Ceramide inhibits L-type calcium channel currents in GH3 cells

Molecular and Cellular Endocrinology 218 (2004) 175–183

Ceramide inhibits L-type calcium channel currents in GH3 cells C.L. Chik a,∗ , B. Li b , E. Karpinski b , A.K. Ho b b

a Department of Medicine, 7-33 Medical Sciences Building, Edmonton, Alta., Canada T6G 2H7 Department of Physiology, Faculty of Medicine, University of Alberta, Edmonton, Alta., Canada T6G 2H7

Received 18 August 2003; accepted 16 October 2003

Abstract In this study, we investigated the effect of ceramide on the L-type Ca2+ channel (L-channel) in GH3 cells. We found that C6-ceramide, but not C6-dihydroceramide, the inactive analogue, had an inhibitory effect on BayK 8644-stimulated GH release. Using patch clamp analysis, C6- and C2-ceramide, but not C6-dihydroceramide, were found to inhibit the L-channel current. Increasing intracellular ceramide level with sphingomyelinase also inhibited the L-channel current. The inhibitory effect of ceramide on the L-channel current was attenuated by calphostin C, a myristolated pseudosubstrate protein kinase C (PKC) inhibitor, and lavendustin A, a tyrosine kinase inhibitor. Combined treatment with lavendustin A and the myristolated PKC inhibitor blocked the effect of ceramide on the L-channel current. These results indicate that ceramide, a lipid messenger of the sphingomyelin pathway, is an important regulator of the L-channel in GH3 cells and both tyrosine kinase and PKC are involved in this effect of ceramide. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Ceramide; Ca2+ channels; PKC; Tyrosine kinase; GH3 cells

1. Introduction The sphingomyelin cycle is an important lipid signaling pathway that not only regulates cell growth and differentiation, but also cellular functions including Ca2+ homeostasis and secretion (Mathias et al., 1998; Perry and Hannun, 1998). The sphingomyelin cycle is activated by various sphingomyelinases and results in the cleavage of sphingomyelin to ceramide, a second messenger, and phosphorylcholine (Hannun, 1994). Ceramide is in turn converted to sphingosine and sphingosine-1-phosphate, two active metabolites (Hannun and Bell, 1989; Spiegel and Merrill, 1996). Alternatively, ceramide is consumed by sphingomyelin synthase, resulting in the resynthesis of sphingomyelin and completion of the sphingomyelin cycle (Hannun, 1994). Activators of this pathway include inflammatory cytokines such as tumor necrosis factor, interleukin 1␤ and interferon ␥

Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; GHRH, GH releasing hormone; I–V relationship, current–voltage relationship; L-type Ca2+ channel, L-channel; PKC, protein kinase C; PMA, 4␤ phorbol 12-myristate 13-acetate ∗ Corresponding author. Tel.: +1-780-492-7213; fax: +1-780-492-8915. E-mail address: [email protected] (C.L. Chik).

(Kim et al., 1991; Kolesnick and Golde, 1994). Cellular targets of ceramide include specific kinases and phosphatases (Abousalham et al., 1997; Dobrowsky and Hannun, 1992; Huwiler et al., 1996; Raines et al., 1993; Younes et al., 1992) that are known regulators of ion channels (Levitan, 1999). Indeed, ceramide has been shown to modulate several types of ion channels including the L-type Ca2+ channel (L-channel), different K+ channels and Cl− channels (Chik et al., 1999, 2001; Gulbins et al., 1997; Hida et al., 1998; Li et al., 1999; Schreur and Liu, 1997; Szabo et al., 1998; Wu et al., 2001; Yu et al., 1999). A regulatory role of ceramide on Ca2+ homeostasis and secretion (Mathias et al., 1998; Perry and Hannun, 1998) prompted our earlier study on GH release in the rat anterior pituitary cells (Negishi et al., 1999). Two specific actions of ceramide in rat somatotrophs were identified (Negishi et al., 1999). Whereas ceramide reduces GH releasing hormone (GHRH)-stimulated GH release through elevation of intracellular Ca2+ , it also enhances GHRH-stimulated cAMP accumulation (Negishi et al., 1999). Although the effect of ceramide on the adenylyl cyclase pathway was found to be mediated through inhibition of phosphodiesterase (Negishi et al., 1999), the mechanism through which ceramide reduces GHRH-induced elevation of intracellular Ca2+ , hence GH secretion, remains unclear. However, pharmacological

0303-7207/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2003.10.048

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studies with a depolarizing concentration of K+ or BayK 8644, an L-channel agonist, suggest an inhibitory effect of ceramide on the L-channel. Therefore, the objective of this study was to examine whether ceramide had an inhibitory effect on the L-channel. To investigate the effects of ceramide on the L-channel and GH release, GH3 cells, an established rat pituitary tumor cell line that secretes GH (Tashjian et al., 1968), was used. Specifically, we determined the effect of ceramide on GH secretion stimulated by BayK 8644 or a depolarizing concentration of K+ and we also investigated the effect of ceramide on the L-channel current and the intracellular mechanisms involved.

2. Materials and methods 2.1. Materials GH3 cells were obtained from American Type Culture Collection (Rockville, MD). 4␤ Phorbol 12-myristate 13-acetate (PMA), a myristolated pseudosubstrate protein kinase C (PKC) inhibitor (myr-PKC [19–27]) and sphingomyelinase were obtained from Sigma. BayK 8644, C2-ceramide, C6-ceramide, C6-dihydroceramide, calphostin C, lavendustin A and lavendustin B were obtained from Calbiochem Corp. (La Jolla, CA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Biofluids Inc. (Rockville, MD). GH assay kit was obtained from the Pituitary Hormone Distribution Agency of the NIADDK (Baltimore, MD) and 125 I-GH was obtained from Corning Hazelton Biotechnologies Corp. (Vienna, VA). All other chemicals were of the purest grades available. 2.2. Treatment of GH3 cells for GH determinations GH3 cells were maintained in Ham’s F-10 medium with 10% horse serum and 2.5% fetal bovine serum in a humidified atmosphere of 95% air–5% CO2 at 37 ◦ C until confluent. The cells were then dissociated from the flasks by incubating with 0.4% trypsin–EDTA for 5–10 min at room temperature. After washing twice to remove trypsin, cells were plated onto multi-welled dishes at a density of 50,000 cells per well for the GH study. After 48 h, the cells were washed twice with DMEM containing 1% bovine serum albumin and equilibrated for 30 min before performing the experiments. The plated cells were washed a third time, and the medium bathing the cells was replaced by medium in which the drugs were dissolved. Drugs were dissolved in at least 200× concentrated solution in H2 O or dimethylsulfoxide (DMSO) and diluted to the final concentration in DMEM (pH 7.4). The concentration of DMSO never exceeded 0.5%. After 15 min, the medium was removed and assayed for GH release. Medium GH was assayed in duplicate using a double antibody RIA (Akman et al., 1993) and the results were expressed as pg/100,000 cells. Intra- and inter-assay variations

for GH were <10%. Data are presented as the mean±S.E.M. of the amount of GH obtained from three independent experiments, each performed in quadruplicate. Data were analyzed by analysis of variance and the Newman–Keuls test. 2.3. Electrophysiological studies Cells (90% confluent) were replated and used for electrophysiological studies 6–8 h after replating. The cells were spherical in shape after replating and the diameter ranged between 10 and 15 ␮m. Ca2+ channel current recordings were obtained using the whole cell version of the patch clamp technique (Chik et al., 1999; Hamill et al., 1981). Patch electrodes were pulled from borosilicate glass capillary tubes (o.d. = 1.2 mm, i.d. = 0.9 mm, FHC, Brunswick, ME) and heat polished. They were filled with a solution containing (in mM): 70 Cs2 -aspartate, 20 HEPES, 11 EGTA, 1 CaCl2 , 5 MgCl2 ·6H2 O, 5 glucose, 5 ATP-Na2 and 5 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 (in mM): 105 Tris–Cl, 0.8 MgCl2 ·6H2 O, 5.4 KCl, 20 BaCl2 , 0.02 tetrodotoxin and 10 HEPES. Twenty millimoles Ba2+ was used as the charge carrier. All solutions were filtered (0.22 ␮m) before use. The osmolarity was adjusted to 320 mOsm and the pH to 7.4. The membrane currents were measured using an Axopatch 1B 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 analogue-to-digital interface (Axon Instruments). Analysis was performed using the pClamp software. To generate current–voltage (I–V) relationships, 250 ms 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 −40 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. Moreover, T-channel currents, which are present in GH3 cells, were almost completely inactivated at a holding potential of −40 mV. The experiments were performed at room temperature (20–22 ◦ C). GH3 cells were evaluated for current run down before they were used for experiments. After the whole cell configuration was established, the inward 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 95% of cells, the initial run down rate was <5% and a stable current could be recorded for the next 60 min. In 5% of cells, the initial run down rate was >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 after 5 min into the bath solution unless other specified.

C.L. Chik et al. / Molecular and Cellular Endocrinology 218 (2004) 175–183

Data are presented as the mean ± S.E.M. percentages 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. The paired t-test was used for comparison between values of the control and those obtained after drug administration. In the case of multiple comparisons, analysis of variance in conjunction with the Newman–Keuls test was applied.

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3. Results 3.1. Ceramide reduces KCl- and BayK 8644-induced increases in GH release K+

In GH3 cells, a depolarizing concentration of (30 mM KCl) or an L-channel agonist BayK 8644 (1 ␮M) caused a three-fold increase in GH release (Table 1). C6-ceramide (30 ␮M) did not have a significant effect on basal GH release. However, treatment with C6-ceramide was effective in reducing the increase in GH release stimulated by BayK 8644 or a depolarizing concentration of K+ in a concentration-dependent manner (Table 1). In contrast, C6-dihydroceramide (30 ␮M), the inactive analogue, had no effect on GH release stimulated by BayK 8644 or a depolarizing concentration of K+ (Table 1). 3.2. Ceramide inhibits the L-type Ca2+ channel current in GH3 cells The effect of C6-ceramide on the L-channel current is shown in Fig. 1. The current was activated by depolarizing the cell from −40 to 20 mV before and after C6-ceramide (30 ␮M) (Fig. 1A). Application of C6-ceramide (30 ␮M) to the bath solution decreased the peak amplitude of the Table 1 Effects of ceramides on KCl and BayK 8644-stimulated GH release in GH3 cells Treatment

0.5% DMSO

+KCl

+BayK 8644

GH (pg/100,000 cells) Control C6-ceramide (10 ␮M) C6-ceramide (30 ␮M) C6-dihydroceramide (30 ␮M)

55 49 45 52

± ± ± ±

5 6 6 6

160 124 85 145

± ± ± ±

20a 11b 20b 18

175 130 90 165

± ± ± ±

25a 13b 15b 22

GH3 cells were incubated in DMEM with 10% bovine serum albumin and treated with KCl (30 mM) or BayK 8644 (1 ␮M) in the absence or presence of ceramides for 15 min. The medium was collected for GH determination by RIA as described in Section 2. Each value represents the mean ± S.E.M. of determinations done in quadruplicate from three independent experiments. a Significantly different from control; P < 0.05. b Significant inhibition by C6-ceramide; P < 0.05.

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Fig. 1. Effect of C6-ceramide on the L-channel current in GH3 cells. (A) Representative current records from a GH3 cell at a test potential of 20 mV (holding potential −40 mV) before (CONT, 䊉) and 15 min after C6-ceramide (C6, 30 ␮M) was added to the bath (䊊). The corresponding I–V relationship is shown on the right. (B) 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. Each value represents the mean ± S.E.M. The n values are shown in the figure in parentheses above the data points. ∗ P < 0.05, compared with the control current.

L-channel current by about 38% (Fig. 1A). It did not change the inactivation kinetics or shift the peak inward current along its voltage axis, as shown in the I–V relationship (Fig. 1A). The onset of the inhibition caused by C6-ceramide occurred within 4–6 min and maximal inhibition occurred between 10 and 12 min. The effect of C6-ceramide was not reversed by wash out (data not shown). The average current run down at 15 min was 3.1±0.4% (n = 6). Inhibition of the L-channel current by C6-ceramide was dependent on concentration, with an estimated EC50 value of 8 ␮M; a small inhibition was observed at 5 ␮M and the maximal inhibition observed was 41.1% at a concentration of 50 ␮M (Fig. 1B). Addition of C2-ceramide (30 ␮M) to the bath solution caused a 33.2% inhibition of the L-channel current (Fig. 2A). The inhibitory effect of C2-ceramide (30 ␮M) on the L-channel current was similar to that of C6-ceramide (30 ␮M) (Figs. 1B and 2A). In contrast, C6-dihydroceramide (30 ␮M), an inactive analogue, had no effect on the L-channel current (Fig. 2A). 3.3. Effect of sphingomyelinase on the L-channel current Cellular ceramide level can be increased by treatment with sphingomyelinase which causes membrane hydrolysis

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Fig. 2. Effect of ceramides and sphingomyelinase on the L-channel current in GH3 cells. (A) The histogram shows the effects of C2-ceramide (C2, 30 ␮M), C6-dihyroceramide (DC6, 30 ␮M) and SMase (0.1 U/ml) on the L-channel current. The currents were measured 15 min after addition of the agents to the bath. Each value represents the mean ± S.E.M. The n values are shown in parentheses above the histogram. ∗ P < 0.05, significant difference between the two groups. (B) Representative current records from a GH3 cell at a test potential of 20 mV (holding potential −40 mV) before (CONT, 䊉) and 15 min after sphingomyelinase (SMase, 0.1 U/ml) was added to the bath (䊊). The corresponding I–V relationship is shown on the right.

of sphingomyelin (Hannun, 1994). Addition of sphingomyelinase (0.1 U/ml) to the bath solution decreased the peak amplitude of the L-channel current by about 29% (Fig. 2). The onset of inhibition caused by sphingomyelinase occurred within 6–8 min after drug addition and maximal inhibition occurred between 12 and 14 min. These results indicate that increasing membrane hydrolysis of sphingomyelin causes an inhibitory effect on the L-channel current similar to that seen after the addition of C6-ceramide. 3.4. Intracellular mechanisms that mediate the effect of ceramide on the L-channel current One target of ceramide action is PKC (Jones and Murray, 1995; Lee et al., 1996; Muller et al., 1995; Sawai et al., 1997). The involvement of PKC in the inhibitory effect of ceramide on the L-channel current was investigated using calphostin C, a specific inhibitor of PKC that competes with the diacylglycerol binding site (Kobayashi et al., 1989). Application of calphostin C (0.1 ␮M) alone caused a 15% decrease in L-channel current (Fig. 3A). Pretreat-

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Fig. 3. Effect of calphostin C on C6-ceramide-mediated inhibition of the L-channel current in GH3 cells. (A) Representative current records from a GH3 cell at a test potential of 10 mV (holding potential −40 mV) subjected to pretreatment with calphostin C (Cal-C, 0.1 ␮M) for 10 min before addition of C6-ceramide (C6, 30 ␮M) to the bath. The effect of C6 was measured 15 min after its addition. The corresponding I–V relationship is shown on the right. (B) The histogram shows the effect of C6-ceramide (C6, 30 ␮M), Cal-C (0.1 ␮M), and C6 (30 ␮M) in cells pretreated with Cal-C (0.1 ␮M). The currents were measured 15 min after addition of the last treatment. Each value represents the mean ± S.E.M. The n values are shown in parentheses above the histogram. ∗ P < 0.05, significant difference between the two groups.

ment of cells with calphostin C for 10 min attenuated the C6-ceramide (30 ␮M) inhibition of the L-channel current from 35.2 to 20.1% (Fig. 3), suggesting that the effect of ceramide on this current may be partly mediated by PKC. To further investigate the involvement of PKC, the effect of ceramide in the presence of a myristolated PKC inhibitor that inhibits Ca2+ and phospholipid dependent PKC was examined (Eichholtz et al., 1993). Bath application of the myristolated PKC inhibitor (10 ␮M) had no effect on the L-channel current (Fig. 4B). However, in cells pretreated with the myristolated PKC inhibitor, the inhibitory effect of C6-ceramide (30 ␮M) on the L-channel current was reduced from 35.2 to 20.4% (Fig. 4). Although these data support the involvement of PKC in ceramide inhibition of the L-channel current, they also suggest that either the PKC inhibitor used is not effective in totally inhibiting PKC activity or that inhibiting PKC alone is not sufficient to reverse the inhibitory effect of ceramide. To determine whether the PKC inhibitor used was sufficient to inhibit PKC activity, the effectiveness of the myristolated PKC inhibitor on inhibition of PKC was assessed in cells treated with PMA. Treatment with PMA

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Fig. 4. Effect of a myristolated PKC inhibitor on C6-ceramide-mediated inhibition of the L-channel current in GH3 cells. (A) Representative current records from a GH3 cell at a test potential of 20 mV (holding potential −40 mV) subjected to pretreatment with myristolated PKC inhibitor (Myr, 10 ␮M) for 30 min before addition of C6-ceramide (C6, 30 ␮M) to the bath. The effect of C6 was measured 15 min after its addition. The corresponding I–V relationship is shown on the right. (B) The histogram shows the effect of C6 (30 ␮M), Myr (10 ␮M), and C6 (30 ␮M) in cells pretreated with Myr (10 ␮M). The currents were measured 15 min after addition of the last treatment. Each value represents the mean ± S.E.M. The n values are shown in parentheses above the histogram. ∗ P < 0.05, significant difference between the two groups.

(0.1 ␮M) decreased the peak amplitude of the L-channel current by about 28% (Fig. 5A). In cells pretreated with the myristolated PKC inhibitor (10 ␮M), PMA (0.1 ␮M) had no significant effect on the L-channel current (Fig. 5B and C), indicating that the concentration of inhibitor used is effective in blocking the effect of PKC on the L-channel current. The above studies suggest that in addition to PKC, another mechanism is involved in the ceramide inhibition of the L-channel current. Since tyrosine kinase is known to mediate the effect of ceramide on ion channels in other cells (Chik et al., 1999; Gulbins et al., 1997; Szabo et al., 1998), we examined the involvement of tyrosine kinase in ceramide inhibition of the L-channel current in GH3 cells using lavendustin A, a tyrosine kinase inhibitor (Hsu et al., 1991). Treatment with lavendustin A (3 ␮M) alone did not have a significant effect on the L-channel current (Fig. 6). The inhibitory effect of C6-ceramide (30 ␮M) was attenuated (from 35.2 to 18.3%) in lavendustin A pretreated cells (Fig. 6B). The attenuation was not observed in cells pretreated with lavendustin B (3 ␮M), the inactive analogue of lavendustin A (data not shown).

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Fig. 5. Effect of a myristolated PKC inhibitor on PMA-mediated inhibition of the L-channel current in GH3 cells. (A) Representative current records from a GH3 cell at a test potential of 20 mV (holding potential −40 mV) before (CONT, 䊉) and 15 min after PMA (0.1 ␮M) was added to the bath (䊊). The corresponding I–V relationship is shown on the right. (B) Representative current records from a GH3 cell at a test potential of 10 mV (holding potential −40 mV) subjected to pretreatment with a myristolated PKC inhibitor (Myr, 10 ␮M) for 30 min before addition of PMA (0.1 ␮M) to the bath. The effect of PMA was measured 15 min after its addition. The corresponding I–V relationship is shown on the right. (C) The histogram shows the effect of PMA (0.1 ␮M), a myristolated PKC inhibitor (Myr, 10 ␮M), and PMA (0.1 ␮M) in cells pretreated with Myr (10 ␮M). The currents were measured 15 min after addition of the last treatment. Each value represents the mean ± S.E.M. The n values are shown in parentheses above the histogram. ∗ P < 0.05, significant difference between the two groups.

The involvement of both PKC and tyrosine kinase in mediating the effect of ceramide was tested by subjecting GH3 cells to treatment with lavendustin A and the myristolated PKC inhibitor. This experiment was performed with inclusion of the myristolated PKC inhibitor in the pipette solution to reduce the pretreatment time of the inhibitors. The presence of the myristolated PKC inhibitor (0.1 ␮M) and lavendustin A (3 ␮M) did not have a significant effect on the L-channel current (Fig. 7B). However, inhibition of PKC and tyrosine kinase with the two inhibitors was

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Fig. 6. Effect of a tyrosine kinase inhibitor on C6-ceramide-mediated inhibition of the L-channel current in GH3 cells. (A) Representative current records from a GH3 cell at a test potential of 10 mV (holding potential −40 mV) subjected to pretreatment with lavendustin A (3 ␮M) for 10 min before addition of C6-ceramide (C6, 30 ␮M) to the bath. The effect of C6 was measured 15 min after its addition. The corresponding I–V relationship is shown on the right. (B) The histogram shows the effect of C6-ceramide (C6, 30 ␮M), lavendustin A (LA, 3 ␮M), and C6 (30 ␮M) in cells pretreated with LA (3 ␮M). The currents were measured 15 min after addition of the last treatment. Each value represents the mean ± S.E.M. The n values are shown in parentheses above the histogram. ∗ P < 0.05, significant difference between the two groups.

effective in blocking the effect of C6-ceramide (30 ␮M) on the L-channel current (Fig. 7). Inclusion of this concentration of the myristolated PKC inhibitor (0.1 ␮M) in the pipette solution also blocked PMA-mediated inhibition (0.1 ␮M) of the L-channel current (data not shown). These results suggest that ceramide probably uses both PKC and tyrosine kinase simultaneously in mediating its effect on the L-channel current in GH3 cells.

4. Discussion 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 (Cifone et al., 1995; Pettus et al., 2002; Tian et al., 1995). Previously, we have shown that ceramide causes inhibition of GHRH-stimulated GH release through a Ca2+ -dependent mechanism in rat anterior pituitary cells (Negishi et al., 1999). By measuring intracellular Ca2+ concentration directly in fura-2 loaded anterior pituitary cells,

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Fig. 7. Effect of a myristolated PKC inhibitor and lavendustin A on C6-ceramide-mediated inhibition of the L-channel current in GH3 cells. (A) Representative current records from a GH3 cell at a test potential of 10 mV (holding potential −40 mV) subjected to pretreatment with a myristoylated PKC inhibitor (Myr, 0.1 ␮M) in the pipette solution and lavendustin A (LA, 3 ␮M) in the bath solution for 10 min before addition of C6-ceramide (C6, 30 ␮M) to the bath. The effect of C6 was measured 15 min after its addition. The corresponding I–V relationship is shown on the right. (B) The histogram shows the effect of C6 (30 ␮M), Myr (0.1 ␮M) + LA (3 ␮M), and C6 (30 ␮M) in cells pretreated with Myr (0.1 ␮M) and LA (3 ␮M) for 10 min. The currents were measured 15 min after addition of the last treatment. Each value represents the mean ± S.E.M. The n values are shown in parentheses above the histogram. ∗ P < 0.05, significant difference between the two groups.

ceramide is effective in reducing GHRH- and KCl-induced increases in intracellular Ca2+ concentration (Negishi et al., 1999). Moreover, ceramide also inhibits GH release stimulated by a depolarizing concentration of K+ or BayK 8644 (Negishi et al., 1999). In contrast, ceramide has no effect on GH release stimulated by ionomycin, a Ca2+ ionophore. These earlier results suggest that ceramide appears to inhibit GH release through the L-channel. In this study, we found that in GH3 cells, C6-ceramide but not C6-dihydroceramide, the inactiave analogue, is effective in inhibiting GH release stimulated by a depolarizing concentration of K+ or BayK 8644, suggesting that ceramide probably inhibits GH release through inhibition of the L-channel. When the effect of ceramide on the L-channel current was investigated using patch clamp analysis, both C6- and C2-ceramide but not C6-dihydroceramide had an inhibitory effect on the L-channel current. Moreover, treatment with sphingomyelinase, which induces the hydrolysis of sphingomyelin and generates ceramide endogenously

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(Hannun, 1994), also causes a similar inhibition of the L-channel current as the active ceramides. These results suggest that C6- and C2-ceramide, the synthetic ceramides, appear to simulate the effect of endogenously produced ceramide on the L-channel current. One possible mechanism by which ceramide can inhibit the L-channel is through a nonspecific lipid effect on the channels. However, this is an unlikely mechanism because the inactive analogue of C6-ceramide, C6-dihydroceramide, has no effect on the L-channel current. Furthermore, the effect of C6-ceramide can be attenuated by kinase inhibitors, indicating the involvement of intracellular signaling mechanisms. Studies using kinase inhibitors show the involvement of tyrosine kinase and PKC. The attenuating effect of lavendustin A, a tyrosine kinase inhibitor, on the ceramide inhibition of the L-channel current suggests a signaling cascade from ceramide via tyrosine kinase to the L-channel. It is of interest to note that tyrosine kinase has been shown to be involved in the ceramide inhibition of different ion channels including the L-channel and the Ca2+ -activated K+ channel in rat pinealocytes (Chik et al., 1999, 2001) and the voltage-gated K+ channel in T-lymphocytes (Gulbins et al., 1997). In addition to the involvement of tyrosine kinase, our results indicate the simultaneous involvement of PKC in the ceramide inhibition of the L-channel current. This is based on the observations that inhibition of the PKC pathway by either calphostin C or the myristolated PKC inhibitor only reduces the ceramide inhibition of the L-channel current. However, inhibition of the tyrosine kinase pathway by lavendustin A and the PKC pathway by the myristolated PKC inhibitor completely blocks the ceramide inhibition of the L-channel current in GH3 cells. The effect of ceramide on PKC has been reported to be isozyme specific. Whereas ceramide has been shown to cause translocation of novel PKC isozymes, PKC␦ and ∈ (Sawai et al., 1997), and activation of an atypical PKC isozyme, PKC␨ (Muller et al., 1995), its effect on the classical PKC isozyme, PKC␣, is inhibitory (Jones and Murray, 1995; Lee et al., 1996). Because all four PKC isozymes are present in GH3 cells (MacEwan et al., 1999), it will be of interest to determine the specific PKC isozymes through which ceramide mediates its effect on the L-channel and the precise molecular mechanisms through which ceramide causes inhibition of the L-channel current. Although calphostin C, a compound that competes with the diacylglycerol binding site (Kobayashi et al., 1989) has been widely used as a specific PKC inhibitor, the difference between calphostin C and the myristolated PKC inhibitor, a membrane permeable pseudosubstrate peptide that inhibits PKC (Eichholtz et al., 1993) on the L-channel current is of interest. Whereas calphostin C causes a significant inhibition of the L-channel current, the myristolated PKC inhibitor has no effect on the L-channel current. One possible explanation for the difference is that calphostin C may have a direct inhibitory effect on the L-channel current as

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reported in cardiac myocytes (Hartzell and Rinderknecht, 1996). The modulation of L-channels by ceramide represents another signaling mechanism through which these channels can be regulated in neuroendocrine cells. Previously, it has been shown that ceramide has an inhibitory effect on the inwardly rectifying K+ current in GH3 cells while having no effect on the L-channel current (Wu et al., 2001). In contrast, our study shows that addition of active but not inactive ceramide has an inhibitory effect on the L-channel current in GH3 cells. Furthermore, generation of endogenous ceramide by treatment with sphingomyelinase also has an inhibitory effect on the L-channel current. A possible explanation for the lack of effect of ceramide on the L-channel current in the previous study could be related to the relatively low concentration of ceramide used (Wu et al., 2001). Alternatively, the experimental conditions of the studies are not identical. We have previously shown that ceramide has an inhibitory effect on the L-channel current in rat pinealocytes (Chik et al., 1999). In contrast to the effect of ceramide on the L-channel current in GH3 cells, the inhibitory effect of ceramide on the pinealocyte L-channel current was only attenuated by lavendustin A, a tyrosine kinase inhibitor, but not by H7, a serine/threonine kinase inhibitor. Therefore, PKC may not be involved in the effect of ceramide on the L-channel current in rat pinealocytes. Ceramide is known to exert unspecific effects on a number of ion channels including the inwardly rectifying K+ current in GH3 cells (Wu et al., 2001), the Ca2+ and the tetrodotoxin-resistant Na+ current and delayed rectifier K+ current in rat sensory neurons (Zhang et al., 2002). A recent report also showed that the inhibitory effect of ceramide on the K+ channel Kv1.3 is secondary to formation of membrane platforms (Bock et al., 2003). Indeed, ceramide may mediate its effect on cellular signaling processes by modulating membrane rafts or microdomains, which coalesce into larger platforms (reviewed by Cremesti et al., 2002; van Blitterswijk et al., 2003). It will be of interest to determine whether membrane platforms are involved in the effect of ceramide on the L-channel current in GH3 cells. Our results indicate that similar to rat somatotrophs, ceramide has an inhibitory effect on GH release stimulated by activation of the L-channel. This signaling mechanism may represent a common pathway through which cytokines such as interleukin 1␤ and tumor necrosis factor, known activators of this pathway, modulate hormone release in GH3 cells. Since the L-channel is involved in Ca2+ signaling, an important regulatory mechanism for cellular processes including apoptosis, cell growth and differentiation (Abernethy and Soldatov, 2002; Berridge et al., 2000), it will be of interest to determine the involvement of ceramide inhibition of L-channels in these cellular processes in GH3 cells. Moreover, since PKC and tyrosine kinase are involved in the ceramide inhibition of the L-channel current, ceramide may also have an effect on cellular mechanisms regulated by PKC or tyrosine kinase in GH3 cells.

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Acknowledgements This work was supported by grants from the Canadian Institutes of Health Research.

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