Blebbistain, a myosin II inhibitor, as a novel strategy to regulate detrusor contractility in a rat model of partial bladder outlet obstruction

Partial bladder outlet obstruction (PBOO), a common urologic pathology mostly caused by benign prostatic hyperplasia, can coexist in 40-45% of patients with overactive bladder (OAB) and is associated with detrusor overactivity (DO). PBOO that induces DO results in alteration in bladder myosin II type and isoform composition. Blebbistatin (BLEB) is a myosin II inhibitor we recently demonstrated potently relaxed normal detrusor smooth muscle (SM) and reports suggest varied BLEB efficacy for different SM myosin (SMM) isoforms and/or SMM vs nonmuscle myosin (NMM). We hypothesize BLEB inhibition of myosin II as a novel contraction protein targeted strategy to regulate DO. Using a surgically-induced male rat PBOO model, organ bath contractility, competitive and Real-Time-RT-PCR were performed. It was found that obstructed-bladder weight significantly increased 2.74-fold while in vitro contractility of detrusor to various stimuli was impaired ～50% along with decreased shortening velocity. Obstruction also altered detrusor spontaneous activities with significantly increased amplitude but depressed frequency. PBOO switched bladder from a phasic-type to a more tonic-type SM. Expression of 5' myosin heavy chain (MHC) alternatively spliced isoform SM-A (associated with tonic-type SM) increased 3-fold while 3' MHC SM1 and essential light chain isoform MLC(17b) also exhibited increased relative expression. Total SMMHC expression was decreased by 25% while the expression of NMM IIB (SMemb) was greatly increased by 4.5-fold. BLEB was found to completely relax detrusor strips from both sham-operated and PBOO rats pre-contracted with KCl, carbachol or electrical field stimulation although sensitivity was slightly decreased (20%) only at lower doses for PBOO. Thus we provide the first thorough characterization of the response of rat bladder myosin to PBOO and demonstrate complete BLEB-induced PBOO bladder SM relaxation. Furthermore, the present study provides valuable evidence that BLEB may be a novel type of potential therapeutic agent for regulation of myogenic and nerve-evoked DO in OAB.     

The expression and functional activities of smooth muscle myosin and non‐muscle myosin isoforms in rat prostate

Benign prostatic hyperplasia (BPH) is mainly caused by increased prostatic smooth muscle (SM) tone and volume. SM myosin (SMM) and non‐muscle myosin (NMM) play important roles in mediating SM tone and cell proliferation, but these molecules have been less studied in the prostate. Rat prostate and cultured primary human prostate SM and epithelial cells were utilized. In vitro organ bath studies were performed to explore contractility of rat prostate. SMM isoforms, including SM myosin heavy chain (MHC) isoforms (SM1/2 and SM‐A/B) and myosin light chain 17 isoforms (LC_17a/b), and isoform ratios were determined via competitive RT‐PCR. SM MHC and NM MHC isoforms (NMMHC‐A, NMMHC‐B and NMMHC‐C) were further analysed via Western blotting and immunofluorescence microscopy. Prostatic SM generated significant force induced by phenylephrine with an intermediate tonicity between phasic bladder and tonic aorta type contractility. Correlating with this kind of intermediate tonicity, rat prostate mainly expressed LC_17a and SM1 but with relatively equal expression of SM‐A/SM‐B at the mRNA level. Meanwhile, isoforms of NMMHC‐A, B, C were also abundantly present in rat prostate with SMM present only in the stroma, while NMMHC‐A, B, C were present both in the stroma and endothelial. Additionally, the SMM selective inhibitor blebbistatin could potently relax phenylephrine pre‐contracted prostate SM. In conclusion, our novel data demonstrated the expression and functional activities of SMM and NMM isoforms in the rat prostate. It is suggested that the isoforms of SMM and NMM could play important roles in BPH development and bladder outlet obstruction.     

Smooth muscle myosin isoform expression and LC20 phosphorylation in innate rat airway hyperresponsiveness

Four smooth muscle myosin heavy chain (SMMHC) isoforms are generated by alternative mRNA splicing of a single gene. Two of these isoforms differ by the presence [(+)insert] or absence [(-)insert] of a 7-amino acid insert in the motor domain. The rate of actin filament propulsion of the (+)insert SMMHC isoform, as measured in the in vitro motility assay, is twofold greater than that of the (-)insert isoform. We hypothesized that a greater expression of the (+)insert SMMHC isoform and greater regulatory light chain (LC(20)) phosphorylation contribute to airway hyperresponsiveness. We measured airway responsiveness to methacholine in Fischer hyperresponsive and Lewis normoresponsive rats and determined SMMHC isoform mRNA and protein expression, as well as essential light chain (LC(17)) isoforms, h-caldesmon, and alpha-actin protein expression in their tracheae. We also measured tracheal muscle strip contractility in response to methacholine and corresponding LC(20) phosphorylation. We found Fischer rats have more (+)insert mRNA (69.4 +/- 2.0%) (mean +/- SE) than Lewis rats (53.0 +/- 2.4%; P < 0.05) and a 44% greater content of (+)insert isoform relative to total myosin protein. No difference was found for LC(17) isoform, h-caldesmon, and alpha-actin expression. The contractility experiments revealed a greater isometric force for Fischer trachealis segments (4.2 +/- 0.8 mN) than Lewis (1.9 +/- 0.4 mN; P < 0.05) and greater LC(20) phosphorylation level in Fischer (55.1 +/- 6.4) than in Lewis (41.4 +/- 6.1; P < 0.05) rats. These results further support the contention that innate airway hyperresponsiveness is a multifactorial disorder in which increased expression of the fast (+)insert SMMHC isoform and greater activation of LC(20) lead to smooth muscle hypercontractility.     

Increased corpus cavernosum smooth muscle tone associated with partial bladder outlet obstruction is mediated via Rho-kinase

Numerous studies have now demonstrated that lower urinary tract symptoms (LUTS) are associated with erectile dysfunction (ED) in men independent of age or general health. Because one-third of men over the age of 50 will develop LUTS and a recent study showed ED in 62% of patients presenting for LUTS, the importance of determining the mechanistic link between these two pathologies is clear. Using a rabbit model of partial bladder outlet obstruction (PBOO), a primary cause of LUTS, we have identified an increased basal corpus cavernosum smooth muscle (CCSM) tone associated with an elevated level of smooth muscle myosin (SMM) phosphorylation in PBOO compared with sham-operated control rabbits (sham). Results from in vitro kinase and phosphatase assays using purified smooth muscle myosin showed increased kinase and decreased phosphatase activities in cellular extracts from corpora cavernosa isolated from PBOO compared with sham rabbits. Increased Rho-kinase expression in the CCSM of PBOO rabbits was suggested by the observations that Rho-kinase inhibitors attenuated the increased kinase activity and were less effective in relaxing CCSM strips from PBOO vs. sham rabbits. This hypothesis was then confirmed by RT-PCR and Western blotting, which demonstrated increased expression of both isoforms of Rho-kinase (ROKalpha and ROKbeta). Increased SMM basal phosphorylation (necessary for SM contraction) in the CCSM of PBOO rabbits, mediated via an increase in Rho-kinase expression/activity, would be expected to make the CCSM more difficult to relax (necessary for erection), which suggests that the RhoA/Rho-kinase pathway as being involved in the mechanism for LUTS-associated ED.     

cDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in arteriosclerosis

Adult rabbit smooth muscles contain two types of myosin heavy chain (MHC) isoforms, SM1 and SM2 which are generated through alternative RNA splicing from a single gene (Nagai, R., Kuro-o, M., Babij, P. & Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). We previously reported that the expression of SM1 and SM2 during vascular development is differentially regulated at the level of RNA splicing, whereby SM1 is constitutively expressed from early development but SM2 appear after birth (Kuro-o, M., Nagai, R., Tsuchimochi, H., Katoh, H., Yazaki, Y., Ohkubo, A. & Takaku, F. (1989) J. Biol. Chem. 264, 18272-18275). We also demonstrated that embryonic vascular smooth muscles contain a third type of MHC isoform, referred to as SMemb in this report, which comigrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with SM2. In the present study we have isolated and characterized a cDNA clone (FSMHC34) for SMemb. FSMHC34 encodes the light meromyosin region including the carboxyl terminus and showed 70% amino acid sequence identity with SM1 or SM2. SMemb is a nonmuscle-type MHC and identical with brain MHC, but clearly distinct from 196-kDa nonmuscle MHC in cultured smooth muscle cells. The expression of SMemb was predominant in embryonic and perinatal aortas, but down-regulated with vascular development. Interestingly SMemb was reexpressed in proliferating smooth muscle cells of arteriosclerotic neointimas. These results suggest that smooth muscle proliferation is coupled to the expression of SMemb and that dedifferentiation of smooth muscles toward the embryonic phenotype is involved in the mechanisms underlying atherosclerosis.     

Effects of sex and estrogen on myosin COOH-terminal isoforms and contractility in rat aorta

We reported that estrogen treatment of ovariectomized rats increased uterine smooth muscle contractility and the ratio of the COOH-terminal myosin heavy chain isoform SM1 (204 kDa) and SM2 [200 kDa; Hewett TE, Martin AF, Paul RJ. J Physiol (Lond) 460: 351-364, 1993]. We extended this model to study sex and estrogen effects on vascular contractility. Experimental groups included 10- to 14-wk-old male (M), female (F), ovariectomized female (OF), and OF treated with estrogen (OF&E) for 7 days with a subcutaneous pellet delivery system, resulting in 17beta-estradiol of 85 (OF&E) vs. 5 (OF or M) pg/ml. The SM1-to-SM2 ratio increased from 1.8 to 2.6 in thoracic aorta, similar to uterine muscle. Isometric force was measured in 5-mm segments of intact and endothelium-denuded (-endo) aorta. With KCl, the maximum forces were in the order OF approximately M > OF&E, and ED50 OF&E > OF approximately M. Differences in ED50 with estrogen persisted after endothelial denudation. The decreased force in -endo OF aorta was not seen in OF&E, suggesting that estrogen altered an endothelium-dependent effect. No differences in maximum forces were noted with norepinephrine: ED50 OF > OF&E > M. Estrogen treatment, in contrast to KCl, increased sensitivity. Endothelial denudation increased sensitivity but reduced the differences between groups. With ACh relaxation, males were more sensitive than females, and estrogen had no effect. In the abdominal aorta, there were no changes in SM1/SM2 with 17beta-estradiol, and differences in contractility were blunted. In summary, estrogen treatment decreased responses to KCl but increased sensitivity to norepinephrine; male rats always demonstrated the highest contractility. An increase in the COOH-terminal myosin heavy chain isoform SM1-to-SM2 ratio with 17beta-estradiol treatment may underlie the changes observed in contractility.     

Determinants of the contractile properties in the embryonic chicken gizzard and aorta

Smooth muscle is generally grouped into two classes of differing contractile properties. Tonic smooth muscles show slow rates of force activation and relaxation and slow speeds of shortening (V(max)) but force maintenance, whereas phasic smooth muscles show poor force maintenance but have fast V(max) and rapid rates of force activation and relaxation. We characterized the development of gizzard and aortic smooth muscle in embryonic chicks to identify the cellular determinants that define phasic (gizzard) and tonic (aortic) contractile properties. Early during development, tonic contractile properties are the default for both tissues. The gizzard develops phasic contractile properties between embryonic days (ED) 12 and 20, characterized primarily by rapid rates of force activation and relaxation compared with the aorta. The rapid rate of force activation correlates with expression of the acidic isoform of the 17-kDa essential myosin light chain (MLC(17a)). Previous data from in vitro motility assays (Rover AS, Frezon Y, and Trybus KM. J Muscle Res Cell Motil 18: 103-110, 1997) have postulated that myosin heavy chain (MHC) isoform expression is a determinant for V(max) in intact tissues. In the current study, differences in V(max) did not correlate with previously published differences in MHC or MLC(17a) isoforms. Rather, V(max) was increased with thiophosphorylation of the 20-kDa regulatory myosin light chain (MLC(20)) in the gizzard, suggesting that a significant internal load exists. Furthermore, V(max) in the gizzard increased during postnatal development without changes in MHC or MLC(17) isoforms. Although the rate of MLC(20) phosphorylation was similar at ED 20, the rate of MLC(20) dephosphorylation was significantly higher in the gizzard versus the aorta, correlating with expression of the M130 isoform of the myosin binding subunit in the myosin light chain phosphatase (MLCP) holoenzyme. These results indicate that unique MLCP and MLC(17) isoform expression marks the phasic contractile phenotype.     

Akt1 isoform modulates phenotypic conversion of vascular smooth muscle cells

In this study, we investigated the role of Akt1 isoform in phenotypic change of vascular smooth muscle cells (VSMCs) and neointima formation. Laminin-induced conversion of synthetic VSMCs into contractile VSMCs was measured by expression of marker proteins for contractile VSMCs and collagen gel contraction assay. Culture of synthetic VSMCs on laminin-coated plates induced expression of marker proteins for contractile VSMCs and showed contraction in response to angiotensin II (AngII) stimulation. Silencing integrin-linked kinase attenuated activation of Akt and blocked phenotypic conversion of VSMCs resulting in the loss of AngII-dependent contraction. Laminin-induced phenotypic conversion of VSMCs was abrogated by phosphatidylinositol 3-kinase inhibitor or in cells silencing Akt1 but not Akt2. Proliferation of contractile VSMCs on laminin-coated plate was enhanced in cells silencing Akt1 whereas silencing Akt2 did not affect. Promoter activity of myocardin and SM22α was enhanced in contractile phenotype and overexpression of myocardin stimulated promoter activity of SM22α in synthetic phenotype. Promoter activity of myocardin and SM22α was reduced in cells silencing Akt1 and promoter activity of SM22α was restored by overexpression of myocardin in cells silencing Akt1. However, silencing of Akt2 affected neither promoter activity of myocardin nor SM22α. Finally, neointima formation in carotid artery ligation and high fat-diet-induced atherosclerosis was facilitated in mice lacking Akt1. This study demonstrates that Akt1 isoform stimulates laminin-induced phenotypic conversion of synthetic VSMCs by regulating the expression of myocardin. VSMCs become susceptible to shifting from contractile to synthetic phenotype by the loss of Akt1 in pathological conditions.     

Myosin II isoforms in smooth muscle: heterogeneity and function

Both smooth muscle (SM) and nonmuscle class II myosin molecules are expressed in SM tissues comprising hollow organ systems. Individual SM cells may express one or more of multiple myosin II isoforms that differ in myosin heavy chain (MHC) and myosin light chain (MLC) subunits. Although much has been learned, the expression profiles, organization within contractile filaments, localization within cells, and precise roles in various contractile functions of these different myosin molecules are still not well understood. However, data supporting unique physiological roles for certain isoforms continues to build. Isoform differences located in the S1 head region of the MHC can alter actin binding and rates of ATP hydrolysis. Differences located in the MHC tail can alter the formation, stability, and size of the myosin thick filament. In these distinct ways, both head and tail isoform differences can alter force generation and muscle shortening velocities. The MLCs that are associated with the lever arm of the S1 head can affect the flexibility and range of motion of this domain and possibly the motion of the S2 and motor domains. Phosphorylation of MLC(20) has been associated with conformational changes in the S1 and/or S2 fragments regulating enzymatic activity of the entire myosin molecule. A challenge for the future will be delineation of the physiological significance of the heterogeneous expression of these isoforms in developmental, tissue-specific, and species-specific patterns and or the intra- and intercellular heterogeneity of myosin isoform expression in SM cells of a given organ.     

Maternal dexamethasone treatment alters myosin isoform expression and contractile dynamics in fetal arteries

We tested the hypothesis that maternal glucocorticoid treatment modulates 17-kDa myosin light chain (myosin LC17) isoform expression and contractile dynamics in fetal ovine carotid arteries. In the single course group, ewes received 6 mg dexamethasone or placebo over 48 h. In the repeated course group, ewes received 6 mg dexamethasone or placebo weekly for 5 wk. In response to 1 microM phenylephrine, arteries from fetuses of dexamethasone-treated ewes exhibited biphasic contractions, characterized by an intermediate relaxation phase. The relaxation rate constant was significantly higher in arteries from the fetuses of dexamethasone than placebo-treated ewes. The observed biphasic contractions suggest the appearance of functional sarcoplasmic reticulum in the arteries from the fetuses of dexamethasone-treated ewes. The myosin LC17(a) isoform expression was lower in the arteries from the fetuses of the placebo-treated ewes than in those from the ewes. Repeated maternal administration of dexamethasone induced an almost twofold increase in myosin LC17(a) isoform expression in the fetal arteries. In contrast, maternal myosin LC17a isoform expression was not affected by dexamethasone treatment. We speculate that dexamethasone-induced increases in fetal myosin LC17(a) isoform expression represent accelerated differentiation of a subpopulation of vascular smooth muscle cells from the fetal to adult phenotype.     

Differences in contractile protein content and isoforms in phasic and tonic smooth muscles

The basis of tonic vs. phasic contractile phenotypes of visceralsmooth muscles is poorly understood. We used gel electrophoresis andquantitative scanning densitometry to measure the content and isoformcomposition of contractile proteins in opossum lower esophagealsphincter (LES), to represent tonic muscle, and circular muscle of theesophageal body (EB), to represent phasic smooth muscle. The amount ofprotein in these two types of muscles is similar: ~27 mg/g of frozentissue. There is no difference in the relative proportion of myosin,actin, calponin, and tropomyosin in the two muscle types. However, theEB contains ~2.4-times more caldesmon than the LES. The relativeratios of - to -contractile isoforms of actin are 0.9 in the LESand 0.3 in EB. The ratio between acidic (LC17a) and basic (LC17b)isoforms of the 17-kDa essential light chain of myosin is 0.7:1 in theLES, compared with 2.7:1 in the EB. There is no significant differencein the ratios of smooth muscle myosin SM1 and SM2 isoforms in the two muscle types. The level of the myosin heavy chain isoform, which contains the seven-amino acid insert in the myosin head, is about threefold higher in the EB compared with LES. In conclusion, the esophageal phasic muscle in contrast to the tonic LES contains proportionally more caldesmon, LC17a, and seven-amino acid-inserted myosin and proportionally less -actin. These differences may providea basis for functional differences between tonic and phasic smoothmuscles.

     

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.     

Heterogeneity in smooth muscle cell population accumulating in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery.

Rabbit smooth muscles contain at least three types of myosin heavy chain (MHC) isoforms; SM1, SM2 and SMemb (NMHC-B), the expression of which is developmentally regulated. We have recently reported that smooth muscles with the embryonic phenotype accumulate in the neointimas produced by endothelial denudation or high-cholesterol feeding. In this study, we examined MHC isoform expression in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery, and determined the phenotype of the smooth muscle cell in the dilated segment. We report here that neointimal cells in the dilated segment are smooth muscle cells with the embryonic phenotype as previously reported in our ballooning-injury study. The medial smooth muscles, however, are composed of heterogeneous population of smooth muscles which differ in stage of differentiation as determined by the MHC isoform expression. These results indicate that MHC isoforms are useful molecular markers to identify abnormally proliferating smooth muscles in diseased arteries and to understand the process of atherogenesis occurring following vascular injury.     

Developmentally regulated expression of vascular smooth muscle myosin heavy chain isoforms

Two types of smooth muscle myosin heavy chain (MHC) isoforms, SM1 and SM2, were recently identified to have different carboxyl termini (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). SM1 and SM2 are considered to be generated from a single gene through alternative RNA splicing. In this study we investigated expression of vascular MHC isoforms during development in rabbits at the mRNA, protein, and histological levels. In adults, all smooth muscle cells reacted with both anti-SM1 and anti-SM2 antibodies on immunofluorescence, suggesting the coexpression of SM1 and SM2 in a single cell. In fetal and perinatal rabbits, however, only anti-SM1 antibody consistently reacted with smooth muscles. Reactivity with anti-SM2 antibody was negative in the fetal and neonatal blood vessels and gradually increased during 30 days after birth. These developmental changes in SM1 and SM2 expression at the histological level coincided with mRNA expression of each MHC isoform as determined by S1 nuclease mapping, indicating that expression of SM1 and SM2 is controlled at the level of RNA splicing. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin from fetal and perinatal aortas revealed the presence of large amount of SM2. Interestingly, fetal SM2 did not cross-react with our anti-SM2 antibody on immunoblotting. We conclude that expression of SM1 and SM2 are differentially regulated during development and that a third type of MHC isoform may exist in embryonic and perinatal vascular smooth muscles.     

Effects of electrically induced contractile activity on cultured embryonic chick breast muscle cells

Development of chicken breast muscle is characterized by the sequential appearance of six electrophoretically distinct myosin heavy chain (HC) isoforms. Cultured secondary myotubes, derived from 12-day embryonic chick breast muscle, mainly express the early embryonic HC isoform HCemb/e, normally present in 8-day embryonic breast muscle, and the two fast light chain isoforms LC1f and LC2f. Direct low-frequency (2.5 Hz) stimulation of these myotubes via platinum electrodes leads to a shift in myosin HC expression with increases in the late embryonic HC isoform HCemb/l amounting to 35% of total HC in 19-day-stimulated cultures. Measurements of 35S-methionine incorporation and immunohistochemical analyses demonstrate increases in LC3f. This increase is also seen at the mRNA level. These results indicate that induced contractile activity promotes myotube maturation in vitro. The observation that chronic stimulation enhances the expression of the slow isoform LC2s at the RNA, as well as the protein level, suggests an additional effect consisting of a fast-to-slow change in phenotype expression. In view of the fact that muscle maturation and phenotype expression is under neural control during development in vivo, our results on directly stimulated, aneural myotubes indicate that neurally transmitted contractile activity may be an important factor in modulating phenotype expression of secondary myotubes.     

Shortening velocity and myosin heavy- and light-chain isoform mRNA in rabbit arterial smooth muscle cells

In smooth muscle cells (SMCs)isolated from rabbit carotid, femoral, and saphenous arteries, relativemyosin isoform mRNA levels were measured in RT-PCR to test forcorrelations between myosin isoform expression and unloaded shorteningvelocity. Unloaded shortening velocity and percent smooth muscle myosinheavy chain 2 (SM2) and myosin light chain 17b(MLC_17b) mRNA levels were not significantly different insingle SMCs isolated from the luminal and adluminal regions of thecarotid media. Saphenous artery SMCs shortened significantly faster(P < 0.05) than femoral SMCs and had more SM2 mRNA(P < 0.05) than carotid SMCs and lessMLC_17b mRNA (P < 0.001) and higher tissuelevels of SMB mRNA (P < 0.05) than carotid and femoralSMCs. No correlations were found between percent SM2 and percentMLC_17b mRNA levels and unloaded shortening velocity in SMCsfrom these arteries. We have previously shown that myosin heavy chain(MHC) SM1/SM2 and SMA/SMB and MLC_17a/MLC_17b isoform mRNA levels correlate with protein expression for these isoforms in rabbit smooth muscle tissues. Thus we interpret these results to suggest that 1) SMC myosin isoform expression andunloaded shortening velocity do not vary with distance from the lumenof the carotid artery but do vary in arteries located longitudinally within the arterial tree, 2) MHC SM1/SM2 and/orMLC_17a/MLC_17b isoform expression does notcorrelate with unloaded shortening velocity, and 3)intracellular expression of the MHC SM1/SM2 and MLC_17a/MLC_17b isoforms is not coregulated.

     

Time course of soleus muscle myosin expression during hindlimb suspension and recovery

This study examined the time course of adult rodent soleus muscle myofibril and myosin isoform protein expression after 4, 8, 16, 28, and 56 days of hindlimb unweighting by tail suspension (S). The time course of soleus muscle recovery (R) was also examined after 28 days of hindlimb unweighting with an additional 4, 8, 16, and 28 days of unrestricted cage activity. During suspension, soleus muscle myofibril protein rapidly decreased from 34.3 +/- 3.1 (1.96SE) mg/pair in the control (C) group to 6.9 +/- 1.4 (1.96SE) mg/pair in S (t = 56 days). The calculated first-order degradation rate constant for this loss was kd = 0.17 days-1 [half time (t1/2) = 4.1 days]. The estimated slow myosin (SM) isoform content decreased from 13.4 +/- 2.0 (1.96SE) mg/pair in C to 2.1 +/- 0.2 (1.96SE) mg/pair in S (kd = 0.19 days-1, t1/2 = 3.6 days). The relative proportion of other myosin isoforms was increased at 28 and 56 days of suspension, reflecting an apparent de novo synthesis and the loss of SM. Recovery of contractile protein after 28 days of suspension was slower for both the myofibril protein and the SM isoform (kd = 0.07 days-1, t1/2 = 10 days). These data suggest that loss of weight bearing specifically affected the mechanisms of contractile protein expression reflected in soleus muscle protein degradation processes. In addition, the expression of the myosin isoforms were apparently differentially affected by the loss of weight-bearing activity.     

Testosterone regulates the expression and functional activity of sphingosine‐1‐phosphate receptors in the rat corpus cavernosum

The bioactive lipid sphingosine‐1‐phosphate (S1P) regulates smooth muscle (SM) contractility predominantly via three G protein‐coupled receptors. The S1P1 receptor is associated with nitric oxide (NO)‐mediated SM relaxation, while S1P2 & S1P3 receptors are linked to SM contraction via activation of the Rho‐kinase pathway. This study is to determine testosterone (T) modulating the expression and functional activity of S1P receptors in corpus cavernosum (CC). Adult male Sprague‐Dawley rats were randomly divided into three groups: sham‐operated controls, surgical castration and T supplemented group. Serum S1P levels were detected by high‐performance liquid chromatography. The expression of S1P1‐3 receptors and sphingosine kinases was detected by real‐time RT‐PCR. In vitro organ bath contractility and in vivo intracavernous pressure (ICP) measurement were also performed. T deprivation significantly decreased ICP rise. Meanwhile, surgical castration induced a significant increase in serum S1P level and the expression of S1P2‐3 receptors by twofold (P < 0.05) but a decrease in the expression of S1P1 receptor. Castration also augmented exogenous phenylephrine (PE), S1P, S1P1,3 receptor agonist FTY720‐P contractility and S1P2‐specific antagonist JTE013 relaxation effect. T supplemented could restore the aforementioned changes. We provide novel data that castration increased serum S1P concentration and up‐regulated the expression of S1P2‐3 receptors in CC. Consistently, agonizing S1P receptors induced CCSM contraction and antagonizing mediated relaxation were augmented. This provides the first clear evidence that S1P system dysregulation may contribute to hypogonadism‐related erectile dysfunction (ED), and S1P receptors may be expected as a potential target for treating ED.