Spontaneous and nicotine-induced Ca2+ oscillations mediated by Ca2+ influx in rat pinealocytes

Abstract

The pineal gland regulates circadian rhythm through the synthesis and secretion of melatonin. The rise of intracellular Ca2+ concentration ([Ca2+]i) following nicotinic acetylcholine receptor (nAChR) stimulation due to parasympathetic nerve activity downregulates melatonin production. Important characteristics and roles of Ca2+ mobilization due to nAChR stimulation remain to be clarified. We report here that spontaneous Ca2+ oscillations can be observed in ∼15% of the pinealocytes in slice preparations from rat pineal glands when this dissociation procedure is done within 6 h from a dark-to-light change. The frequency and half-life of [Ca2+]i rise were 0.86 min−1 and 19 s, respectively. Similar spontaneous Ca2+ oscillations were recorded in 17% of rat pinealocytes that were primary cultured for several days. Simultaneous measurement of [Ca2+]i and membrane potential revealed that spontaneous Ca2+ oscillations were triggered by periodic membrane depolarizations. Spontaneous Ca2+ oscillations in cultured pinealocytes were abolished by extracellular Ca2+ removal or application of nifedipine, a blocker of voltage-dependent Ca2+ channel (VDCC). In contrast, blockers of intracellular Ca2+-release channels, 2-aminoethoxydiphenylborate and ryanodine, have no effect. Our results also reveal that, in 23% quiescent pinealocytes, Ca2+ oscillations were observed following the withdrawal of nicotine. Norepinephrine-induced melatonin secretion from whole pineal glands was significantly decreased by the coapplication of acetylcholine (ACh). This inhibitory effect of ACh was attenuated by nifedipine. In conclusion, both spontaneous and evoked Ca2+ oscillations are due to membrane depolarization following activation of VDCCs. This consists of VDCC α1F subunit, and the associated Ca2+ influx can strongly regulate melatonin secretion in pineal glands.