| Abstrakt: |
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 ([Ca2+]ext), and rMTC6-23 cells, a rat medullary thyroid carcinoma cell line whose secretion is stimulated by an increase in [Ca2+]ext. Parathyroid cells were dispersed from parathyroid adenomas removed from 18 patients with primary hyperparathyroidism. In both cell types, incubation with FM1-43 (2µM) resulted in staining of the plasma membranes, which was rapidly increased following changes in [Ca2+]ext known to stimulate secretion. A high [Ca2+]ext and lanthanum (La3+) decreased the membrane-associated FM1-43 fluorescence. Prolonged incubation (5–30min) 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) [Ca2+]ext if the cytoplasmic calcium concentration ([Ca2+]i) was decreased by the calcium chelator BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,Nƀ,Nƀ-tetra-acetic acid tetrakis(acetoxymethyl ester)] (10–50µM). Alternatively, the expected rise in FM1-43 fluorescence did not occur during incubation in a low (stimulatory) [Ca2+]ext if [Ca2+]i was increased by addition of the calcium ionophore A23187 (10–25µM). These data suggest that [Ca2+]i, rather than the absolute value of [Ca2+]ext, is the main modulator of secretion from parathyroid cells. |
