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Endocrinology Vol. 140, No. 12 5682-5690
Copyright © 1999 by The Endocrine Society

ARTICLES

Ceramide Inhibits L-Type Calcium Channel Currents in Rat Pinealocytes1

C. L. Chik, B. Li, T. Negishi, E. Karpinski and A. K. Ho

Departments of Physiology and Medicine (C.L.C.), Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Address all correspondence and requests for reprints to: Dr. C. L. Chik, Department of Medicine, 7–33 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7. E-mail: cchik{at}ualberta.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In rat pinealocytes, ceramide can inhibit the KCl- and BayK 8644-mediated potentiation of cAMP and cGMP accumulation, suggesting that the L-type Ca2+ channel is a target of ceramide action. This was examined in the present study using intracellular Ca2+ measurement and patch-clamp studies. In fura-2-loaded pinealocytes, C2- and C6-ceramide inhibited the Ca2+ increase caused by BayK 8644 and KCl, but not that caused by norepinephrine, suggesting an inhibitory effect of ceramide on the L-type Ca2+ channels. Patch-clamp analysis confirmed that C2- and C6-ceramide, but not C2-dihydroceramide (the inactive analog) inhibited the L-type Ca2+ channel current. Furthermore, treatments known to increase cellular ceramide levels, including a glucosylceramide synthase inhibitor and sphingomyelinase, also inhibited this current. The inhibitory effect of ceramide on the current was attenuated by lavendustin A, a tyrosine kinase inhibitor, but not by H7, a serine/threonine kinase inhibitor. The effect of ceramide was mimicked by interleukin-1ß, a cytokine highly expressed in the pineal that is known to activate the sphingomyelin pathway. These results indicate that the sphingomyelin pathway is another important signaling mechanism that regulates the L-type Ca2+ channel, and tyrosine kinase appears to be involved in the effect of ceramide.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
L-TYPE Ca2+ channels (L-channels) play an important role in the control of pineal function, including melatonin synthesis (1, 2). Using the whole cell version of the patch-clamp technique, we have shown that rat pinealocytes express the L-type, but not the T- or N-type, Ca2+ channel (3, 4). These channels are activated by the nicotinic subtype of cholinergic receptors (1, 2). They are also regulated by adrenergically mediated mechanisms through cAMP- and cGMP-dependent protein kinases (3, 4). Growth factors, including insulin-like growth factor I and insulin, have also been shown to modulate these channels through a tyrosine kinase-dependent mechanism (5).

Another signaling mechanism that could modulate Ca2+ channel activity is the sphingomyelin pathway (6). This pathway mediates the action of cytokines such as interleukin-1ß, interferon-{gamma}, and tumor necrosis factor-{alpha} (7, 8, 9). Ceramide is produced after sphingomyelin hydrolysis by activation of a sphingomyelinase (9). The ceramide generated can function as a second messenger (9). Direct targets of ceramide that have been identified include specific kinases and phosphatases that are coupled to signaling mechanisms, including mitogen-activated protein kinases and phospholipase D (10, 11, 12, 13, 14). The importance of kinases and phosphatases in the modulation of L-channels in rat pinealocytes (3, 4, 5, 15) suggests that L-channels are probably regulated by ceramide in rat pinealocytes. This was also supported by our recent observation that ceramide selectively inhibits the KCl- and BayK 8644-mediated potentiation of ß-adrenergically stimulated cAMP and cGMP accumulation (16). In this study, we investigated whether ceramide had an acute effect on the pineal L-channels using intracellular Ca2+ measurement and patch-clamp analysis. We found that ceramide inhibits the L-channels in rat pinealocytes and that this inhibition is mediated in part by tyrosine kinases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Sphingomyelinase was obtained from Sigma Chemical Co. (St. Louis, MO). BayK 8644, C2- and C6-ceramide, C2-dihydroceramide, H89, human recombinant interleukin-1ß, KT5823, lavendustin A, lavendustin B, and 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP) were obtained from Calbiochem (La Jolla, CA). Fura-2/AM was obtained from Molecular Probes, Inc. (Eugene, OR). Cs2-aspartate was prepared by Dr. H. J. Liu (Department of Chemistry, University of Alberta, Edmonton, Canada). [125I]cAMP and [125I]cGMP were obtained from ICN Immunobiologicals (Lisle, IL). All other chemicals were of the purest grade available and were obtained commercially. Antibodies for the RIAs of cAMP and cGMP were gifts from Dr. A. Baukal (NICHHD, NIH, Bethesda, MD).

Cell preparations
All procedures were reviewed and approved by the health sciences animal and welfare committee of the University of Alberta. Male Sprague Dawley rats (150 g) were decapitated after cervical dislocation. Pinealocytes were then prepared by trypsinization as described previously (17). The cells were suspended in DMEM containing 10% FCS and were maintained overnight at 37 C in a mixture of 95% air and 5% CO2.

Ca2+ channel current recordings were obtained using the whole cell version of the patch-clamp technique (18). Patch electrodes were pulled from borosilicate glass capillary tubes (od, 1.2 mm; id, 0.9 mm; FHC, Brunswick, ME) and heat polished. They were filled with a solution containing 70 mM Cs2-aspartate, 20 mM HEPES, 11 mM EGTA, 1 mM CaCl2, 5 mM MgCl2·6H2O, 5 mM glucose, 5 mM ATP-Na2, and 5 mM K-succinate. Creatine phosphokinase (50 U/ml) and phosphocreatine-Na2 (20 mM) were added to the pipette solution to reduce current run down. The bath solution contained 105 mM Tris-Cl, 0.8 mM MgCl2·6H2O, 5.4 mM KCl, 20 mM BaCl2, 0.02 mM tetrodotoxin, and 10 mM HEPES. Ba2+ (20 mM) was used as the charge carrier. All solutions were filtered (0.22 µm) before use. The osmolarity was adjusted to 320 mosmol, and the pH was adjusted to 7.4. The membrane currents were measured using an Axopatch 1B whole cell patch-clamp amplifier (Axon Instruments, Foster City, CA). The data were filtered at 5 kHz and sampled at 10 kHz using pClamp software (pClamp 7) and a Digidata 1200B analog to digital interface (Axon Instruments, Foster City, CA). Analysis was performed using the pClamp software. To generate current-voltage (I-V) relationships, 250-msec depolarizing test pulses of increasing amplitude were applied at a frequency of 0.3 Hz. On-line leakage subtraction was implemented using the P/2 protocol in pClamp software. At a holding potential of -50 mV and with Cs+ in the internal solution, hyperpolarizing pulses did not activate any currents, and identical results were obtained with the P/2 or P/4 protocol. The experiments were performed at room temperature (20–22 C).

Pineal cells were evaluated for current run down before they were used for experiments. After the whole cell configuration was established, the current amplitude increased for 2–3 min due to inhibition of the outward K+ current by intracellular Cs+. When the current reached its peak amplitude, it was monitored for an additional 5 min to estimate the run down rate. In 90% of cells, the initial run down rate was less than 5%, and a stable current could be recorded for the next 30 min. In 10% of cells, the initial run down rate was more than 5%. These cells tended to continue to run down and were not used for experiments. If the initial run down rate was less than 5%, the drugs were added to the bath solution after 5 min.

The steady state inactivation and activation as described by the normalized conductance was obtained from deactivation tail current amplitude, normalized, and fitted to a Boltzman function of the form G = Gmax(1/1 + exp(V1/2 - Vx)/k), where G is the normalized conductance, Gmax is the maximal conductance, V1/2 is the voltage at which half of the macroscopic current is activated, Vx is the test voltage, and k is the slope. The magnitude of the tail currents was obtained by fitting the data to two exponential components. The fitting of exponentials to whole cell tail currents was performed using Clampfit (version 6.05). A simplex algorithm was used to find the sum of least square error fits. Iterative fits converged when the simplex fractional error became less than 0.0001. In all fits, the first 250 µsec were ignored to avoid artifacts produced by the settling of the voltage clamp. The amplitude values that were used to construct the normalized conductance curves were extrapolated to the time of repolarization. The tail currents for activation curves were activated from a holding potential of -50 mV to various test potentials and repolarized to -50 mV. To obtain the steady state inactivation, the holding potential was varied from -90 to +25 mV. The test pulse was +50 mV, with repolarization to -50 mV.

Data are presented as the mean ± SEM percentage of control values. At least three different cell preparations were used for each study. The pretreatment I-V relationship was plotted and used as a control. The effects of the drugs were monitored continuously using depolarizing pulses at a frequency of 0.03 Hz, except when generating I-V relationships. Paired t test was used for comparison between control values and those obtained after drug administration. All histograms were corrected for average current rundown at 15 min, which was 5.7 ± 0.4% (n = 6). In the case of multiple comparisons, ANOVA in conjunction with the Newman-Keuls test was applied.

cAMP and cGMP assays
cAMP and cGMP measurements were made on samples of cells (15,000 cells/400 µl) treated with various agents for 15 min; the RIA method of measurement has been described in detail previously (19, 20).

Determination of intracellular Ca2+[Ca2+]i
[Ca2+]i was determined using a fluorescent Ca2+ indicator, fura-2 (21, 22). Briefly, 5 x 105 cells were pelleted and resuspended in culture medium (DMEM with 25 mM HEPES, pH 7.4). The cells were loaded with fura-2 by incubation with fura-2 (5 µM) for 45 min at 37 C. After washing twice with DMEM, the pinealocytes were suspended in a fresh buffered salt solution (buffer B; 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.2 mM MgCl2, 1.2 mM KH2PO4, 25 mM HEPES, and 6 mM glucose, pH 7.4). Fura-2-loaded cells (3 x 105 cells/1.5 ml) were transferred to a cuvette for fluorescence signal determination, using an SLM Aminco DMX-1000 fluorescence spectrophotometer (SLM Instruments, Inc., Urbana, IL) with a thermostatically controlled cell holder fitted with a magnetic stirrer. The excitation wavelengths used were 340 and 380 nm, and emission was monitored at 510 nm. Paired t test was used for analysis of [Ca2+]i measurements. Statistical significance was set at 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
C6-ceramide inhibits KCl and BayK 8644 potentiation of isoproterenol-stimulated cyclic nucleotide accumulation
Treatment of pinealocytes with isoproterenol (1 µM) caused a significant increase in cAMP and cGMP accumulation; the addition of depolarizing concentrations of K+ potentiated the isoproterenol-stimulated cAMP and cGMP accumulation in a concentration-dependent manner as reported previously (16, 23) (Fig. 1Go). C6-ceramide (30 µM) had no effect on basal cAMP and cGMP accumulation (Table 1Go). However, C6-ceramide caused an increase in the EC50 value of the potentiation by KCl without affecting the maximal potentiation (Fig. 1Go). Similar to our previous study (16), C2-ceramide (30 µM) also inhibited the KCl- and BayK 8644-potentiation of isoproterenol-stimulated cyclic nucleotide accumulation (Table 1Go). In contrast, C2-dihydroceramide (30 µM), the inactive analog, was ineffective (Table 1Go). These results suggest that the L-channel may be a target of ceramide action.



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  		Figure 1. Effect of C6-ceramide on isoproterenol (ISO)- and KCl-stimulated cAMP and cGMP accumulation in rat pinealocytes. Pinealocytes (1.5 x 104 cells/400 µl) were incubated in DMEM with 10% FBS in the absence or presence of C6-ceramide (C6; 30 µM) for 5 min. The cells were then stimulated with ISO (1 µM) in the presence of varying concentrations of KCl (10–50 mM) for an additional 15 min, and cellular cAMP and cGMP contents were determined. Each value represents the mean ± SEM of determinations performed in quadruplicate from three independent experiments. For further details, see Materials and Methods. *, P < 0.05 compared with the corresponding treatment without ceramide.

 

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  		Table 1. Effects of ceramides on the potentiating effect of K+ and BayK 8644 on ISO-stimulated cAMP and cGMP responses

 
Ceramide reduces KCl- and BayK 8644-induced increases in [Ca2+]i, but not that induced by norepinephrine
In fura-2-loaded pinealocytes, a depolarizing concentration of K+ (30 mM KCl) or BayK 8644 (1 µM) caused an increase in [Ca2+]i, as in previous studies (21) (Fig. 2Go and Table 2Go). C6-ceramide, C2-ceramide, or C2-dihydroceramide (30 µM) caused a small increase in the fluorescent signal (Fig. 2Go and Table 2Go), which was caused by autofluorescence, as similar increases were observed in a cell-free system. Treatment with C6- or C2-ceramide was effective in reducing the increase in [Ca2+]i caused by BayK 8644 or a depolarizing concentration of K+, whereas C2-dihydroceramide was ineffective (Fig. 2Go and Table 2Go). In contrast, neither C6- nor C2-ceramide had an effect on the increase in [Ca2+]i caused by norepinephrine (1 µM; Fig. 2Go and Table 2Go).



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  		Figure 2. Effects of ceramides on KCl- and norepinephrine-mediated increases in intracellular Ca2+ in rat pinealocytes. Rat pinealocytes were prepared and loaded with the fluorescent Ca2+ indicator fura-2. The ratio of the fluorescence emission signal at 510 nm, excited at 340 and 380 nm, was continuously recorded and calibrated as described. The traces are representative of at least three experiments. a, Addition of norepinephrine (NE; 1 µM) alone; b, addition of C6-ceramide (C6; 30 µM) followed by NE (1 µM); c, addition of C2-dihycroceramide (DH-C2; 30 µM) followed by NE (1 µM); d) addition of KCl (30 mM) alone; e) addition of C6 (30 µM) followed by KCl (30 mM); and f) addition of DH-C2 (30 µM) followed by KCl (30 mM). C6-ceramide decreased the KCl-stimulated, but not the NE-stimulated, increases in intracellular Ca2+. For further details, see Materials and Methods.

 

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  		Table 2. Effect of ceramides and PPMP on KCl-, BayK 8644-, and norepinephrine-mediated increases in intracellular Ca2+

 
Treatment with a glucosylceramide synthase inhibitor, PPMP, has been shown to increase cellular ceramide levels (24, 25). PPMP (10 µM), like C6- and C2-ceramide, was effective in reducing the increase in [Ca2+]i caused by BayK 8644 or by a depolarizing concentration of K+. However, PPMP (10 µM) had no effect on the increase in [Ca2+]i caused by norepinephrine (Table 2Go).

Ceramide inhibits the L-type Ca2+ channel current in rat pinealocytes
As reported previously, the only voltage-dependent Ca2+ channel current found in dissociated pinealocytes is the dihydropyridine-sensitive L-channel current (3, 4, 5). The effect of C6-ceramide on the L-channel current is shown in Fig. 3Go. The current was activated by depolarizing the cell from -50 to 10 mV before and after C6-ceramide (30 µM; Fig. 3AGo). C6-ceramide (30 µM) decreased the peak amplitude of the L-channel current by about 30% (Fig. 3AGo). The data from eight cells were normalized to the peak inward current and plotted as the I-V relationship, as shown in Fig. 3BGo. Inhibition of the L-channel current by C6-ceramide was dependent on concentration, with an estimated EC50 value of 14 µM; a small inhibition was observed at 5 µM, and the maximal inhibition observed was about 30% at a concentration of 50 µM (Fig. 3CGo).



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  		Figure 3. Effect of C6-ceramide on the L-channel current in rat pinealocytes. A, The L-channel current activated by depolarizing the pinealocyte from -50 to 10 mV in the absence (•) or presence ({circ}) of C6-ceramide (C6; 30 µM). B, The combined I-V relationship from a group of cells in the absence (•) or presence ({circ}) of C6 (30 µM; n = 8). The data were normalized to the peak inward control current. C, The effect of C6 on the L-channel current as a function of concentration. The inhibition 15 min after the addition of C6 was used to obtain the data points. The n values are shown in parentheses above the data points. *, P < 0.05 compared with the control current.

 
The effect of C6-ceramide (30 µM) on the L-channel current as a function of time is shown in Fig. 4AGo. The onset of the inhibition caused by C6-ceramide occurred within 7–10 min, and maximal inhibition was observed between 12–15 min. The effect of C6-ceramide was not reversed by wash out (data not shown). The current run down from a group of control cells is included for comparison (Fig. 4AGo). The average current run down at 15 min was 5.7 ± 0.4% (n = 6; Fig. 4AGo). Although less effective than C6 ceramide, C2-ceramide (30 µM) also inhibited the L-channel current (Fig. 4AGo). The inhibition by C2- and C6-ceramide persisted in the presence of BayK 8644 (Fig. 4BGo). In contrast, C2-dihydroceramide (30 µM), an inactive analog, had no effect on basal or BayK 8644-stimulated L-channel current (Fig. 4BGo).



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  		Figure 4. Effect of ceramides on basal and BayK 8644-activated L-channel current in rat pinealocytes. A, The effect of C2-ceramide (C2; 30 µM) and C6-ceramide (C6; 30 µM) on the L-channel current as a function of time. The peak L-channel current at 1-min intervals was normalized to the current 5 min after the whole cell configuration was established and plotted as a percentage of the control current. Ceramides were applied as shown, and the maximal effect was observed 15 min later. Also shown for comparison is the L-channel current rundown (CTRL). B, The combined data 15 min after the addition of C6 (30 µM), C2 (30 µM), or C2-dihydroceramide (DH-C2; 30 µM). Also shown in B is the decrease in L-channel current caused by ceramides in cells pretreated with BayK 8644 (1 µM) for 5 min. The n values are shown in parentheses above the data point. *, P < 0.05 compared with the corresponding treatment without ceramide.

 
Further characterization of the effect of C6-ceramide on the L-channel current was carried out by measuring its effect on deactivation tail currents. The deactivation tail currents before and after C6-ceramide (30 µM) treatment are shown in Fig. 5AGo. The solid lines are the exponential fits to the data. C6-ceramide decreased both the amplitude of the peak current and the deactivation tail current (Fig. 5AGo). In Fig. 5BGo, steady state inactivation and activation as described by normalized conductance are shown. C6-ceramide (30 µM) had no effect on the steady state inactivation, but shifted the activation curve toward more positive membrane potentials. The data points after C6-ceramide at 0, 10, and 20 mV are significantly different from those before treatment with C6-ceramide (P < 0.05; Fig. 5BGo).



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  		Figure 5. Effect of C6-ceramide on L-channel steady state activation and inactivation. A, The deactivation tail currents before and after C6-ceramide (C6; 30 µM) were obtained by depolarizing the cell from -50 to 10 mV and then repolarizing to -50 mV. The solid lines are the curve fits extrapolated to the time of repolarization. C6 decreased the magnitude of the inward current and the deactivation current tail. B, Using the peak extrapolated amplitude of the deactivation tails, normalized steady state inactivation and activation functions were constructed. The control data points (• and {blacksquare}) were fitted to a Boltzman function (solid lines) of the type G = Gmax(1/1 + exp(V1/2 - Vx)k), where G is the normalized conductance, Gmax is the maximal conductance, V1/2 is the voltage at which half of the macroscopic current is activated, Vx is the test voltage, and k is the slope (V1/2 = -18.0 mV; k = -8.0 for the inactivation curve and V1/2 = -8.0 mV; k = 7.5 for the activation curve; n = 6). The data points after C6 are shown as {circ} and {square}. C6 had no significant effect on the steady state inactivation. However, C6 shifted the steady state activation toward more positive potentials, with significant differences at 0, 10, and 20 mV (*, P < 0.05).

 
Effects of PPMP and sphingomyelinase on the L-channel current
Cellular ceramide levels can be increased by treatment with PPMP, a glucosylceramide synthase inhibitor, which prevents the breakdown of ceramide (24, 25), or sphingomyelinase, which causes membrane hydrolysis of sphingomyelin. PPMP (10 µM), like C6-ceramide, caused an inhibition of the L-channel current as a function of time (Fig. 6AGo). The onset of inhibition caused by PPMP occurred within 4–6 min after drug addition, and maximal inhibition was observed between 14–16 min. The inhibition of the L-channel current persisted in BayK 8644-treated cells (Fig. 6BGo).



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  		Figure 6. Effect of PPMP and sphingomyelinase on the L-channel current in rat pinealocytes as a function of time. A, The effect of PPMP (a glucosylceramide synthase inhibitor; 10 µM) and sphingomyelinase (SMase; 0.1 U/ml) on L-channel current as a function of time. The data were analyzed as described in Fig. 4Go. Shown for comparison is the L-channel current run down (CTRL). Both SMase and PPMP were effective in decreasing the L-channel current. The time course of the inhibition was similar to that produced by C2- and C6-ceramide. B, The data obtained 15 min after the addition of PPMP and SMase. Also shown in B is the decrease in L-channel current caused by PPMP and SMase in cells pretreated with BayK 8644 (1 µM) for 5 min. The n values are shown in parentheses above the bars. *, P < 0.05 compared with the corresponding control current.

 
Sphingomyelinase (0.1 U/ml), like PPMP, inhibited the L-channel current (Fig. 6Go, A and B). The onset of inhibition caused by sphingomyelinase occurred within 6–8 min after drug addition, and a 30% reduction was observed after 17 min. These results indicate that preventing the breakdown of ceramide or increasing membrane hydrolysis of sphingomyelin causes an inhibitory effect on the L-channel current similar to that seen after the addition of C6-ceramide.

Lavendustin A, but not H7, attenuates the effect of ceramide on the L-channel current
Ceramides have been shown to stimulate different enzymes, including a ceramide-activated proline-directed protein kinase, Jun N-terminal kinase, Raf-K, and tyrosine kinase (26, 27, 28). Therefore, it is possible that the effect of ceramide can be attenuated by a kinase inhibitor. This was examined by pretreating the cells with different serine/threonine kinase inhibitors or lavendustin A, a tyrosine kinase inhibitor (29). Treatment with H7 (100 µM) alone, a nonspecific serine/threonine kinase inhibitor, for 10 min caused a small inhibition of the L-channel current (Fig. 7AGo). Pretreatment with H7 for 10 min had no effect on the inhibitory action of C6-ceramide (30 µM) on the L-channel current (Fig. 7AGo).



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  		Figure 7. Effect of H7 and lavendustin A on the C6-ceramide-mediated inhibition of L-channel currents in rat pinealocytes. A, H7 (100 µM; n = 7) caused a small inhibition of the L-channel current. In cells pretreated with H7 (100 µM; n = 7) for 10 min, C6-ceramide (C6; 30 µM) was effective in reducing the L-channel current. Shown for comparison is the effect of C6 (30 µM; n = 8; see Materials and Methods). B, Lavendustin A (LA; 3 µM; n = 8) caused a small decrease in the L-channel current. In cells pretreated with LA (3 µM; n = 6) for 10 min, the inhibition caused by C6 was attenuated. Shown for comparison is the effect of C6 (30 µM; n = 8). The n values are shown in parentheses above the bars. *, P < 0.05, compared with treatment without the kinase inhibitor.

 
The effects of two selective protein kinase inhibitors were also determined. Pretreatment with KT5823 (1 µM), a selective protein kinase G inhibitor, for 10 min did not affect the inhibition by C6-ceramide (71.5 ± 3.4% vs. 72.1 ± 5.1% of control current in the presence or absence of KT5823; n = 4). A selective protein kinase A inhibitor, H89 (0.5 µM), also had no effect on the inhibition by C6-ceramide (data not shown). In contrast, treatment with lavendustin A (3 µM) alone reduced the L-channel current by 11.5% (Fig. 7BGo). The inhibitory effect of C6-ceramide was attenuated (from 26.7% to 16.9%) in lavendustin A-pretreated cells (Fig. 7BGo). The attenuation was not observed in cells pretreated with lavendustin B (3 µM), the inactive analog of lavendustin A (data not shown).

Interleukin-1ß inhibits the L-channel current
Several cytokines, including interleukin-1ß, tumor necrosis factor-{alpha}, and interferon-{gamma}, are known to induce sphingomyelin hydrolysis to ceramide (8, 9). As abundant expression of interleukin-1ß and its specific receptor has been demonstrated in the rat pineal gland (30), the effect of interleukin-1ß on the L-channel current was examined. Interleukin-1ß (10 ng/ml), like C6-ceramide, also inhibited the L-channel current (Fig. 8AGo). The onset of inhibition caused by interleukin-1ß occurred within 8–10 min, and the maximal inhibition was observed between 14–16 min (Fig. 8BGo). Heat-inactivated interleukin-1ß had no effect on the L-channel current (data not shown).



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  		Figure 8. Effect of interleukin-1ß on the L-channel current in rat pinealocytes. A, The L-channel current is activated by depolarizing a pinealocyte from -50 to 0 mV in the absence or presence of interleukin-1ß (IL-1ß; 10 ng/ml). The I-V relationships obtained from the same cell are shown on the right. IL-1ß was effective in decreasing the L-channel current. B, The effect of IL-1ß (10 ng/ml) on the L-channel current as a function of time. The data were analyzed as described in Fig. 4Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Signaling through the sphingomyelin pathway, which is present in most mammalian cells, has generally been accepted as an important mechanism in regulating cellular processes such as growth, differentiation, apoptosis, and cell cycle arrest (31, 32). Activation of this pathway involves generation of ceramide through hydrolysis of sphingomyelin by sphingomyelinase. The ceramide produced, which acts a second messenger, has been shown to modulate the activities of different kinases as well as phosphatases in mediating its biological responses (10, 11, 12, 13). However, results from the present study suggest that ceramide, probably through activation of tyrosine kinases, also acts as an important modulator of the L-channel current.

Our earlier study on the effect of ceramide indicated that it inhibited the potentiation of cAMP and cGMP accumulation caused by [Ca2+]i-elevating agents but not that caused by an activator of protein kinase C or an {alpha}1-adrenergic agonist (16). Among the different [Ca2+]i-elevating agents, C6-ceramide was only effective in inhibiting the potentiation caused by a depolarizing concentration of K+ or by BayK 8644, agents that act specifically on the voltage-gated Ca2+ channels. C6-ceramide did not inhibit the potentiation caused by phenylephrine, ionomycin, or thapsigargin, three agents that elevate [Ca2+]i through different mechanisms. In this study, it was found that C6-ceramide caused an increase in the EC50 of the potentiation caused by depolarizing concentrations of K+, suggesting that ceramide may affect the responsiveness of the L-channels to depolarization.

By measuring [Ca2+]i directly, we were able to show that only [Ca2+]i changes mediated through the voltage-gated Ca2+ channels are affected by ceramide. In contrast, ceramide has no effect on the norepinephrine-mediated increase in [Ca2+]i, which is independent of the L-channel (33) but dependent on release from intracellular stores (34) and influx through a receptor-mediated mechanism (33). Together these data provide indirect evidence that in its inhibition of the potentiation of the cyclic nucleotide responses, ceramide is selectively preventing the elevation of [Ca2+]i via the L-type Ca2+ channel rather than inhibiting a Ca2+-mediated event.

Using the whole cell version of the patch-clamp technique, we have confirmed that ceramide has an inhibitory effect on the L-type Ca2+ channel current. This is the first report of an effect of ceramide on the {alpha}1D-subtype of Ca2+ channels (4) that are commonly found in neuroendocrine cells (35). Although ceramide caused a decrease in the peak L-channel current, there was no detectable shift in the potential at which the peak inward current occurred after treatment with C6-ceramide. However, studies with deactivation tail currents showed that C6-ceramide shifted the activation toward more positive potentials, suggesting that C6-ceramide may modify gating of these channels.

Even though our results with ceramide are obtained with C2- and C6-ceramide, two synthetic structural analogs of the naturally occurring ceramide, additional data suggest that generating ceramide endogenously has a similar effect on the L-type Ca2+ channel. This is based on the observations that treatment with PPMP, which inhibits the metabolism of ceramide and elevates cellular ceramide level (24, 25) is effective in inhibiting the L-channel current. Furthermore, treatment with exogenous sphingomyelinase, which induces the hydrolysis of sphingomyelin, also has the same effect as C2- or C6-ceramide. These results suggest that C2- and C6-ceramide, the synthetic ceramides, are simulating the effects of endogenously produced ceramide, a second messenger generated in the sphingomyelin cycle.

With regard to the mechanism through which ceramide inhibits the L-type Ca2+ channel current, ceramide has been shown to activate a novel serine/threonine protein kinase (27). However, our result argues against the involvement of this kinase in the effect of ceramide on the L-channel current, as pretreatment with a serine/threonine kinase inhibitor, H7, has no effect on the inhibition by ceramide. Although we have previously shown that the effect of ceramide on cyclic nucleotide accumulation is mediated through phosphodiesterases (16), this mechanism cannot explain its effect on the L-channel current. This is based on the observations that neither H89, a selective protein kinase A inhibitor, nor KT5823, a selective protein kinase G inhibitor, influences the effect of ceramide. Another downstream effect of ceramide is the stimulation of a cytosolic protein phosphatase (10). Although the L-channel current in rat pinealocytes can be regulated by protein phosphatases, inhibition of protein phosphatase activities reduces rather than enhances the L-channel current (15). Therefore, it is unlikely that activation of protein phosphatase is mediating the inhibitory effect of ceramide on the channel.

Another possible mechanism is that increasing the level of ceramide in the lipid bilayer may have a nonspecific lipid effect on the channels. However, this is unlikely, because both C2- and C6-ceramide produce similar effects on the current, but C2-dihydroceramide, an inactive analog of ceramide, is without effect. Furthermore, the effect of C6-ceramide can be blocked by a tyrosine kinase inhibitor. Indeed, the attenuation of the effect of ceramide by lavendustin A, a tyrosine kinase inhibitor, points toward a signaling cascade from ceramide via tyrosine kinase to the L-channel. It is of interest that the effect of ceramide on the channels in T lymphocytes is also mediated by tyrosine kinases (36). Although the identity of the specific tyrosine kinase involved remains unclear, our result suggests that ceramide is using a specific signaling pathway in mediating its effect on the L-channel. As treatment with lavendustin A also causes a small reduction of the L-channel current, tyrosine kinase appears to have a tonic effect on the basal activity of the L-channel current.

Extracellular messengers that are capable of signaling through the sphingomyelin cycle in different cell types include tumor necrosis factor-{alpha}, interleukin-1ß, interferon-{gamma}, and nerve growth factor (7, 8, 9). Among these messengers, there is a high expression of interleukin-1ß and its receptors in the rat pineal gland (30). In this study, treatment with interleukin-1ß was found to have an inhibitory effect on the L-channel current in this tissue, suggesting that interleukin-1ß may be an activator of the sphingomyelin pathway in the rat pineal gland. Although nerve growth factor is also present in the pineal gland, it has no effect on the L-channel current (5).

The pineal gland predominantly expresses the {alpha}1D-subtype of Ca2+ channels (4). This channel is commonly found in neuronal and neuroendocrine cells (35). The modulation of L-channels by ceramide represents another signaling mechanism through which these channels can be regulated in the rat pineal gland and probably other neuronal tissue. Within the pinealocytes, this regulation may be of physiological importance, as these channels are involved in the control of pineal function, including melatonin synthesis (2). It has recently been shown that acetylcholine, which depolarizes these channels, has an inhibitory effect on melatonin synthesis through the vesicular release of glutamate (2). However, it remains to be determined whether the sphingomyelin cycle has a role to play in melatonin synthesis. Nonetheless, [Ca2+]i has been shown to modulate the adrenergically mediated induction of N-acetyltransferase, the rate-limiting enzyme in melatonin production (37).

The observed effect of ceramide on the L-channels may have a more general implication apart from its contribution to pineal physiology. Our observations suggest that the sphingomyelin pathway may play an important role in the regulation of any cellular events that involve the L-channels. Considering the widespread distribution of L-channels and the physiological processes regulated by these channels (38), the potential exits that the sphingomyelin cycle may regulate cellular processes other than the established ceramide-mediated physiological events (32). In this regard, ceramide also regulates the L-channels in rat ventricular myocytes (6). Indeed, the ceramide effect on L-channels may represent one of the downstream mechanisms through which ceramide mediates its established effects on apoptosis, cell growth, and differentiation (32).

In summary, based on [Ca2+]i measurement and patch-clamp studies, our results support an inhibitory effect of ceramide on the {alpha}1D-subtype of Ca2+ channels, the specific subtype of L-channels expressed in rat pinealocytes (4). Furthermore, tyrosine kinase appears to be involved in this effect of ceramide. Although interleukin-1ß can inhibit this channel, it remains to be determined whether this cytokine can activate the sphingomyelin cycle in the rat pineal gland. Our results are of importance not only to the understanding of pineal physiology, but also to neuroendocrinology in general.


   Acknowledgments
 
The authors thank Dr. Albert Baukal (NICHHD, NIH) for the supply of antisera for the RIA.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada. Back

Received June 2, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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