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Independent Mechanisms (independent + mechanism)
Selected AbstractsPIOGLITAZONE INHIBITS HOMOCYSTEINE-INDUCED MIGRATION OF VASCULAR SMOOTH MUSCLE CELLS THROUGH A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ,-INDEPENDENT MECHANISMCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 12 2008Li Li SUMMARY 1Peroxisome proliferator-activated receptor (PPAR)-, agonists have been demonstrated to exert protective effects against homocysteine (Hcy)-induced pathogenesis. However, the effects of PPAR-, agonists on Hcy-induced migration are unknown. In the present study, we examined the effect of pioglitazone on the migration of vascular smooth muscle cells (VSMC) induced by Hcy and the possible mechanism involved. 2Vascular smooth muscle cells were isolated from the thoracic aortas of male Sprague-Dawley rats. The migration of VSMC was examined using a transwell technique. The generation of intracellular reactive oxygen species (ROS) was measured using the ROS-sensitive fluoroprobe 2,,7,-dichlorodihydrofluorescein diacetate. The activity of NAD(P)H oxidase was assessed by lucigenin enhanced chemiluminescence. Activation of p38 mitogen-activated protein kinase (MAPK) was determined by western blotting. 3The results showed that pioglitazone dose-dependently inhibited the migration of VSMC induced by Hcy. This was not reversed by the PPAR-, antagonist GW9662. In addition, pretreatment with the NAD(P)H oxidase inhibitor diphenylene iodonium (DPI), the free radical scavenger N -acetylcysteine and the p38 MAPK inhibitor SB202190 blocked Hcy-induced VSMC migration. Furthermore, we observed that pioglitazone suppressed Hcy-induced intracellular ROS production; similar effects were observed with DPI and NAC. Pioglitazone attenuated Hcy-induced activation of NAD(P)H oxidase. Moreover, pioglitazone blocked Hcy-induced p38 MAPK phosphorylation; similar effects were observed for DPI, NAC and SB202190. 4The data demonstrate that pioglitazone inhibits Hcy-induced VSMC migration that is independent of PPAR-,. Furthermore, part of the biological effect of pioglitazone involves a decrease in the levels of NAD(P)H oxidase derived-ROS and p38 MAPK activation. [source] Independent mechanisms for ventriloquism and multisensory integration as revealed by theta-burst stimulationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2010Caterina Bertini Abstract The visual and auditory systems often concur to create a unified perceptual experience and to determine the localization of objects in the external world. Co-occurring auditory and visual stimuli in spatial coincidence are known to enhance performance of auditory localization due to the integration of stimuli from different sensory channels (i.e. multisensory integration). However, auditory localization of audiovisual stimuli presented at spatial disparity might also induce a mislocalization of the sound towards the visual stimulus (i.e. ventriloquism effect). Using repetitive transcranial magnetic stimulation we tested the role of right temporoparietal (rTPC), right occipital (rOC) and right posterior parietal (rPPC) cortex in an auditory localization task in which indices of ventriloquism and multisensory integration were computed. We found that suppression of rTPC excitability by means of continuous theta-burst stimulation (cTBS) reduced multisensory integration. No similar effect was found for cTBS over rOC. Moreover, inhibition of rOC, but not of rTPC, suppressed the visual bias in the contralateral hemifield. In contrast, cTBS over rPPC did not produce any modulation of ventriloquism or integrative effects. The double dissociation found in the present study suggests that ventriloquism and audiovisual multisensory integration are functionally independent phenomena and may be underpinned by partially different neural circuits. [source] The death of cardiotonic steroid-treated cells: evidence of Na+i,K+i -independent H+i -sensitive signallingACTA PHYSIOLOGICA, Issue 1-2 2006S. N. Orlov Abstract Na/K-ATPase is the only known target of cardiotonic steroids (CTS) identified in plants, amphibians and later on in several mammalian species, including human. We focus our review on recent data implicating CTS in the tissue-specific regulation of cell survival and death. In vascular smooth muscle cells, CTS inhibited cell death triggered by apoptotic stimuli via a novel Na+i -mediated, Ca2+i -independent mechanism of expression of antiapoptotic genes, including mortalin. In contrast, exposure to CTS in vascular endothelial and renal epithelial cells led to cell death, showing combined markers of apoptosis and necrosis. This mode of cell death, termed oncosis, is caused by CTS interaction with Na/K-ATPase but is independent of the inhibition of Na/K-ATPase-mediated ion fluxes and inversion of the [Na+]i/[K+]i ratio. The intermediates of intracellular signalling involved in Na+i, K+i -independent oncosis of CTS-treated cells remain unknown. Recently, we found that this mode of cell death can be protected by modest intracellular acidification via the expression of H+i -sensitive genes. The molecular origin of intracellular Na+ and H+ sensor involvement in the development of apoptosis and oncosis is currently under investigation. [source] Role of intracellular Ca2+ and calmodulin/MAP kinase kinase/extracellular signal-regulated protein kinase signalling pathway in the mitogenic and antimitogenic effect of nitric oxide in glia- and neurone-derived cell linesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2006Antonella Meini Abstract To elucidate the mechanism of cell growth regulation by nitric oxide (NO) and the role played in it by Ca2+, we studied the relationship among intracellular Ca2+ concentration ([Ca2+]i), mitogen-activated protein kinases [extracellular signal-regulated protein kinase (ERK)] and proliferation in cell lines exposed to different levels of NO. Data showed that NO released by low [(z)-1-[2-aminiethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2diolate (DETA/NO) concentrations (10 µm) determined a gradual, moderate elevation in [Ca2+]i (46.8 ± 7.2% over controls) which paralleled activation of ERK and potentiation of cell division. Functionally blocking Ca2+ or inhibiting calmodulin or MAP kinase kinase activities prevented ERK activation and antagonized the mitogenic effect of NO. Experimental conditions favouring Ca2+ entry into cells led to increased [Ca2+]i (189.5 ± 4.8%), ERK activation and cell division. NO potentiated the Ca2+ elevation (358 ± 16.8%) and ERK activation leading to expression of p21Cip1 and inhibition of cell proliferation. Furthermore, functionally blocking Ca2+ down-regulated ERK activation and reversed the antiproliferative effect of NO. Both the mitogenic and antimitogenic responses induced by NO were mimicked by a cGMP analogue whereas they were completely antagonized by selective cGMP inhibitors. These results demonstrate for the first time that regulation of cell proliferation by low NO levels is cGMP dependent and occurs via the Ca2+/calmodulin/MAP kinase kinase/ERK pathway. In this effect the amplitude of Ca2+ signalling determines the specificity of the proliferative response to NO possibly by modulating the strength of ERK activation. In contrast to the low level, the high levels (50,300 µm) of DETA/NO negatively regulated cell proliferation via a Ca2+ -independent mechanism. [source] Dehydroepiandrosterone inhibits the proliferation and induces the death of HPV-positive and HPV-negative cervical cancer cells through an androgen- and estrogen-receptor independent mechanismFEBS JOURNAL, Issue 19 2009Roma A. Girón Dehydroepiandrosterone (DHEA) has a protective role against epithelial-derived carcinomas; however, the mechanisms remain unknown. We determined the effect of DHEA on cell proliferation, the cell cycle and cell death in three cell lines derived from human uterine cervical cancers infected or not with human papilloma virus (HPV). We also determined whether DHEA effects are mediated by estrogen and androgen receptors. Proliferation of C33A (HPV-negative), CASKI (HPV16-positive) and HeLa (HPV18-positive) cells was evaluated by violet crystal staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction. Flow cytometry was used to evaluate the phases of the cell cycle, and cell death was detected using a commercially available carboxyfluorescein apoptosis detection kit that determines caspase activation. DNA fragmentation was determined using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Flutamide and ICI 182,780 were used to inhibit androgen and estrogen receptors, respectively, and letrozol was used to inhibit the conversion of DHEA to estradiol. Our results show that DHEA inhibited cell proliferation in a dose-dependent manner in the three cell lines; the DHEA IC50 doses were 50, 60 and 70 ,m for C33A, CASKI and HeLa cells, respectively. The antiproliferative effect was not abrogated by inhibitors of androgen and estrogen receptors or by an inhibitor of the conversion of testosterone to estradiol, and this effect was associated with an increase in necrotic cell death in HPV-negative cells and apoptosis in HPV-positive cells. These results suggest that DHEA strongly inhibits the proliferation of cervical cancer cells, but its effect is not mediated by androgen or estrogen receptor pathways. DHEA could therefore be used as an alternative in the treatment of cervical cancer. [source] Preferential Interface Nucleation: An Expansion of the VLS Growth Mechanism for NanowiresADVANCED MATERIALS, Issue 2 2009Brent A. Wacaser Abstract A review and expansion of the fundamental processes of the vapor,liquid,solid (VLS) growth mechanism for nanowires is presented. Although the focus is on nanowires, most of the concepts may be applicable to whiskers, nanotubes, and other unidirectional growth. Important concepts in the VLS mechanism such as preferred deposition, supersaturation, and nucleation are examined. Nanowire growth is feasible using a wide range of apparatuses, material systems, and growth conditions. For nanowire growth the unidirectional growth rate must be much higher than growth rates of other surfaces and interfaces. It is concluded that a general, system independent mechanism should describe why nanowires grow faster than the surrounding surfaces. This mechanism is based on preferential nucleation at the interface between a mediating material called the collector and a crystalline solid. The growth conditions used mean the probability of nucleation is low on most of the surfaces and interfaces. Nucleation at the collector-crystal interface is however different and of special significance is the edge of the collector-crystal interface where all three phases meet. Differences in nucleation due to different crystallographic interfaces can occur even in two phase systems. We briefly describe how these differences in nucleation may account for nanowire growth without a collector. Identifying the mechanism of nanowire growth by naming the three phases involved began with the naming of the VLS mechanism. Unfortunately this trend does not emphasize the important concepts of the mechanism and is only relevant to one three phase system. We therefore suggest the generally applicable term preferential interface nucleation as a replacement for these different names focusing on a unifying mechanism in nanowire growth. [source] Specificity of a new lipid mediator produced by testicular and peritoneal macrophages on steroidogenesisINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 5 2000Lukyanenko Macrophage-derived factor (MDF) is a lipophilic factor produced by rat testicular and peritoneal macrophages that maximally stimulates testosterone production by rat Leydig cells through a steroidogenic acute regulatory protein independent mechanism. The purpose of the present study was to determine whether MDF is also produced by human macrophages, and/or if it acts on human steroidogenic cells. We also studied the tissue-specific functions of MDF by determining if it also acts on steroidogenic cells of the ovary and adrenal glands and, if so, does it require new protein synthesis. It was found that MDF was produced by human peritoneal macrophages, and was capable of stimulating human steroidogenic cells. In terms of tissue specificity, it was found that primary cultures of rat adrenocortical cells respond to MDF with increased secretion of aldosterone and corticosterone, as did rat granulosa cells by producing progesterone. MDF acted in the presence of cycloheximide, indicating that it does not require new protein synthesis. These results indicate that MDF may have significant therapeutic potential and provide a basis for future studies concerning its physiological role in humans. These results further suggest that MDF is not only involved in paracrine regulation of Leydig cells, but also has the potential for the local regulation of steroidogenesis in both granulosa and adrenal cortical cells. [source] Disease-associated casein kinase I , mutation may promote adenomatous polyps formation via a Wnt/,-catenin independent mechanismINTERNATIONAL JOURNAL OF CANCER, Issue 5 2007I-Chun Tsai Abstract The Wnt signaling pathway is critical for embryonic development and is dysregulated in multiple cancers. Two closely related isoforms of casein kinase I (CKI, and ,) are positive regulators of this pathway. We speculated that mutations in the autoinhibitory domain of CKI,/, might upregulate CKI,/, activity and hence Wnt signaling and increase the risk of adenomatous polyps and colon cancer. Exons encoding the CKI, and CKI, regulatory domains were sequenced from DNA obtained from individuals with adenomatous polyps and a family history of colon cancer unaffected by familial adenomatous polyposis or hereditary nonpolyposis colorectal cancer (HNPCC). A CKI, missense mutation, changing a highly conserved residue, Arg324, to His (R324H), was found in an individual with large and multiple polyps diagnosed at a relatively young age. Two findings indicate that this mutation is biologically active. First, ectopic ventral expression of CKI,(R324H) in Xenopus embryos results in secondary axis formation with an additional distinctive phenotype (altered morphological movements) similar to that seen with unregulated CKI,. Second, CKI,(R324H) is more potent than wildtype CKI, in transformation of RKO colon cancer cells. Although the R324H mutation does not significantly change CKI, kinase activity in an in vitro kinase assay or Wnt/,-catenin signal transduction as assessed by a ,-catenin reporter assay, it alters morphogenetic movements via a ,-catenin-independent mechanism in early Xenopus development. This novel human CKI, mutation may alter the physiological role and enhance the transforming ability of CKI, through a Wnt/,-catenin independent mechanism and thereby influence colonic adenoma development. © 2006 Wiley-Liss, Inc. [source] Role of atypical protein kinase C isozymes and NF-,B in IL-1,-induced expression of cyclooxygenase-2 in human myometrial smooth muscle cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Sara V. Duggan Increased myometrial expression of cyclooxygenase-2 (Cox-2) at term results from elevated local levels of inflammatory cytokines, and its inhibition provides a potential route for intervention in human pre-term labor. We have identified a role for atypical protein kinase C (PKC) isozymes in IL-1,-induced Cox-2 expression in human myometrial smooth muscle cells (HMSMC). The PKC inhibitor GF109203X (10 µM) inhibited IL-1,-induced Cox-2 protein and RNA expression, which were also reduced by MAPK and nuclear factor ,B (NF-,B) inhibitors. GF109203X did not affect MAPK activities, and neither did it replicate the effect of p38 MAPK inhibition on Cox-2 mRNA stability, suggesting that PKC operates through an independent mechanism. The effect of GF109203X remained intact after depletion of conventional and novel PKC isozymes by phorbol ester pre-treatment. In contrast LY379196 (10 µM), which at micromolar concentrations inhibits all but atypical PKCs, did not affect Cox-2 expression. A peptide corresponding to the pseudosubstrate sequence of atypical PKCs blocked Cox-2 protein expression, whereas the sequence from conventional PKCs was ineffective. GF109203X did not affect NF-,B binding to nuclear proteins, but strongly reduced NF-,B-dependent transcription in luciferase reporter assays. Our findings indicate that IL-1,-induced Cox-2 expression in HMSMC in culture requires p38-MAPK-mediated mRNA stabilization and an independent activation of Cox-2 transcription which is dependent on the action of atypical PKCs, probably through direct stimulation of the transactivating activity of NF-,B. J. Cell. Physiol. 210: 637,643, 2007. © 2006 Wiley-Liss, Inc. [source] Pituitary adenylate cyclase-activating polypeptide regulates forebrain neural stem cells and neurogenesis in vitro and in vivoJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2006Shigeki Ohta Abstract Recent studies suggest that adult neurogenesis can contribute significantly to recovery from brain damage. As a result, there is strong interest in the field in identifying potentially therapeutic factors capable of promoting increased expansion of endogenous neural stem cell (NSC) populations and increased neurogenesis. In the present study, we have investigated the effects of PACAP on the NSC populations of the embryonic and adult forebrain. Our results demonstrate that the PACAP receptor, PAC1-R, is expressed by both embryonic and adult NSCs. The activation of PACAP signaling in vitro enhanced NSC proliferation/survival through a protein kinase A (PKA)-independent mechanism. In contrast, PACAP promoted NSC self-renewal and neurogenesis through a mechanism dependent on PKA activation. Finally, we determined that the intracerebroventricular infusion of PACAP into the adult forebrain was sufficient to increase neurogenesis significantly in both the hippocampus and the subventricular zone. These results demonstrate PACAP is unique in that it is capable of promoting NSC proliferation/survival, self-renewal, and neurogenesis and, therefore, may be ideal for promoting the endogenous regeneration of damaged brain tissue. © 2006 Wiley-Liss, Inc. [source] Moderate Alcohol Intake in Humans Attenuates Monocyte Inflammatory Responses: Inhibition of Nuclear Regulatory Factor Kappa B and Induction of Interleukin 10ALCOHOLISM, Issue 1 2006Pranoti Mandrekar Background: In contrast to the deleterious effects of chronic excessive alcohol consumption on the liver and cardiovascular system, modest alcohol intake, such as 1 to 2 drinks per day, has benefits on cardiovascular mortality. Little is known about the length of time or the amounts of alcohol consumed that may cause alterations in inflammatory cells such as monocytes that are crucial to atherosclerotic vascular disease. Here, we determine in vivo effects of acute alcohol consumption on inflammatory cytokine production and nuclear regulatory factor ,B (NF- ,B) binding in human monocytes. Methods: Human blood monocytes were isolated by plastic adherence before and after acute alcohol consumption (2 ml vodka/kg body weight). Lipopolysaccharide (LPS)- and superantigen-induced tumor necrosis factor , (TNF ,), interleukin (IL)-1,, and IL-10 production were then determined in monocytes by ELISA. Nuclear regulatory factor- ,B activity of monocytes before and after alcohol consumption was estimated by electromobility shift assay and promoter-driven reporter activity. I,B, was determined by Western blotting in the cytoplasmic extracts. Results: Eighteen hours after moderate alcohol consumption, we found a significant reduction in monocyte production of inflammatory mediators, TNF- , and IL-1,, in response to LPS or staphylococcal enterotoxin B stimulation. Acute alcohol consumption inhibited LPS-induced DNA binding of the p65/p50 NF- ,B in monocytes that regulates the expression of both the TNF- , and the IL-1, genes. Consistent with this, acute alcohol treatment (25 mM) significantly reduced LPS-induced activation of an NF- ,B-driven reporter gene suggesting inhibition of this proinflammatory signaling pathway. Further, LPS-induced I,B, degradation was not affected by acute alcohol consumption indicating an I,B, -independent mechanism, as observed earlier in the in vitro acute alcohol studies. In contrast, monocyte production of the anti-inflammatory cytokine, IL-10, was augmented by acute alcohol intake. Conclusions: Our findings suggest that acute alcohol consumption has dual anti-inflammatory effects that involve augmentation of IL-10 and attenuation of monocyte inflammatory responses involving inhibition of NF- ,B. These mechanisms may contribute to the beneficial effects of moderate alcohol use on atherosclerosis. [source] The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritisJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2006M. VAN DE WOUWER Summary.,Background: Thrombomodulin (TM) is predominantly a vascular endothelial cell plasma membrane glycoprotein that, via distinct structural domains, interacts with multiple ligands, thereby modulating coagulation, fibrinolysis, complement activation, inflammation and cell proliferation. We previously reported that by mediating signals that interfere with mitogen-activated protein kinase and nuclear factor ,B pathways, the amino-terminal C-type lectin-like domain of TM has direct anti-inflammatory properties. Methods: In the current study, we use murine models of acute inflammatory arthritis and biochemical approaches to assess the mechanism by which the lectin-like domain of TM modifies disease progression. Results: Mice lacking the lectin-like domain of TM (TMLeD/LeDmice) develop inflammatory arthritis that is more rapid in onset and more severe than that developed in their wildtype counterparts. In two models of arthritis, treatment of mice with recombinant soluble lectin-like domain of TM significantly suppresses clinical evidence of disease and diminishes monocyte/macrophage infiltration into the synovium, with weaker expression of the pro-inflammatory high mobility group box chromosomal protein 1. While thrombin-TM mediated activation of thrombin activatable fibrinolysis inhibitor inactivates complement factors C3a and C5a, we show that TM has a second independent mechanism to regulate complement: the lectin-like domain of TM directly interferes with complement activation via the classical and lectin pathways. Conclusions: These data extend previous insights into the mechanisms by which TM modulates innate immunity, and highlight its potential as a therapeutic target for inflammatory diseases. [source] Endocytosis of plasma-derived factor V by megakaryocytes occurs via a clathrin-dependent, specific membrane binding eventJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 3 2005B. A. BOUCHARD Summary., Megakaryocytes were analyzed for their ability to endocytose factor V to define the cellular mechanisms regulating this process. In contrast to fibrinogen, factor V was endocytosed by megakaryocytes derived from CD34+ cells or megakaryocyte-like cell lines, but not by platelets. CD41+ex vivo -derived megakaryocytes endocytosed factor V, as did subpopulations of the megakaryocyte-like cells MEG-01, and CMK. Similar observations were made for fibrinogen. Phorbol diester-induced megakaryocytic differentiation of the cell lines resulted in a substantial increase in endocytosis of both proteins as compared to untreated cells that did not merely reflect their disparate plasma concentrations. Factor IX, which does not associate with platelets or megakaryocytes, was not endocytosed by any of the cells examined. Endocytosis of factor V by megakaryocytes proceeds through a specific and independent mechanism as CHRF-288 cells endocytosed fibrinogen but not factor V, and the presence of other plasma proteins had no effect on the endocytosis of factor V by MEG-01 cells. Furthermore, as the endocytosis of factor V was also demonstrated to occur through a clathrin-dependent mechanism, these combined data demonstrate that endocytosis of factor V by megakaryocytes occurs via a specific, independent, and most probably receptor-mediated, event. [source] A molecular basis for the increased vulnerability of substantia nigra dopamine neurons in aging and Parkinson's disease,MOVEMENT DISORDERS, Issue S1 2010C. Savio Chan PhD Abstract Parkinson's disease (PD) is a common neurodegenerative disorder of unknown etiology. There is no cure or proven strategy for slowing the progression of the disease. Although there are signs of pathology in many brain regions, the core symptoms of PD are attributable to the selective degeneration of dopaminergic neurons in the substantia nigra pars compacta. A potential clue to the vulnerability of these neurons is an increasing reliance with age upon L-type Ca2+ channels with a pore-forming Cav1.3 subunit to support autonomous activity. This reliance could pose a sustained stress on mitochondrial ATP generating oxidative phosphorylation, accelerating cellular aging and death. Systemic administration of isradipine, a dihydropyridine blocker of these channels, forces dopaminergic neurons in rodents to revert to a juvenile, L-type Ca2+ channel independent mechanism to generate autonomous activity. This "rejuvenation" confers protection against toxins that produce experimental Parkinsonism, pointing to a potential neuroprotective strategy for PD. Their decades-long track record of safe use in the treatment of hypertension makes dihydropyridines particularly attractive as a therapeutic tool in PD. © 2010 Movement Disorder Society [source] IL-10 Does not Play a Role in Cutaneous Photofrin® Photodynamic Therapy-induced Suppression of the Contact Hypersensitivity Response,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2001Sandra O. Gollnick ABSTRACT Photodynamic therapy (PDT) treatment of both malignant and benign skin diseases has proven to be effective, and its use is increasing worldwide. However, preclinical studies using murine models have shown that PDT of the skin inhibits cell-mediated immune reactions, as measured by the suppression of the contact hypersensitivity (CHS) reaction. We have previously demonstrated that PDT enhances IL-10 expression in treated skin, and that the kinetics of induction of IL-10 is similar to the kinetics of suppression of systemic CHS reactions by cutaneous PDT. In the following report we have expanded upon these studies to demonstrate that cutaneous PDT, using Photofrin®, induces elevated levels of systemic IL-10 that persist for at least 28 days following treatment. The increase in systemic IL-10 correlates to a prolonged suppression of CHS of at least 28 days following cutaneous PDT. IL-10 has been implicated as the causative agent in the suppression of cell-mediated immune reactions by UVB and transdermal PDT. However, in the studies reported here we demonstrate that the suppression of CHS by cutaneous PDT occurs via an IL-10 independent mechanism, as administration of anti,IL-10 antibodies had no effect on the ability of PDT to induce CHS suppression. These results were further confirmed using IL-10 knockout (KO) mice. Cutaneous PDT of IL-10 KO mice resulted in CHS suppression that was not significantly different from suppression induced in wild-type mice. Thus, it appears as though IL-10 does not play a role in CHS suppression by cutaneous PDT. Suppression of cell-mediated immune reactions by UVB and transdermal PDT is reversible by IL-12, which is critical for the development of these reactions. We show that administration of exogenous IL-12 is also able to reverse CHS suppression induced by cutaneous PDT, suggesting that whereas suppression of cell-mediated immune reactions by UVB, transdermal PDT and cutaneous PDT occurs via different mechanisms, a common regulatory point exists. [source] Ca2+ -independent hypoxic vasorelaxation in porcine coronary arteryTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005Min Gu To demonstrate a Ca2+ -independent component of hypoxic vasorelaxation and to investigate its mechanism, we utilized permeabilized porcine coronary arteries, in which [Ca2+] could be clamped. Arteries permeabilized with ,-escin developed maximum force in response to free Ca2+ (6.6 ,m), concomitant with a parallel increase in myosin regulatory light chain phosphorylation (MRLC-Pi), from 0.183 ± 0.023 to 0.353 ± 0.019 MRLC-Pi (total light chain),1. Hypoxia resulted in a significant decrease in both force (,31.9 ± 4.1% prior developed force) and MRLC-Pi (from 0.353 to 0.280 ± 0.023), despite constant [Ca2+] buffered by EGTA (4 mm). Forces developed in response to Ca2+ (6.6 ,m), Ca2+ (0.2 ,m) + GTP,S (1 mm), or in the absence of Ca2+ after treatment with ATP,S (1 mm), were of similar magnitude. Hypoxia also relaxed GTP,S contractures but importantly, arteries could not be relaxed after treatment with ATP,S. Permeabilization with Triton X-100 for 60 min also abolished hypoxic relaxation. The blocking of hypoxic relaxation after ATP,S suggests that this Ca2+ -independent mechanism(s) may operate through alteration of MRLC-Pi or of phosphorylation of the myosin binding subunit of myosin light chain phosphatase. Treatment with the Rho kinase inhibitor Y27632 (1 ,m) relaxed GTP,S and Ca2+ contractures; but the latter required a higher concentration (10 ,m) for consistent relaxation. Relaxations to N2 and/or Y27632 averaged 35% and were not additive or dependent on order. Our data suggest that the GTP-mediated, Rho kinase-coupled pathway merits further investigation as a potential site of this novel, Ca2+ -independent O2 -sensing mechanism. Importantly, these results unambiguously show that hypoxia-induced vasorelaxation can occur in permeabilized arteries where the Ca2+ is clamped at a constant value. [source] Alloimmune Activation Enhances Innate Tissue Inflammation/Injury in a Mouse Model of Liver Ischemia/Reperfusion InjuryAMERICAN JOURNAL OF TRANSPLANTATION, Issue 8 2010X. Shen The deleterious sensitization to donor MHC Ags represents one of the most challenging problems in clinical organ transplantation. Although the role of effector/memory T cells in the rejection cascade has been extensively studied, it remains unknown whether and how these ,Ag-specific' cells influence host innate immunity, such as tissue inflammation associated with ischemia and reperfusion injury (IRI). In this study, we analyzed how allogeneic skin transplant (Tx) affected the sequel of host's own liver damage induced by partial warm ischemia and reperfusion. Our data clearly showed that allo-Tx recipients had increased inflammatory response against IR insult in their native livers, as evidenced by significantly more severe hepatocelluar damage, compared with syngeneic Tx recipient controls, and determined by serum ALT levels, liver histology (Suzuki's score) and intrahepatic proinflammatory gene inductions (TNF-,, IL-1, and CXCL10). The CD4 T cells, but neither CD8 nor NK cells, mediated the detrimental effect of allo-Ag sensitization in liver IRI. Furthermore, CD154, but not IFN-,, was the key mechanism in allo-Tx recipients to facilitate IR-triggered liver damage. These results provide new evidence that alloreactive CD4 T cells are capable of enhancing innate tissue inflammation and organ injury via an Ag-nonspecific CD154-dependent but IFN-, independent mechanism. [source] Osteoblasts stimulated with pulsed electromagnetic fields increase HUVEC proliferation via a VEGF-A independent mechanism,BIOELECTROMAGNETICS, Issue 3 2009Richard A. Hopper Abstract The clinically beneficial effect of low frequency pulsed electromagnetic fields (ELF-PEMF) on bone healing has been described, but the exact mechanism of action remains unclear. A recent study suggests that there is a direct autocrine mitogenic effect of ELF-PEMF on angiogenesis. The hypothesis of this study is that ELF-PEMF also has an indirect effect on angiogenesis by manipulation of vascular endothelial growth factor (VEGF)-A-based paracrine intercellular communication with neighboring osteoblasts. Conditioned media experiments measured fetal rat calvarial cell (FRC) and human umbilical vein endothelial cell (HUVEC) proliferation using tritiated thymidine uptake. We demonstrate that ELF-PEMF (15 Hz, 1.8 mT, for 8 h) has an indirect effect on the proliferation rate of both endothelial cells and osteoblasts in vitro by altering paracrine mediators. Conditioned media from osteoblast cells stimulated with ELF-PEMF increased endothelial proliferation 54-fold, whereas media from endothelial cells stimulated with ELF-PEMF did not affect osteoblast proliferation. We examined the role of the pro-angiogenic mediator VEGF-A in the mitogenic effect of ELF-PEMF-stimulated osteoblast media on endothelial cells. The production of VEGF-A by FRC as measured by ELISA was not changed by exposure to PEMF, and blocking experiments demonstrated that the ELF-PEMF-induced osteoblast-derived endothelial mitogen observed in these studies was not VEGF-A, but some other soluble angiogenic mediator. Bioelectromagnetics 30:189,197, 2009. © 2008 Wiley-Liss, Inc. [source] Characterization of calcium-independent purinergic receptor-mediated apoptosis in hormone-refractory prostate cancerBJU INTERNATIONAL, Issue 3 2008Majid Shabbir OBJECTIVE To investigate the nature of purinergic signalling in hormone-refractory prostate cancer (HRPC) cells in vitro, as extracellular ATP inhibits the growth of HRPC in vitro via the activation of P2 purinergic receptors, and to characterize which P2 receptors subtypes and secondary mechanisms are involved. MATERIALS AND METHODS The effect of extracellular ATP on HRPC cell lines PC-3 and DU-145, and the normal prostate cell line PNT-2, were investigated. Reverse-transcription polymerase chain reaction was used to assess P2 purinergic receptors, which were pharmacologically characterized using various receptor agonists and antagonists. The effect of ATP on intracellular Ca2+ concentration ([Ca2+]i) was examined to asses its role in growth inhibition. The effect of combining ATP with the chemotherapeutic drug mitoxantrone was also assessed. RESULTS PC-3 cells expressed mRNA for P2X4,5,7, P2Y1,2,4,6; DU-145 cells expressed mRNA for P2X4,5, P2Y1,2,4,6,11; PNT-2 cells expressed mRNA for P2X4,5,7 and P2Y1,2,4,6,11. ATP (10,4m) inhibited HRPC PC-3 cell growth by ,,90%, an effect partially inhibited by the nonselective P2 receptor antagonists pyridoxal-5,-phosphate-6-azophenyl-2,,4, disulphonic acid (PPADS) and suramin. The order of potency of agonists was: adenosine 5,-O-(3 thiotriphosphate) > ATP > benzoyl benzoyl ATP >> 2-methylthio ATP. DU-145 cells responded similarly. Pharmacological profiling implicated P2X5 and/or P2Y11 receptors in the antineoplastic response in HRPC. ATP induced apoptosis in a [Ca2+]i -independent mechanism. ATP was significantly less effective on PNT-2 cells, which also had a different order of agonist potency. ATP combined with mitoxantrone in an additive manner in HRPC. CONCLUSIONS ATP effectively reduces growth of HRPC cells via calcium-independent apoptosis. Pharmacological profiling indicates P2X5 and/or P2Y11 receptors in this process, with a different functional purinergic receptor profile and sensitivity in normal vs cancer cells. [source] Age-dependent differential expression of genes involved in steroid signalling pathway in the brain of protandrous black porgy, Acanthopagrus schlegeliDEVELOPMENTAL NEUROBIOLOGY, Issue 5 2009Sherly Tomy Abstract The mechanisms underlying brain sex differentiation in animals are poorly understood. In the present study, using black porgy, Acanthopagrus schlegeli, as primary experimental model, we investigated the temporal expression patterns of receptors for androgen (ar) and estrogen (esr1 and esr2a) in the brain during posthatching ages and analyzed them against the timing of gonadal germ cell development. We hypothesized that endogenous estrogens naturally masculinize the brain of black porgy. The expression of sex steroid receptors was studied in relation to a wider suite of other related genes (nr5a2, nr0b1, star, and cyp19a1b) to provide some insight into the monomale sex differentiation pattern observed in this species. Our results revealed a highly significant increase in esr1 together with the increase in esr2a at 120 dph (days posthatching), suggesting a significant role for esr in sex differentiation in this species. Temporal expression patterns of nr5a2, nr0b1, star, sex steroid receptors, and cyp19a1b in the brain provided evidence for their physiological roles in the monomale sex differentiation in this species. The expression of nr5a2, star, ar, esr1, esr2a, and cyp19a1b increased at 120 dph, a period when brain sex differentiation probably occurs in this species. The study also suggests that neurosteroidogenesis in black porgy may be regulated by both nr5a2 -dependent and nr5a2 -independent mechanisms. The results demonstrated striking differences in the abundance of the gene transcripts in discrete brain region throughout ontogeny. In addition, the sex steroid hormone levels and aromatase activity in brain at different developmental states and the changes in the gene expression patterns in response to aromatase inhibitor treatment are also discussed. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source] Hypertrophic cardiomyopathy: from genetics to treatmentEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 4 2010Ali J. Marian Eur J Clin Invest 2010; 40 (4): 360,369 Abstract Background, Hypertrophic cardiomyopathy (HCM) is the prototypic form of pathological cardiac hypertrophy. HCM is an important cause of sudden cardiac death in the young and a major cause of morbidity in the elderly. Design, We discuss the clinical implications of recent advances in the molecular genetics of HCM. Results, The current diagnosis of HCM is neither adequately sensitive nor specific. Partial elucidation of the molecular genetic basis of HCM has raised interest in genetic-based diagnosis and management. Over a dozen causal genes have been identified. MYH7 and MYBPC3 mutations account for about 50% of cases. The remaining known causal genes are uncommon and some are rare. Advances in DNA sequencing techniques have made genetic screening practical. The difficulty, particularly in the sporadic cases and in small families, is to discern the causal from the non-causal variants. Overall, the causal mutations alone have limited implications in risk stratification and prognostication, as the clinical phenotype arises from complex and often non-linear interactions between various determinants. Conclusions, The clinical phenotype of ,HCM' results from mutations in sarcomeric proteins and subsequent activation of multiple cellular constituents including signal transducers. We advocate that HCM, despite its current recognition and management as a single disease entity, involves multiple partially independent mechanisms, despite similarity in the ensuing phenotype. To treat HCM effectively, it is necessary to delineate the underlying fundamental mechanisms that govern the pathogenesis of the phenotype and apply these principles to the treatment of each subset of clinically recognized HCM. [source] Caspase-dependent and -independent apoptosis of mast cells induced by withdrawal of IL-3 is prevented by Toll-like receptor,4-mediated lipopolysaccharide stimulationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2003Hideshi Yoshikawa Abstract IL-3-dependent mucosal-like mast cells undergo apoptosis upon withdrawal of IL-3. Generally, the apoptosis is mediated by the activation of caspases and inhibited by addition of the pan-caspase inhibitors z-VAD-FMK or BOC-D-FMK. However, DNA fragmentation, a typical characteristic of apoptosis, is not inhibited by z-VAD-FMK or BOC-D-FMK in mast cell apoptosis. In this study, we demonstrate that the apoptosis of mast cells is mediated by both caspase-dependent and -independent mechanisms. The caspase-independent apoptosis is mediated by the translocation of endonuclease,G from mitochondria into nuclei. Withdrawal of IL-3 caused down-regulation of Bcl-xL, resulting in a drop in mitochondrial membrane transition potential followed by the release of cytochrome,c and endonuclease,G from mitochondria. However, stimulation of mast cells through Toll-like receptor,4 (TLR4) by lipopolysaccharide prevented mast cell apoptosis by inducing expression of Bcl-xL. Moreover, the activation of mast cells by LPS is enhanced in the presence of IFN-,, which up-regulates the expression of cell surface TLR4. Taken together, these observations provide evidence that mast cells play importantroles not only in allergic reactions but also in innate immunity recognizing enterobacteria through TLR4, and are regulated differently from allergic inflammation by Th1 cytokines. [source] Ganglioside GD3 expression on target cells can modulate NK cell cytotoxicity via siglec-7-dependent and -independent mechanismsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2003Gavin Nicoll Abstract Siglec-7 is a sialic acid binding receptor with inhibitory potential, expressed on human NK cells and monocytes. It has an unusual binding preference for ,2,8-linked disialic acids, such as those displayed by ganglioside GD3. Here we have investigated whether siglec-7-GD3 interactions are able to modulate NK cell cytotoxicity. Using synthetic polyacrylamide glycoprobes, siglec-7 was found to be masked at the NK cell surface but it could be unmasked by sialidase treatment of NK cells. GD3 synthase-transfected P815 target cells expressed high levels of GD3 and bound strongly to recombinant siglec-7-Fc protein. Surprisingly, GD3 synthase-transfected P815 cells were killed more effectively by untreated cells in a siglec-7-independent manner. However, following sialidase treatment of NK cells, a siglec-7-dependent inhibition of killing was observed. These findings have important implications for NK cell cytotoxicity against tumor cells like melanoma that express high levels of GD3 ganglioside. [source] BDNF,triggered events in the rat hippocampus are required for both short- and long-term memory formationHIPPOCAMPUS, Issue 4 2002Mariana Alonso Abstract Information storage in the brain is a temporally graded process involving different memory types or phases. It has been assumed for over a century that one or more short-term memory (STM) processes are involved in processing new information while long-term memory (LTM) is being formed. Because brain-derived neutrophic factor (BDNF) modulates both short-term synaptic function and activity-dependent synaptic plasticity in the adult hippocampus, we examined the role of BDNF in STM and LTM formation of a hippocampal-dependent one-trial fear-motivated learning task in rats. Using a competitive RT-PCR quantitation method, we found that inhibitory avoidance training is associated with a rapid and transient increase in BDNF mRNA expression in the hippocampus. Bilateral infusions of function-blocking anti-BDNF antibody into the CA1 region of the dorsal hippocampus decreased extracellular signal,regulated kinase 2 (ERK2) activation and impaired STM retention scores. Inhibition of ERK1/2 activation by PD098059 produced similar effects. In contrast, intrahippocampal administration of recombinant human BDNF increased ERK1/2 activation and facilitated STM. The infusion of anti-BDNF antibody impaired LTM when given 15 min before or 1 and 4 hr after training, but not at 0 or 6 hr posttraining, indicating that two hippocampal BDNF-sensitive time windows are critical for LTM formation. At the same time points, PD098059 produced no LTM deficits. Thus, our results indicate that endogenous BDNF is required for both STM and LTM formation of an inhibitory avoidance learning. Additionally, they suggest that this requirement involves ERK1/2-dependent and -independent mechanisms. Hippocampus 2002;12:551,560. © 2002 Wiley-Liss, Inc. [source] Analysis of differential immune responses induced by innate and adaptive immunity following transplantationIMMUNOLOGY, Issue 2 2003Hongzhen He Summary The roles of innate and adaptive immunity in allograft rejection remain incompletely understood. Previous studies analysing lymphocyte deficient or syngeneic graft recipients have identified subsets of inflammatory chemokines and cytokines induced by antigen independent mechanisms. In the current study, we analysed a panel of 60 inflammatory parameters including serum cytokines, intragraft chemokines and cytokines, receptors, and cellular markers. Our results confirmed the up-regulation of a subset of markers by innate mechanisms and also identified a subset of parameters up-regulated only in the context of an adaptive response. Thus, we successfully differentiated markers of the innate and adaptive phases of rejection. Current paradigms emphasize that innate signals can promote a subsequent adaptive response. Interestingly, in our studies, expression of the markers induced by innate mechanisms was markedly amplified in the allogeneic, but not syngeneic or lymphocyte deficient, recipients. These results suggest that inflammatory mediators can have functional overlap between the innate and adaptive responses, and that the adaptive component of the rejection process amplifies the innate response by positive feedback regulation. [source] Granulocyte,macrophage colony-stimulating factor regulates cytokine production in cultured macrophages through CD14-dependent and -independent mechanismsIMMUNOLOGY, Issue 2 2000A. Bergamini Summary Granulocyte,macrophage colony-stimulating factor (GM-CSF) has multiple effects on the antigen phenotype and function of macrophages. In this study we investigated the effect of GM-CSF on cytokine production by macrophages. We found that GM-CSF may modify the tumour necrosis factor-, (TNF-,) and interleukin-6 (IL-6) response to lipopolysaccharide (LPS) through two different mechanisms. Relatively early in culture, GM-CSF increases the amount of cytokines synthesized by responding cells; this effect appears to be unrelated to modulation of CD14 expression and LPS-binding capacity. After prolonged incubation, GM-CSF up-regulates both CD14 expression and LPS-binding capacity, and the frequency of cytokine-producing cells. Release of CD14 in the culture supernatant was decreased in the presence of GM-CSF, suggesting that a reduced shedding was responsible for the effect of GM-CSF on CD14 expression. Enhancement of cytokine production was also observed in GM-CSF-treated macrophages after stimulation by phorbol 12-myristate 13-acetate (PMA), thus indicating that GM-CSF affects both CD14-dependent and -independent cytokine production. Finally, GM-CSF did not modulate the LPS- and PMA-induced production of IL-10 and IL-12. We conclude that GM-CSF may play a role in manipulating the activation-induced expression of pro-inflammatory cytokines by macrophages. Enhanced production of these cytokines could play an important role in the pathogenesis of Gram-negative septic shock syndrome and in defence against infectious agents. [source] Suppression of the TIG3 tumor suppressor gene in human ovarian carcinomas is mediated via mitogen-activated kinase-dependent and -independent mechanismsINTERNATIONAL JOURNAL OF CANCER, Issue 6 2005Kristina Lotz Abstract The TIG3 gene is a retinoic acid inducible class II tumor suppressor gene downregulated in several human tumors and malignant cell lines. Diminished TIG3 expression correlates with decreased differentiation whereas forced expression of TIG3 suppresses oncogenic signaling pathways and subsequently induces differentiation or apoptosis in tumor cells. Analysis of TIG3 mRNA expression in a large set of cDNA pools derived from matched tumor and normal human tissues showed a significant downregulation of TIG3 in 29% of the cDNA samples obtained from ovarian carcinomas. Using in situ hybridization, we demonstrated expression of TIG3 in the epithelial lining of 7 normal ovaries but loss of TIG3 expression in 15/19 of human ovarian carcinoma tissues. In SKOV-3, CAOV-3 and ES-2 ovarian carcinoma cell lines, downregulation of TIG3 mRNA was reversible and dependent on an activated MEK-ERK signaling pathway. Re-expression of TIG3 mRNA in these cells upon specific interference with the MEK-pathway was correlated with growth inhibition of the cells. In OVCAR-3 and A27/80 ovarian carcinoma cells, TIG3 suppression is MEK-ERK independent, but expression could be reconstituted upon interferon gamma (IFN,) induction. Overexpression of TIG3 in A27/80 ovarian carcinoma cells significantly impaired cell growth and despite increased mRNA levels, TIG3 protein was hardly detectable. These results suggest that TIG3 is negatively regulated by an activated MEK-ERK signaling pathway. Further mechanisms must interfere with TIG3 expression that are independent of MEK and partially include interferon-responsive components. © 2005 Wiley-Liss, Inc. [source] Hyperglycemia and glucosamine-induced mesangial cell cycle arrest and hypertrophy: Common or independent mechanisms?IUBMB LIFE, Issue 7 2006Elodie Masson Abstract The Hexosamine Pathway (HP) is one hypothesis proposed to explain glucose toxicity and the alterations observed during the course of diabetic microvascular complication development. Glucosamine is a precursor of UDP-N-Acetylglucosamine (UDP-GlcNAc), the main product of the HP that has often been used to mimic its activation. The transfer of a UDP-GlcNAc residue onto proteins (O-GlcNAc modification) represents the final step of the HP and is considered as a major mechanism by which this pathway exerts its signalling effects. While it is well accepted that the HP promotes extracellular matrix accumulation in the context of diabetic nephropathy, its involvement in the perturbations of cell cycle progression and hypertrophy of renal cells has been poorly investigated. Nevertheless, in a growing number of studies, the HP and O-GlcNAc modification are emerging as important regulators of cell cycle progression. This review will focus on the role of glucosamine and O-GlcNAc modification in cell cycle regulation in the context of diabetic nephropathy. Special emphasis will be given into the role of the HP as a potential mediator of the effects of high glucose on the perturbations of renal cell growth. iubmb Life, 58: 381-388, 2006 [source] Regulatory Mechanisms and Physiological Relevance of a Voltage-Gated H+ Channel in Murine Osteoclasts: Phorbol Myristate Acetate Induces Cell Acidosis and the Channel Activation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2003Hiroyuki Mori Abstract The voltage-gated H+ channel is a powerful H+ extruding mechanism of osteoclasts, but its functional roles and regulatory mechanisms remain unclear. Electrophysiological recordings revealed that the H+ channel operated on activation of protein kinase C together with cell acidosis. Introduction: H+ is a key signaling ion in bone resorption. In addition to H+ pumps and exchangers, osteoclasts are equipped with H+ conductive pathways to compensate rapidly for pH imbalance. The H+ channel is distinct in its strong H+ extrusion ability and voltage-dependent gatings. Methods: To investigate how and when the H+ channel is available in functional osteoclasts, the effects of phorbol 12-myristate 13-acetate (PMA), an activator for protein kinase C, on the H+ channel were examined in murine osteoclasts generated in the presence of soluble RANKL (sRANKL) and macrophage-colony stimulating factor (M-CSF). Results and Conclusions: Whole cell recordings clearly showed that the H+ current was enhanced by increasing the pH gradient across the plasma membrane (,pH), indicating that the H+ channel changed its activity by sensing ,pH. The reversal potential (Vrev) was a valuable tool for the real-time monitoring of ,pH in clamped cells. In the permeabilized patch, PMA (10 nM-1.6 ,M) increased the current density and the activation rate, slowed decay of tail currents, and shifted the threshold toward more negative voltages. In addition, PMA caused a negative shift of Vrev, suggesting that intracellular acidification occurred. The PMA-induced cell acidosis was confirmed using a fluorescent pH indicator (BCECF), which recovered quickly in a K+ -rich alkaline solution, probably through the activated H+ channel. Both cell acidosis and activation of the H+ channel by PMA were inhibited by staurosporine. In ,80% of cells, the PMA-induced augmentation in the current activity remained after compensating for the ,pH changes, implying that both ,pH-dependent and -independent mechanisms mediated the channel activation. Activation of the H+ channel shifted the membrane potential toward Vrev. These data suggest that the H+ channel may contribute to regulation of the pH environments and the membrane potential in osteoclasts activated by protein kinase C. [source] Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004L. Formigli We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca2+ mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca2+ -independent mechanisms of cell contraction have been the focus of numerous studies on Ca2+ sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca2+ -independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca2+, by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca2+ transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca2+ and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKC, and PKC,, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca2+ -independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction. J. Cell. Physiol. 198: 1,11, 2004© 2003 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||||||||