Off-target effect of the cPLA2α inhibitor pyrrophenone: Inhibition of calcium release from the endoplasmic reticulum.
Cytosolic phospholipase A2α (cPLA2α) mediates agonist-induced release of arachidonic acid from membrane phospholipid for production of eicosanoids. The activation of cPLA2α involves increases in intracellular calcium, which binds to the C2 domain and promotes cPLA2α translocation from the cytosol to membrane to access substrate. The cell permeable pyrrolidine-containing cPLA2α inhibitors including pyrrophenone have been useful to understand cPLA2α function. Although this serine hydrolase inhibitor does not inhibit other PLA2s or downstream enzymes that metabolize arachidonic acid, we reported that it blocks increases in mitochondrial calcium and cell death in lung fibroblasts. In this study we used the calcium indicators G-CEPIA1er and CEPIA2mt to compare the effect of pyrrophenone in regulating calcium levels in the endoplasmic reticulum (ER) and mitochondria in response to A23187 and receptor stimulation. Pyrrophenone blocked calcium release from the ER and concomitant increases in mitochondrial calcium in response to stimulation by ATP, serum and A23187. In contrast, ER calcium release induced by the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin was not blocked by pyrrophenone suggesting specificity for the calcium release pathway. As a consequence of blocking calcium mobilization, pyrrophenone inhibited serum-stimulated translocation of the cPLA2α C2 domain to Golgi. The ability of pyrrophenone to block ER calcium release is an off-target effect since it occurs in fibroblasts lacking cPLA2α. The results implicate a serine hydrolase in regulating ER calcium release and highlight the importance of careful dose-response studies with pyrrophenone to study cPLA2α function.
Department of Pediatrics, National Jewish Health, 1400 Jackson St., Denver, CO 80206, USA.
Bogeon
B
Yun
Department of Pediatrics, National Jewish Health, 1400 Jackson St., Denver, CO 80206, USA.
HeeJung
H
Lee
Departments of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195, USA.
Heather
H
Ewing
Departments of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195, USA.
Michael H
MH
Gelb
Department of Pediatrics, National Jewish Health, 1400 Jackson St., Denver, CO 80206, USA. Electronic address: lesliec@njhealth.org.
Christina C
CC
Leslie
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D061207:Calcium Ionophores / Q000494:pharmacology; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D054513:Group IV Phospholipases A2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000166:cytology; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011759:Pyrrolidines / Q000494:pharmacology*; D044967:Serum / Q000737:chemistry; D019284:Thapsigargin / Q000494:pharmacology; D059008:Time-Lapse Imaging / Q000379:methods
D061207:Calcium Ionophores; D003470:Culture Media; D004791:Enzyme Inhibitors; D011759:Pyrrolidines; C455981:pyrrophenone; D000001:Calcimycin; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D054513:Group IV Phospholipases A2; D002118:Calcium
Calcium; Endoplasmic reticulum; Mitochondria; Pyrrophenone; cPLA(2)α