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 outlet obstruction of the urinary bladder induce similar dysfunctions with regard to the contractile responses to both field (neuronal) stimulation and postsynaptic receptor stimulation. Circumstantial evidence indicates that the pathologic response to both conditions is related to two connected processes-tissue ischemia and reperfusion injury-that result in a marked increase in intracellular calcium ([Ca2+]i), followed by the activation of the Ca(2+)-dependent neutral protease calpain. Calpain activation results in the proteolysis of specific membrane proteins, including those of neuronal membranes (resulting in progressive denervation of the detrusor) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), resulting in the previously reported decrease in SERCA. The current study is designed to generate direct support for the theory that both ischemia and partial outlet obstruction result in the activation of calpain.
METHODS: Separate sets of rabbits were subjected to 1 or 2 hours of ischemia, followed by reperfusion for different lengths of time, or partial outlet obstruction for different lengths of time. We determined the state of calpain activation by quantitating tissue proteolysis of alpha-spectrin by Western blot analysis. Correlative organ bath studies were conducted to observe the contractile responses of bladder strips to field stimulation and bethanechol administration.
RESULTS: (1) Sixty minutes of ischemia followed by 30 minutes of reperfusion 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) Twenty-four hours after the creation of a partial obstruction, alpha-spectrin BDP levels were increased 330%, then gradually fell to 130% of control levels by 14 days after obstruction. Concomitantly, the native alpha-spectrin level was decreased 74% 24 hours after obstruction and remained low through 7 days after obstruction. At 14 days after obstruction, the alpha-spectrin levels had recovered to 75% of control levels.
CONCLUSIONS: These findings suggest that Ca(2+)-dependent proteolysis of the preferred calpain substrate alpha-spectrin in urinary bladder tissues is increased significantly by both ischemia/reperfusion and partial outlet obstruction. Temporally, proteolysis precedes the reduced muscle function resulting from these pathologic conditions.