Resealing of transected myelinated mammalian axons in vivo: evidence for involvement of calpain.
The mechanisms underlying resealing of transected myelinated rat dorsal root axons were investigated in vivo using an assay based on exclusion of a hydrophilic dye (Lucifer Yellow-biocytin conjugate). Smaller caliber axons (<5 microm outer diameter) resealed faster than larger axons. Resealing was Ca2+ dependent, requiring micromolar levels of extracellular [Ca2+] to proceed, and further accelerated in 1 mM Ca2+. Two hours after transection, 84% of axons had resealed in saline containing 2 mM Ca2+, 28% had resealed in saline containing no added Ca2+ and only 3% had resealed in the Ca2+ buffer BAPTA (3 mM). The enhancing effect of Ca2+ could be overcome by both non-specific cysteine protease inhibitors (e.g., leupeptin) and inhibitors specific for the calpain family of Ca2+ -activated proteases. Resealing in 2 mM Ca2+ was not inhibited by an inhibitor of phospholipase A2. Resealing in low [Ca2+] was not enhanced by agents which disrupt microtubules, but was enhanced by dimethylsulfoxide (0.5-5%). These results suggest that activation of endogenous calpain-like proteases by elevated intra-axonal [Ca2+] contributes importantly to membrane resealing in transected myelinated mammalian axons in vivo.
Howard
M J
MJ
Department of Physiology and Biophysics, University of Miami School of Medicine, FL 33101, USA.
David
G
G
Barrett
J N
JN
D000818:Animals; D001369:Axons / Q000502:physiology* / Q000648:ultrastructure; D002118:Calcium / Q000502:physiology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology*; D048429:Cell Size; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes; D005727:Ganglia, Spinal / Q000166:cytology / Q000648:ultrastructure; D007091:Image Processing, Computer-Assisted; D007546:Isoquinolines; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives; D008297:Male; D018613:Microscopy, Confocal; D008870:Microtubules / Q000187:drug effects; D009186:Myelin Sheath / Q000502:physiology* / Q000648:ultrastructure; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D051381:Rats; D017207:Rats, Sprague-Dawley
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007546:Isoquinolines; C017475:lucifer yellow; D010741:Phospholipases A; D054467:Phospholipases A2; D002154:Calpain; C013411:biocytin; D008239:Lysine; D002118:Calcium; D004121:Dimethyl Sulfoxide