Cyclic AMP Regulation of Lactate Dehydrogenase

Cyclic AMP Regulation of Lactate Dehydrogenase ISOPROTERENOL AND N6,02’-DIBUTYRYL CYCLIC AMP INCREASE THE LEVELS OF LACTATE DEHYDROGENASE-5 ISOZYME AND ITS MESSENGER RNA IN RAT C6 GLIOMA CELLS* (Received for publication,May 29, 1980)

Dennis F. Derda, Michael F. Miles, John S. Schweppe, and Richard A. JungmannS From the Cancer Center, Biochemistry Division, Northwestern UniversityMedical School, Chicago, Illinois 60611

The mechanism of isoproterenol and W,O“’-dibutyryl adenosine 3‘:5‘-monophosphate (dibutyryl CAMP) induction of lactate dehydrogenase (EC 1.1.1.27) was investigated in the C6 rat glioma cell line. [3H]Leucinelabeled lactate dehydrogenase in noninduced and induced cells wasquantitativelyimmunoprecipitated with rabbit anti-rat lactate dehydrogenase-5antiserum. The immunoprecipitates were analyzed for 3Hlabeled lactate dehydrogenase by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and isoelectrofocusing. Using this technique, it was shownthat isoproterenol + 3-isobutyl-1-methyhanthineand dibutyryl cAMP cause an increase of the [3H]leucineincorporationinto glioma cell lactate dehydrogenase. Analysis of the kinetics of induction and deinduction revealed no change the in rateof degradation of lactate dehydrogenase inthe presence and absence of inducing agent, indicating that the induction was due to an increase in the rate of synthesis of the enzyme. The increased rate of synthesis was prevented by actinomycin D. Isoproterenol + 3-isobutyl-1-methylxanthine increased only the specific rate of synthesis of lactate dehydrogenase-5 isozyme and of the M subunit. The mechanism was further studied by assaying the level of functional mRNA coding for lactate dehydrogenase in a reticulocyte cell-free protein-synthesizing system using glioma cell poly(A)-containing RNA isolated from either isoproterenol or dibutyryl CAMP-induced cells. Analysis of the immunoprecipitated translation product by isoelectrofocusing revealed that isoproterenol or dibutyryl cAMP produced an approximately 8-fold stimulation of the poly(A)+RNA-directed synthesis of thelactate dehydrogenase M subunit. These data demonstrate that isoproterenol and dibutyryl CAMPcontrol the level of functionally active lactate dehydrogenase mRNA in glioma cells which, in turn, determines the extent of synthesis of the lactate dehydrogenase M subunit.

number of enzymes and other proteins (1-5). Although the mechanism underlying enzyme induction by cAMP has been the focus of considerable researchactivity,the issuestill appears very complex and unresolved. Wicks and co-workers (6, 7) and Van de Poll et al. (8), using inhibitors of protein and RNA synthesis, observed that cAMP appeared to affect eukaryoticprotein synthesis a t a post-transcriptional step. Recently, however, a number of investigators have shown that dibutyryl cAMP increases the functionallevels of mRNA for tyrosine aminotransferase (9, lo), phosphoenolpyruvate carboxykinase (ll),and albumin (12). In order to moreclearly define the mechanism involved in CAMP-mediated enzyme induction, our laboratory has recently begun an investigation of the mechanism of induction of lactate dehydrogenase by cAMP in the rat C6 glioma cell line. It hasbeen known for several years that lactate dehydrogenase activity increases in rat C6 glioma cells after catecholamine or dibutyryl cAMP stimulation (13, 14). The levels of cAMP increase rapidly in glioma cells after catecholamine stimulation (14) and this effect and the subsequent increase of lactate dehydrogenase activity are prevented by ,&adrenergic blocking agents (15). Both cycloheximide and actinomycin D block the catecholamine- and dibutyryl CAMP-mediated rise of lactate dehydrogenase activity (14), suggesting that enhanced synthesis of lactate dehydrogenase occurs as the result of an effect of cAMP at the level of transcription. Using specific immunoprecipitation of lactate dehydrogenase after its labeling with radioactive amino acid, Kumar et al. (16) with the C6 glioma cell 2B subclone and our laboratory using the original C6 glioma cell line (17) have recently been able to show that a stimulation of the synthesis of lactate dehydrogenase accounts for the catecholamine-mediated increase of lactate dehydrogenase activity. We have extended these studies and present a detailed report of the effect of isoproterenol and dibutyryl cAMP on the synthesis and degradation of lactate dehydrogenase-5 isozyme and on the functional levels of lactate dehydrogenase M subunit mRNA. We present evidence demonstrating that theinduction of lactate dehydrogenase-5 isozyme is due to an increase in the rate of Within recent years, evidence has accumulated indicating synthesis of the lactate dehydrogenase M subunit with little thatCAMP’ is capable of stimulatingthe induction of a or no change in the rate of degradation. Furthermore, we show that isoproterenol and dibutyryl CAMP cause a rapid * This studywas supported in part by National Institutes of Health Grant GM23895, by the Researchand Education Fund, Northwestern increase of the functional levels of lactate dehydrogenase M Memorial Hospital, andby a Predoctoral Fellowship to M. F. M. from subunit mRNA over a time course that correlates well with the Prudential InsuranceCo. through the Insurance Medical Scientist the time course of lactate dehydrogenase induction. Scholarship Fund. The costs of publication of this article were deEXPERIMENTALPROCEDURES frayed in part by the payment of page charges. This article must Chemicals-[”HJLeucine (specific activity 110 to 145 Ci/mmol), therefore be hereby marked “adoertzsement”in accordance with 18 [‘’Hluridine(specific activity 25 Ci/mmol), and [”SS]methionine (speU.S.C. Section 1734 solely to indicate this fact. __ ~_~________ $ To whom correspondence should be addressed. ’ The abbreviations used are: CAMPor cyclic AMP, adenosine 3’: containing a polyadenylic acid sequence; Hepes, 4-(2-hydroxymethyl)1-piperazineethanesulfonicacid; MIX, 3-isobutyl-1-methylxanthine; 5’-monophosphate; dibutyryl CAMP,N”,O”-dibutyryladenosine 3‘:5‘NaCI/P,, phosphate-buffered saline. monophosphate; SDS, sodium dodecyl sulfate; poly(A)+RNA, RNA

11112

Cyclic AMP Induction of Lactate Dehydrogenase cific activity 950 Ci/mmol) were purchased from New England Nuclear. Oligo(dT)-cellulose (type 3) was from Collaborative Research, and actinomycin D was obtained from Calbiochem. Dulbecco's phosphate-buffered saline (NaCI/P,), Hanks' balanced salt solution, and Eagle's minimumessential medium were from GIBCO. All other biochemicals of analytical grade were purchased fromSigma Chemical c o . Cell Cultures-The original rat C6 glioma cell line was obtained from the American Type Culture Collection (Rockville, Md.) in the 37th passage. Monolayercultures were grown in 150 cm' Bellco tissue culture flaskscontaining 30 ml of Ham's F-10 nutrientmedium (GIBCO or Flow Laboratories) buffered with 35 mM NaHC03 and supplemented with 10% dialyzed fetal calf serum (GIBCO or Flow Laboratories), 50 units/ml of penicillin, and 50 pg/ml of streptomycin. The cells were plated at a density of 5.8 X 10' cells/flask and grown to confluence (between Days 10 and 12) at 37°C in an atmosphere of 5% CO,. Stock cultures were split 1:lO weekly and refed every 2 to 3 days. Larger quantities of cells (for the isolation of RNA) were grown to confluence (in 10 days) in 890 cm2 roller bottles (Corning Glass Works)containing 150 ml of theculturemedium at 37°C inan atmosphere of 5% COP.Twenty-four hoursbefore an induction experiment, the mediumwas replaced with fresh culture medium, and the cells were maintained a t 37°C until harvest. Assay of Lactate DehydrogenaseActivity-Lactate dehydrogenase was assayed either by a spectrophotometric or by afluorometric method. In the spectrophotometric assay, total lactate dehydrogenase activity in a 100,OOO X g supernatant fraction was assayed by converting pyruvate into lactate and measuring the decrease of absorbance of NADH at 340 nm (18). The assay wascarried out at a pyruvate concentration of 0.45 mM at which concentration both the H and M subunit exhibit equal fractions of their enzymatic activity (19). For the determination of the individual lactate dehydrogenase isozymes after their separation on agarose gels, a fluorometric method was employed (20). Specific activity is expressed as units of enzyme/ mg of protein. One unit of enzyme is defined as the amountcatalyzing the transformation of 1 pmol of substrate/min a t 25'C. Induction of Lactate Dehydrogenase-Twenty-four hours before induction, the culture medium was decanted andreplaced with 45 ml of fresh Ham's F-10 medium. Cells were stimulated a t 37°C by the addition of either 10 p~ isoproterenol + 0.5 mM MIX (final concentrations) or of dibutyryl CAMP at the concentrations listed in the text. After the induction, the culture medium was aspirated off, and the cell monolayer was rinsed twice with 30 ml of NaCl/P,. The cells were harvested by scraping with a rubber policeman in 2 to 5 ml of 100 mM potassium phosphate, 1 mM EDTA, and 1 mM mercaptoethanol, pH 7.4. The cell suspension was transferred into a 10-ml test tube and subsequently sonicated at 2°C twice for 10-s periods using a MSE ultrasonic power unit at 1.8A. The sonicated homogenate was centrifuged for 20 min at 20,000 X g. The 20,000 X g supernatant fraction was again centrifuged for 45 min a t 100,000 X g. The 100,OOO X g supernatant fraction was filter sterilized using Millex filters (0.45 p pore size). Theresulting cytosol was used for lactate dehydrogenase activity assays, immunoprecipitation, andtrichloroacetic acid precipitation. The protein concentration in the cytosols varied between 2 and 10 mg/ml. and Incorporation of [3H]Leucine into Total Glioma Cell Protein into Immunoprecipitable Lactate Dehydrogenase-Topulse label cell protein, 150 pCi of ["Hlleucine was added perflask (containing 45 ml of medium)andincubation was continued for 1 h at 37OC. Radioactive labeling was terminated by the addition of 0.3 ml of 0.15 M leucine. The culture medium was aspirated off, and the cell monolayer was rinsed twice with ice-cold NaCI/P, containing 2.4 mM leucine. The cellsweresonicated and acytosol was preparedas described above. Measurement of Half-Life a n d Degradation of Lactate Dehydrogenase-To label protein, 200 pCi of ["Hlleucine was added without or together with 10 p~ isoproterenol + 0.5 mM MIX (final concentration) to each culture flask (containing 45 ml of medium). Incubation was carried out at 37'C for 24 h. To terminateradioactive labeling, a solution of leucine in Hanks' balanced salt solutionwas added to each flask to a final concentration of 2.4 mM leucine. The culture medium was aspirated off, andthe cell monolayer was rinsed twice with Eagle's minimum essential medium containing2.4 mM leucine. In Fig. 7, this point corresponds to time zero. After this final rinse, 45 ml of Ham's F-10 medium containing 2.4 mM leucine were added to each flask and incubation a t 37°C was continued for the time periods indicated in Fig. 7 (chase period). At the endof the chase period, the culture medium was aspirated off, the cells were rinsed twice with

11113

ice-cold NaCI/P, containing 2.4 mM leucine, harvested, and a cytosol was prepared for immunoprecipitation of lactate dehydrogenase as described above. RNA Extraction a n d Isolation of Poly(A)-containing RNA-Total cellular RNA from control and inducedcells was extracted by the guanidinium hydrochloride/ethanol precipitation methodof Cox (21) as modified by Strohman et al. (22). Accordingly, the medium of two roller bottles was decanted and the monolayers (4 to 6 X 10' cells/ bottle) were rinsed once with ice-cold KaCl/P,. Thecells were scraped off, collected in 30 ml of NaCI/Pi, and sedimented by Centrifugation. The cell pellet was suspended in 15 to 20 volumes of 7 M guanidinium hydrochloride and homogenized with a Brinkmann Polytron homogenizer for three 20-s periods. After the addition of sodium acetate (final concentration 0.1 M, pH 5.4) and absolute alcohol (1 volume of ethanol to2 volumesof homogenate), RNAwas allowed to precipitate for 18 h at -20°C. The precipitated RNA was pelleted by centrifugation for 20 min at 12.000 X g, resuspended in 10ml of 7 M guanidinium hydrochloride and 25 mM EDTA, and precipitatedagain by the addition of sodium acetate and absolute ethanol.After 2 to 3 h a t -2O"C, theRNA was pelleted, andthe abovepurification procedure was repeated for a t,otal of three precipitations. The final RNApellet was dissolved in 5 ml of30 mM EDTA, pH 7.0, and extracted with ?hvolume of chloroform/l-butanol (4:1, v/v). After a wash of the organic solvent phase with 1 volume of 30 mM EDTA and pooling of the aqueousphases, RNA was precipitated for 3 h at -20°C by the addition of 2.5volumes of absolute ethanol andsodium acetate to a final concentration of 0.2 M. The RNA was pelleted, redissolved in 5 ml of distilled water, and precipitatedfor 18 h at -20°C after the addition of sodium acetate to a final concentration of 3 M, pH 5.4. The RNA was recovered by centrifugation, rinsed with 70% ethanol, dissolved in 5 ml of distilled water, and either immediately subjected to chromatography on oligo(dT)-cellulose or quick frozen in liquid nitrogen and stored a t -70°C. In a typical experiment, 5 to 6 mg of total RNA was isolated from two roller bottles based on a quantity of 43 pg of RNA/&'" optical unit. The RNA contained less than 0.1% DNA as assayed by the diphenylamine method (23) and exhibited a 260/280 nm ratio of 1.9 to 2.1. T o isolate poly(A)+ RNA, total RNAwas subjected to chromatography on oligo(dT)-cellulose as described by Bantle et al. (24) with minor modifications. Columns containing 0.5 g of oligo(dT)-cellulose were prepared by washing the resin with 10 ml of 0.1 N NaOH and 0.1% diethylpyrocarbonate, followed by equilibration with 10 ml of binding buffer (0.01 M Tris, 0.4 M NaC1, 1 mM EDTA, and 0.5% SDS, pH 7.5). RNA, dissolved in binding buffer (0.5 mg/ml), was applied onto the equilibrated oligo(dT)-cellulose column at 150 to 200 A?(;,, units/g of resin. The column was washed with 20 bed volumes of binding buffer. Elution was carried out with 5 ml of 0.01 M Tris, 1 mM EDTA, and 0.1% SDS, pH 7.4. The eluted RNA was heated to 70°C for 5 min, cooled on ice, and adjusted toa final NaClconcentration of 0.4 M. The solution was then reapplied onto oligo(dT)-cellulose. After a wash with 5 bed volumes of binding buffer, elution was carried out with 1-ml portions of elution buffer. RNA in the eluatewas monitored by measuring the absorbance a t 260 nm or by measuring tritium radioactivity (in the case of ['HJuridine-labeled RNA). The fractions containing poly(A)' RNA were pooled. Poly(A)' RNA was precipitated after theaddition of sodium acetate (0.2 M final concentration) and 2.5 volumes of ethanol for 18 h at -20°C. The precipitate was collected by centrifugation at 15,000 X g for 1 h, lyophilized, and redissolved in sterile water a t a concentration of 0.5 mg of RNA/ml. The RNA was kept in liquidnitrogen until used for translation. Typical yields of poly(A)+ RNAwere 60 to 80 pg recovered from 5 to 6 mg of total glioma cell RNA. Cell-free Protein Synthesis-Rabbit reticulocyte lysate was prepared according to I'almiter (25) with minor modifications. Rabbits (Lancershire Farms),weighing 2 to 4 kg, were injected subcutaneously daily for 6 days with a neutralized solution of 2.5% phenylhydrazine (0.25 ml/kg body weight). When the reticulocyte count was above 90%, the rabbits were exsanguinated by heart puncture. The blood was collected into ice-cold reticulocytesaline (1.5 mM magnesium acetate, 0.14 M NaC1, 5 mM KCI, and 10 mM EDTA, pH 7.0). Heticulocytes were sedimented and washed four times with 5 volumes of reticulocyte saline. The final two washes contained no EDTA, and the final centrifugation was carried out for 5 min at 8,000 X g.After the addition of an equal volume of 2 mM MgCI2, 0.1 mM EDTA, and 1mM dithiothreitol, pH 7.0, the cell pellet was lysed for 5 min at 2"C, and thecell debris was removed by centrifugation for 20 min a t 30,000 X g. Creatine phosphokinase (50 pg/ml) and hemin (40 pg/ml) were added to the supernatant fraction which was aliquoted and kept at

11114

Cyclic AMP Induction of Lactate Dehydrogenase

-70°C. All lysates were depleted of endogenous mRNA activity by treatment with micrococcal nuclease (26).Lysate (1ml) wasincubated for 10 min at 23°C in the presence of 1 mM CaCL and 50 pg of micrococcal nuclease (Sigma Chemical Co., 15,000 units/mg). After incubation, the lysate was cooled on ice, ethylene glycol bis(,LI-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTAj was added to a final concentration of 2.5 mM, and the lysate was quick frozen and stored a t -7O'C until used for translation assay. Cell-free protein synthesis was performed in a total volume of 0.1 ml containing 33 pl of reticulocyte lysate, 1.0 pg of poly(A)+ RNA,25 mM Hepes, 10 mM creatine phosphate, 0.16 M potassium acetate, and 0.05 mCi of ["S]methionine, pH 7.0. All incubations were performed at 30°C for 30 min, after which they were stopped by chilling on ice and by the additionof 75 p1 of NaCI/P, containing 0.02%sodium azide and 1.0 mg/ml of methionine. The mixturewas centrifuged for 20 min at 130,000 X g in a Beckman airfuge. The supernatant fraction was used for the determinationof total [J"S]methionine incorporation into trichloroacetic acid-precipitable protein (26) and for immunoprecipitation. Immunoprecipitation of Lactate Dehydrogenase-Quantitative immunoprecipitationwasperformed by mixing cytosol (approximately 25 to 350 pl) containing 2 units of lactate dehydrogenase or 0.14 ml of supernatant fraction after cell-free protein synthesis with 0.05 ml of 20% Triton X-100 in NaCI/P,, pH 7.4, to a total volume of 0.5 ml. Thesupernatantfraction from cell-free proteinsynthesis contained, inaddition, 10 pg of purified carrier lactatedehydrogenase5 from rat liver and 0.5 mg of methionine. Rabbit anti-rat lactate dehydrogenase-5 antiserum was added to ensure an excess of about 50%. The mixture was incubated at 25°C for 30 min and then for 18 h a t 4°C. The mixture with the immunoprecipitate was layered onto 0.75 ml of immunoprecipitation buffer (NaCI/P, containing 2% Triton X-100 and 0.2% sodium azide) containing 1M sucrose and centrifuged for 2 min a t 10,400 X g in an Eppendorf microfuge. This purification procedure was repeated once, after which the immunoprecipitatewas washed twice with 1 ml of immunoprecipitation buffer andthen suspended in 50 p1 of SDS gel sample buffer (0.05 M Tris, 1%SDS, 1% mercaptoethanol, and10%glycerol, pH 6.8). The sampleswere heated at 90°C for 2 min and then subjected to polyacrylamide gel electrophoresis. SDS-Polyacrylamide Gel Electrophoresis-Electrophoresiswas carried out on 10% polyacrylamide slab gels according to Laemmli (27). The gel was fixed overnight in methanol/acetic acid/water (5~1: 5, v/v/vj, stained with 0.25% Coomassiebrilliantblue R-250, and destained in 5% methanol. The gels were sliced into I-mm sections. The slices were incubated for 24 h at 37°C in I0 ml of Econofluor containing 3% Protosol. The amount of radioactivity incorporated into lactatedehydrogenase was computed by adding theradioactivity of the fractionscorresponding to the lactatedehydrogenase band and subtracting a background estimated from the immunoprecipitationof translations containing no exogenous poly(A) RNA or from an average of areas not containing the dehydrogenase. Zsoelectrofocusing-Immunoprecipitated lactate dehydrogenase was analyzed by isoelectrofocusing under denaturing conditions in urea as described by Wilson et al. (28). The gels were fixed, stained, destained,and sliced into1-mm sections. TwoI-mm slices were placed into a vial and digested with 10ml of 3%Protosol in Liquifluor/ toluene for 24 h at 37°C after which radioactivity was determined. Preparation ofAntiserum against Lactate Dehydrogenase-5-Rat liver lactate dehydrogenase-5 isozyme was purified to homogeneity as described by Ryan andVestling (29) to a specific activity of 322 units/ mg of protein. Enzyme (0.33 mg in 0.25 ml of 100 mM ammonium sulfate, 100 mM potassium phosphate, and 1 mM mercaptoethanol, pH 7.6) was mixed withanequal volume of complete Freund's adjuvant (Difco Laboratories) and injected into a White New Zealand rabbit in about four subcutaneous sites.The procedure was repeated after 2 weeks, and antiserum was collected when a titer of greater value than 50 units of antibody/ml of serum was obtained. One unit of antibody isdefined as that amountnecessary to inhibit completely 1 unit of lactate dehydrogenase. The rabbit was bled by ear vein or cardiacpuncture,andthesera were titrated for their ability to inactivate rat lactate dehydrogenase-5 isozyme by incubating different amounts of the serum with a constant amount of enzyme at 25°C for 20 min (see Fig. 1). Antibodywasalsocheckedfor specificity by Ouchterlony double diffusion analysis (30). The antiserum did not cross-react with lactate dehydrogenase-1 isozyme or with proteins from the rabbit reticulocyte lysate. Analytical Methods-Protein was determined according to Lowry et al.(31) and RNAwas determined spectrophotometricallyusing the molar extinction coefficient at 259 nm of15.4 X M/cm.

RESULTS

Analysis ofAntiserurn against Rat Liver Lactate Dehydrogenase-5 Isozyme-Antiserum against rat liver lactate dehydrogenase-5 isozyme was raised in rabbits by injecting them with the purified antigen as described under "Experimental Procedures." Double immunodiffusion Ouchterlony analysis of the antiserum andpurified rat liver lactate dehydrogenase5, crude rat liver homogenate, and the cytosol from noninduced and induced C6 glioma cells showed a single continuous immunoprecipitation band (results not shown). This suggests the presence of a single antibody-antigensystemandthe identity of the antigen from induced and noninduced glioma cells. The specificity and titer of the antiserum were next determined by (a)immunotitration in which a constant amount of highly purified antigen was incubated withincreasing amounts of rabbit anti-rat lactatedehydrogenase-5 antiserum, and ( b ) by quantitativeimmunoprecipitation in which a constant amount of antiserum was incubated with increasing amounts of cytosol from induced and noninduced cells. The results of the immunotitration are shown in Fig. 1. When the data are plotted as thelactate dehydrogenase activity uersus the amount of antiserum present, both lactate dehydrogenase activities from noninduced and induced C6 glioma cells decrease linearly with increasing antibody concentration.On the basis of this experiment, it was determined that 1 ml of rabbit anti-rat lactatedehydrogenase-5 antiserum is capable of completely inhibiting 62 units of glioma cell lactate dehydrogenase. The results of the quantitative immunoprecipitation are shown in Fig. 2. The equivalence point with respect to the maximum amount of protein in the precipitate occurs at 80 pl of glioma cell cytosol. In the figure, we have also plotted the unitsof lactate dehydrogenase activity added to the assay and the enzymatic activity remaining after immunoprecipitation.

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FIG. 1. Immunotitration of rabbit anti-rat lactate dehydrogenase-5 antiserum. Highly purified rat liver lactate dehydrogenase-5 isozyme, or glioma cell cytosol, equivalent to 2 unit of lactate dehydrogenase, was incubated with theindicated amounts of antiserum in a total volume of 0.5 ml of NaCI/P,. Incubation was carried out for 20 min at 25°C followed by 18 h at 4°C. The samples were then centrifuged for 10 min at 10,400 X g. Supernatant fractionswere assayed for lactate dehydrogenase activity as described under "Experimental Procedures." A, Rat liver lactate dehydrogenase-5 isozyme; 0,cytosol from noninducedglioma cells; 0,cytosol from glioma cells induced for 24 h with isoproterenol + MIX.

Cyclic AMP Induction of Lactate Dehydrogenase P

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FIG. 2. Quantitative immunoprecipitationof glioma cell lactate dehydrogenase. Increasing amounts of cytosolfrom glioma cells, induced for 12 h with isoproterenol + MIX, were incubated with a constant amount of rabbit anti-rat lactatedehydrogenase-5 antiserum for 20 min at 25°C and for 18 h at 4°C. The amountof protein in each immunoprecipitate (A)is plotted uersus the amount of cytosol present in each reaction. The units of lactate dehydrogenase added (0)and the units of activity remaining in the supernatant fraction (0)after immunoprecipitation are also shown.

To measure the turnover of lactate dehydrogenase, we have developed a n immunochemical procedure consisting of labeling of cells withradioactive amino acid, preparation of a 100,000 X g supernatantfraction,immunoprecipitation of radioactively labeled lactate dehydrogenase with specific antiserum against the M subunit of lactate dehydrogenase, and analysis of the immunoprecipitateby SDS-polyacrylamidegel electrophoresis. The results of this procedure demonstrate that thereis only one peakof radioactivity which co-migrates with highly purified rat liver lactate dehydrogenase-5 isozyme (Fig. 3). No radioactive proteins, other than lactate dehydrogenase, are detected. Induction of Lactate Dehydrogenase-The effects of isoproterenol + MIXon ["Hlleucineincorporation intototal trichloroacetic acid-precipitable protein and into immunoprecipitable lactatedehydrogenaseas afunction of timeare shown in Fig. 4.A slight butsignificant increase of "H-labeled lactate dehydrogenase is measured as early as 3 h after the induction (Fig. 4B).The maximum rate of ['Hlleucine incorporation into lactate dehydrogenase is observed a t 12 h, after which time the labeling rate declines but remains elevated at 18 and 24 h.Twelve hoursaftertheinduction,therate increases about 10-fold, resulting in a 5-fold increase in the relativerate' of synthesis of lactatedehydrogenase (from 0.75% in control to 4.58% in induced cells). The rate of ["HIleucineincorporation into lactate dehydrogenase in noninduced cells remains relatively constant between990- and 1490 cpm of "H incorporated/mg of protein during the24-h experimental period. Incorporation of ["Hlleucine into total trichloroacetic acid-precipitable protein is increased between 5 and 24 h after the induction (Fig. 4A). Dibutyryl CAMP, at concentrations ranging from 10-~to IO-'' M, is similarly effective in increasinglactate dehydrogenase activity and in stimulating [,"H]leucine incorporation into lactate dehydrogenase(Fig. 5). Sodiumbutyrateat M is ineffective ininducing either lactatedehydrogenaseactivityorsynthesis(resultsnot shown).

11115

+

Effect of Isoproterenol MIX on the Activity a n d Synthesis of Lactate DehydrogenaseIsozymes a n d on the Synthesisof the M a n d H Subunits-Careful analysis of C6 glioma cells for the presence of the individual lactate dehydrogenase isozymes has revealed the presence of isozymes-3, -4, and -5 but not of isozymes-1 and -2. These observations areconsistently made regardless of the methods applied for the separation of isozymes and measurement of enzymatic activity. Evaluation of the effect of isoproterenol + MIX on the activity of the individual isozymes demonstrates a stimulatory effect of isoproterenol + MIX on the lactate dehydrogenase-5 isozyme only and a slight decrease of the activityof isozymes-3 and -4 (Table I). Analysis of the ["Hlleucine incorporation into isozymes-3, -4, and-5 similarlyrevealsincreasedradioactive labeling of lactate dehydrogenase-5 isozyme and aslightdecrease -of ["Hlleucine incorporation into isozymes-3 and -4 (Table I). ["HILeucine labeling of glioma cell lactate dehydrogenase, immunoprecipitation of all isozymes, and analysis of the radioactive labeling of the M and H subunits after their separation by isoelectrofocusing demonstratethe selective increased synthesis of only the M subunit after induction, and no effect on the H subunit is observed (Table 11, Fig. 1OA). Effect of Isoproterenol + MIX on Lactate Dehydrogenase Degradation-Since the observed increased ["Hlleucine incorporation into lactatedehydrogenase may also be explained by a mechanism which decreases the rate of degradation of lactate dehydrogenase rather than increases the rate of synthesis, we tested whether or not there was a change in the rate of degradation of "H-labeledenzyme. Since the time course of increase of enzyme molecules from basal level to its "

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FIG. 3. Analysis of immunoprecipitated lactate dehydrogenase by SDS-polyacrylamide gel electrophoresis. A, homogeneous rat liver lactate dehydrogenase-5 was subjected to SDS-polyacrylamide gel electrophoresis. After staining with Coomassie blue,the gel was scanned at 600 nm. B , glioma cells were induced for 41/L h with isoproterenol + MIX and pulse-labeled for 1 h with ['Hlleucine. 'HT h e percentage of the relative rate of synthesis (counts per minute labeled lactate dehydrogenase was immunoprecipitated. The immuof "H-labeled lactate dehydrogenase/mg of protein + cpm of total ,"H/ noprecipitate was subjected to SDS-polyacrylamide gel electrophoresis. For details, see "Experimental Procedures." The radioactivity mg of protein X 100) expresses the rate of lactate dehydrogenase in sliced gel sections was determined and is plotted in B. synthesis relative to the rateof total soluble protein synthesis.


11116

TABLEI Effect of isoproterenol MIX on the relative enzymatic activity and rateof synthesis of lactate dehydrogenase isozymes To measure enzymatic activity, glioma cells were induced for 24 h with isoproterenol + MIX under the conditionsdescribed under “Experimental Procedures.” A cytosolwas prepared and subjectedto agarose gel electrophoresis, followed by fluorometric determination of the isozyme activities (20). The activity of the isozymes is given as the percentage of the total activity present in the cytosol. T o determine synthesis, glioma cells were induced with isoproterenol + MIX and labeled with 200 pCi of [“H]leucine for a 24-h period. The “Hlabeled cytosol, equivalent to 1.5 units of lactate dehydrogenase,was subjected to affinity chromatography on oxamate Sepharose (36). Fractions with lactate dehydrogenase activity were pooled and subjectedto polyacrylamide gel electrophoresisunder nondenaturing conditions (38). The radioactivity co-eluting with the individual isozymes was determined after slicing the gel. For details see “Experimental Procedures.”

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FIG. 4. Effect of isoproterenol + MIX on the time course of incorporation of [3H]leucine into total protein and into immunoprecipitable lactate dehydrogenase. Isoproterenol + MIX were added toglioma cell cultures at theindicated times before cell harvest. One hour before cell harvest, [’H]leucine was added. For more experimental details, see “Experimental Procedures.”A, [’H]leucine incorporation into totaltrichloroacetic acid-precipitable protein in induced (0)and noninduced (0)cells. B , [JH]leucine incorporation into immunoprecipitable lactate dehydrogenase in induced (A)and noninduced (A) cells. The results are representative of those obtained in three other identical experiments.

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TABLEI1 Rate of synthesis of lactate dehydrogenase M a n d H subunits Glioma cells were induced with isoproterenol + MIX and labeled with 200 pCi of [’H]leucine for a 24-h period under the conditions described in Table I. ‘H-labeled lactate dehydrogenase was isolated from cytosol, equivalent to 1.5 units of lactate dehydrogenase, by immunoprecipitation. The immunoprecipitate was subjected to isoelectrofocusing under denaturing conditions as described in legend of Fig. 10. For details see “Experimental Procedures.”Radioactivity coeluting with the individual subunits was determined after slicing the pel. Lactate dehydrogenase synthesis ~-

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FIG. 5. Dose response of dibutyryl CAMPon [3H]leucine incorporation into immunoprecipitable lactate dehydrogenase and on lactate dehydrogenase activity. Dibutyryl CAMP, a t the indicated final concentrations, was added 4% h before cell harvest. When activity was measured, dibutyryl CAMPwas added 24 h before cell harvest. Labeling of cells, isolation of cvtosol, determination of enzymaticactivity, andimmunoprecipitation were carried out as described under “Experimental Procedures.” The specific activity of immunoprecipitated lactate dehydrogenase in noninduced the control sample(shown as 100%) was 1300 cpm of :’H/mg of protein. The values shown are means k S.D. from three experiments with three flasks each. 0, lactate dehydrogenase synthesis; 0, lactate dehydrogenase activity.

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~

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new induced steady state level is determined by its rate of degradation (32), it is possible to experimentally estimate the half-life of an enzyme by analyzing its timecourse of increase to a new steady state level and compare it with its half-life determined during the deinduction phase anddecay of radioactively labeled enzyme. If there has been no change in the rate of degradation, then the half-life of t.he enzyme should be identical during induction and deinduction following removal of the inducer. The data of the corresponding experiments obtained during induction and deinduction of lactate dehydrogenase are illustratedin Figs. 6 and 7. During the induction phase, a half-life3 of lactate dehydrogenase of approximately 6Y2 h is determined with a rate constant of degradation ( K t ) of 0.1066 h” (Fig. 6 ) .Similarly, after removal of the inducing agent (Fig. 7 ) , the half-life is 7Y2 h (Kd = 0.0924 h”), a value ” T h e half-life wascalculatedfrom theequation where K,r is the rateof constant degradation.

t % = (ln2/K,i)

~

~


F -

\

-i-

2-

Y

I

*

W

X 0

1;

I

I

7

0

4 Hours

1

1

I

6

8

9

FIG. 6. Analysis of rate of degradation of lactate dehydrogenase during induction. Glioma cells were induced and assayed for immunoprecipitable "H-labeled lactate dehydrogenase as described in the legend of Fig. 4. E, represents the maximum specific activity (counts per minute of ,"H/mg of protein) of the immunoprecipitated enzyme, and E, is the specific activity at any given t,ime.

11117

and synthesis. Addition of actinomycin D to induced cells results ina reduction of the stimulatory effect. At the highest dose of actinomycin D, both the isoproterenol- and dibutyryl CAMP-induced increases of lactate dehydrogenase synthesis and activity are completely prevented. Also, actinomycin D does not significantly affect the uninducedof level the enzyme. These data suggest that lactate dehydrogenase induction appears to require de nouo protein and RNA synthesis. The induction period, during which RNA synthesis necessaryfor lactatedehydrogenaseinductionoccurs,canbeestimated from the data of Table IV. Actinomycin D was added for various time periods during a 3-h induction period. When actinomycin D is added together with isoproterenol + M I X (at 180 min before cell harvest), blockage of' the increase of lactate dehydrogenase synthesis is almost complete. At later times, the inhibitory effect is diminished, and it disappears when actinomycinD is added2 h after the addition of inducing agent. These studies suggest that during the first 2 h of t h e RNA total 180-min induction period, isoproterenol-stimulated synthesis takes place which is necessary for lactate dehydrogenase induction. TABLE111 Effect of actinomycinD on lactate dehydrogenase activity and synthesis induced by isoproterenol MIX or dibutyryl CAMP Glioma cells were induced for 4 h with isoproterenol + MIX or

+

dibutyryl cAMP without and with actinomycin D as indicated and pulse-labeled with ["Hlleucine. When activity was measured, the inducing agents were added 24 h before cell harvest. Cytosol was prepared and lactate dehydrogenase activity and synthesis were determined as described under "Experimental Procedures." The specific activity of immunoprecipitated lactate dehydrogenase in the noninduced control (shown as 1004) was 1250 cpm of ,"H/mg of protein. The values shown are means f S.D. from three seoarateexueriments. Treatment ~

~~~

7 contrrd None 100 100 Isoproterenol (10 p ~ )MIX (0.5 mM) 250 f 12 175 f 3 Actinomycin D (5 pg/ml) f3 108 f 2109 Isoproterenol MIX + actinomycin D (0.05 140 f 2

+

+

dml) Isoproterenol + MIX + actinomycin D (1.0 fig/

120 -+ 5

ml)

99 & 3101 Isoproterenol + MIX + actinomycin D (5.0 pg/ f4 ml) Dibutyryl cAMP (1 mM) 175 & 15 170 f 10 Dibutyryl cAMP + actinomycin D (5.0 wg/ml) 101 2 103 f 5

*

TABLE IV 0

20

10

30

40

Hours

FIG. 7. Analysis of decay of ['HJleucine-labeled lactate dehydrogenase following removal of inducing agent from induced glioma cells. For experimental details see "Experimental Procedures." essentially identical withthat found during induction. Effect of Actinomycin D-It had previously been shown thattheactivation of lactatedehydrogenasebycatecholamines in C6 glioma cells is prevented by inhibitors of R N A and protein synthesis(14). To further investigatethe possible the effect transcriptional dependenceof the enzyme induction, of actinomycin D on lactate dehydrogenase synthesis was investigated. Table 111 summarizes the effect of various concentrations of actinomycin D onisoproterenol + M I X o r dibutyrylCAMP-stimulatedlactatedehydrogenaseactivity

Temporal effect of actinomycin D on isoproterenol-induced lactate dehydrogenase synthesis Isoproterenol + MIX was added at 180 min, and actinomycin D (5 pg/ml) was added at the indicated times beforecell harvest. Cell

cultures were labeled with [JH]leucine, and immunoprecipitable lactate dehydrogenase was determined as described in the legend of Fig. 4. The specific activity of immunoprecipitated lactate dehydrogenase in the induced cells(shown as 100%) was 3600 cpm of .'H/mg of protein. The values shown are means f S.D. from three separate experiments. Time of actinomycin D addition Lactate dehydrogenase synthesis before cell harvest isoproterenol-stimulated value) min

0 60 90 120 150 180

100 100 43 f 3

26 f 1 5 f 0.3 3 f 0.1

( V of'


11118 r

Poly (*)’RNA

Minu1.s

(pg/ml)

FIG. 8. Characterization of the reticulocyte lysate system. A , the reticulocytelysatewas incubated for protein synthesis in the presence of 1.0 pgof purified globin mRNA (O),1.0 pgof poly(A)’ RNA from noninduced glioma cells (A),and in the absence of exogenous RNA (A). For details, see “Experimental Procedures.” Protein was precipitated by the addition of 5% trichloroacetic acid, and ”“S radioactivity was determined. B , the reticulocyte lysate was incubated for protein synthesis in the presence of the indicated amounts of poly(A)’ RNA from noninduced glioma cells. After incubation, prowas precipitated tein (in aliquots representing 1%of the total mixture) by the addition of 5% trichloroacetic acid (01,and the amount of ”’S radioactivity in lactate dehydrogenase in the remaining aliquot was determined after immunoprecipitation and SDS-polyacrylamide gel as described under “Experimental Procedures.” electrophoresis (0)

Distance

Imm)

FIG. 9. SDS-polyacrylamide gel electrophoresis of the immunovreciDitate obtained after translation in a reticulocyte lysate. The reticulocyte lysate was incubated as described under “Experimental Procedures” in the presence of [:‘SS]methionine and 10 pg/ml of poly(A)+ RNAisolated from glioma cells induced for 4 h with 10 PM isoproterenol ( A ) ,noninduced glioma cells ( B ) ,and without exogenous RNA (C). After translation, the [”5S]methionine-labeled lactate dehydrogenase was immunoprecipitated and subjected to SDS-polyacrylamide gel electrophoresis. The gels were subjected to autoradiography. Optical density of the autoradiographs was determined by densitometry. ”

mental approach to quantitate mRNA for lactate dehydrogenase, whichrepresents at themost only0.3%of the cytoplasmic protein in noninduced cells, we have utilized the guanidinium hydrochloride/ethanolprocedure, followed byisolation of poly(A)+-containing RNAusing affinity chromatography on oligo(dT)-cellulose. With these methods, an enriched poly(A)+-containing RNA fraction is obtained withonly minor contamination by 18 and 28 S RNA as analyzed by formamide/polyacrylamide gel electrophoresis (results not shown). Stimulation of glioma cells with isoproterenol has no apparent effect on the recovery of poly(A) RNA or on the RNA electrophoretic profile. In order to optimize the reticulocyte lysate for translation of rat glioma cell mRNA, mRNA activitywas assessed under various experimental conditions. The time course of protein synthesis directedby either endogenous mRNA, purified globin mRNA, or poly(A)RNA from noninduced glioma cells is shown in Fig. 8A. Whereas purified globin mRNA supports protein synthesis linearly over a 60-min incubation period, protein synthesis directed by glioma cell poly(A) RNA levels off after 30 min. Addition of exogenous amino acids, ATP, or GTP has no effect on the kinetics of protein synthesis by glioma cell poly(A) RNA.Optimal concentrationsof Mg2+and

A

E, o

14 L 0

‘,L?L 10

20 Distance

30

40

50

60

(mm)

FIG. 10. Isoelectrofocusing of immunoprecipitated lactate dehydrogenase. 0, Induced cells; 0, noninduced cells. A , lactate dehydrogenase was induced with isoproterenol + MIX for 12 h and

pulse-labeled with C3H]leucine under the conditions described in the legend of Fig. 4. Immunoprecipitated 3H-labeled lactate dehydrogenase was subjected to isoelectrofocusing under denaturing conditions as described under “Experimental Procedures.” B , glioma cells were induced for 4 h by the addition of isoproterenol to a final concentra. RNA isolated from induced and noninduced tion of 10 p ~ Poly(A)’ for RNA and Characterization Of the cells was translated, and lactate dehydrogenase was immunoprecipiReticulocyte Lysate Translation Sy.qtem-Severa1 methods tated. The immunoprecipitated [”SImethionine-labeled lactate deare available for theisolation of active poly(A)+ RNA fractions hydrogenase was subjected to isoelectrofocusing under denaturing from whole cells or cell homogenates. In devising an experi- conditions. For details, see “Experimental Procedures.”


11119

K' were determined as 1 mM Mg'+ and 0.16 M K'. Additionat varying concenally, the response of the translation system trations of poly(A) RNA from noninduced cells is shown in Fig. 8B.The total trichloroacetic acid-precipitable and immunoprecipitable radioactivity is linear up to 20 p g / d of poIy(A)+ RNA. Poly(A) RNA from induced cells gives similar results (not shown). Specificity of Assay for Lactate Dehydrogenase and Its Subunits Synthesized in the Reticulocyte Lysate SystemWhen poly(A)-containing RNA is translated in reticulocyte lysates, the lactatedehydrogenase subunits which are synthesized can be isolated byimmunoprecipitation with rabbit antirat lactate dehydrogenase-5 antiserum. Analysis of the immunoprecipitate fromnoninduced cells onSDS-polyacrylamide gels reveals the presence of a single major peak (of an apparent molecular weight of 35,000) of radioactivity (Fig.9 B ) which co-migrates with the M subunit of rat liver lactate dehydrogenase-5 isozyme (not shown). WhenC6 glioma cells are induced with isoproterenol under conditions that resultin increased synthesis of lactate dehydrogenase, translation of the poly(A)' RNA results in a marked increase of translated lactate dehydrogenase (Fig. 9 A ) . The absenceof radioactivity in the lactate dehydrogenase peak when poly(A)-containing RNA is deleted from the translation assay(Fig. 9C) illustrates the low background observed with the system. Since the antiserum used is specific for the M subunit, it will react with any lactate dehydrogenase isozyme containing 12 8 16 2 4 the M subunit (isozymes-2, -3, -4, and - 5 ) ,resulting in the co"Ours after isoproterenol Addition precipitation of the H subunit. Since SDS-polyacrylamidegel FIG. 11. Time course of induction of mRNA levels for lactate H and M subunits, electrophoresisdoes notseparatethe application of thisseparationmethod doesnot indicate dehydrogenase. Glioma cells were induced by the addition of isowhether our quantitation method detects only M type subunit. proterenol to a final concentration of 10 ELM.At the indicated times, cells were harvested and poly(A)' RNA was isolated, translated, and To evaluate this, the immunoprecipitate was subjected to lactate dehydrogenase was immunoprecipitated as described under isoelectrofocusing underdenaturing conditionsin order to "Experimental Procedures." A, induced glioma cells; A, noninduced separate the two subunits. Analysis of immunoprecipitated cells. The results are representative of those obtained in three other lactatedehydrogenaseprepared by translation of poly(A) identical experiments. RNA from induced and noninduced glioma cells reveals only one major peak of radioactivity at PI8.6 (Fig. 10B) co-migrating with the M subunit. Noradioactivity is detected at PI 5.9, corresponding to theposition of the H subunit (for comparison see Fig. 1OA).This observation indicates that the immunoprecipitable translation product is identical with the M subunit of lactate dehydrogenase and that no appreciable precipitation conditions. of the H subunit occurs under the experimental Induction of Lactate Dehydrogenase M Subunit mRNATo determine the levels of functional mRNA for lactate dehydrogenase M subunit during the period of lactate dehydrogenase induction, poly(A)' RNA was isolated from glioma cells at various times after isoproterenol stimulation. The template activity of the poly(A)+ RNA preparation was then assayed by its capacity to direct total trichloroacetic acidprecipitable protein and lactatedehydrogenase synthesis. The effect of isoproterenol on the functional levels of lactate dehydrogenase mRNA asa function of time is shown in Fig. ll. The rate of lactate dehydrogenase synthesis is presented as the ratioof [""S]methionine incorporated into immunoprecipitable lactate dehydrogenase to ["'S]methionine incorporated into total trichloroacetic acid-precipitable protein. This presentation of data eliminates error due to random variation in 0 LL I I I 0 the overall translation efficiency. The dataof Fig. 11 illustrate 10-8 10" 10-6 10~' a marked rapid increase of the lactatedehydrogenase mRNA Isoproterenol I \II activity after isoproterenol stimulation. The activity peaks8 FIG. 12. Dose response of isoproterenol on the induction of h after stimulation and thereafter declines rapidly to near control levels at 16 h. At the time of maximum induction (8 mRNA levels for lactate dehydrogenase. Glioma cells were induced with isoproterenol for 4 h. Isolation of poly(A)' RNA, transh), lactate dehydrogenase mRNA activity approaches 1%of lation, anddetermination of immunoprecipitated ["S]methioninethe total expressed template activity of the poly(A) RNA labeled lactate dehydrogenase were carried out under the conditions fraction (as measured by the [''%]methionine incorporation described in the legend of Fig. 1.


11120

TABLE V Effect of isoproterenol, dibutyryl CAMP, and sodium butyrate on induction of mRNA levels for lactate dehydrogenase Glioma cells were stimulated for6 h with the listed agents. Isolation of poly(A)' RNA, translation, and determination of immunoprecipitated [:'iS]methionine-labeled lactate dehydrogenasewere carried out under the conditions describedin the legend of Fig. 11.

follows a transient rise of the specific rate of lactate dehydrogenase synthesis (Fig. 4) with an earlysignificantincrease detected by 3 h and with a maximalrate of synthesis occurring by 12 h after the initial induction stimulus. The time course of lactate dehydrogenase synthesis is preceded by increased levels of lactate dehydrogenase mRNA activity (Fig. 11). A significantly elevated level of lactate dehydrogenase mRNA dpm in immunopreis observed as early as2 h after addition of isoproterenol. cipitated lactate dehydrogenase/dpm Since theincreased radioactive labelingof lactate dehydroAddition in trichloroacetic genase may not only be caused by an increased rate of synacid-precipitated protein X IO-.' thesis but by a decrease of the rate of degradation of the _ _ ~ enzyme, we have determined the half-lives of the enzyme None 0.852 Sodium butyrate (2 X 10 .' M ) 0.860 during the induction and deinduction phases. The similarity Isoproterenol (10 M ) 3.76 of the half-lives during induction and deinduction confirms Dibutyryl CAMP ( l o - ' M ) 3.63 that there is no alterationin the rateof degradation of lactate dehydrogenase, and that, therefore, induction of lactate deinto total trichloroacetic acid-precipitable protein). The sig- hydrogenase is caused by an alterationof the rateof synthesis nificance of the induction time course is strongly emphasized of new enzyme molecules. by the relatively constant level of lactatedehydrogenase Kumar et al. (16) using the 2B subclone of the original C6 mRNA activity identified in the noninduced control cells. As glioma cell line have recently reported the norepinephrineindicated by the incorporation of ["S]methionine into total induced synthesis of lactate dehydrogenase. The rate of enprotein, the overall template activity is identical in noninzyme synthesis observed by them was maximalat 9 hand had duced and induced poly(A)+ RNAs (data not shown). returned to basal levels by 24 h. The half-life for decay of ,'HThe dependency of the lactate dehydrogenase mRNA in- labeled lactate dehydrogenasewas determined to be 41 h. duction on the concentrationof isoproterenol is illustrated in Although their reported time course is similar, but not idenFig. 12. Maximal levels of mRNA activity are observed a t lo-' tical, with our reported time course of induction, there is a M isoproterenol and higher concentrations. Dibutyryl cAMP considerable difference between theirreported half-life of at 10"' M is equally effective as isoproterenol in increasingthe lactate dehydrogenase of 41 h inthe 2B subclone and thehalflactate dehydrogenase mRNA activity. Sodium butyrate is life of about 7 h determined by us in the original C6 glioma ineffective as inducing agent (Table V). cell line. Furthermore, Kumar et al. (16) reported the induction of the H subunit after norepinephrine stimulationof the DISCUSSION 2B subclone. In our studies, we were consistently unable to The induction of several enzymes by effector agents that observe an isoproterenol-mediatedmodulation of the activity generate cAMP or by cAMP itself is well recognized (1-5). of lactate dehydrogenase-3 and -4 isozymes (isozyme-1 and -2 The molecular mechanism of cAMP action and the locus at activities are not detectablein the original C6 glioma cell line). which cAMP exerts its regulatory effect remain in most in- This together with our failure to detect increased ["Hlleucine stances hypothetical. Themechanism may comprise selected labeling of the H subunit (Table11) suggests that no induction of the H subunit occurs in the original C6 glioma cell line. actions of CAMP affecting transcriptionalortranslational events, altering post-transcriptional and -translationalmodi- The reasons for these discrepancies are not known but very fications, enzyme turnover, and other mechanisms affecting likely reside in the use of different clones of the C6 glioma cells and differing experimental conditions used during inducenzyme activity. It has beenour working hypothesis that CAMP, through activation of nuclear CAMP-dependent pro- tion and deinduction. Actinomycin D added together with the inducing agent teinkinase and phosphorylation of nuclearproteins, may effectively control the selective transcription of enzyme completely abolished the increase of the [3H]leucine incorporation into immunoprecipitable lactate dehydrogenase. This mRNA by an as yet undefined mechanism. Catecholamines, through CAMP, have been shown to increase the activity of finding suggests a relationship between inhibitionof poly(A)' lactate dehydrogenase in C6 glioma cells (13, 14). These cells, RNA synthesis and suppression of enzyme induction. Furtherefore, provide asuitable model in which to test the role of thermore, since the poly(A)-containing RNA isolated from CAMP during lactate dehydrogenase induction and to eluci- induced glioma cells exhibited increased template activity for date the molecular locus of cAMP action. Recently, Derda the synthesis of lactate dehydrogenase, one could conclude and Jungmann (17) and Kumar et al. (16) have shown that that the induction of lactate dehydrogenase is dependent on the synthesis of new mRNA. However, our results do not catecholamine induction of lactate dehydrogenase is due to a net increasein the an increase in the synthesis of new enzyme protein. In the allow a conclusion whetherornot present study, we have extended our previous findings and synthesis of lactate dehydrogenase mRNA, an activation of show that ( a )the de nouo synthesis of lactate dehydrogenase- preformed mRNA, orstabilization of mRNA accountsfor the 5 isozyme molecules is induced by isoproterenol as well as by increased mRNA activity. Other investigators, using different experimental systems, dibutyryl CAMP,providing a firm basis for the centralrole of cAMP in the induction process; (6) the induction of lactate have similarly been able to demonstrate a CAMP-mediated dehydrogenase-5 isozyme is dueto increased synthesis of modulation of the levels of specific mRNA activity (9-12). ( c ) the Unlike CAMP-modulated enzyme inductionin prokaryotes enzyme molecules rather than altered degradation; induction of lactate dehydrogenase synthesis by isoproterenol which is well characterized (33), regulation by cAMP of proor dibutyryl cAMP is blocked by actinomycin D suggesting, tein synthesis at the transcriptional level in eukaryotes has but not proving, a transcriptional induction mechanism; and been postulated to involve activation of nuclear CAMP-de( d ) isoproterenol as well as dibutyryl cAMP increases the pendent protein kinase and phosphorylative modification of levels of functional mRNA for the glioma cell lactate dehy- nuclear regulatory proteins which participate in the controlof genetic expression (34, 35). Recent studies in our laboratory drogenase M subunit. The temporal course of induction of lactate dehydrogenase have demonstrated a CAMP-mediated activation of nuclear ~~~~

~

I'

" "

~~

Cyclic AMP Induction of Lactate Dehydrogenase CAMP-dependent protein kinase and phosphorylative modification of several histonesin isoproterenol-induced C6 glioma cells (37). Whether or not these events are part of a sequence of molecular events controlling the inductionof lactate dehydrogenase M subunit mRNA remains tobe established. This possibility is presently being investigated using hybridization analysis of nuclear glioma cell transcripts with lactate dehydrogenase cDNAand furtheranalysis of nuclear protein phosphorylation. REFERENCES 1. Wicks, W. D. (1974)Adu. Cyclic Nucleotide Res. 4, 335-438 2. Rosenfeld, M. G., and Barrieux, A. (1979) Adu. Cyclic Nucleotide Res. 11,206-264 3. Lamartiniere, C. A., and Feigelson, M. (1977) J. Biol. Chem.252, 3234-3239 4. Suleiman, A. S., and Vestling, C. S. (1979) J. Biol. Chem. 254, 10621-10628 5. Prahshad, N., and Rosenberg, R. N. (1978) Biochim. Biophys. Acta 539,459-469 6. Wicks, W. D., and McKibbin, J. B. (1972) Biochim. Biophys. Res. Commun. 48, 205-21 1 7. Wicks, W. D., Van Wijk, R., and McKibbin, J . B.(1973) Adu. Enzyme Regul. 11, 117-135 8. Van de Poll, K. W., van Aken, J. M., and van Wijk, R. (1979) Cell Biol. Znt. Reports 3, 247-255 9. Noguchi, T., Diesterhaf, M., andGranner, D. (1978) J . Biol. Chern. 253, 1332-1335 10. Ernest, M. J., and Feigelson, P. (1978) J. Biol. Chem. 253, 319322 11. Iynedjian, P. B., and Hanson, R. W. (1977) J . Biol. Chem. 252, 655-662 12. Brown, P. C., and Papaconstantinou, J. (1979)J. Biol. Chem.254, 9379-9384 13. de Vellis, J.,and Brooker, G. (1974)Science 186, 1221-1223 14. de Vellis, J., and Brooker, G . (1973) in TissueCulture of the Nervous System (Sato, G., ed), pp. 231-245, Plenum Press, New York ~

11121

15. Bottenstein, J. E., and de Vellis, J. (1978)Life Sci. 23, 821-834 McCinnis, J . F., and de Vellis, J. (1980) J. Riol. Chem. 16. Kumar. S., 255, 2315-2321 17. Derda, D. F., and Jungmann, R. A. (1979) Fcd. Proc. 38,230 18. de Vellis, J., Schjeide, 0. A,, and Clemente, C. P. (1967) J . Neurochem. 14,499-511 19. Schweitzer, E. S., Farron, F., and Knox, W. E. (1972) Enzyme 14, 173-184 20. Elevitch, F. R. (1973)Fluorometric Techniquesin Clinical Chemistry, Little Brown & Co., Boston, Mass. 21. Cox, R. A. (1967) Methods Enzymol. 12B, 120-129 22. Strohman, R. C.,Moss, P. S., Mieon-Eastman, J.,Spector, D., Przybjla, A., and Paterson, B. (1977) Cell 10, 265-273 23. Burton, K. (1956) Biochem. J. 62,315-323 24. Bantle, J. A., Maxwell, I. H., and Hahn, W. E. (1976) Anal. Biochem. 72,413-427 25. Palmiter, R. D. (1973)J. Biol. Chem. 248, 2095-2106 26. Pelham, H. R.R., and Jackson, R.