Measurement of protein synthesis in rat lungs perfused in situ

Abstract

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs-Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6-690mum-[U-(14)C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O(2)/CO(2) (19:1) or O(2)/N(2)/CO(2) (4:15:1)]. [U-(14)C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t((1/2)), 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [(14)C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [(14)C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.

Reversible inhibition of protein synthesis in lung by halothane

“…Extracts of tissue or perfusate were neutralized with KOH, the soluble fraction was freeze-dried, and the residue was taken up in 0.05 M-lithium citrate buffer, pH 2.2, and analysed with a Beckman 119Cl amino acid analyser, as described by Rannels et al (1975). Radioactivity was determined by using a portion of the citrate-buffer sample and was assumed to be present only in phenylalanine, as shown by previous studies (Watkins & Rannels, 1980). This assumption was validated periodically.…”

“…The acid-insoluble fraction was washed for determination of the incorporation of radioactive amino acid into lung proteins as described previously Watkins & Rannels, 1980 (Watkins & Rannels, 1980) showed that, when extracellular phenylalanine was present at high concentration, the specific radioactivity of the amino acid in these pools rapidly reached a steady state and remained constant throughout at least 3 h of perfusion.…”

“…Because phenylalanine was not metabolized by perfused lungs, except in the pathways of protein synthesis and degradation (Watkins & Rannels, 1980), net changes in the concentration of the free amino acid reflected the balance between these pathways. In the presence of cycloheximide, net release of phenylalanine provided a direct index of the rate of proteolysis.…”

“…Unless otherwise stated, the perfusion buffer was a modified Krebs-Henseleit bicarbonate buffer containing 5.6 mM-glucose, 4.5% (w/v) bovine serum albumin (fraction V, Pentex; Miles Laboratories), 19 amino acids at concentrations measured in rat plasma (Watkins & Rannels, 1980) and radioactive phenylalanine (690#M). High concentrations of phenylalanine (10 times those found in plasma) were used to minimize dilution of the specific radioactivity of the amino acid with non-radioactive phenylalanine released from protein degradation.…”

“…High concentrations of phenylalanine (10 times those found in plasma) were used to minimize dilution of the specific radioactivity of the amino acid with non-radioactive phenylalanine released from protein degradation. Increasing extracellular phenylalanine to this concentration did not affect the rate of protein synthesis (Watkins & Rannels, 1980). In all experiments, the lungs were ventilated and the perfusate was equilibrated with warmed (370C) humidified 02/N2/CO2 (4:15: 1).…”