Off-target effect of the cPLA2α inhibitor pyrrophenone: Inhibition of calcium release from the endoplasmic reticulum

Abstract

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 Ca2+-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.

Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2

“…We reported that pyrrophenone blocks ER calcium release induced by receptor stimulation but not by thapsigargin suggesting that ABHD2 regulates IP 3 -mediated ER calcium release pathway that couples to mitochondrial calcium uptake, and not pathways that control basal calcium release [22]. A study that directly relates to our findings showed that ABHD2 knockdown blocks hepatitis B virus propagation [47].…”

“…KT195 inhibits by covalent, irreversible carbamoylation of the active site serine. We have uncovered a novel mechanism involving the serine hydrolase ABHD2 for regulating ER calcium release [18, 22]. …”

“…Imaging of ER and mitochondrial calcium following stimulation with A23187 (1 μg/ml) was carried out in control and ABHD2 knockdown IMLF expressing calcium-measuring organelle-entrapped protein indicators (CEPIA) that target to ER (G-CEPIAer) or mitochondria (CEPIA2mt) as described [22, 29]. …”

“…We reported that serine hydrolase inhibitors, pyrrophenone and the triazolurea inhibitor KT195 inhibit CyD-dependent necrotic cell death in lung fibroblasts by blocking ER calcium release, mitochondrial calcium uptake and MPTP formation [18–22]. Pyrrophenone and KT195 do not block cell death by inhibiting their known targets, cytosolic phospholipase A 2 (cPLA 2 α) and α/β-hydrolase domain-containing 6 (ABHD6), respectively but by off-target effects [18–20].…”

“…Pyrrophenone and KT195 do not block cell death by inhibiting their known targets, cytosolic phospholipase A 2 (cPLA 2 α) and α/β-hydrolase domain-containing 6 (ABHD6), respectively but by off-target effects [18–20]. They also inhibit ER calcium release triggered by receptor stimulation with ATP and serum, which act by increasing IP 3 production [22]. The results suggest that the inhibitors target a serine hydrolase that regulates ER calcium release through IP 3 R that is transferred to mitochondria.…”