1 Division of Urology and 2 Department of Pathobiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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%
-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
[
-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 -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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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% -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.
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RESULTS |
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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants P50 DK-52620, DK-39740, and DK-47514.
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FOOTNOTES |
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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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