Effects of buthionine sulfoximine treatment on diaphragm contractility and SR Ca2+ pump function in rats.
The purpose of this study was to examine the effects of glutathione (GSH) depletion and cellular oxidation on rat diaphragm contractility and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) function in vitro under basal conditions and following fatiguing stimulation. Buthionine sulfoximine (BSO) treatment (n = 10) for 10 days (20 mM in drinking water) reduced (P < 0.05) diaphragm GSH content (nmol/mg protein) and the ratio of GSH to glutathione disulfide (GSH/GSSG) by 91% and 71%, respectively, compared with controls (CTL) (n = 10). Western blotting showed that Hsp70 expression in diaphragm was not increased (P > 0.05) with BSO treatment. As hypothesized, basal peak twitch force (g/mm(2)) was increased (P < 0.05), and fatigability in response to repetitive stimulation (350-ms trains at 100 Hz once every 1 s for 5 min) was also increased (P < 0.05) in BSO compared with CTL. Both Ca(2+) uptake and maximal SERCA activity (mumol.g protein(-1).min(-1)) measured in diaphragm homogenates that were prepared at rest were increased (P < 0.05) with BSO treatment, an effect that could be partly explained by a twofold increase (P < 0.05) in SERCA2a expression with BSO. In response to the 5-min stimulation protocol, both Ca(2+) uptake and maximal SERCA activity were increased (P < 0.05) in CTL but not (P > 0.05) in BSO diaphragm. We conclude that 1) cellular redox state is more optimal for contractile function and fatigability is increased in rat diaphragm following BSO treatment, 2) SERCA2a expression is modulated by redox signaling, and 3) regulation of SERCA function in working diaphragm is altered following BSO treatment.
Tupling
A R
AR
Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada. rtupling@uwaterloo.ca
Vigna
C
C
Ford
R J
RJ
Tsuchiya
S C
SC
Graham
D A
DA
Denniss
S G
SG
Rush
J W E
JW
D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D019328:Buthionine Sulfoximine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D003964:Diaphragm / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D007700:Kinetics; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects*; D018763:Muscle Fatigue / Q000187:drug effects; D053580:Muscle Strength / Q000187:drug effects; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation
D000975:Antioxidants; C506945:Atp2a1 protein, rat; C506948:Atp2a2 protein, rat; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D019328:Buthionine Sulfoximine; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D002118:Calcium