Thapsigargin-induced secretion is dependent on activation of a cholera toxin-sensitive and phosphatidylinositol-3-kinase-regulated phospholipase D in a mast cell line.
Release of secretory granules by rat RBL-2H3 mast cells is mediated primarily through activation of protein kinase C (PKC) and elevation of cytosolic free calcium ([Ca++]I). Here, we show that secretion was also dependent on the activation of a cholera toxin-sensitive phospholipase (PL) D in cells stimulated with thapsigargin. Wortmannin, LY294002, butanol, propranolol and Ro31-7549 inhibited responses to variety of secretagogues in a manner consistent with the notion that secretion was regulated by both PLD and PKC in a phosphatidylinositol-3-kinase-dependent manner. The effects of these inhibitors, however, were especially pronounced in cells activated by thapsigargin. This stimulant induced minimal stimulation of PLC but measurable activation of PLD, as assessed by formation of phosphatidylethanol in the presence of ethanol. The activation of PLD was suppressed by inhibitors of phosphatidylinositol-3-kinase and was dependent on a rise in [Ca++]i because thapsigargin failed to activate PLD and secretion when elevation of [Ca++]i was blocked. Treatment of cells with cholera toxin resulted in selective and similar enhancements in the activation of PLD and secretion by thapsigargin, whereas stimulation of PLC and PLA2 was unaffected. A role for PKC was indicated by the blockade of secretory response to thapsigargin by the PKC inhibitor Ro31-7549 and by the ability of the PKC agonist phorbol-12-myristate-13-acetate to reverse the inhibition of secretion by inhibitors of PLD. Such results suggested that thapsigargin, by causing substantial increases in [Ca++]I, induced secondary signals via PLD and PKC that synergized a calcium signal for secretion.
Cissel
D S
DS
Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1760, USA. cisseld@gwgate.nhlbi.nih.gov
Fraundorfer
P F
PF
Beaven
M A
MA
D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000440:Butanols / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured / Q000187:drug effects; D002772:Cholera Toxin / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D008297:Male; D008407:Mast Cells / Q000187:drug effects* / Q000201:enzymology; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010739:Phospholipase D / Q000187:drug effects* / Q000378:metabolism; D011433:Propranolol / Q000494:pharmacology; D051381:Rats; D019284:Thapsigargin / Q000494:pharmacology*; D000077191:Wortmannin
D000319:Adrenergic beta-Antagonists; D000730:Androstadienes; D000440:Butanols; D002118:Calcium; D002772:Cholera Toxin; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010739:Phospholipase D; D011433:Propranolol; D019284:Thapsigargin; D000077191:Wortmannin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one