Regulation of intrasynaptosomal free calcium concentrations: studies with the fluorescent indicator, fluo-3

Intracellular free calcium concentrations in synaptosomes from mouse forebrain were determined fluorometrically with the recently developed calcium indicator, fluo-3. Resting cytosolic calcium concentrations fell within the range of those previously reported in synaptosomes with quin 2 and fura-2. Both the resting and KCl-induced rise in cytosolic calcium were dependent on the concentration of extracellular calcium. Although both 1 mM norepinephrine and carbachol stimulated phospholipase C activity as assessed by the production of inositol mono- and bisphosphates, neither was able to elevate resting cytosolic free calcium. In the absence of extracellular calcium, the addition of Br A(23187), a non-fluorescing calcium ionophore, elicited a significant rise in cytosolic calcium indicating that the fluo-3 method employed in the present investigation was sufficiently sensitive to detect release of membrane bound calcium. It is concluded that fluo-3 is a highly suitable dye for determining cytosolic calcium concentrations in synaptosomal preparations, and secondly, that receptor stimulation of synaptosomal phospholipase C activity is insufficient to alter resting cytosolic calcium.