Use of mitochondrial inhibitors to differentiate kinetic properties of the ATP-dependent Ca2+ uptake system in synaptic membranes |
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Authors: | David H Ross Kennon M Garrett H Lee Cardenas |
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Institution: | (1) Division of Molecular Pharmacology Department of Pharmacology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Driye, 78284 San Antonio, Texas;(2) Present address: CNS Research, American Cyanamid Company, 10965 Pearl River, New York |
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Abstract: | Synaptosomal membranes accumulate 3–6 times more Ca2+ in the presence of ATP (50–1000 M) than basal Ca2+ accumulation (-ATP). The location of this Ca2+ accumulation appears to reside on the cytosolic face of the synaptosome since lysed synaptosomes accumulate 4-times more Ca2+ than intact synaptosomes. The inclusion of mitochondrial inhibitors, oligomycin (0.7 g/ml), sodium azide (100 M) and dinitrophenol (100 M) differentiate mitochondrial from nonmitochondrial Ca2+ accumulation under conditions that are Ca2+]- and ATP-dependent. In the presence of low concentrations of ATP (<150 M) and Ca
free
2+
(2.5 or 6.8 M), Ca2+ accumulation occurs as one process in both lysed synaptosomal membranes and purified synaptic plasma membranes in the presence and/or absence of MI. When ATP levels are increased (>200 M), the Ca2+ accumulation process remains independent of the presence of mitochondrial inhibitors when Ca
free
2+
=2.5 M. When Ca
free
2+
is increased to 6.8 M, mitochondrial inhibitors differentiate mitochondrial from nonmitochondrial accumulation. These studies suggest that optimal conditions for the measurement of Ca2+ accumulating mechanisms in synaptosomal membranes depend on both Ca2+] and ATP. Use of these assay conditions provide evidence that ATP-dependent Ca2+ uptake may be a viable mechanism for the regulation of synaptosomal Ca2+ levels. |
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