Intracellular free Ca 2+ in dissociated cells of the chick pineal gland: regulation by membrane depolarization, second messengers and neuromodulators, and evidence for release of intracellular Ca 2+ stores

Abstract

The regulation of intracellular free Ca 2+ concentration was examined in single dissociated chick pineal cells using the fura-2 technique. ∼ 10% of cells examined exhibited spontaneous Ca 2+ oscillations while the rest were quiescent. Application of salines containing 80 mM KCl evoked large increases in intracellular free Ca 2+ that were dependent upon external Ca 2+ ions. These responses were inhibited by 10 μM nifedipine indicating involvement of L-type Ca 2+ channels. Application of the tumor promoter thapsigargin (2 μM) evoked increases in intracellular free Ca 2+. These responses could be observed in the absence of external Ca 2+ indicating mobilization of internal stores. In the absence of external Ca 2+, the responses to thapsigargin gradually decayed due to depletion of internal Ca 2+ pools. A subsequent exposure to saline containing 5.8 mM CaCl 2 caused a rapid increase in intracellular Ca 2+ that was consistently larger than the peak response to thapsigargin. Applicatio of 100 nM vasoactive intestinal peptide (VIP), a neurohormone that stimulates melatonin secretion from pineal cells, induced a sustained increase in intracellular free Ca 2+ in a subpopulation of cells. In a small number of cells, VIP evoked Ca 2+ oscillations. Approximately half of the cells examined showed no response to VIP. Application of 200 μM norepinephrine, which inhibits melatonin secretion from the chick pineal, had no effect on intracellular free Ca 2+ in any quiescent or spontaneously oscillating cells. Application of 5 mM 8-Br-cAMP evoked sustained increases in intracellular Ca 2+ Similar effects were obtained with the phosphodiesterase inhibitors papaverine (50 μM) or isobutylmethylxanthine (100 μM). Application of 200 nM forskolin, an activator of adenylate cyclase, evoked increases in intracellular free Ca 2+ that could be detected in the presence of 10 μM nifedipine. The responses to forskolin gradually in Ca 2+-free external salines due to depletion of intracellular Ca 2+ stores. Subsequent exposure to external Ca 2+ caused a rapidly developing increase in intracellular Ca 2+ that was larger than the peak response to forskolin. These results indicate that the regulation of intracellular free Ca 2+ in chick pineal cells is complex. These cells exhibit Ca 2+ oscillations and can mobilize both external and internal Ca 2+ pools. Agents that increase intracellular cAMP cause mobilization of internal Ca 2+ stores, possibly secondary messenger systems. Chick pineal cells, like many other cell types, possess mechanisms to allow for refilling of depleted internal Ca 2+ stores. These results suggest New mechanisms for the regulation of melatonin synthesis and secretion and possible sites of action for the intrinsic circadian oscillator.