Calcium binding to the cytosol and calcium extrusion mechanisms in intact synaptosomes and their alterations with aging.
A simple method to measure cytosolic calcium binding in intact presynaptic nerve terminals (synaptosomes) from rat brain, which is based on the simultaneous determination of [Ca2+]i and total [45Ca2+] in quin2-loaded synaptosomes undergoing a switch from high- to low-calcium containing medium, is presented. Binding to the cytosolic compartment alone was obtained following depletion of calcium storing organelles in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone/oligomycin plus caffeine. Synaptosomes, as compared to various cells types, have a high calcium binding capacity to the cytosolic compartment; maximum binding, Ca.Bmax, was 4.76 mM and calculated s0.5 was 218 nM. Calcium binding to the cytosolic compartment as a function of aging was also determined; Ca.Bmax was reduced to 1.84 mM and s0.5 increased to 492 nM in 30-month-old rats, indicating that the buffering of high calcium loads is impaired in old animals. The results obtained for binding of calcium to mitochondria and caffeine-sensitive calcium stores are consistent with an age-dependent reduction in calcium bound to mitochondria, whereas caffeine-sensitive calcium stores were unaffected. Finally, we have estimated the net rates of calcium extrusion in intact synaptosomes, and found that calcium efflux through the Na/Ca exchanger and Ca(2+)-ATPase was markedly reduced in old rats.
Martinez-Serrano
A
A
Departamento de Biologia Molecular, Universidad Autónoma de Madrid, Spain.
Blanco
P
P
Satrústegui
J
J
D000375:Aging / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002110:Caffeine / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000737:chemistry; D002352:Carrier Proteins / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D008297:Male; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism; D009840:Oligomycins / Q000737:chemistry; D051381:Rats; D011919:Rats, Inbred Strains; D019831:Sodium-Calcium Exchanger; D013574:Synaptosomes / Q000378:metabolism* / Q000502:physiology
D002352:Carrier Proteins; D008565:Membrane Proteins; D009840:Oligomycins; D019831:Sodium-Calcium Exchanger; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D002110:Caffeine; D000252:Calcium-Transporting ATPases; D002118:Calcium