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

D. MacMillan, J.G. McCarron

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Abstract

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.
LanguageEnglish
Pages1295–1301
Number of pages7
JournalBritish Journal of Pharmacology
Volume160
Issue number6
DOIs
Publication statusPublished - Jul 2010

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Sarcoplasmic Reticulum
Type C Phospholipases
Smooth Muscle
Phosphoinositide Phospholipase C
Carbachol
Inositol 1,4,5-Trisphosphate Receptors
Photolysis
Caffeine
Ryanodine Receptor Calcium Release Channel
Pharmaceutical Preparations
Muscle Cells
1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione
Cytoplasm
Enzymes

Keywords

  • phospholipase c
  • smooth muscle
  • calcium
  • pharmacology
  • U-73122

Cite this

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title = "The phospholipase C inhibitor U-73122 inhibits Ca2+ release from the intracellular sarcoplasmic reticulum Ca2+ store by inhibiting Ca2+ pumps in smooth muscle",
abstract = "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.",
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AB - 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.

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