Changes in cytoplasmic calcium determine the secretary response to extracellular cations in human parathyroid cells: a confocal microscopy study using FM1-43 dye

Whether activation of the calcium receptor (CaR) modulates secretory events was investigated by real-time fluorescence and confocal microscopy using f ura 2 and FM1-43 fluorescent dye. Two paradigms were used: human parathyroi d cells, which are stimulated by a step from a high to a low extracellular calcium concentration ([Ca2+](ext)), and rMTC6-23 cells, a rat medullary th yroid carcinoma cell line whose secretion is stimulated by an increase in [ Ca2+](ext). Parathyroid cells were dispersed from parathyroid adenomas remo ved from 18 patients with primary hyperparathyroidism. In both cell types, incubation with FM1-43 (2 muM) resulted in staining of the plasma membranes , which was rapidly increased following changes in [Ca2+](ext) known to sti mulate secretion. A high [Ca2+](ext) and lanthanum (La3+) decreased the mem brane-associated FM1-43 fluorescence. Prolonged incubation (5-30 min) in th e presence of FM1-43 resulted in accumulation of the dye in the cytoplasm, its granular distribution suggesting targeting of the secretory compartment . These data suggest that FM1-43 fluorescence is determined by: (i) changes in cell membrane surface area associated with secretion-associated events, (ii) displacement/quenching by extracellular cations and (iii) endocytosis of the dye. In parathyroid cells, a rise in FM1-43 fluorescence occurred d uring incubation in a high (inhibitory) [Ca2+](ext) if the cytoplasmic calc ium concentration ([Ca2+](i)) was decreased by the calcium chelator BAPTA/A M [bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxym ethyl ester)] (10-50 muM). Alternatively, the expected rise in FM1-43 fluor escence did not occur during incubation in a low (stimulatory) [Ca2+](ext) if [Ca2+](i) was increased by addition of the calcium ionophore A23187 (10- 25 muM). These data suggest that [Ca2+](i), rather than the absolute value of [Ca2+](ext), is the main modulator of secretion from parathyroid cells.