Dora B. Goldstein

Alcohol Dependence Produced in Mice by Inhalation of Ethanol: Grading the Withdrawal Reaction

Intoxicating blood levels of ethanol are maintained for several days in mice housed in an atmosphere of ethanol vapor. On removal from the alcohol, all the mice develop withdrawal signs. The signs can be graded to indicate the time course and intensity of the withdrawal reaction.

Increased cholesterol content of erythrocyte and brain membranes in ethanol-tolerant mice.

Mice were treated with ethanol for eight or nine days, using a liquid diet regimen known to produce physical dependence. In previous experiments, synaptosomal plasma membranes and erythrocyte ghosts from such ethanol-treated animals were found to be resistant to the fluidizing effects of ethanol in vitro, as measured by electron paramagnetic resonance. In the present experiments, corresponding membranes were analysed for phospholipid and cholesterol. The ratio of cholesterol to phospholipid was found to be significantly increased in both types of membrane after chronic ethanol treatment. The changed ratio was produced by an increase in cholesterol. There was little or no change in phospholipid content of the membranes. Increased cholesterol may explain the previously observed alteration of physical properties of the membranes.

A method for assay of catalase with the oxygen cathode.

Abstract Catalase can be assayed very simply with the oxygen cathode, by measuring the initial rate of oxygen release from a solution of hydrogen peroxide or sodium perborate. The substrate concentration is high enough, and the time short enough, to prevent decreasing rates during the reaction. The method is suitable for use with crude tissue preparations, as well as with purified enzyme. The initial reaction follows Michaelis-Menten kinetics. An apparent K m of 0.1 M for H 2 O 2 and 0.08 M for perborate was observed.

Protein synthesis at 0°C in Escherichia coli*

Leucine-starved cells of an Escherichia coli auxotroph will incorporate radioactive leucine into protein at 0°C. The incorporation is linear for the first 10 to 15 min. It appears that all the essential steps in protein synthesis go on at 0°C, and a wide variety of proteins is synthesized. The rate of synthesis is about 350 times slower than at 37°C. In this system the polarity of assembly of polypeptide chains, from N-terminal to C-terminal, can be demonstrated by treating the proteins with carboxy-peptidase. A quantitative analysis of the release of incorporated leucine by carboxypeptidase permits estimation of the average time required to add a single amino acid residue to a nascent protein chain at 0°C, whence the number of nascent chains can be derived. This number has also been estimated by direct measurement of nascent protein in sucrose density-gradient centrifugation. A cell contains about 5000 chains of nascent protein, and 18,000 70 s ribo-somes. The assembly time of a protein of 400 residues is 5 sec at 37°C.

The rapid onset of tolerance to ataxic effects of ethanol in mice.

We have developed a precise quantal method for assessing the sensitivity to ethanol in the mouse. Mice placed on a clamped stationary horizontal dowel are scored ataxi or not ataxic depending on whether they are able to remian on the dowel during a 30-s observation period. A threshold blood ethanol concentration is determined by assaying tail blood drawn immediately upon recovery from ethanol-induced ataxia. This threshold is quite reproducible within a population of Swiss-Webster mice (coefficient of variation 9%). The precision of this method allowed us to follow the onset of rapid tolerance during a series of sequential IP ethanol doses. Tolerance persisted overnight in the absence of ethanol, and was found not to increase further with additional ethanol exposure on 2 subsequent days. The observed tolerance was shown not to be due to circadian changes in ethanol sensitivity or repeated practice on the task, indicating a true tissue tolerance.

Alcohol withdrawal reactions and reserpine effects in inbred strains of mice

Abstract Mice of different inbred strains were treated with ethanol for 3 days, by inhalation of alcohol vapor and daily injections of pyrazole. Within strains, “alcohol-adapted” mice were compared with controls. The alcohol-adapted mice received 3.8% (w/v) alcohol in their drinking water for one week and 7.5% alcohol for the next 16 or 19 weeks. During the inhalation period, C57BL mice had lower blood alcohol levels than DBA mice, and alcohol-adapted mice had slightly lower blood levels than controls. On withdrawal the mice were examined repeatedly for convulsions elicited by handling, a measure of the intensity of withdrawal reactions. The withdrawal scores of C57BL mice were significantly lower than those of DBA, BALB or Swiss-Webster mice, more so than could be accounted for by the difference in blood alcohol levels. Mice of 3 strains were treated with reserpine and observed for behavioral effects, including convulsions on handling. Strain differences in reserpine effects closely paralleled the strain differences in alcohol withdrawal seizures.

d-AMINO ACID OXTDASE IN BRAIN: DISTRIBUTION IN SEVERAL SPECIES AND INHIBITION BY PENTOBARBITONE

A STUDY of barbiturate effects in Escherichiu coli (GOLDSTEIN, 1965) showed that alanine oxidation was inhibited by barbitone. L-Alanine oxidation in E. coli requires racemization, followed by oxidation of D-alanine. Barbitone inhibits the D-amino acid oxidase competitively with the substrate. If cells are grown for a few generations in a medium containing L-amino acids and barbitone, the levels of alanine racemase and D-amino acid oxidase are increased. Such cells oxidize alanine abnormally fast. This system represents a possible model for tolerance and the withdrawal syndrome in animals. It was therefore of interest to know whether similar enzymes exist in brain. Mammalian n-amino acid oxidase (I.U.B., 1.4.3.3) of kidney and liver has been known for some decades (KREBS, 1935). There have also been reports of the enzyme in brain (EDLBACHER and WISS, 1944; BURCH, LOWRY, PADILLA and COMBS, 1956; YAGI, NAGATSU and OZAWA, 1956; DUNN and PERKOFF, 1963). The recent development of a sensitive assay (CORRIGAN, WELLNER and MEISTER, 1963) allows further study of the brain enzyme, which is much less active than that of the kidney. This, paper shows that the enzyme is present in the brains of several vertebrate species, and that in mammals it is concentrated in the hindbrain. Pentobarbitone, in relatively high concentration, inhibits the enzyme competitively with substrate.

Effect of alcohol on cellular membranes

Ethanol disrupts the physical structure of cell membranes. The most fluid membranes, including those that are low in cholesterol, are the most easily disordered by ethanol. Although the membrane-disordering effect is small, there is pharmacological, temporal, and genetic evidence that it is important. Animals that are resistant to ethanol intoxication because of their genetic background or because of previous exposure to ethanol are found to have brain membranes that are not easily disordered in vitro. An exception is the increased behavioral sensitivity in aging animals, which is not matched by changes in their membranes. When animals are treated chronically with ethanol, their membranes become stiffer, a response that can be regarded as adaptive. Ethanol may favor the uptake of cholesterol or saturated fatty acids into membranes, thus reducing its own effect.

Alcohol withdrawal reactions in mouse strains selectively bred for long or short sleep times.

Abstract Two lines of mice that differ in their sensitivity to acute hypnotic effects of ethanol were tested for alcohol withdrawal reactions after a standard 3-day exposure to ethanol. The “long-sleep” strain showed a much milder withdrawal reaction than did the “short-sleep” strain. The two strains did not differ in sensitivity to convulsions elicited by pentylenetetrazol. Sleep times and withdrawal reactions did not correlate in individual mice of the genetically heterogeneous stock from which the two lines were derived.

Cholesterol blocks the disordering effects of ethanol in biomembranes

To assess the relation between the physical order of a membrane and its sensitivity to ethanol, we enriched biomembranes with cholesterol, both in vivo and in vitro. Japanese quail of the SEA line (selectively bred for susceptibility to experimental atherosclerosis) were treated for 9 to 16 weeks with a diet that contained 2% cholesterol. This regimen increased the cholesterol content of serum and erythrocytes. The cholesterol content of brain synaptosomal plasma membranes (SPM) was unaffected by the high cholesterol diet. In other experiments, isolated mouse synaptosomal plasma membranes were incubated with cholesterol/phospholipid (C/P) vesicles; different amounts of cholesterol were transferred according to the sterol content of the donor vesicles. Membrane order was determined in both types of membranes by a sensitive electron paramagnetic resonance (EPR) technique. The order parameter with 5- and 12-doxylstearic acid increased along with the cholesterol content. As expected, ethanol disordered membranes (decreased the order parameter) in a concentration-related manner. The slope of the concentration response curve was less steep in high cholesterol than low cholesterol membranes, indicating that cholesterol enrichment partially blocks the membrane action of ethanol in both types of membranes.