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Am J Physiol Regul Integr Comp Physiol 291: R1076-R1084, 2006. First published May 11, 2006; doi:10.1152/ajpregu.00118.2006
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DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY

Regional differences in myosin heavy chain isoform expression and maximal shortening velocity of the rat vaginal wall smooth muscle

Maureen Basha,1 Shaohua Chang,1 Elaine M. Smolock,2 Robert S. Moreland,2 Alan J. Wein,1 and Samuel Chacko1,3

1Division of Urology, University of Pennsylvania, 2Department of Pharmacology and Physiology, Drexel University College of Medicine, and 3Department of Pathobiology, University of Pennsylvania, Philadelphia, Pennsylvania

Submitted 15 February 2006 ; accepted in final form 1 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Contractility of the proximal and distal vaginal wall smooth muscle may play distinct roles in the female sexual response and pelvic support. The goal of this study was to determine whether differences in contractile characteristics of smooth muscle from these regions reside in differences in the expression of isoforms of myosin, the molecular motor for muscle contraction. Adult female Sprague-Dawley rats were killed on the day of estrus, and the vagina was dissected into proximal and distal segments. The Vmax at peak force was greater for tissue strips of the proximal vagina compared with that of distal (P < 0.01), although, at steady state, the Vmax for the muscle strips from the two regions was not different. Furthermore, at steady state, muscle stress was higher (P < 0.001) for distal vaginal strips (n = 5). Consistent with the high Vmax for the proximal vaginal strips, RT-PCR results revealed a higher %SM-B (P < 0.001) in the proximal vagina. A greater expression of SM-B protein (P < 0.001) was also detected by Western blotting (n = 4). Interestingly, there was no regional difference noted in SM-1/SM-2 isoforms (n = 6). The proximal vagina had a higher expression of myosin heavy chain protein (P < 0.01) and a greater percentage of smooth muscle bundles (P < 0.001). The results of this study are the first demonstration of a regional heterogeneity in Vmax and myosin isoform distribution in the vagina wall smooth muscle and confirm that the proximal vaginal smooth muscle exhibits phasic contractile characteristics compared with the distal vaginal smooth muscle, which is tonic.

vagina; smooth muscle; female sexual response

ANIMAL MODELS OF THE FEMALE sexual arousal response suggest the proximal and distal vagina may differ in their contractile response to pelvic nerve (33, 38) and urethral (31) stimulation. It has also been demonstrated that the proximal and distal vagina differ histologically (13), functionally (37), and in nitric oxide synthase and arginase activities (27, 47). The different embryological origin of the proximal and distal parts of the vagina might account for some of these differences (28). Despite these noted differences, many muscle strip studies of the vaginal muscularis (the nonvascular smooth muscle of the vaginal wall) have been performed solely on the distal vagina (2, 13, 14, 25, 26, 50). In light of the evidence of regional differences in aspects of vaginal development and physiology, it is important to determine the mechanisms regulating contraction in both the proximal (upper region adjacent to the uterus) and distal (lower region adjacent to the introitus) vagina.

Relatively little is known of the molecular profile of contractile proteins in the vaginal muscularis. Myosin, the molecular motor of contraction, is a hexamer consisting of two heavy chains, a pair of phosphorylatable 20-kDa light chains, and a pair of essential 17-kDa light chains. The NH2-terminal head of myosin heavy chain (MHC) contains an actin-activated Mg2+-ATPase, which converts chemical energy into the mechanical work required for the sliding of actin filaments past myosin filaments during muscle contraction. Four isoforms of the MHC are formed by alternative splicing of a single MHC gene (3, 4, 48). Alternative splicing of pre-mRNA at the 3' end results in the formation of isoforms SM2 (200 kDa) and SM1 (204 kDa) that differ in their COOH-terminal region (4, 36, 41). Alternative splicing at the 5' end of pre-mRNA has been shown to form two additional isoforms (SM-A, SM-B) of MHC that differ in their NH2-terminal region. The SM-B isoform contains a 7-amino acid insert near the ATP binding site in the myosin head, whereas the SM-A isoform lacks this insert (3, 16, 48). Smooth muscle distribution of these MHC isoforms has been shown to be tissue specific (8, 12, 24, 42), developmentally regulated (48, 49), under the control of steroid hormones (7, 21, 44), and altered in hypertrophied muscle (9, 35, 42).

There has been considerable evidence to suggest that NH2-terminal isoform expression influences shortening velocity of smooth muscle. Higher expression levels of SM-B have been identified in phasic type smooth muscle and are correlated with a greater unloaded shortening velocity (8, 12, 24, 45) and ATPase activity (8, 40) compared with tonic smooth muscle with low or absent levels of SM-B. Furthermore, studies with SM-B knockout mice have determined that shortening velocity is decreased in bladder smooth muscle of mice lacking the SM-B isoform (5, 22).

The functional significance of heterogeneous tissue expression of SM1 and SM2 MHC isoforms has been less conclusive. Although some studies have demonstrated a positive correlation between the SM-1 isoform and unloaded shortening velocity (35, 46), others have failed to detect a difference in shortening velocity that could be attributed to COOH-terminal isoform expression (23, 39, 45). Results are also conflicting regarding a possible role of the carboxyl terminal isoforms on filament assembly (18, 20, 39).

Interpretation of the pattern of isometric force tracings from the proximal and distal vagina have lead to the suggestion that the proximal vagina may be phasic in nature compared with the tonic distal vagina (37). Smooth muscle with a relatively high velocity of shortening, poor maintenance of force, and a tendency to display spontaneous activity are considered phasic, and muscle with a slow velocity of shortening and a superior ability to maintain force are considered as tonic. One goal of this study was to define regional differences in shortening velocities and force generation of the vaginal muscularis to further elucidate contractile differences between the proximal and distal vagina. Given the compelling evidence for a functional significance of different MHC isoforms, a second goal of this study was to determine the MHC isoform expression in the proximal and distal vagina.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Animal use and the experimental protocol were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. Female Sprague-Dawley rats (4–5 mo old, 250–300 g) were obtained from a commercial supplier and housed in a temperature (25°C) and light-controlled (12:12-h light-dark) room with free access to food and water. Histological examination of daily vaginal smears using the Papinocolu method was performed to monitor the estrus cycle of rats through visual assessment of sloughed epithelial cell morphology. To control for possible effects of estrus cycle stage and to ensure animals were cycling normally, all animals were killed the day of estrus following two consecutive cycles by an intraperitoneal injection of an overdose of ketamine (>75 mg/kg) and xylazine (>10 mg/kg).

Tissue preparation. The vaginal tube was isolated and dissected from the urogenital tract, cleaned of connective tissue, and cut open longitudinally. For histological and immunohistochemical procedures, sections from the intact vagina (n = 3) were placed in Histochoice fixative (Amresco, Solon, OH). For functional and molecular studies, the vagina was divided into a proximal (approximately upper two-thirds) and distal segment (approximately lower one-third) under a dissecting microscope (Fig. 1). The vaginal segments were either snap frozen in liquid nitrogen and stored at –80°C or placed in ice-cold MOPS-buffered physiological salt solution (PSS) for same-day physiological studies. The PSS solution contained (in mM) 140 NaCl, 4.7 KCl, 1.2 MgSO4, 1.6 CaCl2, 1.2 Na2HPO4, 2.0 MOPS, 5.0 D-glucose, and 0.02 Na2-EDTA.


Figure 1
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  		Fig. 1. Photograph taken with a stereomicroscope (x0.8) of the mucosal surface of the rat vagina demonstrating the division of the vagina into a proximal and distal segment. The distal vagina, as indicated by the dashed line, was considered the lower third of the vagina containing abundant longitudinal folds.

 
RNA extraction/quantification. Frozen tissue (50–100 mg) was ground into a fine powder with a liquid nitrogen prechilled mortar and pestle (n = 6). RNA was extracted from tissue powder using TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol. Briefly, 1 ml of TRIzol reagent was added to vaginal tissue powder, and the mixture was homogenized with a minielectric homogenizer. Following centrifugation, the resultant supernatant was mixed with 0.2 ml of chloroform and centrifuged for phase separation. The aqueous phase was then isolated, and RNA was precipitated by the addition of isopropyl alcohol. The RNA pellet formed following centrifugation was washed with 75% alcohol and subsequently dissolved in 0.1% diethyl pyrocarbonate-treated water. RNA concentration was quantified at 260/280 with a UV-visible spectrophotometer (BioSpec-mini, Shimadzu).

Reverse transcription. Three micrograms of RNA were reverse transcribed with oligo(dT) primer (Promega, Madison, WI) and Moloney murine leukemia virus reverse transcription (RT) (Invitrogen, Carlsbad, CA) at 37°C for 60 min. The reaction was stopped by heating to 90°C, and the cDNA was stored at –20°C.

Polymerase chain reaction. Polymerase chain reaction (PCR) was performed with 1 µl of cDNA in 49-µl reaction mixture containing the following: upstream and downstream primer, 10x PCR buffer (Applied Biosystems), dNTP (Roche Applied Science, Germany), and Taq polymerase (Perkin-Elmer, Foster City, CA). The 5' to 3' sequences of the upstream and downstream primers were as follows. SMA/SMB: CCA CCA GGG CAA GAA GAC AGC (upstream); TCC GGC GAG CAG GTA GAA GA (downstream). SM1/SM2: AGC AGG CAG AGA AAG GAA ACA CCA (upstream); GAA GTC TGA GTC CCG AGC GTC CAT (downstream).

Western blot analysis. Protein was extracted from 50–100 mg of pulverized tissue from frozen vaginal segments (n = 4) in a buffer containing 0.05 M Tris, 20% glycerol, 1% SDS, 25 mM dithiothreitol, 2% bromophenol blue, and protease inhibitors (0.8 mM phenylmethylsulfonyl fluoride, 10 µM pepstatin, 1 µM antipain, and 0.1 ng/ml soybean trypsin inhibitor). Before the addition of dithiothreitol and bromophenol blue, which interfere with the Bio-Rad DC assay, 25 µl of the supernatant were set aside for protein quantification. To verify accurate protein concentration measurements and confirm that no proteolytic breakdown of protein occurred, 40 µg of protein were separated by 10% SDS-PAGE and stained with Coomassie blue for scanning with a GS800 Bio-Rad calibrated densitometer (Hercules, CA). For total myosin, SM-B, and beta-actin analysis, 5 µg of protein were separated by SDS-PAGE with a highly porous gel (7.5%), and the protein was transferred to an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was then blocked with 5% nonfat milk for 2 h in PBS containing 0.1% Tween 20 (PBST). As an internal control, the membrane was incubated in a 1:5,000 dilution of mouse anti-beta-actin monoclonal antibody (A5316, Sigma, St. Louis, MO) for 1 h followed by incubation in a 1:3,000 dilution of peroxidase-linked sheep anti-mouse antibody (NA931V, Amersham Biosciences, Buckinghamshire, UK) at room temperature for 1 h. For analysis of MHC, the membrane was incubated for 1 h in a 1:50,000 dilution of a polyclonal antibody generated in rabbits against bovine aorta (a generous gift of Dr. R. Adelstein) followed by incubation in a 1:3,000 dilution of peroxidase-linked donkey anti-rabbit antibody (NA934V, Amersham Biosciences) at room temperature for 1 h. For SM-B analysis, the membrane was incubated in a 7-amino acid-specific SM-B antibody generated in our laboratory (10) for 2 h followed by incubation in a 1:3,000 dilution of sheep anti-mouse antibody at room temperature for 1 h. Membranes were washed five times for 3–5 min in PBST between incubations. MHC, SM-B, and beta-actin protein were detected using an enhanced chemiluminescence kit from Amersham Biosciences. Resultant autoradiograms were analyzed by scanning densitometry with quantitation software (Quantity One, Biorad, Hercules, CA).

Measurement of Vmax. Maximal shortening velocity was measured as follows. Longitudinal strips measuring ~1.5 mm x 6 mm were cut from the proximal and distal vagina (n = 5). Each strip was mounted on one end to a micrometer via a plastic clip, for control of muscle length. The other end was attached to an aluminum foil tube connected to a servo-lever (model 300H, Cambridge Technology) interfaced to a Linux operating system-based microcomputer. The preparation was immersed in a large water-jacketed muscle chamber containing PSS at 37°C and aerated with 100% O2. Strips were equilibrated for 90 min, during which time they were repeatedly contracted by the addition of 110 mM KCl (equimolar substitution for NaCl) to the water bath. Between contractions, the strips were stretched to a length that approximates the optimal length for maximal active contraction. For estimation of maximal velocity of shortening, each strip was stimulated with 110 mM KCl and then subjected to a series of isotonic quick releases to afterloads equaling 5, 10, 15, and 20% of the force at the time of release. The change in length at each afterload was fit by a double exponential equation, and a tangent to the fit at 100 ms after release was taken as the isotonic shortening velocity at that afterload. Isotonic shortening velocities at the various afterloads listed above were used to extrapolate velocity at zero load for calculation of Vmax. Vmax was estimated at both the point of peak force of contraction and during steady-state conditions (~6 min following KCl stimulation).

Force/cross-sectional area. At the end of each experiment, strips were placed in Histochoice fixative (Amresco, Solon, OH) and embedded in paraffin. For each muscle strip, 10 cross sections, 5 µm thick, were taken at intervals of 50 µm and stained with hematoxylin and eosin. Images of cross sections were captured with a RT slider SPOT camera (Diagnostic Instruments, Sterling Heights, MI) and analyzed for cross-sectional area of muscle and total cross-sectional area of strip using Image Pro Plus software, version 4.5 (Media Cybernetics, Silver Spring, MD). The amplitude of each contraction at peak force and during steady state was then normalized to force/cross-sectional area of tissue strip (tissue stress) and force/cross-sectional area of smooth muscle (muscle stress) using the values obtained for percent muscle mass.

Histology/immunohistochemistry. Cross sections of 5-µm thickness were taken from the proximal and distal end of the paraffin-embedded vaginal tube. Morphometric analysis of Masson's Trichrome-stained cross sections of the vaginal tube was carried out as described for morphometric analysis of tissue strips. For immunohistochemistry, sections were deparaffinized in a xylene substitute (Shandon, Pittsburgh, PA), followed by descending grades of ethanol, and washed three times in PBS. Tissue sections were first incubated for 1 h in 1% BSA at room temperature to block nonspecific binding. Subsequently, sections were incubated in a 1:300 dilution of anti-{alpha}-smooth muscle actin antibody generated in mouse (M-7786, Sigma) for 1 h, washed 3x in PBST, followed by a 1-h incubation in a 1:400 dilution of anti-mouse IgG-Cys3 (C2181, Sigma). Following a final washing (3x PBST), a drop of mounting medium (Aqua-Mount, Lerner Laboratories, Pittsburgh, PA) and a cover slip were placed on the section, and the edges sealed. Images viewed under a Nikon (Melville, NY) Eclipse E800 fluorescence microscope were captured using a RT Slider SPOT camera, described earlier.

Statistical analysis. All data are expressed as means ±SD. Paired t-tests were used to compare differences between proximal and distal vaginal measurements except for Vmax and amplitude of force which were analyzed using a mixed effects model to consider repeated measures within each rat (SAS, version 9.1). Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Masson's trichrome staining and {alpha}-actin immunostaining demonstrated that the vaginal muscularis was not uniformly distributed and did not form a continuous layer in the vaginal wall (Fig. 2). Where two layers of muscularis were present, it appeared that the inner layer was oriented in a longitudinal arrangement, and the outer layer of the muscularis was arranged circularly (Fig. 2, B and C). In the distal vagina, the thin muscularis layer projected into the mucosal folds (Fig. 2D). Despite these irregularities, there were differences observed between the proximal and distal vagina, most notably that the muscularis was more abundant in the proximal vagina compared with the distal vagina. Morphometric analysis of cross sections indicated that the muscularis comprised 8.80 ± 2.71% of the proximal vaginal wall compared with 3.13 ± 0.21% of the distal vagina.


Figure 2
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  		Fig. 2. Masson's trichrome staining (A and D) and {alpha}-actin immunostaining (B, C, E, and F) of 5-µm sections of proximal and distal vagina demonstrate a thicker vaginal muscularis in the proximal region of the vagina compared with the distal region (n = 3).

 
Similarly, morphometric analysis of tissue strips used for physiological studies indicated a significantly greater percentage (P < 0.001) of smooth muscle in the proximal vagina wall compared with the distal vagina (Table 1). The proximal vagina's phasiclike response to KCl stimulation compared with a toniclike response of the distal vagina was strikingly consistent (Fig. 3). During the period of peak force following KCl stimulation, force/cross-sectional area of tissue strip was almost three times greater (P < 0.001) in strips from the proximal vagina compared with the distal vagina. However, normalization of force to cross-sectional area of the smooth muscle in the strip indicated no regional difference in contractile strength at peak force generation. Force generation remained virtually unchanged (~107 mN/mm2 muscle) for distal strips from peak force to steady state. On the other hand, force generation of proximal strips decreased from 95.6 ± 6.6 mN/mm2 muscle at peak force to 51.9 ± 17.9 mN/mm2 muscle during steady-state conditions. Accordingly, although force was not different between regions at peak force, the distal vagina demonstrated greater force/mm2 muscle (P < 0.05) at steady state compared with the proximal vagina (Fig. 4).


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  		Table 1. Isometric force of contraction of proximal and distal rat vaginal strips detected using a dual-mode servo-lever (model 300H, Cambridge Technology)

 

Figure 3
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  		Fig. 3. Representative tracing of isometric contractile response of longitudinal tissue strips from the proximal (A) and distal (B) rat vagina to 110 mM KCl.

 

Figure 4
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  		Fig. 4. Peak and steady-state force in response to KCl stimulation of muscle from proximal and distal vaginal wall. Values are means ± SD; n = 5. Although no significant difference in peak force was detected, steady-state force was significantly higher (*P < 0.05) for distal strips compared with proximal strips.

 
Vmax was almost threefold higher in the proximal vagina (0.11 ± 0.04 muscle lengths/s) compared with the distal vagina (0.04 ± 0.01 muscle lengths/s) at peak force (P < 0.01). The distal vagina Vmax decreased by ~10%, whereas the Vmax of the proximal vagina decreased 37% from peak force to steady-state conditions, and no significant regional difference in Vmax was detected during steady-state conditions (Fig. 5).


Figure 5
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  		Fig. 5. Vmax of longitudinal strips of proximal and distal vagina estimated using isotonic quick releases of a KCl-stimulated contraction during a period approximating initial force generation (~1 min following KCl addition) and a period approximating steady-state conditions (~6 min following KCl addition). Values are means ± SD; n = 5. The Vmax of the proximal vagina was significantly faster (**P < 0.01) than the distal vagina during the period of force generation.

 
MHC protein detected by Western blotting was ~1.6x higher in the proximal vagina compared with the distal vagina (P < 0.01). The protein expression of the SM-B isoform of MHC was also shown to be regionally different in the vagina (Fig. 6). There was a threefold higher (P < 0.001) expression of SM-B protein in the proximal vagina compared with the distal vagina.


Figure 6
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  		Fig. 6. Western blot analysis of myosin heavy chain (MHC), SM-B (NH2-terminal isoform of MHC with a 7-amino acid insert) and beta-actin (internal control). A: isolated protein (5 µg) was separated using a 7.5% SDS-polyacrylamide gel, blotted to an Immobilon P membrane, and probed with either a rabbit polyclonal antibody against MHC (gift of R. S. Adelstein), a mouse monoclonal antibody against SM-B generated in our laboratory, or a mouse monoclonal antibody against beta-actin (Sigma, A-5316). B: Student's paired t-test analysis of optical density (OD) indicated a significantly lower expression of MHC in distal vagina (*P < 0.05). C: an even greater reduction (***P < 0.001) in SM-B expression was detected in the distal vagina compared with the proximal vagina. Values are means ± SD; n = 4.

 
RT-PCR analysis of mRNA transcripts for SM-A and SM-B isoforms of MHC indicated a predominance of the relative expression of SM-B isoform in the proximal vagina compared with an approximately equal expression level of SM-A and SM-B in the distal vagina (Fig. 7A). Semiquantitative analysis of the mRNA transcripts for MHC isoforms indicated that the percent SM-B [SM-B/(SM-B + SM-A)] in the proximal vagina (73.81 ± 4.44) was significantly greater (P < 0.001) than that of the distal vagina (50.59 ± 4.45) (Fig. 7B).


Figure 7
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  		Fig. 7. RT-PCR analysis of mRNA transcripts for NH2-terminal isoforms (SM-A and SM-B) of MHC in the proximal and distal vagina of Sprague-Dawley rats. A: representative ethidium-bromide stained agarose gel of SM-A and SM-B amplification. B: results of a Student's paired t-test indicated a significantly higher (***P < 0.01) ratio of the OD of SM-B/(SM-A + SM-B) in the proximal vagina compared with the distal vagina. Values are means ± SD; n = 5.

 
RT-PCR analysis of the mRNA transcripts for the SM-1 and SM-2 isoforms of MHC indicated a predominance of the SM-1 isoform in both the proximal (69.41 ± 1.57%) and distal vagina (71.82 ± 1.24%). The percent SM-1 [SM-1/(SM-1 + SM-2)] in the proximal vagina was not statistically different than that in the distal vagina.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of this study are the first demonstration of a regional heterogeneity of Vmax and MHC isoform expression of the vaginal wall smooth muscle. We report that the proximal vagina of the rat displays a significantly higher Vmax and a greater abundance of the SM-B isoform of myosin, which has a higher ATPase activity and a 7-amino acid insert in the NH2-terminal. We have shown the higher content of the SM-B isoform in the proximal vagina compared with the distal vagina at both the message and protein levels.

Animal models of female sexual arousal have illustrated in vivo differences in the contractility of the proximal and distal vagina, demonstrating a functional significance of our findings (15, 31, 34, 38). Decreased intraluminal pressure of the distal vagina following pelvic nerve stimulation of rabbits was first reported by Park et al. (38). In addition to a decrease in distal luminal pressure, Min et al. (34) reported that pelvic nerve stimulation induced an increase in luminal pressure and repeated contractions of the proximal vagina. The data presented in our report indicate a myogenic contribution to the regional difference in the contractility of the vaginal wall smooth muscles. It is noteworthy that luminal pressure within the vagina may be influenced by changes in muscle contractility within the wall of the vagina and also by changes in activity of the surrounding striated muscle of the pelvic floor and abdominal and rectal pressure. However, it has been demonstrated that the measured increase in wall tension and luminal pressure of the proximal vagina of the rat in response to pelvic nerve stimulation was blocked by atropine, suggesting that striated muscles, at least, are not involved in the noted increase (15). Furthermore, in an earlier study of female sexual arousal, McKenna et al. (31) recorded bursts of electrical activity within the vaginal wall (location unspecified), corresponding to an increase in intravaginal pressure in response to urethral stimulation, providing more direct evidence of vaginal contraction.

Previous studies have demonstrated that phasic muscle such as bladder (48) and intestine (16, 24) contain predominantly the SM-B MHC isoform. In vitro motility assays (12, 24, 40) also have indicated that the presence or relative abundance of a 7-amino acid insert in the motor domain near the ATP binding site of the SM-B isoform (3, 16) is correlated with a high velocity of shortening of smooth muscle. Furthermore, regional differences in MHC isoform expression have also been detected in the vasculature, bladder, and stomach (8, 12, 19, 45). Similarly, we have shown that the proximal vagina, with a greater maximal velocity of shortening, also displays a predominance of the SM-B isoform at the mRNA level, whereas the distal vagina was found to express approximately equal amounts of SM-B and the SM-A isoform (Fig. 7). Furthermore, semiquantitative analysis of densitometric readings of Western blots indicated significantly greater (P < 0.001) amounts of SM-B protein in the proximal vagina compared with the distal vagina (Fig. 6). The greater expression of SM-B in the proximal vagina compared with the distal vagina provides a second line of evidence that the proximal vagina is a phasic tissue, distinct from the tonic qualities of the distal vagina.

SM-1/SM-2 expression in the proximal and distal vagina did not parallel changes in SM-A/SM-B expression. This is in agreement with other investigations, suggesting that the NH2-terminal- and COOH-terminal-based isoforms of myosin are independently regulated (8, 12, 32). We report that the vagina displays a predominance of the SM-1 isoform in both regions of the vagina. Additionally, the percent SM-1 was similar in the proximal (69.41 ± 1.57%) and the distal (71.82 ± 1.24%) vagina. This would suggest that SM-1/SM-2 expression does not contribute to differences in shortening velocity or contractile profile in the vagina. A similar lack of correlation between SM-1/SM-2 and contractile properties has been suggested in other smooth muscle tissues (23, 39, 45). In contrast, a positive relationship between percent SM-1 and Vmax has been reported in the myometrium of estrogen-treated ovariectomized rats (17). Morano et al. (35) demonstrated an increase in expression of myosin light chain 17a and concommitant increase in Vmax in the hypertrophied myometrium of pregnant women. Thus it is possible that the results of Hewett et al. (17) may reflect differences in myosin light chain expression rather than heavy chain differences. SM-1/SM-2 isoform expression is under ovarian hormone control in the myometrium (7, 17, 35, 42) and bladder (44). This information, coupled with the fact that mechanical properties of the vagina are altered by ovariectomy (26), makes it interesting to speculate if SM-1/SM-2 expression is altered by ovarian hormones.

Stimulation of the distal vagina produced a monotonic increase in force to a stable value (107.32 ± 19.66 mN/mm2 muscle). Steady-state force of the distal vagina was significantly higher than that produced by the proximal vagina, which contracted with a high transient peak before falling to a lower stable level (Fig. 4). Vmax of the proximal vagina also transiently increased, followed by a decline of 37% to achieve steady-state levels that were similar to those estimated during contraction of the distal vagina (Fig. 5). Peak force levels were similar during membrane depolarization-induced contractions of the proximal and distal vaginal smooth muscle when normalized to cross-sectional area of muscle. Therefore, if total myosin content were similar, one would not expect that the levels of SM-B or SM-A expression would influence peak force. This is in agreement with studies on purified myosin (29) and Triton X-100 permeabilized strips of bladder smooth muscle from SM-B knockout mice (22), all of which produced similar levels of force, regardless of the myosin isoform. However, in other studies using the same transgenic animals, it has been reported that loss of SM-B reduced maximal force generation of bladder (5), while increasing force generation of mesenteric artery and aorta (6). Differences in tissue preparation and modes of activation may be responsible for these conflicting results. There are a variety of normalizing parameters used to express isometric force. We determined the percent muscle mass of the vaginal tissues and therefore used muscle stress (force/cross-sectional area muscle) rather than the more typical tissue stress (force/cross-sectional area of total tissue). Normalization of force to total cross-sectional area of tissue strip indicated a significantly greater peak force of the proximal vagina, a reflection of the greater abundance of smooth muscle in the proximal vagina (Table 1). Using the normalizing parameter of muscle stress, we found no difference in peak force generation between the proximal and distal vagina, indicating that isoform composition did not influence peak force, as mentioned earlier. This clearly supports the importance of determining percent muscle mass when comparing the contractile properties of two smooth muscles or two areas within a single smooth muscle.

Distinct functional and molecular characteristics of the proximal and distal vaginal wall smooth muscle lend support to the theory of a dual origin of the vagina. It is widely accepted that the upper two-thirds of the vagina arises from the Müllerian ducts, whereas the distal vagina arises from the sinovaginal bulbs of the urogenital sinus (28). However, our findings of the predominance of the SM-B isoform within the proximal vagina compared with the distal does not correlate with the predominance of SM-A isoform expression of the uterus (30, 48), which also arises from the Müllerian ducts. Furthermore, our report of an approximately equal expression of SM-A/SM-B isoform expression in the distal vagina does not correlate with the predominant SM-B isoform expression of the urethra (19). Given that SM-A/SM-B isoform expression has been shown to be developmentally regulated in a tissue-specific manner (49), studies of myosin isoform expression of the vagina at different stages of development are required to more precisely relate vaginal SM-A/SM-B expression to its embryological source. Furthermore, recent research has suggested that the sinovaginal bulbs are actually the caudal tips of the Wolffian ducts and raises the possibility that the embryological pathway of vaginal development may need to be reconsidered (1, 11, 43).

In summary, our data provide a molecular explanation for some of the differences in contractile behavior of the proximal and distal vagina and strongly indicate that the proximal vagina is phasic compared with a tonic distal vagina. Furthermore, the results of this study highlight the inappropriateness of extrapolating data from one region of the vagina to the organ as a whole. Although many researchers emphasize the importance of vaginal relaxation during female sexual arousal, much of the evidence for relaxation stems from results of the distal vagina. The phasic nature of the proximal vagina raises the possibility that there may be a contractile component to the female sexual response, which warrants further investigation.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants P50 DK-052620 and T32-DK007708.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Robert S. Adelstein for the generous gift of MHC antibody, Dr. Yongmu Zheng for providing the SM-B antibody, Xianqun Luan for statistical assistance, and Brian Siano for preparation of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Chacko, 3005 Ravdin-Courtyard, HUP, Univ. of Pennsylvania, 3400 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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Acien P, Acien M, and Sanchez-Ferrer M. Complex malformations of the female genital tract. New types and revision of classification. Hum Reprod 19: 2377–2384, 2004.[Abstract/Free Full Text]
  2. Al-Hijji J, Larsson B, and Batra S. Nitric oxide synthase in the rabbit uterus and vagina: hormonal regulation and functional significance. Biol Reprod 62: 1387–1392, 2000.[Abstract/Free Full Text]
  3. Babij P, Kelly C, and Periasamy M. Characterization of a mammalian smooth muscle myosin heavy-chain gene: complete nucleotide and protein coding sequence and analysis of the 5' end of the gene. Proc Natl Acad Sci USA 88: 10676–10680, 1991.[Abstract/Free Full Text]
  4. Babij P and Periasamy M. Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing. J Mol Biol 210: 673–679, 1989.[CrossRef][ISI][Medline]
  5. Babu GJ, Loukianov E, Loukianova T, Pyne GJ, Huke S, Osol G, Low RB, Paul RJ, and Periasamy M. Loss of SM-B myosin affects muscle shortening velocity and maximal force development. Nat Cell Biol 3: 1025–1029, 2001.[CrossRef][ISI][Medline]
  6. Babu GJ, Pyne GJ, Zhou Y, Okwuchukuasanya C, Brayden JE, Osol G, Paul RJ, Low RB, and Periasamy M. Isoform switching from SM-B to SM-A myosin results in decreased contractility and altered expression of thin filament regulatory proteins. Am J Physiol Cell Physiol 287: C723–C729, 2004.[Abstract/Free Full Text]
  7. Capriani A, Chiavegato A, Franch R, Azzarello G, Vinante O, and Sartore S. Oestrogen-dependent expression of the SM2 smooth muscle-type myosin isoform in rabbit myometrium. J Muscle Res Cell Motil 18: 413–427, 1997.[CrossRef][ISI][Medline]
  8. Disanto ME, Cox RH, Wang Z, and Chacko S. NH2-terminal-inserted myosin II heavy chain is expressed in smooth muscle of small muscular arteries. Am J Physiol Cell Physiol 272: C1532–C1542, 1997.[Abstract/Free Full Text]
  9. Disanto ME, Stein R, Chang S, Hypolite JA, Zheng Y, Zderic S, Wein AJ, and Chacko S. Alteration in expression of myosin isoforms in detrusor smooth muscle following bladder outlet obstruction. Am J Physiol Cell Physiol 285: C1397–C1410, 2003.[Abstract/Free Full Text]
  10. Disanto ME, Wang Z, Menon C, Zheng Y, Chacko T, Hypolite J, Broderick G, Wein AJ, and Chacko S. Expression of myosin isoforms in smooth muscle cells in the corpus cavernosum penis. Am J Physiol Cell Physiol 275: C976–C987, 1998.[Abstract/Free Full Text]
  11. Drews U, Sulak O, and Schenck PA. Androgens and the development of the vagina. Biol Reprod 67: 1353–1359, 2002.[Abstract/Free Full Text]
  12. Eddinger TJ and Meer DP. Single rabbit stomach smooth muscle cell myosin heavy chain SMB expression and shortening velocity. Am J Physiol Cell Physiol 280: C309–C316, 2001.[Abstract/Free Full Text]
  13. Giraldi A, Alm P, Werkstrom V, Myllymaki L, Wagner G, and Andersson KE. Morphological and functional characterization of a rat vaginal smooth muscle sphincter. Int J Impot Res 14: 271–282, 2002.[CrossRef][ISI][Medline]
  14. Giraldi A, Persson K, Werkstrom V, Alm P, Wagner G, and Andersson KE. Effects of diabetes on neurotransmission in rat vaginal smooth muscle. Int J Impot Res 13: 58–66, 2001.[CrossRef][ISI][Medline]
  15. Giuliano F, Allard J, Compagnie S, Alexandre L, Droupy S, and Bernabe J. Vaginal physiological changes in a model of sexual arousal in anesthetized rats. Am J Physiol Regul Integr Comp Physiol 281: R140–R149, 2001.[Abstract/Free Full Text]
  16. Hamada Y, Yanagisawa M, Katsuragawa Y, Coleman JR, Nagata S, Matsuda G, and Masaki T. Distinct vascular and intestinal smooth muscle myosin heavy chain mRNAs are encoded by a single-copy gene in the chicken. Biochem Biophys Res Commun 170: 53–58, 1990.[CrossRef][ISI][Medline]
  17. Hewett TE, Martin AF, and Paul RJ. Correlations between myosin heavy chain isoforms and mechanical parameters in rat myometrium. J Physiol 460: 351–364, 1993.[Abstract/Free Full Text]
  18. Hodge TP, Cross R, and Kendrick-Jones J. Role of the COOH-terminal nonhelical tailpiece in the assembly of a vertebrate nonmuscle myosin rod. J Cell Biol 118: 1085–1095, 1992.[Abstract/Free Full Text]
  19. Hypolite JA, Disanto ME, Zheng Y, Chang S, Wein AJ, and Chacko S. Regional variation in myosin isoforms and phosphorylation at the resting tone in urinary bladder smooth muscle. Am J Physiol Cell Physiol 280: C254–C264, 2001.[Abstract/Free Full Text]
  20. Ikebe M, Hewett TE, Martin AF, Chen M, and Hartshorne DJ. Cleavage of a smooth muscle myosin heavy chain near its C terminus by alpha-chymotrypsin. Effect on the properties of myosin. J Biol Chem 266: 7030–7036, 1991.[Abstract/Free Full Text]
  21. Ishibashi K, Evans A, Shingu T, Bian K, and Bukoski RD. Differential expression and effect of 1,25-dihydroxyvitamin D3 on myosin in arterial tree of rats. Am J Physiol Cell Physiol 269: C443–C450, 1995.[Abstract/Free Full Text]
  22. Karagiannis P, Babu GJ, Periasamy M, and Brozovich FV. Myosin heavy chain isoform expression regulates shortening velocity in smooth muscle: studies using an SMB KO mouse line. J Muscle Res Cell Motil 25: 149–158, 2004.[CrossRef][ISI][Medline]
  23. Kelley CA, Sellers JR, Goldsmith PK, and Adelstein RS. Smooth muscle myosin is composed of homodimeric heavy chains. J Biol Chem 267: 2127–2130, 1992.[Abstract/Free Full Text]
  24. Kelley CA, Takahashi M, Yu JH, and Adelstein RS. An insert of seven amino acids confers functional differences between smooth muscle myosins from the intestines and vasculature. J Biol Chem 268: 12848–12854, 1993.[Abstract/Free Full Text]
  25. Kim NN, Min K, Huang YH, Goldstein I, and Traish AM. Biochemical and functional characterization of alpha-adrenergic receptors in the rabbit vagina. Life Sci 71: 2909–2920, 2002.[CrossRef][ISI][Medline]
  26. Kim NN, Min K, Pessina MA, Munarriz R, Goldstein I, and Traish AM. Effects of ovariectomy and steroid hormones on vaginal smooth muscle contractility. Int J Impot Res 16: 43–50, 2004.[CrossRef][ISI][Medline]
  27. Kim SW, Jeong SJ, Munarriz R, Kim NN, Goldstein I, and Traish AM. Role of the nitric oxide-cyclic GMP pathway in regulation of vaginal blood flow. Int J Impot Res 15: 355–361, 2003.[CrossRef][ISI][Medline]
  28. Koff AK. Development of the vagina in the human fetus. Contrib Embryol 24: 59–91, 1933.[Medline]
  29. Lauzon AM, Tyska MJ, Rovner AS, Freyzon Y, Warshaw DM, and Trybus KM. A 7-amino-acid insert in the heavy chain nucleotide binding loop alters the kinetics of smooth muscle myosin in the laser trap. J Muscle Res Cell Motil 19: 825–837, 1998.[CrossRef][ISI][Medline]
  30. Leguillette R, Gil FR, Zitouni N, Lajoie-Kadoch S, Sobieszek A, and Lauzon AM. (+)Insert smooth muscle myosin heavy chain (SM-B) isoform expression in human tissues. Am J Physiol Cell Physiol 289: C1277–C1285, 2005.[Abstract/Free Full Text]
  31. McKenna KE, Chung SK, and McVary KT. A model for the study of sexual function in anesthetized male and female rats. Am J Physiol Regul Integr Comp Physiol 261: R1276–R1285, 1991.[Abstract/Free Full Text]
  32. Meer DP and Eddinger TJ. Expression of smooth muscle myosin heavy chains and unloaded shortening in single smooth muscle cells. Am J Physiol Cell Physiol 273: C1259–C1266, 1997.[Abstract/Free Full Text]
  33. Min K, Kim NN, McAuley I, Stankowicz M, Goldstein I, and Traish AM. Sildenafil augments pelvic nerve-mediated female genital sexual arousal in the anesthetized rabbit. Int J Impot Res 12, Suppl 3: S32–S39, 2000.
  34. Min K, Munarriz R, Berman J, Kim NN, Goldstein I, Traish AM, and Stankovic MR. Hemodynamic evaluation of the female sexual arousal response in an animal model. J Sex Marital Ther 27: 557–565, 2001.[ISI][Medline]
  35. Morano I, Koehlen S, Haase H, Erb G, Baltas LG, Rimbach S, Wallwiener D, and Bastert G. Alternative splicing and cycling kinetics of myosin change during hypertrophy of human smooth muscle cells. J Cell Biochem 64: 171–181, 1997.[CrossRef][ISI][Medline]
  36. Nagai R, Kuro-o M, Babij P, and Periasamy M. Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis. J Biol Chem 264: 9734–9737, 1989.[Abstract/Free Full Text]
  37. Oh SJ, Hong SK, Kim SW, and Paick JS. Histological and functional aspects of different regions of the rabbit vagina. Int J Impot Res 15: 142–150, 2003.[CrossRef][ISI][Medline]
  38. Park K, Goldstein I, Andry C, Siroky MB, Krane RJ, and Azadzoi KM. Vasculogenic female sexual dysfunction: the hemodynamic basis for vaginal engorgement insufficiency and clitoral erectile insufficiency. Int J Impot Res 9: 27–37, 1997.[CrossRef][ISI][Medline]
  39. Rovner AS, Fagnant PM, Lowey S, and Trybus KM. The carboxyl-terminal isoforms of smooth muscle myosin heavy chain determine thick filament assembly properties. J Cell Biol 156: 113–123, 2002.[Abstract/Free Full Text]
  40. Rovner AS, Freyzon Y, and Trybus KM. An insert in the motor domain determines the functional properties of expressed smooth muscle myosin isoforms. J Muscle Res Cell Motil 18: 103–110, 1997.[CrossRef][ISI][Medline]
  41. Rovner AS, Thompson MM, and Murphy RA. Two different heavy chains are found in smooth muscle myosin. Am J Physiol Cell Physiol 250: C861–C870, 1986.[Abstract/Free Full Text]
  42. Sakurai H, Imamura S, Furutani Y, Takao A, Momma K, and Matsuoka R. Unique expression patterns of myosin heavy chain genes in the ductus arteriosus and uterus of rabbits. J Vet Med Sci 61: 1049–1054, 1999.[CrossRef][ISI][Medline]
  43. Sanchez-Ferrer ML, Acien MI, Del Campo FS, Mayol-Belda MJ, and Acien P. Experimental contributions to the study of the embryology of the vagina. Hum Reprod 21: 1623–1628, 2006.[Abstract/Free Full Text]
  44. Sanchez-Ortiz RF, Wang Z, Menon C, Disanto ME, Wein AJ, and Chacko S. Estrogen modulates the expression of myosin heavy chain in detrusor smooth muscle. Am J Physiol Cell Physiol 280: C433–C440, 2001.[Abstract/Free Full Text]
  45. Sherwood JJ and Eddinger TJ. Shortening velocity and myosin heavy- and light-chain isoform mRNA in rabbit arterial smooth muscle cells. Am J Physiol Cell Physiol 282: C1093–C1102, 2002.[Abstract/Free Full Text]
  46. Sparrow MP, Mohammad MA, Arner A, Hellstrand P, and Ruegg JC. Myosin composition and functional properties of smooth muscle from the uterus of pregnant and non-pregnant rats. Pflügers Arch 412: 624–633, 1988.[CrossRef][ISI][Medline]
  47. Traish AM, Kim NN, Huang YH, Min K, Munarriz R, and Goldstein I. Sex steroid hormones differentially regulate nitric oxide synthase and arginase activities in the proximal and distal rabbit vagina. Int J Impot Res 15: 397–404, 2003.[CrossRef][ISI][Medline]
  48. White S, Martin AF, and Periasamy M. Identification of a novel smooth muscle myosin heavy chain cDNA: isoform diversity in the S1 head region. Am J Physiol Cell Physiol 264: C1252–C1258, 1993.[Abstract/Free Full Text]
  49. White SL, Zhou MY, Low RB, and Periasamy M. Myosin heavy chain isoform expression in rat smooth muscle development. Am J Physiol Cell Physiol 275: C581–C589, 1998.[Abstract/Free Full Text]
  50. Ziessen T, Moncada S, and Cellek S. Characterization of the non-nitrergic NANC relaxation responses in the rabbit vaginal wall. Br J Pharmacol 135: 546–554, 2002.[CrossRef][ISI]




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