INTRACELLULAR FREE CA2+ IN DISSOCIATED CELLS OF THE CHICK PINEAL-GLAND - REGULATION BY MEMBRANE DEPOLARIZATION, 2ND-MESSENGERS AND NEUROMODULATORS, AND EVIDENCE FOR RELEASE OF INTRACELLULAR CA2+ STORES

The regulation of intracellular free Ca2+ concentration was examined in single dissociated chick pineal cells using the fura-2 technique. similar to 10% of cells examined exhibited spontaneous Ca2+ oscillations while the rest were quiescent. Application of salines containing 80 mM KC1 evoked large increases in intracellular free Ca2+ that were dependent upon external Ca2+ ions. These responses were inhibited by 10 mu M nifedipine indicating involvement of L-type Ca2+ channels. Application of the tumor promoter thapsigargin (2 mu M) evoked increases in intracellular free Ca2+. These responses could be observed in the absence of external Ca2+ indicating mobilization of internal stores. In the absence of external Ca2+, the responses to thapsigargin gradually decayed due to depletion of internal Ca2+ pools. A subsequent exposure to saline containing 5.8 mM CaCl2, caused a rapid increase in intracellular Ca2+ that was consistently larger than the peak response to thapsigargin. Application of 100 nM vasoactive intestinal peptide (VIP), a neurohormone that stimulates melatonin secretion from pineal cells, induced a sustained increase in intracellular free Ca2+ in a subpopulation of cells. In a small number of cells, VIP evoked Ca2+ oscillations. Approximately half of the cells examined showed no response to VIP. Application of 200 mu M norepinephrine, which inhibits melatonin secretion from the chick pineal, had no effect on intracellular free Ca2+ in any quiescent or spontaneously oscillating cells. Application of 5 mM 8-Br-cAMP evoked sustained increases in intracellular Ca2+ Similar effects were obtained with the phosphodiesterase inhibitors papaverine (50 mu M) or isobutylmethylxanthine (100 mu M) Application of 200 nM forskolin, an activator of adenylate cyclase, evoked increases in intracellular free Ca2+ that could be detected in the presence of 10 mu M nifedipine. The responses to forskolin gradually decayed in Ca2+-free external salines due to depletion of intracellular Ca2+ stores. Subsequent exposure to external Ca2+ caused a rapidly developing increase in intracellular Ca2+ that was larger than the peak response to forskolin. These results indicate that the regulation of intracellular free Ca2+ in chick pineal cells is complex. These cells exhibit Ca2+ oscillations and can mobilize both external and internal Ca2+ pools. Agents that increase intracellular cAMP cause mobilization of internal Ca2+ stores, possibly secondary to effects on other second messenger systems. Chick pineal cells, like many other cell types, possess mechanisms to allow for refilling of depleted internal Ca2+ stores. These results suggest new mechanisms for the regulation of melatonin synthesis and secretion and possible sites of action for the intrinsic circadian oscillator.