Introduction

We have recently identified the Ca2+ channels expressed in the rat pineal gland (1,2). Electrophysiological studies have demonstrated that rat pinealocytes express the L-type but not the T- or N-type Ca2+ channel. RT-PCR analysis of the Ca2+ channel subunit mRNAs revealed that rat pinealocytes express the ttm, tt2M P2 and P4 subunits (2). These channels can be inhibited by activation of the adrenergic mechanism through the involvement of cAMP- and cGMP-dependent protein kinases (1,2). Activation of insulin growth factor-1 receptors also has an inhibitory effect on these channels through a tyrosine kinase-dependent mechanism (3). Furthermore, these channels are under tonic phosphorylation control through phosphoprotein phos-phatase 1 (4).

Another signalling pathway which has received much attention recently is the sphingomyelin cycle (5,6) (Figure 1). Ceramide, which is produced following sphingomyelin hydrolysis, has been shown to regulate intracellular enzymes such as protein kinase C (7), tyrosine kinases (8), diacylglycerol kinase (9), phosphatases (10) and phospholipases (11). Since both kinases and phosphatases can modulate the cyclic nucleotide responses (12,13) as well as the L-type Ca2+ channel in the rat pinealocyte, we investigated whether the sphingomyelin cycle could modulate pineal function through its effect on cyclic nucleotide accumulation and/or the L-type Ca2+ channel.

Figure 1. A schematic representation of the sphinogomyelin cycle. H, hormone or other ligand; MAPKinase, mitogen-activated protein kinase; PKC, protein kinase C; PKCZ, the Z; isozyme of PKC; PLD, phospholipase D; R, receptor; SMase, sphingomyelinase.

2. EFFECT OF CERAMIDE ON PINEAL CYCLIC NUCLEOTIDE RESPONSES

Pinealocytes were prepared from male Sprague-Dawley rats (150 gm, University of Alberta Animal unit) by trypsinization as previously described (14,15). The cells were suspended in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and maintained at 37°C for 24hr in a gas mixture of 95% air and 5% CO2 before experimental treatment. Aliquots of cells (1.5 x 104 cells/0.4ml) were treated with drugs which had been prepared in concentrated solutions in water or dimethylsulfoxide. The duration of the drug treatment period was 15 min for cAMP and cGMP accumulation. Cellular cAMP and cGMP contents were determined using a radioimmunoassay procedure in which samples were acetylated prior to analysis (16).

2.1. Effect of Ceramide on Adrenergic-Stimulated cAMP and cGMP Responses (17)

Treatment with isoproterenol (ISO, 1 (M) alone caused a significant increase in cAMP and cGMP accumulation; the addition of a depolarizing concentration ofK+(30 mM KCI) potentiated these responses by 8-10fold (Figure 2). Pretreatment with C2-ceramide for 5min had no effect on basal or ISO-stimulated cAMP or cGMP accumulation. However, C2-ceramide inhibited the ISO + KCI-stimulated responses in a dose-dependent manner. Significant inhibition was observed at 10 (M of C2-ceramide and at 100(M the reduction was more than 70% for both cAMP and cGMP responses. C6- and C8-ceramide were also effective in reducing the ISO + KCI-stimulated cAMP and cGMP responses (Figure 3). In contrast, C2-dihydroceramide, an inactive analogue, had no effect.

2.2. Effects of Ceramide on Cyclic Nucleotide Accumulation Stimulated by ISO in the Presence of Other Potentiating Agents

The inhibitory effect of C2-ceramide was dependent on the treatments used to potentiate the P-adrenergic-stimulated cAMP and cGMP accumulation (17). Pre-

Figure 2. Effect of C2-ceramide on ISO- and ISO + KCI-stimulated cAMP and cGMP accumulation in rat pinealocytes. Pinealocytes (1.5 x 104 cells/ 400^1) were incubated in DMEM with 10% fetal bovine serum and pre-treated with or without C2-ceramide (C2, 3-100^M) for 5 min. The cells were then stimulated with ISO (1 |^M) in the presence or absence of KCI (30 mM) for an additional 15 min. Each value represents the mean ± SEM of determinations done in duplicate on three samples of cells. *Significantly different from treatment with ISO + KCI.

Figure 2. Effect of C2-ceramide on ISO- and ISO + KCI-stimulated cAMP and cGMP accumulation in rat pinealocytes. Pinealocytes (1.5 x 104 cells/ 400^1) were incubated in DMEM with 10% fetal bovine serum and pre-treated with or without C2-ceramide (C2, 3-100^M) for 5 min. The cells were then stimulated with ISO (1 |^M) in the presence or absence of KCI (30 mM) for an additional 15 min. Each value represents the mean ± SEM of determinations done in duplicate on three samples of cells. *Significantly different from treatment with ISO + KCI.

cAMP cGMP

Figure 3. Effect of ceramides on the potentiating effect of K+ on ISO-stimulated cAMP and cGMP responses. Pinealocytes (1.5 x 104 cells/ 400 ^1) were incubated in DMEM with 10% fetal bovine serum and pre-treated with or without C2-, C2-dihydro(C2-dihy)-, C6- and C8-ceramide (30 ^M) for 5min. The cells were then stimulated with ISO (1 |^M) and KCI (30mM) for an additional 15min. Each value represents the mean ± SEM of determinations done in duplicate on three samples of cells. *Significantly different from treatment with ISO + KCI.

+ISO+KC1 +ISO+KC1

Figure 3. Effect of ceramides on the potentiating effect of K+ on ISO-stimulated cAMP and cGMP responses. Pinealocytes (1.5 x 104 cells/ 400 ^1) were incubated in DMEM with 10% fetal bovine serum and pre-treated with or without C2-, C2-dihydro(C2-dihy)-, C6- and C8-ceramide (30 ^M) for 5min. The cells were then stimulated with ISO (1 |^M) and KCI (30mM) for an additional 15min. Each value represents the mean ± SEM of determinations done in duplicate on three samples of cells. *Significantly different from treatment with ISO + KCI.

treatment with C2-ceramide for 15min had no effect on the potentiation by phenylephrine [an a -adrenergic agonist] (18), 40-phorbol 12-myristate 13-acetate [a protein kinase C activator] (18-20), genistein [a tyrosine kinase inhibitor] (21,22), calyculin A [a serine/threonine phosphatase inhibitor] (13) or isobutylmethylxanthine [a phosphodiesterase inhibitor]. These results suggest that the inhibitory effect of C2-ceramide may be specific to intracellular Ca2+ elevating agents.

Additional studies showed that the inhibitory effect of C2-ceramide was dependent on the mechanism by which intracellular Ca2+ was elevated. Pre-treatment with C2-ceramide for 5min inhibited only the BayK 8644 (an L-type Ca2+ channel agonist)-mediated potentiation of the ISO-stimulated cAMP and cGMP responses (17). In contrast, C2-ceramide had no effect on the potentiating effects of ionomycin [a Ca2+ ionophore] (23) or thapsigargin [an intracellular Ca2+-ATPase inhibitor] (24).

Treatment with 1 -phenyl - 2 - hexadecanoylamino - 3 - morpholino - 1 -propanol (PPMP), a glucosylceramide synthase inhibitor, has been shown to increase cellular ceramide levels (25,26). Pre-treatment with PPMP for 15min had an effect similar to that of C2-ceramide: PPMP had no effect on basal or ISO-stimulated cAMP and cGMP accumulation, but selectively inhibited the ISO + KCl- or ISO + BayK 8644-stimulated cAMP and cGMP accumulation (Figure 4).

Taken together, these results indicate that ceramide selectively inhibits the poten-tiation mediated by elevation of intracellular Ca2+. Ceramide has no effect on the poten-tiation mediated by an activator of protein kinase C or inhibitors of tyrosine kinase and phosphatases. Furthermore, this inhibitory effect of ceramide is highly specific and depends on the mechanism through which intracellular Ca2+ is elevated. For example,

Figure 4. Effect of PPMP on the cAMP and cGMP responses stimulated by ISO in the absence or presence of potentiating agents. Pinealocytes (1.5 x 104 cells/ 400^l) were incubated in DMEM with 10% fetal bovine serum and pre-treated with or without PPMP (10 ^M) for 15 min. The cells were then stimulated with ISO (1 |^M) in the presence or absence of potentiating agents as indicated, for an additional 15 min. Each value represents the mean ± SEM of determinations done in duplicate on three samples of cells. *Significantly different from corresponding treatment without PPMP.

Figure 4. Effect of PPMP on the cAMP and cGMP responses stimulated by ISO in the absence or presence of potentiating agents. Pinealocytes (1.5 x 104 cells/ 400^l) were incubated in DMEM with 10% fetal bovine serum and pre-treated with or without PPMP (10 ^M) for 15 min. The cells were then stimulated with ISO (1 |^M) in the presence or absence of potentiating agents as indicated, for an additional 15 min. Each value represents the mean ± SEM of determinations done in duplicate on three samples of cells. *Significantly different from corresponding treatment without PPMP.

ceramide inhibits only the potentiation caused by agents such as depolarizing concentrations of K+ or BayK 8644 which elevate intracellular Ca2+ through activation of the L-type Ca2+ channel. Ceramide, however, has no effect on the potentiation mediated by ionomycin or thapsigargin, two agents which elevate intracellular Ca2+ in rat pinealocytes (24). Our results therefore suggest that an increase in ceramide levels significantly inhibits L-type Ca2+ channels in rat pinealocytes.

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