Sphingosine Inhibits Voltage-operated Calcium Channels in GH4C1 Cells*

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 (I Ba) 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 GDPβS (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.

In the present study we investigated the mechanism of inhibitory action of sphingosine (SP) on voltage-activated calcium channels (VOCCs) in pituitary GH 4 C 1 cells. Using the patch-clamp technique in the whole-cell mode, we show that SP inhibits Ba 2؉ currents (I Ba ) 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 Ba 2؉ 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 GH 4 C 1 cells, and that calcium may be a cofactor in this inhibition.
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 depolariza-tion-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, VOCCdependent 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 ([Ca 2ϩ ] i ) are of crucial importance for the regulation of hormone synthesis and secretion (18,19). The increase in [Ca 2ϩ ] 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)(24)(25)(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 [Ca 2ϩ ] i (31).
Stimulating GH 3 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 GH 4 C 1 pituitary cells (34). This effect was independent of an action of PKC. In GH 4 C 1 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 GH 4 C 1 cells. Using whole-cell, single-channel, and outside-out patch-clamp methods, we show that SP inhibits the gating of VOCCs in GH 4 C 1 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). Cell Culture-Clonal rat pituitary GH 4 C 1 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-10ϩ medium) in a water-saturated atmosphere of 5% CO 2 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-10ϩ 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 Hepesbuffered salt solution (HBSS-II buffer, containing (in mM): CsCl, 120; MgCl 2 , 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 Ϫ90 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 l), and the cells were extensively perfused with HBSS-I (containing (in mM): BaCl 2 , 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 Ϫ70 mV with test pulses to Ϫ20 mV and ϩ10 mV made throughout the experiment to avoid additional rundown of the channels. For the calculations, we used data derived from pulses to ϩ10 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 GH 4 C 1 cells.
To calculate the half-inactivation time (IT 50 ), 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).
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; MgCl 2 , 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): BaCl 2 , 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 singlechannel 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 CaCl 2 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 Ϫ50 mV to obtain more stable patches than those obtained at Ϫ70 mV, with test pulses to Ϫ10 mV and ϩ10 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 GH 4 C 1 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 patchclamp technique, we now found that 10 M 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 (IT 50 ) for the rundown was 267 Ϯ 42 s, whereas in the presence of 10 M SP, IT 50 was 143 Ϯ 8 s (p Ͻ 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.
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 I Ba in a manner similar to that observed in control cells (in these experiments, the IT 50 value was 149 Ϯ 36 s, n ϭ 3; p Ͻ 0.05).

FIG. 3. G proteins do not modify I Ba in GH 4 C 1 cells.
A, double-pulse protocol to release functional inhibition of VOCCs by G proteins. A1, pulse protocol. The cell was held at Ϫ70 mV and then depolarized to ϩ10 mV. The cell was then depolarized first to ϩ100 mV and then to ϩ10 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 M GTP␥S. B, the cell was held at Ϫ70 mV and then step depolarizations to ϩ10 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 M SP. In this experiment, the IT 50 was 118 s. 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 GH 4 C 1 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 Ca 2ϩ channels by G protein (45) ; Fig. 3). In these experiments, we could not detect any difference in I Ba . 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 IT 50 value was 87 Ϯ 14 s (n ϭ 4; p Ͻ 0.05, Fig. 3). Taken together, the above results exclude G proteins as the mediators of the observed SP-evoked inhibition of the VOCCs.
Effects of SP on Single Ca 2ϩ Channels in GH 4 C 1 Cells-To test whether SP has a direct effect on the VOCCs, singlechannel analyses using the cell-attached mode of the patch clamp technique were performed with SP in the pipette solution. SP rapidly inhibited the Ba 2ϩ 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 M 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 M SP, no openings of the VOCCs were observed in 7 out of 9 cells.
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 Ca 2ϩ concentration was buffered to 100 nM. This Ca 2ϩ concentration is below the resting intracellular Ca 2ϩ concentration, which in our GH 4 C 1 cells was 204 Ϯ 15 nM (mean Ϯ S.E., n ϭ 6) as determined with Fura 2. After depolarization of the patch membrane to Ϫ10 mV, frequent openings of one or, rarely, two Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ channels, which was not observed in cell-attached recordings (Table II and Fig. 6). Presently, we do not have an explanation for this observation.
Application of 10 M 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.

DISCUSSION
In the present study, we have investigated the effect of SP on VOCCs in GH4C1 cells. Our results suggest that SP inhibits

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 Ն3 M SP, it proved difficult to measure two current amplitudes. When 10 M 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 Ϯ SE, if not indicated differently. 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 Ca 2ϩ 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 [Ca 2ϩ ] i in intact GH 4 C 1 cells using Fura 2 (34); thus, we think it unlikely that SP could inhibit the channels through the mobilization of intracellular Ca 2ϩ . Nev- FIG. 6. Calcium currents in an excised outside-out patch from GH 4 C 1 cells. A, the trace shows a 500 ms pulse to Ϫ10 mV from a holding potential of Ϫ50 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   ertheless, our study showed that in the presence of strong intracellular Ca 2ϩ 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 IT 50 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 M 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 ϩchannels 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 Ltype 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 [ 3 H]PN 200-110 to GH 4 C 1 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 GH 4 C 1 cells by increasing the closed time probability of the channels. The action of SP apparently requires free intracellular Ca 2ϩ . 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 GH 4 C 1 cells. 3 Considering that the regulation of pituitary hormone synthesis and secretion is critically dependent on intracellular Ca 2ϩ dynamics, SP may be an important regulator of pituitary cell function.