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

The regulation of intracellular free Ca2+ concentration was examined i n single dissociated chick pineal cells using the fura-2 technique. si milar to 10% of cells examined exhibited spontaneous Ca2+ oscillations while the rest were quiescent. Application of salines containing 80 m M KC1 evoked large increases in intracellular free Ca2+ that were depe ndent upon external Ca2+ ions. These responses were inhibited by 10 mu M nifedipine indicating involvement of L-type Ca2+ channels. Applicat ion of the tumor promoter thapsigargin (2 mu M) evoked increases in in tracellular free Ca2+. These responses could be observed in the absenc e of external Ca2+ indicating mobilization of internal stores. In the absence of external Ca2+, the responses to thapsigargin gradually deca yed due to depletion of internal Ca2+ pools. A subsequent exposure to saline containing 5.8 mM CaCl2, caused a rapid increase in intracellul ar Ca2+ that was consistently larger than the peak response to thapsig argin. Application of 100 nM vasoactive intestinal peptide (VIP), a ne urohormone that stimulates melatonin secretion from pineal cells, indu ced a sustained increase in intracellular free Ca2+ in a subpopulation of cells. In a small number of cells, VIP evoked Ca2+ oscillations. A pproximately half of the cells examined showed no response to VIP. App lication of 200 mu M norepinephrine, which inhibits melatonin secretio n from the chick pineal, had no effect on intracellular free Ca2+ in a ny quiescent or spontaneously oscillating cells. Application of 5 mM 8 -Br-cAMP evoked sustained increases in intracellular Ca2+ Similar effe cts were obtained with the phosphodiesterase inhibitors papaverine (50 mu M) or isobutylmethylxanthine (100 mu M) Application of 200 nM fors kolin, an activator of adenylate cyclase, evoked increases in intracel lular free Ca2+ that could be detected in the presence of 10 mu M nife dipine. The responses to forskolin gradually decayed in Ca2+-free exte rnal salines due to depletion of intracellular Ca2+ stores. Subsequent exposure to external Ca2+ caused a rapidly developing increase in int racellular Ca2+ that was larger than the peak response to forskolin. T hese results indicate that the regulation of intracellular free Ca2+ i n chick pineal cells is complex. These cells exhibit Ca2+ oscillations and can mobilize both external and internal Ca2+ pools. Agents that i ncrease intracellular cAMP cause mobilization of internal Ca2+ stores, possibly secondary to effects on other second messenger systems. Chic k pineal cells, like many other cell types, possess mechanisms to allo w for refilling of depleted internal Ca2+ stores. These results sugges t new mechanisms for the regulation of melatonin synthesis and secreti on and possible sites of action for the intrinsic circadian oscillator .