Multiple calcium mobilization pathways in single avian salt gland cells.

Agonist-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in individual secretory cells from the avian salt gland were detailed using dual-wavelength microspectrofluorimetry of the Ca2(+)-sensitive fluorescent probe fura-2. Resting [Ca2+]i averaged 42 +/- 5 nM. Stimulation with the cholinergic agonist carbachol (1 microM) resulted in a rapid increase in [Ca2+]i to 308 +/- 26 nM, which was sustained at a nearly constant elevated level (328 +/- 31 nM) throughout agonist application. In the absence of extracellular Ca2+ or in the presence of an inorganic blocker of Ca2+ entry (Ni2+, 1 mM), only a transient increase in [Ca2+]i occurred on agonist stimulation, whereas subsequent readmission of Ca2+ or washout of Ni2+ reinitiated a sustained increase in [Ca2+]i. The initial transient response results from Ca2+ release from intracellular stores, whereas the sustained phase represents entry of extracellular Ca2+ into the cytoplasm. Repetitive stimulations in Ca2(+)-free medium alternating with Ca2(+)-containing medium were performed to examine the mechanisms involved in refilling of the agonist-sensitive intracellular pool. After depletion of the intracellular pool by stimulation in Ca2(+)-free medium, removal of the agonist and readmission of Ca2+ resulted in a rapid transient increase in [Ca2+]i that could be blocked by Ni2+, La3+, or elevated K+. Subsequent removal of extracellular Ca2+ and restimulation nonetheless showed that complete refilling of the intracellular pool had occurred in each case. These results suggest that two separate Ca2(+)-entry mechanisms, one sensitive to Ni2+, La3+, and elevated K+ and responsible for the agonist-induced increase in [Ca2+]i and one insensitive to the blockers and involved in refilling of the intracellular pool, may exist in salt gland cells. Spontaneous oscillations of [Ca2+]i that are independent of extracellular Ca2+ have also been observed in 10% of the cells. The abolition of the oscillations by depletion of the agonist-sensitive pool suggests this pool as the Ca2+ source for the oscillations.