The uptake and fate of pinocytosed fluid were investigated in monolayers of pulmonary alveolar macrophages and fetal lung fibroblasts using the fluid-phase marker, [14 C]sucrose . Initial experiments revealed that cellular accumulation of chromatographically repurified [14 C]sucrose was not linear with incubation time . Deviation from linearity was shown to be due to constant exocytosis of accumulating marker . Chromatographic analysis revealed that the cells were unable to metabolize sucrose and were releasing it intact by a process that was temperature-sensitive but not dependent on extracellular calcium and magnesium . A detailed analysis of the kinetics of exocytosis was undertaken by preloading cells with [ 1 'C] sucrose for various lengths of time and then monitoring the appearance of radioactivity into isotope-free medium . Results indicated that modeling the process of fluidphase pinocytosis and subsequent exocytosis required at least two intracellular compartments in series, one compartment being of small size and turning over very rapidly (t 1/2 = 5 min in macrophages, 6-8 min in fibroblasts) and the other compartment being apparently larger in size and turning over very slowly (t 1/2 = 180 min in macrophages, 430-620 min in fibroblasts) . Computer-simulation based on this model confirmed that the kinetics of efflux faithfully reflected the kinetics of influx and that the rate of efflux completely accounted for the deviation from linearity of accumulation kinetics . Moreover, the sizes of the compartments and magnitude of the intercom partment fluxes were such that the majority of fluid internalized in pinocytic vesicles was rapidly returned to the extracellular space via exocytosis . This result provides direct experimental evidence for a process previously thought necessary based solely on morphological and theoretical considerations . Furthermore, the turnover of pinocytosed fluid was so dynamic that accumulation deviated from linearity even within the first few minutes of incubation . We were able to show that the kinetics of exocytosis allowed calculation of the actual pinocytic rate, a rate that was nearly 50% greater than the apparent initial rate obtained from the slope of the uptake curve over the first 10 min .Pinocytosis may represent the afferent arm of a homeostatic mechanism for recycling the plasmalemma . The foundation for this hypothesis was laid by the work of Steinman et al . (37) . Their stereological analysis of pinocytosis in macrophages and L-cells revealed that the volume and surface area of incoming pinocytic vesicles was ten times greater than that which the secondary lysosomal compartment was shown to accommodate and, therefore, they postulated : (a) that interiorized pinocytic vesicle fluid must rapidly egress from the vacuolar system and (b) that pinocytic vesicle membrane is recycled back to the cell surface . Recently, the latter prediction has received experimental support both in mammalian (25, 34) and nonmammalian (6, 42) cells . These studies provided evide...

The concept that a general intracellular pool serves as the sole precursor of amino acids for protein biosynthesis has been vigorously debated in recent years. To help resolve this controversy, we followed the distribution of intraperitoneally administered [(3)H]valine in the tRNA and the extracellular and intracellular compartments of rat liver. The specific radioactivity of the valine released from isolated tRNA was 2-3 times higher than that of intracellular valine, suggesting that the intracellular pool cannot be the sole precursor of amino acids used for charging tRNA. In addition, the specific radioactivity of the tRNA was only half that of the extracellular valine. Therefore it is unlikely that the valyl-tRNA is charged exclusively with amino acids derived from the extracellular pool. A model is proposed which stipulates that both extracellular and intracellular amino acids contribute to a restricted compartment that funnels amino acids towards protein biosynthesis.

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The effects of acute exposure to insulin on protein synthesis were examined in primary, differentiated cultures of embryonic chick heart and skeletal muscle cells. Synthetic rates were calculated using the specific activity of tRNA-bound leucine as precursor, a specific activity that was significantly less than that of extracellular leucine but greater than that of free, intracellular leucine at 0.2 mM external leucine. Insulin did not alter these relationships. Doses of insulin in the physiological range produced significant stimulation of protein synthesis in both cell types. Maximal responses, involving approximately 30% increases in both absolute and fractional rates, were observed at higher insulin concentrations. Significant stimulation by insulin was seen in cardiac cells after only 1 h of insulin treatment, and the effects of the hormone were observed both in the presence and absence of serum in the culture medium.

Knowledge of the dynamics of collagen turnover requires information regarding rates of synthesis of this group of connective-tissue proteins. The relationship of various amino acid pools to the tRNA precursor pool used for protein synthesis is known to vary between different cell types and tissues, even for essential amino acids. We studied extracellular, intracellular and tRNA-proline pools in cultured human lung IMR-90 fibroblasts to determine the relationship between them as candidate proline precursor pools for total protein and collagen synthesis. Time-course experiments showed that the three proline pools attained distinctly different steady-state specific radioactivities (extracellular greater than intracellular greater than tRNA) at the extracellular proline concentration of 0.2 mM. The kinetics of radioisotope incorporation into cell protein and collagenase-digestible protein indicated that the intracellular free proline pool could not be used reliably as a precursor for calculating synthetic rates. However, tRNA-proline behaved isotopically as if it were the precursor and provided synthesis rates 2-3-fold higher than those calculated by using either free proline pool. The incorporation of labelled lysine and leucine was constant over a wide range of extracellular proline concentrations. Fractional rates of protein synthesis based on tRNA-amino acid were the same with [3H]phenylalanine as with [3H]proline. The specific radioactivity of cell-associated hydroxyproline reached a steady-state value 8-10h after radioisotope administration which matched the mean tRNA-proline specific radioactivity, suggesting that tRNA-proline is not isotopically compartmentalized. A model of cellular proline-pool relationship is presented and discussed.