C2-Ceramide Increases Cytoplasmic Calcium
Concentrations in Human Parathyroid Cells
Radu Mihai, Teresa Lai, George Schofield,* and John R. Farndon
Department of Surgery, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom; and *Department of Biochemistry,
School of Medical Sciences, University of Bristol, Bristol, United Kingdom
Received December 29, 1999
Effects of extracellular calcium ([Ca
2
]
ext
) on para-
thyroid 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
]
ext
activates the sphingomyelin pathway
in bovine parathyroid cells, generating ceramides
and sphingosine. This study explored the direct ef-
fects of synthetic ceramides on [Ca
2
]
i
in human
parathyroid cells. Cells from five parathyroid adeno-
mas removed from patients with primary hyperpara-
thyroidism were dispersed and maintained in pri-
mary culture. Intracellular calcium concentration
([Ca
2
]
i
) [Ca
2
]
i
was monitored using standard quan-
titative fluorescence microscopy in Fura-2/AM-
loaded cells. Laser scanning microscopy was used to
monitor the intracellular distribution of a fluores-
cent ceramide analogue (BODIPY-C5). After addi-
tion of 10 M C2-ceramide (N-acetyl-D-erythro-
sphingosine), [Ca
2
]
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 ap-
peared 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
]
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 [Ca
2
]
i
nor
the responses to an increase in [Ca
2
]
ext
and to neo-
mycin. 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 poten-
tial to modulate changes in [Ca
2
]
i
and secretion in
response to [Ca
2
]
ext
in human parathyroid cells.
© 2000 Academic Press
Key Words: human parathyroid cells; hyperparathy-
roidism; C2-, C6-ceramide; sphingosine; Fura-2/AM;
C5-BODIPY; fluorescence microscopy; confocal
microscopy.
Extracellular calcium concentration ([Ca
2+
]
ext
) is the
main factor controlling secretion from parathyroid
cells. Changes in [Ca
2+
]
ext
are sensed by a plasma mem-
brane calcium receptor (CaR) (1). High [Ca
2+
]
ext
and
other agents (e.g. neomycin and trivalent cations La
3+
and Gd
3+
) stimulate the CaR, which is coupled to the
G-protein-phospholipase C pathway that leads to gen-
eration of inositol 1,4,5-trisphosphate (IP
3
) and release
of Ca
2+
from intracellular stores (1). In this way, high
[Ca
2+
]
ext
is translated into high [Ca
2+
]
i
and this inhibits
secretion through an as yet unknown mechanism.
In bovine parathyroid cells high [Ca
2+
]
ext
increases the
concentration of sphingosine (2), one of the intermediates
in the sphingomyelin pathway. In many cell types, the
sphingomyelin pathway is involved in the regulation of
cell proliferation, cell-cell interaction, differentiation and
oncogenesis (3). Its activation begins with hydrolysis of
membrane sphingomyelin by a neutral sphingomyeli-
nase, leading to increased ceramide levels in target cells.
The hydrolysis of ceramide by a ceramidase produces free
sphingosine, which can be further converted to
sphingosine-1-phosphate (by a sphingosine-kinase) or to
sphingosine-phosphorylcholine. Although very little is
known about the effects of sphingolipids in the parathy-
roid cells, the increase in sphingosine concentration in
response to [Ca
2+
]
ext
(2) raises the possibility that the
sphingomyelin pathway is functionally important in
parathyroid cell.
The aim of this study was to explore the possibility
that compounds generated through the sphingomyelin
pathway modulate the response of human parathyroid
cells to changes in [Ca
2+
]
ext
. The effects of synthetic
ceramides on [Ca
2+
]
i
were investigated in human para-
thyroid cells from patients with primary hyperparathy-
roidism. [Ca
2+
]
i
was measured in individual cells using
the widely-accepted method of quantitative fluores-
cence microscopy in cells loaded with the calcium-
marker Fura-2/AM. Confocal microscopy was used to
monitor the intracellular distribution of a fluorescent
ceramide analogue (C5-BODIPY).
Biochemical and Biophysical Research Communications 268, 636 – 641 (2000)
doi:10.1006/bbrc.2000.2159, available online at http://www.idealibrary.com on
636 0006-291X/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.