Cholera toxin induces synthesis of phospholipase A2-activating protein.
The mechanism of cholera toxin (CT)-stimulated arachidonate metabolism was evaluated. CT caused rapid in vitro synthesis of prostaglandin E2 (PGE2) in murine smooth muscle-like cells (BC3H1), reaching maximal levels within 3 to 4 min. In comparison, cyclic AMP (cAMP) levels were unchanged, and addition of dibutyryl cAMP did not affect PGE2 synthesis. CT-induced PGE2 synthesis was prevented by actinomycin D or cycloheximide, indicating a need for de novo protein synthesis. Northern blot analysis of total RNA from BC3H1 cells revealed that exposure to CT resulted in an increase in abundance of mRNA encoding phospholipase A2 (PLA2)-activating protein (PLAP). PLAP is a regulatory protein that increases the enzymatic activity of cellular PLA(2), which in turn causes increased hydrolysis of arachidonate from membrane phospholipids. Furthermore, CT evoked the accumulation of PLAP mRNA in J774 (murine monocyte/macrophage) and Caco-2 (human intestinal epithelial) cells in vitro, but the responses were more delayed than that of BC3H1 cells. A protein band of approximately 35 kDa, which corresponded to the size of PLAP, was observed in sodium dodecyl sulfate extracts of Caco-2 cells by Western blot (immunoblot) analysis using affinity-purified antibodies to PLAP synthetic peptides. Synthesis of PLAP protein was increased after 2 h of exposure to CT. Exposure of mouse intestinal loops to either CT or live Salmonella typhimurium for 3 h increased mucosal PLAP mRNA levels. The role of PLAP in CT-induced PGE2 synthesis provides an attractive explanation for the reported suppression of CT-induced intestinal secretion by inhibitors of protein synthesis.
Peterson
J W
JW
Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston 77555-1019, USA.
Saini
S S
SS
Dickey
W D
WD
Klimpel
G R
GR
Bomalaski
J S
JS
Clark
M A
MA
Xu
X J
XJ
Chopra
A K
AK
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology*; D015232:Dinoprostone / Q000096:biosynthesis; D004789:Enzyme Activation; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis
D011506:Proteins; D012333:RNA, Messenger; C055324:phospholipase A2-activating protein; D002772:Cholera Toxin; D010741:Phospholipases A; D054467:Phospholipases A2; D015232:Dinoprostone