reperfusion or partial outlet obstruction-induced spectrin proteolysis by calpain with contractile dysfunction in rabbit bladder

ELSEVIER

CORRELATION OF ISCHEMIAIREPERFUSION OR PARTIAL OUTLET OBSTRUCTION-INDUCED SPECTRIN PROTEOLYSIS BY CALPAIN WITH CONTRACTILE DYSFUNCTION IN RABBIT BLADDER YANG

ZHAO,

SHEILA

S. LEVIN,

ALAN

J. WEIN,

AND

ROBERT

M. LEVIN

ABSTRACT 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 ([Ca”]J, followed by the activation of the Ca2+-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 Ca2+-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 rictivation 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) Twentyfour 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. of the preferred calpain substrate alConclusions. These findings suggest that Ca2+ -dependent proteolysis pha-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. Copyright 1997 by Elsevier Science Inc. UROLOGY 49: 293-300, 1997.

This work was supported in part by grantsfrom the Veterans Administration Medical Center and NIH Grants RO-I-DK 26508, RO-l-DK-33559, RO-1 -DK 44689, and RO-1 -DK 39740. From the Division of Urology, University of Pennsylvania, Philadelphia, Pennsylvania; Department of Biological Science, Albany College of Pharmacy, Albany, New York; Division of Urology, Albany Medical College and Veterans Administration Medical Centers, Stratton, Albany, New York and Philadelphia, Pennsylvania. Reprint requests: Robert M. Levin, M.D., Department of Biological Science, Albany College of Pharmacy, Albany, NY 12208 Submitted: July 16, 1996, accepted (with revisions): August 28, 1996 COPYRIGHT 1997 BY ELSEVIER SCIENCE INC. ALL

RIGHTS

RESERVED

linically, ischemia and partial outlet obstruction (secondary to benign prostatic hyperplasia [BPH]) of the urinary bladder are associated with a variety of pathologic conditions that result in a dysfunctional bladder.le3 In experimental rabbit models, both ischemia and partial outlet obstruction have similar effects on bladder morphology, biochemistry, urodynamics, and contractile function.4-7 The response to partial outlet obstruction can be characterized as follows: There is a significant increase in bladder mass that begins at 1 day and reaches a maximum be-

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tween 7 and 14 days after obstruction.4l5 Metabolically, there is a shift from oxidative to anaerobic metabolism and a marked reduction in mitochondrial enzyme activity associated with the decreased contractile responses to various forms of stimulation, including field stimulation and direct receptor stimulation.5’7-‘0 Unilateral ischemia results in a very similar sequence of events.7’11’12 On a molecular level, both partial outlet obstruction and ischemia result in a very similar pattern of gene activation.13’14 The common factor for both experimental ischemia and partial outlet obstruction is a reduction of blood flow (bladder wall perfusion) and the resulting hypoxia. It has been demonstrated in both dog and rabbit models of partial outlet obstruction that both bladder wall perfusion and oxygen tension decrease with bladder filling and contraction and that reperfusion and reoxygenation occur on bladder emptying.15-l7 That reperfusion injury in the rabbit bladder occurs after release of urethral ligation was evidenced by the presence of malondialdehyde (MDA), a marker for lipid peroxidation, in both bladder muscle and mucosa.l* Elevated concentrations of mtracellular calcium ([ Ca’+]J during and/or after transient ischemia have been shown to trigger cellular events that lead to neural degeneration and death.19-2’ During ischemia-induced hypoxia, calcium enters vulnerable neurons through voltage-sensitive and receptor-operated channels and is released from intracellular stores.22 Consequently, calpain, a calcium-activated neutral protease, degrades several prominent cytoskeletal proteins, including spectrin, microtubuleassociated protein, and neurofilament proteins.23-25 Spectrin is a prominent component of the plasma membranes of neurons and other cells of the central nervous system, where it comprises 3% of the total membrane protein.26 This mechanism of ischemiainduced elevation of [Ca’+]i to above physiologic levels and the subsequent activation of calpain proteolysis of spectrin have been observed in the brain and heart and are thought to be a major pathologic pathway.26)27 Reperfusion after ischemia causes a further increase in [Ca’+]i and further calpain activation.27 The current study was designed to determine whether the contractile and cellular dysfunctions induced by ischemia (with reperfusion) and partial outlet obstruction are related to calpain activation. As our initial investigation, we quantitated the effect of ischemia/reperfusion and partial outlet obstruction on spectrin proteolysis. MATERIAL

AND

METHODS

MATERIAL Purified human erythrocyte spectrin, mouse monoclonal anti-spectrin antibody, and polyclonal goat anti-mouse im-

294

munoglobulin ical Co.

G (IgG) were purchased

from Sigma Chem-

Fifty-two male New Zealand White rabbits (3.0 to 3.5 kg) were used for this study. Each rabbit was anesthetized with ketamine/xylazine (25 mg/lO mg intramuscularly); surgical anesthesia was maintained with intravenous pentobarbital. Partial Obstruction of Bladder Outlet (20 Rabbits). The urinary bladder was catheterized with an 8F catheter, and the bladder was exposed through a midline incision. A partial obstruction was created by placing a O-silk ligature loosely around the catheterized urethra. The catheter was removed and the incision closed with 3-O silk. Control sham-operated rabbits underwent the same procedures (anesthesia, catheterization, and surgery) except for placement of the ligature. At 1,3,5, 7, and 14 days after obstruction (4 rabbits at each time period), rabbits were anesthetized again. The bladders were rapidly removed, frozen in liquid nitrogen, and stored at -80°C. Ischemia. The vesical arteries and veins were dissected away from the bladder at the level of the bladder neck, and vessel clamps were placed bilaterally to occlude the vasculature. The bladder of each animal was emptied to ensure that overdistention did not play a role in this study. The exposed bladder was then covered with Tyrode’s solution-soaked sponges. Sham operation of the control animals (4 rabbits) included anesthesia, bladder exposure, and bladder coverage but no vessel clamping. After 10, 20, 40, or 60 minutes of vessel occlusion followed by 30 minutes of reperfusion via removal of the vessel clamps, one-half of each bladder tissue specimen was used for Western blot studies and the other half for muscle bath experiments. In an extended reperfusion group, after 60 minutes of occlusion, the vessel clamps were removed, and the incision was closed with 3-O silk; at 24 and 72 hours after surgery, these rabbits were anesthetized again and their bladders removed. Four rabbits were studied at each time point.

MUSCLE BATH STUDIES Four longitudinal strips measuring 0.3 X 1.0 cm were cut from each bladder sample and suspended individually in an oxygenated (95% Oz, 5% COz) muscle bath chamber containing 30 mL of Tyrode’s solution with dextrose. Each strip was then allowed to equilibrate for 60 minutes at l-g tension. The contractile response to field stimulation was determined using stimulation parameters of 80 V, 1-ms duration, 20-second train, and 1,2,4,8, 16, and 32 Hz. Over 90% of the contractile responses could be blocked by preincubation with tetrodotoxin (1 PM). Only the maximal response to 64 Hz stimulation is presented. The effect of ischemia on the response to all frequencies of stimulation was similar. Dose-response curves to bethanechol (2 to 250 PM) were generated via a cumulative addition of the drug to the muscle baths at 5-minute intervals. The maximal response of the bladder strips to bethanechol (250 PM) is presented. There was no effect of ischemia on the median effective dose (ED,,) for bethanechol. The contractile responses were monitored using a Grass 79D polygraph.

EXTRACTION OFPROTEIN Frozen tissue was homogenized with a Polytron homogenizer in extraction buffer (10 mM Tris-acetate, 10 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 1 mM phenylmethylsulfonyl fluoride [PMSF], and 2 PM leupeptin) for 1 minute, and the tissue homogenate was then centrifuged (39OOg) for 10 minutes. The supernatant containing the tissue extract was collected, and the concentration of total soluble protein was measured by the Bradford method. UROLOGY

49 (2), 1997

SODIUMDODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS Protein extracts were separated by 6% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Tissue extracts were thawed on ice and mixed 1:l with SDS sample buffer. After boiling at 100°C for 1 minute, a lO+L aliquot of each sample was loaded, and electrophoresis was carried out at 10 mA per gel for 2 hours at room temperature. Purified spectrin was run on the same gel each time.

WESTERNBLOTTING After electrophoretic separation, electroblotting of the proteins to nitrocellulose membranes was performed. Before the electrophoresis was completed, the transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol, pH 8.3) was prepared and placed in the refrigerator to equilibrate at 4°C. Nitrocellulose membranes, filter pads, and filter papers were soaked in a cold transfer buffer for at least 10 minutes. Transfer was carried out at 30 V overnight (more than 15 hours) and kept in a cold box (4°C) to prevent denaturation of sample proteins.

After blotting, the nitrocellulose membranes were placed in Tris-buffered blocking solution (0.5 M NaCl, 50 mM Tris, pH 7.5) containing 5% nonfat dry milk and gently agitated for 1 hour at room temperature. After the blocking procedure, blots were washed three times for 10 minutes with Tris-buffered saline containing 0.1% Tween-20 (TTBS) and incubated with a mouse anti-spectrin monoclonal antibody overnight (more than 15 hours). Blots were washed three times for 10 minutes in TTBS and then incubated for 2 hours at room temperature with the second antibody, a goat anti-mouse IgG peroxidase conjugate diluted 1:400 in Tris-buffered saline buffer (TBS). Blots were removed from the secondary antibody solution, washed twice for 10 minutes in TTBS, then 10 minutes in TBS. Blots were immersed in 100 mL of color developing solution containing 40 mg of 3’-diaminobenzidine tetrahydrochloride (DAB), 10 mL of 0.5 M Tris, 1 mL of 1 M imidazole, and 25 PL of Hz02 until a light brown color developed.28

DENSITOMETRK QUANTITATION After air drying at room temperature, nitrocellulose membranes were scanned directly with an Ultra Scan XL laser scanner (LKB Pharmacia Co.) at 633 nm. The amounts of the protein in each sample were quantified from the intensity of the bands. Preliminary studies confirmed that the intensity of spectrin bands was linear to the amounts of pure spectrin applied to the gel. The intensity of bands was expressed in arbitrary units. Statistical difference was evaluated by the Newman-Keuls post hoc method. A probability value of 0.05 was considered statistically significant.

~MMLJNOREACTIVITYOFk4BBIT URINARYBLADDER PROTEINSTO MOUSE ANTI-SPECTMNANTIBODY To detect and quantify spectrin and its breakdown products (BDPs) from normal rabbit urinary bladder tissue, cross-reactivity between mouse anti-spectrin antibody and rabbit urinary bladder proteins was assessed via Western blotting.28 Similarly, to demonstrate spectrin proteolysis in ischemic and obstructed bladder tissue, cross-reactivity between mouse anti-spectrin antibody and rabbit proteins was assessed in immunoblots of protein in the extracts from rabbit urinary bladder tissue subjected to these experimental pathologies. Although the antibody reacts with pure spectrin protein that contains both 240- and 235-kilodalton (kDa) components, UROLOGY

49 (Z), 1997

only the 240-kDa alpha-spectrin band was evident on Western blots of rabbit bladder tissues. The spectrin degradation products from rabbit bladder tissues (normal, ischemic, and obstructed) appeared as 70- and 80-kDa bands.

RESULTS EFFECTOF bCHEMJA/&PERFUSIONON CONTRACTILEFLINCT~ONOF~BBITBLADDER SMOOTHMUSCLE

At the end of 60 minutes of ischemia, the experimental bladders were noticeably blue in color; they gradually returned to their original color approximately 15 minutes after the vessel clamps were removed. Figure 1 shows the contractile responses of bladder strips to 32-Hz field stimulation. There was no significant inhibition of muscle contractility exhibited by bladder strips from rabbits subjected to 10,20, or 40 minutes of ischemia followed by 30 minutes of reperfusion. However, a significant 89% decrease in muscle contraction was observed in tissue strips from bladders subjected to 60 minutes of ischemia followed by 30 minutes of reperfusion. The effect of ischemia on the response of the bladder to all frequencies was similar. Figure 2 shows the responses to bethanechol stimulation (250 ,uM). There was no effect of ischemia on the ED5,,. A similar time-dependent inhibition of muscle contraction was exhibited by bladder strips from animals subjected to 10, 20, 40, and 60 minutes of ischemia followed by 30 minutes of reperfusion. TIME-COLJRSEOFSPECTRINPROTEOLYSLSAFTER COMPLETEBLADDERISCHEMIAAND REPERFLJSION

The levels of both native alpha-spectrin and its BDPs were consistent among control animals (data not shown). Figure 3 shows that the level of BDPs in the ischemic bladder was increased 260% after 10 minutes of ischemia followed by 30 minutes of reperfusion and did not increase further (significantly) through 60 minutes. Figure 4 shows that native alpha-spectrin was decreased by 25% after 10 minutes of ischemia followed by 30 minutes of reperfusion and progressively decreased by 75% after 60 minutes of ischemia followed by 30 minutes of reperfusion. To determine the effect of extended reperfusion on spectrin proteolysis, some ischemic rabbits were allowed to recover at 24 or 72 hours. After 24 or 72 hours, the levels of BDPs were, respectively, 120% or 123% of control levels (Fig. 5), whereas the levels of native alpha-spectrin remained at only 40% or 45% of control levels (Fig. 6) over the same time period.

TrheCoum

OF SPECTRINPROTEOLYS~S AFTER PARTIAL BLADDER OUTLET OBsTRucTroN

A significant increase in bladder mass was evident at 1 day after surgery and reached a maxi295

T

-i

Control

40 60 20 Duration of &hernia (min)

FIGURE 1. Effect of ischemia on muscle contraction in response to field stimulation. Samples were subjected to 10, 20, 40, and 60 minutes of complete ischemia followed by 30 minutes of reperfusion. Field stimulation was conducted using 80 Vat 32 Hz. Each point is the average of eight individual preparations isolated from 4 rabbits ? SEM.

mum of fivefold greater than control levels at 14 days after obstruction. The soluble protein level was not significantly changed over this time (data not shown). Figure 7 shows a 330% increase in BDPs during the first 24 hours after obstruction, which gradually decreased to 130% of control levels over the next 14 days. Correspondingly, the level of native alpha-spectrin was decreased 74% at 24 hours and remained at this low level until 7 days after obstruction. At the end of 14 days, the alpha-spectrin level had returned to 75% of control levels (Fig. 8). Although no contractile studies were performed in association with these studies on partial outlet obstruction, previous studies using this same model have demonstrated that the contractile responses to field stimulation and bethanechol were maximally decreased at 1 day after obstruction and recovered progressively to a stable level by 14 days.29 COMMENT

The specific aim of the current study was to identify the cellular mechanisms that relate to the contractile dysfunctions observed in our severe models of partial outlet obstruction and ischemia in the rabbit. These studies show that the timecourse of calpain activation is consistent with the time-course of the contractile dysfunctions in both the ischemic and obstruction models. Because of 296

40 60 20 Duration of &hernia (min)

Contml

10 I

10 I

FIGURE 2. Effect of ischemia on muscle contraction in response to bethanechol stimulation. Samples were subjected to 10, 20, 40, and 60 minutes of complete ischemia followed by 30 minutes of reperfusion. Dose-response curves to bethanechol(250 PM) were generated. Each point is the average of eight individual preparations isolated from 4 rabbits + SEM.

i

60

40

20

0 Control

10 I

20

40

60

Duration of &hernia (min)

FIGURE 3. Effect of ischemia

on the concentration of breakdown products. Bladder samples were subjected to IO to 60 minutes of complete ischemia followed by 30 minutes of reperfusion. Total soluble protein extraction was analyzed by SDS-PAGE and Western blot with mouse anti-spectrin monoclonal antibody. The spectrin breakdown product bands were quantified by densitometric analysis. Each bar is the average of eight individual preparations isolated from 4 rabbits + SEM. “P ~0.05, statistically different from control (no ischemia). UROLOGY

49 (3, 1997

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Duration of ischemia (min) FIGURE 4. Effect of ischemia on the concentration of spectrin. Bladder samples were subjected to 10 to 60 minutes of complete ischemia followed by 30 minutes of reperfusion. Total soluble protein extraction was analyzed by SDS-PAGE and Western blot with mouse antispectrin monoclonal antibody. The native spectrin bands were quantified by densitometric analysis. Each bar is the average of eight individual preparations isolated from 4 rabbits + SEM. * P ~0.05, statistically different from control (no ischemia).

the acute nature of these studies, no direct correlation with micturition dysfunctions in men with obstructive dysfunction secondary to BPH should be made.” However, we have studied both rabbit and cat models of mild bladder outlet obstruction for periods of time up to 1 year. These models develop contractile dysfunctions slowl~.~~,~~ At the end of 1 year, a significant number of rabbits and a higher percentage of cats show increased pressure generation and only minor problems with emptying. These groups of animals have enlarged bladders in the presence of the partial outlet obstruction and compensated bladder function. Other animals show various levels of decompensation and functional impairment. The animals that show decompensation after 1 year of obstruction have characteristics very similar to the dysfunctions observed in the severly obstructed rabbits after 2 weeks, and thus our hypothesis is that decompensation, independent of when it occurs, is mediated by the same cellular dysfunctions. In our severe model, we believe that the contractile and cellular dysfunctions associated with partial outlet obstruction result from ischemia (hypoxia) and ischemia followed by reperfusion (superoxide generation). Both hypoxia- and superoxide-generated reactive oxygen species (eg, UROLOGY 49 (21,1997

-r Control 30min 24hrs 12hrs I I Duration of reperfusion

FIGURE 5. Effect of reperfusion on the concentration of breakdown products. Bladder samples were subjected to 60 minutes of complete ischemia followed by 24 and 72 hours of recovery (reperfusion). Total soluble protein extraction was analyzed by SDS-PAGE and Western blot with mouse anti-spectrin monoclonal antibody. The spectrin breakdown product bands were quantified by densitometric analysis. Each bar is the average of eight individual preparations isolated from 4 rabbits L SEM.

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FIGURE 6.

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Duration of reperfbsion

of reperfusion on the concentration of spectrin. Bladder samples were subjected to 60 minutes of complete ischemia followed by 24 and 72 hours of recovery (reperfusion). Total soluble protein extraction was analyzed by SDS-PAGE and Western blot with mouse anti-spectrin monoclonal antibody. The native spectrin bands were quantified by densitometric analysis. Each bar is the average of eight individual preparations isolated from 4 rabbits 2 SEM. *P ~0.05, statistically different from control (no ischemia). Effect

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Duration of obstruction (days) FIGURE 7. Effect of partial outlet obstruction on the concentration of breakdown products. Bladder samples were subjected to 1 to 14 days of partial outlet obstruction. Total soluble protein extraction was analyzed by SDS-PAGE and Western blot with mouse anti-spectrin monoclonal antibody. Spectrin breakdown product bands were quantified by densitometric analysis. Each bar is the average of eight individual preparations isolated from 4 rabbits I SEM. *P ~0. OS, statistically different from control (no obstruction).

FIGURE 8. Effect of partial outlet obstruction on the concentration of spectrin. Bladder samples were subjected to 1 to 14 days of partial outlet obstruction. Total soluble protein extraction was analyzed by SDS-PAGE and Western blot with mouse anti-spectrin monoclonal antibody. The native spectrin bands were quantified by densitometric analysis. Each bar is the average of eight individual preparations isolated from 4 rabbits +- SEM. *P ~0.05, statistically different from control (no obstruction).

H,Oz) disrupt mitochondrial Ca2+ homeostasis (by different mechanisms), thereby initiating a cascade of events, which leads to pathologically elevated cytosolic Ca2+ accumulation.33-35 Calcium ion overload triggers signaling pathways that activate hydrolytic enzymes, such as endonucleases, phospholipases A2 and C, and proteases (eg, calpain). Calcium ion-activated endonucleases modulate DNA strand breaks and altered gene expression. 37,38Phospholipase A2 activity results, ultimately, in modification of the permeability of the inner mitochondrial membrane by creating defects in the membrane lipid phase; it also disrupts plasmalemmal integrity.3g These membrane disturbances further serve to deregulate [Ca’+]i homeostasis. Calpains are a family of at least six Ca2+ -dependent neutral endopeptidases whose mode of action is nondigestive, limited proteolysis. These cytoplasmic enzymes act at cell membranes on protein substrates that are closely associated with or that translocate to the cell membrane on stimulation. Calpain substrates include cytoskeletal proteins (spectrin, MAPS, neurofilaments); membrane proteins (EGF, integrin, Ca2+/MgZf-ATPase); enzymes (PKC, MLCK, and calcineurin); and transcription factors (Fos, Jun) .40,41

Significantly enhanced alpha-spectrin proteolysis in ischemic/reperfused and obstructed bladder tissues was evidenced by both decreased native alpha-spectrin content and increased alpha-spectrin BDPs. This suggests that both bladder ischemia/ reperfusion and partial outlet obstruction cause pathologic changes via a similar intracellular pathway. Studies of the cellular responses to partial outlet obstruction and ischemia demonstrate three major cellular dysfunctions that result in the observed reductions in bladder contractility and function (ie, ability to empty). These include proreductions in mitochongressive denervation42; drial oxidative phosphorylation8~43; and a selective decrease in the activity of SERCA.44,45 The premise that these dysfunctions can result from ischemia/ reperfusion-induced disruptions in Ca2+ homeostasis is supported by the following observations: (1) In vitro experimental hypoxia of normal bladder tissue results in a rapid and excessive increase in free [Ca2+]i.46 (2) The time-course of calpain activation correlates with the time-course of the contractile and cellular dysfunctions. (3) The contractile response to neuronal and receptor stimulation is dependent on the release of stored Ca2+ from the sarcoplasmic reticulum (SR)47,48; the contractile dysfunctions associated with partial outlet obstruction correlate with disrupted SR Ca2+ stor-

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age/release mechanisms.47’48 (4) The degree of contractile dysfunction is directly proportional to the degradation of SERCA (a preferred calpain substrate), as shown using both enzymatic activity assays and Western blot analysis.44%45 (5) Limiting Ca2+ entry into the cell by treatment with Ca2+ channel blockers results in protection of the bladder after partial outlet obstruction in rats.4g Further support comes from studies that showed that in both canine and rabbit models of partial outlet obstruction, bladder wall perfusion (blood flow) and oxygen tension decrease with bladder filling and that reperfusion and reoxygenation occur on bladder emptying. Bladder filling and emptying in nonobstructed animals are not associated with altered blood perfusion or the level of tissue oxygenation.15-l7 In the rabbit model, reperfusion injury in rabbit bladder was evidenced by the presence of MDA, a marker for lipid peroxidation.l’ The concentration of BDPs in normal bladder was relatively low compared with the concentration of native spectrin. This is consistent with the demonstration that the initial response to both ischemia and outlet obstruction is a substantial increase in BDPs that corresponds to a relatively small decrease in native spectrin. Because the BDPs can continue to degrade beyond the ability of the antibodies to bind, the concentrations of native spectrin and BDPs do not necessarily relate to each other at the longer time periods. In conclusion, we hypothesize that, similar to ischemic disorders of other organ systems (eg, brain and heart), the etiology for bladder dysfunction secondary to partial outlet obstruction and ischemia is related directly to calpain activation by hypoxia-induced phasic increases in [ Ca’+], and subsequent superoxide-induced Ca2+ cycling (and further [ Ca’+]i increase) on reperfusion. These processes cause disruption of neuronal, mitochondrial, and SR membranes, resulting in the observed detrusor denervation, decreased rate of mitochondrial oxidative phosphorylation, and decreased SERCA activity. REFERENCES 1. Sterling AM, Ritter RC, and Zinner NR: The physical basis of obstructive uropathy, in Hinman F Jr (Ed): Benign Prostatic Hypertrophy. New York, Springer-Verlag, 1983, pp 433-442. 2. Grayhack JT, and Kozlowski JM: Benign prostatic hyperplasia, in Gillenwater JY, Grayhack JT, Howards SS, and Duckett JW (Eds): Adult and Pediatric Urology. Chicago, Yearbook Medical Publishers, 1987, pp 1062-1126. 3. Wein AJ, Levin RM, and Barrett DM: Voiding function and dysfunction. Relevant anatomy, physiology, and pharmacology, in Gillenwater JY, Grayhack JT, Howards SS, and Duckett JW (Eds): Adult and Pediatric Urology. St. Louis, Mosby Year Book, 1991, pp 933-999. 4. Malkowicz SB, Wein AJ, Elbadawi A, Van Arsdalen K, Ruggieri MR, and Levin RM: Acute biochemical and funcUROLOGY

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