A procedure for detecting changes in the internal Ca2+ concentration in isolated nerve endings using the metallochromic dye arsenazo III.
A method for detecting changes in the internal concentration of Ca2+ in synaptosomes from mouse brain is described. Synaptosomes were fused with phosphatidylcholine-phosphatidylserine unilamellar liposomes previously loaded with the metalochromic, Ca2+ -sensitive dye arsenazo III, in order to introduce the dye into the synaptosomes. The fusion was promoted by La3+. Changes in the differential absorption between 660 and 690 nm, which indicate changes in Ca2+ concentration, were followed in a double beam spectrophotometer. It was found that both in the dye-loaded liposomes and in the fused synaptosomes, the addition of the Ca2+ ionophore A23187 to the medium containing Ca2+ produced a notable change in the differential absorbance 660-690 nm. When the depolarizing alkaloid veratridine was added, there was no response in the liposomes, whereas a change in the differential absorbance 660-690 nm was detected in the fused synaptosomes containing arsenazo but only when Na+ was present in the medium. These fused synaptosomes were able to release labeled gamma-aminobutyric acid as a response to veratridine, in a Na+ -dependent manner, similarly to control non-fused synaptosomes. These results demonstrate the feasibility of fusion to introduce Ca2+ -sensitive dyes into isolated nerve endings from the mammalian brain and therefore to detect changes in their internal Ca2+ concentration.
D000818:Animals; D001150:Arsenazo III; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D006652:Histological Techniques; D051379:Mice; D013194:Staining and Labeling; D009435:Synaptic Transmission / Q000187:drug effects; D013574:Synaptosomes / Q000378:metabolism / Q000648:ultrastructure*; D014701:Veratridine / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D001150:Arsenazo III; D000001:Calcimycin; D005680:gamma-Aminobutyric Acid; D014701:Veratridine; D002118:Calcium