The phospholipase C inhibitor U-73122 inhibits Ca2+ release from the intracellular sarcoplasmic reticulum Ca2+ store by inhibiting Ca2+ pumps in smooth muscle

The sarcoplasmic reticulum (SR) releases Ca2+ via inositol 1,4,5-trisphosphate receptors (IP3R) in response to IP3-generating agonists. Ca2+ release subsequently propagates as Ca2+ waves. To clarify the role of IP3 production in wave generation, the contribution of a key enzyme in the production of IP3 was examined using a phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U-73122. Single colonic myocytes were voltage-clamped in whole-cell configuration and cytosolic Ca2+ concentration ([Ca2+]cyto) measured using fluo-3. SR Ca2+ release was evoked either by activation of IP3Rs (by carbachol or photolysis of caged IP3) or ryanodine receptors (RyRs; by caffeine). U-73122 inhibited carbachol-evoked [Ca2+]cyto transients. The drug also inhibited [Ca2+]cyto increases, evoked by direct IP3R activation (by photolysis of caged IP3) and RyR activation (by caffeine), which do not require PI-PLC activation. U-73122 also increased steady-state [Ca2+]cyto and slowed the rate of Ca2+ removal from the cytoplasm. An inactive analogue of U-73122, U-73343, was without effect on either IP3R- or RyR-mediated Ca2+ release. U-73122 inhibited carbachol-evoked [Ca2+]cyto increases. However, the drug also reduced Ca2+ release when evoked by direct activation of IP3R or RyR, slowed Ca2+ removal and increased steady-state [Ca2+]cyto. These results suggest U-73122 reduces IP3-evoked Ca2+ transients by inhibiting the SR Ca2+ pump to deplete the SR of Ca2+ rather than by inhibiting PI-PLC.