Effect of partial outlet obstruction on rabbit urinarybladder smooth muscle func - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:35 |只看该作者 |倒序浏览 |打印

作者：XiaolingSu, RaymundStein, Michaela C.Stanton, StephenZderic,  Robert S.Moreland作者单位：1 Department of Pharmacology and Physiology, DrexelUniversity College of Medicine, Philadelphia 19102; and Department of Urology, The Children‘s Hospital ofPhiladelphia, Philadelphia, Pennsylvania 19101
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      【摘要】
   Bladder outlet obstructionsecondary to benign prostate hyperplasia is associated with manycellular changes. This study was designed to determine whether thesechanges involve the contractile apparatus. Bladder smooth muscles fromrabbits subjected to partial outlet obstruction for 2 wk were mountedfor isometric force, isotonic shortening velocity, and myosin lightchain (MLC) phosphorylation levels. Muscle strips from obstructedbladders exhibited spontaneous phasic activity; muscle strips fromcontrol bladders did not. Muscle strips from obstructed bladdersexhibited increased sensitivity and higher levels of stress in responseto the cumulative addition of KCl or carbachol compared with control.During noncumulative addition of KCl or carbachol, no differences insensitivity were noted. Muscle strips from obstructed bladders hadelevated basal MLC phosphorylation levels and stimulation producedsmall increases in MLC phosphorylation compared with control. V max during KCl stimulation of muscle stripsfrom obstructed bladders was 10-fold lower than control. Our resultssuggest that bladder outlet obstruction produces a muscle cell thatdevelops higher levels of force but with greatly reduced cross bridgecycling rates. 4 t: |5 Z& {7 [3 m
      【关键词】 benign prostatic hyperplasia shortening velocity myosin lightchain phosphorylation carbachol! O! ^+ ^/ ]; ~' ?- u# Q
   INTRODUCTION( r$ i1 z6 H. y7 a8 N
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THE PATHOLOGICAL PROGRESSION of untreated bladder outlet obstruction has beenexplained clinically by the concept of a detrusor muscle compensatoryresponse. This concept assumes that in response to obstruction, thebladder smooth muscle hypertrophies to produce the elevated pressuresnecessary to maintain effective emptying. However, if the obstructionis left untreated, the bladder becomes dysfunctional, leading to asignificant loss of contractile ability and an increase in postvoidresidual volume. This is presumably due to an imbalance between thepassive and active mechanical properties of the detrusor muscle and themagnitude of the resistance to flow. Removal of the obstruction beforea state of severe dysfunction reverses the hypertrophic response, andnormal function may be regained ( 7, 26 ).; h2 O+ ~& h! u" H

In general, results from studies using animal models of bladder outletobstruction report rapid and marked morphological and functionalchanges in the detrusor muscle, similar to those reported in humanclinical studies ( 3, 6, 11, 17, 20, 23 ). The majority ofthese animal studies report a decrease in several parameters ofdetrusor contractility. This is also true using the model we employedin the present study, the acute partially obstructed rabbit urinarybladder model ( 15 ). In this acute animal model, partialobstruction results in 1 ) significant hypertrophy of thesmooth muscle with a several-fold increase in bladder mass; 2 ) a decrease in the sensitivity to cholinergic stimulationof both the isolated whole bladder and isolated mucosal intact smooth muscle strips from the bladder body; 3 ) a decrease inabsolute isometric force development using mucosal intact strips ofbladder wall; and 4 ) an increase in postvoiding residualvolume ( 14, 27, 29, 30 ). Our goal for the present studywas to verify whether outlet obstruction decreases smooth musclecontractility, using a preparation containing primarily smooth musclecells (devoid of both serosal and mucosal layers), and to ascertain thestep(s) involved in the excitation process that may account for theobstruction induced changes.

More specifically, it is known that the time course of isometric forcedevelopment of the normal intact rabbit bladder or isolated strips ofbladder smooth muscle to agonist activation consists of two phases: aninitial transient phase, in which force rises rapidly to a peak(phasic) then decaying slowly before attaining a steady level that ismaintained for a prolonged period, and the tonic phase( 24 ). In the hypertrophied bladder, both the phasic andtonic components of the isometric force response have been shown to bedepressed, using mucosal intact bladder wall strips ( 15, 16, 27 ). In particular, it has been suggested that a reduced rate offorce development and a significantly reduced ability to maintain forceduring the tonic phase occur in mucosal intact muscle strips from theobstructed bladder ( 15, 21 ). This present study wasdesigned to verify these findings in a bladder strip preparationcontaining primarily smooth muscle cells and then examine themechanism(s) potentially responsible for this altered mechanical performance.

MATERIALS AND METHODS
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Animal model. Four-month-old male New Zealand White rabbits weighing 2.5-3.5 kgwere used in this study. All animal studies were approved by theChildren's Hospital of Philadelphia Animal Care and Use Committee.Partial bladder-outlet obstruction was created as previously reported( 21 ). Briefly, after the animal was anesthetized, an 8 French catheter was inserted into the bladder via the urethra, and thebladder neck was exposed through a small vertical extraperitoneal abdominal incision. The ureters and vas deferens were identified, and a2-0 silk suture was placed below the bladder neck. To maximize standardization of the partial outlet obstruction, a second 8 Frenchcatheter was placed outside the urethra, and the silk suture was tiedaround both catheters. Both catheters were then removed. In thesham-operated group, the silk suture was placed around the catheterizedurethra but not tied and then the catheter was removed. Data collectedfrom sham-operated rabbits along with rabbits that did not undergo anysurgical intervention were used as the control. The rabbits were housedin metabolic cages and monitored for voiding frequency and volume.Fourteen days after surgery to induce partial outlet obstruction, theanimals were euthanized using IACUC-approved techniques, and thebladders were quickly removed., r4 ?+ v8 }4 Q+ s0 c

Tissue preparation. The bladder neck, trigone, and base region were removed, leaving onlymiddle detrusor body for experimentation. In all but one set ofexperiments, the mucosa and serosa were carefully removed under adissecting microscope. In one set of experiments, the mucosal layer wasretained. Muscle strips (~1.5 × 6 mm) were cut along thecentral axis of the bladder in the longitudinal orientation. At leastfour to eight strips were obtained from each bladder. The bladderstrips were mounted in water-jacketed muscle chambers containing aMOPS-buffered physiological salt solution ( 18 ) at 37°Cand aerated with 100% O 2. The strips were equilibrated forat least 90 min. After an equilibration period, a partial length-tension curve was performed to determine the optimal length foractive stress development ( L o ).- t3 N+ U4 `* A  r5 E- d; b

Tissues to be used for histological examination were fixed at L o in buffered 10% formalin. The tissues wereembedded in paraffin from which 5-µm longitudinal and transversesections were cut and stained with hematoxylin-eosin and with Massontrichrome. Histological sections from the center of the embedded tissuestrips were used for determination of proportion of smooth muscle to avoid any end effects from tissue clamps. Sections were magnified andprojected onto a sheet of heavy paper. The total and smooth musclespecific areas were outlined, cut, and weighed. The ratio of the smoothmuscle area to the total area was used to estimate the proportion ofmuscle in the tissue sections.

Measurement of contraction. Bladder strips used for isometric force recording were mounted betweentwo plastic clips, one attached to a micrometer for length adjustmentand the other to a Grass FT.03 force transducer and a Grass model 7Dpolygraph. Concentration-response curves were constructed by either thecumulative or noncumulative addition of KCl (equimolar substitution forNaCl) or carbachol. Each muscle strip was subjected to one of fourprotocols. Data obtained in these protocols are expressed as activestress (stress = force/cross-sectional area) or normalized as apercentage of the maximal response to 110 mM KCl. Cross-sectional areawas determined using tissue length and wet weight as previouslydescribed ( 18 ).
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Estimates of maximal velocity of shortening were performed bysubjecting the bladder strips to a series of isotonic quick releases toafterloads ranging from 0.12 to 0.4 times the force at the instant ofrelease as previously described for vascular smooth muscle( 18 ). Strips were mounted on one end by a plastic clipattached to a micrometer for control of muscle length and on the otherend by an aluminum foil tube connected to a Cambridge Technology 300Hservo lever interfaced to Northstar Horizon computers. Isotonicshortening velocity at each afterload was estimated using the lengthchange between 1 and 2 s after the release. A linearization of thehyperbolic force-velocity equation was used to estimate the maximalvelocity of shortening during zero load.2 b9 X/ L. R1 V/ m+ b8 A$ P+ h2 a$ T
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Biochemical studies. For measurement of myosin light chain (MLC) phosphorylation levels, allstrips were mounted, equilibrated, and then rapidly frozen atappropriate time points during a contractile event in a dry ice/acetoneslurry containing 6% trichloroacetic acid and 10 mM DTT. The stripswere then slowly thawed at room temperature. The tissues were rinsed inacetone and air-dried, and then dry weights were recorded. Theacetone-dried tissues were homogenized in a solution containing 1%SDS, 10% glycerol, and 1 mM DTT using glass/glass homogenizers. Thehomogenates were clarified by centrifugation and then subjected totwo-dimensional gel electrophoresis, followed by transfer tonitrocellulose membranes as previously described ( 19 ).Proteins were visualized using AuroDye forte colloidal gold proteinstain (Amersham) and quantified using laser scanning densitometry(Molecular Dynamics). MLC phosphorylation levels were calculated as apercentage of the sum of the densitometric analysis of both thephosphorylated and unphosphorylated forms of the MLC.

Bladder strips were also processed for the determination ofmyosin-to-actin ratios with minor modifications of techniques previously reported ( 5 ). Briefly, aliquots of thehomogenized tissues were subjected to SDS-PAGE. The separating gel wasdivided into two components; the bottom 5 cm of the gel contained 12% acrylamide, whereas the upper 7 cm contained 7.5% acrylamide. Thestacking gel was the typical 4% acrylamide. The use of two distinctacrylamide concentrations provided better resolution for myosin heavychain and actin on a single gel. Three different dilutions of eachtissue homogenate were loaded onto the gels to ensure linearity ofquantitation. After electrophoresis the gels were stained withCoomassie blue R-250. Band identity of myosin and actin was confirmedby immunoblots. Aliquots of the homogenized tissue samples that wereused for determination of MLC phosphorylation were also used fordetermining total protein content by the Bradford method using bovineserum albumin as a standard.* |6 W4 P2 c9 C+ H, n. z3 V
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Data analysis. All data are presented as means ± SE. Student's t -test and ANOVA were used when it was appropriate. Valuesof P taken as significant.

RESULTS4 x* T% e1 i& i# K$ E$ O0 l

Partial outlet obstruction of the rabbit bladder produces severalsignificant changes in both the structure and function of the organ. Interms of urinary output, Table 1 showsthe data obtained from control and outlet-obstructed animals housed in metabolic cages. Animals subjected to outlet obstruction for 2 wk hadsignificantly higher number of voids/day and significantly loweraverage volume/void. It is noteworthy that total void volume/day is notdifferent between the two animal groups. Bladder weights fromobstructed animals were also significantly elevated compared with thosefrom control animals, suggestive of obstruction-induced bladderhypertrophy.
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Table 1. Functional and anatomic properties of control rabbits and rabbitssubjected to partial bladder outlet obstruction% x( v3 j! M  j. j0 y# g; Z9 p! h
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It has been generally believed that bladder smooth muscles from animalssubjected to partial outlet obstruction develop lower levels of maximalforce ( 7, 14, 15 ). To confirm those results in our ownlaboratory, we measured the time course of maximal force development inbladder strips from control and obstructed animals. In theseexperiments, we used the typical bladder muscle strip in which theserosal layer but not the mucosal layer had been removed. The results,presented in Fig. 1, show thatsignificantly less stress (force/cross-sectional area) is developed bythe muscle strips from an obstructed animal (wet wt = 3.11 ± 0.35 mg; n = 6); compared with control (wet wt = 1.98 ± 0.23 mg; n = 6). This depression of stressdevelopment is noted in response to either agonist activation (10 µMcarbachol; Fig. 1 A ) or membrane depolarization (stimulationby 110 mM KCl; Fig. 1 B ).

Fig. 1. Contraction of bladder wall dissected free of serosallayer. Urinary bladder from control ( ) and partialoutlet-obstructed ( ) rabbits was dissected free of theserosal layer and strips were mounted for isometric force recording. A : bladder smooth muscle strips were contracted by theaddition of 10 µM carbachol. B : bladder smooth musclestrips were contracted by the addition of 110 mM KCl. Strips of bladderwall from animals subjected to outlet obstruction developedsignificantly less stress than strips from control animals. Stripweights were 1.98 ± 0.23 (control) and 3.11 ± 0.35 mg(obstructed). Values are means ± SE; n = 6.

A decrease in stress can be due to either an increase in bladder wallnonmuscle mass or a decrease in muscle cell contractility. To determinewhether muscle mass was altered in bladders from obstructed animals, wedeveloped a tissue preparation devoid of both the serosal and mucosallayers and subjected the tissue containing predominantly a smoothmuscle layer to histological examination as described in MATERIALS AND METHODS. Representative sections from a stripof control bladder smooth muscle and one from an animal subjected topartial outlet obstruction are presented in Fig. 2. Compared with thesmooth muscle strip from a control animal, the tissue from theobstructed bladder shows gross changes in smooth muscle orientation.However, obstruction of the bladder did not alter the content of musclearea (40.4 ± 2.5% muscle in control tissues; 45.2 ± 8.0%muscle in tissue from obstructed bladders; n = 4 bothgroups). These qualitative changes in bladder wall cellular orientationdemonstrate that partial outlet obstruction alters the cytoarchitectureof the tissue but not the percentage of muscle mass, at least in thesmooth muscle layer. In terms of intracellular components, the datalisted in Table 1 show that the ratio of myosin heavy chain to actin isalso not altered by partial outlet obstruction.

Fig. 2. Histological presentation of bladder smooth musclepreparations from control and outlet-obstructed animals. A :longitudinal section of bladder smooth muscle strip from controlanimals stained with hematoxylin-eosin. Section demonstrates parallelarrangement of smooth muscle cells. B : cross section ofbladder smooth muscle strip from control animal stained with Massontrichrome. C : cross section of bladder smooth muscle stripfrom outlet-obstructed animal stained with Masson trichome. Thesections from outlet-obstructed animals appear to have more collagenand less smooth muscle organization.+ Q  a+ L, g- z
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The results obtained from Fig. 2 demonstrate that the percent musclemass does not appear to be significantly different in tissues from thetwo animals groups. Therefore, a decrease in the contractility of thesmooth muscle cells induced by partial outlet obstruction could accountfor the depression in force noted in Fig. 1. Cumulative andnoncumulative concentration-response curves were constructed inresponse to KCl (Fig. 3, A and B ) and carbachol (Fig. 4, A and B ). In these andall subsequent experiments, we used thedissection technique that removed both the serosal and mucosal layers,resulting in a tissue strip with a higher percentage of smooth musclecells. Strips of urinary bladder from partial outlet-obstructed animalsproduced more stress to the cumulative addition of either KCl orcarbachol compared with strips from control animals. Strips of bladderfrom obstructed animals were also more sensitive to either KCl(EC 50 : 18 mM obstructed; 29.5 mM control) or carbachol(EC 50 : 0.27 µM obstructed; 0.84 µM control) during thecumulative response experiments compared with tissue from controlanimals (Figs. 3 A and 4 A ). There were nosignificant differences in the maximal levels of stress developed orsensitivity of response to KCl during the noncumulative response experiments using bladder strips from obstructed compared with controlanimals. There were also no significant differences in the sensitivityto carbachol during the noncumulative response between smooth muscletissues from the two animal groups. The level of stress developed atthe highest carbachol concentration (100 µM) was significantly lessin smooth muscle from obstructed compared with control animals.- H- w7 @# ^9 X9 l! e5 h/ a

Fig. 3. KCl concentration-response curves using rabbit bladdersmooth muscle. A : strips of bladder smooth muscle dissectedfree of both serosal and mucosal layers were subjected to thecumulative addition of KCl from 4.7 to 110 mM. Smooth muscle stripsfrom control bladders ( ) produced lower levels ofstress and were less sensitive in response to the addition KCl comparedwith smooth muscle strips from partially obstructed bladders( ). Maximal levels of stress were developed at 30 mM instrips from partially obstructed bladders and 80 mM in strips fromcontrol bladders. The calculated EC 50 values were 18 mM forstrips from partially obstructed bladders and 29.5 mM for control.Values are means ± SE; n = 5-8. B : strips of bladder smooth muscle from control( ) and partial bladder-obstructed animals( ) were subjected to the noncumulative addition of KCl.There were no differences in either maximal levels of stress attainedor in the sensitivity to KCl between strips from control compared withthose from partial bladder-obstructed animals. The calculatedEC 50 values were 54.1 mM for strips from control bladdersand 37.7 mM for those from obstructed bladders. Values are means ± SE; n = 5-8.

Fig. 4. Carbachol concentration-response curves using rabbitbladder smooth muscle. A : strips of bladder smooth muscledissected free of both the serosal and mucosal layers were subjected tothe cumulative addition of carbachol from 0.01 to 100 µM. Smoothmuscle strips from control bladders ( ) produced lowerlevels of stress and were less sensitive in response to the additioncarbachol compared with smooth muscle strips from partialoutlet-obstructed animals ( ). Maximal levels of stresswere developed at 3 µM in strips from partial outlet-obstructedanimals and 100 µM in strips from control animals. The calculatedEC 50 values were 0.27 µM for strips from partialoutlet-obstructed animals and 0.84 µM for control. Values aremeans ± SE; n = 5-8. B : strips ofbladder smooth muscle from control ( ) and partialoutlet-obstructed animals ( ) were subjected to thenoncumulative addition of carbachol. There was no difference in thesensitivity to carbachol between strips from control compared withthose from partially obstructed bladders. There was a significantdifference in the maximal stress developed only at the highestcarbachol concentration. The calculated EC 50 values 0.87 µM for strips from control and 0.42 µM for those from obstructedanimals. Values shown are means ± SE; n = 5-8.* f: [3 d  ?" f( x) p6 t

During the collection of data for the construction of the noncumulativeconcentration-response curves, one difference in the smooth musclesfrom the two animal sources was striking, that being the temporalprofile of a single contractile event. Figure 5 shows the averaged results of severalcontractions of the bladder strips in response to 110 mM KCl. The rateof the initial phasic force development is significantly slower inbladder smooth muscle strips from control compared with muscle stripsfrom partial outlet-obstructed animals (Table 1 ). Moreover, in contrastto the typical initial phasic contraction followed by the lower butsuprabasal steady-state maintenance of force in bladder strips fromcontrol animals, bladders strips from the outlet-obstructed animalsmaintained peak forces longer and decayed significantly more slowly. Itis also of interest to point out that the time course of a contractionin the mucosa-intact strip is prolonged compared with that in thestrips dissected free of the mucosal layer. We believe this is mostlikely due to enhanced diffusional delays in the thicker mucosa-intacttissues. Figure 6 shows that thequasi-steady-state levels of force, as a percentage of peak force, aresignificantly higher in bladder strips from outlet-obstructed animalscompared with those from control in response to several concentrationsof carbachol. Thus the alteration of contractile profile is notstimulus dependent and instead is a fundamental change in the behaviorof the muscle strip after partial outlet obstruction.5 d+ O- ^  u$ Y) b

Fig. 5. Time course of bladder smooth muscle in strips fromcontrol and partial outlet-obstructed animals. Muscle strips werecontracted in response to 110 mM KCl, and the temporal profile wasmonitored as a percentage of maximal force developed by strips fromcontrol ( ) and obstructed ( ) animals.Muscle strips from both sources demonstrated a rapid increase in stressto high levels followed by a decrease to significantly lower levels ofsteady-state stress maintenance. However, the fall in stress in stripsfrom obstructed animals was significantly slower and steady-statelevels of stress were significantly higher compared with strips fromcontrol bladders. Values are means ± SE; n = 5-7.9 w8 B- d2 F* H. I6 ~. I3 N& w
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Fig. 6. Difference in peak and steady-state forces in musclestrips from control and partial outlet-obstructed animals. Experimentssuch as that shown in Fig. 5 were performed at several carbacholconcentrations. The magnitude of steady-state force as a percentage ofpeak force was measured, and the results are shown. Steady-state levelsof force as a percentage of peak force were significantly higher instrips from obstructed (gray bars) compared with strips from control(filled bars) animals. Slow relaxation from the peak of the phasiccomponent of the contraction to significantly higher levels ofsteady-state force was noted in all strips from partialoutlet-obstructed animals compared with those from control animals.Values are means ± SE; n = 12-14.  [! ^+ t9 X- V1 f( \, a

The primary step in the initiation of a smooth muscle contraction isthe calcium- and calmodulin-dependent phosphorylation of the 20,000-DaMLC ( 9, 12 ). Thus it was important to determine whetherthe partial outlet obstruction-induced alterations in contraction werecorrelated to a change in MLC phosphorylation levels. We stimulatedbladder strips from control and obstructed animals with 110 mM KCl,rapidly froze the tissues at various times during the contractileevent, and processed the tissues for quantitation of MLCphosphorylation levels. The results of these experiments are shown inFig. 7. Surprisingly, basal levels of MLCphosphorylation were significantly elevated in bladder strips fromobstructed animals compared with those from control. Stimulated levelsof MLC phosphorylation were not different from the two animal groupseven though the temporal profile of force was significantly different.Of potential importance in terms of the elevated basal levels of MLCphosphorylation was the finding that all muscle strips from obstructedanimals exhibited spontaneous phasic activity, whereas this was notedin from control animals.
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Fig. 7. Time course of force and myosin light chain (MLC)phosphorylation levels in KCl-stimulated bladder smooth muscle. Musclestrips were stimulated with 110 mM KCl and then frozen at specifictimes for quantitation of MLC phosphorylation levels ( A ) andthe concomitant increase in force ( B ). Stimulation of musclestrips from control ( ) and outlet-obstructed( ) animals increased force. However, force developmentwas significantly slower and was maintained for longer time in musclestrips from the outlet-obstructed animals. Basal levels of MLCphosphorylation were elevated in muscle strips from outlet-obstructedanimals, but stimulation-induced levels were similar in tissues fromboth animal groups. Values are means ± SE; n = 6-9.$ u9 A# g3 [# E( r' {
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Several groups have shown that after partial bladder outletobstruction, the primary isoform of myosin in the smooth muscle cellschanges from SM-B to SM-A ( 2, 10, 28 ). The SM-A isoform ofmyosin is characterized by a slower actin-activated myosin ATPaseactivity. We were therefore interested in determining whether theshortening velocities of bladder strips were similarly altered afterpartial outlet obstruction. Bladder strips from control andoutlet-obstructed animals were stimulated with 110 mM KCl and thensubjected to several isotonic releases at 5, 15, and 30 s ofcontraction. The maximal velocities of shortening( V o ) were estimated as described in MATERIALS AND METHODS and are shown in Fig. 8. V o of themuscle strips from control animals declines with time of stimulation,as has been shown in most smooth muscles examined ( 18 ). V o of the muscle strips from obstructed animals was more than an order of magnitude lower than that from control. Wealso performed a limited number of force redevelopment experiments using carbachol as the stimulus. Force redevelopment during carbachol stimulation was consistently slower in strips from outlet-obstructed animals compared with strips from control animals (data not shown). These results are consistent with the biochemical studies demonstrating a change in SM-A, the myosin isoform with a lower actin-activated myosin ATPase activity ( 13, 22 ).4 X* X% c7 s; f4 g, z7 c- X: }1 V$ N
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Fig. 8. Maximal velocity of shortening in bladder smooth musclestrips from control and partial outlet-obstructed animals. Isotonicshortening velocities were measured at 5, 15, and 30 s ofstimulation in response to 110 mM KCl. Shortening velocities weremeasured at afterloads ranging from 0.1 to 0.4 times the force at timeof release. Linearization of the hyperbolic force-velocity relationshipprovided an estimate of the maximal velocity of shortening. Maximalvelocities of shortening were more than an order of magnitude higher atall time points measured in stimulated muscle strips from controlanimals (filled bars) compared with those from partialoutlet-obstructed (gray bars) animals. Values are means ± SE; n = 6-7./ X# o. k* t( h! K. o

DISCUSSION+ O3 B, Y. \5 g5 s5 {) G3 o# p

The results presented in this study clearly show that smoothmuscle tissue of the rabbit urinary bladder undergoes significant functional alterations in response to partial outlet obstruction. Inour opinion, the most striking of these alterations is the significantincrease in spontaneous phasic activity and the maintenance of highlevels of force after stimulation in bladder smooth muscle from partialoutlet-obstructed animals.

Also of potential interest is the finding that bladder wall strips fromanimals subjected to partial outlet obstruction dissected free of onlythe serosal layer developed less stress compared with strips fromcontrol animals. This is in contrast to the finding that bladder wallstrips from both control and outlet-obstructed animals dissected freeof both serosal and mucosal layers developed similar levels of stress.Because stress is calculated as force/cross-sectional area, it ispossible that the hypertrophied mucosal layer in the bladders frompartial outlet-obstructed animals increased cross-sectional area andthus decreased stress. However, levels of actual force were also lowerin those tissues from obstructed animals containing a mucosal layer. Amore plausible explanation is that an altered matrix within the mucosallayer impedes contractile activity, resulting in lower levels of forcedevelopment. Due to this possibility and because we were interested inexamining the smooth muscle cells as directly as possible, allsubsequent studies were performed using a mucosal and serosal-freepreparation. It is important to note that it is well documented thatpartial bladder outlet obstruction induces numerous changes incontractile protein isoform, changes in expression levels ofcontractile regulatory proteins, and changes in calcium handling in thebladder smooth muscle cell ( 2, 7, 10, 21, 26, 29 ). Thusbased on the finding that the stress generation of the muscles from thetwo sources is similar, we interpret this to suggest that the numerousand widespread changes may be compensatory in nature and important inmaintaining bladder function in the face of an obstruction. Based onthe categories of partial outlet obstruction as suggested by Levin etal. ( 7 ), we would classify our results as applying to thelate compensated or early decompensated state. This compensated stateis one in which contractile function and bladder weight have stabilizedbefore going into the failing or severely decompensated state. However,bladder function was compromised, as shown by the data in Table 1,hence the placement in the early decompensated state.) |# W) o+ B7 U" e. A8 b
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Bladder smooth muscle strips from partial outlet-obstructed animalsshowed no significant differences in either the sensitivity ormagnitude of contraction in response to the noncumulative addition ofKCl and only a small difference in magnitude with the noncumulative addition of carbachol. In contrast, smooth muscle from the obstructed animals showed enhanced sensitivity and higher levels of force to bothKCl and carbachol during the cumulative additions. We believe theresults presented in Figs. 3 A and 4 A demonstratethat partial outlet obstruction produces a significant loss of the mechanism(s) responsible for desensitization of smooth muscle contraction. We also propose that bladder smooth muscle fromoutlet-obstructed rabbits may be an excellent model for the study ofthe mechanism(s) underlying receptor and contractile desensitization.7 S8 {7 f# m/ i* r
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McConnell and colleagues ( 4 ) have shown thatstimulation-induced maximal levels of MLC phosphorylation are similarin bladder smooth muscle strips from partial outlet-obstructed andcontrol animals. Our results support these earlier findings. Inaddition, we provide results showing that basal values of MLCphosphorylation are elevated in bladder smooth muscle from obstructedanimals compared with those from control. The impact of an elevatedbasal value of MLC phosphorylation with no change instimulation-induced values is a decrease in the MLC phosphorylationdependence of contraction. The elevated basal values of MLCphosphorylation may also provide insight into the significant increasein spontaneous phasic activity in smooth muscle from obstructed animalscompared with control. MLC phosphorylation and contraction are bothcalcium-dependent events. It is reasonable to assume that the increasein basal values of MLC phosphorylation and spontaneous activity isrelated and that both may be due to an increased calcium leak acrossthe smooth muscle plasma membrane. This would be consistent with the well-described changes that occur in vascular smooth muscle during mostforms of hypertension. Tonic vascular tissue from hypertensive animalshas been shown to produce spontaneous phasic contractions and that thisis the result of an increase in calcium influx from the extracellularspace ( 8 ).# U  F. l/ _) S0 R* D3 i
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Biochemical studies have shown that, after partial outlet obstruction,the smooth muscle cells undergo a change in the predominant isoform ofmyosin ( 2 ). Wang et al. ( 28 ) have presentedevidence demonstrating that bladder smooth muscle from control animals contains predominately the SM-A isoform of myosin, whereas bladder smooth muscle from obstructed animals contains predominately the slowerSM-B isoform of myosin. It has long been accepted that maximal velocityof shortening measurements provides an excellent estimate of myosinATPase activity ( 1 ). This information provided therationale for performing the mechanical characterization of the intactbladder smooth muscle from the two animal groups. Our results onmaximal isotonic shortening velocity in intact tissue are consistentwith the biochemical evidence that smooth muscle from the bladder ofpartial outlet-obstructed animals contains a slower isoform of myosincompared with that from control rabbits.& S* U9 c- G% l* V& F

Typically, stimulation of bladder smooth muscle produces an initialphasic contraction followed by a significantly lower sustained tonicphase. Bladder smooth muscles from animals subjected to partial outletobstruction express a significantly altered contractile profile( 21, 29 ). The phasic portion of a contraction of smooth muscle from the outlet-obstructed animals is prolonged to the point ofapproaching a tonic contraction. As shown in Figs. 5 and 6,quasi-steady-state levels of force are close to that developed at thepeak of the phasic contraction. Our present studies do not address themechanism(s) responsible for this high level of maintained force afterpartial outlet obstruction. However, depending on how one looks at theproblem, it is possible to suggest plausible speculations. Thetransient nature of a contraction of bladder smooth muscle from controlanimals may be due to an active relaxation process or as a result of arapid transient increase in activator calcium. The simplest explanationfor the slow decrease in force in bladder strips from obstructedanimals is higher intracellular calcium levels at any time during thecontractile event. If the transient increase in calcium is prolonged,then one would expect a prolonged transient contraction. Thispossibility is supported by the higher basal values of MLCphosphorylation and the increase in spontaneous contraction. Thispossibility is not supported by the lack of change in either peakvalues or temporal profile of stimulation-induced increases in MLCphosphorylation. It is also interesting that the time course of acontraction in the mucosal intact strip is prolonged compared with thatin the strips dissected free of the mucosal layer. We believe this ismost likely due to enhanced diffusional delays in the thicker mucosalintact tissues.
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A more complex explanation for the maintenance of force in muscle fromobstructed animals could be a decrease in an active relaxation process.If one assumes that the transient nature of the bladder musclecontraction is the result of active relaxation, then any loss in thismechanism would produce a more tonic-like contraction. If this were thecase, then the maintained force in muscles from obstructed animalscould be due to either an alteration in the mechanism(s) responsiblefor active relaxation or a change in the tissue that opposesrelaxation. This present study does not address these possibilities.However, we have presented preliminary information suggesting that thePKC-dependent pathway for contraction of bladder smooth muscle iseither absent or constitutively active in tissues from obstructedbladders ( 25 ). If constitutively active, then this mayexplain the maintained contraction. Alternatively, it is well acceptedthat after partial outlet obstruction, the bladder matrix significantlyincreases in content. Any increased stiffness due to matrix materialsonce contracted would oppose an active muscle relaxation. What is clearhowever, is that temporal profile of a smooth muscle contraction fromthe partial outlet-obstructed animals is significantly different fromthat of control muscles.

Significant changes in smooth muscle have been shown to occur in mostif not all pathophysiological states involving hollow organs. Ourpresent study confirms and expands on the previous studies, showingthat partial outlet obstruction secondary to benign prostatehyperplasia alters the functional status of the bladder smooth muscle.These changes include a prolonged contractile response to normalstimulation, a change in the mechanism of contractile desensitization,an alteration in basal MLC phosphorylation levels, and a decrease inthe cross bridge cycling rate. It is now important to direct attentionto determine how these alterations impact on micturition, whethercontinued obstruction induces a severely decompensated state, andwhether removal of the obstruction reverses the change.3 {* G; j3 I- f: T
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ACKNOWLEDGEMENTS
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This work was supported in part by funds from National Institute ofDiabetes and Digestive and Kidney Diseases O'Brien Center GrantsDK-52620 (University of Pennsylvania Medical Center) and DK-57252(R. S. Moreland).! u+ D' T) d' B! G3 J/ J
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