Regulation Of Smooth Muscle Contraction Research Articles

Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE.

Phosphorylation of the myosin-targeting subunit 1 of myosin light chain phosphatase (MYPT1) plays an important role in the regulation of smooth muscle contraction, and several sites of phosphorylation by different protein Ser/Thr kinases have been identified. Furthermore, in some instances, phosphorylation at specific sites affects phosphorylation at neighboring sites, with functional consequences. Characterization of the complex phosphorylation of MYPT1 in tissue samples at rest and in response to contractile and relaxant stimuli is, therefore, challenging. We have exploited Phos-tag SDS-PAGE in combination with Western blotting using antibodies to MYPT1, including phosphospecific antibodies, to separate multiple phosphorylated MYPT1 species and quantify MYPT1 phosphorylation stoichiometry using purified, full-length recombinant MYPT1 phosphorylated by Rho-associated coiled-coil kinase (ROCK) and cAMP-dependent protein kinase (PKA). This approach confirmed that phosphorylation of MYPT1 by ROCK occurs at Thr(697)and Thr(855), PKA phosphorylates these two sites and the neighboring Ser(696)and Ser(854), and prior phosphorylation at Thr(697)and Thr(855)by ROCK precludes phosphorylation at Ser(696)and Ser(854)by PKA. Furthermore, phosphorylation at Thr(697)and Thr(855)by ROCK exposes two other sites of phosphorylation by PKA. Treatment of Triton-skinned rat caudal arterial smooth muscle strips with the membrane-impermeant phosphatase inhibitor microcystin or treatment of intact tissue with the membrane-permeant phosphatase inhibitor calyculin A induced slow, sustained contractions that correlated with phosphorylation of MYPT1 at 7 to ≥10 sites. Phos-tag SDS-PAGE thus provides a suitable and convenient method for analysis of the complex, multisite MYPT1 phosphorylation events involved in the regulation of myosin light chain phosphatase activity and smooth muscle contraction.

Is Erectile Dysfunction an Example of Abnormal Endothelial Function?

Erectile dysfunction (ED) affects approximately half of men during middle age. Erectile dysfunction is often an early symptom of systemic vascular disease, which may precipitate significant cardiac events. The pathophysiology of ED and cardiovascular disease is closely linked. Endothelial dysfunction occurs at an early stage in ED and cardiovascular disease (CVD). In normal conditions, nitric oxide dependent and independent mechanisms regulate penile vascular tone ensuring an appropriate balance of vasoconstriction and vasodilatation. A normal endothelium is responsible for mediating the effect of pro-erectile mediators derived from the endothelium and is critical in normal erectile function. Endothelial dysfunction disrupts the homeostatic mechanisms responsible for regulation of smooth muscle contraction and penile vascular tone. Reduced bioavailability of nitric oxide (NO) occurs as a response to endothelial damage. Phosphodiesterases further degrade levels of cyclic guanosine monophosphate (cGMP) and impair smooth muscle relaxation and erectile function. A number of endothelium derived NO independent mediators of erectile function have been described and are known to contribute to ED in the presence of endothelial damage. This review provides an up to date analysis of the role of the endothelium in ED describing the pathways involved and how these represent current and potential therapeutic targets.

Mice with Double Knockout of Calponin 1 and Calponin 2 Genes Demonstrate Contractility Modifications in Vascular Smooth Muscle

Calponin is an actin filament associated regulatory protein in smooth muscle and non-muscle cells. Calponin-1 is specifically expressed in smooth muscle cells and calponin-2 is expressed in both smooth muscle and multiple non-muscle cell types. The role of calponin-1 in the regulation of smooth muscle contraction has been extensively investigated. Calponin-1 gene knockout mice demonstrated minor but clearly detectable changes in smooth muscle contractility, especially in the urogenital system. Here we compared the impacts of single and double knockout of calponin-1 and calponin-2 genes in mice on smooth muscle contractility. The results showed that either calponin-1 or calponin-2 knockout did not significantly alter the passive tension-active tension relationship in aortic smooth muscle and the mice exhibited normal blood pressure. The double knockout mice survived well into adulthood in the absence of physiological stress. However, the double knockout mice showed higher passive tension and less KCl- and norepinephrine-induced active tension in endothelium-removed aorta rings. The double knockout mice also showed lower systolic and diastolic blood pressures compared to gender-matched wild type controls. The results suggest that calponin-1 and −2 both contribute to the regulation of vascular smooth muscle contractility. The minimized phenotypic effects of calponin-1 and calponin-2 single knockout mice indicate a mutual compensation between the two isoforms in smooth muscle cells. Supporting this notion, analysis of protein expression in various types of smooth muscle showed up-regulations of one isoform when the other was deleted. The results implicate that calponins are molecular targets for modifications of smooth muscle contractility and especially blood pressure. Simultaneously targeting the expression and function of both two calponins may provide a potential therapeutic approach to the development of new treatment of hypertension.

Highly sensitive myosin phosphorylation analysis in the renal afferent arteriole.

The regulation of smooth muscle contraction and relaxation involves phosphorylation and dephosphorylation of regulatory proteins, particularly myosin. To elucidate the regulatory mechanisms, analyzing the phosphorylation signal transduction is crucial. Although a pharmacological approach with selective inhibitors is sensitive and a useful technique, it leads to speculation regarding a signaling pathway but does not provide direct evidence of changes at a molecular level. We developed a highly sensitive biochemical technique to analyze phosphorylation by adapting Phos-tag SDS-PAGE. With this technique, we successfully analyzed myosin light chain (LC20) phosphorylation in tiny renal afferent arterioles. In the rat afferent arterioles, endothelin-1 (ET-1) induced diphosphorylation of LC20 at Ser19 and Thr18 as well as monophosphorylation at Ser19 via ETB receptor activation. Considering that LC20 diphosphorylation can decrease the rate of dephosphorylation and thus relaxation, we concluded that LC20 diphosphorylation contributes, at least in part, to the prolonged contraction induced by ET-1 in the renal afferent arteriole.

A Small Molecule Pyrazolo[3,4-d]Pyrimidinone Inhibitor of Zipper-Interacting Protein Kinase Suppresses Calcium Sensitization of Vascular Smooth Muscle.

A novel inhibitor of zipper-interacting protein kinase (ZIPK) was used to examine the involvement of ZIPK in the regulation of smooth muscle contraction. Pretreatment of de-endothelialized rat caudal arterial smooth muscle strips with the pyrazolo[3,4-d]pyrimidinone inhibitor 2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo [3,4-d]-pyrimidin-6-yl)thio)propanamide (HS38) decreased the velocity of contraction (time to reach half-maximal force) induced by the phosphatase inhibitor calyculin A in the presence of Ca(2+) without affecting maximal force development. This effect was reversed following washout of HS38 and correlated with a reduction in the rate of phosphorylation of myosin 20-kDa regulatory light chains (LC20) but not of protein kinase C-potentiated inhibitory protein for myosin phosphatase of 17 kDa (CPI-17), prostate apoptosis response-4, or myosin phosphatase-targeting subunit 1 (MYPT1), all of which have been implicated in the regulation of vascular contractility. A structural analog of HS38, with inhibitory activity toward proviral integrations of Moloney (PIM) virus 3 kinase but not ZIPK, had no effect on calyculin A-induced contraction or protein phosphorylations. We conclude that a pool of constitutively active ZIPK is involved in regulation of vascular smooth muscle contraction through direct phosphorylation of LC20 upon inhibition of myosin light chain phosphatase activity. HS38 also significantly attenuated both phasic and tonic contractile responses elicited by phenylephrine, angiotensin II, endothelin-1, U46619, and K(+)-induced membrane depolarization in the presence of Ca(2+), which correlated with inhibition of phosphorylation of LC20, MYPT1, and CPI-17. These effects of HS38 suggest that ZIPK also lies downstream from G protein-coupled receptors that signal through both Gα12/13 and Gαq/11.

Targeted Deletion of all Caldesmon Splice Variants in Mice Results in Abdominal Wall Closure Defects

Ample evidence obtained in vitro indicates that the thin filament linked protein, caldesmon (CaD) is involved in regulation of smooth muscle contraction. Homozygous h‐CaD deficient mice were reported to be viable and exhibit a but small slowing of relaxation (Guo et al., 2013). However, the results are confounded by upregulation of non‐muscle l‐CaD in smooth muscle. Thus, the in vivo function of CaD is still not clear. Here we report that deletion of all splice variants of Cald1 results in perinatal lethality whereas the life span of heterozygotes (hets) is normal. Gene targeting was achieved by deleting exons 2‐13 in the murine Cald1 gene. Homologous recombination was confirmed on DNA level by Southern blots and PCR, and on protein level by Western Blotting. Homozygous offspring followed Mendelian rules at E15.5 but was abrogated at E19.5. The bodyweight of the ‐/‐ fetuses at E18.5 was reduced by ~20%. The most prominent feature of the ‐/‐ fetuses is the persistence of the physiological umbilical hernia whereby the size of the abdominal wall defect is variable. Bladder contractility of adult hets was not significantly affected. In conclusion, our results indicate that deletion of CaD does not result in early embryonic lethality but it may be essential for organ maturation. Moreover, lack of h‐CaD but not lack of all splice variants including non‐muscle l‐CaD is compatible with life.

Modulation of conformations of myosin subfragment-1 (S-1) and inhibition of S-1 ATPase by mussel calponin

Although the role of calponin in physiological processes in smooth muscles has been studied for a long time, it remains obscure in many respects. Despite the large number of experimental data, the role of calponin in the regulation of smooth muscle contraction has not yet been established. The study of calponin-like proteins (i.e., proteins with homologous sequence and structure functioning in the cells of animals of different taxonomic groups) can help to solve this problem. In this work, we studied the newly identified calponin of the mussel Crenomytilus grayanus with a molecular weight of 40 kDa (CaP-40). First, we tested its inhibitory effect on the actin-activated S-1 ATPase in the presence of calcium ions. Second, we studied the effect of CaP-40 on the conformational changes of the myosin head in modeling different stages of the ATPase cycle in glycerinated muscle fibers by polarization fluorometry. It is shown that Cap-40 inhibits the activity of rabbit S-1 ATPase. Inhibition reached 80% at a CaP-40-to-actin ratio of 1: 2. In addition, it is shown that Cap-40 modulates the S-1 conformation in such a way that the formation of strong types of binding (stages A · M and A · M · ADP) between S-1 and actin is inhibited and they switch to a weak bond. However, CaP-40 had almost no effect on the weak binding state (stage A · M · ADP · Pi). These results indicate that the interaction of CaP-40 with actin leads to the conformational changes in actin and myosin head that prevent the formation of the strong bond between S-1 and actin in the ATP hydrolysis cycle.

In vivo roles for myosin phosphatase targeting subunit‐1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction

Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction. Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle.

Abstract 4994: E2F1 deficiency causes increased migration through upregulation of MYLK

Abstract Malignant melanoma is an extremely aggressive disease with high metastatic potential. Several studies have shown that the transcription factor E2F1 is overexpressed in melanoma. E2F1 belongs to the family of E2F transcription factors; is more abundant in melanoma cells than in normal melanocytes, which highlights its relevance to melanoma biology. E2F1 participates in a variety of biological processes such as proliferation, apoptosis, development, differentiation, DNA damage and repair, oxidative metabolism, and self-renewal by virtue of its transcriptional activator functions. However, its role in many of these biological processes in melanoma is not yet fully addressed. In this study, we show that E2F1 deficiency increased migration and invasion of melanoma cells, which was associated with increased MYLK at both RNA and protein level. MYLK is a calcium-calmodulin (Ca2+//CaM)-dependent myosin light chain kinase, which phosphorylates Ser-19 of myosin II after activation. This process is implicated in the regulation of smooth muscle contraction. In non-smooth muscle, MYLK is known to be involved in many inflammatory diseases through regulation of tight junction and intestinal epithelial barrier function. MYLK is also involved with proliferation and migration in cancers of the breast, prostate and colon, but its expression and role in melanoma cells is unknown. ML-7, the specific inhibitor for MYLK abrogated the increased migration and invasion due to E2F1 deficiency. Transient knockdown of MYLK also significantly abrogated the increased migration and invasion from E2F1 deficiency. Our results strongly suggest that E2F1 deficiency enhanced migration and invasion through regulating MYLK expression. We also found increased pERK in E2F1 knockdown cells, which suggest that ERK activation may be involved in the up-regulation of MYLK. All of our data taken together suggests that E2F1-mediated inhibition of melanoma cell migration occurs through a complex cross-talk between E2F1-ERK-MYLK and is not through a simple linear relationship between signaling molecules. Supported by R21 CA125719 (RG). Citation Format: Peng Meng, Rita Ghosh. E2F1 deficiency causes increased migration through upregulation of MYLK. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4994. doi:10.1158/1538-7445.AM2014-4994

Inhibition of smooth muscle force generation by focal adhesion kinase inhibitors in the hyperplastic human prostate.

Smooth muscle contraction may be critical for lower urinary tract symptoms (LUTS) in patients with benign prostate hyperplasia and requires stable anchorage of the cytoskeleton to the cell membrane. These connections are regulated by focal adhesion kinase (FAK). Here, we addressed the involvement of FAK in the regulation of smooth muscle contraction in hyperplastic human prostate tissues. Prostate tissues were obtained from radical prostatectomy. Expression of FAK and focal adhesion proteins was assessed by Western blot analysis and immunohistochemical stainings. Effects of the FAK inhibitors PF-573228 and Y-11 on contraction of prostate strips were examined in the organ bath. Expression of FAK and focal adhesion proteins (integrin-5α, paxilin, and c-Src) was detected by Western blot analysis in prostate samples. By double immunofluorescence staining with calponin and pan-cytokeratin, expression of FAK was observed in stromal and epithelial cells. Immunoreactivity for FAK colocalized with integrin-5α, paxilin, talin, and c-Src. Stimulation of prostate tissues with the α1-adrenergic agonist phenylephrine increased the phosphorylation state of FAK at Tyr³⁹⁷ and Tyr⁹²⁵ with different kinetics, which was blocked by the α1-adrenoceptor antagonist tamsulosin. Norepinephrine and phenylephrine induced concentration-dependent contractions of prostate strips. Both FAK inhibitors PF-573228 and Y-11 significantly inhibited norepinephrine- and phenylephrine-induced contractions. Finally, PF-573228 and Y-11 inhibited contractions induced by electric field stimulation, which was significant at the highest frequency. In conclusion, α1-adrenergic smooth muscle contraction or its regulation involves FAK in the human prostate. Consequently, FAK may be involved in the pathophysiology of LUTS and in current or future LUTS therapies.

Histone deacetylase 8 regulates cortactin deacetylation and contraction in smooth muscle tissues.

Histone deacetylases (HDACs) are a family of enzymes that mediate nucleosomal histone deacetylation and gene expression. Some members of the HDAC family have also been implicated in nonhistone protein deacetylation, which modulates cell-cycle control, differentiation, and cell migration. However, the role of HDACs in smooth muscle contraction is largely unknown. Here, HDAC8 was localized both in the cytoplasm and the nucleus of mouse and human smooth muscle cells. Knockdown of HDAC8 by lentivirus-encoding HDAC8 shRNA inhibited force development in response to acetylcholine. Treatment of smooth muscle tissues with HDAC8 inhibitor XXIV (OSU-HDAC-44) induced relaxation of precontracted smooth muscle tissues. In addition, cortactin is an actin-regulatory protein that undergoes deacetylation during migration of NIH 3T3 cells. In this study, acetylcholine stimulation induced cortactin deacetylation in mouse and human smooth muscle tissues, as evidenced by immunoblot analysis using antibody against acetylated lysine. Knockdown of HDAC8 by RNAi or treatment with the inhibitor attenuated cortactin deacetylation and actin polymerization without affecting myosin activation. Furthermore, expression of a charge-neutralizing cortactin mutant inhibited contraction and actin dynamics during contractile activation. These results suggest a novel mechanism for the regulation of smooth muscle contraction. In response to contractile stimulation, HDAC8 may mediate cortactin deacetylation, which subsequently promotes actin filament polymerization and smooth muscle contraction.

Hypoxic pulmonary hypertension: the paradigm is changing.

Hypoxic pulmonary hypertension: the paradigm is changing.

Purinergic receptor stimulation induces Ca2+ oscillations and smooth muscle contraction in small pulmonary veins (1175.6)

The small intrapulmonary veins (SPV) are thought to participate in the control of pulmonary blood flow and its excessive contraction has been associated with the development of pulmonary edema. However, the regulation of smooth muscle contraction by intracellular signaling in SPV is poorly understood. We used rat lung slices with phase‐contrast and confocal microscopy to investigate Ca2+ signaling and contraction during purinergic receptor activation in small (100‐250 µm) pulmonary veins. We found that ATP induced strong, concentration‐dependent, and reversible vein contraction. Vein contraction was accompanied by intracellular Ca2+ oscillations that propagated along the vein smooth muscle cell as Ca2+ waves. In the absence of extracellular Ca2+, ATP induced Ca2+ oscillations that were attenuated in time and transient vein contraction. The InsP3 receptor inhibitor 2‐APB inhibited the Ca2+ oscillations and contraction induced by ATP in absence of extracellular Ca2+. In contrast, Ca2+ oscillations and contraction were resistant to ryanodine. Vein contraction was inhibited by phospholipase Cβ inhibitor U73122 and by purinergic receptor antagonist suramin. These results suggest that ATP is a strong vasoconstrictor of SPV and that ATP activate purinergic P2Y receptors in vein smooth muscle to induce InsP3 receptor‐mediated Ca2+ oscillations and vein contraction.Grant Funding Source: American Heart Association

Calponin1 inhibits dilated cardiomyopathy development in mice through the εPKC pathway

BackgroundCalponin1 (CNN1) is involved in the regulation of smooth muscle contraction in physiological situation and it also expresses abnormally in a variety of pathological situations. We found that the expression of CNN1 decreased significantly in the heart tissue of a cTnTR141W transgenic dilated cardiomyopathy (DCM) mouse model and an adriamycin (ADR)-induced DCM mouse model, suggesting that CNN1 is involved in the pathogenesis of DCM. However, the role of CNN1 on cardiac function, especially on pathogenesis of DCM, has not been clarified. In this study, we tested whether rescued expression of CNN1 could prevent the development of DCM and investigated its possible mechanisms. Methods and resultsThe DCM phenotypes were significantly improved with the transgenic expression of CNN1 in the cTnTR141W×CNN1 double transgenic (DTG) mice, which was demonstrated by the survival, cardiac geometry and function analyses, as well as microstructural and ultrastructural observations based on echocardiography and histology examination. The expression of CNN1 could also resist the cardiac geometry breakage and dysfunction in the ADR-induced DCM mice model. Meanwhile, the epsilon isoform of protein kinase C (εPKC) activator and inhibitor could reverse the activation of εPKC/ERK/mTOR pathway and DCM phenotypes in the cTnTR141W and cTnTR141W×CNN1 double transgenic (DTG) mice. ConclusionsεPKC/ERK/mTOR pathway activation induced by the rescued expression of CNN1 contributed to the improvement of cardiac dysfunction and pathological changes observed in the DTG mice. CNN1 could be a therapeutic target to prevent the development of DCM and heart failure (HF).

PKC activation increases Ca2+sensitivity of permeabilized lymphatic muscle via myosin light chain 20 phosphorylation-dependent and -independent mechanisms

The contractile activity of muscle cells lining the walls of collecting lymphatics is responsible for generating and regulating flow within the lymphatic system. Activation of PKC signaling contributes to the regulation of smooth muscle contraction by enhancing sensitivity of the contractile apparatus to Ca(2+). It is currently unknown whether PKC signaling contributes to the regulation of lymphatic muscle contraction. We hypothesized that the activation of PKC signaling would increase the sensitivity of the lymphatic myofilament to Ca(2+). To test this hypothesis, we determined the effects of PKC activation with phorbol esters [PMA or phorbol dibutyrate (PDBu)] on the contractile behavior of α-toxin-permeabilized rat mesenteric and cervical lymphatics or the thoracic duct. The addition of PMA or PDBu induced a significant increase in the contractile force of submaximally activated α-toxin-permeabilized lymphatic muscle independent of a change in intracellular Ca(2+) concentration, and the Ca(2+)-force relationship of lymphatic muscle was significantly left shifted, indicating greater myofilament Ca(2+) sensitivity. Phorbol esters increased the maximal rate of force development, whereas the rate of relaxation was reduced. Western blot and immunohistochemistry data indicated that the initial rapid increase in tension development after stimulation by PDBu was associated with myosin light chain (MLC)20 phosphorylation; however, the later, steady-state Ca(2+) sensitization of permeabilized lymphatic muscle was not associated with increased phosphorylation of MLC20 at Ser(19), 17-kDa C-kinase-potentiated protein phosphatase-1 inhibitor at Thr(38), or caldesmon at Ser(789). Thus, these data indicate that PKC-dependent Ca(2+) sensitization of lymphatic muscle may involve MLC20 phosphorylation-dependent and -independent mechanism(s).

EP2 Receptor Activates Dual G Protein Signaling Pathways that Mediate Contrasting Proinflammatory and Relaxatory Responses in Term Pregnant Human Myometrium

Prostaglandin (PG) E2 (PGE(2)) plays a central role in the regulation of smooth muscle contractions. Classically, PGE(2) stimulates contractions via EP1 and EP3 receptors, whereas EP2 and EP4 maintain quiescence. Labor involves a change from myometrial quiescence to contractions with a shift from anti- to proinflammatory pathways. EP2, a Gαs-coupled receptor, is known to mediate its actions via cAMP signaling. However, we have recently shown that EP2 also activates the proinflammatory PG G/H synthase-2 (PGHS-2). Here, we identify the mechanism underlying the ability of EP2 to maintain uterine quiescence and activate a proinflammatory/prolabor response in term-pregnant human myometrium. Human myometrial biopsies for in vivo and in vitro studies were taken at cesarean section at term, before or after the onset of labor. Activation of EP2 increased intracellular levels of cAMP and reduced contractility. Contrastingly, EP2 stimulation increased levels of PGHS-2, membrane-associated PGE synthase-1, and PGE(2). This was entirely dependent on EP2-mediated activation of calcium signaling. Both calcium signaling and up-regulation of PGHS-2 were insensitive to the Gαi inhibitor pertussis toxin but inhibited by small interfering RNA knockdown of Gαq/11. There were no differences in EP2 mRNA or protein levels between upper or lower segment myometrium or between pre- and postlabor myometrium. However, in myocytes taken after the onset of labor, cAMP signaling was markedly attenuated, whereas activation of calcium and PGHS-2 was preserved. Overall, the dual coupling of EP2 to Gαs-cAMP and Gαq/11-calcium pathways underlies its ability to mediate contrasting functions in term pregnancy and the "switching" to a prolabor receptor.

Role of Abl in airway hyperresponsiveness and airway remodeling

BackgroundAsthma is a chronic disease that is characterized by airway hyperresponsiveness and airway remodeling. The underlying mechanisms that mediate the pathological processes are not fully understood. Abl is a non-receptor protein tyrosine kinase that has a role in the regulation of smooth muscle contraction and smooth muscle cell proliferation in vitro. The role of Abl in airway hyperresponsiveness and airway remodeling in vivo is largely unknown.MethodsTo evaluate the role of Abl in asthma pathology, we assessed the expression of Abl in airway tissues from the ovalbumin sensitized and challenged mouse model, and human asthmatic airway smooth muscle cells. In addition, we generated conditional knockout mice in which Abl expression in smooth muscle was disrupted, and then evaluated the effects of Abl conditional knockout on airway resistance, smooth muscle mass, cell proliferation, IL-13 and CCL2 in the mouse model of asthma. Furthermore, we determined the effects of the Abl pharmacological inhibitors imatinib and GNF-5 on these processes in the animal model of asthma.ResultsThe expression of Abl was upregulated in airway tissues of the animal model of asthma and in airway smooth muscle cells of patients with severe asthma. Conditional knockout of Abl attenuated airway resistance, smooth muscle mass and staining of proliferating cell nuclear antigen in the airway of mice sensitized and challenged with ovalbumin. Interestingly, conditional knockout of Abl did not affect the levels of IL-13 and CCL2 in bronchoalveolar lavage fluid of animals treated with ovalbumin. However, treatment with imatinib and GNF-5 inhibited the ovalbumin-induced increase in IL-13 and CCL2 as well as airway resistance and smooth muscle growth in animals.ConclusionsThese results suggest that the altered expression of Abl in airway smooth muscle may play a critical role in the development of airway hyperresponsiveness and airway remodeling in asthma. Our findings support the concept that Abl may be a novel target for the development of new therapy to treat asthma.

Role of the Adapter Protein Abi1 in Actin-associated Signaling and Smooth Muscle Contraction

Actin filament polymerization plays a critical role in the regulation of smooth muscle contraction. However, our knowledge regarding modulation of the actin cytoskeleton in smooth muscle just begins to accumulate. In this study, stimulation with acetylcholine (ACh) induced an increase in the association of the adapter protein c-Abl interactor 1 (Abi1) with neuronal Wiskott-Aldrich syndrome protein (N-WASP) (an actin-regulatory protein) in smooth muscle cells/tissues. Furthermore, contractile stimulation activated N-WASP in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer efficiency of an N-WASP sensor. Abi1 knockdown by lentivirus-mediated RNAi inhibited N-WASP activation, actin polymerization, and contraction in smooth muscle. However, Abi1 silencing did not affect myosin regulatory light chain phosphorylation at Ser-19 in smooth muscle. In addition, c-Abl tyrosine kinase and Crk-associated substrate (CAS) have been shown to regulate smooth muscle contraction. The interaction of Abi1 with c-Abl and CAS has not been investigated. Here, contractile activation induced formation of a multiprotein complex including c-Abl, CAS, and Abi1. Knockdown of c-Abl and CAS attenuated the activation of Abi1 during contractile activation. More importantly, Abi1 knockdown inhibited c-Abl phosphorylation at Tyr-412 and the interaction of c-Abl with CAS. These results suggest that Abi1 is an important component of the cellular process that regulates N-WASP activation, actin dynamics, and contraction in smooth muscle. Abi1 is activated by the c-Abl-CAS pathway, and Abi1 reciprocally controls the activation of its upstream regulator c-Abl.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Prostate-apoptosis response-4 phosphorylation in vascular smooth muscle

The protein prostate-apoptosis response (Par)-4 has been implicated in the regulation of smooth muscle contraction, based largely on studies with the A7r5 cell line. A mechanism has been proposed whereby Par-4 binding to MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase, MLCP) blocks access of zipper-interacting protein kinase (ZIPK) to Thr697 and Thr855 of MYPT1, whose phosphorylation is associated with MLCP inhibition. Phosphorylation of Par-4 at Thr155 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition and contraction. We tested this “padlock” hypothesis in a well-characterized vascular smooth muscle system, the rat caudal artery. Par-4 was retained in Triton-skinned tissue, suggesting a tight association with the contractile machinery, and indeed Par-4 co-immunoprecipitated with MYPT1. Treatment of Triton-skinned tissue with the phosphatase inhibitor microcystin (MC) evoked phosphorylation of Par-4 at Thr155, but did not induce its dissociation from the contractile machinery. Furthermore, analysis of the time courses of MC-induced phosphorylation of MYPT1 and Par-4 revealed that MYPT1 phosphorylation at Thr697 or Thr855 preceded Par-4 phosphorylation. Par-4 phosphorylation was inhibited by the non-selective kinase inhibitor staurosporine, but not by inhibitors of ZIPK, Rho-associated kinase or protein kinase C. In addition, Par-4 phosphorylation did not occur upon addition of constitutively-active ZIPK to skinned tissue. We conclude that phosphorylation of Par-4 does not regulate contraction of this vascular smooth muscle tissue by inducing dissociation of Par-4 from MYPT1 to allow phosphorylation of MYPT1 and inhibition of MLCP.