Lidocaine inhibits migration of tenocytes by downregulating focal adhesion kinase and paxillin phosphorylation.

Lidocaine is the most frequently applied local infiltration anesthetic agent for treating tendinopathies. However, studies have discovered lidocaine to negatively affect tendon healing. In the current study, the molecular mechanisms and effects of lidocaine on tenocyte migration were evaluated. We treated tenocytes intrinsic to the Achilles tendons of Sprague-Dawley rats with lidocaine. The migration ability of cells was analyzed using electric cell-substrate impedance sensing (ECIS) and scratch wound assay. We then used a microscope to evaluate the cell spread. We assessed filamentous actin (F-actin) cytoskeleton formation through immunofluorescence staining. In addition, we used Western blot analysis to analyze the expression of phospho-focal adhesion kinase (FAK), FAK, phospho-paxillin, paxillin, and F-actin. We discovered that lidocaine had an inhibitory effect on the migration of tenocytes in the scratch wound assay and on the ECIS chip. Lidocaine treatment suppressed cell spreading and changed the cell morphology and F-actin distribution. Lidocaine reduced F-actin formation in the tenocyte during cell spreading; furthermore, it inhibited phospho-FAK, F-actin, and phospho-paxillin expression in the tenocytes. Our study revealed that lidocaine inhibits the spread and migration of tenocytes. The molecular mechanism potentially underlying this effect is downregulation of F-actin, phospho-FAK, and phospho-paxillin expression when cells are treated with lidocaine.

Autophagy-related gene PXN as a prognostic marker: Promotion of ovarian cancer progression by suppressing the p110ß/Vps34/Beclin1 pathway.

Among gynecological malignancies, ovarian cancer has the highest mortality rate and has sparked widespread interest in studying the mechanisms underlying ovarian cancer development. Based on TCGA and GEO databases, we investigated the highly expressed autophagy-related genes that determine patient prognosis using limma differential expression and Kaplan-Meier survival analyses. The biological processes associated with these genes were also predicted using GO/KEGG functional enrichment analysis. CCK-8, cell scratch, and transwell assays were used to investigate the effects of PXN on the proliferation, migration, and invasion abilities of ovarian cancer cells. Transmission electron microscopy was used to observe the autophagosomes. The expression of autophagy proteins and the PI3K/Akt/mTOR and p110ß/Vps34/Beclin1 pathway proteins in ovarian cancer cells was detected using western blot; autophagy protein expression was further detected and localized using cellular immunofluorescence. A total of 724 autophagy-related genes were found to be overexpressed in ovarian -cancer tissues, with high expression of PEX3, PXN, and RB1 associated with poor prognosis in patients (p < .05). PXN activates and regulates signaling pathways related to cellular autophagy, ubiquitination, lysosomes, PI3K-Akt, and mTOR. Autophagosomes were observed in all cell groups. The increase in PXN gene expression promoted the proliferation, migration, and invasion of ovarian cancer cells, increased the expression of SQSTM1/p62 protein, decreased LC3II/LC3Ⅰ, inhibited the phosphorylation of Akt and mTOR proteins, and suppressed the expression of PI3K(p110ß) and Beclin1 proteins. The decrease in PXN expression also confirmed these changes. Thus, PXN is highly expressed during ovarian cancer and is associated with poor patient prognosis. It may promote ovarian cancer cell proliferation, migration, and invasion by inhibiting cellular autophagy via suppression of the p110ß/Vps34/Beclin1 pathway.

[Lutein inhibits the adhesion, invasiveness and metastasis of human prostate cancer PC-3M cells].

OBJECTIVE: To explore the effects of lutein on the adhesion, invasiveness and metastasis of human prostate cancer PC-3M cells and its action mechanism. METHODS: We divided human prostate cancer PC-3M cells into a control, a low-dose lutein, a medium-dose lutein and a high-dose lutein group, and treated them with 0, 10, 20 and 40 µmol/L lutein, respectively. Then we examined the adhesion of the cells to matrix by cell adhesion assay and the changes in cell pseudopodia by Phalloidin staining, detected the expressions of paxillin, matrix metalloproteinase 2 (MMP-2), MMP-9, recombinant tissue inhibitors of metalloproteinase 1 (TIMP-1), E-cadherin, N-cadherin and vimentin by Western blot, determined the invasiveness and migration of the cells by scratch and Transwell assays, and observed their dynamic movement by high-intension imaging. RESULTS: Compared with the control, the lutein intervention groups showed significant reduction in the number of the cells adhered to matrix, the number of cell pseudopodia, the expressions of paxillin, MMP-2, MMP-9, N-cadherin and vimentin, the rates of migration, invasion and metastasis, and the distances of displacement and movement of the cells. However, the expressions of TIMP-1 and epithelial-mesenchymal transition-related E-cadherin were upregulated significantly. CONCLUSION: Lutein can inhibit cell adhesion, reduce the expressions of MMPs, and suppress cell invasion and migration by inhibiting the process of epithelial-mesenchymal transition.

The Promotion of Migration and Myogenic Differentiation in Skeletal Muscle Cells by Quercetin and Underlying Mechanisms.

Aging and muscle disorders frequently cause a decrease in myoblast migration and differentiation, leading to losses in skeletal muscle function and regeneration. Several studies have reported that natural flavonoids can stimulate muscle development. Quercetin, one such flavonoid found in many vegetables and fruits, has been used to promote muscle development. In this study, we investigated the effect of quercetin on migration and differentiation, two processes critical to muscle regeneration. We found that quercetin induced the migration and differentiation of mouse C2C12 cells. These results indicated quercetin could induce myogenic differentiation at the early stage through activated p-IGF-1R. The molecular mechanisms of quercetin include the promotion of myogenic differentiation via activated transcription factors STAT3 and the AKT signaling pathway. In addition, we demonstrated that AKT activation is required for quercetin induction of myogenic differentiation to occur. In addition, quercetin was found to promote myoblast migration by regulating the ITGB1 signaling pathway and activating phosphorylation of FAK and paxillin. In conclusion, quercetin can potentially be used to induce migration and differentiation and thus improve muscle regeneration.

Phyllanthus taxodiifolius Beille Disrupted N-cadherin, Vimentin, Paxillin and Actin Stress Fibers in Glioblastoma.

OBJECTIVE: Glioblastoma is the most aggressive and lethal brain tumor in adults with highly invasive properties. In this present study, we explored the effects of Phyllanthus taxodiifolius Beille extract on molecules known to be hallmarks of aggressive glioblastoma including N-cadherin and vimentin, mesenchymal markers, as well as paxillin, a major adaptor protein that regulates the linking of focal adhesions to the actin cytoskeleton. METHODS: P. taxodiifolius were air-dried, powdered and percolated with methanol, filtered, concentrated and lyophilized to yield a crude methanol extract. C6 glioblastoma cell line was used in this study. The expression of N-cadherin and vimentin, as well as the activation of paxillin was determined using Western blot analysis. The effect of the extract on focal adhesions and actin cytoskeleton were investigated using immunofluorescence staining and confocal imaging. RESULTS: In the presence of 40 µg/ml Phyllanthus taxodiifolius Beille extract, the expression of N-cadherin and vimentin were significantly decreased (p<0.001 and p<0.05, respectively). Activation of paxillin was also diminished as indicated by a reduction of phosphorylated-paxillin (p<0.01). Consequently, actin stress fibers in glioblastoma cells were abolished as evidenced by the decrease in focal adhesion (p<0.001) and stress fibers numbers (p<0.001). CONCLUSION: Our study demonstrates for the first time that P. taxodiifolius interferes with multiple key molecules related to pathological hallmarks of glioblastoma. These molecules are involved with cell contacts, focal adhesions, and the formation and stabilization of actin stress fibers, which are required for glioblastoma metastatic behavior. These results provide further evidence supporting the potential of P. taxodiifolius and its bioactive compounds as anti-cancer agents.

IP3R-mediated activation of BK channels contributes to mGluR5-induced protection against spinal cord ischemia-reperfusion injury.

Spinal cord ischemia-reperfusion injury (SCIRI) can cause dramatic neuron loss and lead to paraplegia in patients. In this research, the role of mGluR5, a member of the metabotropic glutamate receptors (mGluRs) family, was investigated both in vitro and in vivo to explore a possible method to treat this complication. In vitro experiment, after activating mGluR5 via pretreating cells with (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG) and 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB), excitotoxicity induced by glutamate (Glu) was attenuated in primary spinal cord neurons, evidenced by higher neuron viability, decreased lactate dehydrogenase (LDH) release and less detected TUNEL-positive cells. According to Western Blot (WB) results, Glu treatment resulted in a high level of large-conductance Ca2+- and voltage-activated K+ (BK) channels, with activation relying on the mGluR5-IP3R (inositol triphosphate) pathway. In vivo part, a rat model of SCIRI was built to further investigate the role of mGluR5. After pretreating them with CHPG and CDPPB, the rats showed markedly lower spinal water content, attenuated motor neuron injury in the spinal cord of L4 segments, and better neurological function. This effect could be partially reversed by paxilline, a blocker of BK channels. In addition, activating BK channels alone using specific openers: NS1619 or NS11021 can protect spinal cord neurons from injury induced by either SCIRI or Glu. In conclusion, in this research, we proved that mGluR5 exerts a protective role in SCIRI, and this effect partially works via IP3R-mediated activation of BK channels.

Inhibition of Potassium Channels Affects the Ability of Pig Spermatozoa to Elicit Capacitation and Trigger the Acrosome Exocytosis Induced by Progesterone.

During capacitation, sperm undergo a myriad of changes, including remodeling of plasma membrane, modification of sperm motility and kinematic parameters, membrane hyperpolarization, increase in intracellular calcium levels, and tyrosine phosphorylation of certain sperm proteins. While potassium channels have been reported to be crucial for capacitation of mouse and human sperm, their role in pigs has not been investigated. With this purpose, sperm samples from 15 boars were incubated in capacitation medium for 300 min with quinine, a general blocker of potassium channels (including voltage-gated potassium channels, calcium-activated potassium channels, and tandem pore domain potassium channels), and paxilline (PAX), a specific inhibitor of calcium-activated potassium channels. In all samples, acrosome exocytosis was induced after 240 min of incubation with progesterone. Plasma membrane and acrosome integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and total and progressive sperm motility were evaluated after 0, 120, and 240 min of incubation, and after 5, 30, and 60 min of progesterone addition. Although blocking potassium channels with quinine and PAX prevented sperm to elicit in vitro capacitation by impairing motility and mitochondrial function, as well as reducing intracellular calcium levels, the extent of that inhibition was larger with quinine than with PAX. Therefore, while our data support that calcium-activated potassium channels are essential for sperm capacitation in pigs, they also suggest that other potassium channels, such as the voltage-gated, tandem pore domain, and mitochondrial ATP-regulated ones, are involved in that process. Thus, further research is needed to elucidate the specific functions of these channels and the mechanisms underlying its regulation during sperm capacitation.

Extra Virgin Olive Oil Phenols Vasodilate Rat MesentericResistance Artery via Phospholipase C (PLC)-CalciumMicrodomains-Potassium Channels (BKCa) Signals.

Recent evidence suggests that the reason Extra Virgin Olive Oil (EVOO) lowers blood pressure and reduces the risk of developing hypertension is partly due to minor components of EVOO, such as phenols. However, little is still known about the mechanism(s) through which EVOO phenols mediate anti-hypertensive effects. The aim of the present study was to investigate the mechanisms of action of EVOO phenols on mesenteric resistance arteries. A pressure myograph was used to test the effect of EVOO phenols on isolated mesenteric arteries in the presence of specific inhibitors of: 1) BKca channels (Paxillin, 10-5 M); 2) L-type calcium channels (Verapamil, 10-5 M); 3) Ryanodine receptor, RyR (Ryanodine, 10-5 M); 4) inositol 1,4,5-triphosphate receptor, IP3R, (2-Aminoethyl diphenylborinate, 2-APB, 3 × 10-3 M); 5) phospholipase C, PLC, (U73122, 10-5 M), and 6) GPCR-Gαi signaling, (Pertussis Toxin, 10-5 M). EVOO phenols induced vasodilation of mesenteric arteries in a dose-dependent manner, and this effect was reduced by pre-incubation with Paxillin, Verapamil, Ryanodine, 2-APB, U73122, and Pertussis Toxin. Our data suggest that EVOO phenol-mediated vasodilation requires activation of BKca channels potentially through a local increase of subcellular calcium microdomains, a pivotal mechanism on the base of artery vasodilation. These findings provide novel mechanistic insights for understanding the vasodilatory properties of EVOO phenols on resistance arteries.

Paxillin regulated genomic networks in prostate cancer.

Paxillin is extensively involved in focal adhesion signaling and kinase signaling throughout the plasma membrane and cytoplasm. However, recent studies in prostate cancer suggest that paxillin also plays a critical role in regulating gene expression within the nucleus, serving as a liaison between cytoplasmic and nuclear MAPK and Androgen Receptor (AR) signaling. Here we used RNA-seq to examine the paxillin-regulated transcriptome in several human prostate cancer cell lines. First, we examined paxillin effects on androgen-mediated transcription in control or paxillin-depleted AR-positive LNCaP and C4-2 human prostate cancer cells. In androgen-dependent LNCaP cells, we found over 1000 paxillin-dependent androgen-responsive genes, some of which are involved in endocrine therapy resistance. Most paxillin-dependent AR-mediated genes in LNCaP cells were no longer paxillin-dependent in androgen-sensitive, castration-resistant C4-2 cells, suggesting that castration-resistance may markedly alter paxillin effects on genomic AR signaling. To examine the paxillin-regulated transcriptome in the absence of androgen signaling, we performed RNA-seq in AR-negative PC3 human prostate cancer cells. Paxillin enhanced several pro-proliferative pathways, including the CyclinD/Rb/E2F and DNA replication/repair pathways. Additionally, paxillin suppressed pro-apoptotic genes, including CASP1 and TNFSF10. Quantitative PCR confirmed that these pathways are similarly regulated by paxillin in LNCaP and C4-2 cells. Functional studies showed that, while paxillin stimulated cell proliferation, it had minimum effect on apoptosis. Thus, paxillin appears to be an important transcriptional regulator in prostate cancer, and analysis of its transcriptome might lead to novel approaches toward the diagnosis and treatment of this important disease.

Potassium channels-mediated electrophysiologic responses are inhibited by cytosolic phospholipase A2α ablation.

Cytosolic phospholipase A2α (cPLA2α) is implicated in the progression of excitotoxic neuronal injury and cerebral ischemia. Previous work suggests that cPLA2α increases aberrant electrophysiologic events through attenuating K channel functions. Nevertheless, which K channels are affected by cPLA2α needs to be determined. Here we examined K channels-mediated electrophysiologic responses in hippocampal CA1 pyramidal neurons from wild-type and cPLA2α mice using simultaneous patch-clamp recording and confocal Ca imaging. After the exposure to the blockers of Ca-sensitive and A-type K channels, all CA1 neurons developed spike broadening and increased dendritic Ca transients. These effects were occluded in CA1 neurons from cPLA2α mice. Therefore, cPLA2α modulates the functions of Ca-sensitive and A-type K channels in neurotoxicity.

Nerve growth factor sensitizes adult sympathetic neurons to the proinflammatory peptide bradykinin.

Levels of nerve growth factor (NGF) are elevated in inflamed tissues. In sensory neurons, increases in NGF augment neuronal sensitivity (sensitization) to noxious stimuli. Here, we hypothesized that NGF also sensitizes sympathetic neurons to proinflammatory stimuli. We cultured superior cervical ganglion (SCG) neurons from adult male Sprague Dawley rats with or without added NGF and compared their responsiveness to bradykinin, a proinflammatory peptide. The NGF-cultured neurons exhibited significant depolarization, bursts of action potentials, and Ca(2+) elevations after bradykinin application, whereas neurons cultured without NGF showed only slight changes in membrane potential and cytoplasmic Ca(2+) levels. The NGF effect, which requires trkA receptors, takes hours to develop and days to reverse. We addressed the ionic mechanisms underlying this sensitization. NGF did not alter bradykinin-induced M-current inhibition or phosphatidylinositol 4,5-bisphosphate hydrolysis. Maxi-K channel-mediated current evoked by depolarizations was reduced by 50% by culturing neurons in NGF. Application of iberiotoxin or paxilline, blockers of Maxi-K channels, mimicked NGF treatment and sensitized neurons to bradykinin application. A calcium channel blocker also mimicked NGF treatment. We found that NGF reduces Maxi-K channel opening by decreasing the activity of nifedipine-sensitive calcium channels. In conclusion, culture in NGF reduces the activity of L-type calcium channels, and secondarily, the calcium-sensitive activity of Maxi-K channels, rendering sympathetic neurons electrically hyper-responsive to bradykinin.

Pharmacological blockade of the MaxiK channel attenuates experimental acute pancreatitis and associated lung injury in rats.

Increasing evidence has recently demonstrated that soluble heparan sulfate (HS), a degradation product of extracellular matrix produced by elastase, plays a key role in the aggravation of acute pancreatitis (AP) and associated lung injury. However little is known about the detailed mechanism underlying HS-induced inflammatory cascade. Our previous work has provided a valuable clue that a large-conductance K(+) channel (MaxiK) was involved in the HS-stimulated activation of murine macrophages. Here we attempted to ask whether pharmacological inhibition of the MaxiK channel will exert beneficial effects on the treatment of AP and secondary lung injury. The protective effects of paxilline, a specific blocker of MaxiK, on rats against sodium taurocholate induced AP were evaluated. Our data showed that paxilline substantially attenuated AP and resultant lung injury, mainly by limiting the burst of inflammatory responses, as proven by decreased plasma concentrations of tumor necrosis factor-α and macrophage inflammatory protein-2, together with unimpaired pancreatic enzyme activities in rats suffering from AP. Compared with the therapeutic administration, pre-treatment of paxilline showed superior potential to slow down the progress of AP. Furthermore, AP rats received paxilline exhibited improved histopathologic alterations both in the pancreas and the lungs, and even lower lung MPO activity. Taken together, our study provides evidence that MaxiK is involved in the spread of inflammatory responses and the following lung injury during the attack of AP, indicating that this ion channel is a promising candidate as a therapeutic target for AP.

SAH-induced suppression of voltage-gated K(+) (K (V)) channel currents in parenchymal arteriolar myocytes involves activation of the HB-EGF/EGFR pathway.

Potassium channels play an important role in the regulation of arterial tone, and decreased activity of these ion channels has been linked to pial artery vasospasm after subarachnoid hemorrhage (SAH). Our previous work has shown that acute application of a blood component, oxyhemoglobin, caused suppression of voltage-gated K(+) (K(V)) channels through heparin-binding epidermal growth factor-like growth factor (HB-EGF)-mediated activation of epidermal growth factor receptor (EGFR). Using patch clamp electrophysiology, we have now examined whether this pathway of K(V) channel suppression is activated in parenchymal arteriolar myocytes following long-term in vivo exposure to subarachnoid blood. We have found that K(V) currents, but not large conductance Ca(2+) activated or inwardly rectifying K(+) channel currents, were decreased in parenchymal arteriolar myocytes freshly isolated from day 5 SAH model rabbits. Interestingly, parenchymal arteriolar myocytes from control animals were more sensitive to exogenous HB-EGF (half-maximal inhibitory concentration [IC(50)] 0.2 ± 0.4 ng/ml) compared to pial arterial myocytes (IC(50) 2.4 ± 1.3 ng/ml). However, HB-EGF and oxyhemoglobin failed to decrease K(V) currents in parenchymal arteriolar myocytes from SAH animals, consistent with EGFR activation and K(V) current suppression by SAH. These data suggest that HB-EGF/EGFR pathway activation contributes to K(V) current suppression and enhanced parenchymal arteriolar constriction after SAH.

Paxillin contracts the osteoclast cytoskeleton.

Osteoclastic bone resorption depends upon the cell's ability to organize its cytoskeleton via the αvß3 integrin and osteoclastogenic cytokines. Because paxillin associates with αvß3, we asked if it participates in skeletal degradation. Unlike deletion of other αvß3-associated cytoskeleton-regulating molecules, which impairs the cell's ability to spread, paxillin-deficient (Pax(-/-) ) osteoclasts, generated from embryonic stem cells, "superspread" in response to receptor activator of NF-κB ligand (RANKL) and form large, albeit dynamically atypical, actin bands. Despite their increased size, Pax(-/-) osteoclasts resorb bone poorly, excavating pits approximately one-third normal depth. Ligand-occupied αvß3 or RANKL promotes paxillin serine and tyrosine phosphorylation, the latter via cellular sarcoma (c-Src). The abnormal Pax(-/-) phenotype is rescued by wild-type (WT) paxillin but not that lacking its LD4 domain. In keeping with the appearance of mutant osteoclasts, WT paxillin, overexpressed in WT cells, contracts the cytoskeleton. Most importantly, the abnormal phenotype of Pax(-/-) osteoclasts likely represents failed RANKL-mediated delivery of myosin IIA to the actin cytoskeleton via the paxillin LD4 domain but is independent of tyrosine phosphorylation. Thus, in response to RANKL, paxillin associates with myosin IIA to contract the osteoclast cytoskeleton, thereby promoting its bone-degrading capacity.

Src kinases catalytic activity regulates proliferation, migration and invasiveness of MDA-MB-231 breast cancer cells.

SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27(Kip1) expression and reduced c-Myc levels, restraining G1­S transition. In contrast, SU6656 did not modify p27(Kip1) expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer.

Intermittent hypoxia in rats increases myogenic tone through loss of hydrogen sulfide activation of large-conductance Ca(2+)-activated potassium channels.

RATIONALE: Myogenic tone, an important regulator of vascular resistance, is dependent on vascular smooth muscle (VSM) depolarization, can be modulated by endothelial factors, and is increased in several models of hypertension. Intermittent hypoxia (IH) elevates blood pressure and causes endothelial dysfunction. Hydrogen sulfide (H(2)S), a recently described endothelium-derived vasodilator, is produced by the enzyme cystathionine γ-lyase (CSE) and acts by hyperpolarizing VSM. OBJECTIVE: Determine whether IH decreases endothelial H(2)S production to increase myogenic tone in small mesenteric arteries. METHODS AND RESULTS: Myogenic tone was greater in mesenteric arteries from IH than sham control rat arteries, and VSM membrane potential was depolarized in IH in comparison with sham arteries. Endothelium inactivation or scavenging of H(2)S enhanced myogenic tone in sham arteries to the level of IH. Inhibiting CSE also enhanced myogenic tone and depolarized VSM in sham but not IH arteries. Similar results were seen in cerebral arteries. Exogenous H(2)S dilated and hyperpolarized sham and IH arteries, and this dilation was blocked by iberiotoxin, paxilline, and KCl preconstriction but not glibenclamide or 3-isobutyl-1-methylxanthine. Iberiotoxin enhanced myogenic tone in both groups but more in sham than IH. CSE immunofluorescence was less in the endothelium of IH than in sham mesenteric arteries. Endogenouse H(2)S dilation was reduced in IH arteries. CONCLUSIONS: IH appears to decrease endothelial CSE expression to reduce H(2)S production, depolarize VSM, and enhance myogenic tone. H(2)S dilatation and hyperpolarization of VSM in small mesenteric arteries requires BK(Ca) channels.

Force augmentation and stimulated actin polymerization in swine carotid artery.

The phenomenon of posttetanic potentiation, in which a single submaximal contraction or series of submaximal contractions strengthens a subsequent contraction, has been observed in both skeletal and cardiac muscle. In this study, we describe a similar phenomenon in swine carotid arterial smooth muscle. We find that a submaximal K(+) depolarization increases the force generation of a subsequent maximal K(+) depolarization; we term this "force augmentation." Force augmentation was not associated with a significant increase in crossbridge phosphorylation or shortening velocity during the maximal K(+) depolarization, suggesting that the augmented force was not caused by higher crossbridge phosphorylation or crossbridge cycling rates. We found that the characteristics of the tissue before the maximal K(+) depolarization predicted the degree of force augmentation. Specifically, measures of stimulated actin polymerization (higher prior Y118 paxillin phosphorylation, higher prior F-actin, and transition to a more solid rheology evidenced by lower noise temperature, hysteresivity, and phase angle) predicted the subsequent force augmentation. Increased prior contraction alone did not induce force augmentation since readdition of Ca(2+) to Ca(2+)-depleted tissues induced a partial contraction that was not associated with changes in noise temperature or with subsequent force augmentation. These data suggest that stimulated actin polymerization may produce a substrate for increased crossbridge mediated force, a process we observe as force augmentation.

Pressure-induced vascular oxidative stress is mediated through activation of integrin-linked kinase 1/betaPIX/Rac-1 pathway.

High blood pressure induces a mechanical stress on vascular walls and evokes oxidative stress and vascular dysfunction. The aim of this study was to characterize the intracellular signaling causing vascular oxidative stress in response to pressure. In carotid arteries subjected to high pressure levels, we observed not only an impaired vasorelaxation, increased superoxide production, and NADPH oxidase activity, but also a concomitant activation of Rac-1, a small G protein. Selective inhibition of Rac-1, with an adenovirus carrying a dominant-negative Rac-1 mutant, significantly reduced NADPH oxidase activity and oxidative stress and, more importantly, rescued vascular function in carotid arteries at high pressure. The analysis of molecular events associated with mechanotransduction demonstrated at high pressure levels an overexpression of integrin-linked kinase 1 and its recruitment to plasma membrane interacting with paxillin. The inhibition of integrin-linked kinase 1 by small interfering RNA impaired Rac-1 activation and rescued oxidative stress-induced vascular dysfunction in response to high pressure. Finally, we showed that betaPIX, a guanine-nucleotide exchange factor, is the intermediate molecule recruited by integrin-linked kinase 1, converging the intracellular signaling toward Rac-1-mediated oxidative vascular dysfunction during pressure overload. Our data demonstrate that biomechanical stress evoked by high blood pressure triggers an integrin-linked kinase 1/betaPIX/Rac-1 signaling, thus generating oxidative vascular dysfunction.

Oncogenic NRAS has multiple effects on the malignant phenotype of human melanoma cells cultured in vitro.

Activating mutations in the NRAS gene, which occur predominantly in codon 61 (Q61R, Q61K) are among the most common genetic events in malignant melanoma. NRAS protein with oncogenic codon 61 mutations may therefore be good therapeutic targets. In the present study, we used gene expression profiling as a method for global characterization of gene expression alterations that resulted from treatment of melanoma cells with siRNA specifically targeting NRAS(Q61R). Sixteen probe sets representing 15 unique genes were identified whose expression was significantly altered by siRNA against NRAS(Q61R) in 2 melanoma cell lines. The genes with altered expression are involved in several functions, including modulation of cell growth, invasion and migration. The results suggest that downregulation of cyclin E2 and cyclin D1 and also upregulation of the negative cell-cycle regulator HBP1 in NRAS(Q61R) knockdown cells contribute to the inhibition of cell proliferation. Furthermore, suppression of oncogenic NRAS results in reduced migration and invasion, which is accompanied by downregulation of EphA2 (a receptor tyrosine kinase), uPAR (urokinase receptor) and cytoskeleton proteins such as leupaxin, paxillin and vinculin. These studies support the concept that suppression of oncogenic NRAS by siRNA can induce growth arrest and inhibit invasion of human melanoma cells by modulating the levels of these gene products.

Functional and molecular consequences of ionizing irradiation on large conductance Ca2+-activated K+ channels in rat aortic smooth muscle cells.

AIMS: The goal of this study was to evaluate the influence of gamma-irradiation on Ca(2+)-activated K(+) channel (BK(Ca)) function and expression in rat thoracic aorta. MAIN METHODS: Aortic cells or tissues were studied by the measurement of force versus [Ca(2+)](i), patch-clamp technique, and RT-PCR. KEY FINDINGS: Stimulation of smooth muscle cells with depolarizing voltage steps showed expression of outward K(+) currents. Paxilline, an inhibitor of BK(Ca) channels, decreased outward K(+) current density. Outward currents in smooth muscle cells obtained from irradiated animals 9 and 30 days following radiation exposure demonstrated a significant decrease in K(+) current density. Paxilline decreased K(+) current in cells obtained 9 days, but was without effect 30 days after irradiation suggesting the absence of BK(Ca) channels. Aortic tissue from irradiated animals showed progressively enhanced contractile responses to phenylephrine in the post-irradiation period of 9 and 30 days. The concomitant Ca(2+) transients were significantly smaller, as compared to tissues from control animals, 9 days following irradiation but were increased above control levels 30 days following irradiation. Irradiation produced a decrease in BK(Ca) alpha- and beta(1)-subunit mRNA levels in aortic smooth muscle cells suggesting that the vasorelaxant effect of these channels may be diminished. SIGNIFICANCE: These results suggest that the enhanced contractility of vascular tissue from animals exposed to radiation may result from an increase in myofilament Ca(2+) sensitivity in the early post-irradiation period and a decrease in BK(Ca) channel expression in the late post-irradiation period.