Premium
Diacylglycerol and calcium induce rapid enhancement of A‐system amino acid transport by independent mechanisms in human T‐lymphocytes
Author(s) -
Woodlock Timothy J.,
Segel George B.,
Lichtman Marshall A.
Publication year - 1989
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041410106
Subject(s) - ionomycin , diacylglycerol kinase , cycloheximide , protein kinase c , calcium , amino acid , biochemistry , chemistry , intracellular , biology , protein biosynthesis , kinase , organic chemistry
Sn‐1,2‐diacylglycerols (DAG) and ionized‐free calcium can act as intracellular second messengers for cell activation. Traditionally, T‐lymphocyte activation is assessed by measurements of DNA synthesis or lymphokine production, but these responses require several days to occur and involve multiple intermediary regulatory steps. In contrast, we have found that T‐lymphocytes demonstrate rapid enhancement of A‐(alanine‐favoring) system amino acid uptake when treated with DAG or ionomycin. A 30–40% increase in the initial velocity of uptake (v i ) of the synthetic A‐system specific amino acid, methylamino‐isobutyric acid (MeAIB), was measured following 5 min of exposure to DAG or ionomycin. The v i was enhanced 60% from 12 to 19 μmol/liter cell water per min after 30 min exposure of T‐cells to optimal concentrations of dioctanoylglycerol (30 μM), oleoylacetylglycerol (30 μM), or ionomycin (5 μM) ( P < .01 for each agent). A 50‐fold excess of non‐radioactive MeAIB inhibited 80% of [ 14 C]MeAIB uptake in both unstimulated and stimulated cells, indicating that uptake remained largely carrier‐mediated on treatment with these agents. Cycloheximide, 100 μg/ml, inhibited protein synthesis but did not block the A‐system amino acid transport enhancement induced by DAG or ionomycin. The DAG‐induced increase in the v i was blocked 40% with 100 μM H‐7, an inhibitor of protein kinase C. H‐7 treatment did not inhibit the ionomycin‐induced A‐system enhancement. A marked increase in cytoplasmic free calcium was measured when T‐lymphocytes were exposed to ionomycin but not on DAG exposure, and the A‐system effect of ionomycin but not DAG was blocked by extracellular EGTA. These data are compatible with two pathways for rapid enhancement of A‐system amino acid uptake in T‐lymphocytes. DAG stimulation is mediated via protein kinase C whereas ionomycin produces an A‐system effect of similar magnitude independent of protein kinase C by an increase in cytoplasmic calcium.