The Molecular Effects of Alcohol : Clues to the Enigmatic Action of Alcohol

Ann. N.Y. Acad. Sci.

993: 82–94 (2003). ©2003 New York Academy of Sciences.

The Molecular Effects of Alcohol

Clues to the Enigmatic Action of Alcohol

MEENA KUMARI,

a

ANTJE ANJI,

a

HENRI WOODS, JR.,

b

AND MAHARAJ K. TICKU

c

a

Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas, USA

b

Wildlife Division, Connecticut Department of Environmental Protection, Burlington, Connecticut, USA

c

Department of Pharmacology, University of Texas Health Science Center, San Antonio, Texas, USA

A

BSTRACT

: Chronic ethanol treatment (50mM, five days) induces stabilizationof NR1 receptor subunit mRNA in cultured fetal cortical neurons. In thispaper, we investigate the mechanism(s) by which ethanol mediates its effectson NR1 mRNA. Specifically, we have determined if cellular localization ofNR1 mRNA in cortical neurons and/or

de novo

protein synthesis is essential forethanol-mediated stabilization of NR1 mRNA. Subcellular fractionation stud-ies show that all detectable NR1 mRNA is associated with rough endoplasmicreticulum, indicating that subcellular distribution of NR1 mRNA in fetal corti-cal neurons does not play a role in ethanol-mediated NR1 mRNA stabilization.However, inhibition of protein synthesis by cycloheximide abolished the mRNAstabilizing effect of ethanol on NR1 mRNA, thus suggesting

de novo

proteinsynthesis is crucial for the action of ethanol on NR1 mRNA stabilization.

K

EYWORDS

: NMDA receptors; fetal cortical neurons; chronic ethanol treatment;

de novo

protein synthesis; mRNA stability; NR1 receptor subunit; rough endoplasmic reticulum; mRNA subcellular distribution

INTRODUCTION

N

-Methyl-

D

-aspartate (NMDA) receptors, one of the three major subtypes ofglutamate receptors, are composed of three subunits, namely NMDA R1, R2, andR3,

1–3

that are products of three gene families.

2,4

These subunits differ in their ana-tomic distribution, functional properties, and regulation.

2,3,5–8

The exact subunitcomposition of the NMDA receptors is not known; however, it is believed that nativeNMDA receptors comprise combinations of NR1, the key component for functionalreceptors, and NR2 subunits

1

or NR3 subunits.

2,3

Through NMDA receptor stimu-lated calcium influx, this receptor system plays a crucial role in neuronal plasticitythat underlies memory, learning, and development.

9

Overstimulation of NMDA

Address for correspondence: Dr. M. Kumari, Department of Anatomy and Physiology,Kansas State University, 231 Coles Hall, Manhattan, KS 66506, USA. Fax: 785-532-4557.

[email protected]

83KUMARI

et al.

: PROTEIN SYNTHESIS AND NMDA R1

M

RNA STABILIZATION

receptors can trigger an increase in [Ca

2

+

]

i

above physiologic levels, resulting inneuronal degeneration and cell death.

10

In recent years, the NMDA receptor system has emerged as an important sitefor the action of ethanol. Although we know that alcohol mediates some of its dele-terious effects through the NMDA receptor system, the exact molecular sitesof interaction between NMDA receptors and ethanol have yet to be defined. It isknown that chronic ethanol treatment

in vivo

increases NMDA receptor number andfunction.

11

−

17

Comparable results are obtained when mouse fetal cortical neuronsare exposed to chronic ethanol treatment

in vitro

(50mM, five days).

18

Ethanol-induced upregulation of the NMDA receptors

in vitro

is accompanied by an augmen-tation of NR1 and NR2B polypeptides

19

with a concomitant increase in NR2BmRNA levels.

20

The NR1 polypeptide levels are also upregulated

in vivo

in responseto chronic ethanol treatment.

21

At the molecular level, chronic ethanol treatment(50mM, five days) of cultured fetal cortical neurons increases the rate of NR2B genetranscription and alters the NR1 mRNA stability.

22

Specifically, chronic ethanoltreatment increases the halflife of NR1 mRNA from 16h in control neurons to morethan 24h in neurons grown in the presence of ethanol. In fact, no decay of the NR1mRNA is detectable up to 24h, suggesting that ethanol has a stabilizing effect on theNR1 mRNA.

22

In eukaryotic cells, the halflives of mRNAs vary from a few minutes to severalhours

23

and change in response to a host of stimuli, including cell growth rates,exposure to hormones, and environmental stimuli.

24

This flexibility in mRNA deg-radation rates can in part be accomplished by the fate of the mRNA following itsexport from the nucleus to the cytoplasm. Once the mRNA reaches the cytoplasm, itis never naked and is associated with mRNA-binding proteins. In the cytoplasm,mRNAs can also associate with special proteins forming messenger ribonucleopro-tein complexes (mRNPs). These mRNPs can exist in two subcellular locations: asfree cytoplasmic mRNPs (in this report, referred to as nonmembrane-associated)or be associated with rough endoplasmic reticulum (in this report, referred to asmembrane-associated).

25

Experimental evidence suggest that this subcellular local-ization of mRNAs can alter their decay rate.

26,27

Additional diversity in mRNAdecay rate may be achieved by RNA-binding proteins that act as either mRNA desta-bilizers or stabilizers.

24

In vitro

studies employing protein synthesis inhibitors, suchas cycloheximide (CHX), indicate that in some cases new protein synthesis is a pre-requisite for mRNA stabilization in response to a stimulus.

28,29

In this report we have examined potential molecular mechanism(s) involved inethanol-induced NR1 mRNA stabilization. Because the exact mechanisms that influ-ence mRNA stability in response to various stimuli have yet to be elucidated, weexplored (1) if NR1 mRNA exists in more than one subcellular location and if etha-nol induces translocation of NR1 mRNA from a nonmembrane-associated pool to amembrane-associated pool and, hence, alters the decay rate of NR1 mRNA, and (2)if

de novo

protein synthesis is necessary for the NR1 mRNA stabilization by chronicethanol. Our results showed for the first time that (1) NR1 mRNA was always asso-ciated with rough endoplasmic reticulum (i.e., the membrane-associated pool) irre-spective of ethanol treatment, and (2) ethanol-induced stabilization of NR1 mRNArequired

de novo

protein synthesis.

84 ANNALS NEW YORK ACADEMY OF SCIENCES

EXPERIMENTAL PROCEDURES

DNA Clones and Probes

EcoRI linearized plasmids, namely pFPR1, pFPR2BR, and p15GI, were tran-scribed

in vitro

in the presence of [

32

P]

α

-CTP (specific activity, 3,000Ci/mMol)(DuPont) to generate NR1, NR2B, and cyclophilin (IB15) radiolabeled cRNAs,respectively, as described elsewhere.

22

The amounts of NR1 and NR2B mRNA insamples were quantified by ribonuclease protection assays. Cyclophilin cRNA wasincluded to normalize the results.

Cell Culture and Ethanol Treatment

Cortical neurons were isolated from 14–15-day-old mouse fetuses and cultured asdescribed elsewhere.

22

Time-pregnant mice (strain C57 BL/6) purchased from Har-lan (Indianapolis, IN) were used in accord with institutional guidelines, and proce-dures were approved by the animal welfare committee. Briefly, cerebral hemispheresdissected from fetuses under sterile conditions were minced and cells were dissoci-ated by trituration using a Pasteur pipette. Cell viability was determined by trypanblue exclusion test and a known number of viable cells (2.4

×

10

7

cells/75cm

2

flask)were plated in poly-

L

-lysine coated plastic flasks and grown in minimum essentialmedium (MEM) containing 100

µ

M

L

-glutamine, 10

%

fetal bovine serum, and 10

%

heat-inactivated horse serum under 95

%

O

2

and 5

%

CO

2

at 37

°

C. On day 2, a mix-ture of 5-fluoro-2

′

-deoxyuridine and uridine at a final concentration of 10

µ

g/mL wasadded to the media to inhibit cell proliferation.

30

These cultures contained in excessof 92

%

neuronal cells and the remainder were glial cells, which do not expressNMDA receptors.

31

From day 3, cortical neurons were grown in the presence of 50mM ethanol forfive consecutive days with change of medium every 24h. Where applicable, cellsgrown in the absence of ethanol were used as untreated controls. Cultured fetal cor-tical neurons were processed depending upon the experimental design.

Isolation of the Membrane-Associated and the Nonmembrane-Associated mRNAs

Cortical neurons grown in the presence of 50mM ethanol for five days werewashed twice with ice-cold phosphate buffered saline (PBS) containing 1mM calci-um chloride and 10mM magnesium chloride. Cells grown in the absence of ethanolserved as control. The membrane-associated and nonmembrane-associated mRNAswere separated by differential centrifugation as described elsewhere.

32

Briefly,washed cells were scraped and suspended in buffer A (10mM Tris.HCl [pH7.4],10mM NaCl, 1.5mM MgCl

2

). Cells were allowed to swell by storing them on ice for10min. The swollen cells were homogenized with 10 down and 10 up strokes in atight Dounce homogenizer. Homogenates were mixed with 0.1volume of 2Msucrose prepared in buffer A, layered over a 5mL 12

%

sucrose-buffer A cushion, andcentrifuged at 2,000

g

for three minutes to pellet the nuclei. Supernatants were recov-ered and mixed with 1/4 volume of buffer B (250mM Tris.HCl [pH7.4], 20mMMgCl

2

, 2.5M KCl) and sodium heparin (final concentration 500

µ

g/mL) (includedas RNase inhibitor). The resulting mixtures were layered over a stepwise gradient of

85KUMARI

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: PROTEIN SYNTHESIS AND NMDA R1

M

RNA STABILIZATION

sucrose (3mL of 20

%

sucrose-buffer A layered over 1mL of 2M sucrose-buffer A)and centrifuged at 25,000rpm for 30min at 4

°

C in a Beckman ultracentrifuge usinga SW41 rotor. Supernatants above 20

%

sucrose–2M sucrose interphase containingribonucleoproteins,

t

RNA, protein, free polysomes, ribosomes, and so forth, wererecovered as fraction 1. Membrane fraction containing microsomes, mitochondria,lysosomes at 20

%

sucrose–2M sucrose interphase were also recovered as fraction 2and diluted to 10mL with buffer A. Both fractions were mixed with SDS to 1

%

andEDTA to 25mM, extracted sequentially with phenol–chloroform–isoamyl alcohol(25

:

24

:

1) and chloroform. Aqueous phase containing RNA was recovered and RNAwas precipitated by ethanol. RNA pellets recovered by centrifugation at 14,000rpmfor 30min at 4

°

C were suspended in DEPC water and the concentration was deter-mined by reading absorbance at 260nm.

Twenty micrograms of RNA from each fraction were analyzed by ribonucleaseprotection assay as described elsewhere.

22

Briefly, 20

µ

g of total RNA was suspend-ed in 20

µ

L of hybridization buffer (Ambion, TX) containing 150,000cpm of

32

P-labeled NR1 cRNA probe and 15,000cpm of

32

P-labeled cyclophilin cRNAprobe and incubated at 50

°

C for 16–18h. Next day, samples were RNase digested,and recovered RNA-RNA hybrids were separated on 5

%

sequencing gel under dena-turing conditions. Gels were dried, exposed to PhosphorImager screen, and datawere analyzed using the ImageQuant program (Molecular Dynamics, Sunnyvale,CA, USA).

Effect of Cycloheximide on the Halflife of NR1 mRNA

Cortical neurons were grown in the presence of 50mM ethanol for five days asdescribed above. On day 6, cells were fed with medium containing either cyclohex-imide (10

µ

g/mL) or cycloheximide (10

µ

g/mL) and actinomycin-

D

(10

µ

g/mL) withchange of medium after 24h. Cultures were terminated at 12h, 24h, 36h, and 48hby lysing the cells in Trizol (Gibco, BRL). Total RNA was isolated according to themanufacturer’s instructions. The NR1, NR2B, and cyclophilin mRNA levels werequantified by ribonuclease protection assays as described above. Dried gels wereexposed to PhosphorImager screen and the data were analyzed using theImageQuant program (Molecular Dynamics, Sunnyvale, CA, USA).

Inhibition of Protein Synthesis by Cycloheximide

The extent of inhibition of translation by cycloheximide (CHX) was measured incultured fetal cortical neurons. To determine the extent of inhibition of protein syn-thesis, fetal cortical neurons were cultured for five days in the presence of 50mMethanol with change of medium every 24h as described above. On day 6, cells wereincubated with cycloheximide (10

µ

g/mL) for 12h. At the end of this period, cellswere fed with fresh medium containing CHX and 40

µ

Ci of [

35

S]-methionine–cystine mixture and allowed to grow for three hours at 37

°

C. Cells were washedtwice with ice-cold PBS containing 0.1mM phenylmethylsufonyl floride (PMSF)and 100-fold excess of cold methionine, scraped, homogenized, and centrifuged at14,000rpm for 20min at 4

°

C. Supernatants were recovered and protein content wasmeasured using BCA protein assay reagent (Pierce, Rockford, IL). Aliquots contain-ing 100

µ

g protein were spotted on Whatman 3 MM filter paper circles and air dried.


The filters were washed for five minutes in boiling 10

%

trichloroacetic acid (TCA),2

×

10min in 5

%

cold TCA, rinsed in absolute alcohol, and then in acetone, air dried,and counted in a Beckman scintillation counter. The data are presented as thepercentage of protein synthesis relative to cortical neurons grown in the presence ofethanol but not exposed to cycloheximide.

Quantification of Autoradiograms

The NR1 and NR2B subunit mRNAs and cyclophilin mRNA content were quan-tified using PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and data wereanalyzed using the ImageQuant program (Molecular Dynamics). To control forequal sample loading, the signal densities of the NR1 and NR2B subunit protectedfragments were divided by the signal density of cyclophilin protected fragment fromthe same sample. Results were expressed as percentages of control. Data points aremean ± SE values of three independent experiments.

Statistical Analysis

Where appropriate, statistical significance was analyzed in terms of variance(ANOVA) with a post hoc Scheffe’s test. A value p < 0.05 was considered significant.

RESULTS

Membrane-Associated versus Nonmembrane-Associated NR1 mRNA

To determine if subcellular localization of NR1 mRNA plays a role in ethanol-induced stabilization of NR1 mRNA, we examined the subcellular distribution ofNR1 mRNA in the cytoplasm of cortical neurons. Specifically, we investigated ifNR1 mRNA exists both in membrane-associated and nonmembrane-associatedpools in the cytoplasm of fetal cortical neurons, and if ethanol facilitates transloca-tion of the NR1 mRNA from the nonmembrane-associated pool to the membrane-associated pool. Cortical neurons were cultured for five days in the presence orabsence of 50mM ethanol as described under EXPERIMENTAL PROCEDURES. Cellhomogenates were fractionated on sucrose gradients by differential centrifugation toisolate membrane-associated and nonmembrane-associated mRNAs. The relativeamounts of NR1 mRNA in two pools were determined by ribonuclease protectionassay (RPA). Cyclophilin (1B15) mRNA, which is also stabilized by ethanol,22 wasincluded as an internal control. The results shown in FIGURE 1 indicate that all detect-able NR1 mRNA exist only in the membrane-associated pool, irrespective of wheth-er cortical neurons were grown in the absence or presence of ethanol. In contrast,cyclophilin mRNA was present in both pools, that is, nonmembrane-associated andmembrane-associated, in cortical neurons grown in the absence of ethanol. Follow-ing ethanol treatment (50mM, five days), a redistribution of cyclophilin mRNAoccurred such that a larger proportion of the cyclophilin mRNA was present in themembrane-associated pool (FIG. 1). These results suggest that translocation of NR1mRNA from a nonmembrane-associated to a membrane-associated pool was notinvolved in the stabilization of NR1 mRNA in response to chronic ethanol treatmentof fetal cortical neurons.

87KUMARI et al.: PROTEIN SYNTHESIS AND NMDA R1 MRNA STABILIZATION

De Novo Protein Synthesis and NR1 mRNA Levels

Previous reports from our laboratory indicate that the halflife of NR1 mRNA isprolonged by chronic ethanol treatment of fetal cortical neurons22 and presumablythis effect is caused by the inhibition of NR1 mRNA degradation. Since de novo syn-thesis of protein factors is known to be involved in the process of mRNAdegradation24 we examined the effect of inhibition of ongoing protein synthesis onethanol-induced NR1 mRNA stabilization. The halflives of NR1 and NR2B mRNAswere calculated in the presence of CHX to assess the effect of de novo protein syn-thesis on the ethanol-induced stabilization of NR1 mRNA. Fetal cortical neuronspretreated with ethanol (50mM) for five consecutive days were incubated with theprotein synthesis inhibitor, cycloheximide (CHX) (10µg/mL) or in combination

FIGURE 1. Distribution of NR1 and cyclophilin mRNAs in nonmembrane-associatedand membrane-associated polysome preparations. Cells grown in the presence or absenceof ethanol were lysed and polysomes were separated by sucrose density gradient centrifu-gation as described under EXPERIMENTAL PROCEDURES. Total RNA was purified from bothfractions by phenol–chloroform extraction and analyzed by ribonuclease protection assay.Protected fragments are identified by their name on the left. R1, NR1 subunit; 1B15, cyclo-philin. Lane P, undigested probes; Lane M, end-labeled markers, φX174 HinfI digested(numbers indicate nucleotides); Lane B, NR1 and cyclophilin mRNAs in membrane-asso-ciated fraction; Lane F, NR1 and cyclophilin mRNAs in nonmembrane-associated fraction.Both fractions (F and B) obtained from ethanol treated cells were compared with fractions(F and B) obtained from the control cultures. This experiment was repeated three times withsimilar results.


with the transcription inhibitor, actinomycin-D (10µg/mL) for 0, 12, 24, 36, and 48h.When neurons were incubated with drugs for 48h, neurons were given fresh mediumcontaining appropriate drugs every 24h. Total RNA extracted at each time point wasused to quantify NR1, NR2B, and cyclophilin mRNAs by ribonuclease protectionassay (RPA). Preliminary experiments demonstrated that 10µg/mL concentration ofCHX efficiently inhibited de novo protein synthesis in cortical neurons. Under ourexperimental conditions, inhibition of protein synthesis exceeded 95% when mea-sured by incorporation of labeled methionine into TCA-precipitable proteins (seeFIGURE 2).

Although NR2B mRNA levels in cortical neurons grown in the presence of etha-nol declined with increase in CHX exposure time (see FIGURE 3A), no alteration inthe halflife of NR2B was observed. The calculated halflife of NR2B mRNA in thepresence of CHX and ethanol was 14h (FIG. 3B), which is not significantly differentfrom the halflife of R2B (t1/2 = 15.7h) reported previously in the presence of ethanolalone.22 NR1 mRNA levels also declined progressively when cortical neurons pre-exposed to ethanol (50mM, five days) were cultured in the presence of CHX or CHXand actinomycin-D (FIG. 3A). Inhibition of ongoing protein synthesis by CHX desta-bilized NR1 mRNA, such that the halflife of NR1 mRNA in the presence of ethanolwas 23h (FIG. 3C). The calculated halflife of NR1 mRNA in the presence of CHXand ethanol was not significantly different from that reported in neurons grown inthe absence of ethanol (t1/2 = 16h).22 These observations suggest that inhibitionof de novo protein synthesis in cortical neurons pre-exposed to ethanol (50mM,five days) had no effect on the decay rate of the NR2B mRNA but facilitated thedegradation rate of NR1 to the same extent as seen in cells grown in the absence

FIGURE 2. Effect of CHX on de novo protein synthesis in cells pre-exposed toethanol. Cells were cultured in the presence of CHX for 12 h followed by incubation with[35S]-methionine–cystine mixture for three hours. TCA-precipitable radiolabeled proteinswere detected by scintillation counting. Inhibition of protein synthesis is expressed as per-centage of control.


FIGURE 3. Effect of cycloheximide (CHX) on the decay rate of NR1, NR2B, andcyclophilin mRNAs in cortical neurons grown in the presence of ethanol. Fetal cortical neu-rons cultured in the presence of 50 mM ethanol for five days were exposed to CHX or a mix-ture of CHX and actinomycin-D for various time periods. Total RNA isolated at each of thetime point was used to quantify NR1, NR2B, and cyclophilin mRNA levels by ribonucleaseprotection assays (RPAs). A. A gel autoradiogram showing protected fragments identifiedby their name on the left: R2B, NR2B subunit; 1B15, cyclophilin; R1, NR1 subunit. LaneC, RNase digested probes; Lane tRNA, tRNA control to determine non-specific hybridiza-tion; Lane P, undigested probes; Lane M, end-labeled markers, φX174 HinfI digested (num-bers indicate nucleotides); Lane 0, 0 h control; Lanes 12 to 48, incubation of cells with CHXor CHX and actinomycin-D (indicated above) for 12 h, 24 h, 36 h, and 48 h. Hybridizationsignal of NR2B (B) and NR1 (C) in the presence of CHX and actinomycin-D were quanti-fied on a PhosphorImager as described under EXPERIMENTAL PROCEDURES. Results areexpressed as percentage of the respective zero-time value and plotted on a logarithmic scaleversus time. The halflives of NR1 and NR2B mRNAs were calculated from the slope of thelines. Data points are mean ± SE values of three separate experiments.


of ethanol. It is possible that ethanol-induced protein factor(s) were essential forethanol-induced stabilization of NR1 mRNA in fetal cortical neurons.

DISCUSSION

In recent years, mRNA degradation has been recognized as an important step inthe regulation of gene expression. Although attempts have been made to dissect themechanisms that govern mRNA degradation, there are no general principles that areapplicable to every mRNA in a eukaryotic cell. In this study, we have exploredpotential molecular mechanism(s) by which chronic ethanol treatment stabilizesNR1 mRNA in fetal cortical neurons. We have utilized our in vitro model system offetal cortical neurons to demonstrate that de novo protein synthesis was crucial forethanol-induced stabilization of NR1 mRNA. Our present results also show that, inboth the presence and absence of ethanol, NR1 mRNA is always associated with therough endoplasmic reticulum and, hence, subcellular distribution of NR1 mRNAdoes not influence NR1 mRNA stability.

Previous studies from our laboratory indicate that chronic ethanol treatment offetal cortical neurons results in an increased expression (67%) of NR1 receptor sub-unit polypeptide levels19 with no significant alterations (11%) in the NR1 mRNAlevels.20 A similar ethanol treatment paradigm stabilizes the NR1 mRNA in fetalcortical neurons.22 We reason that perhaps NR1 mRNA, like the vitellogeninmRNA,33 exists in two pools in fetal cortical neurons; that is, (1) a nonmembrane-associated pool that has translationally inactive mRNAs (mRNPs); and (2) a mem-brane-associated pool that is associated with the rough endoplasmic reticulum(RER) and has actively translated mRNAs.25,34 We hypothesize that upon chronicethanol challenge, NR1 mRNA is translocated from its storage form (i.e., mRNPs)to the RER for active translation. This translocation of NR1 mRNA in response tochronic ethanol treatment could not only facilitate translation, thus accounting for anincrease in the NR1 polypeptide levels19,21 as has been documented for myosinheavy chain mRNA34 and TATA-binding protein mRNA,35 but could also protectthe NR1 mRNA from degradation by cytoplasmic nucleases, as demonstrated forphosphoenolpyruvate carboxykinase and histone mRNAs.26,27 To test our hypothe-sis, we employed differential centrifugation to separate membrane-associatedmRNAs from nonmembrane-associated mRNAs from the cytoplasm of cortical neu-rons grown in the absence or presence of ethanol (50mM, five days) and analyzedfor the presence of NR1 mRNA and cyclophilin (an internal control) by RPA. Weobserved that all detectable NR1 mRNAs sedimented in the membrane-associatedfraction irrespective of whether neurons were cultured in the presence or absenceof ethanol (FIG. 1). In contrast, cyclophilin mRNA was present in both fractions;that is, membrane-associated and nonmembrane-associated (FIG. 1). Followingethanol treatment, translocation of cyclophilin mRNA occurred, such that the ratioof membrane-associated versus nonmembrane-associated cyclophilin mRNA washigher in comparison with the control group (FIG. 1). This is an interesting observa-tion and worthy of further investigation, but is beyond the scope of the present studyrelating to ethanol-mediated stabilization of the NR1 mRNA. Taken together, theseresults suggest that (1) differential centrifugation is effective in separating the


membrane-associated mRNAs from the nonmembrane-associated mRNAs and thatthe existence of NR1 mRNA in membrane-associated fraction is not a methodologicartifact; and (2) mechanism(s) other than subcellular distribution are involved inNR1 mRNA stabilization.

In the past, several investigators observed a link between mRNA stability and denovo protein synthesis.24 These studies suggest that labile protein factor(s) inducedin response to various stimuli play a pivotal role in the turnover rate of specificmRNAs in eukaryotic cells.24,28,36–38 For example, estradiol stabilizes the estrogenreceptor mRNA in hepatocytes,29 whereas tumor necrosis factor (TNF) stabilizes thec-kit mRNA in primary acute myelogenous leukemia blasts.28 In both instances, thestabilization effect of estradiol or TNF on the respective mRNA is abolished whenongoing protein synthesis is inhibited by cycloheximide.28,29 These observationssupport the fact that extracellular stimuli induce the synthesis of a labile CHX-sensitive factor(s) that interact(s) with the respective mRNAs to increase their half-lives. To delineate if protein factor(s) are crucial for ethanol-induced NR1 mRNAstabilization in cortical neurons and if such protein factor(s) already existed in cellsor their synthesis was induced by chronic ethanol treatment, we cultured fetal corti-cal neurons in the presence of 50mM ethanol for five days followed by exposure toprotein synthesis inhibitor CHX for 0, 12, 24, 36, and 48hours. Cells were alsosimultaneously incubated with CHX and actinomycin-D to determine the influenceof inhibition of protein synthesis on the half lives of NR1 and R2B mRNAs.Although we included glyceraldehyde-3-phosphate dehydrogenase (data not shown)and cyclophilin mRNAs as internal controls in this study, we could not discern theeffects of CHX and actinomycin-D on these two mRNAs since we do not know theirnormal turnover rates in fetal cortical neurons grown in the presence or absence ofethanol. We, therefore, included NR2B as an internal control since we know that theturnover rate of R2B mRNA is not influenced by chronic ethanol treatment in vitro.22

In the present study, the calculated halflives of NR1 and NR2B mRNAs in fetalcortical cells grown in the presence of ethanol (50mM, five days) and cycloheximidewere 23 and 14h, respectively (FIG. 3). It is noteworthy that the turnover rate ofNR2B mRNA in the presence of CHX and ethanol (t1/2 = 14h) was not significantlydifferent from the turnover rate in cells grown in the presence of ethanol alone(t1/2 = 15.7h).22 On the contrary, inhibition of de novo protein synthesis by CHXhad a marked effect on the halflife of NR1 mRNA in the presence of ethanol. Asreported previously, it was not possible to calculate the halflife of NR1 in corticalneurons grown in the presence of ethanol alone as chronic ethanol exerts a stabilizingeffect on the NR1 mRNA. In fact, no decay of NR1 mRNA is observed after 24h ofactinomycin-D treatment.22 In this study, we observed that the halflife of NR1 mRNAin the presence of ethanol and CHX is 23h, which is not significantly different fromthe decay rate of NR1 mRNA in fetal cortical neurons cultured in the absence ofethanol. Thus, we demonstrate for the first time that ethanol-induced stabilizationof NR1 required de novo protein synthesis. Although these results do not providedirect evidence for the binding of a specific ethanol-inducible protein factor(s) toNR1 mRNA, this study strongly suggests that NR1 mRNA stability is regulatedby a mechanism(s) that involves RNA-protein interaction. Additional ongoing stud-ies will help us elucidate the role of ethanol-induced protein factor(s) in NR1 mRNAstabilization.


In summary, the results of the studies reported here suggest that (1) NR1 mRNAis associated with rough endoplasmic reticulum both in the presence or absence ofethanol, and (2) de novo protein synthesis plays a crucial role in the ethanol-mediatedstabilization of NR1 mRNA. Additional studies are under way to delineate hownewly synthesized proteins stabilize NR1 mRNA in fetal cortical neurons in responseto chronic ethanol treatment.

ACKNOWLEDGMENT

This study was supported by NIH-NIAAA Grants AA10552 and AA12070.

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