C2-ceramide increases cytoplasmic calcium concentrations in human parathyroid cells

Effects of extracellular calcium ([Ca2+](ext)) on parathyroid cells are mainly due to the activation of a plasma membrane calcium receptor (CaR) coupled with release of intracellular calcium. In addition, high [Ca2+](ext) activates the sphingomyelin pathway in bovine parathyroid cells, generating ceramides and sphingosine. This study explored the direct effects of synthetic ceramides on [Ca2+](i) in human parathyroid cells. Cells from five parathyroid adenomas removed from patients with primary hyperparathyroidism were dispersed and maintained in primary culture, Intracellular calcium concentration ([Ca2+](i)) [Ca2+](i) was monitored using standard quantitative fluorescence microscopy in Fura-2/AM-loaded cells. Laser scanning microscopy was used to monitor the intracellular distribution of a fluorescent ceramide analogue (BODIPY-C5). After addition of 10 mu M Ca-ceramide (N-acetyl-D-erythrosphingosine), [Ca2+](i) increased rapidly (30-60 s) to a peak three times above basal levels in 70% of cells (37/55 cells in four experiments). This effect appeared to be due to release of Ca2+ from intracellular stores rather than Ca2+ entry from the extracellular medium. Ca-responsive cells had a smaller [Ca2+](i) response to subsequent stimulation with the CaR agonist-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 responses to an increase in [Ca2+](i), and to neomycin. C5-BODIPY generated intense perinuclear fluorescence, suggesting targeting of the ceramides to the Golgi apparatus. These data demonstrate that endogenous generation of ceramides has the potential to modulate changes in [Ca2+](i) and secretion in response to [Ca2+](ext) in human parathyroid cells. (C) 2000 Academic Press.