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+)](ext)), and rMTC6-23 cells, a rat medullary thyroid carcinoma cell line whose secretion is stimulated by an increase in [Ca(2+)](ext). 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+)](ext) known to stimulate secretion. A high [Ca(2+)](ext) 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+)](ext) if the cytoplasmic calcium concentration ([Ca(2+)](i)) 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+)](ext) if [Ca(2+)](i) was increased by addition of the calcium ionophore A23187 (10-25 microM). These data suggest that [Ca(2+)](i), rather than the absolute value of [Ca(2+)](ext), is the main modulator of secretion from parathyroid cells.
Mihai
R
R
University Department of Surgery, Bristol Royal Infirmary, Bristol BS2 8HW, U.K. r_mihai99@hotmail.com
Lai
T
T
Schofield
G J
GJ
Farndon
J R
JR
D000328:Adult; D000368:Aged; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002412:Cations; D003593:Cytoplasm / Q000378:metabolism*; D005260:Female; D006801:Humans; D007811:Lanthanum / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D008875:Middle Aged; D010280:Parathyroid Glands / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011726:Pyridinium Compounds / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*
D002412:Cations; C073804:FM1 43; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D000001:Calcimycin; D007811:Lanthanum; D002118:Calcium