C2-Ceramide increases cytoplasmic calcium concentrations in human parathyroid cells.

Effects of extracellular calcium (Ca(2+)) 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 Ca(2+) activates the sphingomyelin pathway in bovine parathyroid cells, generating ceramides and sphingosine. This study explored the direct effects of synthetic ceramides on Ca(2+) 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 (Ca(2+)) Ca(2+) 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 microM C2-ceramide (N-acetyl-d-erythro-sphingosine), Ca(2+) 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 Ca(2+) from intracellular stores rather than Ca(2+) entry from the extracellular medium. C2-responsive cells had a smaller Ca(2+) 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 Ca(2+) nor the responses to an increase in Ca(2+) 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 Ca(2+) and secretion in response to Ca(2+) in human parathyroid cells.