Ca2+Clearance at Growth Cones Produced by Crayfish Motor Axons in an Explant Culture

Abstract

Intracellular free Ca2+concentration ([Ca2+]i) plays an important role in the regulation of growth cone (GC) motility; however, the mechanisms responsible for clearing Ca2+from GCs have not been examined. We studied the Ca2+-clearance mechanisms in GCs produced by crayfish tonic and phasic motor axons by measuring the decay of [Ca2+]iafter a high [K+] depolarizing pulse using fura-2AM. Tonic motor axons regenerating in explant cultures develop GCs with more rapid Ca2+clearance than GCs from phasic axons. When Na/Ca exchange was blocked by replacing external Na+with N-methyl-d-glucamine (NMG), [Ca2+]idecay was delayed in both tonic and phasic GCs. Tonic GCs appear to have higher Na/Ca exchange activity than phasic ones since reversal of Na/Ca exchange by lowering external Na+caused a greater increase in [Ca2+]ifor tonic than phasic GCs. Application of the mitochondrial inhibitors, Antimycin A1 (1 μM) and CCCP (10 μM), demonstrated that mitochondrial Ca2+uptake/release was more prominent in phasic than tonic GCs. When both Na/Ca exchange and mitochondria were inhibited, the plasma membrane Ca2+ATPase was effective in extruding Ca2+from tonic, but not phasic GCs. We conclude that Na/Ca exchange plays a prominent role in extruding large Ca2+loads from both tonic and phasic GCs. High Na/Ca exchange activity in tonic GCs contributes to the rapid decay of [Ca2+]iin these GCs; low rates of Ca2+extrusion plus the release of Ca2+from mitochondria prolongs the decay of [Ca2+]iin the phasic GCs.