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

Background and purpose: The sarcoplasmic reticulum (SR) releases Ca2+ via inositol 1,4,5-trisphosphate receptors (IP(3)R) in response to IP(3)-generating agonists. Ca2+ release subsequently propagates as Ca2+ waves. To clarify the role of IP(3) production in wave generation, the contribution of a key enzyme in the production of IP(3) was examined using a phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U-73122. Experimental approach: 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 IP(3)Rs (by carbachol or photolysis of caged IP(3)) or ryanodine receptors (RyRs; by caffeine). Key results: U-73122 inhibited carbachol-evoked [Ca2+](cyto) transients. The drug also inhibited [Ca2+](cyto) increases, evoked by direct IP(3)R activation (by photolysis of caged IP(3)) 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 IP(3)R- or RyR-mediated Ca2+ release. Conclusions and implications: U-73122 inhibited carbachol-evoked [Ca2+](cyto) increases. However, the drug also reduced Ca2+ release when evoked by direct activation of IP(3)R or RyR, slowed Ca2+ removal and increased steady-state [Ca2+](cyto). These results suggest U-73122 reduces IP(3)-evoked Ca2+ transients by inhibiting the SR Ca2+ pump to deplete the SR of Ca2+ rather than by inhibiting PI-PLC. This article is commented on by Hollywood et al., pp. 1293-1294 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00795.x.