiology and Biophysics, Department of Medicine, Univel University of Arka 7220. AR 72205-7199, US/

ElSciences, 4301 West Markham

Received 21 December 1993; revise

ract lb-Adrenergic receptor gene expression was investigated tw rat hepal ated in two Clone 9 and McA-RH7777 ce analysis, Clone 9 cells expressed a 2.7 kb alb-adrenergic receptor rec~ gene transcript whereas two tw transcripts, 3.3 kb a observed wed in total cellular RNA isolated from rat liver. A binding site sit for the al-adrenergic antagoni mtagonist [3H]prazosin was o~ a~ K d = 0.11 _ 0.02 nM, n = 5). 9 celll membrane preparations ( B m a x = 47 _ 7 fmol/mg protein and 5 In contrast, alb-adr pre gene transcripts could not be detected in total cellular RNA prepared from McA-RH7777 cells cell, by either Northern ribonuclease uclease protection assays. However, results from nuclear run-off x assays indicated that the tt alb-adrenergic rec transcribed cribed in McA-RH77" McA-RH7777 cells and alb-adrenergic receptor gen me transcripts were observed in McA-RH7777 1V cell nucl resultsts suggest that oqh-adrenergic receptor gene expression in liv~ liver may be regulated in part post~ost-transcriptionally and l regulation ation may be altered or disrupted in the Clone 9 and McA-Rl McA-RH7777 cell lines. Keywords: ords: Adrenen Adrenergic receptor; Hepatocyte; RNA processing; Transcriptic ~tion

1. I n t r o d u c t i o n In liver, many of the actions ions of epinephrine and norepinephrine, including regulation ration of carbohydrate, lipid and protein metabolism as well as effects on hepatic growth, are mediated by the alb-adrenergic receptor subtype. Hepatic adrenergic res;sponsiveness appears to be regulated in a number of physiological states through alterations in alb-adrenergic receptor gene expression [1]. For example, hepatic alb-adrenergic renergic receptor and m R N A levels are relatively low in fetal and newborn SpragueDawley rats and reach their hi aighest levels in adults [2-5]. In addition, placement of acutel tely isolated adult rat hepatocytes in culture leads to a decr~ ecrease in levels of both the Cqb-adrenergic receptor and its m R N A [6]. Moreover, alb-adrenergic receptor m R NIA A levels transiently increase during hepatic regeneration followin ollowing partial hepatectomy [7]. This observation may bee significant in light of the known mitogenic effects of al-adrc number of tissues including heart [8] ^,

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the demonstration that mutant alb-adrene when transfected into NIH /' 3T3 fibroblast~, mitogenesis and tumoro~~genicity of NIH 3T3 Currently, little information is availa mechanisms involved in regulation of aibceptor gene expression. Of ( interest is that S r rat hepatocytes express two alb-adrenergic transcripts, 3.3 kb and 2.7 kb in approx amounts [12-14]. Both transcripts are de single gene [13], differ only in their 5'-reg appear to arise from the use of altemative p However, the physiologgical significance of adrenergic receptor gene transcripts remains current study, alb-adrem -adrenergic receptor gene e studied in two rat liver cell c lines. Clone 9 i~ cell line derived from normal n rat liver that sity-dependent growth inhibition it [17,18] an1 cal similarities to hepato Ipatocytes [18]. The M c / ~om a chemically induced )ur results demonstrate thal alb-adrenergic receptor ~rgic receptor that can be d ;mbrane preparations. In c

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Deng, L.E. Cornett / Biochimica et Biophysi

anctional receptor despite adrenergic receptor gene. processing of alb-adrenpears to be altered in the ines from that in normal

Materials

U1 reagents used were molecular biology grade or er obtained from commercial sources. Cell culture

¢IcA-RH7777 and Clone 9 cells were obtained from the erican Tissue Culture Collection (ATCC) and were ntained in Dulbecco's minimal essential medium ~co-BRL, Gaithersburg, MD) supplemented with 10% serum and 5% fetal calf serum. DDT1-MF2 cells were ared in Dulbecco's minimal essential medium supple~ted with 2.5% calf serum and 2.5% horse serum. mented tia was changed every 48 h and cells were subcultured Media once a week. 2.3. Radioligand assays [ 33H]Prazosin H was used to identify al-adrenergic recep-

y differential cenpreparations isolated by tors in membrane prel: r77, Clone 9, and DDT1-MF2 trifugation from McA-RH7777 cells as previously describedd [20]. Data from radioligand assays were analyzed using LIGAND [21]. 2.4. cDNA probes

bes were used in these experiFour different cDNA probes ments: (1) a 2 kb hamster a~lb-adrenergic receptor encoding cDNA cloned into the EcoRI site of pTZ18R which ted sequence, a 1545 bp open includes a short 5'-untranslated reading frame and a 543 b p 3'-untranslated region (proniversity) [22]; (2) a 2.1 kb rat vided by M. Caron, Duke Universit' oding cDNA cloned into the alb-adrenergic receptor encodin lich includes a 239 bp 5'-unEcoRI site of pGEM4Z which translated region, a 1545 bp open reading frame and a 301 ovided by H. Chin, NIH) [23]; bp 3'-untranslated region (provided [A cloned into the PstI site of (3) a 2.2 kb full-length cDNA ken fl-actin [24]; and (4) a 0.7 pBR322 which encodes chicken kb cDNA cloned into the PstI site of pTZ18R which encodes the 5'-region of rat a-fetoprotein (provided by T. cnntainina nlasmids were diSargent, NIH) [25]. Insert-containi gested with the appropriate restric inserts were purified by preparat trophoresis. Purified inserts were t

usin t clear cpm

~TP (3000 Ci/mmol, Nex~ hA) to a specific activit 3

2.5.

m and Northern blot analy

T by e tion (Mo]

RNA was prepared from h~ anidine monothiocyanate, ] or by using the TRI Rt arch Center, Cincinnati, C ztrophoresis on 1% agaro~, lowed by transfer to Ma I ron Separations, Westboro, th 20 × SSC using stand; lot analysis was performec

subj(

dehy mer~ lary [27]. desc 2.6.

e protection assays

T sizec

lergic receptor antisense R/' kmbion, Austin, TX) in t] [ Ofo~- 3~= I-']U 11-' t o l llowin owmg the manufacturer' with DsaI of pGE/~ Following linearization a B a m H I / P s t I fragmen ment of the rat alb-adre cDNA, SP6 polymeras~ 'merase was used to gene cleotide anti-sense RNA. RN,a Total cellular RN~ hybridized to the antisense antisez RNA ( ~ 105 cpJ 42°C followed by RNase RN~ T1 (20 U ) / R N :otected fragments were al digestion. The protectet polyacrylamide gels with wi 8 M urea. Polya were dried and were exposed to Kodak with intensifying screens screen at - 7 0 ° C for 15assays 2. 7. Nuclear run-off transcription tra

Cells were washed with ice-cold phos saline (PBS), were scra t)ed into ice-cold PB by centrifu~ policeman, and were collected cc buffer (10 ml~ pellet was lysed with lysis 1 7.4, 2 mM MgC12, 3 nmM CaC12, and 1% and nuclei were collected collectc by centrifugation resuspended in 5 min. Isolated nuclei were w pH 8.3, 40% g buffer (50 mM Tris-HCl, Tris-H and were stor MgC12, 0.1 mM EDTA) EDq use and were ir Nuclei were thawed before beJ 9resence of [ a- 32P]UTF min at 37°C in the pres nucleotides. Elongated RNA transcripts we for 15 min at 1 0 / z g / / z l RNase-free DNase I phenol. The pr~ were extracted with 65°C 65 hybridized to ( 2 . 1 0 6 cpm/sample) was ' immobilized on nylon filters in hybridizati pH 8.0, mM NaC1. 10 mM Tris-HCl, T 1M EDTA, 50 /~g/ml yq aylic acid, and 100 /zg/m] for 18 h. The filters w SDS three times at 25°C

Deng, L.E. Cornett/ Biochimica et Biophysi Table [3H]p memt

,, to McA-RH7777, Clone 9 an

Cell 1

K d (nM)

Bmax

(fmol/mg prot~

•

I

40 50 60 Bound [aH] Prazosin (pM)

70

1. Scatchard analysis of [3H]prazosin binding to partially purified branes prepared from three cell lines. Membranes prepared from -RH7777 (closed circles), Clone 9 (closed squares) and DDT1-MF2 (closed triangles) were incubated with increasing concentrations of 9razosin. Radioligand assays were carried out as described [20]. Data s represent the mean of duplicate determinations from a representaexperiment. This representative experiment was carried out with branes prepared from either five (Clone 9 and DDT1-MF2 cells) or (McA-RH7777) different cell platings.

McAClone DDT1

n.d. 0.11+0.02 0.09+_0.02

Radio meth(

¢¢ere carried out as described ur m constants (K d) and binding aed from binding isotherms that x~ are shown as the mean + S.E. : its. n.d., not detected.

(Bmax

LIGA numb

[3H] obse cells

3.2. and ~/0.1% SDS three times at 65°C with shaking. Washed rs were exposed to Kodak X - O M A T A R film at 0°C with intensifying screens for 7 - 1 0 days.

2.8. Isolation of cytoplasmic and nuclear RNA 2ells were washed and collected as described above. Cells The harvested cells were resuspended in lysis buffer (50 m M i Tris-HC1, p H 8.0, 100 m M NaC1, 5 m M MgC12, and 0.5% Nonidet P-40) and were subjected to Dounce homogenization (ten strokes). Following centrifugation of cell homogenates at 500 × g for: 5 min, nuclear and cytoplasing the T R I - R E A G E N T TM LS mic R N A were isolated usin (Molecular Research Centerr, Cincinnati, OH). R N A was denatured and blotted onto nylon filters which were then hybridized to c D N A probes as described for Northern blot analysis.

n.d. 46_+ 7 150+15

defined by 10 g M phentol nembranes prepared from

"gic receptor gene transcr '7 cells

IOtal cellular R N A was prepared from T 90 male Spragu taken from postnatal day d cells and x~ Clone 9 cells and McA-RH7777 McANorthern blot analysis. A representative I~ shown in Fig. 2. Consistent with previo ,-adrenergic receptor g results [12], two OLlb-a( 3.3 kb and 2.7 kb, were wer~ detected in liver a rved in heart. Clone 9 kb m R N A was observe only the 2.7 kb m R N A while McA-RH775 appear to express either alb-adrenergic rec~ script, alb-Adrenergic receptor gene trans~ d c A - R H 7 7 7 7 cell R N A ew undetectable in McA-R] ern blots were exposed to film for up to thl 9nuclease protection assays not shown). Ribonuclea to be approx. 10-fold more sensitive than

3.Results 3.1. Radioligand assays [3H]Prazosin bound saturabl ably and with high affinity to a single site in membrane pre[ mrations isolated from Clone 9 cells and DDT1-MF2 cells (Fi~ig. 1). The DDT1-MF2 smooth muscle cell line was establi,,~hed from a leimyosarcoma of a Syrian hamster vas deferens :ens and has been previously ~s the Oqb-adrenergic receptor shown to abundantly express [28]. From five separate ex t~eriments with different membrane preparations, the bindin ding site concentrations were ,rl 1150 g O -I.. 47 _ 7 f m o l / m g protein and __5 1 K - F r ~ . l / r a n ~ r ~ * * ; . for Clone 9 and DDT1-MF2 cells, l The dissociation constants were 0.1 -1- 0.02 nM for Clone 9 cells and D [

3.3 kb • 2.7 Ida •

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Fig. 2. Northern blot analysis of oqb-adrenergicrecep in rat tissues and hepatic cell ce lines. Samples (20 ~a RNA isolated from postnatal ostnatal day 90 Sprague-Dawley heart (H-90), and from Clom Clone 9 (Clone 9) and McA.'-F,11~: w P r P P 1 P e f r a n h a r ~~resed ,~Pd in in a 1.0% agarose/6% fort )tted to a nylon membrane as de d with the rat alb-adrenergic re Kodak X-OMAT film for 7 da This autoradiograph is represenl ~hich produced identical results.

Deng, L.E. Cornett / Biochimica et Biophys&

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~. Detection of Otlb-adrenergic receptor mRNA by ribonuclease tion assay. Samples (20 ~g) of total cellular RNA isolated from p O ~ t l l i tttal t i l l day 90 rat liver (L-90), Clone 9 cells (Clone 9), McA-RH7777 RH7777), DDT~-MF2 cells (DDTQ, and yeast (Yeast RNA) were cells (RH7777~ lized to a rat Otlb-adrenergicreceptor cRNA (10 ~ cpm). Undigested hybridized cRNA that was diluted 1:100 was electrophoresed in the outside lane. aarkers were prepared from HinfI digested pBR322 plasmid DNA Size markers rere subsequently 5' end-labeled with [y-a2p]ATP. The dried gel that were w a s e x~posed to Kodak X-OMAT film overnight at - 70°C with intensify:reens. This experiment was repeated three times with identical ing scr results. analysis for the detection off specific messenger RNAs. Therefore, total cellular RNA, isolated from Sprague-Dawills, Clone 9 cells, and DDT xley rat liver, McA-RH7777 cells ibonuclease aclease protection assays MF2 cells was subjected to ribonuclc isense RNA probe for albusing a 271 nucleotide antisense adrenergic receptor mRNA. Inn liver and Clone 9 cells, the ected fragment was observed expected 243 nucleotide protected stent with that obtained from (Fig. 3), a result that is consistent 3ntrast, a protected fragment Northern blot analysis. In contrast. was not observed in total cellular R N A isolated from McA-RH7777 cells (Fig. 3). The subjection of total cellular RNA prepared from DDTT1-MF-2 cells to analysis by ribonuclease protection assay with the antisense RNA probe otected fragments, because of resulted in several smaller protected mismatches between the rat and hamster alb-adrenergic ~s expected, a protected flagreceptor eDNA sequences. As east R N A indicating that the ment was not observed in yeast antisense R N A specifically hybridized to alb-adrenergic receptor transcripts. ,ant, re&: recentor eene tran3.3. Localization o f alb-adrenergic scripts

To determine the level at which a

trans~ was 1 isolat gene that i other Albm entiat expre malia nucle lular script expre script mic t McA. be dc detec of o~ nuclei nucle

ivity of the Otlb-adrenergic nuclear run-off assays (F F1-MF2 cells served as a p( scription of the alb-adren 17777 cells appeared to be 2 cells (Fig. 4). The relativ amin and /3-actin, were or plasma protein synthesi tes [29]. fl-Actin is a cytos ~tively high levels by ne~ Slot blot analysis of RNA lasmic fractions showed di of alb-adrenergic recepl one 9 and McA-RH7777 9 cells, alb-adrenergic etected in both the nuclear g. 5). In contrast, in the r lls alb-adrenergic receptor e nuclear fraction, but w~ toplasmic fraction (Fig. 5). : receptor m R N A in McA is consistent Wltll the nuclear run-off nt with

gene expressic 3.4. alb-Adrenergic receptor rece ence o f cycloheximide

the protein synt Treatment of cells with w in an increased cycloheximide often results resl certain m R N A species, a phenomenon term~

.g .~ ~..;~.~: ~':~ !i~~i~, i(~i ~!!::?i,....

............

- - (~lb-AR (Rat] (~lb-AR (Han ::~,, :i~ ~

Albumin - - ~-Actin

........ ~: :~g --pGEi ........... Fig. 4. aab-Adrenergic receptor recep gene transcriptional RH7777and DDT1-MF2 cell nuclei. Transcript elon$ under Materials an out with 2-107 nuclei as described desc amountsof radiolabeled RNA RN (2.106 cpm) were 1 following cDNAs (5 /zg): rat n Oqb-adrenergic recepl ator (hamster alb-AR), rat a hamster alb-adrenergic receptc chicken fl-actin (fl-actin), and plasmid pGEM4Z (pGE ~GEM vectors with the appropri ~bilization onto the nylon membl ed to Kodak X-OMAT film for ] eens. This experiment was repe~

Deng, L.E. Cornett / Biochimica et Biophysi Yeast RNA

cells,

I 1 16

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Nuclear RNA 8

4

2

Id with 1 0 0 / x g / m l cycloh ~r to change steady-state or gene transcripts (Fig. 6al alysis Otlb-adrenergic receI undetectable in total cell1 RH7777 cells that had be~ 3heximide over 24 h (Fig. ~y ribonuclease protectior or mRNA remained undetq dated from cycloheximide Lta not shown), c~-Fetoprote control and cycloheximide ggesting that the cells rem; le protein synthesis inhibit

di adrer Nortl scrip lated

~.gofRNA

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or gene transcripts by slot blot isolated from hepatic cell lines, lated from McA-RH7777 and rials and methods. RNA was ared and blotted onto the nylon membrane in the indicated amounts. RNA (16 /~g) was used as a negative control. The probe used was it a]b-adrenergic receptor cDNA that had been random primerd with [a-32p]dATP to a specific activity of 4-108 c p m / / x g . The was exposed to Kodak X-OMAT film for 5 days at - 7 0 ° C with ;ifying screens. This autoradiograph is representative of experiments d out with nuclear and cytoplasmic RNA isolated from five differ~11platings.

100

whe~ adrer cellu RH7' detec RH7' the p

4. Di [31-34]. Superinduction has been attributed to in;ed transcription rate as well as increased stability of essed mRNAs [31,33,35]. Therefore, experiments were ed out to determine whether c~b-adrenergic receptor transcript levels could be increased in Clone 9 and t-RH7777 cells with cycloheximide treatment. Clone 9 McA-RH7777

S~ of evidence suggest a rol adrer or in the growth of hepatc larly generative phase following ctom ctomy, l"~orepmepnrlne phrine stimulates DNA sy tured rat hepatocytes, an effect that is blockq ergic receptor antagoni~ onists [9]. Moreover,

A. Cyeloheximide Treated

Control

I

I

0

1

2

4

8

I

24 Liver

I 0

1

2

4

8

24

Hours

2.7 I ~

2.0~,.

,~

G-FP

B. Cycloheximide Treated

0

1

3 . 3I~> ~ I 2.7 Fig. 6. Effectof cycloheximideon oqb-adrener with (Cycloheximide-labeledlanes) and witbot points and 30 ~g were subjectedto Northernb [ ~_32P]dATPto a specificactivityof 4 • l0 s c

Control I

2

4

8

24

I Liver

I 0

1

2

4

8

24

j

Hours

alb-AR

?d-I7777 cells (A) and Clone 9 cell atal cellular RNA was isolated at etoprotein cDNAs were random p~ 'or either 7 days (Oqb-adrenergic r ad identical results were obtained

Deng, L.E. Cornett / Biochimica et Biophysic

ative DNA synthesis folParadoxically, a number nan [7] hepatic cell lines trenergic receptors• The of these cell lines to is not entirely clear• t cell line an alb-adrenresent which apparently protein [7]. Preliminary riments from our laboratory suggested that the Clone ll line expressed and the McA-RH7777 cell line did xpress the alb-adrenergic receptor subtype and thereboth cell lines would be useful in exploring alblergic receptor gene expression in hepatic cell lines• H]Prazosin, an cq-adrenergic receptor antagonist, d specifically and with high affinity to membranes tred from Clone 9 cells• In contrast, specific binding H]prazosin was not observed to membranes prepared McA-RH7777 cells• Results from Northern blot analand ribonuclease protection assays using total cellular isolated from both cell lines confirmed the concluregarding alb-adrenergic receptor gene expression in ~'o cell lines reached from radioligand assays• Clone 9 cells expressed a 2.7 kb mRNA that presumably encodes the C~lb-adrenergic receptor subtype, while the McA777 cell line did not contain detectable alb-adrenergic RH7777 rece[~tor gene transcripts• Surprisingly, results from nuclear )ff assays which measure specific gene transcription run-off rates indicated that McA-RH7777 cells express the O~ablergic receptor gene at a relatively high rate. Moreadrener receptor mRNA was detected in nuo v e r , c~h-adrenergic lb aic RNA isolated from McAclear RNA, but not cytoplasmic RH7777 cells• It has been estimated that nuclear RNA tal cellular RNA [39]. Thus, comprises approx. 1% of total ~richment of a RNA species there can be considerable enrk an occur with fractionation of confined to the nucleus that can ~. Therefore, it is predictable nuclear and cytoplasmic RNA. that C~lb-adrenergic receptor gene transcripts could be den McA-RH7777 cell nuclear tected by slot blot analysis in otal cellular RNA by either RNA (Fig. 5), but not in total bonuclease clease protection assays Northern blot analysis or ribonucle (Figs. 2 and 3). Gene expression in eukar 3yotic organisms is frequently lly. In senescent human firegulated post-transcriptionall) broblasts, expression of the 1:proliferating-cell-nuclear-antieel of maturation of precursor gen gene is blocked at the level amples of regulation of gene into mature mRNA [40]• Exam lscript turnover in the nucleus expression at the level of transcri are relatively rare• Alkaline t)hosphatase mRNA levels are regulated by intron sequences that destabilize nascent tranTransport of RNA molecules scripts within the nucleus [41]•• Trans ~lasm c~ can also be a target for from the nucleus to the cytoplasm regulation• For example, a human im protein, rev, promotes nucleus to c viral mRNA containing a rev-resp

stabil flz-ad onist Ac proviq failur ergic Since tectec woul( script rapid] unabl gene in vit~ copy this c turno' intact

demonstrated, for example eptor gene expression duriI 3] or hepatic development [ )eriments were carried ou garding the mechanism(s) 1 zA-RH7777 cells to expre ~spite apparent transcriptio~ :gic receptor gene transcril~ Jlated from McA-RH7777 t the al~-adrenergic recep not transported out of the in the cytosol. Thus far,' re transport of alb-adren ut of McA-RH7777 cell n lease assay [45], probably t qb-adrenergic receptor gem ~wise, relatively little is kn adrenergic receptor gene DDT1-MF2 smooth muscl zeptor gene transcript is re OLlb-a 6 to 8 h [46,47]. Treatment with CellS With g l u c o c o r)rticoids t l C O l O tends to stabilize, cells receptor gene transcripts [47]. In cultured ad cytes, both the 3.3 kb aJ md 2.7 kb alb-adre~ display similar half-lives gene transcripts displa) data). In general, several cis- and trans-acti~ fluence the stability of ml been shown to influence malian cells. The length of a transcript's poll 9rotein (PABP) have b a poly(A) + binding pro! with the stability of spec•ific mRNAs. For ex not polyadenyla mRNA, which is normally norma short half-life, becomes more stable when polyadenylated [48,49]. The presence of st~ atranslated region of som~ tures in the 3'-untransla known to be important in regulating tran~, [50]. Finally, many unst able transcripts hav to have one or more co~pies of an AU-rich 3'-untranslated regions [[51]. Based on the and gene, none of tt quence of the cDNA al elements appear to be present in the rat additional expe receptor mRNA. Clearly, Clearl necessary to establish the 1 mechanism(s) t absence of cytoplasmic alb-adrenergic rece] McA-RH7777 cells desp,ite apparent gene tr 'tes have been shown Finally, rat hepatocyt~ alb-adrenergic receptor gene transcripts, a[ are derived 2.7 kb [12-14]. Both transcripts tra of RNase H m gene [13] and from results resl 5'-regions [15]. appear to differ only in their t significance of the two alb-adrenergic rec ablished, but each could has not been establishe regulated f scriots which are differentially differ tively, putative short open J gion of the larger alb-adrel 1 give rise to one or more 1: strated during expression

Deng, L.E. Cornett / Biochimica et Biophys

]. Interestingly, Clone 9 2.7 kb m R N A . Careful ,~shly isolated hepatocytes ~ic stimulation could shed 1 importance of the two a l b - a d r e n e r g i c receptor ined in two hepatic cell ted hepatocytes which by t h e m blot analysis express two C~lb-adrenergic receptor e transcripts, Clone 9 cells express only the 2.7 kb .adrenergic receptor m R N A while M c A - R H 7 7 7 7 cells not appear to express either transcript. Surprisingly, by lear run-off assays M c A - R H 7 7 7 7 cells appear to tranbe the a l b - a d r e n e r g i c receptor gene and a l b - a d r e n e r g i c :ptor m R N A can be detected in M c A - R H 7 7 7 7 nuclear A preparations. These results suggest complex postscriptional regulation of a l b - a d r e n e r g i c receptor gene ression in liver which m a y be altered or disrupted in Clone 9 and M c A - R H 7 7 7 7 cell lines. Further study of -adrenergic receptor gene expression in the Clone 9 and ~,-RH7777 cell lines should provide insight into mechans i n v o l v e d in altered hepatic responsiveness to adrenergic stimulation.

Acknowledgements This ['his work was supported b y N I H R01 G M 3 0 6 6 9 , N S F RII9122108 )122108 and a research grant from the University of zes Graduate School. Arkansas for Medical Sciences

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[13]]

, Jr. and Cornett, L.E. (1991) J

[14] ]] l [1511 [16]~

, Cotecchia, S., Lorenz, W., Leun tg, T.L., Brownstein, M., Lefkowi Biol. Chem. 266, 6365-6369. :1 Cornett, L.E. (1993) J. Recept. nos, G. (1993) Gene 131, 243-2, nail-Beigi, F. and Loeb, J.N. (19

[17]] [18]]

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e Nechaud, B. and Potter, V.R. ( :ne Expression (Fishman, W.H. ,cademic Press, New York. , Yamaguchi, N., Orenstein, J.b, 1974) in Mechanisms of Chemi ~t Liver and Hepatoma Cell Cult }son, E.B., eds.), pp. 441-459, Ac

[20],

~nd Norris, J.S. (1985) Mol. (

[21]] [22],

d Rodbard, D. (1980) Anal. Biocl7 chwinn, D.A., Randall, R.R., Left ilka, B.K. (1988) Proc. Natl. A~

[23] •

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