AJP - Cell Physiology, Vol 258, Issue 2 C289-C298, Copyright © 1990 by American Physiological Society

ARTICLES

Multiple calcium mobilization pathways in single avian salt gland cells

E. L. Stuenkel and S. A. Ernst
Department of Physiology, University of Michigan, Ann Arbor 48109.

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.