C2-ceramide increases cytoplasmic calcium concentrations in human parathyroid cells

Effects of extracellular calcium ([Ca2+](ext)) on parathyroid cells are mai nly due to the activation of a plasma membrane calcium receptor (CaR) coupl ed with release of intracellular calcium. In addition, high [Ca2+](ext) act ivates the sphingomyelin pathway in bovine parathyroid cells, generating ce ramides and sphingosine. This study explored the direct effects of syntheti c ceramides on [Ca2+](i) in human parathyroid cells. Cells from five parath yroid adenomas removed from patients with primary hyperparathyroidism were dispersed and maintained in primary culture, Intracellular calcium concentr ation ([Ca2+](i)) [Ca2+](i) was monitored using standard quantitative fluor escence microscopy in Fura-2/AM-loaded cells. Laser scanning microscopy was used to monitor the intracellular distribution of a fluorescent ceramide a nalogue (BODIPY-C5). After addition of 10 mu M Ca-ceramide (N-acetyl-D-eryt hrosphingosine), [Ca2+](i) increased rapidly (30-60 s) to a peak three time s above basal levels in 70% of cells (37/55 cells in four experiments). Thi s effect appeared to be due to release of Ca2+ from intracellular stores ra ther than Ca2+ entry from the extracellular medium. Ca-responsive cells had a smaller [Ca2+](i) response to subsequent stimulation with the CaR agonis t-neomycin (1 mM). These responses were specific to C2 since C6-ceramide (N -hexanoyl-D-erythro-sphingosine) did not affect basal [Ca2+](i) nor the res ponses to an increase in [Ca2+](i), and to neomycin. C5-BODIPY generated in tense perinuclear fluorescence, suggesting targeting of the ceramides to th e Golgi apparatus. These data demonstrate that endogenous generation of cer amides has the potential to modulate changes in [Ca2+](i) and secretion in response to [Ca2+](ext) in human parathyroid cells. (C) 2000 Academic Press .