Ceramide modulates nicotinic receptor-dependent Ca²⁺ signaling in rat chromaffin cells

Ceramide, which is an integral component of the sphingomyelin signaling pathway, can attenuate voltagegated Ca²⁺ channel (VGCC) activity in a number of cell types. The aim of the present study was to determine whether ceramide can also modulate VGCC activity, and as a consequence nicotinic receptor-dependent Ca²⁺ signaling and catecholamine secretion, in rat adrenal chromaffin cells. Short-term C₆-ceramide (CER) treatment dose-dependently inhibited nicotine (NIC)-induced peak intracellular Ca²⁺ transients. Sphingomyelinase elicited similar responses, whereas the inactive ceramide analog C₂-dihydroceramide had no effect on NICinduced Ca²⁺ transients. CER suppressed KCl- and NICinduced Ca²⁺ transients to a similar extent, suggesting that the voltage-gated Ca²⁺ channel was a primary site of inhibition. In direct support of this concept, whole-cell patch-clamp analysis demonstrated that CER and sphingomyelinase significantly reduced peak Ca²⁺ currents. Pretreatment with staurosporine significantly attenuated CER-dependent inhibition of both NIC-induced Ca²⁺ transients and peak Ca²⁺ current, suggesting that the effects of CER are mediated at least in part by protein kinase C. Consistent with suppressed Ca²⁺ signaling, CER also significantly inhibited NIC-induced catecholamine secretion measured at the single-cell level by carbon fiber amperometry. This effect of CER was also significantly attenuated by pretreatment with staurosporine These data demonstrate that the sphingomyelin signaling pathway can modulate nicotinic receptor-dependent Ca²⁺ signaling and catecholamine secretion in rat chromaffin cells.