Sphingosine Inhibits Voltage-operated Calcium Channels in
GH4C1 Cells*
Alexey
Titievsky
,
Ira
Titievskaya
,
Michael
Pasternack
,
Kai
Kaila
, and
Kid
Törnquist
§¶
From the
Department of Biosciences, Division of
Animal Physiology, University of Helsinki, Helsinki, the
§ Minerva Foundation Institute for Medical Research,
Helsinki, and the Department of Biology, Åbo Akademi University,
Turku, Finland
 |
ABSTRACT |
In the present study we investigated the
mechanism of inhibitory action of sphingosine (SP) on voltage-activated
calcium channels (VOCCs) in pituitary
GH4C1 cells. Using the patch-clamp
technique in the whole-cell mode, we show that SP inhibits
Ba2+ currents (IBa) when 0.1 mM BAPTA is included in the patch pipette. However, when
the BAPTA concentration was raised to 1-10 mM, SP was
without a significant effect. The effect of SP was apparently not
mediated via a kinase, as it was not inhibited by staurosporine. By
using the double-pulse protocol (to release possible functional inhibition of the VOCCs by G proteins), we observed that G proteins apparently evoked very little functional inhibition of the VOCCs. Furthermore, including GDP
S (guanyl-5
-yl thiophosphate) in the patch pipette did not alter the inhibitory effect of SP on the Ba2+ current, suggesting that SP did not modulate the VOCCs
via a G protein-dependent pathway. Single-channel
experiments with SP in the pipette, and experiments with excised
outside-out patches, suggested that SP directly inhibited VOCCs. The
main mechanism of action was a dose-dependent prolongation
of the closed time of the channels. The results thus show that SP is a
potent inhibitor of VOCCs in GH4C1 cells, and
that calcium may be a cofactor in this inhibition.
 |
INTRODUCTION |
Sphingosine (SP)1 and
related sphingolipids are considered potent endogenous inhibitors of
protein kinase C (PKC) (1), as well as activators of proliferation
(2-4). Recently, it has been shown that SPs stimulate the
release of sequestered calcium (3, 5-8) in many cell types. A role for
sphingosine and sphingosine 1-phosphate as possible second messengers
has been postulated (9, 10).
An interesting observation made recently is that sphingosines regulate
the gating of a novel type of calcium channel located in membranes of
intracellular calcium stores in endothelial cells (11). Furthermore,
sphingosines inhibit calcium entry through voltage-operated calcium
channels (VOCCs) in cardiac cells (12), and capacitative calcium entry
in Jurkat T cells (13). An inhibitory effect of sphingosines on
depolarization-evoked calcium entry has also been observed in
synaptosomes (14). In brain microsomes, the sphingosine derivative
sphingosine phosphorylcholine stimulated calcium release via the
ryanodine receptor (15), whereas sphingosine blocked activation of the
ryanodine receptor in cardiac cells (16). Another interesting
observation is that SP inhibits sustained, VOCC-dependent
calcium gradients in hippocampal neurons after stimulation with either
glutamate or N-methyl-D-aspartate (17). This
SP-evoked inhibition was attributed to inhibition of PKC, but in the
light of recent observations, it may be the result of SP-evoked
blockade of VOCCs. Thus, SPs appear to be potent regulators of calcium
signaling. However, the mechanisms involved are not yet known.
In pituitary cells, changes in intracellular free calcium
([Ca2+]i) are of crucial importance for the
regulation of hormone synthesis and secretion (18, 19). The increase in
[Ca2+]i may be the result of agonist-evoked
activation of phospholipase C and the hydrolysis of
phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate,
and the concomitant release of sequestered calcium (20-22).
Agonist-evoked activation of phospholipase C also results in the
activation of VOCCs (23-26). In pituitary cells, the L- and the T-type
VOCCs have been identified (27, 28). In particular, L-type VOCCs are
considered important for the stimulation of hormone synthesis and
secretion (23, 24, 29, 30) but also for the steady-state regulation of
[Ca2+]i (31).
Stimulating GH3 pituitary cells with diacylglycerols
activates sphingomyelinase, resulting in the production of sphingosine and ceramides (32, 33). In a recent study, we observed that SPs
potently inhibited calcium entry via VOCCs in
GH4C1 pituitary cells (34). This effect was
independent of an action of PKC. In GH4C1
cells, PKC is assumed to participate in the regulation of VOCCs (20,
23, 35, 36) (see also Refs. 37 and 38), and thus both PKC and SPs may
be important modulators of VOCCs in these cells. In the present study,
we have investigated the mechanisms of action of SP on VOCCs in
GH4C1 cells. Using whole-cell, single-channel,
and outside-out patch-clamp methods, we show that SP inhibits the
gating of VOCCs in GH4C1 cells. This mechanism may, in part, be dependent on intracellular calcium.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Fura 2-AM was obtained from Molecular Probes
(Eugene, OR). Sphingosine, staurosporine, GTP
S, GDP
S, and
tetrodotoxin were acquired from Sigma. Ham's F-10 nutrient mixture was
from Life Technologies, Inc., and serum was from Biological
Industries (Beth Haemek, Israel). All other reagents were of analytical
grade. Culture dishes were obtained from Falcon Plastics (Oxnard, CA). [3H]PN 200-110 (81 Ci/mmol) was from Amersham (Little
Chalfont, Buckinghamshire, UK).
Cell Culture--
Clonal rat pituitary
GH4C1 cells were generously given by Dr. Armen
H. Tashjian, Jr. (Harvard University, Boston, MA). The cells were grown
in monolayer culture in Ham's F-10 nutrient mixture with 15% (v/v)
horse serum and 2.5% fetal bovine serum (Ham's F-10+ medium) in a
water-saturated atmosphere of 5% CO2 and 95% air at
37 °C, as described previously (39, 40). Before an experiment, the
cells from a single donor culture were harvested with 0.02% EDTA
solution and subcultured in 35-mm or 100-mm culture dishes for 7-9
days. For the patch-clamp experiments, the cells were grown on
coverslips in 35-mm dishes. The cells were fed with Ham's F-10+ medium
every 2-3 days.
Patch-Clamp Experiments--
The studies were performed using
the patch-clamp technique in the whole-cell mode, in the cell-attached
mode, and in the excised outside-out mode (41). The electrodes were
made from GC150TF glass micropipettes (Clark Electromedical
Instruments, Reading, United Kingdom). In the whole-cell experiments,
the electrodes had a resistance of about 4-5 M
when filled with
Hepes-buffered salt solution (HBSS-II buffer, containing (in
mM): CsCl, 120; MgCl2, 5; Mg-ATP, 2; BAPTA,
0.1-10; glucose, 10; Hepes, 20 (pH 7.15), adjusted with CsOH). Leak
and capacitative currents in the whole-cell recordings were compensated
by the P/4 routine from a holding potential of
90 mV. The access
resistance was 7-15 M
. No series resistance compensation was made.
Before an experiment, the coverslips were attached to a perfusion
chamber (volume approximately 300 µl), and the cells were extensively perfused with HBSS-I (containing (in mM):
BaCl2, 60; TEA-Cl, 75; glucose, 10; Hepes, 10 (pH 7.2),
adjusted with TEA-OH) or sphingosine using a peristaltic pump
(ISMATEC®) at 1.5 ml/min. Sphingosine was applied by
switching the channel of the pump. The recordings were made with an
EPC-9 patch clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany)
at room temperature. The current signals were filtered at 2.3 kHz,
sampled at 2 kHz, and stored on an Atari Mega/Ste computer. The cells were held at
70 mV with test pulses to
20 mV and +10 mV made throughout the experiment to avoid additional rundown of the channels. For the calculations, we used data derived from pulses to +10 mV. A
stock solution of sphingosine (10 mM) was made in ethanol. The final concentration of the solvent did not exceed 0.2%. This concentration of the solvent did not have any effects on VOCCs in
GH4C1 cells.
To calculate the half-inactivation time (IT50),
i.e. defined as the 50% run-down of the initial steady
current, a logistic equation was used, where t is the time
from rupturing the cell membrane, b is the slope, and
d is the residual current after full inactivation (Equation 1).
|
(Eq. 1)
|
This normalization of the results, where the steady state
current before application of test compound was considered 100%, was
made to make a comparison between different measurements feasible.
In the cell-attached single-channel recordings, the cells were bathed
in a high potassium HBSS-III buffer (containing (in mM):
KCl, 140; MgCl2, 1.13; glucose, 10; HEPES, 10 (pH 7.2),
adjusted with KOH) to abolish the membrane potential. In these
experiments, the pipettes were filled with HBSS-IV buffer (containing
(in mM): BaCl2, 110; TEA-Cl, 10; HEPES,
10 (pH 7.2), adjusted with TEA-OH), and had a resistance of 15-20
M
. Leak and capacitive currents in the single-channel experiments
were subtracted and compensated for in all recordings. Data analysis
was made using the pCLAMP6 program (Axon Instruments).
The external solution used for the excised outside-out patch recordings
was HBSS-I. The internal pipette solution in these experiments was
HBSS-II lacking BAPTA but containing 4 mM CaCl2 and 10 mM EGTA to give a free calcium concentration of 100 nM. This resting calcium concentration was chosen because
of the apparent calcium-dependent effects of sphingosine
observed in the whole-cell and cell-attached experiments. Thick-walled
glass pipettes were used, which had a resistance of 9.5-11.5 M
. The
outside-out patches were obtained using standard techniques (41).
Patches with a resistance less than 5 G
were rejected from the
experiments. Usually, the patches had a resistance higher than 20 G
.
The excised patches were clamped at
50 mV to obtain more stable
patches than those obtained at
70 mV, with test pulses to
10 mV and
+10 mV. The current signals were sampled at 5 kHz, and filtered at 1 kHz using an Axoclamp-1A amplifier and Clampex software (Axon
Instruments). After the sampling, no additional digital filtering was
used. Data analysis was made using pClamp 6 software and Microcal
Origin software.
 |
RESULTS |
Effects of Sphingosine on VOCCs in GH4C1
Cells--
In a recent study, we showed that sphingosine derivatives
inhibit the activation of VOCCs in GH4C1 cells, but the mechanism remained unclear (34). Using the whole-cell mode of the patch-clamp technique, we now found that 10 µM SP potently suppressed
the current amplitude without any profound shift in the current-voltage relationship, suggesting that there is no voltage dependence of the
inhibition (Fig. 1). VOCCs are prone to
inhibition via a calcium-dependent mechanism (see Ref. 42).
To evaluate whether SP could modulate VOCCs via this mechanism,
different concentrations of BAPTA were included in the pipette
solution. With 0.1 mM BAPTA in the pipette solution, SP
rapidly inhibited IBa (Fig. 1). In control
cells, the half-time value (IT50) for the
rundown was 267 ± 42 s, whereas in the presence of 10 µM SP, IT50 was 143 ± 8 s (p < 0.05). However, when the pipette solution
contained 1 mM or 10 mM BAPTA, the run-down in
the presence of SP proceeded at a rate equal to that observed in
control cells (Fig. 2). This result
suggests that the inhibitory effect of SP on the VOCCs is mediated via
a calcium-dependent mechanism.

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Fig. 1.
The inhibitory effect of SP on calcium
channel currents in GH4C1 cells.
Representative traces of Ba2+ currents obtained by
depolarizing the cells from 70 mV to the potentials indicated.
A1, control currents. A2, currents obtained after
the application of 10 µM SP. B,
current-voltage relationship of the Ba2+ current. Panel
shows control currents ( ) and currents obtained after the
application of 10 µM SP ( ). The data shown are from the same cell. In this experiment, the patch pipette contained 0.1 mM BAPTA.
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Fig. 2.
BAPTA attenuates the inhibitory effect of SP
on VOCCs in whole-cell recordings. Time course of the run-down of
normalized individual currents
(I/Imax) measured at the end of a
200-ms depolarizing pulse, where Imax is the
maximal current measured immediately prior to addition of test
compound, and I the current measured at time points after
addition of test compound. The cells were held at 70 mV and then step
depolarizations to +10 mV were made. In these experiments, the patch
pipette contained 0.1 mM (A1 and B1)
or 1 mM (A2 and B2) BAPTA. Control
cells (A1 and A2), and cells perfused with 10 µM SP (B1 and B2). The
crossed lines indicate the calculated time where the current
was decreased by 50% (IT50, see Equation 1
under "Experimental Procedures"). In A1 and
B1, the data shown are from 5 separate cells, and in
A2 and B2 from 7 separate cells each.
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In some studies, BAPTA has been shown to act as a kinase inhibitor
(43). To test whether the effect of SP was mediated via activation of
protein kinases, the cells were treated with 200 nM
staurosporine. Following a 15-min pretreatment of the cells with
staurosporine, SP still (in the continuous presence of staurosporine) inhibited IBa in a manner similar to that
observed in control cells (in these experiments, the
IT50 value was 149 ± 36 s,
n = 3; p < 0.05).
It is well known that G proteins may have a constitutive inhibitory
effect on VOCCs, and that agonist-evoked inhibition of VOCCs may be
mediated via a G protein-dependent mechanism (see Ref. 44).
However, in GH4C1 cells, G proteins seem to
have a very modest effect on the VOCCs, as evaluated using the
double-pulse protocol (to release the possible functional inhibition of
Ca2+ channels by G protein (45); Fig.
3). In these experiments, we could not
detect any difference in IBa. Addition of 2 mM GDP
S (to inhibit G protein activation) to the pipette
solution had no observable effects on the inhibitory action of SP on
the calcium channel current. In these experiments, the
IT50 value was 87 ± 14 s
(n = 4; p < 0.05, Fig. 3). Taken
together, the above results exclude G proteins as the mediators of the
observed SP-evoked inhibition of the VOCCs.

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Fig. 3.
G proteins do not modify
IBa in GH4C1 cells.
A, double-pulse protocol to release functional inhibition of
VOCCs by G proteins. A1, pulse protocol. The cell was held
at 70 mV and then depolarized to +10 mV. The cell was then
depolarized first to +100 mV and then to +10 mV. The interval between
the pulses in the double-pulse experiment was 1 ms. A2,
current traces obtained using the protocol depicted in A1.
The patch pipette contained 0.1 mM BAPTA. A3, an
experiment identical to that shown in A2, except that the
pipette also contained 300 µM GTP S. B, the
cell was held at 70 mV and then step depolarizations to +10 mV were made as described in Fig. 2. The patch pipette contained 0.1 mM BAPTA and 2 mM GDP S. When a stable
current was obtained, the cell was perfused with 10 µM
SP. In this experiment, the IT50 was 118 s.
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Effects of SP on Single Ca2+ Channels in
GH4C1 Cells--
To test whether SP has a
direct effect on the VOCCs, single-channel analyses using the
cell-attached mode of the patch clamp technique were performed with SP
in the pipette solution. SP rapidly inhibited the Ba2+
current (Fig. 4), suggesting a direct
effect of SP on the VOCCs. An analysis of the kinetic characteristics
of the single channels showed that SP inhibited the VOCCs mainly
by increasing the closed time of the channels (Fig.
5 and Table
I). For the control cells, two
conductance states were found. Using 3 µM and higher
concentrations of SP, only one conductance amplitude could usually be
detected. The values of the single-channel dwell times and amplitudes
are summarized in Table I. When the pipette solution contained 10 µM SP, no openings of the VOCCs were observed in 7 out of
9 cells.

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Fig. 4.
Effect of SP as seen in single-channel
recordings in GH4C1 cells. The experiments
were performed using the cell-attached patch-clamp configuration.
A1, consecutive sweeps from recordings in a control cell.
A2, calculated open probability
(P(open)) of a single L-type Ca2+
channel during a 200-ms depolarization to +10 mV for the cell shown in
A1. The consecutive sweeps shown in A1 are
depicted by a solid bar. B, consecutive sweeps
(B1) and calculated P(open) (B2) from a recording with 3 µM sphingosine in
the pipette. The P(open) in consecutive sweeps
was calculated using the routine provided by the pClamp 6 software. The
results in A and B are from separate cells.
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Fig. 5.
Effect of sphingosine on amplitude histograms
and dwell times in single-channel recordings in
GH4C1 cells. The left panels
depict the results obtained with a control cell, and the right
panels recordings with 5 µM SP in the pipette. The
results are from two separate cells. A, unitary currents. By
F-comparison, a two-unitary current model was chosen for the
best fit of the experimental data. For the control cell, the currents
are 0.417 and 0.575 pA, and for the SP-treated cell 0.544 and 0.672 pA. B, closed time probability histogram. Two closed states were
selected for the best fit of the data by F-comparison. The
closed times are 0.334 and 3.215 ms for the control cell, and 0.509 and
11.283 ms for the SP-treated cell. C, open time histogram. A
single open state was found. For the control cell, the open time was
0.553 ms, and for the sphingosine-treated cell 0.310 ms.
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Table I
Effect of sphingosine on the amplitude and kinetics of single VOCCs
Sphingosine was added to the electrode filling solution at the
concentrations indicated. When the pipettes contained 3
µM SP, it proved difficult to measure two current
amplitudes. When 10 µM SP was tested no openings were
observed in 7 out of 9 cells tested. The results obtained in the two
other cells were impossible to analyze. The values given are the
mean ± SE, if not indicated differently.
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Action of Sphingosine on Excised Outside-out Patches--
To
exclude an effect of cytosolic factors, we tested the action of
sphingosine on excised outside-out patches. In these experiments, the
free Ca2+ concentration was buffered to 100 nM.
This Ca2+ concentration is below the resting intracellular
Ca2+ concentration, which in our
GH4C1 cells was 204 ± 15 nM
(mean ± S.E., n = 6) as determined with Fura 2. After depolarization of the patch membrane to
10 mV, frequent
openings of one or, rarely, two Ca2+ channels were observed
(Fig. 6). We observed a substantial
run-down of the channels during the first 5 min of the recording. Thus, sphingosine was usually applied within 1 min of the recordings. The
calculated dwell times of the channels and their amplitudes (Table
II) were quite similar to those obtained
in the cell-attached recordings, suggesting that the functional
properties of Ca2+ channels were well preserved in the
outside-out recordings. Interestingly, in the outside-out
configuration, we observed a second long-lasting open state of the
Ca2+ channels, which was not observed in cell-attached
recordings (Table II and Fig. 6). Presently, we do not have an
explanation for this observation.

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Fig. 6.
Calcium currents in an excised outside-out
patch from GH4C1 cells. A, the trace
shows a 500 ms pulse to 10 mV from a holding potential of 50 mV.
Single-channel openings to one main conductance and one subconductance
state can be observed. Furthermore, the channels had two open state
dwell time probabilities and two closed states (see Table II).
B, open probability (P(open)) histogram of consecutive
sweeps from the cell shown in A.
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Table II
Characterization of the amplitudes and kinetics of single VOCCs in
outside-out patches
The values given are the mean ± S.E. of 10 patches from separate
cells.
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Application of 10 µM sphingosine led to a dramatic
decrease in the open probability of the channels (Fig.
7). In 7 out of 8 cells, a complete
inhibition of the channel activity was observed within 1 min after
application of sphingosine.

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Fig. 7.
Effect of SP on an excised outside-out patch
from GH4C1 cells. Upper panel, open
probability (P(open)) of a single Ca2+ channel calculated from a 500-ms pulse to -10 mV
before and after application of 10 µM sphingosine.
Bottom panels, the traces show original recordings used for
calculating the open probabilities before (A) and after
(B) application of sphingosine. The arrows in the
upper panel mark the calculated open probabilities
corresponding to respective traces. Similar results were obtained in 9 other patches from 9 other cells.
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 |
DISCUSSION |
In the present study, we have investigated the effect of SP on
VOCCs in GH4C1 cells. Our results suggest that SP inhibits the
VOCC-mediated current by prolonging the closed state of the channels,
apparently in a calcium-dependent manner. Our present study
is the first to show such properties of SP.
An important kind of modulation of VOCCs occurs via G protein-mediated
mechanisms. In some cell types, the effects of SP derivatives have been
shown to be in part mediated by a G protein (8, 46, 47). Furthermore,
several recent reports strongly suggest the existence of membrane
receptors for SP derivatives, and this putative receptor appears to be
coupled to a pertussis toxin-sensitive G protein (48-50). However, by
using several manipulations known to affect G
protein-dependent mechanisms (i.e. the
double-pulse protocol to release the possible functional inhibition of
Ca2+ channels by G protein (45), and by including GDP
S
in the pipette solution), we could not influence the action of SP.
Thus, an effect of SP mediated via a G protein seems unlikely in the
present work.
Sphingosine derivatives have been reported to inhibit the release of
sequestered calcium in excitable cells (Refs. 16 and 51; but see also
Ref. 15). We have been unable to detect an SP-evoked increase in
[Ca2+]i in intact GH4C1
cells using Fura 2 (34); thus, we think it unlikely that SP could
inhibit the channels through the mobilization of intracellular
Ca2+. Nevertheless, our study showed that in the presence
of strong intracellular Ca2+ buffering (achieved by
including 1-10 mM BAPTA in the pipette solution), the
effect of SP was abolished. This result suggests, but does not prove,
that calcium is necessary for the SP-evoked inhibition of VOCCs. In
control cells, the concentration of BAPTA in the pipette did not affect
the IT50 value of the run-down of the VOCCs.
Another possibility is that BAPTA inhibited a kinase in our cells, as
recent studies have indicated that BAPTA may inhibit tyrosine kinases
(43). This explanation appears unlikely, as pretreatment of the cells
with the potent kinase inhibitor staurosporine neither abolished nor
potentiated the effect of SP.
A striking effect of SP was observed on the kinetics of single VOCCs.
Using 5 µM and higher concentrations of SP, we found that
the single channels were inhibited almost immediately after the
gigaseal formation. Also in the excised outside-out experiments, the
effect of SP on the open probability of the channels was almost immediate. These results suggest that SP inhibits the VOCCs directly (or possibly via a membrane-delimited action). We observed that SP
significantly prolonged the closed time of the channels, without significant effects on either the amplitude, or the open time probability. These data suggest that SP is not an open-channel blocker.
Similar results were found recently for the SP-evoked inhibition of
single K+-channels in smooth muscle cells (52). In
addition, Yasui and Palade (53) suggested that the effect of SP on
VOCCs in ventricular myocytes could be due to an effect of SP on
channel gating. However, no single-channel experiments were performed
in their study.
In the present report, we did not evaluate which types of VOCCs were
suppressed by SP. In a recent report, we have shown data suggesting
that SPs apparently inhibited the L-type of VOCCs (34). Theoretically,
the effect of SP could be mediated via binding to the dihydropyridine
binding site in the VOCCs. However, preliminary binding experiments
showed that SP did not inhibit the binding of the dihydropyridine
antagonist [3H]PN 200-110 to
GH4C1
cells.2 Furthermore,
considering that at least four different binding sites for antagonists
to the VOCCs are known (54), we cannot exclude the possibility that SP
could bind to some other known site than that of dihydropyridines.
In conclusion, we have shown that SP, possibly directly or via a
membrane-delimited action, inhibits VOCCs in
GH4C1 cells by increasing the closed time
probability of the channels. The action of SP apparently requires free
intracellular Ca2+. Although the relatively rapid run-down
of the channels in the present study precluded an investigation on the
reversibility of the SP-evoked inhibition, our previous study clearly
showed an almost total recovery of calcium entry after washout of SP (34). Thus, the effects of SP are not the result of an irreversible blockade of the channels. The physiological significance of SP in the
regulation of pituitary cell function is still unclear. However, in
preliminary studies, we have been able to measure significant
endogenous levels of SP in GH4C1
cells.3 Considering that the
regulation of pituitary hormone synthesis and secretion is critically
dependent on intracellular Ca2+ dynamics, SP may be an
important regulator of pituitary cell function.
 |
FOOTNOTES |
*
This work was supported by the Sigrid Juselius Foundation,
the Novo Nordisk Foundation, the Liv och Hälsa Foundation, and the Ella and Georg Ehrnrooth Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Biology,
Åbo Akademi University, BioCity, Artillerigatan 6, 20520 Turku, Finland. Fax: 358-2-265-4748.
1
The abbreviations used are: SP, sphingosine;
PKC, protein kinase C; VOCC, voltage-activated calcium channel;
,
ohm(s); GDP
S, guanyl-5
-yl thiophosphate; GTP
S, guanosine
5
-3-O-(thio)triphosphate; HBSS, Hepes-buffered salt
solution.
3
K. Törnquist and H. Vuorela, unpublished
results.
2
L. Karhapää and K. Törnquist,
unpublished observation.
 |
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