Deletion of SM-B, the high ATPase isoform of myosin, upregulates the PKC-mediated signal transduction pathway in murine urinary bladder smooth muscle

Abstract

Detrusor smooth muscle (DSM) hypertrophy induced by partial bladder outlet obstruction (PBOO) is associated with changes in the NH₂-terminal myosin heavy chain isoform from predominantly SM-B to SM-A, alteration in the Ca²⁺ sensitization pathway, and the contractile characteristics from phasic to tonic in rabbits. We utilized the SM-B knockout (KO) mouse to determine whether a shift from SM-B to SM-A without PBOO is associated with changes in the signal transduction pathway mediated via PKC and CPI-17, which keeps the myosin phosphorylation (MLC₂₀) level high by inhibiting the myosin phosphatase. DSM strips from SM-B KO mice generated more force in response to electrical field stimulation, KCl, carbachol, and phorbol 12,13-dibutyrate than that of age-matched wild-type mice. There was no difference in the ED₅₀ for carbachol but the maximum response was greater for the SM-B KO mice. DSM from SM-B KO mice revealed increased mass and hypertrophy. The KO mice also showed an overexpression of PKC-α, increased levels of phospho-CPI-17, and an elevated level of IP₃ and DAG upon stimulation with carbachol. Two-dimensional gel electrophoresis revealed an increased level of MLC₂₀ phosphorylation in response to carbachol. Together, these changes may be responsible for the higher level of force generation and maintenance by the DSM from the SM-B KO bladders. In conclusion, our data show that ablation of SM-B is associated with alteration of PKC-mediated signal transduction and CPI-17-mediated Ca²⁺ sensitization pathway that regulate smooth muscle contraction. Interestingly, similar changes are also present in PBOO-induced DSM compensatory response in the rabbit model in which SM-B is downregulated.

myosin ii is the major component of the thick filament in smooth muscles from all sources. Smooth muscle myosin II is regulated by alternative splicing at both 3′ and 5′ end of the myosin heavy chain (MHC) pre-mRNA producing COOH-terminal (SM1 and SM2) and NH₂-terminal(SM-A and SM-B) MHC isoforms, respectively. Alternative splicing at the 5′ end inserts a 21-nt insertion that encodes a seven amino acid sequence in the NH₂-terminal head region of the myosin near the ATP-binding site (3, 26). Studies have shown that smooth muscle myosin containing the SM-B isoform (with the 7-amino acid insert) have a higher actin-activated ATPase activity and a higher shortening velocity compared with those smooth muscle which predominantly consist of SM-A, lacking the seven amino acid insert (14, 26, 29). Studies have also shown that smooth muscles containing primarily this high-velocity isoform (SM-B), such as the urinary bladder, tend to be more phasic while those containing primarily SM-A such as aorta and esophageal sphincter tend to be more tonic (14, 26, 38, 47). Thus, the SM-B myosin isoform is present in phasic smooth muscles, although other characteristics such as the membrane properties are different (12, 47). The tonic and phasic properties of smooth muscle and the predominance of various SM isoforms are highly tissue specific and dependent on the function of the organ in which they are present. The functional role of these isoforms is demonstrated in the lower urinary tract where urinary bladder (containing predominantly SM-B) (15) requires a rapid and phasic contraction in the bladder body to initiate bladder emptying while the more tonic urethral smooth muscle (containing more of the SM-A isoform) (8) helps to facilitate urine storage during the filling phase (51, 52).

Changes in the myosin isoforms in hypertrophy associated with various disease processes have been reported in cardiac (30, 37), skeletal (19, 45), and smooth muscle (15, 50). Partial bladder outlet obstruction (PBOO)-induced hypertrophy in both rabbits and mice causes a shift in the NH₂-terminal MHC isoform from predominantly SM-B to SM-A (2, 15). Concomitant with a decrease in SM-B (the high ATPase isoform) and an increase in SM-A (low ATPase isoform), there is a decrease in maximum shortening velocity and the hypertrophied detrusor smooth muscle (DSM) reveals contractile characteristics typical of tonic smooth muscle compared with the phasic contraction shown by normal DSM (46). In addition, PBOO-induced DSM hypertrophy also shows an upregulation of Rho-kinase (5) which is implicated in calcium sensitization of muscle contraction and an increase in the resting myosin light chain (MLC₂₀) phosphorylation level (8, 10, 44).

In addition to the myosin NH₂-terminal isoforms playing a role in controlling the actin-activated myosin ATPase activity, the Ca²⁺/calmodulin-dependant phosphorylation of the myosin regulatory light chain (MLC₂₀) regulates the actin-activated myosin ATPase activity of smooth muscle myosin (9, 20, 40) and cross bridge cycling in smooth muscles (7, 13, 24). The level of myosin phosphorylation is also regulated under certain conditions without an elevation of cytosolic Ca²⁺ by the small GTPase Rho-activated kinase, which regulates myosin light chain phosphatase activity (MLCP), through the membrane-activated second messenger systems such as Rho A (11, 42, 43). The regulation of MLCP is complex, involving a cascade of reactions including second messengers and downstream effectors of membrane and receptor activation. The availability of an SM-B knockout model in which the smooth muscle MHC B isoform is ablated offers a unique opportunity to study the effect of alteration of myosin isoforms on the pathways that regulate contraction in smooth muscle in the absence of bladder outlet obstruction. This study utilized the SM-B knockout mouse to determine whether a shift in the NH₂-terminal MHC isoform from SM-B to SM-A without PBOO is associated with changes in the signal transduction pathway mediated via the PKC and CPI-17, a pathway that lowers the myosin phosphatase activity and keeps the myosin LC₂₀ at a high level of phosphorylation, thus maintaining the smooth muscle tone.

MATERIALS AND METHODS

SM-B null mice.

The method for producing the SM-B null mice has been previously described (4). Mice were killed by exposure to CO₂ in accordance with the ethical treatment of animals under a protocol approved by the University of Pennsylvania Institutional Animal Care and Use Committee. The mice used for these studies were F₂ generation males and females between the ages of 20 and 24 wk old.

Tissue preparation.

DSM strips were obtained from male and female mice. The bladder was surgically removed at the level of the bladder neck and placed into Tyrodes 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 urothelial layer was gently removed by scraping the surface of the open bladder with a sharp scalpel 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 ureteral opening to the bladder).

Force measurements.

Strips of DSM were suspended longitudinally in 15 ml of Tyrodes buffer at 37°C as previously described (30). After a 30-min equilibration, the length of optimal force development (L_o) was determined by increasing the length of each strip in 1.5-mm increments until maximal contractile force to electrical field stimulation (EFS; 70 V, 32 Hz, 1-ms duration) was achieved. After determination of L_o, the bath was replaced with fresh Tyrode's buffer for 15 min to allow stabilization of the muscle at the resting level. Longitudinal strips of bladder (∼1 × 4 mm, 10 mg) were prepared for depolarization with high-KCl solution (125 mM) to evaluate tonic and phasic properties (41). The tissue was then washed three times (10 min each) with Tyrodes buffer and a carbachol concentration-response curve (0–25 μM) was carried out to determine the effect of cholinergic stimulation on the muscle strips. Once again, the strips were washed, as previously described, and 3 μM PDBu was added to evaluate the effect of PKC activation in the smooth muscle. A final wash and addition of high KCl (125 mM) establish that the potency of the muscle strips has not declined disproportionately during the course of the experiment. In separate experiments, we used the specific PKC inhibitor, Bisindolylmaleimide-1 (Bis-1; 10 μM), and the Rho-kinase inhibitor, Y27632 (20 μM), to reverse the contractions caused by PDBu. These inhibitors were also preincubated with muscle strips for 15 min before activation with FS (32 Hz) or carbachol (10 μM) to determine their inhibitory effects. The force was standardized to the cross-sectional area of the muscle strips.

Estimation of myosin light chain phosphorylation.

Analysis to determine the level of myosin light chain phosphorylation in muscle strips was described previously (22). Strips were frozen rapidly at rest or at different levels of force by snapping with clamps previously chilled in dry ice-acetone slurry followed by immersion in dry ice-acetone slurry for 30 s and stored in liquid nitrogen. Strips of bladder smooth muscle were also stimulated with carbachol (25 μM) and rapidly snap-frozen at 100% maximum force and stored in liquid nitrogen. Frozen muscle strips immersed in liquid nitrogen were ground to a fine powder using 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. This mixture was left at room temperature for 30 min until all of the dry ice had evaporated. The sample in acetone was then centrifuged (8,000 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% NP₄₀, and 5% β-mercaptoethanol] and homogenized using a mini-electric homogenizer. After centrifugation, ∼50 μl of the supernatant were then applied to IEF cylindrical gels (1 × 65 mm) and IEF at 350 V was carried out overnight. Gels were then subjected to SDS-PAGE (14%) and stained as previously described (22). Spots corresponding to 20 kDa were identified as smooth muscle LC₂₀ by Western blotting using antibody specific to 20-kDa myosin light chain. The phosphorylated and unphosphorylated LC₂₀ were scanned and analyzed using a Bio-Rad GS-700 imaging densitometer and using a 2D-PAGE Molecular Analyst Software program (Bio-Rad, Hercules, CA).

Measurement of inositol 1,4,5-triphosphate and total IP.

To measure 1,4,5-triphosphate (IP₃), we prelabeled murine bladders with [³H] inositol, treated the tissue with LiCl (10 mM) and carbachol (0.03 mM), and rapidly froze the tissue in liquid N₂. The tissue was then extracted with trichloroacetic acid and the inositol phosphates were separated by Dowex chromatography (28). The radioactivity was measured in a Packard 1500 Tri-Carb liquid scintillation counter and the results were normalized as disintegrations per minute (DPMs) per 15 mg bladder tissue.

Measurement of diacylglycerol.

Diacylglycerol (DAG) is extracted and measured according to the method of Bligh and Dyer (6). Briefly, DAG is extracted with a 1:1 mixture of chloroform/methanol for 1 to 2 h on ice. Next, 0.2 M KCl is added to the mixture with vortexing to break the phase and allow the chloroform to separate from the methanol. The mixture is centrifuged briefly which aids the separation and the lower chloroform phase is transferred to a new test tube. The sample is then dried under nitrogen and the lipid extract made soluble in octyl-β-glucoside/cardiolipin solution. The DAG in the sample is then assayed using DAG kinase in the presence of [γ-³²P]ATP, further separated by thin layer chromatography (TLC) and the radioactivity is measured in a scintillation counter.

SDS-PAGE and Western blot analysis.

Proteins were separated by electrophoresis of denatured protein on SDS-polyacrylamide gels (7.5%). Protein amounts used for quantitation as well as for 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% β-mercaptoethanol). After being blocked with 5% milk for 1 h, the membrane was incubated with a 1:10,000 dilution of primary antibody for PKC-α (Sigma) and 1:3,000 dilution for CPI-17 and phospho-CPI-17 (Upstate), 1:1,000 dilution of primary antibody for ROK-α, 1:1,000 dilution for ROK-β, and 1:2,000 dilution for RhoA (Cytoskeleton) for 1 h at room temperature, washed three times with 10 ml PBS containing 0.05% Tween 20 (PBST), and further incubated with corresponding secondary antibody (goat anti-mouse IgG; 1:4,000) or (donkey anti-rabbit; 1:4,000) for 1 h at room temperature. All antibody solutions were diluted in PBST and membranes were washed thoroughly with PBST between incubations. Target proteins were detected using enhanced chemiluminescence kit from Amersham Life Sciences. The amount of the target proteins was determined by scanning densitometry using Bio-Rad GS-800 Calibrated Densitometer and Quantity 1 software (Bio-Rad).

RESULTS

Bladder weight.

Figure 1A shows the bladder weights for wild-type (WT; n = 10) and SM-B knockout (KO; n = 10) bladders. The mean bladder weights in milligrams for age- and weight-matched mice were 25 ± 0.074 and 33.0 ± 0.09 for WT and SM-B KO mice, respectively (P < 0.05). Figure 1B shows the histologic appearance of a representative Masson's trichrome-stained cross section (5 μm) of the bladder strips used for physiological studies. The amount of the bladder smooth muscle layer relative to interstitial connective tissue, respectively, was greater for the SM-B KO (72 vs. 28%) compared with WT (61 vs. 39%) bladder, as determined using imaging software (ImagePro) to quantify the area of smooth muscle and collagen in the histological sections.

Fig. 1.

A: mean bladder weights for wild-type (WT; mean = 25 ± 0.074) and SM-B knockout (KO; mean = 33.0 ± 0.09), n = 10, P < 0.05. The average body weight of WT and KO mice was similar. B: representative photomicrographs (×40) showing 5-μm cross sections stained with Masson's trichrome for both WT and SM-B KO bladders. A much higher density of muscle fibers can be observed in the SM-B KO compared with the WT bladders with significantly less intermuscular connective tissue matrix in the KO.

Force.

Figure 2 shows the carbachol concentration-response curve for WT and KO mice (n = 4). There was no difference in the ED₅₀ (0.7 ± 0.059 and 0.68 ± 0.065 for WT and KO, respectively). However, the maximum response (E_max) for the KO bladder (0.71 ± 0.12 g/mm²) was significantly higher than that for the WT (0.47 ± 0.059 g/mm²), P < 0.05. Figure 2B shows representative tracings of the raw data for WT, KO, and when superimposed.

Fig. 2.

A: summary data for the carbachol concentration-response curve for WT (n = 5) and SM-B KO (n = 5). The force data for each point represent the mean and SE of strips of bladder smooth muscle. There was no difference in the ED₅₀ but the E_max of 0.47 ± 0.059 g/mm² for the WT was significantly less than that of the SM-B KO (0.71 ± 0.12) g/mm², P < 0.05. B: representative tracings of the raw data for WT, SM-B KO, and when superimposed, respectively.

Figure 3A shows the responses to EFS (70 V, 32 Hz, 1-ms duration), KCl (125 mM), PDBu (3 μM), and carbachol. The data indicate that the KO bladder strips produce significantly higher levels of force in response to all forms of stimulation (n = 4; P < 0.05). Representative tracings of the summary data in Fig. 3A are shown in Fig. 3B. It is noteworthy that the plateau phase of the force curve is higher for the KO bladder strips than the WT bladders for all forms of activation, indicating that the force is maintained at a higher level as a percentage of maximum force for the KO bladder strips compared with WT.

Fig. 3.

A: maximal response to electrical field stimulation (EFS; 70 V, 32 Hz, 1-ms duration), KCl (125 mM), PDBU (3 μM), and carbachol (20 μM). In all cases, the SM-B KO bladder strips responded with higher force than the WT bladders, P < 0.05. B: representative tracings of the summary data in A for KCl, EFS, and PDBu. Note the higher levels of force maintenance in the SM-B KO bladders compared with WT.

Table 1 shows the effects of the PKC inhibitor, Bis-1, and the Rho-kinase inhibitor, Y27632, on contractile function of WT and SM-B KO bladders. Bis-1 caused 100% relaxation of the contraction in response to PDBu for both WT and SM-B KO bladders. Preincubation of muscle strips with Bis-1 caused a less dramatic but significant reduction in force generation in response to FS and carbachol. While the Rho-kinase inhibitor, Y27632, seems to cause a decline in force levels in WT bladders in response to all stimulations, this decline was not significant. This was also true for SM-B KO bladders except for the response to carbachol which was significantly inhibited by Y27632.

Table 1.

Effect of Bis-1 (10 μM) and Y27632 (20 μM) on contraction induced by FS (32 Hz), carbachol (10 μM), and PDBu (3 μM) in WT and SM-B KO bladders

	FS	Carbachol	PDBu
	Force, g	Force, g	Force, g
WT	1.13±0.15	1.1±0.53	0.16±0.07
SM-B KO	2.03±0.27*	1.62±0.08*	0.26±0.04*
WT+BIS-1	0.82±0.08	0.74±0.03*	0.02±0.015*
SM-B KO+BIS-1	1.82±0.38	0.74±0.08†	0.01±0.006†
WT+Y27632	0.90±0.22	0.87±0.21	0.11±0.05
SM-B KO+Y27632	1.71±0.31	1.01±0.29†	0.21±0.03

Values are means ± SE, n = 5, P < 0.05. FS, field stimulation; WT, wild-type; KO, knockout.

^*Significantly different from WT.

^†Significantly different from SM-B KO.

PKC-α, CPI-17, and Phospo-CPI-17 at the protein level.

Since the amount of myosin affects the number of cross bridges that can be formed in a muscle, we estimated the myosin content in the DSM from WT and SM-B KO mice. Western blot analysis revealed no significant difference (data not shown). Force maintenance in the DSM from PBOO is associated with overexpression and overactivity of Rho-kinase (5); however, the expression of Rho-kinase and RhoA in the DSM from SM-B null mouse bladders is not different from that of bladders from WT mice (Fig. 4D). Since CPI-17 has been shown to contribute to the calcium sensitization pathway (17), we analyzed the expression of CPI-17 and PKC which is important for the phosphorylation of CPI-17 in the DSM from the SM-B null bladders. Figure 4 shows the Western blot for PKC-α (A), CPI-17 (B), and Phospho-CPI-17 (C) at the resting level after loading 20 μg of protein for each sample. The loading level was determined by doing protein assays of the extracted proteins and loading 20 μg which was within the linear range of the protein standard curve. Scanning densitometry of the bands reveals a significant, P < 0.05, upregulation of PKC-α in the SM-B KO bladders [8.87 ± 0.34 optical density units (ODU) compared with 5.0 ± 0.43 ODU for WT]. There was no difference in the other PKC isoforms (PKC, ɛ and θ) tested; however, the amount of phosphorylated CPI-17 was significantly elevated for SM-B KO bladders as determined from scanning densitometry of the Western blots made using antibody specific to phospho-CPI-17 (6.03 ± 0.9 ODUs, compared with 3.30 ± 0.53 ODUs for WT), P < 0.05.

Fig. 4.

Western blot for PKC-α (A), CPI-17 (B), and Phospho-CPI-17 (C). PKC-α was significantly elevated in SM-B KO bladders (8.87 ± 0.34) ODUs compared with WT bladders (5.0 ± 0.43) ODUs, P < 0.05. There was no difference in the total CPI-17 level (B); however, the level of phospho-CPI-17 (C) was higher in the SM-B KO mice (5.04 ± 0.95) compared with the WT mice (2.74 ± 0.78) ODUs, n = 4, P < 0.05. D: results for ROKα, ROKβ, and RhoA. The data reveal no significant difference between WT and SM-B KO bladders.

MLC phosphorylation.

A time course of phosphorylation at 5, 15, and 30 s after activation with 20 μM carbachol is shown in Fig. 5A. Figure 5B shows representative two-dimensional gels of the unphosphorylated and phosphorylated MLC₂₀ at each time point. The level of MLC₂₀ phosphorylation at each time point (n = 4) was 26.4 ± 2.9, 30.0 ± 3.2, and 30.0 ± 3.5 for WT bladders and 39 ± 5.1, 38 ± 4.4, and 31 ± 3.3 for bladders from SM-B null mice, P < 0.05 for time points at 5.0 and 15 s, respectively. There was no significant difference at 30 s. There was also no difference in the level of MLC₂₀ phosphorylation between WT (19.96 ± 2.7%) and SM-B (22.02 ± 3.4%) null bladders under resting conditions, n = 4, P = 0.061.

Fig. 5.

A: time course of MLC₂₀ phosphorylation at 5, 15, and 30 s after activation with 20 μM carbachol. The mean level of phosphorylation at each time point was 26.4 ± 2.9, 30.0 ± 3.2, and 30.0 ± 3.5%, respectively, for WT bladders and 39 ± 5.1, 38 ± 4.4, and 31 ± 3.3% for SM-B KO bladders, P < 0.05, n = 4. B: representative 2-dimensional gels showing unphosphorylated and phosphorylated MLC₂₀ at each time point for WT and SM-B KO bladders, respectively.

Total IP, IP₃ production, and DAG.

Since our data show that PKC-mediated signaling is involved as shown from the activation of the DSM muscle strips with PDBU, we analyzed the IP₃ production, one of the downstream effects of PKC stimulation. Figure 6A shows the results (n = 4) for total IP₃ production at rest (L₀) and when stimulated with 25 μM carbachol. The IP₃ levels were significantly higher in the KO bladders both at rest and when stimulated with carbachol (25 μM), P < 0.05. Figure 6B shows the results for total IP production under stimulated and unstimulated conditions. There was no significant difference between the WT and KO under unstimulated conditions while carbachol (25 μM) caused a significant (P < 0.05) increase in total IP production in the SM-B KO bladders. The level of DAG was significantly elevated in the KO bladders (1,869 ± 94.1 ODUs compared with 1,397 ± 128 for WT, P < 0.05).

Fig. 6.

A: total IP₃ production at rest (L₀) and when stimulated with 25 μM carbachol. The IP₃ levels were higher in the SM-B KO bladders both at rest and when activated by carbachol, P < 0.05. B: results for total IP production. There was no difference in the IP production in the absence of stimulation while carbachol (25 μM) caused an increase in the SM-B KO compared with the WT, P < 0.05.

DISCUSSION

The role of the NH₂-terminal smooth muscle myosin isoforms, SM-A and SM-B, in the regulation of smooth muscle myosin ATPase activity and shortening velocity has been well-documented (4, 14, 26). These studies established a compelling relationship between the presence of the SM-B isoform, which contains a 7-amino acid insert in the head region of the SMMHC close to the ATP binding site, and the high actin-activated Mg-ATPase activity and shortening velocity of the smooth muscle in which it is present. Smooth muscles lacking the insert (SM-B) contain myosin with low ATPase activity and low velocity of shortening. Thus, it has been demonstrated that the regulation of smooth muscle function is highly dependent on the nature and composition of the myosin isoforms present within the smooth muscle bundles.

Increasingly, however, a link has been established between the regulation of smooth muscle function and other pathways such as RhoA/Rho-kinase and PKC-CPI-17 (23, 39, 43). Regulation through these pathways has been implicated in the calcium sensitization of smooth muscle, an increase in tone without a corresponding rise in intracellular calcium (18, 42). Thus, it is clear that the absence or presence of specific myosin isoforms may play a role in the regulation of tone and contraction through these pathways also. In this study, we report that bladders in which the SM-B myosin has been knocked out (bladders containing only SM-A) show an elevated plateau phase in response to KCl stimulation (Fig. 3B) and peak contraction, an increase in mass, and a heightened contractile response to the PKC activator, PDBu, compared with WT mice. An increased level of tone is typically present in smooth muscle containing high levels of SM-A such as aorta, urethra, esophageal sphincter, and corpus cavernosum smooth muscle (16, 22, 47). However, the mechanism responsible for high levels of tone and force maintenance in smooth muscle has not been fully understood. Previous studies on bladder tissue from the SM-B KO mouse indicated that the insert can affect the cooperativity between myosin heads and modulate the kinetics of crossbridge cycling (25).

One of the changes in the contractile proteins associated with obstruction-induced bladder hypertrophy is a shift in the NH₂-terminal isoform from SM-B to SM-A (15). Thus, the SM-B KO mouse provides a unique opportunity to investigate the changes in the signal transduction regulation and pathophysiology of the smooth muscle following a switch in the myosin from SM-B to SM-A in the absence of PBOO. Our initial observations reveal that the bladders from SM-B null mice were significantly heavier than bladders from WT mice and that the DSM strips from SM-B null produced higher levels of force when normalized for cross-sectional area. A representative Trichrome stain shows the difference in cross-sectional area of smooth muscle relative to collagen (Fig. 1B). This indicates that the bladder wall from SM-B null mice may indeed have more smooth muscle relative to collagen than that of WT bladders which may contribute to the higher force produced by the DSM from SM-B null bladders when normalized for cross-sectional area. Of particular interest was the effect of EFS, carbachol, and PKC activator. These three forms of smooth muscle activation converge at a common intersection in the signal transduction pathway that involves the activation of PKC. EFS has been shown to release neurotransmitters such as acetylcholine from nerve terminals which can then bind cholinergic receptors on the smooth muscle cell membrane, while carbachol can directly bind to, and activate these receptors when added to a bath solution bathing the SM strip. These G protein-coupled receptors when activated lead to phosphoinositide turnover with the release of the phosphoinositide hydrolysis products IP₃ and DAG. IP₃ has been shown to release calcium from intracellular Ca²⁺ stores while DAG can directly activate PKC. Thus, both of these hydrolytic products of phosphoinositide can further amplify contraction in smooth muscle (21, 48). To probe the enhanced force seen in the SM from SM-B null bladders, we used the synthetic PKC activator PDBu to stimulate the smooth muscle in our physiological studies. PDBu is a well-known activator of PKC signaling and contraction in a variety of smooth muscles (1, 12, 33, 53). Our results indicate a remarkable difference with bladders from SM-B null, recording substantially higher force levels than WT bladders when corrected for cross-sectional area. To gain additional insight into the remarkable contractile response and high level of force of DSM from bladders from SM-B null (containing only SM-A) to both carbachol and PDBu, we studied PKC-α expression, CPI-17, and phospho-CPI-17 at the protein level. Carbachol can activate PKC indirectly through G protein-coupled receptors leading to DAG formation (31, 36), while PDBu can directly activate PKC as indicated previously. Our data indicate significant upregulation of PKC-α and phospho-CPI-17 but no change in the total CPI-17 expression level. CPI-17 is a substrate for PKC and when phosphorylated becomes active and inhibits MLCP. Thus, an increased level of phospho-CPI-17 can further inhibit MLCP leading to a shift toward continued and persistent MLC₂₀ phosphorylation and force in the absence of a rise in intracellular calcium (18, 49). This phenomenon is referred to as calcium sensitization and may further explain the high maximum force and force maintenance seen in the KO bladders. Interestingly, a high level of force maintenance and upregulation of Rho-kinase have also been reported in rabbit bladders subjected to PBOO. Rho-kinase has also been shown to phosphorylate the regulatory subunit of phosphatase (MYPT) and can also inhibit MLCP activity. Rho-kinase expression was similar in SM-B KO and WT detrusor (Fig. 4D). In addition, the human myometrium undergoes hypertrophic changes during pregnancy (34). Studies by Ozaki et al. (35) also reveal a heightened contractile response of the pregnant myometria to PDBu and upregulation of PKC-β and CPI-17 at the mRNA level. PKC-β expression in the DSM was similar for SM-B KO and WT.

As indicated in a recent paper by Eto et al. (17), CPI-17 phosphorylation induced conformational changes in CPI-17, making it a potent inhibitor of MLCP. Thus, phosphorylated CPI-17 caused increased inhibition of MLCP activity (27). The inhibited MLCP is unable to dephosphorylate the MLC which is required for relaxation of the smooth muscle, and as a consequence, this may promote higher levels of force. Since our data showed elevated levels of phospho-CPI-17, we studied the MLC₂₀ phosphorylation level both under resting and stimulated conditions in WT and SM-B KO bladders. While the data showed no difference in the resting phosphorylation level, the SM-B KO bladders showed a significant increase in MLC₂₀ phosphorylation compared with WT bladders after activation with carbachol. This increased phosphorylation was persistent at 5 and 15 s after activation and coincided at 30 s. This suggests that the higher phosphorylation levels in the SM-B KO bladders may contribute to the elevated force levels seen in these bladders due to increased number of activated cross bridges. Prior studies from this lab reveal that after PBOO, force levels were maintained at a high level compared with unobstructed bladders, and this increased force was accompanied by higher levels of MLC₂₀ phosphorylation in the PBOO bladders (46). In conclusion, our data show that ablation of SM-B is associated with smooth muscle hypertrophy, alteration of PKC-mediated signal transduction, and CPI-17-mediated Ca²⁺ sensitization pathway that regulate smooth muscle contraction. Interestingly, similar changes are present also in PBOO-induced DSM compensatory response in the rabbit model in which SM-B is downregulated. Thus, there appears to be a cross-talk between the smooth muscle myosin isoforms and the signal transduction pathway in the SM-B KO bladders which contribute to higher force levels in these tissues.

GRANTS

This work was supported by George O'Brien Urology Research Center Grant P50 DK-52620.