The phospholipase C inhibitor U-73122 inhibits Ca(2+) release from the intracellular sarcoplasmic reticulum Ca(2+) store by inhibiting Ca(2+) pumps in smooth muscle.
OBJECTIVE
The sarcoplasmic reticulum (SR) releases Ca(2+) via inositol 1,4,5-trisphosphate receptors (IP(3)R) in response to IP(3)-generating agonists. Ca(2+) release subsequently propagates as Ca(2+) 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.


METHODS
Single colonic myocytes were voltage-clamped in whole-cell configuration and cytosolic Ca(2+) concentration ([Ca(2+)](cyto)) measured using fluo-3. SR Ca(2+) release was evoked either by activation of IP(3)Rs (by carbachol or photolysis of caged IP(3)) or ryanodine receptors (RyRs; by caffeine).


RESULTS
U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) transients. The drug also inhibited [Ca(2+)](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 [Ca(2+)](cyto) and slowed the rate of Ca(2+) removal from the cytoplasm. An inactive analogue of U-73122, U-73343, was without effect on either IP(3)R- or RyR-mediated Ca(2+) release.


CONCLUSIONS
U-73122 inhibited carbachol-evoked [Ca(2+)](cyto) increases. However, the drug also reduced Ca(2+) release when evoked by direct activation of IP(3)R or RyR, slowed Ca(2+) removal and increased steady-state [Ca(2+)](cyto). These results suggest U-73122 reduces IP(3)-evoked Ca(2+) transients by inhibiting the SR Ca(2+) pump to deplete the SR of Ca(2+) rather than by inhibiting PI-PLC.
Macmillan
D
D
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK. d.macmillan@strath.ac.uk
McCarron
J G
JG
D000814:Aniline Compounds / Q000737:chemistry; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D003106:Colon / Q000166:cytology; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology*; D006168:Guinea Pigs; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000187:drug effects / Q000378:metabolism; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000187:drug effects; D018408:Patch-Clamp Techniques; D054801:Phosphoinositide Phospholipase C / Q000037:antagonists & inhibitors*; D011760:Pyrrolidinones / Q000494:pharmacology*; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D014966:Xanthenes / Q000737:chemistry
D000814:Aniline Compounds; D004963:Estrenes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011760:Pyrrolidinones; D019837:Ryanodine Receptor Calcium Release Channel; D014966:Xanthenes; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C059715:Fluo-3; D002110:Caffeine; D002217:Carbachol; D054801:Phosphoinositide Phospholipase C; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium