$39
Biochimica et Biophyslca Acts, 442 (1976) 289--250 © Elsevier Scientific Publishing Company, Amste~m~l -- Printed in The Netherlands
BBA 98667 MECHANISM OF D-AMPHETAMINE INHIBITION OF PROTEIN SYNTHESIS
B. SUREN BALIGA, JOSEF Z~tIRINGER, MITCHELL TRACHTENBERG, MICH.~L A. MOSKOWITZ and HAMISH N. M U N R O Laboratory o f Physiological Chem/stry, Dep~vtment o f NutrRicn ~nd Food Sc~aee, Massachusetts Institute o f Technology, Cambridge, Ma~. 02139 (U.S.A.)
(Received February 3rd, 1976)
Summary
At 1 h after intraperitoneal administration of D-mnphetamine sulphate (15 mg/kg), rat brain polyribosomes show disaggregation accompanied by reduced capacity for in vitro peptide chain elongation. The direct action of amphetamine on cell-freeprotein-synthesizing systems was therefore explored. W h e n brain or liverpolyribosomes from untreated ratswere incubated with p H 5 enzyme, peptide chain elongation was not inhibited by the addition of 4 m M amphetamine to the medium. O n the other hand, an initiation-dependent system consisting of rat liveror brain m R N A and wheat germ S-30 fraction show-ed inhibition of [~Hlleucine incorporation by 5 0 % when 4 m M amphet~nine were added. The metabolites of amphetamine, p-hydroxyamphetamine and phydroxynorephedrine, had no inhibitory action in either system, but the potent neurotoxin p-chloroamphetamine was a more powerfulinhibitor o f initiation than amphetamine. By using [~H]ampheta~ine, it was shown that amphetamine binds to the 80.S ribosornes of the wheat germ system. This binding depended on the presence in the system of natural liveror brain m R N A or several synthetic m R N A s , but was not promoted by polyufidylic acid as the messenger. Significantly,polyuridylic acid-dependent polyphenylalanine synthesis by the wheat germ system was not inhibited by amphetamine or p-chloroamphefamine. Therefore, it was concluded that amphetamine inhibitsprotein synthesis by interfering with initiation through a step related to formation of the m R N A ribosome complex. Introduction Amphetamine, a well-known sympathomimetic drug, acts on the central nervous system to induce locomotor activity [~.,2],hypo- and hyperthermh |3],
240
anorexia [4] and stereotypic movements [5]. These actions are due, in part, to the ability of amphetamine to stimulate catecholamine receptors indirectly by promoting release or by blocking synaptic uptake of endogenous catecholamine neurotransmitters [6]. Catecholamine release triggered by amphetamine can also affect cerebral glycogen metabolism [7] and may be an/mportant factor in the psychotic reaction that develops in human beings with long-term amphetamine use [8]. Recently, we reported that t.he administration of V-amphetamine sulfate (10 mg/kg) causes disaggregation of rat brain polysomes [9] and a parallel decrease in the rate of protein synthesis in the brain [10]. Pretreatment with drugs that block dopamine receptors prevents amphetamine-induced disaggregation of polysomes [9]. L-Dopa, a precursor of catecholamines, also induces disaggregation of brain polysomes, and brain dopamine receptors appear to mediate this phenomenon [11]. Since L-dopa inhibits brain protein synthesis in young rats more effectively than amphetamine, and since amphetamine, but not L-dopa, causes polysome diaggregation in liver in vivo (unpublished observations), amphetarnine may act on protein synthesis in more ways than those mediated through the dopamine mechan/sm. The direct action of amphetamine on protein synthesis at the translational level was therefore investigated. The action of amphetamine was studied in two cell-free systems. In the first system, polyribosomes bearing nascent chains were incubated in a cell-free system to elongate the preexisting peptide chains. The second system consisted of ribosomal subunits and soluble factors from wheat germ, the activity of which is dependent on added messenger RNA, and thus involves initiation followed by elongation. Our results demonstrate that Damphetamine inhibits protein synthesis only in the wheat germ system and thus indicate that D.amphetamine blocks initiation but not elongation. A preliminary report of this work has been presented [12]. Materials and Methods Male Sprague-Dawiey rats (Charles River Breeding Laboratories, Wilmington, MA), weighing 100--120 g, were used in all experiments. Fresh commercial wheat germ (Bar Ray Mill, Tel-Aviv, Israel) was obtained as a gLft from Dr. Bryan Roberts (M.I.T.). L-[4,5-SH]Leucine (spec. act. 50 Ci/mmol) and D-[G3H]amphetamine sulphate (spec. act. 2--10 Ci/mmol) were purchased from New England Nuclear, Boston, MA. The poly(U), poly(A), poly(AGU) and the trinucleotide codon AUG were supplied by Miles Laboratories, Elkhart, IN. D-Amphetamine sulphate and DL-parachloroamphetamine hydrochloride were obtained from Sigma Chemical Co., St. Louis, Mo., whilep-hydroxynorephedrine • HCI was obtained from the Aldrich Chemical Co., Milwaukee, WI. Parahydroxyamphetamme hydrobromide wa.*. generously supplied by Smith, Kline and French Laboratories, Philadelphia, PA. All other chemicals were reagent grade. Preparation and incubation of polysomes Brain polyribosomes were prepared as previously described [9]. Polysome profiles were analyzed on a linear sucrose gradient (10--40%), and the absorption profiles were recorded at 260 nm on a Gilford spectrophotometer [13].
241 Liver polyribosomes, pH 5 enzyme fractions, and elongation factors I and 2 were prepared as previously described [13] and sto~ed at --70°C before use. The incubation mixture (final volume, 50 ~l) contained 50 ~g polysomes, 25--50 ~g pH 5 enzyme protein, 50 mM TFis. HC1 (pH 7.6}, 5 mM MgCI2, 80 mM NH4C1, 2 mM ATP, 0.2 mM GTP, 2 mM dit~othreitol, 0A ~M L-[3H]leu cine Opec. act. 30--50 Ci/mmol), and 20 ~M ambm acid mixture minus leucine [13]. After incubation for 60 rnin at 37°C, the reaction was terminated by ~ e addition of an equal amount of 10% tfichloroacetic acid and increased to I ml with 5% trichloroacetic acid. It was then heated for I5 rain at 90~C. The f~etion that was insoluble in hot trichloroacetie acid was filtered on Whatrnan GF/A glass fiber paper, washed with 5% cold tr£chlomaeefic acid, and dried. Radioactivity was measured after the addition of 10 ml of toluenelPPO]POFOP in a Nuclear Chicago Liquid Scintillation counter. Incorporation of [~C]ph_enylalanine into the liver polysome system under the d~ection of poly(U} was performed as described previously [14]. Extraction of totaland poly(A )-containingm R N A Total R N A was extracted from eithermicrosomes or polysomes by the dodecyl sulphate/phenol method [15]. The R N A was isolatedby ethmlol precipRation and applied to an oligo(dT)-cellulosecolumn [16]. Poly(A}-~h R N A fraction~swere pooled and stored in small aliquotsat --70°C. Preparation and incubation of the wheat germ system The 30 000 × g supernatant fraction (S.30} of wheat germ was prepared as described by Roberts and Paterson [17], except that the S-30 fractionwas passed through a Sephadex G-25 column without preincubation.The incubation mixture (final volume, 25/~1) contained 28 mM HepeslKOH (pH 7.0), 8{) mM KCI, 3 mM magnesium acetate, 1 mM ATP, 0.2 mM GTP, 2 mM dRhiothreRol, 8 mM creatine phosphate, 8 ~g/ml creatine phosphgkinm~. 20~30 $~M ..~m__~o acid mixture minus leucine, 3.2 pM [SH[leucine (spec. act. 3 ~ 5 0 Ci]mmol}, 10 ~l wheat germ S-30, and 0.5--1 ~g mRNA. The reaction mLxture was incubated for 120 rain at 25°C. Incorporation of [3H|leucine into total protein was measured according to the method of Mans and Novelli [18]. Apart from increasing the concentrations of magnesium acetate (10 raM) and using [~C]phenylalanyl tRNA, poly(U)
Biochimica et Biophyslca Acts, 442 (1976) 289--250 © Elsevier Scientific Publishing Company, Amste~m~l -- Printed in The Netherlands
BBA 98667 MECHANISM OF D-AMPHETAMINE INHIBITION OF PROTEIN SYNTHESIS
B. SUREN BALIGA, JOSEF Z~tIRINGER, MITCHELL TRACHTENBERG, MICH.~L A. MOSKOWITZ and HAMISH N. M U N R O Laboratory o f Physiological Chem/stry, Dep~vtment o f NutrRicn ~nd Food Sc~aee, Massachusetts Institute o f Technology, Cambridge, Ma~. 02139 (U.S.A.)
(Received February 3rd, 1976)
Summary
At 1 h after intraperitoneal administration of D-mnphetamine sulphate (15 mg/kg), rat brain polyribosomes show disaggregation accompanied by reduced capacity for in vitro peptide chain elongation. The direct action of amphetamine on cell-freeprotein-synthesizing systems was therefore explored. W h e n brain or liverpolyribosomes from untreated ratswere incubated with p H 5 enzyme, peptide chain elongation was not inhibited by the addition of 4 m M amphetamine to the medium. O n the other hand, an initiation-dependent system consisting of rat liveror brain m R N A and wheat germ S-30 fraction show-ed inhibition of [~Hlleucine incorporation by 5 0 % when 4 m M amphet~nine were added. The metabolites of amphetamine, p-hydroxyamphetamine and phydroxynorephedrine, had no inhibitory action in either system, but the potent neurotoxin p-chloroamphetamine was a more powerfulinhibitor o f initiation than amphetamine. By using [~H]ampheta~ine, it was shown that amphetamine binds to the 80.S ribosornes of the wheat germ system. This binding depended on the presence in the system of natural liveror brain m R N A or several synthetic m R N A s , but was not promoted by polyufidylic acid as the messenger. Significantly,polyuridylic acid-dependent polyphenylalanine synthesis by the wheat germ system was not inhibited by amphetamine or p-chloroamphefamine. Therefore, it was concluded that amphetamine inhibitsprotein synthesis by interfering with initiation through a step related to formation of the m R N A ribosome complex. Introduction Amphetamine, a well-known sympathomimetic drug, acts on the central nervous system to induce locomotor activity [~.,2],hypo- and hyperthermh |3],
240
anorexia [4] and stereotypic movements [5]. These actions are due, in part, to the ability of amphetamine to stimulate catecholamine receptors indirectly by promoting release or by blocking synaptic uptake of endogenous catecholamine neurotransmitters [6]. Catecholamine release triggered by amphetamine can also affect cerebral glycogen metabolism [7] and may be an/mportant factor in the psychotic reaction that develops in human beings with long-term amphetamine use [8]. Recently, we reported that t.he administration of V-amphetamine sulfate (10 mg/kg) causes disaggregation of rat brain polysomes [9] and a parallel decrease in the rate of protein synthesis in the brain [10]. Pretreatment with drugs that block dopamine receptors prevents amphetamine-induced disaggregation of polysomes [9]. L-Dopa, a precursor of catecholamines, also induces disaggregation of brain polysomes, and brain dopamine receptors appear to mediate this phenomenon [11]. Since L-dopa inhibits brain protein synthesis in young rats more effectively than amphetamine, and since amphetamine, but not L-dopa, causes polysome diaggregation in liver in vivo (unpublished observations), amphetarnine may act on protein synthesis in more ways than those mediated through the dopamine mechan/sm. The direct action of amphetamine on protein synthesis at the translational level was therefore investigated. The action of amphetamine was studied in two cell-free systems. In the first system, polyribosomes bearing nascent chains were incubated in a cell-free system to elongate the preexisting peptide chains. The second system consisted of ribosomal subunits and soluble factors from wheat germ, the activity of which is dependent on added messenger RNA, and thus involves initiation followed by elongation. Our results demonstrate that Damphetamine inhibits protein synthesis only in the wheat germ system and thus indicate that D.amphetamine blocks initiation but not elongation. A preliminary report of this work has been presented [12]. Materials and Methods Male Sprague-Dawiey rats (Charles River Breeding Laboratories, Wilmington, MA), weighing 100--120 g, were used in all experiments. Fresh commercial wheat germ (Bar Ray Mill, Tel-Aviv, Israel) was obtained as a gLft from Dr. Bryan Roberts (M.I.T.). L-[4,5-SH]Leucine (spec. act. 50 Ci/mmol) and D-[G3H]amphetamine sulphate (spec. act. 2--10 Ci/mmol) were purchased from New England Nuclear, Boston, MA. The poly(U), poly(A), poly(AGU) and the trinucleotide codon AUG were supplied by Miles Laboratories, Elkhart, IN. D-Amphetamine sulphate and DL-parachloroamphetamine hydrochloride were obtained from Sigma Chemical Co., St. Louis, Mo., whilep-hydroxynorephedrine • HCI was obtained from the Aldrich Chemical Co., Milwaukee, WI. Parahydroxyamphetamme hydrobromide wa.*. generously supplied by Smith, Kline and French Laboratories, Philadelphia, PA. All other chemicals were reagent grade. Preparation and incubation of polysomes Brain polyribosomes were prepared as previously described [9]. Polysome profiles were analyzed on a linear sucrose gradient (10--40%), and the absorption profiles were recorded at 260 nm on a Gilford spectrophotometer [13].
241 Liver polyribosomes, pH 5 enzyme fractions, and elongation factors I and 2 were prepared as previously described [13] and sto~ed at --70°C before use. The incubation mixture (final volume, 50 ~l) contained 50 ~g polysomes, 25--50 ~g pH 5 enzyme protein, 50 mM TFis. HC1 (pH 7.6}, 5 mM MgCI2, 80 mM NH4C1, 2 mM ATP, 0.2 mM GTP, 2 mM dit~othreitol, 0A ~M L-[3H]leu cine Opec. act. 30--50 Ci/mmol), and 20 ~M ambm acid mixture minus leucine [13]. After incubation for 60 rnin at 37°C, the reaction was terminated by ~ e addition of an equal amount of 10% tfichloroacetic acid and increased to I ml with 5% trichloroacetic acid. It was then heated for I5 rain at 90~C. The f~etion that was insoluble in hot trichloroacetie acid was filtered on Whatrnan GF/A glass fiber paper, washed with 5% cold tr£chlomaeefic acid, and dried. Radioactivity was measured after the addition of 10 ml of toluenelPPO]POFOP in a Nuclear Chicago Liquid Scintillation counter. Incorporation of [~C]ph_enylalanine into the liver polysome system under the d~ection of poly(U} was performed as described previously [14]. Extraction of totaland poly(A )-containingm R N A Total R N A was extracted from eithermicrosomes or polysomes by the dodecyl sulphate/phenol method [15]. The R N A was isolatedby ethmlol precipRation and applied to an oligo(dT)-cellulosecolumn [16]. Poly(A}-~h R N A fraction~swere pooled and stored in small aliquotsat --70°C. Preparation and incubation of the wheat germ system The 30 000 × g supernatant fraction (S.30} of wheat germ was prepared as described by Roberts and Paterson [17], except that the S-30 fractionwas passed through a Sephadex G-25 column without preincubation.The incubation mixture (final volume, 25/~1) contained 28 mM HepeslKOH (pH 7.0), 8{) mM KCI, 3 mM magnesium acetate, 1 mM ATP, 0.2 mM GTP, 2 mM dRhiothreRol, 8 mM creatine phosphate, 8 ~g/ml creatine phosphgkinm~. 20~30 $~M ..~m__~o acid mixture minus leucine, 3.2 pM [SH[leucine (spec. act. 3 ~ 5 0 Ci]mmol}, 10 ~l wheat germ S-30, and 0.5--1 ~g mRNA. The reaction mLxture was incubated for 120 rain at 25°C. Incorporation of [3H|leucine into total protein was measured according to the method of Mans and Novelli [18]. Apart from increasing the concentrations of magnesium acetate (10 raM) and using [~C]phenylalanyl tRNA, poly(U)
