CHOLERA-TOXIN INDUCES SYNTHESIS OF PHOSPHOLIPASE A(2)-ACTIVATING PROTEIN

The mechanism of cholera toxin (CT)-stimulated arachidonate metabolism was evaluated. CT caused rapid in vitro synthesis of prostaglandin E( 2) (PGE(2)) in murine smooth muscle-like cells (BC(3)H1), reaching max imal levels within 3 to 4 min. In comparison, cyclic AMP (cAMP) levels were unchanged, and addition of dibutyryl cAMP did not affect PGE(2) synthesis. CT-induced PGE(2) synthesis was prevented by actinomycin D or cycloheximide, indicating a need for de novo protein synthesis. Nor thern blot analysis of total RNA from BC(3)H1 cells revealed that expo sure to CT resulted in an increase in abundance of mRNA encoding phosp holipase A(2) (PLA(2))-activating protein (PLAP). PLAP is a regulatory protein that increases the enzymatic activity of cellular PLA(2), whi ch in turn causes increased hydrolysis of arachidonate from membrane p hospholipids. Furthermore, CT evoked the accumulation of PLAP mRNA in J774 (murine monocyte/macrophage) and Caco-2 (human intestinal epithel ial) cells in vitro, but the responses were more delayed than that of BC(3)H1 cells. A protein band of approximately 35 kDa, which correspon ded to the size of PLAP, was observed in sodium dodecyl sulfate extrac ts of Caco-2 cells by Western blot (immunoblot) analysis using affinit y-purified antibodies to PLAP synthetic peptides. Synthesis of PLAP pr otein was increased after 2 h of exposure to CT. Exposure of mouse int estinal loops to either CT or live Salmonella tyhimurium for 3 h incre ased mucosal PLAP mRNA levels. The role of PLAP in CT-induced PGE(2) s ynthesis provides an attractive explanation for the reported suppressi on of CT-induced intestinal secretion by inhibitors of protein synthes is.