Sujata Tewari

ALTERATIONS IN BRAIN RNA METABOLISM FOLLOWING CHRONIC ETHANOL INGESTION

The in vivo labelling of various brain RNA fractions was found to be altered due to chronic ethanol ingestion by C57BL/6J mice. Ingestion of ethanol resulted in a marked decrease in the incorporation of intraventricularly injected [5‐3H] orotic acid into tRNA, rRNA and polyribosomal RNA. The inhibition was more pronounced in the polyribosomal fraction than the ribosomal fraction. In the nucleus, a biphasic effect of ethanol was demonstrated in the nRNA fraction where increased incorporation of precursor label at early time points was followed by marked depression. Absence of a similar increase in the cytoplasmic RNA fractions is suggestive of a possible defect in the transport of RNA from nucleus to cytoplasm resulting in the accumulation of RNA in the nucleus. Ethanol ingestion had no effect on the synthesis of acid soluble nucleotides following the in vivo administration of [5‐3H]orotic acid or [5‐3H]uridine into the brain. Over 90 per cent of the radioactivity in either ‘control’ or ‘ethanol’ brain could be recovered in the UMP fraction with negligible conversion to GMP, AMP or CMP. The results suggest that the observed changes in RNA metabolism following chronic ethanol ingestion are due to an alteration in the transcription and/or the processing of RNA in the nucleus rather than a function of reduced availability of nucleotides.

EFFECTS OF CHRONIC ETHANOL INGESTION ON BRAIN AMINOACYL‐tRNA SYNTHETASES AND tRNA

The effects of chronic ethanol ingestion on the in vivo aminoacylation of brain transfer RNA (tRNA) were examined in C57BL/6J mice. A pronounced inhibition in the formation of [14C]leucy]‐tRNA and [14C]phenylalanyl‐tRNA was observed in the ethanol drinking mice. Properties of aminoacyl‐tRNA synthetases and tRNA were examined following their separation and isolation on a DEAE‐cellulose column. Synthesis of [14C]leucyl‐tRNA was found to have a complete dependence on ATP and Mg2+. Incubations were carried out by cross‐matching tRNA from control rat brain with synthetases obtained from the brains of control or ethanol‐drinking mice. Under these conditions, a decreased ability for aminoacylation could be demonstrated when the source of enzyme was derived from ethanol‐treated brain. The data indicate that the major effect of ethanol ingestion on the aminoacylation reaction is exerted on aminoacyl‐tRNA synthetases.

Alterations in cell free brain protein synthesis following ethanol withdrawal in physically dependent rats.

The effects of ethanol withdrawal were determined on cell free brain protein synthesis in physically dependent rats. Following the development of physical dependence, ethanol abstinence for 24 h resulted in decreased protein synthesis in cerebral tissue. The observed inhibition of [14C]leucine incorporation into protein was found to be reversible after 7 days of ethanol withdrawal. Although the ribosomes from control, ethanol-treated and ethanol-withdrawn animals were highly responsive to polyuridylic acid stimulation, the ribosomes from the control group consistently exhibited higher activity. The determination of protein content of the ribosomal fraction showed a significant increase following ethanol administration and was further enhanced by ethanol abstinence. The results suggest that ethanol-induced changes at the ribosomal level may result in defective association of mRNA causing depression of brain protein synthesis.

Ethanol-induced conformational changes in rat brain microsomal membranes

Abstract The conformation of membrane-bound proteins was monitored by the reaction of sulfhydryl (SH) groups with N -ethyl maleimide (NEM) and 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB). Brain microsomes isolated from rats chronically imbibing a 10% (v/v) ethanol solution were found to contain 12–16 per cent more DTNB-reactive SH groups than similarly isolated controls. [ 3 H]NEM reacted with 32 per cent more SH groups in the microsomes obtained from ethanol-treated rats than controls. Since no change in the protein composition was evident from disc electrophoresis, and no significant differences in the total number of SH groups was present between the two samples, these findings suggest a conformational change in the microsomal membrane. Microsomes isolated from ethanol-naive animals, when treated with ethanol in vitro , showed a dose-dependent decrease in DTNB-reactive SH groups. The results indicate that the neural membrane may exist in different morphological states during acute and chronic exposure to ethanol.

Ethanol-induced changes in properties of rat brain ribosomes

Altered in vivo and in vitro brain protein metabolism have been demonstrated in rodents following long-term ethanol ingestion. In the present study, ethanol effects were examined on properties of brain ribosomes of male Sprague-Dawley rats ingesting a specially formulated Lieber-DeCarli liquid diet. The development of physical dependence was demonstrated by the presence of withdrawal reactions within 24 hr of ethanol abstinence. Data showed significant inhibition of in vitro protein synthesis by ribosomes from the “ethanol” and “1-day-withdrawn” groups. Partial reversal of inhibition occurred by using a control brain pH 5 enzymes source instead of the matched source. The observed [14C]leucine-incorporating activity was temperature dependent, with the optimum temperature being 37°C. The determination of the state of ribosomal aggregation showed an increased monosomes-disomes ratio in the “ethanol” group. The ratio was even more increased in the “1-day-withdrawn” group. Data suggest that reduced ribosomal binding to stable mRNA may be a contributing factor in the ethanol-induced effects on protein synthesis.

Chronic Ethanol Ingestion by Rodents: Effects on Brain RNA

Publisher Summary This chapter discusses the effects of chronic ethanol ingestion on the brain of rodents. In brain tissue, besides their role in cellular metabolism, proteins and ribonucleic acids (RNA) are thought to have distinctive functions. There is strong evidence that mnemonic processes are clearly impaired as a function of alcohol ingestion, though the precise nature of this effect still requires further elucidation. Animal studies have also demonstrated impairment in brain functions following chronic ethanol ingestion. In a study described in the chapter, adverse effects of chronic ethanolism were observed in the in vivo labeling of transfer RNA, mitochondrial RNA, and ribosomal RNA. Inhibition of mitochondrial RNA as a result of ethanol is of great interest as most of the intramitochondrial RNA is not nuclear in origin and, therefore, the effects observed on this organelle are independent of the effects demonstrated on the nuclear fraction. Long-term ethanol ingestion was shown to have a differential effect on the labeling of rRNA in two different populations of ribosomes. Labeling of free rRNA was found to be decreased under conditions where labeling of bound rRNA was increased.

Neurophysiological changes in the in vitro rat hippocampus following chronic lithium administration

The effect of chronic lithium exposure on the electrophysiological responses of the Schaffer collateral-commissural (SCC) input to the CA1 pyramidal neurons in the in vitro hippocampus was investigated. Experimental animals were intubated intragastrically with lithium carbonate (150 mg/kg) for 3-4 weeks. This treatment produced lithium levels in serum and hippocampus of 0.3-1.3 mM. During the recording period, the hippocampal slice retained a stable lithium concentration of 53% of the initial value. Chronic lithium exposure had a depressive effect on input/output relationships, paired-pulse facilitation and strength of orthodromic inhibition. The antidromic inhibition was virtually intact. No obvious differences were found between control and lithium slices in amplitude, latency or waveforms, of synaptic and antidromic extracellular potentials. These findings are compatible with a major action of lithium on the excitability of SCC axons and synaptic terminals.

Translational regulation in growing clonal human astrocytoma cells in culture

The in vivo and in vitro protein synthesis by polysomes prepared from Cox astrocytoma cells grown in the presence of 100 mM ethanol were examined during transition from exponential to stationary growth phase. A sharp decline of translational activities of Cox poly (A)+messenger RNAs (mRNAs) occurred during this transition. This decline was accentuated when cells were grown in the presence of ethanol. The observed decline in mRNA translational activity was investigated in vitro in a micrococcal nuclease treated, mRNA depleted postmitochondrial supernatant (PMS) fraction containing [35S]methionine. The formation of the35S-labeled 40S ternary complex in the absence of mRNA and of the35S-labeled 80S initiation complex in the presence of Cox or brain poly (A)+mRNAs were reduced substantially when the source of PMS was from stationary phase or ethanol exposed cells. The sedimentation of peaks containing 40S ternary and 80S initiation complexes following sucrose density gradient analysis showed marked reductions in [35S] methionine labeling during the transition to stationary phase and also following ethanol exposure. The reduced formation of initiation complexes suggests possible functional modifications of eukaryotic initiation factor-2 (eIF-2) present in the PMS fraction and of mRNAs under these conditions. Data suggest that cells initiate adaptive or protective mechanisms by reducing the rate of the initiation reaction following environmental alterations produced by ethanol.

Prenatal Ethanol Studies on Astrocytes

Children born with fetal alcohol syndrome (FAS) or fetal alcohol effects (FAE) exhibit pre and postnatal growth deficiencies with specific damages to the central nervous system (CNS) (27, 43, 44). Clinical and experimental studies show microcephaly, mental retardation and specific biochemical and morphological alterations in cerebral tissue following perinatal alcohol exposure (11, 21, 23, 37, 38). Inhibition of [3H]-thymidine uptake/incorporation into DNA in fetal rat cells and tissues by ethanol (12), or inhibition of protein synthesis in fetal and neonatal brain by prenatal and postnatal alcohol exposure have been reported (47, 48). Significant deficits were observed in the dendritic structure of the hippocampus of 8 to 10 weeks-old adult offspring exposed in uteroto ethanol (2). Furthermore, alterations in hippocampal CAl pyramidal neurons in mice exposed perinatally to alcohol (9, 10) and high sensitivity of hippocampal messenger RNA (mRNA) among other available mRNA species to in utero ethanol exposure (51) have been demonstrated.

Regulation Of Translation In Cox Astrocytoma Cells Grown In The Presence of Ethanol

The use of tow concentrations of fetal calf serum in cultures of di f ferent ages of C 1300 mouse neuroblastoma, clone neuro-2a, has produced morphological developmental alterations which included presynaptic elements in the form of varicosities or terminal swellings. The lat ter ware f i l led with round to oval, clear vesicles, the size ranging from 40-200 nm. The use of a defined medium did not in any way affect the morphological development of these cells. Addition of ~-aminobutyr ic acid (GABA) or sod_~m brorr~ide (NaBr) to the culture medium at concentrations ranging from 10 t o 10-~M over short periods of t ime (2 days), produced increased plast ici ty of the neurites, accompanied by the presence of immature synapticl ike junctions. Application of a mixture of bovine brain gangliosides consisting of 27%GMI, 40%GDla, 16% GDIb and 19% GT to GABA or NaBrcondit ionedcells, induced further morphological di f ferent iat ion which was accompanied by mature synapse-like contacts. No such contacts could be detected when the GMI fract ion was removed from the ganglioside mixture. Time-lapse cinematography revealed that gangliosides contributed elaborate contacts of long duration to the stable system. With the support of German Science Foundation Grant Sp237/2-1 .