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 microM) but not by the inositol 1,4,5-trisphosphate receptor (IP(3)R) blocker, 2-aminoethoxydiphenyl borate (100 microM), identifying them as Ca(2+) sparks. Caffeine (10 mM) and carbachol (10 microM) 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.
White
Carl
C
Smooth Muscle Research Group, School of Medicine, Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK.
McGeown
J Graham
JG
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology*; D002469:Cell Separation; D005753:Gastric Mucosa / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008297:Male; D032342:Muscle Cells / Q000378:metabolism*; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D013270:Stomach / Q000166:cytology
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D002217:Carbachol