Regional variation in myosin isoforms and phosphorylation at the resting tone in urinary bladder smooth muscle

Joseph A. Hypolite1, Michael E. DiSanto1, Yongmu Zheng1, Shaohua Chang1, Alan J. Wein1, and Samuel Chacko1,2

1 Division of Urology and 2 Department of Pathobiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Urinary bladder filling and emptying requires coordinated control of bladder body and urethral smooth muscles. Bladder dome, midbladder, base, and urethra showed significant differences in the percentage of 20-kDa myosin light chain (LC20) phosphorylation (35.45 ± 4.6, 24.7 ± 2.2, 13.6± 2.1, and 12.8 ± 2.7%, respectively) in resting muscle. Agonist-mediated force was associated with a rise in LC20 phosphorylation, but the extent of phosphorylation at all levels of force was less for urethral than for bladder body smooth muscle. RT-PCR and quantitative competitive RT-PCR analyses of total RNA from bladder body and urethral smooth muscles revealed only a slight difference in myosin heavy chain mRNA copy number per total RNA, whereas mRNA copy numbers for NH2-terminal isoforms SM-B (inserted) and SM-A (noninserted) in these muscles showed a significant difference (2.28 × 108 vs. 1.68 × 108 for SM-B and 0.12 × 108 vs. 0.42 × 108 for SM-A, respectively), which was also evident at the protein level. The ratio of COOH-terminal isoforms SM2:SM1 in the urethra was moderately but significantly lower than that in other regions of the bladder body. A high degree of LC20 phosphorylation and SM-B in the bladder body may help to facilitate fast cross-bridge cycling and force generation required for rapid emptying, whereas a lower level of LC20 phosphorylation and the presence of a higher amount of SM-A in urethral smooth muscle may help to maintain the high basal tone of urethra, required for urinary continence.

myosin isoform; urethra; urinary continence


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BOTH THE FILLING AND EMPTYING PHASES of urinary bladder function are dependent on the coordinated control of a storage chamber, the bladder body, and its outlet, the urethra. The essence of this control is the ability of the bladder to increase in volume at relatively low, but sufficient, intravesical pressure to prevent the overdistension of the bladder while the bladder outlet (urethra) remains in a contracted or tonic state to maintain bladder continence. Several reports indicate that the smooth muscle in the bladder body is in a constant state of controlled contraction during the filling stage, while the bladder outlet is closed (15, 34). Bladder emptying is accompanied by a reversal of function in which contractile forces predominate in the bladder body smooth muscle with a concomitant reduction in outlet resistance of the bladder neck and urethra associated with the relaxation of the smooth muscle (26, 28). While there is still considerable controversy regarding the mechanisms that control micturition, there is little question that the mechanical events occur in a coordinated manner to facilitate storage and rapid emptying of the bladder.

Using bladder strips to study contractility, Levin et al. (27) reported a lack of uniformity in bladder contraction in the progression from the bladder dome proximally to the base distally. Those studies indicated a force gradient, in response to pharmacological stimulation, with maximum force production in the most proximal region (dome) and minimum force in the most distal region studied (urethra). Similar studies by Khanna et al. (25) also showed a similar gradation of response to cholinergic stimulation of longitudinal and circular strips of rabbit bladder body, base, and urethra. However, it is not known whether these observations rest in inherent differences in the isoforms of myosin, the molecular motor for contraction, or the regulation of contraction.

During the relatively passive phase of bladder filling, which occurs with little change in intravesical pressure, the bladder neck and urethra must remain in a more or less tonic or contracted state to maintain bladder continence (30, 38). This can be best visualized in a rabbit bladder containing urine by removing the entire bladder, neck, and urethra region from the animal to just below the level of the proximal urethra and holding it up vertically so that the urethra points downward. When this is done, there is no leakage of urine (9), suggesting that a significant portion of the continence mechanism of the neck and urethra may be due to the intrinsic tone of the smooth muscle in this area (19, 40). In general, smooth muscles exhibit either tonic or phasic characteristics, although the presence of both components may exist in a smooth muscle due to a heterogeneous cell population or an intermediate type of muscle cell (reviewed in Ref. 33). The phasic smooth muscle responds to depolarization with high K+ by rapid and transient contractions, whereas tonic smooth muscle exhibits slow, sustained K+-induced contractions (33).

The major regulatory pathway for smooth muscle contraction is generally thought to be myosin mediated through phosphorylation of the 20-kDa myosin light chain (LC20) by a Ca2+/calmodulin-dependent kinase (MLCK). Phosphorylation of LC20 is required for actin activation of the myosin ATPase activity (6, 18, 32), the enzymatic activity that hydrolyzes ATP and provides the energy for the sliding of actin filaments past the myosin filaments during muscle shortening (see review, Ref. 1). Physiological studies on a variety of smooth muscles show a correlation between myosin phosphorylation and force development (reviewed in Ref. 22). However, factors in addition to myosin phosphorylation can regulate cross-bridge cycling rates in both tonic and phasic contractions (16, 37). Force maintenance is achieved at low levels of phosphorylation and with non- or slow-cycling cross bridges (5, 11).

Smooth muscle tissues from all sources have been shown to contain the myosin isoforms SM1 and SM2, which are different at the COOH-terminal tail regions (7). However, some visceral type smooth muscles contain myosin with a 7-amino acid insert in the NH2-terminal head region near the ATP-binding site (8). These isoforms are encoded by different myosin heavy chain (MHC) mRNAs, which are formed by alternative splicing of the pre-mRNA transcripts from a single myosin gene (2, 3, 41). Alternative splicing at the 5' end of the MHC pre-mRNA transcript results in an insertion of 21 nucleotides (encoding 7 amino acids) at the NH2-terminal region close to the ATP-binding site of the smooth muscle MHC (2). Kelley et al. (23) showed that myosin with this 7-amino acid insert (SM-B) has a higher actin-activated Mg2+-ATPase activity and moves actin filaments faster in the in vitro motility assay than myosin without the insert (SM-A). The SM-B and SM-A isoforms are found in the phasic (i.e., urinary bladder) and tonic (i.e., aorta) types of smooth muscles, respectively. In addition, DiSanto et al. (13) reported that the prevalence of inserted myosin increases as the aorta branches to form the muscular arteries and that the actin-activated ATPase activity of the myosin isolated from the distributing arteries (femoral and saphenous) is twofold higher than that of aortic smooth muscle, which is composed entirely of the isoform without the 7-amino acid insert (13). The maximum shortening velocity (Vmax) of smooth muscle containing the 7-amino acid insert is also higher than that of aortic smooth muscle containing the noninserted myosin (13). In contrast, Haase and Morano (20) reported that, although the amount of inserted MHC is 50% less in the rat myometrium compared with the bladder, the apparent Vmax of these smooth muscle tissues were comparable. The reason for this discrepancy regarding the expression of the myosin with the NH2-terminal insert and the contractile characteristics is not known.

In this report, we show that there are inherent differences between the bladder body and urethral smooth muscle tissues in the expression of myosin isoforms and the relative levels of LC20 phosphorylation. Thus differences in the myosin isoforms or regulation of myosin phosphorylation, or both, may explain the coordinated contraction of the lower urinary system that is crucial for bladder filling and continence until the stimulus occurs for micturition.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Tissue preparation. Rabbit bladder smooth muscle tissues were obtained from male New Zealand White rabbits weighing 2.2-2.4 kg. The animals were sedated with ketamine and xylazine (25 mg/kg ketamine, 6 mg/kg xylazine im) and further anesthetized with Nembutal (25 mg/kg iv). The bladder was surgically removed at the level of the urethra and placed into Tyrode buffer (125 mM NaCl, 2.7 mM KCl, 23.8 mM NaHCO3, 0.5 mM MgCl2 · 6H2O, 0.4 mM NaH2PO4 · H2O, 1.8 mM CaCl2, and 5.5 mM dextrose). The mucosal and serosal layers were gently removed, and the rest of the bladder, comprised primarily of smooth muscle tissue, was equilibrated at 37°C in 95% O2-5% CO2. Samples of smooth muscle tissue were obtained from the bladder body (region extending proximally above the ureters) and the urethra (tubular area below the bladder neck).

Force measurements. Strips of bladder muscle (~50 mg) were suspended longitudinally in 10 ml of Tyrode buffer at 37°C as previously described (27). After a 30-min equilibration, the length of optimal force development (Lo) was determined by increasing the length of each strip in 1.5-mm increments until maximal contractile force to 70 V, 32 Hz, and 1-ms duration was achieved. After equilibration at Lo in Tyrode buffer for 15 min to allow stabilization of the muscle at the resting level, muscle strips were rapidly frozen in liquid nitrogen for analysis of phosphorylation levels at the resting tone. Longitudinal strips of bladder (~2 × 10 mm, 50 mg) were used for depolarization with high-KCl solution (125 mM) to evaluate tonic and phasic properties (33). All strips were prepared according to the protocol above to reach Lo before stimulation with KCl, and force was measured as described (27). To determine the relationship between active force and myosin phosphorylation levels for bladder body and urethra, force in response to bethanecol was also measured.

Estimation of myosin light chain phosphorylation. Analysis to determine the level of myosin light chain (MLC) phosphorylation in muscle strips was performed as described previously (14, 29). Strips were frozen rapidly at rest or at different levels of force by snapping with clamps previously chilled in a dry ice-acetone slurry, followed by immersion in dry ice-acetone slurry for 30 s, and were stored in liquid nitrogen. Strips of bladder smooth muscle and urethra were also stimulated with bethanechol (500 µM) and rapidly snap-frozen at 50, 80, and 100% maximum force and stored in liquid nitrogen. Frozen muscle strips immersed in liquid nitrogen were ground to a fine powder with the use of a prechilled mortar and pestle. The powder was then added to a mixture of dry ice-acetone to inactivate any residual endogenous kinases and phosphatases remaining after the first treatment in the dry ice-acetone slurry (4). This mixture was left at room temperature for 30 min until all of the dry ice had evaporated, as previously described (14, 29). In some experiments, 10% trichloroacetic acid was added to the acetone to determine whether this procedure might enhance the inactivation of the kinase and phosphatase over that achieved with nitrogen and acetone alone. However, this step was eliminated because two-dimensional (2-D) gel electrophoresis revealed no change in phosphorylation levels with this additional treatment. The sample in acetone was then centrifuged (6,000 g for 10 min), and the acetone was removed. The pellet was mixed with isoelectric focusing (IEF) sample buffer [50 µl/10 mg tissue containing 9.5 M urea, 1.6% ampholyte (pH 5-7), 0.4% ampholyte (pH 3-10), 2% NP-40, and 5% beta -mercaptoethanol] and homogenized with a mini-electric homogenizer. After centrifugation, ~50 µl of the supernate was then applied to IEF cylindrical gels (1 × 65 mm), and IEF at 350 V was carried out overnight (31). Gels were then subjected to SDS-PAGE (14%) and stained. Spots corresponding to the phosphorylated and unphosphorylated LC20 were scanned and analyzed with a Bio-Rad GS-700 imaging densitometer and a 2D-PAGE Molecular Analyst Software program (Bio-Rad, Hercules, CA). The identity of these spots was confirmed by Western blotting of the two-dimensional slab gels with the use of an anti-LC20 monoclonal antibody. As a control for the possibility of enzymatic activity (kinase/phosphatase) during the preparation of samples for IEF, pure 32P-phosphorylated myosin was added to the frozen powder in some experiments; ~ 95% of the added 32P-myosin was recovered in the LC20 spots, indicating <5% nonspecific loss. Similarly, addition of [gamma -32P]ATP to the tissue during homogenization in the IEF buffer during preparation of the sample for isoelectric focusing did not result in 32P incorporation into LC20, indicating the absence of detectable MLCK activity under these conditions.

RNA extraction and RT-PCR. RNA was extracted by using the guanidinium thiocyanate method with a kit (Stratagene, La Jolla, CA) as described previously (13). Briefly, pieces of tissue (~50 mg) were crushed to a fine powder with the use of a prechilled mortar and pestle, transferred to another prechilled tube, and homogenized in denaturing buffer with a mini-electric homogenizer. RNA was extracted from the homogenate by using a water-saturated phenol solution and was quantified by ultraviolet spectrophotometry. RNA quality was determined by electrophoresis through formaldehyde-agarose gels, followed by ethidium bromide staining. Approximately 3.5 µg RNA was then reverse transcribed with oligo(dT) primer (Promega, Madison, WI) and Moloney murine leukemia virus reverse transcriptase (RT) (GIBCO BRL, Gaithersburg, MD) at 37°C for 60 min. The cDNA was heated to 90°C for 5 min to inactivate the RT, and 2 µl of this sample were subjected to PCR amplification according to DiSanto et al. (13). PCR amplification of the mRNA for the 5' end (NH2-terminal insert) and 3' end (SM1 and SM2) were performed with the use of specific primers (13). An upstream and a downstream primer that anneal with the 3' untranslated region of rabbit smooth muscle alpha -actin cDNA was included in the PCR as an internal standard (13).

Quantitative competitive RT-PCR. A competitive internal standard for the quantitative determination [quantitative competitive RT-PCR (QC RT-PCR)] of rabbit smooth muscle myosin heavy chain (SMMHC) transcript was generated by using an RT-PCR competitor construction kit (Ambion, Austin, TX). Briefly, a pair of primers was designed to amplify a region of the rabbit SMMHC cDNA encoding a sequence common to all four myosin heavy chain isoforms (SM1/SM2/SM-A/SM-B). Locations of the primers are shown in the schematic drawing of the SMMHC cDNA shown in Fig. 1. The sequence of the upstream primer was 5'-CGCTAATACGACTCACTATAGGGAGAGGAGGGCCCTCAAAGTCCAGTTCGGCAGAACGAGGAGAAGAGGAG-3' (Q3), where nucleotides (nt) 1-3 serve as a GC clamp, nt 4-20 represent a core T7 promoter sequence, and nt 31-50 and 51-70 correspond to nt 4806-4825 and 4851-4871, respectively, of the rabbit uterus SMMHC full-length cDNA coding sequence. The sequence of the downstream primer was 5'-TGGAAGTCCTTCATCTGAGCC-3' (Q4), representing nt 5049-5069 of the rabbit uterus SMMHC sequence. In vitro transcription of the PCR product produced by this primer pair yields a competitor RNA molecule spanning nt 4806-5069 but lacking 25 residues from nt 4826-4850. A second primer pair was designed in which the upstream primer 5'-GGCCCTCAAAGTCCAGTTCG-3' (Q1) corresponded to nt 4806-4825 of the rabbit SMMHC and the downstream primer 5'-CTTCCTCCCGCCCTTTGATG-3' (Q2) corresponded to nt 5004-5023. Thus amplification of the competitor using the Q1, Q2 primer pair in the presence of rabbit smooth muscle total RNA produced two bands. Endogenous myosin cDNA produces a band at 266 bp, while the competitor RNA produces a band at 241 bp due to the 25-nt deletion. To determine SMMHC copy number, total RNA was extracted from rabbit bladder body and urethra as described above, six aliquots of each sample of rabbit total RNA (0.1 µg) were co-reverse transcribed with various concentrations of competitor RNA (determined by measuring [32P]ATP incorporation), and PCR was carried out. The bands obtained in the gels were scanned with a densitometer, and the exact number of SMMHC copies was determined by graphical analysis of the data.


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Fig. 1.   Schematic drawing of the smooth muscle myosin heavy chain (SMMHC) cDNA. The hatched bar represents the 6,644-bp rabbit uterus SMMHC SM1/SM-A complete cDNA. Solid triangles indicate regions where the 21-nt sequence and the 39-nt sequence are alternatively spliced in for the SM-B and SM2 isoforms, respectively. Small solid bars indicate the locations of the P1 and P2 primers used to amplify the region containing the 21-nt alternative splice site and the P3 and P4 primers used to amplify the region containing the 39-nt alternative splice site, respectively. Additional small solid bars indicate the location of the Q1-Q4 primers used for the construction and amplification of the SMMHC competitor cRNA. The competitor cRNA is constructed from a region of the SMMHC cDNA common to all four SMMHC isoforms. The sequences and exact positions of the primers as well as the detailed procedure for the relative RT-PCR, competitor cRNA construction, and the quantitative competitive (QC) RT-PCR are given in the text.

Antibodies. A 7-amino acid peptide with the sequence (QGPSLAY) identical to that deduced from the 21-nt insert in the 5' end of the MHC mRNA was conjugated to keyhole limpet hemocyanin, and a polyclonal antibody was raised in BALB/c mice. The IgG fraction was purified from mouse serum on Ultralink Affinity Pak Immobilized Protein A columns (Pierce, Rockford, IL) and used for Western blotting. Immunoblotting and immunofluorescence studies of aortic and bladder smooth muscles have confirmed the specificity of this antibody (12). Antibodies against the whole myosin heavy chain (catalog no. M7786), 20-kDa light chain (catalog no. M4401), and secondary antibodies were purchased from Sigma Chemical (St. Louis, MO).

Quantitation of heavy chain isoforms and Western blot analysis. The SM1 and SM2 myosin heavy chains were separated by slow electrophoresis of denatured protein on highly porous SDS-polyacrylamide gels (4.5%) as previously described (36). After having been stained with Coomassie blue, proteins were quantitated by scanning densitometry with a Bio-Rad GS-700 imaging densitometer. Protein amounts used to determine the SM1:SM2 protein ratio as well as those in the Western blots were in the linear range for absorbance. Identical gels were run and transferred to an Immobilon-P membrane (Millipore, Bedford, MA) overnight at 30 V (Bio-Rad mini-transfer unit) in buffer (25 mM Tris, 192 mM glycine, 0.005% SDS, and 0.056% beta -mercaptoethanol). After blocking with 10% Sea Block (Pierce) for 1 h, the membrane was incubated with a 1:500 dilution of primary antibody (1:500 dilution of 7-amino acid-specific antibody or 1:5,000 dilution of antibody to the whole smooth muscle myosin molecule) overnight at 4°C, washed, and further incubated with secondary antibody (goat anti-mouse IgG) for 1 h at room temperature. All antibody solutions were diluted in 10% Sea Block, and membranes were washed thoroughly with PBS containing 0.05% Tween 20 between incubations. Immunoreactivity was visualized with the use of 3,3'-diaminobenzidine. Two-dimensional electrophoresis and Western blotting of the 20-kDa myosin light chain LC20 were carried out as described above. LC20 recognized by the antibody were visualized on X-ray film by using an enhanced chemiluminescence kit from Amersham (Arlington Heights, IL).

Statistical analysis. Data were analyzed by statistical analysis with Student's t-test. Differences were considered significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Contractile characteristics of smooth muscle strips from bladder body and urethra. The contractile profiles produced by muscle strips from the bladder body and urethra in response to 0.125 M KCl are depicted in Fig. 2. The strips from the bladder body developed a transient force, followed by a partial relaxation and a second rise in tension, as shown previously (21). The tension decreased slowly and reached the basal level in 4 min (Fig. 2, top). Unlike the muscle strips from the bladder body, those from the urethra did not show the initial force transient, but after reaching a maximum, the force was maintained at a slightly higher level during the 4-min measurement period (Fig. 2, bottom). Thus force development in the urethral muscle tends to be slower, and the maximal level of force achieved is maintained over a long period of time, whereas force development by bladder body muscle strips is rapid but quickly dissipated. These results suggest the presence of more phasic characteristics in the bladder body and more tonic characteristics in the urethra.


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Fig. 2.   Contractile responses of strips to 125 mM KCl. Strips of bladder (10 × 2 mm), weighing 50 mg (wet weight), were equilibrated with 95% O2-5% CO2 in Tyrode buffer for 1 h. Strips were then attached to a force transducer and stretched incrementally (1.25 mm each time) until the response to electrical field stimulation (80 V, 32 Hz, 1 ms) was maximal (Lo ). The bath solution was changed, and the strips were allowed to equilibrate for a further 45 min. Strips of bladder body (top) and urethra (bottom) were then subjected to 125 mM K+, and force was recorded.

MLC phosphorylation at the resting tone in the bladder and urethra. In our initial experiment, phosphorylation of LC20 was determined in different regions of the bladder and urethra at Lo (optimal length) by 2-D PAGE. Figure 3 shows representative 2-D gels of proteins extracted from the bladder dome (A) and the urethra (B). Percent basal phosphorylation was 35.5 ± 4.6, 24.7 ± 2.2, 13.6 ± 2.1, and 12.8 ± 2.7% for dome, midbladder, base, and urethra, respectively (means ± SE for 3 different animals) as determined by scanning densitometry of the 2-D gels. These data clearly indicate a significant (P < 0.05) difference in the level of LC20 phosphorylation in different regions of the lower urinary tract, particularly between the bladder body (dome and midbladder body) and the outlet areas (base and urethra). Western blot analysis of the 2-D gels with antibody specific to smooth muscle LC20 revealed reactivity of both phosphorylated and unphosphorylated LC20 spots in the gel (data not shown), confirming that the spots identified as LC20 were derived from smooth muscle myosin.


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Fig. 3.   Two-dimensional (2-D) gel electrophoresis of extracts from bladder dome and urethra. 2-D gel electrophoresis was performed as described in MATERIALS AND METHODS. The relative levels of 20-kDa phosphorylated (P) and unphosphorylated (U) myosin are shown in protein extracted from the dome (A) and urethra (B). The pH range of the isoelectric focusing gel and the molecular size markers are indicated. For orientation purposes, actin, tropomyosin, and the 17-kDa essential light chain are also labeled. Densitometric scanning was used to quantify unphosphorylated and phosphorylated myosin.

Figure 4 shows the relationship between agonist-mediated force and LC20 phosphorylation. Because the major difference in phosphorylation was observed between urethra and the bladder body, these two tissues were used for these experiments. The level of LC20 phosphorylation was 16 and 29% for the urethra and bladder body, respectively, at the resting tone before stimulation. With an increase to 50% of maximum force, the phosphorylation increased to 28 and 45% for urethra and bladder body, respectively. Interestingly, the increase in force to 80% did not induce a proportional increase in LC20 phosphorylation for either tissue. At 100% force, phosphorylation levels for the bladder body declined significantly, whereas levels for the urethra remained constant. At each force, the level of LC20 phosphorylation for the urethral smooth muscle at the resting tone was significantly lower than in the bladder body.


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Fig. 4.   Effect of agonist-mediated force on 20-kDa myosin light chain (LC20) phosphorylation. Bladder (black-lozenge ) and urethra () strips were prepared as described in MATERIALS AND METHODS for force measurements. In control experiments (no stimulation), each strip was removed from the bath after 30 min of equilibration, rapidly frozen in a dry ice-acetone slurry, and stored in liquid nitrogen. For stimulation of bladder body and urethra, bethanechol was used at a concentration (500 µM) known to induce maximal contraction of bladder smooth muscle. Strips were stimulated at 50, 80, and 100% maximum force, rapidly frozen in a dry ice-acetone slurry, and stored in liquid nitrogen until analysis by 2-D gel electrophoresis.

Expression of MHC isoform mRNA. To determine whether myosin isoforms in the bladder smooth muscle exhibit an expression pattern that is relevant to the physiological force measurements described earlier, total RNA from different regions of the rabbit bladder was reverse transcribed with oligo(dT) primer, and the resulting cDNA was PCR amplified using upstream (P1; 5'-TACAGGAGCATGCTG-3' corresponding to nt 580-594) and downstream primers (P2; 5'-TGGCGGATGGCTCGTGA-3' corresponding to nt 901-917) that span the region encoding the 25- to 50-kDa junction of rabbit smooth muscle MHC ATP-binding regions (locations of P1 and P2 are indicated in Fig. 1) . The PCR products (cDNA) obtained with these primers represent the relative level of mRNA expression of the 21-nt insert present in the smooth muscle. As shown in Fig. 5, the PCR products corresponding to the noninserted and inserted MHC mRNA transcripts migrated as a 337- and 358-bp band, respectively, in a 2% agarose gel. Expression of the noninserted SM-A was clearly greater in the urethra (Fig. 5, lane 5) than in the dome (lane 2), midbody (lane 3), or base (lane 4), where virtually 100% of the myosin contains the 7-amino acid insert in the head of the myosin close to the ATP-binding region (SM-B). Densitometric scanning of the agarose gels revealed that only 81 ± 2.4% of the myosin in the urethral region was inserted and confirmed our gross observations that the rest of the bladder had fully inserted myosin (~100%). The specificity of this set of primers for SM-B mRNA transcripts has been documented (12, 13). Thus these data indicate that, at the level of the proximal urethra, there is an abrupt decrease in myosin mRNA containing the 21-nt insert at the 5' end, which encodes a 7-amino acid insert present in the SM-B MHC near the ATP-binding site.


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Fig. 5.   Analysis of MHC mRNA transcripts for the NH2-terminal inserted myosin. PCR primers were used to amplify bladder smooth muscle cDNA fragments obtained by reverse transcription of mRNA present in the total RNA extracted from smooth muscle tissue from different regions of the bladder. Fragments were separated by electrophoresis on 2% agarose gels, followed by ethidium bromide staining. Specific downstream and upstream primers (sequences given in RESULTS) were used to competitively amplify the 5' end region of the mRNA transcript containing the MHC SM-A/SM-B alternatively spliced site, which encodes the NH2-terminal region near the ATP-binding site. Lane 1, 100-bp DNA ladder used as marker; lane 2, dome; lane 3, midbladder; lane 4, base; lane 5, urethra. Rabbit smooth muscle alpha -actin cDNA was amplified in each reaction as an internal control.

The mRNAs encoding the SM1 and SM2 myosin isoforms that differ at the COOH-terminal tail region are also produced by alternative splicing, but at the 3' end. Figure 6 shows RT-PCR analysis using a set of upstream (P3; 5'-GCTGGAGGAGGCCGAGGAGGAGTC-3' corresponding to nt 5751-5774) and downstream oligonucleotide primers (P4; 5'-GAGCCATCTGCGTTTTCAATAA-3' corresponding to nt 5936-5957) that span the 3'-terminal coding regions of both SM1 and SM2 (locations of P3 and P4 are indicated in Fig. 1). Two PCR products of 203- and 245-bp, representing SM1 and SM2 cDNA fragments, respectively, were obtained for all bladder and urethral smooth muscle samples analyzed. There was no significant difference in the SM2:SM1 isoform ratio at the mRNA level among the dome (2.0:1), midbody (1.95:1), and base (1.90:1). However, a moderate but significant (P < 0.05) difference was found in the SM2:SM1 isoform ratio at the mRNA level within the urethra (1.5:1) compared with the dome region (2.0:1) of the bladder body.


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Fig. 6.   RT-PCR analysis of MHC SM1/SM2 mRNA transcripts. PCR primers were used to amplify bladder smooth muscle cDNA fragments, as obtained in Fig. 4, from different regions of the bladder. Upstream and downstream primers (sequences given in RESULTS) were used to competitively amplify the 3' end region of the mRNA transcripts encoding the COOH-terminal tail region of SM1 and SM2. Lane 1, 100-bp DNA ladder; lane 2, dome; lane 3, midbladder; lane 4, base; lane 5, urethra. Rabbit smooth muscle alpha -actin cDNA was amplified in each reaction as an internal control.

To obtain quantitative information on the mRNAs for these isoforms in smooth muscle tissues from bladder body and the urethra, total RNA samples extracted from each tissue were analyzed by QC RT-PCR (Fig. 7, A and B, respectively). Urinary bladder body possessed 2.4 ± 0.1 × 108 copies of the SMMHC transcript per 100 ng of total RNA, whereas the urinary bladder urethra had 2.1 ± 0.1 × 108 copies (Fig. 7C), a slight (~ 12%) but statistically significant difference (P < 0.05), as shown in Fig. 8A.


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Fig. 7.   QC RT-PCR. Known amounts of mutant cRNA corresponding to the nt sequence 4710-5023 for rabbit SMMHC present in all MHC isoforms but differing by deletion of a 25-nt fragment (nt 4730-4754) were mixed with total RNA and used in QC RT-PCR with appropriate primers as described in MATERIALS AND METHODS. Representative ethidium bromide-stained agarose gels shown are from QC RT-PCR performed on bladder body (A) and urethra (B). The competitor fragment runs below the fragment produced from the endogenous SMMHC cDNA. Lanes 1-6: 1 × 109, 5 × 108, 2.5 × 108, 1.25 × 108, 6.25 × 107, and 3.125 × 107 copies of competitor, respectively. Lane 7: no competitor. Lane M: a 100-bp DNA ladder. Bands in the gels were scanned with a densitometer, and the data have been plotted (C) to determine the exact number of SMMHC copies, which is the same as the mutant SMMHC (mSMMHC) copy number when the 2 band intensities are equal.



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Fig. 8.   SMMHC mRNA copy numbers in rabbit bladder body and urethra. Results from the graphical analyses shown in Fig. 7 (n = 4) revealed an average number of mRNA copies of SMMHC gene per 100 ng of total RNA of 2.4 × 108 for the bladder body and 2.1 × 108 for the urethra (A). This difference was small but significant (P < 0.05). Based on this information and on the ratio of the isoforms determined from Figs. 5 and 6, the absolute concentration of alternatively spliced MHC isoforms in the tissues per 100 ng of total RNA was calculated as 2.28 × 108 copies of SM-B and 0.12 × 108 copies of SM-A for the bladder body, compared with 1.68 × 10 8 and 0.42 × 108 copies of SM-B and SM-A, respectively, for the urethra (B). Copy numbers of SM2 are 1.6 × 108 and 1.3 × 108, respectively, for the bladder body and urethral smooth muscles, and the copy number for SM1 in both tissues is around 0.75 × 108 (C).

These data, combined with the results from the relative PCR studies (Figs. 5 and 6), were used to determine the absolute copy number of individual SMMHC isoforms (Fig. 8, B and C). Thus the urinary bladder body smooth muscle consisted of predominantly SM-B (2.28 × 108 copies) with only 0.12 × 108 copies of SM-A mRNA, whereas the urethra contained less SM-B (1.68 × 108 copies) and 3.5 times more SM-A mRNA (0.42 × 108 copies) than the bladder body. Copy numbers for the SM2 mRNAs were 1.6 × 108 and 1.3 × 108, respectively, for the bladder body and urethral smooth muscle. However, the number of mRNA copies for SM1 in both muscles was lower than that for SM2, and the difference in SM1 copy numbers between urethra and bladder body was not remarkable.

Expression of MHC isoform protein. Figure 9 shows expression of the SM1 and SM2 MHC isoforms in the tissue homogenate of the bladder dome (lane 2) and urethra (lane 3) after electrophoresis on 4.5% SDS-PAGE gels. Figure 9B shows SM1 and SM2 electrophoretically separated at 204- and 200-kDa, respectively. Scanning densitometry revealed SM2:SM1 ratios for the dome (~2.2:1) and urethra (~ 1.5:1) to be significantly different (P < 0.05).


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Fig. 9.   SDS-PAGE and Western blot analysis of MHC in smooth muscle tissue from dome and urethra performed by using antibodies against the NH2-terminal insert. Total protein extracted from each tissue was loaded onto a highly porous 4.5% SDS-polyacrylamide gel, and SM1 and SM2 were separated by overnight electrophoresis with the use of a large (16 × 16 cm) gel. Protein concentrations from both samples were adjusted to obtain equal loading of the myosin by trial runs. Lane 1, 200-kDa size marker prestained with Coomassie blue; lane 2, dome; lane 3, urethra. A: Western blots obtained by using antibody against the 7-amino acid insert for dome and urethra. B: protein bands stained with Coomassie brilliant blue. C: immunoreaction with antibody against the whole myosin molecule. Notice the separation of the SM1 and SM2 in the Coomassie blue-stained gel (B). Proteins were quantified by scanning densitometry. The prominence of the molecular size standard in the Western blots is not due to immunoreaction but instead to dye (Coomassie blue) bound to the proteins before electrophoresis.

Because the NH2-terminal inserted and noninserted isoforms (SM-B and SM-A) are not readily separated by gel electrophoresis, an antibody specific for the 7-amino acid insert was used to distinguish them. Proteins were isolated from bladder body (dome) and urethra, and samples containing equal amounts of MHC were electrophoresed and then blotted to Immobilon-P membrane. Western blot analysis revealed antibody against the 7-amino acid peptide reacting with both SM1 and SM2 (Fig. 9A), indicating that the 7-amino acid insert is present in both the COOH-terminal isoforms. Use of an antibody cross-reactive with all myosin isoforms (Fig. 9C) showed that equal amounts of myosin were loaded onto the gel for both dome and urethra.

To determine whether there is a difference in the expression of SM-B isoform in different regions of the bladder, protein samples extracted from the dome, midregion, base, and urethra were electrophoresed and analyzed by Western blotting with the antibody against the 7-amino acid peptide insert. Figure 10B shows the SM-B isoforms in the dome (lane D), midbody (lane M), bladder base (lane B), and urethra (lane U). Although slightly more myosin from the urethra was loaded (Fig. 10A, lane U) compared with other regions (Fig. 10A, lanes D, M, and B), Western blotting showed less reaction with the myosin from the urethra (Fig. 10B). Densitometric scanning showed that reactivity was moderately, but significantly, stronger (despite the lower loading) in the dome (Fig. 10, lane D) than in the urethra (lane U; see also Table 1). Thus the decrease in mRNA transcripts for SM-B MHC isoform in the urethra is also reflected at the protein level. Myosin samples from the midbody and bladder base reacted equally with the antibody against the 7-amino acid peptide compared with samples from the dome. The proportion of SM-B isoform in the smooth muscle tissue of the urethra was decreased by ~20% compared with that of the bladder body.


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Fig. 10.   SDS-PAGE and Western blot analysis of MHC in smooth muscle tissue from dome, midbladder, base, and urethra. Extracts from each tissue containing equal amounts of myosin were loaded onto a highly porous 4.5% SDS-polyacrylamide gel and separated by electrophoresis on a 8 × 8-cm minigel electrophoresis unit for 1 h. Notice that the SM1 and SM2 are not separated in this gel. Lane D, dome; lane M, midbladder; lane B, base; lane U, urethra. A: Coomassie blue staining of protein for the 4 regions. B: Western blot obtained by using antibody against the 7-amino acid insert for dome, midbladder, base, and urethra. All proteins were quantified by scanning densitometry.


                              
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Table 1.   Summary of SDS-PAGE and Western blot analysis for MHC isoforms in the dome and urethra


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies (25-27) have demonstrated a pattern of force generation with maximum contraction in the bladder body and minimum contraction in the base and urethra in response to stimulation by agonists in vitro. This force wave throughout the body, along with relaxation of the urethra, facilitates bladder emptying at low intravesical pressure (34, 35, 39).

Smooth muscle strips from the bladder body showed a phasic response to K+ depolarization (Fig. 2), whereas urethral muscle strips behaved like tonic smooth muscle. This difference might reflect inherent differences in the smooth muscle cells due to a difference in the molecular motor for contraction and/or its regulation via myosin phosphorylation or as mediated through thin filament-associated proteins. In the present study, we analyzed the two major parameters known to affect the contractile characteristics of smooth muscle, MLC phosphorylation, and myosin isoform composition.

The basal levels of phosphorylation of the myosin in the dome and midregion of the bladder body are greater than those of the bladder base and the urethra. The increased phosphorylation levels for both the urethra and bladder body smooth muscles during force generation in response to agonists (shown in Fig. 4) indicate an association between force generation and MLC phosphorylation; thus it appears to be the major regulatory mechanism for force generation in both regions. LC20 phosphorylation levels in the urethral smooth muscle remained constant from 50 to 100% force (Fig. 4). However, phosphorylation levels in the bladder body smooth muscle continued to rise up to 80% force, declining thereafter; at 100% force, phosphorylation levels began to return to basal level. Thus maximum force is maintained at nearly basal levels of phosphorylation in the bladder body. In the urethral muscle, maximum force was achieved with ~50% less phosphorylation, and this level of phosphorylation was maintained at maximal force.

Expression of the SM1 and SM2 isoforms also differed, at both the mRNA and protein levels, in the bladder body and urethral smooth muscle, but the difference was not as remarkable as that of the NH2-terminal isoforms. Nearly all of the MHC mRNA in these regions demonstrated an SM2:SM1 ratio close to 2:1 (Fig. 6) and contained the 21-nt insert (7-amino acids) at the 5' end of the MHC mRNA molecule (Fig. 5). However, QC RT-PCR analysis showed that only 80% of the MHC mRNA in the urethral smooth muscle contained the insert (Fig. 8B). This decreased level of expression of the SM-B mRNA transcripts was also evident at the protein level, where SM-B protein levels in the urethra were 33% less than in the dome (Table 1).

RT-PCR analyses were used to determine the relative expression of the mRNA transcripts for NH2-terminal and COOH-terminal isoforms. The major difference between bladder body and urethral smooth muscle tissues was seen in the expression of SM-A and SM-B. While the bladder body was composed of very little (5%) SM-A, the urethral smooth muscle contains ~20% SM-A transcript. Although the SMMHC copy numbers determined for urinary bladder are higher than those we reported earlier (12), the numbers reported in this paper are more accurate because 1) a radioactive incorporation was used to quantify the competitor; 2) the competitor was made RNase resistant by incorporation of chemically modified nucleotides; 3) there was no insertion of extraneous DNA sequence into the competitor, which might affect the amplification rate; and 4) the competitor was constructed to contain an extra stretch of sequence at the 3' end to equalize the effect of the 25-nt deletion upon reverse transcription.

The presence of the insert in the NH2-terminal head regions correlates with high actin-activated Mg2+- ATPase activity and high velocity of actin filaments in the in vitro motility assays (23). In addition, a correlation between high levels of myosin isoform with the NH2-terminal insert and high maximum velocity of shortening has also been reported for arterial smooth muscle from different regions (13). While our previous studies showed that the bladder smooth muscle contains inserted myosin (13), those analyses did not address the question of regional variations within the bladder.

Using an in vitro whole bladder model, Damaser et al. (10) showed that the work required to completely empty the rabbit bladder is preceded by significant power generation in the bladder body, where SM-B and phosphorylation levels are greatest. It is well established that the underlying mechanism for contraction in smooth muscle is the reversible phosphorylation of the LC20. The Mg2+-ATPase of smooth muscle myosin is activated upon interaction with actin only when the myosin is phosphorylated (6, 16, 29). The MLC phosphorylation is also correlated with an increase in force (5, 11; see review, Ref. 22). Gong et al. (17) showed that phasic smooth muscles have a higher MLC kinase activity than tonic smooth muscle, resulting in higher LC20 phosphorylation levels in phasic smooth muscle. Our contractile measurements (Figs. 2 and 4) demonstrate that the bladder body responds more like a phasic muscle, while the urethra shows more tonic characteristics. These findings are consistent with the high level of phosphorylation found throughout the phasic bladder body and the relatively low level found in the tonic urethra.

The presence of noninserted myosin (SM-A) and the low degree of MLC phosphorylation in the urethra may contribute to slowly cycling cross bridges and a slower rate of contraction, thus helping to maintain a high resting tone in the urethral smooth muscle. Similarly, the rabbit corpus cavernosum is a smooth muscle that also has a high resting tone and contains both inserted (SM-B) and noninserted (SM-A) isoforms (12). The cross bridges in the bladder body smooth muscle, on the other hand, would cycle at a faster rate, because of the presence of myosin (~100%) containing the 7-amino acid insert (SM-B), which has a twofold higher actin-activated Mg2+-ATPase activity than SM-A (13, 23) and a significantly higher level of phosphorylation compared with the urethral smooth muscle. The high resting level of phosphorylation may be important in maintaining the intravesical pressure required to prevent overdistension of the bladder during filling and to develop force rapidly in response to the stimulus for bladder emptying.

Finding SM-A myosin isoform and a low level of resting phosphorylation in the urethral smooth muscle does not rule out the existence of additional regulation through signal transduction via Ca2+-dependent or -independent protein kinase C. Agonist-induced translocation by protein kinase C has been demonstrated in tonic type smooth muscle in ferret aorta (24). This regulation may be mediated through thin filament-associated proteins, such as caldesmon or calponin, and may complement the difference in cross-bridge cycling due to differences in the isoforms of myosin, the molecular motor for force generation.

In conclusion, we find that the urinary bladder and its outlet, the urethra, contain rather unique properties with respect to tonicity, LC20 phosphorylation, and the composition of MHC isoforms. These differences are consistent with the functional requirements of these two tissues. The filling and emptying of the bladder require precise coordination between different regions of the bladder and the urethra. Rapid force development in the phasic bladder body is accompanied by relaxation of the urethra, while the storage of urine in the bladder requires a high resting tone in the tonic urethra, with low intravesical pressure in the bladder body. MLC phosphorylation levels and the composition of the myosin with the 7-amino acid insert at the NH2-terminal region were highest in the bladder body, where maximum force would be required for emptying. In contrast, the low level of phosphorylation, decreased NH2-terminal-inserted myosin, and decreased SM2:SM1 ratio in the urethra may contribute to a unique isoform mixture in this tissue that results in slow cycling, low-energy myosin cross bridges, and a high resting tone during filling while facilitating relaxation during bladder emptying. The high resting tone of the urethral smooth muscle, characterized by the presence of the SM-A isoform and a low level of LC20 phosphorylation, is sufficient to keep the urethra closed to maintain continence during bladder filling. The difference in the level of myosin phosphorylation may be due to a difference between bladder body and urethral smooth muscles in the expression or activities of kinases and phosphatases.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants P50 DK-52620, DK-39740, and DK-47514.


    FOOTNOTES

Address for reprint requests and other correspondence: S. Chacko, Dept. of Pathobiology and Division of Urology, Univ. of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104 (E-mail: chackosk{at}mail.med.upenn.edu).

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.

Received 15 May 1998; accepted in final form 7 September 2000.


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