CORRELATION OF ISCHEMIA REPERFUSION OR PARTIAL OUTLET OBSTRUCTION-INDUCED SPECTRIN PROTEOLYSIS BY CALPAIN WITH CONTRACTILE DYSFUNCTION IN RABBIT BLADDER/

Objectives. In the rabbit, both experimental ischemia and partial outl et obstruction of the urinary bladder induce similar dysfunctions with regard to the contractile responses to both field (neuronal) stimulat ion and postsynaptic receptor stimulation. Circumstantial evidence ind icates that the pathologic response to both conditions is related to t wo connected processes-tissue ischemia and reperfusion injury-that res ult in a marked increase in intracellular calcium ([Ca2+](i)), followe d by the activation of the Ca2+-dependent neutral protease calpain. Ca lpain activation results in the proteolysis of specific membrane prote ins, including those of neuronal membranes (resulting in progressive d enervation of the detrusor) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA), resulting in the previously reported decrease in SERCA. The current study is designed to generate direct support for the theory th at both ischemia and partial outlet obstruction result in the activati on of calpain, Methods. Separate sets of rabbits were subjected to 1 o r 2 hours of ischemia, followed by reperfusion for different lengths o f time, or partial outlet obstruction for different lengths of time. W e determined the state of calpain activation by quantitating tissue pr oteolysis of alpha-spectrin by Western blot analysis. Correlative orga n bath studies were conducted to observe the contractile responses of bladder strips to field stimulation and bethanechol administration. Re sults. (1) Sixty minutes of ischemia followed by 30 minutes of reperfu sion resulted in (a) a reduction in the contractile responses to field stimulation and bethanechol (89% and 57%, respectively), and (b) a 72 % decrease in native alpha-spectrin, with a concomitant 300% increase in its breakdown products (BDPs). Neither alpha-spectrin nor its BDPs had returned to control levels after 72 hours of reperfusion. (2) Twen ty-four hours after the creation of a partial obstruction, alpha-spect rin BDP levels were increased 330%, then gradually fell to 130% of con trol levels by 14 days after obstruction. Concomitantly, the native al pha-spectrin level was decreased 74% 24 hours after obstruction and re mained low through 7 days after obstruction. At 14 days after obstruct ion, the alpha-spectrin levels had recovered to 75% of control levels. Conclusions. These findings suggest that Ca2+-dependent proteolysis o f the preferred calpain substrate alpha-spectrin in urinary bladder ti ssues is increased significantly by both ischemia/reperfusion and part ial outlet obstruction. Temporally, proteolysis precedes the reduced m uscle function resulting from these pathologic conditions. Copyright 1 997 by Elsevier Science Inc.