Functionally separate intracellular Ca2+ stores in smooth muscle

Area of Science:

• Cellular Biology
• Physiology
• Biochemistry

Background:

• Cytosolic Ca(2+) concentrations ([Ca(2+)](c)) in smooth muscle are regulated by inositol 1,4,5-trisphosphate receptors (Ins(1,4,5)P(3)R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR).
• The spatial organization and interplay of Ins(1,4,5)P(3)R and RyR on SR Ca(2+) stores critically influence Ca(2+) signaling dynamics.
• The precise arrangement of these receptors on common or distinct SR stores remains unresolved.

Purpose of the Study:

• To investigate the functional arrangement and Ca(2+) store relationships of Ins(1,4,5)P(3)R and RyR in isolated colonic myocytes.
• To elucidate how the organization of these receptors impacts Ca(2+) release and signaling.

Main Methods:

• Utilized isolated single voltage-clamped colonic myocytes.
• Employing caffeine application to deplete ryanodine-sensitive stores.
• Manipulating external Ca(2+) to assess Ins(1,4,5)P(3)-sensitive store dependence.
• Inhibition of SR Ca(2+) ATPase with thapsigargin and cyclopiazonic acid to study store refilling.

Main Results:

• Depletion of ryanodine-sensitive stores abolished Ins(1,4,5)P(3)-induced Ca(2+) release, suggesting a shared Ca(2+) store.
• Ins(1,4,5)P(3)R-mediated Ca(2+) release did not trigger Ca(2+)-induced Ca(2+) release at RyR.
• Depletion of Ins(1,4,5)P(3)-sensitive stores resulted in a minor reduction in caffeine-evoked transients, indicating not all RyR reside on the common store.
• Ins(1,4,5)P(3)-sensitive stores depend on external Ca(2+) for refilling, unlike a subset of RyR-containing stores refilled from the cytosol.

Conclusions:

• Evidence supports the existence of two functionally distinct Ca(2+) stores within the SR.
• One store appears to contain only RyR and is refilled from the cytosol ([Ca(2+)](c)).
• A second, distinct store contains both Ins(1,4,5)P(3)R and RyR and relies on external Ca(2+) for replenishment.