Partial bladder outlet obstruction alters Ca2 sensitivity of force, but not of - 《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 13:45 |只看该作者 |倒序浏览 |打印

作者：Michaela C. Stanton, Michele Clement, Edward J. Macarak, Stephen A. Zderic,  Robert S. Moreland作者单位：1 Department of Pharmacology and Physiology,Drexel University College of Medicine, Philadelphia 19102; Department of Urology, The Children‘s Hospital ofPhiladelphia, Philadelphia 19101; and Department ofAnatomy and Cell Biology, University of Pennsylvania School of DentalMedicine, Philadelphia
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      【摘要】3 G4 B: b7 x3 j* U. R9 g
   Partial bladder outlet obstruction in the rabbit produces changes inbladder function similar to those seen clinically in patients with obstructiveuropathies. Whole organ function is significantly altered, as are the smoothmuscle cells inside the bladder wall. This study was designed to determinewhether outlet obstruction alters smooth muscle function at the level ofcontractile filaments. Rabbit bladders were partially obstructed for 2 wk.Triton X-100 was used to provide a detergent-skinned bladder smooth musclepreparation that would allow control of the intracellular environment whilethe ability to shorten and develop force is maintained.Ca 2 -force and Ca 2 -myosin lightchain (MLC) phosphorylation relations and maximal velocity of shortening weredetermined. The Ca 2 sensitivity of force wassignificantly lower in tissues from animals subjected to outlet obstruction compared with tissues from control animals. In contrast, no difference wasnoted in the Ca 2 sensitivity of MLC phosphorylation. Maximal levels of stress and MLC phosphorylation were similar in both animalgroups. Maximal velocity of shortening was significantly slower in tissuesfrom outlet-obstructed animals at all Ca 2 concentrations compared with tissues from control animals. Ultrastructurally, detergent skinning had little effect on structural integrity. Moreover,tissues from obstructed animals showed an increase in the number ofsarcolemmal attachment plaque structures. We suggest that partial bladderoutlet obstruction produces deleterious (e.g., decrease inCa 2 sensitivity of force) and compensatory (e.g.,increase in membrane attachment plaques) changes in bladder smooth musclecells. 0 k* ]7 q1 b# ]0 Q% J
      【关键词】 maximal velocity of shortening Triton X detergentskinned fibers attachment plaques electron micrographs) ^! q" x# P, P- u) v0 M2 {7 r
   ANIMAL MODELS ARE USEFUL TOOLS to study the effects of partial bladder outlet obstruction on muscle physiology. Some of the changes in musclefunction that occur in such models appear to mimic some of the changes seen incertain clinical uropathies, such as prostatic hyperplasia( 15, 35 ). The rabbit model has beenused extensively, and information derived from these studies has providedimportant advances in our knowledge of how outlet obstruction impacts bladderfunction ( 16, 36 ). Bladders subjected tooutlet obstruction undergo specific alterations in expression of contractile,regulatory, and structural proteins, leading to a shift in contractileproperties from a phasic to a more tonic-like contraction( 1, 3, 5 ). In addition, we haverecently shown that bladder strips devoid of mucosal and serosal layers develop similar levels of force( 31 ). The similarity in force development in bladder smooth muscle is not due to a change in percent musclemass in the hypertrophied compared with the control bladders. Therefore, oneplausible interpretation is that outlet obstruction-induced changes inintracellular components of the smooth muscle cell are compensatory at thecontractile protein level to maintain normal levels of force in the face ofthe increased resistance.
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Contraction of smooth muscle, including bladder muscle cells, is initiatedby stimulation-induced increases in cytosolic free Ca 2 concentration. The cytosolic Ca 2 binds to calmodulin, and the Ca 2 -calmodulin complex activates the enzymemyosin light chain (MLC) kinase. Active MLC kinase catalyzes phosphorylation of the 20-kDa MLC, which activates the myosin molecule and allows it tointeract with actin, with the resultant increase in cross-bridge cycling andforce development (for review see Ref. 10 ). Dephosphorylation of theMLC by a MLC phosphatase initiates relaxation of the muscle cell. In additionto this primary pathway for excitation-contraction coupling, there are alsoseveral modulatory pathways that both increase and decrease the sensitivity ofthe contractile filaments to Ca 2 ( 26 ) as well as the latchstate, in which high force can be maintained in the absence of proportionallevels of MLC phosphorylation( 21 ). However, it is clearthat Ca 2 -dependent MLC phosphorylation is an importantinitiating step in smooth muscle contraction. Thus an alteration in theCa 2 -MLC phosphorylation-force relation would be onelikely mechanism that could account for the higher levels of force necessaryto overcome increased resistance and to maintain force for longer periods oftime, as in the switch from a phasic to a tonic contractile profile.

Therefore, the goal of this study was to determine whether partial bladderoutlet obstruction has direct effects on the contractile apparatus of thedetrusor smooth muscle cells. To approach this problem, we employed the TritonX-100 detergent-skinned preparation, which allows precise control of themuscle cell intracellular environment while maintaining its ability to develop force and shorten. If partial bladder outlet obstruction alters eitherCa 2 -dependent force or MLC phosphorylation or the MLC dephosphorylation step, then this preparation will allow direct assessment ofthe change. On the other hand, if no alteration in any parameter ofcontraction is noted, then this will suggest that obstruction alters one ofthe modulatory or regulatory pathways not present in the Triton X-100detergent-skinned preparation. Thus, regardless of the actual results, theinformation gained using this preparation will be important. We also examined the detergent-skinned preparation at the ultrastructural level to determinethe effect of partial bladder outlet obstruction or detergent skinning on thestructural integrity of the tissue.
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MATERIALS AND METHODS5 g5 `. f! J" N/ u& l8 Q- |
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Surgical techniques. Mature male New Zealand White rabbits weighing 1.8-2.2 kg were used in this study. All animal studies wereapproved by the Institutional Animal Care and Use Committees of DrexelUniversity College of Medicine and The Children's Hospital of Philadelphia.Partial bladder outlet obstruction was created using an extraperitonealapproach with minimal bladder neck dissection. Catheters (8-Fr) were placed inside and outside the surgically exposed urethra, and 4.0 silk was used toligate the urethra. Both catheters were then removed. Sham surgeries wereidentical, except the ligature was cut and removed before the wound wasclosed. Nonoperated rabbits served as an additional control. All bladders wereharvested after 2 wk of obstruction and immediately placed in ice-cold physiological salt solution containing (in mM) 140 NaCl, 4.7 KCl, 1.2MgSO 4, 1.6 CaCl 2, 1.2 Na 2 HPO 4, 2.03-( N -morpholino)propanesulfonic acid, 5.0 D -glucose, and0.02 Na 2 -EDTA.9 ?" H1 e7 ?, ]: C
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Tissue preparation. The predominantly smooth muscle layer of thebladder wall was dissected free of both serosal and mucosal layers, and stripswere prepared for measurement of isometric force, isotonic shorteningvelocity, and quantification of MLC phosphorylation levels. Longitudinalstrips measuring 1.5 x 6 mm were cut from the middle portion of thedetrusor body.

Isometric force was measured using strips mounted between two plastic clipsin water-jacketed muscle chambers aerated with 100% O 2. One clipwas attached to a micrometer for length control and the other to a forcetransducer (model FT.03, Grass Instrument) and a polygraph (model 7D, GrassInstrument). The strips were allowed to equilibrate for 90 min at 37°C andstretched to a length that approximates the optimal length for maximal activecontraction ( 31 ).: ]! ?# _9 }+ V$ E/ R# Q3 ]

All muscle strips were detergent skinned using a 0.5% Triton X-100solution. The equilibrated intact strips were exposed toCa 2 -free physiological salt solution for 30 min andthen placed in a solution containing 5 mM EGTA, 20 mM imidazole, 50 mMpotassium acetate, 1 mM dithiothreitol (DTT), 150 mM sucrose, and 0.5% TritonX-100 for 60 min. The strips were then exposed for 10 min to high-EGTA (5.0mM) and then low-EGTA (0.2 mM) relaxing solutions, respectively, containing 20mM imidazole (pH 6.8), 50 mM potassium acetate, 6 mM MgCl 2, 6 mM ATP, and 1 mM DTT. All experiments using detergent-skinned strips wereperformed at room temperature, pH 6.8, and at an ionic strength of 120 mM.Ca 2 contracting solutions contained 1 mM freeMg 2  , 4 mM MgATP, 1 mM DTT, 5 mM EGTA, 20 mM imidazole (pH 6.8), sufficient potassium acetate to maintain ionic strength constant,and sufficient CaCl 2 to achieve the appropriate free Ca 2 concentration. The amounts of total compound addedto achieve the appropriate free concentration were calculated using a computerprogram to solve the simultaneous multiequilibrium equations, as previouslydescribed ( 19 ).  f/ V9 }% o7 ~) v* O, Y1 o4 f

Mechanical measurements. Isotonic shortening velocity measurements were performed using strips mounted on one end by a plastic clip attached to amicrometer for control of muscle length and on the other end to an aluminumfoil tube connected to a servo-lever (model 300H, Cambridge Technology)interfaced to a Linux operating system-based personal computer. Thedetergent-skinned strips were exposed to solutions containing, in addition to appropriate compounds to reflect intracellular conditions, 1.0, 3.0, and 20.0µM Ca 2  . After stable force was achieved at eachCa 2 concentration, the strips were subjected toisotonic quick releases to afterloads ranging from 5-20% of the initialforce at the time of release. The change in length at each afterload was fitby a double-exponential equation, and a tangent to the fit at 100 ms afterrelease was taken as the isotonic shortening velocity at that afterload.Isotonic shortening velocities at several afterloads were used to extrapolate velocity at zero load for calculation of maximal shortening velocity at eachCa 2 concentration.
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MLC phosphorylation. For determination of MLC phosphorylation levels, detergent-skinned strips were rapidly frozen by immersion in a dryice-acetone slurry containing 6% (wt/vol) trichloroacetic acid and 10 mM DTT.The muscle strips were allowed to slowly thaw to room temperature, rinsed for30 min in acetone, and homogenized on ice. The homogenization buffer contained1.0% sodium dodecyl sulfate, 10% glycerol, and 20 mM DTT. Homogenized stripswere subjected to two-dimensional electrophoresis followed by transfer tonitrocellulose membranes, as previously described ( 20 ). Proteins were visualizedusing colloidal gold stain (Amersham Pharmacia Biotech). MLC was quantified byscanning densitometry using a scanning densitometer (model GS 800, Bio-Rad).Values are reported as moles of P i per mole of MLC and werecalculated by taking the volume of the densitometric spot representing monophosphorylated MLC as a percentage of the total volume of thedensitometric spots for monophosphorylated and nonphosphorylated MLC.7 Z/ I, Q% N. Y( `
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Electron microscopy. Bladder wall dissected free of both serosal and mucosal layers was detergent skinned as described above. The skinnedstrips were then fixed in place at physiological lengths by immersion in ahigh-EGTA relaxing solution containing 1.5% (vol/vol) glutaraldehyde for 2 h.The tissues were rinsed in high-EGTA relaxing solution, dehydrated in ethanol,and then embedded in LR White resin. Thin sections were collected on grids,and then the sections were contrasted with 3% uranyl acetate and examined in atransmission electron microscope (model 100CX, JEOL).

Data analysis. Values are means ± SE. Student's t -test was used for unpaired data. P
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RESULTS" X, f+ [& @, m$ A; L& ]

The data in Table 1 show thefunctional measurements of the rabbit bladder in control and sham-operatedanimals combined and in animals subjected to partial outlet obstruction for 2 wk. The animals subjected to partial bladder outlet obstruction havesignificantly higher voiding frequencies and lower urinary volumes per void.The bladders from the obstructed group also have significantly greater wetweights.

Table 1. Functional measurements of rabbit bladder in control and sham animalsand animals subjected to partial outlet obstruction

The sensitivity of intact bladder smooth muscle to the noncumulative addition of carbachol or KCl is not different in bladder smooth muscle fromcontrol animals and animals subjected to partial bladder outlet obstruction( 29 ). The intact tissue,however, does not allow a direct examination of the contractile apparatus. Wetherefore performed studies using the Triton X-100 detergent-skinned preparation. Triton X-100 detergent-skinned strips of bladder smooth musclewere subjected to the noncumulative addition of various freeCa 2 concentrations. The results of these experiments are shown in Fig. 1. Theaddition of Ca 2 to detergent-skinned strips produced aconcentration-dependent increase in stress (force/cross-sectional area) insmooth muscle from both animals groups. However, theCa 2 sensitivity of stress development is significantlyless in smooth muscle from animals subjected to partial bladder outletobstruction than in smooth muscle from control animals: EC 50 = 1.2± 0.2 µM ( n = 6) vs. 6.3 ± 0.4 µM ( n =7). The maximal stress generated by the Triton X-100 detergent-skinned fiberswas significantly greater than that developed by the intact preparations: intact control 7.9 ± 0.7 x 10 4 vs. detergent-skinned control 3.1 ± 0.5 x 10 5 N/m 2 (qualitativelysimilar results were obtained in tissues from obstructed animals).% ?- k& s  [% Z& t& @
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Fig. 1. Stress development in Triton X-100 detergent-skinned tissues of bladdersmooth muscle from control animals ( ) and animals subjected to partialbladder outlet obstruction ( ). Detergent-skinned tissues from control andobstructed animals were contracted noncumulatively in response to various freeCa 2 concentrations ([Ca 2 ]) andallowed to reach steady-state levels of force. Force was measured, as wastissue length and then weight, for determination of cross-sectional area. Allvalues are presented as stress (force/cross-sectional area). Detergent-skinnedtissues from obstructed animals exhibited a significant decrease inCa 2 sensitivity of force compared with tissues fromcontrol animals. There were no differences in the maximal value of stressattained. Values are means ± SE for 6 determinations.*Significantly different from control ( P

MLC phosphorylation is the predominant step in the initiation of a smoothmuscle contraction. To fully understand the effect of partial bladder outletobstruction on the Ca 2 -dependent regulation ofcontraction, one needs to know the Ca 2 dependence ofMLC phosphorylation. The tissues in which Ca 2 -dependent stress was obtained for Fig. 1 were frozen after a stable force recording was attained, usually within 10 minof contraction, for quantitation of MLC phosphorylation levels. The resultant data are shown in Fig. 2.Similar to the results of maximal stress development, there were nodifferences in the maximal levels of MLC phosphorylation attained in bladdersmooth muscle strips between control animals compared with those subjected topartial bladder outlet obstruction. In contrast to the results shown in Fig. 1, however, there were notrends, let alone significant differences, in the Ca 2 dependence of MLC phosphorylation between the muscle strips from the twoanimal groups.4 c  I) p, Z$ N. k( e+ T
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Fig. 2. Myosin light chain (MLC) phosphorylation in Triton X-100 detergent-skinnedtissues of bladder smooth muscle from control animals ( ) and animalssubjected to partial bladder outlet obstruction ( ). Detergent-skinnedtissues from control and obstructed animals were contracted in response tovarious free Ca 2 concentrations and allowed to reachsteady-state levels of force. Tissues were then frozen and processed forquantitation of MLC phosphorylation levels. There were no significantdifferences in Ca 2 sensitivity of MLC phosphorylationor maximal values of MLC phosphorylation between tissues from control andobstructed animals. Values are means ± SE for 6 determinations.( ?0 Z0 n+ ?  E0 L! q+ s! _( E
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Several biochemical studies have clearly shown that partial bladder outletobstruction changes the isoform of myosin from the faster SM-B isoform to theslower SM-A isoform ( 2, 9 ), which translates to adecrease in the maximal velocity of shortening ( 31 ). Figure 3 shows the results ofexperiments performed to measure maximal velocity of shortening in TritonX-100 detergent-skinned strips from control and outlet-obstructed animals. Atevery Ca 2 concentration examined, detergent-skinnedtissues from outlet-obstructed animals exhibited significantly lower levels ofmaximal velocity of shortening compared with tissues from control animals.
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Fig. 3. Maximal velocity of shortening in Triton X-100 detergent-skinnedpreparation of bladder smooth muscle. Detergent-skinned strips of muscle fromcontrol animals (solid bars) and animals subjected to partial bladder outletobstruction (open bars) were mounted for isotonic force recording. Strips werecontracted to 1, 3, or 20 µM Ca 2 and, whensteady-state force was attained, subjected to several quick releases tovarious afterloads. Maximal velocity of shortening was calculated usingisotonic shortening velocities at each afterload taken at 100 ms after thequick release and extrapolating to zero load. Maximal velocity of shorteningis significantly lower in detergent-skinned strips from outlet-obstructedanimals compared with those from the control animals at allCa 2 concentrations. Values are means ± SE for 6 determinations. *Statistically different from control, P

Exposure of a tissue to Triton X-100 can be quite damaging. It is thereforepossible that tissue from a pathological state such as partial outletobstruction may have more damage after Triton X-100, and this may account for,in part, the decreased Ca 2 sensitivity of force. Toaddress this possibility, we fixed tissues from control and outlet-obstructedanimals and processed them for examination at the level of the electron microscope. Despite detergent treatment, structural preservation is quitegood. In normal ( Fig.4 A ) and obstructed tissue ( Fig. 4 B ), nuclear andcytoplasmic structures are evident. The sarcolemma and intracellularcompartments are evident in Fig.4. Note the close apposition of adjacent smooth muscle cells, thestructural detail of the sarcolemma, dense bodies within the cytoplasm, andcytoskeletal filaments.0 t" C9 z; M0 Q. f1 }) @
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Fig. 4. Electron micrograph of Triton X-100 detergent-skinned bladder smooth musclefrom control animals and animals subjected to outlet obstruction. A :control bladder tissue. Black arrows indicate the presence of plaque-likedense bodies at the level of the plasma membrane, the structure of which hasbeen obviously disrupted by Triton X-100 treatment. White arrows denote thepresence of cytoplasmic dense bodies. B : tissue fromoutlet-obstructed animals. Note extensive dense bodies associated withdisrupted plasma membranes in obstructed bladder tissue (black arrows) andduplicated basement membranes extracellularly (white arrow).$ n5 V8 m7 n( E9 g) d

Of particular interest are the junctional structures that exist betweensmooth muscle cells consisting of electron-dense plaque-like structuresimmediately beneath the sarcolemma. These plaque-like structures are sometimespaired, being present in both adjacent smooth muscle cells whereas in otherinstances, they exist as single structures present in only one cell. Tissuefrom control animals ( Fig. 4A )and tissue from obstructed animals ( Fig.4 B ) contain these plaque-like structures. Of interest isthe apparent increase in the plaque-like structures in tissue from obstructedanimals compared with control animals. These regions are where cytoskeletalproteins come into close apposition with the sarcolemma, likely providingstructural rigidity to these sites, where tensional forces are transferredfrom the smooth muscle cell to both the adjacent smooth muscle cells and theextracellular matrix. This is shown most clearly in Fig. 5 (obstructed animal),where an interstitial fibroblast nucleus can be seen in the extracellularmatrix between the two smooth muscle cells. Note also the very extensivesingle plaque-like structures directly adjacent to the extracellular matrix.( K/ y5 K+ |+ ?) C' t' j0 f

Fig. 5. Electron micrograph of Triton X-100 detergent-skinned bladder smooth musclefrom obstructed bladder tissue. Extensive dense bodies (plaques) are present(large black arrows) at the cell surface at the interface between the smoothmuscle cell and the extracellular matrix. Note also the fibroblast cell withinthe extracellular matrix (white arrow).

DISCUSSION! v$ i! z5 q& V% D
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It is well documented that partial bladder outlet obstruction producesnumerous alterations in the smooth muscle of the bladder wall( 2, 7, 9, 23, 34 ). Not as well understood iswhich alterations are compensatory to maintain force output in the face of anincreased resistance to flow and which alterations are simply deleteriousoutcomes of the obstruction. Additionally, a large amount of attention hasbeen given to extracellular stimulation and the resultant development of forceby the smooth muscle cells( 18, 22 ). In contrast, the cascadeof events termed excitation-contraction coupling of the smooth muscle cellsafter outlet obstruction has received significantly less attention. In thisstudy, we present evidence to suggest that partial bladder outlet obstructionhas direct effects on the contractile apparatus of bladder wall smooth muscle.By using a Triton X-100 detergent-skinned smooth muscle preparation, we wereable to study the contractile apparatus without interference fromreceptor-mediated modulatory pathways and Ca 2 handling. The results obtained are, we believe, interesting on several levels. In termsof the pathophysiology of partial bladder outlet obstruction, one of the moststriking findings in our study was the decrease in theCa 2 sensitivity of force without a concomitant decreasein the Ca 2 sensitivity of MLC phosphorylation. In termsof the cell biology of a smooth muscle cell, the fact that partial bladderoutlet obstruction alters the isoform of myosin, as evidenced by a change inmaximal shortening velocity, is of interest. In terms of the smooth musclecells functioning together as a coordinated organ, the increase in attachment plaque-like structures after the obstruction may be important in transmissionof the force signal. And last, in terms of using the Triton X-100detergent-skinned smooth muscle cell as an experimental model, our resultsshow that bladder smooth muscle is an excellent source of tissue for thispurpose.
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The differential effect of partial bladder outlet obstruction on force andMLC phosphorylation can be accounted for by at least two possibilities. Thefirst possibility is that the obstruction produces an uncoupling between MLCphosphorylation and force development. Any cell swelling that alters thespacing between thin and thick filaments could alter the ability of actin toactivate a phosphorylated myosin. Such an event has been used experimentallyto inhibit contraction of smooth muscle while maintaining the ability tophosphorylate the MLC ( 14 ). Whether cell swelling or changes in the filament lattice structure occur inbladder smooth muscle after outlet obstruction is, to our knowledge, notknown. However, the fact that higher levels of stress were developed in thepermeabilized compared with the intact tissue tends to discount thispossibility.

The second possibility to account for the decrease inCa 2 sensitivity of force, and not of MLCphosphorylation, is an outlet obstruction-induced loss of any regulatorypathway acting in parallel with MLC phosphorylation. We have previously shown that Ca 2 stimulates protein kinase C (PKC) activity inTriton X-100 detergent-skinned vascular smooth muscle( 12 ). Additionally, we havealso demonstrated that phorbol ester-, and presumably, PKC-, dependentcontractions of bladder smooth muscle are attenuated after partial bladderoutlet obstruction ( 27 ).Because PKC has been implicated in most smooth muscle thin filament regulatory hypotheses, it is possible that the loss of this contractile pathwaycontributes to the decrease in force at each submaximal Ca 2 concentration. Whether the decrease inCa 2 sensitivity of force seen in the present study isdue to a change in the filament lattice structure or loss of a parallelpathway for contractile activation cannot be definitively determined. What isdefinite is that partial bladder outlet obstruction has direct effects on thecontractile filaments.3 Z/ I3 [9 \9 {( s% E
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Biochemical studies have clearly shown that the isoform of myosin inbladder smooth muscle changes from the faster SM-B to the slower SM-A isoformin response to partial bladder outlet obstruction ( 2, 9 ). We have also shown thatthis change translates into a slower maximal velocity of shortening in intactstrips of bladder smooth muscle from obstructed animals( 31 ). Our present resultsextend this information to directly demonstrate that maximal velocity ofshortening is slower in muscle cells from animals subjected to outletobstruction at every Ca 2 concentration examined. Thusthe activation of cross-bridge cycling in smooth muscle from the obstructedanimals remains Ca 2 dependent, but the rate of cyclingis significantly slower. How this change in cross-bridge cycling rates affectsthe contractility of the muscle is not known. Given the many steps involved inexcitation-contraction coupling in smooth muscle( 26 ), it is most likely notimportant in the slower rate of force development in smooth muscle from obstructed animals. This is also supported by the fact that in the TritonX-100 detergent-skinned fiber, where more direct activation of the contractileapparatus can be elicited, there were no significant differences in the rateof force development between tissues from control and outlet-obstructedanimals (data not shown). Where the slower rate of shortening velocity mayimpact bladder smooth muscle contractions is in relation to the tonic-likecontractile event of muscle from obstructed animals compared with the typicalphasic contraction of muscle from control animals. If the cross-bridgedetachment rate is significantly slower in muscles containing the SM-A isoform compared with the SM-B isoform of myosin, then it is entirely possible thatforce would be maintained for longer periods of time. Thus the switch inisoform of myosin may be responsible, in part, for the switch from a phasic toa tonic contractile profile.$ F% {! M3 g; ]) `: W* k( `+ e6 C# S
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We have previously shown that maximal force development in intact bladdersmooth muscle from animals subjected to partial bladder outlet obstruction issimilar to that developed by muscle from control animals( 31 ). This is consistent withthe similar levels of maximal force developed in the two groups of Triton X-100 detergent-skinned muscle. Considering the myriad of changes in theactivity and content of intracellular components that have been shown to occurin bladder smooth muscle after partial outlet obstruction( 2, 7, 9, 23, 33 ), no change in maximal force output is surprising. The significant increase in plaque-like structuresin smooth muscle from the outlet-obstructed animals may, in part, account forthe higher than expected levels of force revealed in the skinned fiberpreparation. If these plaque-like structures are associated with attachmentpoints for the transmission of force from the contractile apparatus to thecell membrane, then an increase in force development may not be unexpected. Gunst and her colleagues ( 32 )have shown that alterations in the proteins associated with attachment plaquescan alter the magnitude of a contraction. It is interesting to speculate that one compensatory alteration the smooth muscle cell undergoes in response tothe increased resistance to flow is an increase in the number of contactpoints for the transduction of force from the contractile filaments to thecell membrane.

Rather than an increase in the number of points for transduction of force,the plaque-like structures may represent an increase in the number of gapjunctions in smooth muscle from the outlet-obstructed animals. Christ and hiscolleagues ( 4 ) and Haefligerand co-workers ( 8 ), using amodel of rat bladder outlet obstruction, demonstrated a specific increase inconnexin43. Moreover, Fry et al.( 6 ) suggested that an increasein electrical coupling between smooth muscle cells of the human bladder mayaccount for, in part, localized aberrant contractions. Our results do notdirectly address this possibility, but the time frame of our partial outletobstruction may fit the experimental results found by at least Christ et al.( 4 ) and Haefliger et al.( 8 ). These two groups useddifferent severity of outlet obstruction, with an increase in connexin43demonstrable after 9 h of severe and 6 wk of moderate obstruction. Our studiesused a 2-wk time frame of obstruction. An increase in the number of electrical connections could account for the similar levels of force generated in anintact bladder smooth muscle preparation( 31 ) but would not be expectedto have any influence in a detergent-skinned preparation. Future studies usingintact tissues should address the specific junctional components of theseplaque-like structures.

A very unexpected result, at least to those who have used smooth muscleskinned fiber preparations, was the high level of stress (force/cross-sectional area) developed by the Triton X-100 detergent-skinned fibers. On average, most detergent-skinned smooth muscle preparations develop 40-80% of their preskinning force or stress ( 13, 17, 19 ). In our study, the TritonX-100 detergent-skinned bladder smooth muscle preparations developed nearlyfour times the level of stress developed by the intact smooth musclepreparation. It is possible that, even with supramaximal levels of KCl or carbachol, maximal levels of activator Ca 2 and,therefore, stress cannot be attained in the intact preparation, whereas thedirect addition of micromolar levels of Ca 2 can beintroduced into the detergent-skinned fiber. On the other hand, relative toother smooth muscles, possibly the bladder smooth muscle has a more extensivecytoskeletal structure that aids in maintaining cellular integrity after thefairly harsh exposure to Triton X-100.

Other investigators have also used skinning and permeabilizing procedureson urinary bladder smooth muscle, but the results are mixed. Using TritonX-100 detergent-skinned smooth muscle from rat urinary bladder, Arner and hisco-workers ( 25 ) found thatpartial outlet obstruction reduced the force output compared with tissues fromcontrol animals by 25%. Consistent with our results, they also found thatshortening velocity was significantly reduced. Kanaya( 11 ) found similar levels offorce development in a saponinpermeabilized preparation of bladder smoothmuscle compared with the intact state. In these studies( 11, 25 ) as well as others( 17, 34 ), theCa 2 sensitivity of force (EC 50 ) was found tobe 1 µM Ca 2  , similar to that found in our studyusing tissue from control animals.* A1 Q( z; A% F

As stated above, one complicating problem in the interpretation of theseresults is determining which alterations are compensatory to maintain normalfunction in the face of the obstruction and which are deleterious as a resultof the obstruction. It seems intuitively obvious that the high levels ofstress that can be developed by smooth muscle tissues from both animal groups, but especially the outlet-obstructed animals, helps to at least initially,maintain normal bladder function. Considering the greater the contractileability, the better the bladders would be expected to perform, resulting inlower residual volumes. The fact that bladders from outlet-obstructed animalshave a greater post-void volume, then one can assume that the force generatedby the smooth muscle in the obstructed bladder is still insufficient toproduce complete emptying. A similar argument could be made for the increasednumber of apparent attachment points for transduction of force from thecontractile proteins to the membrane. In contrast, the decrease in the Ca 2 sensitivity of force in smooth muscle fromoutlet-obstructed animals would seem to be a deleterious alteration. If ittakes a greater level of stimulus to produce the required magnitude of forceto completely empty the bladder, then one can understand why bladders fromoutlet-obstructed animals have a higher post-void volume. The change in myosinisoform to one with a slower actin-activated ATPase activity can be viewed intwo modes: compensatory as well as deleterious. If less force is developed atany given level of stimulation, as evidenced by the decrease in theCa 2 sensitivity of force, then this may be countered bya longer contraction. The slower ATPase activity of the SM-A isoform, due mostlikely to a slower off rate, may provide a prolonged contractile event. On theother hand, if stimulation durations are not changed but it takes longer toempty the bladder, then this may lead to a greater post-void volume. Futurestudies aimed at comparing in vivo urodynamics and in vitro biochemistry andphysiology should shed valuable light on these speculations.; y! l! J, U  Q) ~% @9 {
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Bladder function after partial outlet obstruction has been categorized ascompensated (mildly dysfunctional) or decompensated (severely dysfunctional)by Levin and his colleagues( 36 ) and by our group( 28 - 30 ).As we have discussed in a previous report ( 31 ), our animal modelpresents bladder function consistent with a decompensated state. However, ourresults obtained specifically from smooth muscle, as discussed above, showfunctional aspects that would be consistent with a compensated bladder andothers that would be consistent with a decompensated bladder. The apparent discrepancy between the obstruction-induced changes as measured in wholebladder and those measured in the smooth muscle layer may have structural andtemporal components. It is very possible that the obstruction-induced changesin the mucosal layer of the bladder( 24 ) exacerbate the early butsmall changes in the smooth muscle, and, after longer periods of obstruction, more significant deleterious changes in the smooth muscle layer aid thepathological progression to a failing bladder. It also must be kept in mindthat different species and different modes of obstruction may producedifferent effects on bladder smooth muscle function. Studies comparing theeffects of obstruction in different animal models would be beneficial inanswering this possibility.

In summary, using a Triton X-100 detergent-skinned preparation that allowsthe direct examination of contractile protein function in a tissue thatmaintains the ability to contract, we found that bladder smooth muscle fromrabbits subjected to partial outlet obstruction has a reducedCa 2 sensitivity to force without a concomitant changein the Ca 2 sensitivity of MLC phosphorylation. Themaximal velocity of shortening was also significantly lower in smooth musclefrom the obstructed animals, consistent with a change in the isoform ofmyosin. Electron-microscopic examination showed that the detergent-skinnedcells showed excellent structural integrity and, more importantly, asignificant increase in the number of sarcolemmal attachment plaquelike structures in muscle from the outlet-obstructed animals. We interpret theseresults to suggest that partial bladder outlet obstruction produces severalalterations at the level of contractile activation and regulation that arecompensatory to maintain normal force in the face of the increased resistanceto flow and deleterious to bladder function that, with time, most likely aidin the deterioration of bladder function and the switch from a compensated toa decompensated state.
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DISCLOSURES
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This work was supported, in part, by funds from National Institute ofDiabetes and Digestive and Kidney Diseases O'Brien Center Grant DK-52620 (E.J. Macarak, R. S. Moreland, and S. A. Zderic) and Grant DK-57252 (R. S.Moreland).
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