Carbachol triggers RyR‐dependent Ca 2+ release via activation of IP 3 receptors in isolated rat gastric myocytes

Possible interactions between different intracellular Ca 2+ release channels were studied in isolated rat gastric myocytes using agonist‐evoked Ca 2+ signals. Spontaneous, local Ca 2+ transients were observed in fluo‐4‐loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 μ m ) but not by the inositol 1,4,5‐trisphosphate receptor (IP 3 R) blocker, 2‐aminoethoxydiphenyl borate (100 μ m ), identifying them as Ca 2+ sparks. Caffeine (10 m m ) and carbachol (10 μ m ) initiated Ca 2+ release at sites which co‐localized with each other and with any Ca 2+ spark sites. In fura‐2‐loaded cells extracellular 2‐aminoethoxydiphenyl borate and intracellular heparin (5 mg ml −1 ) both inhibited the global cytoplasmic [Ca 2+ ] transient evoked by carbachol, confirming that it was IP 3 R‐dependent. 2‐Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca 2+ store content since 2‐aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP 3 Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca 2+ release through co‐operation between IP 3 Rs and RyRs, and implicate IP 3 Rs in the regulation of Ca 2+ store content.