Interaction between thapsigargin and ATP4− in the regulation of the intracellular calcium in rat submandibular glands

Rat submandibular glands were digested with crude collagenase, and the intracellular calcium concentration of the cellular suspension was measured using fura-2. In the absence of extracellular magnesium and calcium ([Ca2+]o), ATP had no effect; the response to ATP peaked at 1-2.5 mM [Ca2+]o and was inhibited at 5 mM. One millimolar (mM) extracellular ATP did not increase the leak of LDH or fura-2; 10 microM Coomassie brilliant blue G specifically inhibited the effect of ATP on [Ca2+]in. Depleting intracellular calcium pools with thapsigargin did not affect the response to ATP. Using a Ca(2+)-free/Ca2+ reintroduction protocol, it was shown that ATP and thapsigargin increase the uptake of extracellular calcium. The effect of the two agonists was synergistic. Removal of extracellular sodium inhibited the effect of carbachol on [Ca2+]in and the calcium uptake but potentiated the response to ATP. These results suggest that, after binding to purinergic receptors, extracellular ATP4- increases [Ca2+]in. ATP4- does not mobilize thapsigargin-sensitive intracellular calcium pools (among which is the IP3-sensitive calcium pool) but stimulates the uptake of extracellular calcium by a mechanism inhibited by extracellular sodium, probably by opening a nonselective cation channel.