Galectin-1 inhibits PDGF-BB-induced proliferation and migration of airway smooth muscle cells through the inactivation of PI3K/Akt signaling pathway.

Childhood asthma is one of the most common chronic childhood diseases. Platelet-derived growth factor BB (PDGF-BB) induced airway smooth muscle cell (ASMC) proliferation and migration are involved in the pathogenesis of asthma. Galectin-1 (Gal-1) is a glycan-binding protein that has been found to be involved in the progression of asthma. However, the mechanism remains unclear. In the current study, we aimed to evaluate the role of Gal-1 in regulating the phenotype switching of ASMCs, which is an important mechanism in the pathogenesis of asthma. Our results showed that Gal-1 was markedly down-regulated in the samples from asthma patients. In vitro study also proved that Gal-1 expression was decreased in PDGF-BB-stimulated ASMCs. In addition, Gal-1 overexpression significantly inhibited PDGF-BB-induced ASMCs proliferation and migration, while Gal-1 knockdown exhibits opposite effects of Gal-1 overexpression. The PDGF-BB-caused reductions in expressions of α-smooth muscle actin (α-SMA), specific muscle myosin heavy chain (SM-MHC), and calponin were elevated by Gal-1 overexpression, but were deteriorated by Gal-1 knockdown in ASMCs. Furthermore, overexpression of Gal-1 inhibited PDGF-BB-stimulated PI3K/Akt activation in ASMCs. Notably, treatment with IGF-1, an activator of PI3K, reversed the effects of Gal-1 on ASMCs proliferation, migration, and phenotype switching. In conclusion, these findings showed that Gal-1 exerted inhibitory effects on PDGF-BB-stimulated proliferation, migration, and phenotype switching of ASMCs via inhibiting the PI3K/Akt signaling pathway. Thus, Gal-1 might be a promising target for the treatment of asthma.

The STK16 inhibitor STK16-IN-1 inhibits non-adrenergic and non-neurogenic smooth muscle contractions in the human prostate and the human male detrusor.

Mixed lower urinary tract symptoms (LUTS) (voiding symptoms suggestive of benign prostatic hyperplasia plus storage symptoms, which can be caused by overactive bladder) are common in men. Unwanted contraction of prostate and/or bladder smooth muscle has been implied in the pathophysiology of male LUTS. Here, we examined effects of the serine/threonine kinase 16 (STK16) inhibitor STK16-IN-1 on contraction of human tissues from the prostate and male detrusor. Tissues were obtained from radical prostatectomy and radical cystectomy. Contractions were studied in an organ bath and STK16 expressions by Western blot analyses and fluorescence staining. In prostate tissues, STK16-IN-1 (1 µM) inhibited contractions induced by endothelin-1 and the thromboxane A2 analog U46619. Contractions of prostate tissues induced by noradrenaline, the α1-agonists phenylephrine and methoxamine, or electric field stimulation (EFS) were not changed by STK16-IN-1. In male detrusor tissues, STK16-IN-1 inhibited contractions induced by the cholinergic agonists carbachol and metacholine, and contractions induced by U46619. EFS-induced contractions of detrusor tissues were not changed by STK16-IN-1. Western blot analyses of prostate and detrusor tissues revealed bands matching the molecular weight of STK16. Fluorescence staining of prostate tissues using STK16 antibodies resulted in immunoreactivity in smooth muscle cells. STK16-IN-1 selectively inhibits non-adrenergic/non-neurogenic smooth muscle contractions in the male prostate and to limited extent in the bladder. Because non-adrenergic contractions in the male LUTS may account for limited efficacy of α1-blockers and for α1-blocker-resistant symptoms, studies assessing add-on of STK16-IN-1 to α1-blockers in mixed LUTS appear feasible.

Inhibition of prostatic smooth muscle contraction by the inhibitor of G protein-coupled receptor kinase 2/3, CMPD101.

Alpha1-adrenoceptors induce prostate smooth muscle contraction, and hold a prominent role for pathophysiology and therapy of lower urinary tract symptoms in benign prostatic hyperplasia. G protein-coupled receptors are regulated by posttranslational regulation, including phosphorylation by G protein-coupled receptor kinases 2 and 3 (GRK2/3). Although posttranslational adrenoceptor regulation has been recently suggested to occur in the prostate, this is still marginally understood. With the newly developed CMPD101, a small molecule inhibitor with assumed specificity for GRK2/3 is now available. Here, we studied effects of CMPD101 on smooth muscle contraction of human prostate tissue. Electric field stimulation caused frequency-dependent contractions, which were inhibited concentration-dependently by CMPD101 (5 µM, 50 µM). 50 µM of CMPD101 did not affect myosin light chain (MCL) phosphorylation or Rho kinase activity, and did not alter contractions induced by highmolar KCl. Noradrenaline, the α1-adrenoceptor agonist phenylephrine, endothelin-1, and the thromboxane A2 analogue U46619 induced concentration-dependent contractions, which were inhibited by CMPD101 (50 µM). CMPD101 (50 µM) did not change phosphorylation of ß2-adrenoceptors or ß2-adrenergic relaxation of prostate strips. Molecular detection by Western blot and peroxidase staining suggested expression of GRK2 and GRK3 in human prostates. Double labeling in fluorescence staining confirmed that immunoreactivity for GRK2 and GRK3 was located to smooth muscle cells in the prostate stroma. In conclusion, CMPD101 inhibits adrenergic, neurogenic, and non-adrenergic smooth muscle contractions in the human prostate. Underlying mechanisms may be independent from GRK inhibition, and from inhibition of MLC kinase and Rho kinase. This may point to unknown properties of CMPD101.

Effects of Red Wine Vinegar Beverage on the Colonic Tissue of Rodents: Biochemical, Functional and Pharmacological Analyses.

A beverage made of red wine vinegar and grape juice (Yamanashi-no-megumi™) was developed as a supplemental fluid containing polyphenols, which has been clinically shown to enhance the colonic transit. In this study, we assessed the mechanism of its prokinetic action by analyzing the effects on both the colonic phosphodiesterase activity of rats (n=4) and the isolated colonic strip preparation of guinea pigs (n=4). The 7% (v/v) solution of the beverage significantly decreased the phosphodiesterase activity by 9% (n=4). The beverage in concentrations of 0.7, 2.1 and 7% (v/v) relaxed the colonic strips pre-contracted by 1 µmol/L of carbachol in a concentration-related manner with 50, 58 and 79%, each response of which was diminished to 11, 19 and 46%, respectively in the presence of 100 µmol/L of L-nitro-arginine methyl ester. These results obtained by biochemical, functional and pharmacological analyses suggest that the beverage could relax the colon through both cAMP-associated and nitric oxide-dependent pathways, which may partly explain clinically observed prokinetic effect of the beverage.

FEATURES OF HISTOCHEMICAL CHANGES IN THE ACTIVITY OF SUCCINATE DEHYDROGENASE OF ARTIFICIAL BLADDER IN DYNAMICS (EXPERIMENTAL STUDY).

The gold standard for the treatment of invasive bladder cancer recognized throughout the world is radical cystectomy with orthotopic ileocystoplasty using the ileal intestinal tract. The study of the effect of urine on the adaptation of the mucosa of the artificial bladder continues for the last twenty years. According to the researchers, the results are quite contradictory, as some scientists note the hypersecretion of sulphomucins, sialomucins, progressive atrophy of microvilli, adenomatous hyperplasia and dysplasia. The aim of investigation to study the features of the histochemically revealed activity of succinate dehydrogenase in the wall of the artificial bladder and ileum in experimental animals. The material of the present study were the results obtained from the study of 18 female mini-pigs aged 4-5 months and weighing 8-10 kg. The modeling of the artificial bladder was performed in experimental animals, by cystectomy and subsequent ileo-cystoplasty. Experimental animals with a bladder model in groups of 6 animals were withdrawn from the experiment 3, 6 and 12 months after operational modeling. As for the wall of the official bladder, the changes in the activity of the studied enzymes were significant and showed not only possible changes in the activity of the Krebs cycle, but also about periodic displacements of the accents of substrate maintenance. These changes, in our view, are related to the transformation processes in the structural elements of the ileum wall, from which an unproblem has been formed to fulfill new functional duties. Signs of a violation of energy metabolism indicate the processes of hypoxia in the tissue of the artificial bladder and require further study and observation.

PDE8 Is Expressed in Human Airway Smooth Muscle and Selectively Regulates cAMP Signaling by ß2-Adrenergic Receptors and Adenylyl Cyclase 6.

Two cAMP signaling compartments centered on adenylyl cyclase (AC) exist in human airway smooth muscle (HASM) cells, one containing ß2-adrenergic receptor AC6 and another containing E prostanoid receptor AC2. We hypothesized that different PDE isozymes selectively regulate cAMP signaling in each compartment. According to RNA-sequencing data, 18 of 24 PDE genes were expressed in primary HASM cells derived from age- and sex-matched donors with and without asthma. PDE8A was the third most abundant of the cAMP-degrading PDE genes, after PDE4A and PDE1A. Knockdown of PDE8A using shRNA evoked twofold greater cAMP responses to 1 µM forskolin in the presence of 3-isobutyl-1-methylxanthine. Overexpression of AC2 did not alter this response, but overexpression of AC6 increased cAMP responses an additional 80%. We examined cAMP dynamics in live HASM cells using a fluorescence sensor. PF-04957325, a PDE8-selective inhibitor, increased basal cAMP concentrations by itself, indicating a significant basal level of cAMP synthesis. In the presence of an AC inhibitor to reduce basal signaling, PF-04957325 accelerated cAMP production and increased the inhibition of cell proliferation induced by isoproterenol, but it had no effect on cAMP concentrations or cell proliferation regulated by prostaglandin E2. Lipid raft fractionation of HASM cells revealed PDE8A immunoreactivity in buoyant fractions containing caveolin-1 and AC5/6 immunoreactivity. Thus, PDE8 is expressed in lipid rafts of HASM cells, where it specifically regulates ß2-adrenergic receptor AC6 signaling without effects on signaling by the E prostanoid receptors 2/4-AC2 complex. In airway diseases such as asthma and chronic obstructive pulmonary disease, PDE8 may represent a novel therapeutic target to modulate HASM responsiveness and airway remodeling.

Effect of estrogen on prostaglandin synthetase in bovine oviduct smooth muscle.

Gamete and embryo transport is an important function of the oviduct. This type of transport involves both smooth muscle contraction and epithelial cell secretions, and the former is mediated by prostaglandins (PGs) and their receptors. Our objective was to study the regulation of prostaglandin synthetase (prostaglandin-endoperoxide synthase-1 (PTGS1), prostaglandin-endoperoxide synthase-2 (PTGS2), mPGES-1, mPGES-2, cPGES, and PGFS) by estradiol (E2) in bovine oviduct smooth muscle. Prostaglandin synthetase mRNA and protein expression were investigated using real-time RT-PCR and Western blot analyses, respectively. Prostaglandin synthetase mRNA and protein expression were increased in oviductal smooth muscle tissue after treatment with different concentrations of estradiol for various time periods. The results indicated that there was no increase in expression observed after treatment with fulvestrant, a selective antagonist of the E2 receptor, indicating that E2 interacts with specific E2 nuclear receptors to upregulate PTGS1, PTGS2, mPGES-1, and PGFS expression. In conclusion, E2 increases PTGS1, mPGES-1, and PGFS mRNA and protein expression in bovine oviductal smooth muscle when added for different periods of time and at different concentrations. Additionally, E2 is transported intracellularly and interacts with specific E2 nuclear receptors to increase PTGS1, PTGS2, mPGES-1 and PGFS expression.

CF airway smooth muscle transcriptome reveals a role for PYK2.

Abnormal airway smooth muscle function can contribute to cystic fibrosis (CF) airway disease. We previously found that airway smooth muscle from newborn CF pigs had increased basal tone, an increased bronchodilator response, and abnormal calcium handling. Since CF pigs lack airway infection and inflammation at birth, these findings suggest intrinsic airway smooth muscle dysfunction in CF. In this study, we tested the hypothesis that CFTR loss in airway smooth muscle would produce a distinct set of changes in the airway smooth muscle transcriptome that we could use to develop novel therapeutic targets. Total RNA sequencing of newborn wild-type and CF airway smooth muscle revealed changes in muscle contraction-related genes, ontologies, and pathways. Using connectivity mapping, we identified several small molecules that elicit transcriptional signatures opposite of CF airway smooth muscle, including NVP-TAE684, an inhibitor of proline-rich tyrosine kinase 2 (PYK2). In CF airway smooth muscle tissue, PYK2 phosphorylation was increased and PYK2 inhibition decreased smooth muscle contraction. In vivo NVP-TAE684 treatment of wild-type mice reduced methacholine-induced airway smooth muscle contraction. These findings suggest that studies in the newborn CF pig may provide an important approach to enhance our understanding of airway smooth muscle biology and for discovery of novel airway smooth muscle therapeutics for CF and other diseases of airway hyperreactivity.

The role of S-nitrosoglutathione reductase (GSNOR) in human disease and therapy.

S-nitrosoglutathione reductase (GSNOR), or ADH5, is an enzyme in the alcohol dehydrogenase (ADH) family. It is unique when compared to other ADH enzymes in that primary short-chain alcohols are not its principle substrate. GSNOR metabolizes S-nitrosoglutathione (GSNO), S-hydroxymethylglutathione (the spontaneous adduct of formaldehyde and glutathione), and some alcohols. GSNOR modulates reactive nitric oxide (•NO) availability in the cell by catalyzing the breakdown of GSNO, and indirectly regulates S-nitrosothiols (RSNOs) through GSNO-mediated protein S-nitrosation. The dysregulation of GSNOR can significantly alter cellular homeostasis, leading to disease. GSNOR plays an important regulatory role in smooth muscle relaxation, immune function, inflammation, neuronal development and cancer progression, among many other processes. In recent years, the therapeutic inhibition of GSNOR has been investigated to treat asthma, cystic fibrosis and interstitial lung disease (ILD). The direct action of •NO on cellular pathways, as well as the important regulatory role of protein S-nitrosation, is closely tied to GSNOR regulation and defines this enzyme as an important therapeutic target.

Focal adhesion kinase (FAK) and mechanical stimulation negatively regulate the transition of airway smooth muscle tissues to a synthetic phenotype.

The effects of mechanical forces and focal adhesion kinase (FAK) in regulating the inflammatory responses of airway smooth muscle (ASM) tissues to stimulation with interleukin (IL)-13 were investigated. Canine tracheal tissues were subjected to different mechanical loads in vitro, and the effects of mechanical load on eotaxin secretion and inflammatory signaling pathways in response to IL-13 were determined. Eotaxin secretion by tissues in response to IL-13 was significantly inhibited in muscles maintained at a higher (+) load compared with those at a lower (-) load as assessed by ELISA, and Akt activation was also reduced in the higher (+) loaded tissues. Conversely the (+) mechanical load increased activation of the focal adhesion proteins FAK and paxillin in the tissues. The role of FAK in regulating the mechanosensitive responses was assessed by overexpressing FAK-related nonkinase in the tissues, by expressing the FAK kinase-dead mutant FAK Y397F, or by treating tissues with the FAK inhibitor PF-573228. FAK inactivation potentiated Akt activity and increased eotaxin secretion in response to IL-13. FAK inhibition also suppressed the mechanosensitivity of Akt activation and eotaxin secretion. In addition, FAK inactivation suppressed smooth muscle myosin heavy chain expression induced by the higher (+) mechanical load. The results demonstrate that the imposition of a higher mechanical load on airway smooth muscle stimulates FAK activation, which promotes the expression of the differentiated contractile phenotype and suppresses the synthetic phenotype and the inflammatory responses of the muscle tissue.

Airway smooth muscle NOX4 is upregulated and modulates ROS generation in COPD.

The burden of oxidative stress is increased in chronic obstructive pulmonary disease (COPD). However, whether the intra-cellular mechanisms controlling the oxidant/anti-oxidant balance in structural airway cells such as airway smooth muscle in COPD is altered is unclear. We sought to determine whether the expression of the NADPH oxidase (NOX)-4 is increased in airway smooth muscle in COPD both in vivo and primary cells in vitro and its role in hydrogen peroxide-induced reactive oxygen species generation. We found that in vivo NOX4 expression was up-regulated in the airway smooth muscle bundle in COPD (n = 9) and healthy controls with >20 pack year history (n = 4) compared to control subjects without a significant smoking history (n = 6). In vitro NOX4 expression was increased in airway smooth muscle cells from subjects with COPD (n = 5) compared to asthma (n = 7) and upregulated following TNF-α stimulation. Hydrogen peroxide-induced reactive oxygen species generation by airway smooth muscle cells in COPD (n = 5) was comparable to healthy controls (n = 9) but lower than asthma (n = 5); and was markedly attenuated by NOX4 inhibition. Our findings demonstrate that NOX4 expression is increased in vivo and in vitro in COPD and although we did not observe an intrinsic increase in oxidant-induced reactive oxygen species generation in COPD, it was reduced markedly by NOX4 inhibition supporting a potential therapeutic role for NOX4 in COPD.

Vimentin dephosphorylation at ser-56 is regulated by type 1 protein phosphatase in smooth muscle.

BACKGROUND: The intermediate filament protein vimentin undergoes reversible phosphorylation and dephosphorylation at Ser-56, which plays an important role in regulating the contraction-relaxation cycles of smooth muscle. The protein phosphatases that mediate vimentin dephosphorylation in smooth muscle have not been previously investigated. METHODS: The associations of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) with vimentin in mouse tracheal rings was evaluated by co-immunoprecipitation. Lentivirus-mediated shRNA against PP1 was used to assess the role of PP1 in vimentin dephosphorylation and the vimentin-associated process in smooth muscle. RESULTS: Co-immunoprecipitation analysis showed that vimentin interacted with PP1, but barely with PP2A, in airway smooth muscle. Knockdown of PP1 by lentivirus-mediated shRNA increased the acetylcholine-induced vimentin phosphorylation and smooth muscle contraction. Because vimentin phosphorylation is able to modulate p130 Crk-associated substrate (p130CAS) and actin polymerization, we also evaluated the role of PP1 in the biological processes. Silencing of PP1 also enhanced the agonist-induced the dissociation of p130CAS from vimentin and F/G-actin ratios (an index of actin polymerization). However, PP1 knockdown did not affect c-Abl tyrosine phosphorylation, an important molecule that controls actin dynamics. CONCLUSIONS: Taken together, these findings suggest that PP1 is a key protein serine/threonine phosphatase that controls vimentin Ser-56 dephosphorylation in smooth muscle. PP1 regulates actin polymerization by modulating the dissociation of p130CAS from vimentin, but not by affecting c-Abl tyrosine kinase.

Influence of betaine and arginine supplementation of reduced protein diets on fatty acid composition and gene expression in the muscle and subcutaneous adipose tissue of cross-bred pigs.

The isolated or combined effects of betaine and arginine supplementation of reduced protein diets (RPD) on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig m. longissimus lumborum and subcutaneous adipose tissue (SAT) were assessed. The experiment was performed on forty intact male pigs (Duroc×Large White×Landrace cross-breed) with initial and final live weights of 60 and 93 kg, respectively. Pigs were randomly assigned to one of the following five diets (n 8): 16·0 % of crude protein (control), 13·0 % of crude protein (RPD), RPD supplemented with 0·33 % of betaine, RPD supplemented with 1·5 % of arginine and RPD supplemented with 0·33 % of betaine and 1·5 % of arginine. Data confirmed that RPD increase intramuscular fat (IMF) content and total fat content in SAT. The increased total fat content in SAT was accompanied by higher GLUT type 4, lipoprotein lipase and stearoyl-CoA desaturase mRNA expression levels. In addition, the supplementation of RPD with betaine and/or arginine did not affect either IMF or total fat in SAT. However, dietary betaine supplementation slightly affected fatty acid composition in both muscle and SAT. This effect was associated with an increase of carnitine O-acetyltransferase mRNA levels in SAT but not in muscle, which suggests that betaine might be involved in the differential regulation of some key genes of lipid metabolism in pig muscle and SAT. Although the arginine-supplemented diet decreased the mRNA expression level of PPARG in muscle and SAT, it did not influence fat content or fatty acid composition in any of these pig tissues.

LDH-A promotes malignant progression via activation of epithelial-to-mesenchymal transition and conferring stemness in muscle-invasive bladder cancer.

Lactate dehydrogenase-A(LDH-A) is an important rate-limiting enzyme in the Warburg effect. Survival analysis indicated poor clinical outcomes in MIBC with high LDH-A expression. The results of in vitro experiment indicated that LDH-A promotes MIBC cells proliferation, invasion and migration. The positive relationship between LDH-A expression and CSC/EMT markers was confirmed both in invasive bladder cell line and in 136 MIBC specimens. Thus, we conclude that LDH-A may be a promising target for MIBC.

The influence of heavy metal ions, spermine and sodium nitroprusside on ATP-hydrolases of cell membranes of rat colon smooth muscle.

The specific features of functional lability of the rat colon smooth muscle (CSM) АТР-hydrolases were studied. Na+,K+-AТРase activity is effectively inhibited by divalent ions of both transition (≥ 0,1 µM) and nontransition (≥ 1 µM) heavy metals in succession by efficiency: Cu2+ > Fe2+ ≥ Cd2+ (10 µM). Polyamine spermine (0,5-1,0 mM) is a weak Na+,K+-AТРase inhibitor at saturation concentrations of ions and substrate. Sodium nitroprusside (1 mM) as nitric oxide-generating compound exhibits weak Na+,K+-AТРase inhibition only after prolonged preincubation with membranes. Mg2+-АТР-hydrolase activity in all cases is much more resistant to studied agents. Considering the example of the CSM Na+,K+-AТРase it is assumed that enzyme has specific biochemical features that contribute to its role as a potential target and redox-sensor, mediating the pathological mechanisms of heavy metal intoxication and cell oxidative damage.

[The influence of iron ions on ATP-hydrolases activity of cell membranes of rat colon smooth muscle and kidney].

To elucidate the specific features of the ATP-hydrolases structural resistance in the membrane under the action of the prooxidants: Fe2' and hydrogen peroxide, and N-ethylmaleimide (NEM) the colonic smooth muscle (CSM) Na+, K(+)-ATPase activity was compared with activities of the corresponding Mg(2+)-ATP-hydrolase and ATPases from kidney medullar layer of rats. The inhibition study of the CSM Na+, K(+)-ATPase by divalent iron shows the decrease of the activity by 30% at 0.1 µM FeSO4 and in the range of 0.1-10 µM--to 45% of residual activity. When comparing with kidney enzyme (rep- resents exclusively α1-isozyme) the CSM Na+, K(+)-ATPase sensitivity to Fe2+ is reliably higher at its submicromolar concentration. CSM Mg2+-ATPase is much more resistant to iron ions effect, than kidney one. However for two tissues Mg2(+)-ATPase activity is always more resistant as compared with corresponding Na+, K(+)-ATPase activity. Against 1 mM EGTA Na+,K(+)-ATPase and Mg2(+)-ATPase activities of GMOK and kidneys are equally insensitive to effect of hydrogen peroxide in concentration up to 1 mM. But in the presence of 20 µM FeSO4 in the concentration range of 1 nM-1 mM of H2O2 the Na+, K(+)-ATPase is inhibited to greater extent, than Mg2+-ATPase activity. NEM sensitivity of the two ATP-hydrolase systems corresponds to prooxidant sensitivity that indicates the distinct importance of SH-groups for their functioning. It is concluded that Na+, K+-ATPase can serve as a marker of membrane sensitivity to oxidation, Mg(2+)-ATPase is resistant to oxidation and can be considered as criterion of the oxidation resistance when comparing membrane enzyme complexes, es- pecially in GMOK.

Bladder function in mice with inducible smooth muscle-specific deletion of the manganese superoxide dismutase gene.

Manganese superoxide dismutase (MnSOD) is considered a critical component of the antioxidant systems that protect against oxidative damage. We are interested in the role of oxidative stress in bladder detrusor smooth muscle (SM) in different disease states. In this study, we generated an inducible, SM-specific Sod2(-/-) mouse model to investigate the effects of MnSOD depletion on the function of the bladder. We crossbred floxed Sod2 (Sod2(lox/lox)) mice with mice containing heterozygous knock-in of a gene encoding a tamoxifen-activated Cre recombinase in the SM22α promoter locus [SM-CreER(T2)(ki)(Cre/+)]. We obtained Sod2(lox/lox),SM-CreER(T2)(ki)(Cre/+) mice and injected 8-wk-old males with 4-hydroxytamoxifen to induce Cre-mediated excision of the floxed Sod2 allele. Twelve weeks later, SM-specific deletion of Sod2 and depletion of MnSOD were confirmed by polymerase chain reaction, immunoblotting, and immunohistochemistry. SM-specific Sod2(-/-) mice exhibited normal growth with no gross abnormalities. A significant increase in nitrotyrosine concentration was found in bladder SM tissue of SM-specific Sod2(-/-) mice compared with both wild-type mice and Sod2(+/+), SM-CreER(T2)(ki)(Cre/+) mice treated with 4-hydroxytamoxifen. Assessment of 24-h micturition in SM-specific Sod2(-/-) mice revealed significantly higher voiding frequency compared with both wild-type and SM-specific Cre controls. Conscious cystometry revealed significantly shorter intercontraction intervals and lower functional bladder capacity in SM-specific Sod2(-/-) mice compared with wild-type mice. This novel model can be used for exploring the mechanistic role of oxidative stress in organs rich in SM in different pathological conditions.

Catch muscle myorod modulates ATPase activity of Myosin in a phosphorylation-dependent way.

Myorod is expressed exclusively in molluscan catch muscle and localizes on the surface of thick filaments together with twitchin and myosin. Myorod is an alternatively spliced product of the myosin heavy-chain gene that contains the C-terminal rod part of myosin and a unique N-terminal domain. The unique domain is a target for phosphorylation by gizzard smooth myosin light chain kinase (smMLCK) and, perhaps, molluscan twitchin, which contains a MLCK-like domain. To elucidate the role of myorod and its phosphorylation in the catch muscle, the effect of chromatographically purified myorod on the actin-activated Mg2+-ATPase activity of myosin was studied. We found that phosphorylation at the N-terminus of myorod potentiated the actin-activated Mg2+-ATPase activity of mussel and rabbit myosins. This potentiation occurred only if myorod was phosphorylated and introduced into the ATPase assay as a co-filament with myosin. We suggest that myorod could be related to the catch state, a function specific to molluscan muscle.

Expression and distribution of phosphodiesterase isoenzymes in the human male urethra.

OBJECTIVE: To investigate the expression and distribution of phosphodiesterase (PDE) isoenzymes PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A in human urethral tissue. METHODS: Specimens of penile urethra were obtained from male subjects who had undergone male-to-female sex reassignment surgery. Using immunohistochemistry (immunofluorescence), the occurrence of PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A, the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide was examined in urethral sections. Cytosolic supernatants prepared from isolated human urethral tissue were subjected to Western blot analysis using specific anti-PDE antibodies. RESULTS: Immunosignals specific for PDE1A, 4A, 4B, and 5A were observed in the urethral smooth musculature. The smooth muscle bundles were seen innervated by slender nerve fibers, characterized by the expression of the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide. The expression of the PDE isoenzymes mentioned was confirmed by Western blotting. CONCLUSION: The results provide evidence for a significance of both the cyclic adenosine monophosphate and cyclic guanosine monophosphate signaling in the control of human urethral smooth muscle. The selective inhibition of PDE isoenzymes might represent a pharmacologic option to influence the function of smooth musculature in the human outflow region.

Early inflammatory damage to intestinal neurons occurs via inducible nitric oxide synthase.

Intestinal inflammation affects the enteric nervous system (ENS) that lies adjacent to the smooth muscle layers. Previously, we showed that the loss of ENS neurons in animal models such as tri-nitrobenzene sulphonic acid (TNBS)-induced colitis was a limited and early event despite progressive worsening of inflammation. Here, we demonstrated that the rapid appearance of activated immune cells in the intestinal wall is selectively neurotoxic via iNOS-derived NO, using TNBS-induced colitis in both rats and mice, and a co-culture model of ENS neurons and smooth muscle. An influx of neutrophils and macrophages occurred within hours of initiation of rat colitis, correlating with iNOS expression, acutely elevated NO and neuronal death. In vitro, chemical donors of NO selectively caused axonal damage and neuronal death. These outcomes were similar to those seen with combined culture with either activated peritoneal immune cells or the immune cell lines RAW-264 and RBL-2H3. Immune cell-mediated neurotoxicity was blocked by the iNOS inhibitor L-NIL, and neuronal death was inhibited by the RIP-1 kinase inhibitor necrostatin. In a mouse model, the stereotypic loss of myenteric neurons by Day 4 post-TNBS was abrogated by the selective iNOS inhibitors L-NIL or 1400W without effect on other parameters of intestinal inflammation. Preservation of ENS neurons also ameliorated the hyperplasia of smooth muscle that is characteristic of intestinal inflammation, in line with prior work showing neural regulation of smooth muscle phenotype. This identifies a predominant pathway of immune cell damage to the ENS, where early, acute elevation of NO from iNOS can be cytotoxic to myenteric neurons.