Contractile Signaling Research Articles

Special issue Heidelberg Heart II: Abstracts of oral and poster presentations

s of oral presentations A 1 — Desmosomal molecules in and out of junctions Kathleen J. Green, Adi Dubash, Lisa M. Godsel Departments of Pathology and Dermatology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA kgreen@northwestern.edu Desmosomes are intercellular junctions that anchor the intermediate filament (IF) cytoskeleton to sites of strong intercellular adhesion, and play a critical role in ensuring mechanical integrity of the skin and the heart. Desmosome building blocks come primarily from three protein families. Transmembrane members of the cadherin family, the desmogleins and desmocollins, cooperate to form the adhesive interface. Within the junctional plaque, the cytoplasmic tails of the cadherins provide a scaffold for armadillo family members including plakoglobin and plakophilins (PKPs) 1-3 and the IF-binding protein, desmoplakin (DP), which in turn anchors the strain-bearing IF cytoskeleton to the plaque. In cardiac muscle, desmosome molecules are major architectural components of the intercalated discs, highly organized regions of the plasma membrane comprising components of adherens junctions, gap junctions, and desmosomes that together coordinate mechanical and electrochemical signaling between adjacent cardiac myocytes. In vertebrates, postnatal remodeling of these specialized regions of the plasma membrane occurs, giving rise to the area composita in which desmosomal building blocks are intermixed with components of adherens junctions. The crucial functions of desmosome molecules in epithelial and cardiac tissues are highlighted by the discovery of mutations that cause skin and heart disease. While interference with the architectural roles of desmosome molecules has been assumed to make an important contribution to tissue responses that lead to disease pathogenesis, functions that transcend their well-established roles in adhesion and IF-anchorage are emerging. Desmosome molecules have recently been shown to guide the remodeling of microtubules during epidermal morphogenesis, and also govern actin remodeling by regulating Rho GTPases during junction assembly. Of particular interest is the desmosomal armadillo protein plakophilin 2 (PKP2), which is reported as the most frequent target for mutation in arrhythmogenic right ventricular cardiomyopathy (ARVC), a leading cause of sudden cardiac death in the young. We previously showed that in epithelial cells, PKP2 regulates the localization and activity of RhoA to locally control actomyosin contractile signaling important for cell junction formation, while keeping global RhoA signaling in check. In the heart, signaling through RhoA and its downstream effectors is critical for normal cardiac development and physiology. Indeed, our data show that PKP2 silencing results in elevated RhoA and disruption of actin organization in cardiac myocytes. Further, loss of PKP2 or its fellow armadillo protein plakoglobin leads to elevated expression of target genes of the transcriptional regulator SRF (serum response factor), which is activated in a RhoA-dependent fashion, and along with Rho plays essential roles in cardiac development, hypertrophy and fibrosis. These data suggest a new cellular pathway through which desmosome deficiency could contribute to pathogen-

JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets

AbstractMounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin αIIbβ3. Once platelet activation has occurred, integrin αIIbβ3 stabilizes thrombus formation by providing agonist-independent “outside-in” signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A–deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation.

Regulation of Contractile Proteins and Protein Translational Signaling in Disused Muscle

Background/Aims: Muscle disuse can lead to muscle atrophy and impaired skeletal muscle function. How skeletal muscle modulates protein translational signaling in response to prolonged muscle disuse is not well understood. Using the hindlimb unloading (HU) model of muscle atrophy we examined how hindlimb unweighting affects protein translational signaling, including the activation of Akt/mTOR/p70S6K/S6 signaling and the inhibitory association of 4EBP1 with translation initiation factor eIF4E. Methods: Male F344BN rats were randomized into baseline control, or subjected to HU for 3, 7 or 14 days. Body weight, gastrocnemius muscle, and individual myofiber cross-sectional area were measured to evaluate the degree of muscle atrophy. The amounts of myosin and related muscle contractile proteins were assessed using SDS-PAGE and immunoblotting. Microarray analysis was used to evaluate changes in the mRNA expression of muscle contractile proteins. Total and phosphorylated proteins of Akt/mTOR/p70S6K/S6 pathway were determined via immunoblotting, while the association of 4EBP1 with eIF4E was detected via co-immunoprecipitation. Results: Unloading for 3 days significantly reduced cytosolic myosin content and was associated with increased binding of 4EBP1 to eIF4E, while prolonged unloading (14 days) was associated with the activation of Akt/mTOR/p70S6K/S6 signaling, decreased binding of 4EBP1 to eIF4E, increased cytosolic myosin and elevations in myofibrillar mRNA levels. Conclusion: Taken together, these data suggest that prolonged muscle disuse induces a biphasic translational signaling response that is associated with diminished and then increased muscle contractile protein expression.

Abstract P203: Nuclear PKA Compartmentation Manages Hypertrophic Responses to β-Adrenergic Signaling

Protein kinase A (PKA) directly mediates several cardiac behaviors in response to β-adrenergic stimulation. However, it remains unclear how PKA makes context-dependent decisions to select between contractile and hypertrophic β-adrenergic signaling responses. We have previously shown PKA activation by isoproterenol is spatially heterogeneous in neonatal rat cardiac myocytes, indicating subcellular β-adrenergic signaling compartmentation. We hypothesize hypertrophic β-adrenergic signaling responses are specifically regulated by nuclear compartmentation of PKA activity. We expressed spatially targeted FRET reporters for PKA activity and found PKA responses to isoproterenol to be slower and less sensitive in the nucleus than in the cytosol. To investigate mechanisms underlying these differences, we constructed a biochemically mechanistic computational model of cytosolic and nuclear PKA activity in rat cardiac myocytes. Using this model, we found that nuclear PKA dynamics are limited by diffusional transport barriers, whereas nuclear PKA sensitivity to isoproterenol is limited by protein kinase inhibitor (PKI) inhibition. Moreover, our model predicts these differences in compartmented PKA activity may help PKA select between different substrates, sensitive to the temporal characteristics of β-adrenergic stimulation. In the model, short, transient isoproterenol treatment fully phosphorylates phospholamban while CREB is only partially phosphorylated. In contrast, long, sustained isoproterenol treatment fully phosphorylates both phospholamban and CREB. To test the model prediction that compartmented PKA can regulate different cardiac behaviors, we over-expressed PKA targeted to the cytosol and nucleus. Myocytes over-expressing PKA in the nucleus were 24.8% larger than control (n = 908 cells, p = 0.0002), while myocytes over-expressing PKA in the cytosol were the same size as control cells (n = 987 cells, p = 0.9971). Together, these results highlight an important role for nuclear PKA compartmentation in selecting hypertrophic responses to β-adrenergic stimulation.

Arrestins 2 and 3 differentially regulate ETA and P2Y2 receptor-mediated cell signaling and migration in arterial smooth muscle

Overstimulation of endothelin type A (ET(A)) and nucleotide (P2Y) Gα(q)-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied Gα(q)-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca(2+) levels were assessed in isolated ASMCs loaded with Ca(2+)-sensitive dyes, P2Y(2) and ET(A) receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y(2) receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ET(A) receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ET(A) and P2Y(2) receptor-stimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease.

Abstract 18218: Vascular Dysfunction as a Primary Cause of Hypertension in Mice with Smooth Muscle Specific Deletion of RGS2

The etiology of hypertension in most cases is idiopathic. Current dogma states that all forms of hypertension arise from renal abnormalities of sodium handling. We have hypothesized that a primary abnormality of vascular smooth muscle cell (SMC) contraction can cause hypertension. To directly test this hypothesis we created and characterized inducible, SMC-specific RGS2 knockout mice (RGS2-SMC-KO) harboring a SMC-specific deletion of the PKGIα target the GTPase activating protein Regulator of G-protein Signaling 2. In these studies, SMC-specific deletion of RGS2 was induced at 7 weeks of age and Cre-negative mice treated identically as the RGS2-SMC-KO were used as controls. Telemetric blood pressure (BP) studies in ambulatory mice demonstrated that BP was significantly higher in RGS2-SMC-KO vs. control mice (135 ± 5 vs. 122 ± 7 mmHg, respectively. P <0.01), with no change in heart rate. Ex vivo aortic rings of RGS2-SMC-KO mice were hypercontractile in response to phenylephrine (PE) (138 ± 6 vs. 124±4% of pre-existing tone at 6 mM PE, respectively. P <0.05) Hemodynamic studies in anesthetized RGS2-SMC-KO mice revealed increased total systemic vascular resistance (SVR; 3.22x10 4 ± 0.01x10 4 vs. 1.61x10 4 ± 0.55x10 4 , respectively. P <0.001), and significantly higher SBP (106 ± 5 vs. 93 ± 1 mmHg, respectively. P <0.01). Following treatment with the NOS inhibitor L-NAME, BP decreased less in the RGS2-SMC-KO than in the control mice (⋔SBP: 38 ± 7 vs. 47 ± 3 mmHg, P <0.001). We found identical BP responses to changes in dietary sodium intake in the RGS2-SMC-KO mice vs. control, supporting normal renal function in the RGS2-SMC-KO mice. These data support that SMC-specific deletion of RGS2 leads to hypertension, with accompanying loss of RGS2-dependent attenuation of contractile signaling in blood vessels, with increased SVR and hypercontractile arterial function. The hypertension in RGS2-SMC-KO mice together with normal sodium handling supports the hypothesis that high BP can arise from a primary abnormality of vascular smooth muscle cell contractile regulation. Taken together, these data have potentially important implications for the diagnosis and treatment of human vascular diseases and suggest RGS2 as a new therapeutic target for some forms of hypertension.

Testosterone regulates smooth muscle contractile pathways in the rat prostate: emphasis on PDE5 signaling

Testosterone (T) plays a permissive role in the development of benign prostatic hyperplasia (BPH), and phosphodiesterase 5 inhibitors (PDE5is) have been found to be effective for BPH and lower urinary tract symptoms (LUTS) in clinical trials. This study investigated the effect of T on smooth muscle (SM) contractile and regulatory signaling pathways, including PDE5 expression and functional activity in prostate in male rats (sham-operated, surgically castrated, and castrated with T supplementation). In vitro organ bath studies, real-time RT-PCR, Western blot analysis, and immunohistochemistry were performed. Castration heavily attenuated contractility, including sensitivity to phenylephrine with SM myosin immunostaining revealing a disrupted SM cell arrangement in the stroma. PDE5 was immunolocalized exclusively in the prostate stroma, and orchiectomy signficantly reduced PDE5 immunopositivity, mRNA, and protein expression, along with nNOS and ROKβ mRNA, whereas it increased eNOS plus α(1a) and α(1b) adrenoreceptor expression in castrated animals. The PDE5i zaprinast significantly increased prostate strip relaxation to the nitric oxide donor sodium nitroprusside (SNP) in control but not castrated rats. But SNP alone was more effective on castrated rats, comparable with sham treated with SNP plus zaprinast. T supplementation prevented or restored all above changes, including SNP and zaprinast in vitro responsiveness. In conclusion, our data show that T positively regulates PDE5 expression and functional activities in prostate, and T ablation not only suppresses prostate size but also reduces prostatic SM contractility, with several potential SM contraction/relaxation pathways implicated. Zaprinast findings strongly suggest a major role for PDE5/cGMP in this signaling cascade. PDE5 inhibition may represent a novel mechanism for treatment of BPH.

The promise of inhibition of smooth muscle tone as a treatment for erectile dysfunction: where are we now?

Ten years ago, the inhibition of Rho kinase by intracavernosal injection of Y-27632 was found to induce an erectile response. This effect did not require activation of nitric oxide-mediated signaling, introducing a novel target pathway for the treatment of erectile dysfunction (ED), with potential added benefit in cases where nitric oxide bioavailability is attenuated (and thus phosphodiesterase type 5 (PDE5) inhibitors are less efficacious). Rho-kinase antagonists are currently being developed and tested for a wide range of potential uses. The inhibition of this calcium-sensitizing pathway results in blood vessel relaxation. It is also possible that blockade of additional smooth muscle contractile signaling mechanisms may have the same effect. In this review, we conducted an extensive search of pertinent literature using PUBMED. We have outlined the various pathways involved in the maintenance of penile smooth muscle tone and discussed the current potential benefit for the pharmacological inhibition of these targets for the treatment of ED.

S100A1 Genetically Targeted Therapy Reverses Dysfunction of Human Failing Cardiomyocytes

This study investigated the hypothesis whether S100A1 gene therapy can improve pathological key features in human failing ventricular cardiomyocytes (HFCMs). Depletion of the Ca²⁺-sensor protein S100A1 drives deterioration of cardiac performance toward heart failure (HF) in experimental animal models. Targeted repair of this molecular defect by cardiac-specific S100A1 gene therapy rescued cardiac performance, raising the immanent question of its effects in human failing myocardium. Enzymatically isolated HFCMs from hearts with severe systolic HF were subjected to S100A1 and control adenoviral gene transfer and contractile performance, calcium handling, signaling, and energy homeostasis were analyzed by video-edge-detection, FURA2-based epifluorescent microscopy, phosphorylation site-specific antibodies, and mitochondrial assays, respectively. Genetically targeted therapy employing the human S100A1 cDNA normalized decreased S100A1 protein levels in HFCMs, reversed both contractile dysfunction and negative force-frequency relationship, and improved contractile reserve under beta-adrenergic receptor (β-AR) stimulation independent of cAMP-dependent (PKA) and calmodulin-dependent (CaMKII) kinase activity. S100A1 reversed underlying Ca²⁺ handling abnormalities basally and under β-AR stimulation shown by improved SR Ca²⁺ handling, intracellular Ca²⁺ transients, diastolic Ca²⁺ overload, and diminished susceptibility to arrhythmogenic SR Ca²⁺ leak, respectively. Moreover, S100A1 ameliorated compromised mitochondrial function and restored the phosphocreatine/adenosine-triphosphate ratio. Our results demonstrate for the first time the therapeutic efficacy of genetically reconstituted S100A1 protein levels in HFCMs by reversing pathophysiological features that characterize human failing myocardium. Our findings close a gap in our understanding of S100A1's effects in human cardiomyocytes and strengthen the rationale for future molecular-guided therapy of human HF.

Rho inhibitor prevents ischemia–reperfusion injury in rat steatotic liver

Hepatic stellate cells are thought to play a role in modulating intrahepatic vascular resistance based on their capacity to contract via Rho signaling. We investigated the effect of a Rho-kinase inhibitor on ischemia-reperfusion injury in the steatotic liver. Steatotic livers, induced by a choline-deficient diet in rats, were subjected to ischemia-reperfusion injury. Hepatic stellate cells isolated from steatotic livers were analyzed for contractility and Rho signaling activity. The portal pressure of the perfused rat liver and the survival rate after ischemia-reperfusion were also investigated. Hepatic stellate cells from steatotic livers showed increased contractility and upregulation of Rho-kinase 2 compared with those from normal livers. Furthermore, endothelin-1 significantly enhanced the contractility and phosphorylation level of myosin light chain and cofilin in hepatic stellate cells isolated from steatotic livers. A specific Rho-kinase inhibitor, fasudil, significantly suppressed the contractility and decreased the phosphorylation levels of myosin light chain and cofilin. Serum levels of endothelin-1 were markedly increased after IR in rats with steatotic livers, whereas fasudil significantly decreased endothelin-1 serum levels. Rats with steatotic livers showed a significant increase in portal perfusion pressure after ischemia-reperfusion and a significant decrease in survival rate; fasudil treatment significantly reduced these effects. Activation of Rho/Rho-kinase signaling in hepatic stellate cells isolated from steatotic livers is associated with an increased susceptibility to ischemia-reperfusion injury. A Rho-kinase inhibitor attenuated the activation of hepatic stellate cells isolated from steatotic livers and improved ischemia-reperfusion injury in steatotic rats.

Update on Corpus Cavernosum Smooth Muscle Contractile Pathways in Erectile Function: A Role for Testosterone?

Normal erectile function (EF) involves a coordinated relaxation of the arteries that supply the penis and the corpus cavernosum smooth muscle (CCSM), resulting in expansion of the sinusoids and increased intracavernous pressure. But the CCSM spends the majority of its time in the contracted state which is mediated by norepinephrine released from nerve endings and other vasoconstrictors like endothelins released from the endothelium. These agents cause smooth muscle myosin (SMM) phosphorylation by elevating intracellular calcium. When calcium returns to basal levels, the calcium sensitivity increases and prevents myosin dephosphorylation, which involves the RhoA/Rho-kinase (ROK) mechanism, thus maintaining force. Although mounting evidences demonstrate that androgens have a major influence on EF that is not just centrally mediated, this notion remains quite controversial. To summarize the current knowledge on CCSM contractile pathways, the role they play in modulating EF, and the influence of androgens. The article reviews the literature and contains some previously unpublished data on CCSM contraction signaling including the role that androgens are known to play in modulating these pathways. Data from peer-reviewed publications and previously unpublished observations. In addition to downregulation of many pro-erectile molecular mechanisms, decreased testosterone (T) levels upregulate CCSM contractility, including hyperresponsiveness to α-adrenergic agonists, increased SMM phosphorylation, alteration of SMM isoform composition, activation of RhoA/ROK signaling and modulation of sphingosine-1-phosphate regulation of CCSM tone. Decreased T levels upregulate CCSM contractile signaling. Meanwhile, it downregulates CCSM relaxation pathways synergizing to produce erectile dysfunction (ED). Although some urologists and researchers are still skeptical of the influence of androgens on penile erection, understanding these molecular control mechanisms as well as the influence that androgens have on these pathways should provide new evidence supporting the roles of androgens in EF and enhance the discovery of novel targets for drug development to treat ED.

Abstract LB-96: Extrinsic and intrinsic force and GBM aggression

Abstract Despite concerted effort the mean survival time for patients with aggressive GBM remains unchanged with a mean of 14 months. Indeed, high grade GBMs are notoriously resistant to therapy and typically disseminate throughout the brain tissue, severely compromising patient treatment. In this respect, the most lethal GBMs arise within the SVZ region of the brain which is rich in stem cells. This has lead to speculation that GBM aggression is related to their stem-like origin. Consistently, using AFM indentation we determined that analogous to neural stem cells (NSCs), high grade GBMs are very soft and express high levels of the Notch regulated gene HES-1. We also found that high grade GBMs and NSCs both express elevated levels of the stemness-promoting repressor NCoR2. We further determined that NCoR2 expression correlates with poor GBM patient prognosis, which is an observation that accords with our recent finding that NCoR2 enhances breast tumor survival in response to radiation, chemotherapy and immune activators in vitro and in vivo. These findings are consistent with a stem-like origin for GBMs and identify NCoR2 as a putative unique survival mechanism that could explain the treatment resistance of this disease. Interestingly, AFM measurements of freshly excised murine brain tumor slices showed that GBMs arising within the SVZ region are also quite stiff. Preliminary findings also showed that isolated high grade GBM cells but not low grade oligodendroglioma cells have a greatly enhanced mechano-responsiveness e.g. they spread more on soft gels and possess a vastly altered glycocalyx; traits that predict elevated contractility and integrin signaling. Given MRI data indicating very aggressive GBMs in the SVZ region are highly vascularized and experience elevated compression the findings support the idea that tumors that arise within this highly mechanically-challenged SVZ microenvironment have a unique mechano-behavior that could facilitate their growth, survival and dissemination. Indeed, the high compliance, contractility and mechanical resistance of GBMs would facilitate their growth, survival and expansion within compressed brain tissue and would foster their ability to navigate and disseminate into the dense GBM-associated ECM. Because reducing tumor force can revert the malignant phenotype of cancerous tissue and will reduce tumor cell invasion, growth, and survival and inhibit tumor progression and decrease tumor incidence we predict that the altered force microenvironment and intrinsic mechanobehavior of GBMs contribute to their aggression. Consequently, studies are now underway to clarify the interplay between cell and tissue force and GBM behavior and to determine the relevance of NCoR2 to GBM treatment resistance. (Supp by: NCI grants U54CA143836–01 and NIH/NCI R01 CA138818–01A1 to V.M.W. and a National Science Foundation (GRFP) Fellowship to Y.A.M.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-96. doi:10.1158/1538-7445.AM2011-LB-96

Regulation of contractility and metabolic signaling by the β2-adrenergic receptor in rat ventricular muscle

AimsCardiac function is modulated by the sympathetic nervous system through β-adrenergic receptor (β-AR) activity and this represents the main regulatory mechanism for cardiac performance. To date, however, the metabolic and molecular responses to β2-agonists are not well characterized. Therefore, we studied the inotropic effect and signaling response to selective β2-AR activation by tulobuterol. Main methodsStrips of rat right ventricle were electrically stimulated (1Hz) in standard Tyrode solution (95% O2, 5% CO2) in the presence of the β1-antagonist CGP-20712A (1μM). A cumulative dose–response curve for tulobuterol (0.1–10μM), in the presence or absence of the phosphodiesterase (PDE) inhibitor IBMX (30μM), or 10min incubation (1μM) with the β2-agonist tulobuterol was performed. Key findingsβ2-AR stimulation induced a positive inotropic effect (maximal effect=33±3.3%) and a decrease in the time required for half relaxation (from 45±0.6 to 31±1.8ms, −30%, p<0.001) after the inhibition of PDEs. After 10min of β2-AR stimulation, p-AMPKαT172 (54%), p-PKBT308 (38%), p-AS160T642 (46%) and p-CREBS133 (63%) increased, without any change in p-PKAT197. SignificanceThese results suggest that the regulation of ventricular contractility is not the primary function of the β2-AR. Rather, β2-AR could function to activate PKB and AMPK signaling, thereby modulating muscle mass and energetic metabolism of rat ventricular muscle.

Differences in the Expression of Multi‐Drug Resistant Protein 5 (MRP5) and Regulation of cGMP Levels in Phasic and Tonic Smooth Muscles

Previous studies have identified differences in the expression of contractile proteins and signaling pathways that regulate myosin light chain phosphorylation and contraction in phasic and tonic smooth muscle. cGMP plays a critical role in smooth muscle relaxation and the intracellular levels of cGMP are regulated by its hydrolysis via phosphodiesterase 5 (PDE5) and efflux via MRP5. AIM To determine the role of MRP5 in the regulation of cGMP levels in proximal versus distal stomach. METHODS Expression of MRP5 was analyzed by quantitative RT-PCR and western blot. Increase in cGMP levels in response to sodium nitroprusside (SNP) was measured in the presence of the PDE5 inhibitor, zaprinast. RESULTS Both mRNA and protein expression of MRP5 was 4-fold higher in muscle cells from proximal stomach than distal stomach. MRP5 was co-immunoprecipitated with caveolin-1, the main caveolar protein. In both proximal and distal stomach, SNP caused an increase in intra-and extracellular cGMP levels. The increase in extracellular cGMP was significantly higher than intracellular cGMP in muscle cells of proximal stomach, whereas extra- and intracellular cGMP levels were not significantly different in muscle cells of distal stomach. CONCLUSION Higher expression and function of MRP5, which provides a rapid mechanism for regulation of cGMP levels, may further contribute to the tonic contractile phenotype of proximal stomach.

Agonist‐Induced Rho Kinase and ZIP kinase Activity Levels in Different Regions of the Stomach

The molecular events that regulate the tonic and phasic phenotypes of smooth muscle are attributed to differential expression of contractile proteins and signaling pathway that regulate MLC kinase and MLC phosphatase activity, and thus MLC20 phosphorylation. The activity of MLC phosphatase is inhibited by Rho kinase/ZIP kinase-dependent pathways. AIM To determine whether Rho kinase and ZIP kinase activity levels correlate with the contractile phenotypes in proximal (tonic) versus distal (phasic) stomach. METHODS Muscle cells were isolated separately from proximal and distal regions of rabbit stomach. Acetylcholine (ACh)-induced stimulation of Rho kinase and ZIP kinase activity was determined by immunokinase assay. RESULTS ACh (0.1 μM) caused stimulation of Rho kinase and ZIP kinase activity in muscle cells of both proximal and distal stomach. However, stimulation of both kinases was significantly higher in muscle cells from proximal stomach compared to distal stomach. Y27632, a selective blocker of Rho kinase, inhibited stimulation of ZIP kinase activity suggesting that stimulation of ZIP kinase activity is dependent on Rho kinase activity. CONCLUSION Higher activity levels of Rho kinase and ZIP kinase, which normally inhibit MLC phosphatase and increase MLC20 phosphorylation, in proximal stomach correlate with its tonic phenotype compared to the distal stomach that exhibits phasic phenotype.

Arrestins differentially regulate histamine‐ and oxytocin‐evoked phospholipase C and mitogen‐activated protein kinase signalling in myometrial cells

The uterotonins oxytocin and histamine, mediate contractile signals through specific G protein-coupled receptors, a process which is tightly controlled during gestation to prevent preterm labour. We previously identified G protein-coupled receptor kinase (GRK)2 and GRK6 as respective cardinal negative regulators of histamine H(1) and oxytocin receptor signalling. GRK-mediated phosphorylation promotes arrestin recruitment, not only desensitizing receptors but activating an increasing number of diverse signalling pathways. Here we investigate potential roles that arrestins play in the regulation of myometrial oxytocin/histamine H(1) receptor signalling. Endogenous arrestins2 and 3 were specifically depleted using RNA-interference in a human myometrial cell line and the consequences of this for G protein-coupled receptor-mediated signalling were assessed using Ca(2+) /inositol 1,4,5-trisphophate imaging and standard mitogen-activated protein kinase (MAPK) assays. Depletion of arrestin3, but not arrestin2 enhanced and prolonged H(1) receptor-stimulated Ca(2+) responses, whilst depletion of either arrestin increased oxytocin receptor responses. Arrestin3 depletion decreased H(1) receptor desensitization, whilst removal of either arrestin isoform was equally effective in preventing oxytocin receptor desensitization. Following arrestin3 depletion oxytocin-induced phospho-extracellular signal-regulated kinase1/2 signals were diminished and histamine-stimulated signals virtually absent, whereas depletion of arrestin2 augmented extracellular signal-regulated kinase1/2 responses to each agonist. Conversely, depletion of arrestin3 enhanced p38 signals to each agonist, whilst arrestin2 suppression increased oxytocin-, but not histamine-induced p38 MAPK responses. Arrestin proteins are key regulators of H(1) and oxytocin receptor desensitization, and play integral roles mediating uterotonin-stimulated MAPK-signalling. These data provide insights into the in situ regulation of these receptor subtypes and may inform pathophysiological functioning in preterm labour.

Alterations of Membrane Currents, Contractility and Calcium Signaling in Gq/G11 Single and Double Ko Mice

In contrast to its role in pathophysiology very little is known about the role of the Gq/G11 signaling cassette in the physiology of cardiac myocytes. We have generated transgenic mice combining a G11-KO approach with the Cre-recombinase tool to knock-down (KD) Gq in a tissue-specific inducible manner. Western-blot analysis depict the rapid loss of Gq protein in less than 2 weeks while the use of a reporter mouse substantiated tissue specificity. We analyzed the effects of (i) Cre expression, (ii) vehicle/Tamoxifen injection, (iii) G11-KO and (iv) Gq-KD on resting potential and capacitance, action potentials (AP) and I(to) by patch-clamping ventricular myocytes. Calcium homeostasis was investigated by Fura2 video-imaging and contraction behavior by measuring sarcomere length. While basic cellular parameters were largely unchanged (cell capacitance, resting membrane potential) in all genotypes and treatments, AP, calcium handling and contractile behavior were altered by vehicle/Tamoxifen injection, G11-KO and Gq-KD. Vehicle injection alone caused upregulation of I(to) and shortening of APs similar to G11-KO. In contrast additional Gq-KD, restorated“ AP properties and I(to). Vehicle injection itself into wt mice caused increases in basal calcium that were absent after Tamoxifen injection, Cre expression had mild effects on calcium handling. Tamoxifen injection and Gq/G11-KO caused major changes in calcium handling and contractile behavior. In conclusion, despite the broad application of the Cre-recombinase system, care has to be taken for possible effects of Cre expression and vehicle/Tamoxifen injection. The Gq/G11 signaling system plays an important role in the physiological modulation of proteins involved in electrophysiology and calcium homeostasis most likely regulated by basal levels of neurohormonal stimulation.This work was supported by the DFG (KFO196) and the Medical Faculty (HOMFOR).

Substance P Activates Both Contractile and Inflammatory Pathways in Lymphatics Through the Neurokinin Receptors NK1R and NK3R

The aim of this study was to elucidate the molecular signaling mechanisms by which substance P (SP) modulates lymphatic muscle contraction and to determine whether SP stimulates both contractile as well as inflammatory pathways in the lymphatics. A rat mesenteric lymphatic muscle cell culture model (RMLMCs) and known specific pharmacological inhibitors were utilized to delineate SP-mediated signaling pathways in lymphatics. We detected expression of neurokinin receptor 1 (NK1R) and neurokinin receptor 3 (NK3R) in RMLMCs. SP stimulation increased phosphorylation of myosin light chain 20 (MLC₂₀) as well as p38 mitogen associated protein kinase (p38-MAPK) and extracellular signal regulated kinase (ERK1/2) indicating activation of both a contractile and a pro-inflammatory MAPK pathway. Pharmacological inhibition of both NK1R and NK3R significantly affected the downstream SP signaling. We further examined whether there was any crosstalk between the two pathways upon SP stimulation. Inhibition of ERK1/2 decreased levels of p-MLC₂₀ after SP activation, in a PKC dependent manner, indicating a potential crosstalk between these two pathways. These data provide the first evidence that SP-mediated crosstalk between pro-inflammatory and contractile signaling mechanisms exists in the lymphatic system and may be an important bridge between lymphatic function modulation and inflammation.

Modulation of cardiac contractility by serine/threonine protein phosphatase type 5

BackgroundProtein phosphatase 5 (PP5) a serine/threonine phosphatase is ubiquitously expressed in mammalian tissues including the heart, but its physiological role in the heart is still unknown. Therefore, we used a transgenic mouse model to get a first insight into the cardiac role of PP5. Methods and resultsWe generated transgenic mice with cardiac myocyte specific overexpression of PP5. Successful overexpression of PP5 was demonstrated by Western blotting, immunohistochemistry and enhanced arachidonic acid-stimulated protein phosphatase activity in transgenic hearts. Cardiac function was examined on the level of isolated cardiac myocytes, isolated organs and in intact animals. Whereas Ca2+ transients and cell shortening remained unchanged, L-type Ca2+ currents were decreased in isolated cardiac myocytes from transgenic mice. Ventricular contractility was reduced in isolated perfused hearts under basal conditions and after β-adrenergic stimulation. In intact animals, echocardiography revealed increased left ventricular diameters and decreased contractility and invasively measured hemodynamic performance by left ventricular catheterization demonstrated a reduced response to β-adrenergic stimulation in transgenic mice compared to wild type. ConclusionsOverexpression of PP5 affected contractility and β-adrenergic signaling in the hearts of transgenic mice. Taken together, these findings are indicative of a regulatory role of PP5 in cardiac function.

Src-dependent phosphorylation of ROCK participates in regulation of focal adhesion dynamics

When a cell migrates, the RhoA-ROCK-mediated contractile signal is suppressed in the leading edge to allow dynamic adhesions for protrusion. However, several studies have reported that RhoA is indeed active in the leading edge of a migrating cell during serum stimulation. Here, we present evidence that regulation of ROCKII phosphorylation at the Y722 site in peripheral focal contacts is crucial for controlling the turnover of the focal adhesion (FA) complex uncoupled from RhoA activation during serum-stimulated migration. However, this phosphorylation control is dispensable for migration when RhoA is downregulated in cells treated with platelet-derived growth factor (PDGF). We further present evidence that ROCKII is phosphorylated by Src in FAs and this phosphorylation event decreases RhoA binding activity of ROCKII. Lack of this regulatory control leads to sustained myosin-mediated contractility and FA elongation during lysophosphatidic acid (LPA) stimulation. Altogether, our data suggest that Src-dependent ROCKII phosphorylation provides a means of tuning contractility required for FAs dynamics when RhoA is active.