1Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia 19102; 2Department of Urology, The Children's Hospital of Philadelphia, Philadelphia 19101; and 3Department of Anatomy and Cell Biology, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania 19101
Submitted 21 May 2003 ; accepted in final form 4 June 2003
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ABSTRACT |
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maximal velocity of shortening; Triton X-100 detergent-skinned fibers; attachment plaques; electron micrographs
Contraction of smooth muscle, including bladder muscle cells, is initiated by stimulation-induced increases in cytosolic free Ca2+ concentration. The cytosolic Ca2+ binds to calmodulin, and the Ca2+-calmodulin complex activates the enzyme myosin light chain (MLC) kinase. Active MLC kinase catalyzes phosphorylation of the 20-kDa MLC, which activates the myosin molecule and allows it to interact with actin, with the resultant increase in cross-bridge cycling and force development (for review see Ref. 10). Dephosphorylation of the MLC by a MLC phosphatase initiates relaxation of the muscle cell. In addition to this primary pathway for excitation-contraction coupling, there are also several modulatory pathways that both increase and decrease the sensitivity of the contractile filaments to Ca2+ (26) as well as the latch state, in which high force can be maintained in the absence of proportional levels of MLC phosphorylation (21). However, it is clear that Ca2+-dependent MLC phosphorylation is an important initiating step in smooth muscle contraction. Thus an alteration in the Ca2+-MLC phosphorylation-force relation would be one likely mechanism that could account for the higher levels of force necessary to overcome increased resistance and to maintain force for longer periods of time, as in the switch from a phasic to a tonic contractile profile.
Therefore, the goal of this study was to determine whether partial bladder outlet obstruction has direct effects on the contractile apparatus of the detrusor smooth muscle cells. To approach this problem, we employed the Triton X-100 detergent-skinned preparation, which allows precise control of the muscle cell intracellular environment while maintaining its ability to develop force and shorten. If partial bladder outlet obstruction alters either Ca2+-dependent force or MLC phosphorylation or the MLC dephosphorylation step, then this preparation will allow direct assessment of the change. On the other hand, if no alteration in any parameter of contraction is noted, then this will suggest that obstruction alters one of the modulatory or regulatory pathways not present in the Triton X-100 detergent-skinned preparation. Thus, regardless of the actual results, the information gained using this preparation will be important. We also examined the detergent-skinned preparation at the ultrastructural level to determine the effect of partial bladder outlet obstruction or detergent skinning on the structural integrity of the tissue.
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MATERIALS AND METHODS |
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Tissue preparation. The predominantly smooth muscle layer of the
bladder wall was dissected free of both serosal and mucosal layers, and strips
were prepared for measurement of isometric force, isotonic shortening
velocity, and quantification of MLC phosphorylation levels. Longitudinal
strips measuring 1.5 x 6 mm were cut from the middle portion of the
detrusor body.
Isometric force was measured using strips mounted between two plastic clips in water-jacketed muscle chambers aerated with 100% O2. One clip was attached to a micrometer for length control and the other to a force transducer (model FT.03, Grass Instrument) and a polygraph (model 7D, Grass Instrument). The strips were allowed to equilibrate for 90 min at 37°C and stretched to a length that approximates the optimal length for maximal active contraction (31).
All muscle strips were detergent skinned using a 0.5% Triton X-100 solution. The equilibrated intact strips were exposed to Ca2+-free physiological salt solution for 30 min and then placed in a solution containing 5 mM EGTA, 20 mM imidazole, 50 mM potassium acetate, 1 mM dithiothreitol (DTT), 150 mM sucrose, and 0.5% Triton X-100 for 60 min. The strips were then exposed for 10 min to high-EGTA (5.0 mM) and then low-EGTA (0.2 mM) relaxing solutions, respectively, containing 20 mM imidazole (pH 6.8), 50 mM potassium acetate, 6 mM MgCl2, 6 mM ATP, and 1 mM DTT. All experiments using detergent-skinned strips were performed at room temperature, pH 6.8, and at an ionic strength of 120 mM. Ca2+ contracting solutions contained 1 mM free Mg2+, 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 CaCl2 to achieve the appropriate free Ca2+ concentration. The amounts of total compound added to achieve the appropriate free concentration were calculated using a computer program to solve the simultaneous multiequilibrium equations, as previously described (19).
Mechanical measurements. Isotonic shortening velocity measurements were performed using strips mounted on one end by a plastic clip attached to a micrometer for control of muscle length and on the other end to an aluminum foil tube connected to a servo-lever (model 300H, Cambridge Technology) interfaced to a Linux operating system-based personal computer. The detergent-skinned strips were exposed to solutions containing, in addition to appropriate compounds to reflect intracellular conditions, 1.0, 3.0, and 20.0 µM Ca2+. After stable force was achieved at each Ca2+ concentration, the strips were subjected to isotonic quick releases to afterloads ranging from 520% of the initial force at the time of release. The change in length at each afterload was fit by a double-exponential equation, and a tangent to the fit at 100 ms after release 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 each Ca2+ concentration.
MLC phosphorylation. For determination of MLC phosphorylation levels, detergent-skinned strips were rapidly frozen by immersion in a dry ice-acetone slurry containing 6% (wt/vol) trichloroacetic acid and 10 mM DTT. The muscle strips were allowed to slowly thaw to room temperature, rinsed for 30 min in acetone, and homogenized on ice. The homogenization buffer contained 1.0% sodium dodecyl sulfate, 10% glycerol, and 20 mM DTT. Homogenized strips were subjected to two-dimensional electrophoresis followed by transfer to nitrocellulose membranes, as previously described (20). Proteins were visualized using colloidal gold stain (Amersham Pharmacia Biotech). MLC was quantified by scanning densitometry using a scanning densitometer (model GS 800, Bio-Rad). Values are reported as moles of Pi per mole of MLC and were calculated by taking the volume of the densitometric spot representing monophosphorylated MLC as a percentage of the total volume of the densitometric spots for monophosphorylated and nonphosphorylated MLC.
Electron microscopy. Bladder wall dissected free of both serosal and mucosal layers was detergent skinned as described above. The skinned strips were then fixed in place at physiological lengths by immersion in a high-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 a transmission electron microscope (model 100CX, JEOL).
Data analysis. Values are means ± SE. Student's t-test was used for unpaired data. P < 0.05 was taken as significant.
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RESULTS |
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The sensitivity of intact bladder smooth muscle to the noncumulative addition of carbachol or KCl is not different in bladder smooth muscle from control animals and animals subjected to partial bladder outlet obstruction (29). The intact tissue, however, does not allow a direct examination of the contractile apparatus. We therefore performed studies using the Triton X-100 detergent-skinned preparation. Triton X-100 detergent-skinned strips of bladder smooth muscle were subjected to the noncumulative addition of various free Ca2+ concentrations. The results of these experiments are shown in Fig. 1. The addition of Ca2+ to detergent-skinned strips produced a concentration-dependent increase in stress (force/cross-sectional area) in smooth muscle from both animals groups. However, the Ca2+ sensitivity of stress development is significantly less in smooth muscle from animals subjected to partial bladder outlet obstruction than in smooth muscle from control animals: EC50 = 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 fibers was significantly greater than that developed by the intact preparations: intact control 7.9 ± 0.7 x 104 vs. detergent-skinned control 3.1 ± 0.5 x 105 N/m2 (qualitatively similar results were obtained in tissues from obstructed animals).
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MLC phosphorylation is the predominant step in the initiation of a smooth muscle contraction. To fully understand the effect of partial bladder outlet obstruction on the Ca2+-dependent regulation of contraction, one needs to know the Ca2+ dependence of MLC phosphorylation. The tissues in which Ca2+-dependent stress was obtained for Fig. 1 were frozen after a stable force recording was attained, usually within 10 min of 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 no differences in the maximal levels of MLC phosphorylation attained in bladder smooth muscle strips between control animals compared with those subjected to partial bladder outlet obstruction. In contrast to the results shown in Fig. 1, however, there were no trends, let alone significant differences, in the Ca2+ dependence of MLC phosphorylation between the muscle strips from the two animal groups.
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Several biochemical studies have clearly shown that partial bladder outlet obstruction changes the isoform of myosin from the faster SM-B isoform to the slower SM-A isoform (2, 9), which translates to a decrease in the maximal velocity of shortening (31). Figure 3 shows the results of experiments performed to measure maximal velocity of shortening in Triton X-100 detergent-skinned strips from control and outlet-obstructed animals. At every Ca2+ concentration examined, detergent-skinned tissues from outlet-obstructed animals exhibited significantly lower levels of maximal velocity of shortening compared with tissues from control animals.
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Exposure of a tissue to Triton X-100 can be quite damaging. It is therefore possible that tissue from a pathological state such as partial outlet obstruction may have more damage after Triton X-100, and this may account for, in part, the decreased Ca2+ sensitivity of force. To address this possibility, we fixed tissues from control and outlet-obstructed animals and processed them for examination at the level of the electron microscope. Despite detergent treatment, structural preservation is quite good. In normal (Fig. 4A) and obstructed tissue (Fig. 4B), nuclear and cytoplasmic structures are evident. The sarcolemma and intracellular compartments are evident in Fig. 4. Note the close apposition of adjacent smooth muscle cells, the structural detail of the sarcolemma, dense bodies within the cytoplasm, and cytoskeletal filaments.
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Of particular interest are the junctional structures that exist between smooth muscle cells consisting of electron-dense plaque-like structures immediately beneath the sarcolemma. These plaque-like structures are sometimes paired, being present in both adjacent smooth muscle cells whereas in other instances, they exist as single structures present in only one cell. Tissue from control animals (Fig. 4A) and tissue from obstructed animals (Fig. 4B) contain these plaque-like structures. Of interest is the apparent increase in the plaque-like structures in tissue from obstructed animals compared with control animals. These regions are where cytoskeletal proteins come into close apposition with the sarcolemma, likely providing structural rigidity to these sites, where tensional forces are transferred from the smooth muscle cell to both the adjacent smooth muscle cells and the extracellular matrix. This is shown most clearly in Fig. 5 (obstructed animal), where an interstitial fibroblast nucleus can be seen in the extracellular matrix between the two smooth muscle cells. Note also the very extensive single plaque-like structures directly adjacent to the extracellular matrix.
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DISCUSSION |
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The differential effect of partial bladder outlet obstruction on force and MLC phosphorylation can be accounted for by at least two possibilities. The first possibility is that the obstruction produces an uncoupling between MLC phosphorylation and force development. Any cell swelling that alters the spacing between thin and thick filaments could alter the ability of actin to activate a phosphorylated myosin. Such an event has been used experimentally to inhibit contraction of smooth muscle while maintaining the ability to phosphorylate the MLC (14). Whether cell swelling or changes in the filament lattice structure occur in bladder smooth muscle after outlet obstruction is, to our knowledge, not known. However, the fact that higher levels of stress were developed in the permeabilized compared with the intact tissue tends to discount this possibility.
The second possibility to account for the decrease in Ca2+ sensitivity of force, and not of MLC phosphorylation, is an outlet obstruction-induced loss of any regulatory pathway acting in parallel with MLC phosphorylation. We have previously shown that Ca2+ stimulates protein kinase C (PKC) activity in Triton X-100 detergent-skinned vascular smooth muscle (12). Additionally, we have also demonstrated that phorbol ester-, and presumably, PKC-, dependent contractions of bladder smooth muscle are attenuated after partial bladder outlet obstruction (27). Because PKC has been implicated in most smooth muscle thin filament regulatory hypotheses, it is possible that the loss of this contractile pathway contributes to the decrease in force at each submaximal Ca2+ concentration. Whether the decrease in Ca2+ sensitivity of force seen in the present study is due to a change in the filament lattice structure or loss of a parallel pathway for contractile activation cannot be definitively determined. What is definite is that partial bladder outlet obstruction has direct effects on the contractile filaments.
Biochemical studies have clearly shown that the isoform of myosin in bladder smooth muscle changes from the faster SM-B to the slower SM-A isoform in response to partial bladder outlet obstruction (2, 9). We have also shown that this change translates into a slower maximal velocity of shortening in intact strips of bladder smooth muscle from obstructed animals (31). Our present results extend this information to directly demonstrate that maximal velocity of shortening is slower in muscle cells from animals subjected to outlet obstruction at every Ca2+ concentration examined. Thus the activation of cross-bridge cycling in smooth muscle from the obstructed animals remains Ca2+ dependent, but the rate of cycling is significantly slower. How this change in cross-bridge cycling rates affects the contractility of the muscle is not known. Given the many steps involved in excitation-contraction coupling in smooth muscle (26), it is most likely not important in the slower rate of force development in smooth muscle from obstructed animals. This is also supported by the fact that in the Triton X-100 detergent-skinned fiber, where more direct activation of the contractile apparatus can be elicited, there were no significant differences in the rate of force development between tissues from control and outlet-obstructed animals (data not shown). Where the slower rate of shortening velocity may impact bladder smooth muscle contractions is in relation to the tonic-like contractile event of muscle from obstructed animals compared with the typical phasic contraction of muscle from control animals. If the cross-bridge detachment rate is significantly slower in muscles containing the SM-A isoform compared with the SM-B isoform of myosin, then it is entirely possible that force would be maintained for longer periods of time. Thus the switch in isoform of myosin may be responsible, in part, for the switch from a phasic to a tonic contractile profile.
We have previously shown that maximal force development in intact bladder smooth muscle from animals subjected to partial bladder outlet obstruction is similar to that developed by muscle from control animals (31). This is consistent with the similar levels of maximal force developed in the two groups of Triton X-100 detergent-skinned muscle. Considering the myriad of changes in the activity and content of intracellular components that have been shown to occur in 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 structures in smooth muscle from the outlet-obstructed animals may, in part, account for the higher than expected levels of force revealed in the skinned fiber preparation. If these plaque-like structures are associated with attachment points for the transmission of force from the contractile apparatus to the cell 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 plaques can alter the magnitude of a contraction. It is interesting to speculate that one compensatory alteration the smooth muscle cell undergoes in response to the increased resistance to flow is an increase in the number of contact points for the transduction of force from the contractile filaments to the cell 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 gap junctions in smooth muscle from the outlet-obstructed animals. Christ and his colleagues (4) and Haefliger and co-workers (8), using a model of rat bladder outlet obstruction, demonstrated a specific increase in connexin43. Moreover, Fry et al. (6) suggested that an increase in electrical coupling between smooth muscle cells of the human bladder may account for, in part, localized aberrant contractions. Our results do not directly address this possibility, but the time frame of our partial outlet obstruction may fit the experimental results found by at least Christ et al. (4) and Haefliger et al. (8). These two groups used different severity of outlet obstruction, with an increase in connexin43 demonstrable after 9 h of severe and 6 wk of moderate obstruction. Our studies used 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 an intact bladder smooth muscle preparation (31) but would not be expected to have any influence in a detergent-skinned preparation. Future studies using intact tissues should address the specific junctional components of these plaque-like structures.
A very unexpected result, at least to those who have used smooth muscle
skinned 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
4080% of their preskinning force or stress
(13,
17,
19). In our study, the Triton
X-100 detergent-skinned bladder smooth muscle preparations developed nearly
four times the level of stress developed by the intact smooth muscle
preparation. It is possible that, even with supramaximal levels of KCl or
carbachol, maximal levels of activator Ca2+ and,
therefore, stress cannot be attained in the intact preparation, whereas the
direct addition of micromolar levels of Ca2+ can be
introduced into the detergent-skinned fiber. On the other hand, relative to
other smooth muscles, possibly the bladder smooth muscle has a more extensive
cytoskeletal structure that aids in maintaining cellular integrity after the
fairly harsh exposure to Triton X-100.
Other investigators have also used skinning and permeabilizing procedures
on urinary bladder smooth muscle, but the results are mixed. Using Triton
X-100 detergent-skinned smooth muscle from rat urinary bladder, Arner and his
co-workers (25) found that
partial outlet obstruction reduced the force output compared with tissues from
control animals by 25%. Consistent with our results, they also found that
shortening velocity was significantly reduced. Kanaya
(11) found similar levels of
force development in a saponinpermeabilized preparation of bladder smooth
muscle compared with the intact state. In these studies
(11,
25) as well as others
(17,
34), the
Ca2+ sensitivity of force (EC50) was found to
be
1 µM Ca2+, similar to that found in our study
using tissue from control animals.
As stated above, one complicating problem in the interpretation of these results is determining which alterations are compensatory to maintain normal function in the face of the obstruction and which are deleterious as a result of the obstruction. It seems intuitively obvious that the high levels of stress 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 contractile ability, the better the bladders would be expected to perform, resulting in lower residual volumes. The fact that bladders from outlet-obstructed animals have a greater post-void volume, then one can assume that the force generated by the smooth muscle in the obstructed bladder is still insufficient to produce complete emptying. A similar argument could be made for the increased number of apparent attachment points for transduction of force from the contractile proteins to the membrane. In contrast, the decrease in the Ca2+ sensitivity of force in smooth muscle from outlet-obstructed animals would seem to be a deleterious alteration. If it takes a greater level of stimulus to produce the required magnitude of force to completely empty the bladder, then one can understand why bladders from outlet-obstructed animals have a higher post-void volume. The change in myosin isoform to one with a slower actin-activated ATPase activity can be viewed in two modes: compensatory as well as deleterious. If less force is developed at any given level of stimulation, as evidenced by the decrease in the Ca2+ sensitivity of force, then this may be countered by a longer contraction. The slower ATPase activity of the SM-A isoform, due most likely to a slower off rate, may provide a prolonged contractile event. On the other hand, if stimulation durations are not changed but it takes longer to empty the bladder, then this may lead to a greater post-void volume. Future studies aimed at comparing in vivo urodynamics and in vitro biochemistry and physiology should shed valuable light on these speculations.
Bladder function after partial outlet obstruction has been categorized as compensated (mildly dysfunctional) or decompensated (severely dysfunctional) by Levin and his colleagues (36) and by our group (2830). As we have discussed in a previous report (31), our animal model presents bladder function consistent with a decompensated state. However, our results obtained specifically from smooth muscle, as discussed above, show functional aspects that would be consistent with a compensated bladder and others that would be consistent with a decompensated bladder. The apparent discrepancy between the obstruction-induced changes as measured in whole bladder and those measured in the smooth muscle layer may have structural and temporal components. It is very possible that the obstruction-induced changes in the mucosal layer of the bladder (24) exacerbate the early but small changes in the smooth muscle, and, after longer periods of obstruction, more significant deleterious changes in the smooth muscle layer aid the pathological progression to a failing bladder. It also must be kept in mind that different species and different modes of obstruction may produce different effects on bladder smooth muscle function. Studies comparing the effects of obstruction in different animal models would be beneficial in answering this possibility.
In summary, using a Triton X-100 detergent-skinned preparation that allows the direct examination of contractile protein function in a tissue that maintains the ability to contract, we found that bladder smooth muscle from rabbits subjected to partial outlet obstruction has a reduced Ca2+ sensitivity to force without a concomitant change in the Ca2+ sensitivity of MLC phosphorylation. The maximal velocity of shortening was also significantly lower in smooth muscle from the obstructed animals, consistent with a change in the isoform of myosin. Electron-microscopic examination showed that the detergent-skinned cells showed excellent structural integrity and, more importantly, a significant increase in the number of sarcolemmal attachment plaquelike structures in muscle from the outlet-obstructed animals. We interpret these results to suggest that partial bladder outlet obstruction produces several alterations at the level of contractile activation and regulation that are compensatory to maintain normal force in the face of the increased resistance to flow and deleterious to bladder function that, with time, most likely aid in the deterioration of bladder function and the switch from a compensated to a decompensated state.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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