Diminution of cerebral protein synthesis diminished the cerebral responses of mice to some neuroactive drugs, while an increase in synthesis increased the responses. Protein synthesis in whole brains (tested in vitro ) was diminished by giving living mice different inhibitors by different routes. The inhibitors tested (chloramphenicol, cycloheximide, and puromycin) diminished the behavioral responses of the mice to levodopa without affecting either its cerebral uptake or its conversion to dopamine. A diminution of the reactions of dopaminergic receptors was suggested by the diminished responses to the dopaminergic drug, apomorphine, while participation of cholinergic ones was suggested by experiments with oxotremorine. Proof that receptors had been specifically involved was secured on homogenized caudate nuclei from chloramphenicol-treated mice, in which the dopamine-activated production of cyclic AMP was markedly diminished. A stimulator of cerebral protein synthesis, the artificial double-stranded RNA, poly(I)·poly(C), increased the behavioral responses to these three drugs while it increased the dopamine-activated production of cyclic AMP. Since all these experimental increases or decreases in the responses to drugs required the lapse of only a few hours, proteins with rapid turnover rates must be critical in the activation of several kinds of cerebral receptors.

Cells dissociated with 0.25% crude trypsin from the muscle tissue of 9-day-old chick embryos were employed to investigate the effect of puromycin on cellular metabolism. Parallel studies were also made, using the gyratory shaker, to confirm the effectiveness of puromycin in inhibiting cell aggregation and protein synthesis. Puromycin when introduced at a concentration of 10µg/ml into a suspension of cells in Eagle's MEM did not completely inhibit cell aggregation. Small aggregates were formed in the first 4 h of the experiment. Protein synthesis of the rotated cells, as measured by the incorporation of L-[α-14C]leucine into proteins, was arrested by 91.7% within 15 min of introducing puromycin into a cell suspension. The antibiotic retained its inhibitory effect on protein synthesis for the 24-h period of rotation. Puromycin inhibited the cellular oxygen uptake and carbon dioxide evolution of the rotated cells by 40% within 4 h of its introduction. However, treated cells were still respiring, though at a much reduced rate, at the end of the 24-h experimental period. The release of radioactive carbon dioxide by puromycin-treated cells was also inhibited by 40% at the 4-h stage but after 8 h no further 14CO2 was evolved. The presence of the antibiotic markedly inhibited the uptake of glucose by trypsin-dissociated cells. The level of glycogen and lactate in cells suspended in Eagle's MEM was reduced very considerably over a 24-h period. The presence of puromycin accelerated glycogen utilization over the first 6 h of rotation but at 24 h there was a difference of only 0.6% between the glycogen content of treated cells and controls. At 24 h 11.3% less lactate remained in the puromycin-treated cells than in the controls. The ATP/ADP ratio of trypsin-dissociated cells decreased from an initial value of 2.59 to 1.45 after rotation for 24 h. In the presence of puromycin the ATP/ADP ratio was 0.62 at 4 h and had further declined to 0.48 by 24 h. The effects of puromycin on the aggregation, protein synthesis and cellular metabolism of trypsin-dissociated cells are discussed in relation to cellular adhesive mechanisms.

Trypsin-dissociated cells from the muscle tissue of 9-day-old chick embryos were employed to investigate the effects of cycloheximide and a puromycin-cycloheximide mixture on cell aggregation, protein synthesis and respiratory metabolism. Cycloheximide when introduced at a concentration of 10 µg/ml into a suspension of cells in Eagle's MEM inhibited aggregation by 25% at 24 h. At this time an inhibition of 40% was apparent in the presence of a mixture of cycloheximide and puromycin both at a concentration of 10 µg/ml. Both cycloheximide and the cycloheximide-puromycin mixture arrested protein synthesis of rotated cells by 90% within 15 min of introducing the antibiotics into cell suspensions. The antibiotics retained their inhibitory effects on protein synthesis for the 24-h period of rotation. Cycloheximide inhibited cellular oxygen uptake and carbon dioxide evolution of rotated cells by 25% at the end of the 24-h experimental period. At this time an inhibition of 30% was observed in the presence of the cycloheximide-puromycin mixture. The release of radioactive carbon dioxide by cycloheximide-treated cells was inhibited by 46% at 24 h. In the presence of the antibiotic mixture, 14CO2 release was inhibited by 30% at 4 h, but after 8 h very little further 14CO2 was evolved. As a control, puromycin (10 µg/ml) inhibited cell aggregation and respiration to an extent similar to that previously reported. The results are discussed in terms of puromycyl peptides producing a metabolic effect on cell aggregation. It is considered that this is additional to the effect of puromycin inhibiting aggregation through the arrest of protein synthesis.

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