PHA stimulation of human lymphocytes during amino acid deprivation. Protein, RNA and DNA synthesis

Abstract

Resting lymphocytes are in the Go phase of the cell cycle. Upon activation by PHA, they progress into GI with accompanying increased protein and RNA synthesis, initiate DNA synthesis and divide. We have studied the kinetics of inhibition of macromolecular synthesis during activation in the absence of single amino acids. Three types of kinetics are observed. In the absence of tryptophan or isoleucine, stimulated lymphocytes show a normal increase in protein and RNA synthesis during the first 30 hours of stimulation, initiate DNA synthesis but are subsequently inhibited. In phenylalaninedeficient medium, no DNA synthesis occurs in spite of a slight increase in protein synthesis. No increase in macromolecular synthesis is observed in medium lacking any one of the other essential amino acids (eg: lysine).Our results indicate that the kinetics of macromolecular synthesis in tryptophan-deficient medium is the result of a limited reserve of protein-bound tryptophan which becomes exhausted after 30 hours. On the other hand, delayed inhibition of synthesis in isoleucine-deficient medium probably reflects an initially low requirement for this amino acid followed by inhibition of the synthesis of isoleucine-rich proteins involved in some late event of stimulation. Partial deprivation of lysine results in kinetics of protein synthesis similar to that in tryptophan-or isoleucine-deficient media. The results indicate that the kinetics of macromolecular synthesis during activation of lymphocytes in the absence of an essential amino acid is a function of the quantitative requirement for that amino acid, a t a given time during stimulation.Upon replacement of lysine, lymphocytes inhibited by lysine deficiency begin RNA and protein synthesis immediately and a t a rate faster than that of unstimulated cultures to which PHA is added. They also initiate DNA synthesis earlier and therefore, are closer to the S phase than resting lymphocytes. I t is concluded that lymphocytes stimulated in the absence of lysine are activated even though no overall increase in macromolecular synthesis is observed. Furthermore, the kinetics of DNA synthesis following reversal of inhibition by phenylalanine suggests that lymphocytes stimulated during phenylalanine deprivation become arrested a t most six hours before S. These results indicate that amino acid deficiencies lead to arrest of activated lymphocytes a t various stages of stimulation, depending on how stringent these deficiencies are. With the exception of tryptophan (TRY), isoleucine (ILE) and phenylalanine (PHE), omission of an essential amino acid from the culture medium results in a low, constant rate of protein synthesis similar to that of unstimulated cultures. Amino acid deprivation may block the early events that lead to activation of lymphocytes. On the other hand, the lack of response may represent the outcome of an "abortive stimulation" i.e., activation does occur but subsequent macromolecular synthesis is prevented. In this paper, we present the kinetics of RNA and DNA synt...

Phytohaemagglutinin Stimulation of Human Lymphocytes during Amino-Acid Deprivation. RNA Polymerase I Activity of Isolated Nuclei

“…The data also provided evidence that lymphocytes stimulated in lysinedeficient medium are closer to the S phase (DNA synthesis) of the cell cycle than resting lymphocytes [6]. The results presented in the present paper show that during the early response to phytohaemagglutinin, there is a component of increased transcriptional activity which, although sensitive to cycloheximide, is unaffected by lysine deprivation and which remains stable for at least 24 h.…”

“…When lymphocytes are stimulated in the absence of an essential amino acid, the normal increase in the rate of RNA synthesis in response to phytohaemagglutinin does not occur [6]. Fig.3 shows the kinetics of activation of RNA polymerase I during the first 8 h of stimulation in complete medium and in medium lacking lysine.…”

“…lysine) from the culture medium prevents the changes in RNA, protein and DNA synthesis induced by phytohaemagglutinin [5,h]. The kinetics of macromolecular synthesis during reversal of inhibition by replacement of the amino acid, however, suggested that amino acid deficiency does not prevent activation [6].…”

Extracellular Branched-Chain Amino Acids, Especially Valine, Regulate Maturation and Function of Monocyte-Derived Dendritic Cells

“…In previous cohort studies, the BCAA-supplemented groups demonstrated elevations in the absolute lymphocyte count (9,10). In previous in vitro studies, the omission of a single BCAA from the medium of cultured lymphocytes resulted in the complete abolition of protein synthesis or proliferation (11)(12)(13). These findings simply reflect the fact that BCAA are essential cell components.…”

Beneficial effects of supplementation with branched-chain amino acids on postoperative bacteremia in living donor liver transplant recipients

“…Bassit et al29 reported that BCAA supplementation restored the ability of peripheral blood mononuclear cells to proliferate in response to mitogens after long‐distance intensive exercise. Meanwhile, in vitro studies30‐32 have shown that the omission of a single BCAA from a medium of cultured lymphocytes completely abolishes protein synthesis and cellular proliferation.…”