Myosin Light Chain Kinase Pathway Research Articles

Loganin exerts neuroprotective effect by inhibiting neuronal pyroptosis in rat with cerebral haemorrhage.

Intracerebral haemorrhage (ICH) presents significant challenges in clinical management because of the high morbidity and mortality, necessitating novel therapeutic approaches. This study aimed to assess the neuroprotective effects of loganin in a rat ICH model. Sprague-Dawley rats were used, subjected to a collagenase-induced ICH model, followed by loganin treatment at doses of 2.5, 5 and 10 mg/kg. Neurological functions were evaluated using the modified neurological severity score (mNSS) and a rotarod test. Results indicated a significant improvement in neurological functions in loganin-treated groups, evident from the mNSS and rotarod tests, suggesting dose-dependent neuroprotection. Loganin also effectively reduced the blood-brain barrier (BBB) permeability and cerebral oedema. Additionally, it mitigated cellular pyroptosis, as shown by terminal deoxynucleotidyl transferase dUTP nick-end labelling staining and western blot analysis, which indicated reduced levels of pyroptosis markers in treated rats. Furthermore, loganin's regulatory effects on the adenosine A2A receptor and myosin light chain kinase pathways were observed, potentially underpinning its protective mechanism against ICH. The study concludes that loganin exhibits significant neuroprotective properties in a rat ICH model, highlighting its potential as a novel therapeutic strategy. Despite promising results, the study needs further research to determine loganin's therapeutic potential in human ICH patients. This research paves the way for further exploration into loganin's clinical applications, potentially revolutionizing treatment strategies for patients suffering from intracerebral haemorrhage.

Electroacupuncture protects the intestinal mucosal barrier in diarrhea-predominant Irritable Bowel Syndrome rats by regulating the MCs/Tryptase/PAR-2/MLCK pathway.

The pathogenesis of diarrhea-predominant irritable bowel syndrome (IBS-D) is related to damage to the intestinal mucosal barrier function. Based on the Mast cell (MC)/Tryptase/Protease-activated receptor-2 (PAR-2)/Myosin light chain kinase (MLCK) pathway, this study explored the effect of electroacupuncture (EA) on IBS-D rats and its possible mechanism of protecting the intestinal mucosal barrier. The IBS-D rat model was established by mother-offspring separation, acetic acid enema, and chronic restraint stress. The efficacy of EA on IBS-D rats was evaluated by observing the rate of loose stool (LSP) and the minimum volume threshold of abdominal withdrawal reflex (AWR) in rats. Mast cells and the ultrastructure of intestinal mucosa were observed by H&E staining, toluidine blue staining, and transmission electron microscopy. The expression levels of Tryptase, PAR-2, MLCK, zonula occludens-1 (ZO-1), and Occludin in rats were detected by ELISA, qRT-PCR, and western blot. After 7 days of intervention, compared to the IBS-D group, the loose stool rates of rats in IBS-D + EA group and IBS-D + ketotifen group were decreased (P < 0.01), the minimum volume thresholds of AWR were improved (P < 0.01), the inflammation of colon tissue decreased, the number of MCs were decreased (P < 0.01), the expression of Tryptase, PAR-2, and MLCK were lowered (P < 0.01, P < 0.05), and the expression of ZO-1 and Occludin were enhanced (P < 0.01, P < 0.05). Compared to the EA group, there was no significant difference in each index between the ketotifen groups (P > 0.05). EA has a good therapeutic effect on IBS-D rats. Regulating the MCs/Tryptase/PAR-2/MLCK pathway may be a mechanism to protect the intestinal mucosal barrier.

Oenothera biennis cell culture produce lignans activating Piezo1 triggering the Myosin Light Chain Kinase depending pathways

Piezo1 and Piezo2 are mechanoreceptors involved in sensing both internal and external mechanical forces converting them in electrical signals to the brain.Piezo1 is mainly expressed in the endothelial system and in epidermis sensing shear stress and light touch. The internal traction forces generated by Myosin Light Chain Kinase (MYLK) activate Piezo1, regulating cell contraction.We observed Oenothera biennis cell culture hydro-soluble extract (ObHEx) activated MYLK regulating cell contraction ability. The aim of this work was to test the hypothesis that ObHEx activates Piezo1 through MYLK pathway using CHO cell overexpressing Piezo1, HUVEC and SHSY5Y cells endogenously expressing high levels of Piezo1. Results showed that ObHEx extracts were able to activate Piezo1 and the effect is due to Liriodendrin and Salvadoraside, the two most abundant lignans produced by the cell culture. The effect is lost in presence of MYLK specific inhibitors confirming the key role of this pathway and providing indication about the mechanism of action in Piezo1 activation by lignans.In summary, these results confirmed the connection between Piezo1 and MYLK, opening the possibility of using lignans-containing natural extracts to activate Piezo1.

The Probiotic Lactobacillus paracasei Ameliorates Diarrhea Cause by Escherichia coli O8via Gut Microbiota Modulation1

IntroductionKoumiss is a fermented horse milk food containing abundant probiotics. Lactobacillus paracasei is a bacterial strain isolated from koumiss that helps regulate the intestinal microbiota. One of the major cause of diarrhea is an imbalance of the intestinal flora. The aim of this study was to investigate whether Lactobacillus paracasei can ameliorate E. coli-induced diarrhea and modulate the gut microbiota.MethodsMouse models of diarrhea were established via intragastric E. coli O8 administration. We then attempted to prevent or treat diarrhea in the mice via intragastric administration of a 3 × 108 CFU/mL L. paracasei cell suspension. The severity of diarrhea was evaluated based on the body weight, diarrhea rate, and index, fecal diameter, ileum injury, hematoxylin-eosin (H&E) staining, and diamine oxidase (DAO) and zonulin expression. Expression of the tight junction (TJ) proteins claudin-1, occludin, and zona occludens (ZO-)1 were detected by immunohistochemistry (IHC). Gastrointestinal mRNA expression levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were detected by real-time polymerase chain reaction (RT-PCR). The microbial composition was analyzed by 16s rRNA sequencing.ResultsThe L. paracasei demonstrated excellent therapeutic efficacy against diarrhea. It elevated the TJ protein levels and downregulated proinflammatory cytokines IL-6, IL-1β, TNF-α, and p65, myosin light chain 2 (MLC2), myosin light chain kinase (MLCK). Moreover L. paracasei increased those bacteria, which can product short-chain fatty acid (SCFA) such Alistipes, Odoribacter, Roseburia, and Oscillibacter.ConclusionL. paracasei ameliorated diarrhea by inhibiting activation of the nuclear factor kappa B (NF-κB)-MLCK pathway and increasing the abundance of gut microbiota that produce SCFA.

Lanthanum chloride causes blood-brain barrier disruption through intracellular calcium-mediated RhoA/Rho kinase signaling and myosin light chain kinase.

Rare earth elements (REEs) have caused bioaccumulation and adverse health effects attributed to extensive application. The penetrability of REEs across the blood-brain barrier (BBB) contributes to their neurotoxicity process, but potential mechanisms affecting BBB integrity are still obscure. The present study was designed to investigate the effects of lanthanum on BBB adheren junctions and the actin cytoskeleton in vitro using bEnd.3 cells. After lanthanum chloride (LaCl3, 0.125, 0.25 and 0.5 mM) treatment, cytotoxicity against bEnd.3 cells was observed accompanied by increased intracellular Ca2+. Higher paracellular permeability presented as decreased TEER (transendothelial electrical resistance) and increased HRP (horse radish peroxidase) permeation, and simultaneously reduced VE-cadherin expression and F-actin stress fiber formation caused by LaCl3 were reversed by inhibition of ROCK (Rho-kinase) and MLCK (myosin light chain kinase) using inhibitor Y27632 (10 μM) and ML-7 (10 μM). Moreover, chelating overloaded intracellular Ca2+ by BAPTA-AM (25 μM) remarkably abrogated RhoA/ROCK and MLCK activation and downstream phosphorylation of MYPT1 (myosin phosphatase target subunit 1) and MLC2 (myosin light chain 2), therefore alleviating LaCl3-induced BBB disruption and dysfunction. In conclusion, this study indicated that lanthanum caused endothelial barrier hyperpermeability accompanied by loss of VE-cadherin and rearrangement of the actin cytoskeleton though intracellular Ca2+-mediated RhoA/ROCK and MLCK pathways.

High-fat diet promotes experimental colitis by inducing oxidative stress in the colon.

Diets high in animal fats are associated with increased risks of inflammatory bowel disease, but the mechanism remains unclear. In this study, we investigated the effect of high-fat diet (HFD) on the development of experimental colitis in mice. Relative to mice fed low-fat diet (LFD), HFD feeding for 4 wk increased the levels of triglyceride, cholesterol, and free fatty acids in the plasma as well as within the colonic mucosa. In an experimental colitis model induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS), mice on 4-wk HFD exhibited more severe colonic inflammation and developed more severe colitis compared with the LFD counterparts. HFD feeding resulted in higher production of mucosal pro-inflammatory cytokines, greater activation of the myosin light chain kinase (MLCK) tight junction regulatory pathway, and greater increases in mucosal barrier permeability in mice following TNBS induction. HFD feeding also induced gp91, an NADPH oxidase subunit, and promoted reactive oxygen species (ROS) production in both colonic epithelial cells and lamina propria cells. In HCT116 cell culture, palmitic acid or palmitic acid and TNF-α combination markedly increased ROS production and induced the MLCK pathway, and these effects were markedly diminished in the presence of a ROS scavenger. Taken together, these data suggest that HFD promotes colitis by aggravating mucosal oxidative stress, which rapidly drives mucosal inflammation and increases intestinal mucosal barrier permeability.NEW & NOTEWORTHY This study demonstrates high-fat diet feeding promotes colitis in a 2,4,6-trinitrobenzenesulfonic acid-induced experimental colitis model in mice. The underlying mechanism is that high-fat diet induces oxidative stress in the colonic mucosa, which increases colonic epithelial barrier permeability and drives colonic mucosal inflammation. These observations provide molecular evidence that diets high in saturated fats are detrimental to patients with inflammatory bowel diseases.

Lactobacillus reuteri LR1 Improved Expression of Genes of Tight Junction Proteins via the MLCK Pathway in IPEC-1 Cells during Infection with Enterotoxigenic Escherichia coli K88.

Intestinal epithelial barrier damage disrupts immune homeostasis and leads to many intestinal disorders. Lactobacillus reuteri strains have probiotic functions in their modulation of the microbiota and immune system in intestines. In this study, the effects of L. reuteri LR1, a new strain isolated from the feces of weaning piglets, on intestinal epithelial barrier damage in IPEC-1 cells caused by challenge with enterotoxigenic Escherichia coli (ETEC) K88 were examined. It was found that L. reuteri LR1, in large part, offset the ETEC K88-induced increase in permeability of IPEC-1 cell monolayers and decreased the adhesion and invasion of the coliform in IPEC-1 cells. In addition, L. reuteri LR1 increased transcript abundance and protein contents of tight junction (TJ) proteins zonula occluden-1 (ZO-1) and occludin in ETEC K88-infected IPEC-1 cells, whereas it had no effects on claudin-1 and F-actin expression. Using colloidal gold immunoelectron microscopy, these effects of L. reuteri LR1 on ZO-1 and occludin content in IPEC-1 cells were confirmed. By using ML-7, a selective inhibitor of myosin light-chain kinase (MLCK), the beneficial effect of L. reuteri LR1 on contents of ZO-1 and occludin was shown to be dependent on the MLCK pathway. In conclusion, L. reuteri LR1 had beneficial effects on epithelial barrier function consistent with increasing ZO-1 and occludin expression via a MLCK-dependent manner in IPEC-1 cells during challenge with ETEC K88.

1,25-Dihydroxyvitamin D Protects Intestinal Epithelial Barrier by Regulating the Myosin Light Chain Kinase Signaling Pathway.

The myosin light chain kinase (MLCK) pathway controls intestinal epithelial barrier permeability by regulating the tight junction. 1,25-dihydroxyvitamin D (1,25(OH)2D3)-vitamin D receptor (VDR) signaling protects the epithelial barrier, but the molecular mechanism is incompletely understood. MLCK activation and barrier permeability were studied using monolayers of HCT116, Caco-2, and SW480 cells treated with tissue necrosis factor α with or without 1,25(OH)2D3. The MLCK pathway was analyzed in normal and inflamed colonic biopsies from patients with ulcerative colitis. Colonic mucosal barrier permeability and MLCK activation were also investigated using trinitrobenzene sulfonic acid-induced colitis models in vitamin D analog paricalcitol-treated wild-type mice and mice carrying VDR deletion in colonic epithelial cells. Tissue necrosis factor α increased cell monolayer permeability and induced long isoform of MLCK expression and myosin II regulatory light chain (MLC) phosphorylation, and 1,25(OH)2D3 blocked tissue necrosis factor α-induced increases in monolayer permeability and MLCK-MLC pathway activation by a VDR-dependent fashion. 1,25(OH)2D3 directly suppressed long MLCK expression by attenuating NF-κB activation, and chromatin immunoprecipitation assays confirmed that 1,25(OH)2D3 disrupted p65 binding to 3 κB sites in long MLCK gene promoter. In human ulcerative colitis biopsies, VDR reduction was associated with increases in long MLCK expression and MLC phosphorylation. In trinitrobenzene sulfonic acid colitis models, paricalcitol ameliorated colitis, attenuated the increase in mucosal barrier permeability, and inhibited long MLCK induction and MLC phosphorylation. In contrast, mice with colonic epithelial VDR deletion exhibited more robust increases in mucosal barrier permeability and MLCK activation compared with wild-type mice. These data demonstrate that 1,25(OH)2D3-VDR signaling preserves the mucosal barrier integrity by abrogating MLCK-dependent tight junction dysregulation during colonic inflammation.

Rho‑associated kinase inhibitor, Y‑27632, inhibits the invasion and proliferation of T24 and 5367 bladder cancer cells.

The serine/threonine kinases, Rho‑associated protein kinase I and II (ROCK I and II), regulate the cytoskeleton by acting downstream of the small GTPase, Rho, and have been implicated in tumorigenesis and cancer metastasis. Inhibition of ROCK signaling has been shown to suppress the invasion and migration of several types of cancer cells. In this study, the effect of the ROCK inhibitor, Y‑27632, on the proliferation and invasion of T24 and 5637 bladder cancer cells was investigated. In the proliferation assays, the cells were exposed to 0, 10, 25, 50, 75, 100, 125 or 150 µmol/l Y‑27632 and proliferation was determined using Cell Counting kit‑8 after 24, 48 and 72 h. In the invasion assays, the cells were placed in the upper chamber of transwell plates and subjected to 0, 25, 50 or 75 µmol/l Y‑27632 for 24 h, after which invasion was measured. Y‑27632 significantly suppressed the cell proliferation of T24 and 5637 cells in a concentration- and time‑dependent manner. Y‑27632 also inhibited the invasion of T24 and 5637 cells in a concentration‑dependent manner (P<0.001). In addition, Y‑27632 suppressed myosin light chain kinase (MLCK) phosphorylation in T24 and 5637 cells, confirming that it is also a downstream effector of the Rho/ROCK pathway in T24 and 5637 bladder cancer cells. In conclusion, the Rho/ROCK/P‑MLCK pathway may be important in tumor cell metastasis in bladder cancer.

Tu1816 1,25-Dihydroxyvitamin D Protects Intestinal Epithelial Barrier by Regulating the Myosin Light Chain Kinase Pathway

Tu1816 1,25-Dihydroxyvitamin D Protects Intestinal Epithelial Barrier by Regulating the Myosin Light Chain Kinase Pathway

Higher expression of phosphorylated myosin regulatory light chain in the common bile duct in pancreaticobiliary maljunction accompanied by bile duct dilatation in children: a post-mortem observational study

PurposeTo examine the content of phosphorylated myosin regulatory light chain (P-MLC20) and myosin light-chain kinase (MLCK) in the common bile duct of pediatric patients with pancreaticobiliary maljunction (PBM) accompanied by bile duct dilatation (BDD), and investigate their potential role in PBM accompanied by BDD.MethodsTwenty-one specimens of the common bile duct from pediatric patients with PBM accompanied by BDD were collected. P-MLC20 was examined with immunohistochemistry. The expression of P-MLC20 and MLCK was also examined with Western blot. Twenty-one specimens of the common bile duct from pediatric patients without PBM and BDD were used as controls.ResultsThe mean optical density (MOD), mean labeling intensity (MLI) and minimum qualifying scores (MQS) of P-MLC20 were 115.6856 ± 58.1634, 21.7125 % ± 9.6555 and 21.3531 ± 6.5255, respectively. In the control group, MOD, MLI and MQS were 96.5581 ± 9.7859, 11.1813 % ± 3.6208 and 10.7819 ± 3.5323, respectively. There was no significant difference in MOD between the two groups (P > 0.05), whereas there was a significant difference in MLI and MQS between the two groups (P < 0.05). The expression of P-MLC20 and MLCK, as determined with Western blot, was also significantly higher in the PBM group than in the control group (P < 0.05).ConclusionP-MLC20 is associated with increased contractile force of the smooth muscle of the common bile duct in pediatric patients with PBM accompanied by BDD. The enhanced expression of P-MLC20 in the common bile duct probably contributes to increased bile duct pressure in PBM via the MLCK pathway.

Carbachol ameliorates lipopolysaccharide-induced intestinal epithelial tight junction damage by down-regulating NF-κβ and myosin light-chain kinase pathways

Carbachol is a cholinergic agonist that protects the intestines after trauma or burn injury. The present study determines the beneficial effects of carbachol and the mechanisms by which it ameliorates the lipopolysaccharide (LPS)-induced intestinal barrier breakdown. Rats were injected intraperitoneally with 10mg/kg LPS. Results showed that the gut barrier permeability was reduced, the ultrastructural disruption of tight junctions (TJs) was prevented, the redistribution of zonula occludens-1 and claudin-2 proteins was partially reversed, and the nuclear factor-kappa beta (NF-κβ) and myosin light-chain kinase (MLCK) activation in the intestinal epithelium were suppressed after carbachol administration in LPS-exposed rats. Pretreatment with the α7 nicotinic acetylcholine receptor (α7nAchR) antagonist α-bungarotoxin blocked the protective action of carbachol. These results suggested that carbachol treatment can protect LPS-induced intestinal barrier dysfunction. Carbachol exerts its beneficial effect on the amelioration of the TJ damage by inhibiting the NF-κβ and MLCK pathways in an α7nAchR-dependent manner.

Signaling pathways in cerebral vasospasm

AbstractCerebral vasospasm is a deadly complication following the rupture of intracranial aneurysms. One new development in the experimental treatment of cerebral vasospasm is the looming target of signaling pathways. The pathogenesis of cerebral vasospasm involves multiple signaling pathways in proliferation, inflammation, cell death, smooth muscle phenotype changes, vascular remodeling, and contraction. A review of all of these areas is beyond the scope of this article, and as such, three systems that mediate these vascular responses have been selected: the tyrosine kinase-MAP kinase pathway, the sphingosine-1-Rho myosin light chain kinase pathway and protein kinase C.

Significance of α-SMA in myofibroblasts emerging in renal tubulointerstitial fibrosis.

Myofibroblast transdifferentiation plays a crucial role in the development and progression of renal tubulointerstitial fibrosis. However, the significance of α-smooth muscle actin (α-SMA) expression, which is the major morphological characteristic of myofibroblasts, remains to be determined in detail. The effect of α-SMA expression on fibrosis tissue was examined by using a fibrosis model (collagen gel) in vitro. The transdifferentiation of fibroblasts into myofibroblasts was triggered in the culture medium with 0.5% fetal bovine serum (FBS)+transforming growth factor (TGF)-β1, but not with 10% FBS+TGF-β1. The TGF-β1-induced gel contraction caused by myofibroblasts was greater than that by fibroblasts. Gel contraction by myofibroblasts involved the Ca²+-dependent myosin light chain kinase pathway, as well as the activation of Rho kinase and p38 mitogen-activated protein kinase (MAPK). Taken together, these findings suggest that α-SMA expression in renal interstitial fibroblasts, i.e., myofibroblast transdifferentiation, accelerates the contraction of the tubulointerstitial fibrosis tissue via the Ca²+-dependent pathway, in addition to the pathways involved in fibroblast contraction; this event may lead to renal atrophy and renal failure.

Berberine Ameliorates Intestinal Epithelial Tight-Junction Damage and Down-regulates Myosin Light Chain Kinase Pathways in a Mouse Model of Endotoxinemia

This study aimed to examine the protective effect of berberine in endotoxin-induced intestinal tight-junction injury in a mice model of endotoxinemia. Endotoxinemia was induced by intraperitoneal injection of lipopolysaccharide (10 mg/kg). Mice were randomized to 5 groups: control mice, berberine-treated mice, lipopolysaccharide (LPS)-injected mice, mice pretreated with berberine, and mice administered berberine following LPS injection. Samples were collected 12 h after LPS treatment. Ileal mucosal permeability to fluorescein isothiocyanate dextran assay indicated that berberine reduced the permeability of the gut barrier in endotoxinemia. Transmission electron microscopy revealed that pretreatement with berberine partly prevented ultrastructural disruption of tight junctions by LPS. Immunofluorescence and Western blot analysis were performed, and the results demonstrated that pretreatement with berberine partially reversed the redistribution of tight-junction proteins in colon epithelium and in membrane microdomains. Our data also indicated that pretreatement with berberine could suppress translocation, from cytoplasm to the nucleus, of nuclear factor-κB and myosin light chain kinase activation in the intestinal epithelium. Pretreatement with berberine attenuates disruption of tight junctions in intestinal epithelium in a mice model of endotoxinemia. This may possibly have been mediated through down-regulation of the nuclear factor-κB and myosin light chain kinase pathway.

Increased contractility of hepatic stellate cells in cirrhosis is mediated by enhanced Ca2+-dependent and Ca2+-sensitization pathways

Activation of hepatic stellate cells (HSCs) results in cirrhosis and portal hypertension due to intrahepatic resistance. Activated HSCs increase their contraction after receptor agonist stimulation; however, the signaling pathways for the regulation of contraction are not fully understood. The aim of this study was to elucidate the change in contractile mechanisms of HSCs after cirrhotic activation. The expression pattern of contractile regulatory proteins was analyzed with quantitative RT-PCR and Western blotting. The phosphorylation levels of myosin light chain (MLC), 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), and MLC phosphatase targeting subunit 1 (MYPT1) after endothelin-1 (ET-1) stimulation in culture-activated HSCs were measured using phosphorylation-specific antibodies. In vivo-activated HSCs were isolated from rats subjected to bile duct ligation and repeated dimethylnitrosoamine injections. HSCs showed increased expression of not only α-smooth muscle actin, but also the contractile regulatory proteins MLC kinase (MLCK), Rho kinase 2 (ROCK2), and CPI-17 during HSC activation in vitro. In culture-activated HSCs, ET-1 increased phosphorylation of CPI-17 at Thr18, which was markedly inhibited by the PKC inhibitor Ro-31-8425. ET-1 induced phosphorylation of MYPT1 at Thr853, which was suppressed by the ROCK inhibitor Y-27632. ET-1 induced sustained phosphorylation of MLC at Thr18/Ser19, which was inhibited by both Ro-31-8425 and Y-27632. Consistent with the data obtained from the in vitro study, HSCs isolated from cirrhotic rats showed increased expression of α-smooth muscle actin, MLCK, CPI-17, and ROCK2 compared with HSCs from nontreated rats. Furthermore, MLC phosphorylation in in vivo-activated HSCs was increased, according to enhanced phosphorylation of CPI-17 and MYPT1 in the presence of ET-1. These results suggest that activated HSCs may participate in constriction of hepatic sinusoids in the cirrhotic liver through both Ca(2+)-dependent (MLCK pathway) and Ca(2+)-sensitization mechanism (CPI-17 and MYPT1 pathways).

Characterization of Cytoskeleton-Enriched Protein Fraction of the Trabecular Meshwork and Ciliary Muscle Cells

To understand the molecular basis for the known distinct contractile characteristics of trabecular meshwork (TM) and ciliary muscle (CM) cells, the cytoskeleton-enriched protein fractions of the TM and CM cells were isolated and characterized. The nonionic surfactant insoluble fraction enriched for cytoskeletal proteins was isolated from human and porcine TM tissue and cells and from CM cells and was characterized by SDS-PAGE, mass spectrometry, and immunoblotting techniques. The cytoskeleton-enriched protein fraction derived from both human and porcine TM cells contained Plectin 1, Filamin A, non-muscle myosin IIA, clathrin, α-actinin, vimentin, actin, caldesmon, myosin IC, and annexin A2 as major proteins and was noted to exhibit compositional similarity with the cytoskeletal protein fraction isolated from TM tissue. Importantly, the cytoskeletal protein composition of the TM cells was also found to be similar to that noted for CM and vascular endothelial cells. Although the activity of myosin II, a crucial regulator of cellular contraction and a major component of the cytoskeletal protein fraction in TM and CM cells, was regulated predominantly by Rho kinase in both cell types, myosin light chain kinase (MLCK) also appeared to control myosin II activity in CM cells. These data reveal that the activity of non-muscle myosin II, a critical molecule of cellular contraction, was found to be regulated differentially in TM and CM cells by the Rho kinase and the MLCK pathways despite their compositional similarity in cytoskeletal protein profile.

Regulation of ANP secretion from isolated atria by prostaglandins and cyclooxygenase-2

Cyclooxygenase (COX) is a key enzyme regulating the production of various prostaglandins (PGs) from arachidonic acid. Angiotensin II has been reported to be an important inflammatory mediator, which increases COX-2. The aim of this study was to determine the role of various PGs and COX-2 in the regulation of atrial natriuretic peptide (ANP) secretion. PGF2α and PGD2 caused dose-dependent increases in ANP release and intra-atrial pressure. The potency for the stimulation of ANP secretion by PGF2α was higher than that by PGD2. In contrast, PGE2, PGI2, PGJ2, and thromboxane A2 did not show any significant effects. The increases in intra-atrial pressure and ANP secretion induced by PGF2α and PGD2 were significantly attenuated by the pretreatment with an inhibitor of PGF2α receptor. By the pretreatment with an inhibitor for phospholipase C (PLC), inositol 3-phosphate (IP3) receptor, protein kinase C (PKC), or myosin light chain kinase (MLCK), PGF2α-mediated increase in ANP secretion and positive inotropy were attenuated. Inhibitor for COX-1 or COX-2 did not cause any significant effects on atrial parameters. In hypertrophied rat atria, PGF2α-induced positive inotropy and ANP secretion were markedly attenuated whereas COX-2 inhibitor stimulated ANP secretion. The expression of COX-2 increased and the expression of PGF2α receptor mRNA decreased in hypertrophied rat atria. These results suggest that PGF2α increased the ANP secretion and positive inotropy through PLC–IP3–PKC–MLCK pathway, and the modulation of ANP secretion by COX-2 inhibitor and PGF2α may partly relate to the development of renal hypertension.

TNF induces caspase-dependent inflammation in renal endothelial cells through a Rho- and myosin light chain kinase-dependent mechanism

The pathogenesis of LPS-induced acute kidney injury (AKI) requires signaling through tumor necrosis factor-alpha (TNF) receptor 1 (TNFR1), which within the kidney is primarily located in the endothelium. We showed previously that caspase inhibition protected mice against LPS-induced AKI and in parallel significantly inhibited LPS-induced renal inflammation. Therefore we hypothesized that caspase activation amplifies TNF-induced inflammation in renal endothelial cells (ECs). In cultured renal ECs, TNF induced apoptosis through a caspase-8-dependent pathway. TNF caused translocation of the p65 subunit of NF-kappaB to the nucleus, resulting in upregulation of inflammatory markers such as adhesion molecules ICAM-1 and VCAM-1. However, the broad-spectrum caspase inhibitor Boc-d-fmk reduced NF-kB activation as assessed by gel shift assay, reduced phosphorylation of subunit IkappaBalpha, and significantly inhibited TNF-induced expression of ICAM-1 and VCAM-1 as assessed by both real-time PCR and flow cytometry. Broad-spectrum caspase inhibition markedly inhibited neutrophil adherence to the TNF-activated endothelial monolayer, supporting the functional significance of this effect. Specific inhibitors of caspases-8 and -3, but not of caspase-1, reduced TNF-induced NF-kappaB activation. Caspase inhibition also reduced TNF-induced myosin light chain (MLC)-2 phosphorylation, and activation of upstream regulator RhoA. Consistent with this, MLC kinase (MLCK) inhibitor ML-7 reduced TNF-induced NF-kappaB activation. Thus caspase activation influences NF-kappaB signaling via its affect on cytoskeletal changes occurring through RhoA and MLCK pathways. These cell culture experiments support a role for caspase activation in TNF-induced inflammation in the renal endothelium, a key event in LPS-induced AKI.

PKC and MLCK-Dependent, Cytokine-Induced Rat Coronary Endothelial Dysfunction

Heart disease is one of the leading causes of death in the United States, killing nearly one million people every year. Inflammatory mediators or cytokines are released following myocardial infarction and ischemia/reperfusion injury. These cytokines, of which interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha (TNF-alpha) are among the most important, propagate the activation of a multitude of signaling pathways, such as the protein kinase C (PKC) and myosin light chain kinase (MLCK) pathways, which lead to deleterious changes in the structure and function of the coronary microvascular endothelium. The effects of cytokines on rat heart microvascular endothelial cell monolayer integrity, PKC activity, and adherens junction protein alteration were examined. Further, an in vivo rat coronary ischemia/reperfusion injury model was used to determine vascular leakage and TNF-alpha release. Administration of the above mentioned cytokines to cell monolayers resulted in significant increases in PKC activation, gap formation, and hyperpermeability across the monolayer and beta-catenin phosphorylation/reorganization. Inhibition of conventional PKC and MLCK attenuated permeability increases. Ischemia/reperfusion injury to the left ventricle resulted in TNF-alpha release as well as conventional PKC- and MLCK-dependent protein extravasation from the circulation to the heart tissue. These results identify the conventional PKC and MLCK pathways as important factors in coronary endothelial dysfunction elicited by IR injury and cytokine release. Further examination of these molecular signaling cascades has the potential of identifying targets for therapeutic intervention following ischemic events in the heart.