Effect Of Ceramide On The Ltype Ca2 Channels

3.1. Patch Clamp Analysis

Ca2+ channel current recordings were obtained using the whole cell version of the patch clamp technique (1-4,27). Patch electrodes were filled with a solution containing (in mM): 70 Cs2-aspartate, 20 HEPES, 11 EGTA, 1 CaCL, 5 MgC^. 6H2O, 5 glucose, 5 ATP-Na2 and 5 K-succinate. Creatine phosphokinase (50U/ml) and phos-phocreatine-Na, (20 mM) were added to the pipette solution to reduce current run down. The bath solution contained (in mM): 105 Tris Cl, 0.8 MgCL. 6H2O, 5.4 KCl, 20 BaCl2, 0.02 tetrodotoxin and 10 HEPES. 20mM Ba2+ was used as the charge carrier. The membrane currents were measured using an Axopatch 1B whole cell patch clamp amplifier (Axon Instruments, Foster City, CA). The data were sampled using pClamp software (pClamp 5.7) and a Digidata 1200 analogue-to-digital interface (Axon Instruments). Analysis was performed using the pClamp software. At a holding potential of -50mV and with Cs+ in the intracellular solution, hyperpolarizing pulses did not activate any currents. Data are presented as the mean ± SEM percentages of control values. The pretreatment I-V relationship was plotted and used as a control.

3.2. Effect of Ceramide on 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-type Ca2+ channel current (1-4). The effect of C2-ceramide on the L-type Ca2+ channel current is shown in Figure 5A, 5B. C2-ceramide (30^M) decreased the peak amplitude of the L-type Ca2+ channel current in a pinealocyte by about 25% (Figure 5A). It did not shift the peak inward current along its voltage axis, as shown in the I-Vrelationship (Figure 5A). The onset of inhibition caused by C2-ceramide occurred within 8 to 10min and the maximal inhibition was observed between 15 and 20min. The inhibition by C2-ceramide persisted in the presence of BayK 8644 (Figure 5B). Similar to C2-ceramide, C6-ceramide also inhibited basal and BayK 8644-stimulated the L-type Ca2+ channel current (data not shown). In contrast, C2-dihydroceramide, an inactive analogue, had no effect on the basal and BayK 8644-stimulated L-type Ca2+ channel current.

A similar inhibitory effect on the L-type Ca2+ channel current was observed when endogenous ceramide level was increased by PPMP, a glucosylceramide synthase inhibitor (Figure 5B). The onset of inhibition caused by PPMP occurred within 8 to 10min and the maximal inhibition was observed between 15 and 20min. PPMP also inhibited the BayK 8644-mediated increase in the L-type Ca2+ channel current (Figure 5B).

Figure 5. Effect of C2-ceramide on the L-type Ca2+ channel current in rat pinealocytes. (A) The L-type Ca2+ channel current is activated by depolarizing a pinealocyte from -40mV to l0mV in the absence (•) or presence (o) of C2-ceramide (C2, 30^M). The I-V relationships obtained from the same cell are shown on the right. (B) The combined data showing the effect of C2-ceramide (30^M) and PPMP (10^M) in the absence or presence of BayK 8644 (Bay K, 1 ^M), n = 5 for each treatment group.

Figure 5. Effect of C2-ceramide on the L-type Ca2+ channel current in rat pinealocytes. (A) The L-type Ca2+ channel current is activated by depolarizing a pinealocyte from -40mV to l0mV in the absence (•) or presence (o) of C2-ceramide (C2, 30^M). The I-V relationships obtained from the same cell are shown on the right. (B) The combined data showing the effect of C2-ceramide (30^M) and PPMP (10^M) in the absence or presence of BayK 8644 (Bay K, 1 ^M), n = 5 for each treatment group.

3.3. Interleukin 1p Inhibits the L-Type Ca2+ Channel Current

Several cytokines, including interleukin lp, tumor necrosis factor- a, and interferon-y are known to induce sphingomyelin hydrolysis to ceramide (6). Since abundant expression of interleukin lp and its specific receptor has been demonstrated in the rat pineal gland (28), the effect of interleukin ip on the L-type Ca2+ channel current was examined. Similar to ceramides, interleukin lp (10ng/ml) inhibited the amplitude of the L-type Ca2+ channel current by about 23% (data not shown). The onset of inhibition caused by interleukin 1p occurred within 8 to 10min and the maximal inhibition was observed between 15 and 20min. Interleukin lp also inhibited the BayK 8644-mediated increase in the L-type Ca2+ channel current.

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