Protein synthesis in pulmonary alveolar macrophages. Source of amino acids for leucyl-tRNA.

Extracellular, intracellular and tRNA-bound leucine pools of the adherent pulmonary alveolar macrophage were examined to determine the relationships between them and the precursor for protein synthesis. When cells were cultured in media of various leucine concentrations, the patterns of isotope distribution in intracellular and extracellular leucine did not correlate with the patterns seen in protein-bound leucine. hence, the free leucine pools cannot be used reliably as precursors for calculating rates of protein synthesis. tRNA-bound leucine, however, behaved isotopically as if it were the precursor. Constant synthetic rates were calculated using the tRNA specific activity over a wide range of leucine concentrations. In addition, by measuring the tRNA-bound specific activities of three different amino acids, leucine, valine and phenylalanine, and their respective specific activities in protein, we were able to calculate independently three separate but identical synthetic rates. At physiological amino acid concentrations, the macrophage intracellular leucine pool and the tRNA-bound leucine pool received less than half of their amino acids from extracellular sources. At 5 mM external leucine, the intracellular specific activity was indistinguishable from that of the medium leucine, but the specific activity of the tRNA-bound leucine pool remained only about 50% that of the extracellular value. The most straightforward interpretation of why the tRNA-bound leucine did not flood with external label under conditions where the intracellular pool has reached equilibrium is to propose that some portion of the leucine for protein synthesis is derived directly from protein turnover before the degradation products have mixed with the common amino acid pool.

Effects of kaolinite on amino acid transport and incorporation into protein by rabbit pulmonary alveolar macrophages.

A study was made of the effects of kaolinite, an aluminum silicate found in cigarette smoke and in alveolar macrophages of cigarette smokers, on the in vitro function of rabbit alveolar macrophages. Macrophages lavaged by standard procedures were incubated as adherent monolayers in the presence or absence of kaolinite, and amino acid incorporation into protein and transport subsequently measured. In the presence of dialyzed serum, kaolinite slightly inhibited incorporation into protein during the first 2 to 3 hr of incubation, after which incorporation ceased and a large percentage of newly synthesized protein was released (50% effect at approximately 0.5 mg/ml kaolinite). A dual-isotope experiment indicated that any change in the synthesis of protein which may have occurred was not selective for any protein or group of proteins. Kaolinite also stimulated noncompetitive amino acid accumulation after 2 to 3 hr in the presence of serum. The effects of kaolinite were immediate when incubations were conducted in the absence of serum. Control experiments showed all of the effects of the aluminum silicate to be on the cells and not on the incubation medium. These results suggest that kaolinite is cytotoxic and exerts its effects by a mechanism similar to that proposed for magnesium silicates and silica, in which the naked silicate is immediately cytotoxic, but if coated with serum protein must first be uncoated by lysozomal enzymes before destroying the cells.

Acute stress and the brain norepinephrine uptake mechanism in the rat.

The kinetic constants for norepinephrine uptake in cerebral cortical homogenates were determined in vitro immediately following an acute stress consisting of either forced immobilization, cold-wet exposure, combined cold-plus-restraint, swim stress, or electric footshock in the rat. The kinetic constants, apparent Km and Vmax, for uptake of 3H-l-norepinephrine were significantly increased only following 10 min swim at 22 degrees or following 5 min electric footshock. When severe hypothermia accompanied the stress, the findings suggested that a profound reduction in body temperature was associated with depressed responsiveness of brain noradrenergic mechanisms to stress including decreased uptake kinetic constants. In a series in which the duration of electric footshock was varied from 2 to 30 min, it was noted that the NE uptake kinetic constants were increased at 5 min, but were similar to paired controls at 2, 10 and 30 min following the onset of footshock. It was concluded that various acute stresses did not elicit a generalized response of the cortical NE uptake mechanism to stress in the rat. Furthermore, when uptake kinetic constants did change with stress, the values were often within the range of normal values seen in the rat.