Changes in cytoplasmic calcium determine the secretory 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 fura 2 and FM1-43 fluorescent dye. Two paradigms were used: human parathyroid cells, which are stimulated by a step from a high to a low extracellular calcium concentration (Ca(2+)), and rMTC6-23 cells, a rat medullary thyroid carcinoma cell line whose secretion is stimulated by an increase in Ca(2+). Parathyroid cells were dispersed from parathyroid adenomas removed from 18 patients with primary hyperparathyroidism. In both cell types, incubation with FM1-43 (2 microM) resulted in staining of the plasma membranes, which was rapidly increased following changes in Ca(2+) known to stimulate secretion. A high Ca(2+) and lanthanum (La(3+)) decreased the membrane-associated FM1-43 fluorescence. Prolonged incubation (5-30 min) in the 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 during incubation in a high (inhibitory) Ca(2+) if the cytoplasmic calcium concentration (Ca(2+)) was decreased by the calcium chelator BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] (10-50 microM). Alternatively, the expected rise in FM1-43 fluorescence did not occur during incubation in a low (stimulatory) Ca(2+) if Ca(2+) was increased by addition of the calcium ionophore A23187 (10-25 microM). These data suggest that Ca(2+), rather than the absolute value of Ca(2+), is the main modulator of secretion from parathyroid cells.