INTRODUCTION

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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

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Materials-- Fura 2-AM was obtained from Molecular Probes (Eugene, OR). Sphingosine, staurosporine, GTPS, GDPS, 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.

(Eq. 1)

	RESULTS

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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 I_Ba (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.

View larger version (14K): [in this window] [in a new window]   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.

View larger version (30K): [in this window] [in a new window]   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.

View larger version (16K): [in this window] [in a new window]   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 GTPS. 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 GDPS. When a stable current was obtained, the cell was perfused with 10 µM SP. In this experiment, the IT50 was 118 s.

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.

View larger version (42K): [in this window] [in a new window]   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.

View larger version (22K): [in this window] [in a new window]   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.

View this table: [in this window] [in a new window]   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.

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.

View larger version (16K): [in this window] [in a new window]   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.

View larger version (19K): [in this window] [in a new window]   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.

	DISCUSSION

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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 GDPS 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.