Calcium dependence of damage to mouse motor nerve terminals following oxygen/glucose deprivation.
Motor nerve terminals are especially sensitive to an ischemia/reperfusion stress. We applied an in vitro model of this stress, oxygen/glucose deprivation (OGD), to mouse neuromuscular preparations to investigate how Ca(2+) contributes to stress-induced motor terminal damage. Measurements using an ionophoretically-injected fluorescent [Ca(2+)] indicator demonstrated an increase in intra-terminal [Ca(2+)] following OGD onset. When OGD was terminated within 20-30min of the increase in resting [Ca(2+)], these changes were sometimes reversible; in other cases [Ca(2+)] remained high and the terminal degenerated. Endplate innervation was assessed morphometrically following 22min OGD and 120min reoxygenation (32.5°C). Stress-induced motor terminal degeneration was Ca(2+)-dependent. Median post-stress endplate occupancy was only 26% when the bath contained the normal 1.8mM Ca(2+), but increased to 81% when Ca(2+) was absent. Removal of Ca(2+) only during OGD was more protective than removal of Ca(2+) only during reoxygenation. Post-stress endplate occupancy was partially preserved by pharmacological inhibition of various routes of Ca(2+) entry into motor terminals, including voltage-dependent Ca(2+) channels (ω-agatoxin-IVA, nimodipine) and the plasma membrane Na(+)/Ca(2+) exchanger (KB-R7943). Inhibition of a Ca(2+)-dependent protease with calpain inhibitor VI was also protective. These results suggest that most of the OGD-induced motor terminal damage is Ca(2+)-dependent, and that inhibition of Ca(2+) entry or Ca(2+)-dependent proteolysis can reduce this damage. There was no significant difference between the response of wild-type and presymptomatic superoxide dismutase 1 G93A mutant terminals to OGD, or in their response to the protective effect of the tested drugs.
Department of Physiology and Biophysics, University of Miami Miller School of Medicine, P.O. Box 016430, Miami, FL 33101, USA. jtalbot@med.miami.edu
Janet D
JD
Talbot
Gavriel
G
David
Ellen F
EF
Barrett
John N
JN
Barrett
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002038:Bungarotoxins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D004195:Disease Models, Animal; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics; D005947:Glucose / Q000172:deficiency*; D006801:Humans; D000860:Hypoxia / Q000473:pathology*; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009045:Motor Endplate / Q000473:pathology*; D016472:Motor Neuron Disease / Q000235:genetics / Q000473:pathology*; D009046:Motor Neurons / Q000378:metabolism / Q000473:pathology; D009469:Neuromuscular Junction / Q000503:physiopathology*; D011485:Protein Binding / Q000187:drug effects; D013482:Superoxide Dismutase / Q000235:genetics; D013890:Thiourea / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D013997:Time Factors
C101670:2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate; D001426:Bacterial Proteins; D002038:Bungarotoxins; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D004533:Egtazic Acid; C109415:SOD1 G93A protein; D013482:Superoxide Dismutase; D013890:Thiourea; D005947:Glucose; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium