Lactic Acid Dehydrogenase Release Research Articles

TargeTron Inactivation of Chlamydia trachomatis gseA Results in a Lipopolysaccharide 3-Deoxy-d-Manno-Oct-2-Ulosonic Acid-Deficient Strain That Is Cytotoxic for Cells.

All members of the family Chlamydiaceae have lipopolysaccharides (LPS) that possess a shared carbohydrate trisaccharide antigen, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) that is functionally uncharacterized. A single gene, genus-specific epitope (gseA), is responsible for attaching the tri-Kdo to lipid IVA. To investigate the function of Kdo in chlamydial host cell interactions, we made a gseA-null strain (L2ΔgseA) by using TargeTron mutagenesis. Immunofluorescence microscopy and immunoblotting with a Kdo-specific monoclonal antibody demonstrated that L2ΔgseA lacked Kdo. L2ΔgseA reacted by immunoblotting with a monoclonal antibody specific for a conserved LPS glucosamine-PO4 epitope, indicating that core lipid A was retained by the mutant. The mutant strain produced a similar number of inclusions as the parental strain but yielded lower numbers of infectious elementary bodies. Transmission electron microscopy of L2ΔgseA-infected cells showed atypical developmental forms and a reduction in the number of elementary bodies. Immunoblotting of dithiothreitol-treated L2ΔgseA-infected cells lysates revealed a marked reduction in outer membrane OmcB disulfide cross-linking, suggesting that the elementary body outer membrane structure was affected by the lack of Kdo. Notably, lactic acid dehydrogenase release by infected cells demonstrated that L2ΔgseA was significantly more cytotoxic to host cells than the wild type. The cytotoxic phenotype may result from an altered outer membrane biogenesis structure and/or function or, conversely, from a direct pathobiological effect of Kdo on an unknown host cell target. These findings implicate a previously unrecognized role for Kdo in host cell interactions that facilitates postinfection host cell survival.

MicroRNA‐17‐3p protects against excessive posthypoxic autophagy in H9C2 cardiomyocytes via PTEN–Akt–mTOR signaling pathway

The activity of phosphatase and tensin homolog (PTEN) can be inhibited by miR-17-3p, which results in attenuating myocardial ischemia/reperfusion injury (IRI), however, the mechanism behind this phenomenon is still elusive. Suppression of PTEN leads to augmented protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling strength and constrained autophagy activation, which might be the one mechanism for the ameliorated myocardial IRI. Thus, we tested the hypothesis that miR-17-3p attenuated hypoxia/reoxygenation (H/R)-mediated damage in cardiomyocytes by downregulating excessive autophagy via the PTEN-Akt-mTOR axis. The expression of miR-17-3p was remarkably increased after H/R treatment (6-h hypoxia followed by 6-h reoxygenation; H6/R6), which was concomitant with the increase of the release of lactic acid dehydrogenase (cell injury marker) and the enhancement LC3II/I ratio (autophagy markers) in H9C2 cardiomyocytes. Ectoexpression of miR-17-3p agomir led to remarkable augmentation of miR-17-3p expression and evidently attenuated H/R-mediated cell damage and excessive autophagy. Furthermore, an increase in miR-17-3p expression elicited constrained phosphorylation of PTEN (Ser380 ) while enhanced the phosphorylation of Akt (Thr308 , Ser473 ) and mTOR (Ser536 ) after H/R stimulation. In addition, pretreatment with LY-294002 (an Akt selective inhibitor) and rapamycin (an mTOR selective inhibitor) significantly abrogated the protective function of miR-17-3p on H/R-mediated cell damage and autophagy in H9C2 cardiomyocytes. Taken together, these observations indicated that the enhancement of the PTEN/Akt/mTOR axis and the consequent suppression of autophagy overactivation might represent an underlying mechanism by which miR-17-3p attenuated H/R-mediated damage in H9C2 cells.

Induction of multiple subroutines of regulated necrosis in murine macrophages by natural BH3-mimetic gossypol.

Macrophages are critical sentinel cells armed with multiple regulated necrosis pathways, including pyroptosis, apoptosis followed by secondary necrosis, and necroptosis, and are poised to undergo distinct form(s) of necrosis for tackling dangers of pathogenic infection or toxic exposure. The natural BH3-mimetic gossypol is a toxic phytochemical that can induce apoptosis and/or pyroptotic-like cell death, but what exact forms of regulated necrosis are induced remains largely unknown. Here we demonstrated that gossypol induces pyroptotic-like cell death in both unprimed and lipopolysaccharide-primed mouse bone marrow-derived macrophages (BMDMs), as evidenced by membrane swelling and ballooning accompanied by propidium iodide incorporation and lactic acid dehydrogenase release. Notably, gossypol simultaneously induces the activation of both pyroptotic and apoptotic (followed by secondary necrosis) pathways but only weakly activates the necroptosis pathway. Unexpectedly, gossypol-induced necrosis is independent of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, as neither inhibitor for the NLRP3 pathway nor NLRP3 deficiency protects the macrophages from the necrosis. Furthermore, necrotic inhibitors or even pan-caspase inhibitor alone does not or only partly inhibit such necrosis. Instead, a combination of inhibitors composed of pan-caspase inhibitor IDN-6556, RIPK3 inhibitor GSK'872 and NADPH oxidase inhibitor GKT137831 not only markedly inhibits the necrosis, with all apoptotic and pyroptotic pathways being blocked, but also attenuates gossypol-induced peritonitis in mice. Lastly, the activation of the NLRP3 pathway and apoptotic caspase-3 appears to be independent of each other. Collectively, gossypol simultaneously induces the activation of multiple subroutines of regulated necrosis in macrophages depending on both apoptotic and inflammatory caspases.

Long non-coding RNA LOC366613 alleviates the cerebral ischemic injury via regulating the miR-532-5p/phosphatase and tensin homolog axis

ABSTRACT Cerebral infarction (CI) has become a leading cause of death in China. Long non-coding RNAs (lncRNAs) are intensively involved in the progression of CI. Here, we aimed to investigate the effects of lncRNA LOC366613 (LOC366613) on cerebral I/R injury, as well as its possible mechanism. Transient middle cerebral artery occlusion (MCAO) was used to establish a mouse model of cerebral I/R, and the PC12 cell line was used to establish an in vitro oxygen-glucose deprivation (OGD) injury model. The MTT assay was used to determine cell viability, and qRT-PCR was used to determine RNA levels. Western blotting was conducted to detect protein expression levels. The TUNEL assay and flow cytometry were used to measure cell apoptosis, and 2,3,5-triphenyltetrazolium chloride (TTC) was used to determine cerebral infarct volume. Finally, RNA pull-down and luciferase activity assays were used to examine interactions between miR-532-5p and LOC366613, as well as between miR-532-5p and phosphatase and tensin homolog (PTEN). LOC366613 was overexpressed in patients with cerebral I/R injury. In PC12 cells, knockdown of LOC366613 reduced the apoptosis rate and lactic acid dehydrogenase (LDH) expression, while increasing cell viability. Moreover, miR-532-5p was shown to be a target of LOC366613, as predicted. Downregulation of miR-532-5p reversed the effects of LOC366613 knockdown on PC12 cell apoptosis, LDH release, and cell viability. Finally, PTEN was verified as a target of miR-532-5p. LOC366613 participates in cerebral I/R injury by regulating the miR-532-5p/PTEN axis, potentially providing a new CI treatment target.

Kappa opioid receptors internalization is protective against oxygen-glucose deprivation through β-arrestin activation and Akt-mediated signaling pathway

Hypoxia induces reversible κ-opioid receptor (KOR) internalization similar to the internalization that is induced by KOR agonists. In the current study, we demonstrate that this KOR internalization is a protective mechanism via the β-arrestin specific pathway in an oxygen-glucose deprivation (OGD) model. Mouse neuroblastoma Neuro2A cells were stably transfected with mouse KOR–tdTomato fusion protein (N2A-mKOR-tdT cells). Various concentrations of salvinorin A (SA), a highly selective KOR agonist, were given in the presence and absence of norbinaltorphimine (norBNI), which is a KOR antagonist, or Dyngo-4a (internalization inhibitor) or API-2 (Akt/Protein kinase B signaling inhibitor-2). Various concentrations of SA and RB-64 (22-thiocyanatosalvinorin A, selective for the G protein signaling pathway) were administered both in normoxic and hypoxic conditions. Autophagosomes and ultrastructural components of cells were observed using transmission electron microscopy (TEM). Cell viability, severity of cell injury, and levels of proteins related to the Akt signaling pathway were evaluated using live cell counting (by Cell Counting Kit-8), the lactic acid dehydrogenase (LDH) release rate, and Western blot analysis, respectively. SA promoted cell survival and attenuated OGD-induced cell injury. The Akt signaling pathway is activated by SA. KOR internalization, when blocked by norBNI or Dyngo-4a, increased LDH release and decreased cell viability under OGD. Treatment with SA significantly inhibited autophagy, and the effects of SA on autophagy were reversed by API-2 pretreatment. RB-64 in a low concentration without β-arrestin recruitment did not reduce LDH release and increase cell viability as observed with SA. KOR internalization through β-arrestin activation is a protective mechanism against OGD. The Akt pathway might play a critical role in modulating these protective effects by inhibiting autophagy.

MicroRNA‐181c‐5p enhances NFκB‐mediated inflammation via targeting PTPN4 in H9C2 cardiomyocytes during hypoxia/ reoxygenation

BackgroundActivation of nuclear factor kappa B (NFκB) and subsequent inflammation occurs in various models of myocardial ischemia/reperfusion injury (I/RI). Thus, inhibition of NFκB‐mediated inflammation is of clinical significance for improving post‐ischemic recovery. miR‐181c‐5p was significantly up‐regulated in the early phase of myocardial infarction in patients. However, whether miR‐181c‐5p mediates cardiac I/RI through NFκB‐mediated inflammation is unknown. We aimed to explore the role and mechanism of miR‐181c‐5p in inflammation during I/RI in H9C2 cardiomyocytes under hypoxia/reoxygenation (H/R) injury.Methods and ResultsIn response to H/R (6 hours hypoxia followed by 6 hours reoxygenation), the expression of miR‐181c‐5p was significantly up‐regulated, suggestive of a potential role of miR‐181c‐5p in the pathology of myocardial I/RI. In addition, H/R significantly induced H9C2 cardiomyocytes injury, evidenced by increased release of lactic acid dehydrogenase (LDH, cell injury marker) and reduced cell viability, concomitantly with enhanced phosphorylation and degradation of IκBα, p65 nuclear translocation and increased production of NFκB‐mediated pro‐inflammatory cytokines tumor necrosis factor α (TNFα), interleukin (IL)6 and IL1β. To demonstrate the potential role of miR‐181c‐5p in H/R‐induced cell inflammation and injury, H9C2 cardiomyocytes were transfected with the miR‐181c‐5p agomir or its negative control. Overexpression of miR‐181c‐5p significantly aggravated H/R‐induced cell injury (increased LDH level and reduced cell viability) and exacerbated NFκB‐mediated inflammation [greater phosphorylation and degradation of IκBα, p65 nuclear translocation and increased levels of pro‐inflammatory cytokines (TNFα, IL6, IL1 β)]. By using computational prediction algorithms (TargetScan and miRDB), protein tyrosine phosphatase non‐receptor type 4 (PTPN4) was predicted as a potential target gene of miR‐181c‐5p, as also verified by the luciferase reporter assay. Indeed, overexpression of miR‐181c‐5p significantly attenuated the mRNA and protein expression of PTPN4 in H9C2 cardiomyocytes, while knockdown of PTPN4 significantly aggravated H/R‐induced enhancement of LDH level, phosphorylation and degradation of IκBα and pro‐inflammatory cytokines level, which mimicked the pro‐inflammatory effects of miR‐181c‐5p.ConclusionsmiR‐181c‐5p exacerbates H/R‐induced cardiomyocyte injury and NFκB‐mediated inflammation via targeting PTPN4, and the miR‐181c‐5p/PTPN4 signaling may be a novel target to combat myocardial I/RI.Support or Funding InformationThe work was supported by GRF (17117217M, Research Grants Council of Hong Kong).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Naftopidil reduced the proliferation of lung fibroblasts and bleomycin-induced lung fibrosis in mice.

Naftopidil, an α‐1 adrenoceptor antagonist with few adverse effects, is prescribed for prostate hyperplasia. Naftopidil inhibits prostate fibroblast proliferation; however, its effects on lung fibroblasts and fibrosis remain largely unknown. Two normal and one idiopathic pulmonary fibrosis human lung fibroblast lines were cultured with various naftopidil concentrations with or without phenoxybenzamine, an irreversible α‐1 adrenoceptor inhibitor. We examined the incorporation of 5‐bromo‐2ʹ‐deoxyuridine into DNA and lactic acid dehydrogenase release by enzyme‐linked immunosorbent assay, cell cycle analysis by flow cytometry, scratch wound‐healing assay, and mRNA expressions of type IV collagen and α‐smooth muscle actin by polymerase chain reaction. Effects of naftopidil on bleomycin‐induced lung fibrosis in mice were evaluated using histology, micro‐computed tomography, and surfactant protein‐D levels in serum. Naftopidil, dose‐dependently but independently of phenoxybenzamine, inhibited 5‐bromo‐2ʹ‐deoxyuridine incorporation in lung fibroblasts. Naftopidil induced G1 cell cycle arrest, but lactic acid dehydrogenase release and migration ability of lung fibroblasts were unaffected. Naftopidil decreased mRNA expressions of type IV collagen and α‐smooth muscle actin in one normal lung fibroblast line. Histological and micro‐computed tomography examination revealed that naftopidil attenuated lung fibrosis and decreased serum surfactant protein‐D levels in bleomycin‐induced lung fibrosis in mice. In conclusion, naftopidil may have therapeutic effects on lung fibrosis.

Helper CD4 T cells expressing granzyme B cause glial fibrillary acidic protein fragmentation in astrocytes in an MHCII-independent manner.

During inflammatory processes of the central nervous system, helper T cells have the capacity to cross the blood-brain barrier and injure or kill neural cells through cytotoxic mechanisms. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is part of the astrocyte cytoskeleton that can become fragmented in neuroinflammatory conditions. The mechanism of action by which helper T cells with cytotoxic properties injure astrocytes is not completely understood. Primary human astrocytes were obtained from fetal brain tissue. Human helper (CD4+ ) T cells were isolated from peripheral blood mononuclear cells and activated with the superantigen staphylococcal enterotoxin E (SEE). Granzyme B was detected by enzyme linked immunosorbent assay and intracellular flow cytometry. GFAP fragmentation was monitored by western blotting. Cell death was monitored by lactic acid dehydrogenase release and terminal biotin-dUTP nick labeling (TUNEL). Astrocyte migration was monitored by scratch assay. Adult human oligodendrocytes were cultured with sublethally injured astrocytes to determine support function. Helper T cells activated with SEE expressed granzyme B but not perforin. Helper T cells released granzyme B upon contact with astrocytes and caused GFAP fragmentation in a caspase-dependent, MHCII-independent manner. Sublethally injured astrocytes were not apoptotic; however, their processes were thin and elongated, their migration was attenuated, and their ability to support oligodendrocytes was reduced in vitro. Helper T cells can release granzyme B causing sublethal injury to astrocytes, which compromises the supportive functions of astrocytes. Blocking these pathways may lead to improved resolution of neuroinflammatory lesions.

FOXD3 inhibits SCN2A gene transcription in intractable epilepsy cell models

The expression of sodium voltage-gated channel alpha subunit 2 (SCN2A) is closely related to the development of epilepsy. This study investigated regulatory element of the SCN2A gene involved in epilepsy. An intractable epilepsy cell model was constructed using hippocampal primary neurons and the SH-SY5Y cell line. SCN2A protein and gene expression in cells as well as the level of lactic acid dehydrogenase (LDH) in the cell culture supernatants was detected. Potential regulatory factors of SCN2A and its upstream regulatory elements were identified using the dual-luciferase reporter assay. Finally, the role of the hypothetical transcription factor in epilepsy was examined by using its small interfering RNA (siRNA). Results found that levels of LDH and expression of the hypothetical transcription factor, Forkhead box D3 (FOXD3), was both increased in the model cells, whereas that of SCN2A was decreased. The results of dual-luciferase reporter assays revealed that an upstream region of SCN2A gene spanning from nucleotides −1617 to −1470 was a transcription factor binding region and a trans-acting factor role of FOXD3 was identified in the core region (GGCAAAATTAT). Then the FOXD3 binding site was further verified by the chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA). After SH-SY5Y cells were transfected with FOXD3 siRNA, the release of LDH into culture supernatants and the LDH expression levels in cells were significantly decreased. SCN2A expression in model cells was increased by knockdown of FOXD3. Therefore, this study demonstrated that FOXD3 is a trans-acting factor of SCN2A, and this mechanism may play a role in cell injury after epilepsy.

Mild hypothermia protects hippocampal neurons from oxygen-glucose deprivation injury through inhibiting caspase-3 activation

Mild hypothermia (MH) is thought to be one of the most effective therapeutic methods to treat hypoxic-ischemic encephalopathy (HIE) after cardiac arrest (CA). However, its precise mechanisms remain unclear. In this research, hippocampal neurons were cultured and treated with mild hypothermia and Ac-DEVD-CHO after oxygen-glucose deprivation (OGD). The activity of caspase-3 was detected, in order to find the precise concentration of Ac-DEVD-CHO with the same protective role in OGD injury as MH treatment. Western blot and immunofluorescence staining were conducted to analyze the effects of MH and Ac-DEVD-CHO on the expressions of caspase-3, caspase-8, and PARP. The neuronal morphology was observed with an optical microscope. The lactic acid dehydrogenase (LDH) release rate, neuronal viability, and apoptotic rate were also detected. We found that MH (32 °C) and Ac-DEVD-CHO (5.96 μMol/L) had equal effects on blocking the activation of caspase-3 and the OGD-induced cleavage of PARP, but neither had any effect on the activation of caspase-8, which goes on to activate caspase-3 in the apoptotic pathway. Meanwhile, both MH and Ac-DEVD-CHO had similar effects in protecting cell morphology, reducing LDH release, and inhibiting OGD-induced apoptosis in neurons. They also similarly improved neuronal viability after OGD. In conclusion, caspase-3 serves as a key intervention point of the key modulation site or regulatory region in MH treatment that protects neuronal apoptosis against OGD injury. Inhibiting the expression of caspase-3 had a protective effect against OGD injury in MH treatment, and caspase-3 activation could be applied to evaluate the neuroprotective effectiveness of MH on HIE.

Individualized cryoballoon energy pulmonary vein isolation guided by real-time pulmonary vein recordings, the randomized ICE-T trial

The ideal energy dosing remains unclear in second-generation cryoballoon (CB) pulmonary vein isolation (PVI). We aimed to investigate the effect of an individualized dosing strategy based on time to PVI (TTI). The purpose of this study was to prospectively investigate the safety and efficacy of individualized PVI using the second-generation CB guided by real-time pulmonary vein recordings. Two groups were prospectively randomized: ICE-T group: if TTI <75 seconds, then no bonus freeze; and control group: acute PVI followed by 1 empiric bonus freeze. Freeze duration was set to 240 seconds. The primary end point was single procedure sinus rhythm after 12 months (blanking period 3 months). Secondary end points included procedural data, complications, and biomarker release. In total, 100 patients with paroxysmal atrial fibrillation were randomized. The primary end point was not different (88% vs 82%). Procedure and fluoroscopy times were significantly shorter in the ICE-T group (70 ± 20 minutes vs 89 ± 21 minutes; P < .001 and 10.6 ± 3.9 minutes vs 12.7 ± 5.5 minutes; P = .03). More complications occurred in the control group (n = 9 vs n = 3) (persistent phrenic nerve injury: n = 1 vs n = 0; transient phrenic nerve injury: n = 5 vs n = 2; esophageal lesions: n = 3 vs n = 1). Postablation troponin T levels were not different (ICE-T group vs control group: 1035 ± 402 ng/L vs 1219 ± 509 ng/L; P = .099), whereas a significantly lower lactic acid dehydrogenase release was observed in the ICE-T group (259 ± 47 U/L vs 282 ± 57 U/L; P = .038). Multivariate analysis identified a mean TTI of >43 seconds as the only independent predictor of recurrent atrial tachyarrhythmia. The individualized CB PVI strategy allows faster atrial fibrillation ablation without affecting the favorable clinical outcome. A short TTI appears to predict freedom from recurrent atrial tachyarrhythmia.

Preventing Nitration of Specific Tyrosine Sites in Adenine Nucleotide Translocase Differentially Protects Against Cell Oxidative Stress Injury

The adenine nucleotide translocase (ANT) is the most abundant protein in the inner mitochondrial membrane and the main transporter for ATP and ADP exchange across the membrane. Ischemia reperfusion (IR) injury causes mitochondrial accumulation of reactive O2 and N2 species (ROS/RNS), which can induce irreversible and deleterious tyrosine nitration (NTyr) of protein. In this study, we investigated IR‐induced NTyr of ANT and its impact on cell damage during oxidative stress. Isolated guinea pig hearts were subjected to 35 min ischemia and 20 min reperfusion or no‐IR (control). At the end of reperfusion, ANT was purified from IR or control heart mitochondria and subjected to Western blotting with antibodies to ANT and nitro‐Tyr. We found that IR induced NTyr of ANT. Mass spectrometry analysis identified ANT tyrosines (Y) residues Y81, Y191 and Y195 were nitrated when mitochondria were treated with 100 µM peroxynitrite, an oxidant. To determine the contribution of nitration of ANT Y81, Y191 and Y195 on cellular injury induced by oxidative stress, a single mutant Y81F (phenyalanine) and a double mutant Y192F and Y195F were constructed. After transfection of these mutants into a cardiomyocyte cell line, HL‐1, we found that overexpression of both the single and double mutants of ANT displayed less lactic acid dehydrogenase release induced by hypoxia and reoxygeneration compared to the wild type ANT. Moreover, the association of Voltage‐dependent anion channels with ANT was reduced after IR. In summary, we have identified specific NTyr residues in ANT following oxidative stress, and demonstrated that preventing nitration of these sites attenuated cell oxidative stress injury.

Interaction of cyclic mechanical stretch and toll‐like receptor 4‐mediated innate immunity in rat alveolar type II cells

In cases of infection-induced acute lung injury, mechanical ventilation might be necessary to maintain oxygenation. Although low tidal volume ventilation is applied, alveolar over-distension may occur and result in ventilator-induced lung injury. In this study, we investigate (i) the influence of lipopolysaccharide (LPS) stimulation on high-amplitude stretching; and (ii) the effect of stretching on LPS-mediated immune response in isolated rat alveolar type II cells. Type II cells were incubated with LPS and stretched for 24 h on elastic membranes. Initially we examined apoptosis and lactic acid dehydrogenase release in LPS-treated stretched cells. Furthermore we determined toll-like receptor (TLR) 4 expression, TLR4 signalling by analysis of nuclear factor κB (NF-κB) activation and the secretion of inflammatory cytokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-2, interleukin-1 beta, tumour necrosis factor alpha). Our results show that LPS increases apoptosis and cytotoxicity in high amplitude stretched cells. Stretching and LPS activate NF-κB. The LPS influence is the prevailing one while no synergistic effects were observed by additional stretching. LPS stimulates an increased secretion of the inflammatory mediators only. Stretching had no influence on cytokines secretion. We conclude that activation of TLR4 mediated immunity intensifies cell damage caused by stretching whereas in return stretching had no influence on TLR4 mediated innate immunity.

Protective effects of muscone on ischemia–reperfusion injury in cardiac myocytes

Ethnopharmacological relevanceMusk has been traditionally used in Chinese medicine as the main ingredient of many formulations for the treatment of chest pain and angina pectoris. Aim of the studyTo investigate the protective effects of muscone (the active ingredient of musk) on ischemia–reperfusion (I/R) injury induced by hypoxia and low glucose in primary cultured rat cardiac myocytes. Materials and methodsPrimary cultures of neonatal rat cardiac myocytes were subjected to ischemia–reperfusion in media, with or without muscone. Cell viability, release of lactic acid dehydrogenase (LDH), superoxide dismutase (SOD) activity, malondialdehyde (MDA) levels, creatine kinase (CK) and caspase-3 activities, as well as intracellular free Ca2+ concentrations, were measured. Cellular apoptosis and mitochondrial membrane potential (MMP) were assessed by flow cytometry, and the expression of Bcl-2 and Bax proteins was assessed by Western blotting. ResultsFollowing the exposure of cardiac myocytes to ischemia–reperfusion, there was a marked decrease in pulsating frequency, cell viability, SOD activity, MMP, and the expression of Bcl-2 protein, accompanied by increased LDH release, MDA production, CK and caspase-3 activities, intracellular free Ca2+ concentrations, rate of apoptosis, and expression of Bax protein. Pretreatment with muscone (0.215, 0.43, 0.86μg/mL) prior to I/R injury significantly attenuated the above changes. ConclusionMuscone has a protective effect against I/R injury in cardiac myocytes, indicating that muscone may potentially provide therapeutic benefit in I/R injury by inhibiting cellular oxidative stress and apoptosis.

Sennoside-induced secretion is not caused by changes in mucosal permeability or Na+,K(+)-ATPase activity.

The effect of sennosides (50 mg kg-1) on the rat colon in-situ was studied 6 h after oral treatment when the laxative effect was maximal. In a second experiment, rhein (4 x 10(-3) M), an active sennoside metabolite, was administered into the lumen of the colon for 1 h. Both sennosides and rhein reduced net H2O and Na+ absorption or reversed it to net secretion. Paracellular permeability, as measured using erythritol as a small marker molecule, was increased 2- to 3-fold; permeability to a large molecule, PEG 1000, was unchanged. The activity of Na+,K(+)-ATPase in the colon mucosa was not affected. There was no damage of the epithelial cells as determined by lactic acid dehydrogenase release. These results indicate that neither inhibition of Na+,K(+)-ATPase nor damage of the colon epithelium are involved in the secretory effect of sennosides or rhein. The increased paracellular permeability of small molecules fits into the concept of stimulation of active chloride secretion by sennosides, which is electrochemically and osmotically balanced by an increase in Na+ and H2O flow via the paracellular pathway.

Intrathecal Morphine Suppresses Immune Function in Rats with Inflammatory-induced Pain

Acute and chronic systemic administration of morphine is known to suppress immune function; however, the effect of chronic intrathecal (IT) morphine on immune function in inflammatory-induced pain is still unclear. This study examined the effects on the immune system of IT morphine in rats with formalin-induced pain. Lumbar IT catheters were implanted in rats and saline or 2.5, 5.0 or 10.0 microg/h morphine were administered for 7 days. On the last day, formalin-induced inflammatory pain was induced in rat hind paws and pain intensity was assessed. Rat spleens were then harvested for immune function assay. The IT morphine induced a dose-dependent analgesic effect and lactic acid dehydrogenase release assay showed dose-dependent suppression of natural killer cell activity. Concanavalin-A-induced splenocyte proliferation assay showed IT morphine to suppress T lymphocyte function in a dose-dependent manner. Flow cytometry showed IT morphine significantly to decrease T lymphocyte function and the percentages of T lymphocyte subsets in a dose-dependent manner. Hence, in inflammatory-induced pain IT morphine was found to suppress immune function. Chronic IT morphine should be used cautiously to treat chronic pain in immunocompromised cases.

Causes of excitation-induced muscle cell damage in isometric contractions: mechanical stress or calcium overload?

Prolonged or unaccustomed exercise leads to muscle cell membrane damage, detectable as release of the intracellular enzyme lactic acid dehydrogenase (LDH). This is correlated to excitation-induced influx of Ca2+, but it cannot be excluded that mechanical stress contributes to the damage. We here explore this question using N-benzyl-p-toluene sulfonamide (BTS), which specifically blocks muscle contraction. Extensor digitorum longus muscles were prepared from 4-wk-old rats and mounted on holders for isometric contractions. Muscles were stimulated intermittently at 40 Hz for 15-60 min or exposed to the Ca2+ ionophore A23187. Electrical stimulation increased 45Ca influx 3-5 fold. This was followed by a progressive release of LDH, which was correlated to the influx of Ca2+. BTS (50 microM) caused a 90% inhibition of contractile force but had no effect on the excitation-induced 45Ca influx. After stimulation, ATP and creatine phosphate levels were higher in BTS-treated muscles, most likely due to the cessation of ATP-utilization for cross-bridge cycling, indicating a better energy status of these muscles. No release of LDH was observed in BTS-treated muscles. However, when exposed to anoxia, electrical stimulation caused a marked increase in LDH release that was not suppressed by BTS but associated with a decrease in the content of ATP. Dynamic passive stretching caused no increase in muscle Ca2+ content and only a minor release of LDH, whereas treatment with A23187 markedly increased LDH release both in control and BTS-treated muscles. In conclusion, after isometric contractions, muscle cell membrane damage depends on Ca2+ influx and energy status and not on mechanical stress.

Identification and Characterization of an RTX Toxin in the Emerging Pathogen Kingella kingae

Kingella kingae is an emerging bacterial pathogen that is increasingly recognized as the causative agent of a variety of pediatric diseases, including septic arthritis and osteomyelitis. The pathogenesis of K. kingae disease is believed to begin with colonization of the upper respiratory tract. In the present study, we examined interactions between K. kingae and cultured respiratory epithelial cells and observed potent cytotoxicity, detected by both microscopy and lactic acid dehydrogenase (LDH) release assays. Experiments with synovial and macrophage cell lines revealed cytotoxicity for these cell types as well. Using mariner mutagenesis and a screen for loss of cytotoxicity, a genetic locus encoding an RTX toxin system was identified. Disruption of the K. kingae RTX locus resulted in a loss of cytotoxicity for respiratory epithelial, synovial, and macrophage cell lines. DNA sequence analysis demonstrated that the RTX locus is flanked by insertion elements and has a reduced G+C content compared to that of the whole genome. Two relatively less invasive Kingella species, K. oralis and K. denitrificans, were found to be noncytotoxic and to lack the RTX region, as determined by LDH release assays and Southern blotting. We concluded that K. kingae expresses an RTX toxin that has wide cellular specificity and was likely acquired horizontally. The possible roles for this toxin in the pathogenesis of K. kingae disease include breaching of the epithelial barrier and destruction of target tissues, such as synovium (joint lining).

Anoxia induces Ca2+ influx and loss of cell membrane integrity in rat extensor digitorum longus muscle

Anoxia can lead to skeletal muscle damage. In this study we have investigated whether an increased influx of Ca2+, which is known to cause damage during electrical stimulation, is a causative factor in anoxia-induced muscle damage. Isolated extensor digitorum longus (EDL) muscles from 4-week-old Wistar rats were mounted at resting length and were either resting or stimulated (30 min, 40 Hz, 10 s on, 30 s off) in the presence of standard oxygenation (95% O2, 5% CO2), anoxia (95% N2, 5% CO2) or varying degrees of reduced oxygenation. At varying extracellular Ca2+ concentrations ([Ca2+]o), 45Ca influx and total cellular Ca2+ content were measured and the release of lactic acid dehydrogenase (LDH) was determined as an indicator of cell membrane leakage. In resting muscles, incubated at 1.3 mM Ca2+, 15-75 min of exposure to anoxia increased 45Ca influx by 46-129% (P<0.001) and Ca2+ content by 20-50% (P<0.001). Mg2+ (11.2 mM) reduced the anoxia-induced increase in 45Ca influx by 43% (P<0.001). In muscles incubated at 20 and 5% O2, 45Ca influx was also stimulated (P<0.001). Increasing [Ca2+]o to 5 mM induced a progressive increase in both 45Ca uptake and LDH release in resting anoxic muscles. When electrical stimulation was applied during anoxia, Ca2+ content and LDH release increased markedly and showed a significant correlation (r2=0.55, P<0.001). In conclusion, anoxia or incubation at 20 or 5% O2 leads to an increased influx of 45Ca. This is associated with a loss of cell membrane integrity, possibly initiated by Ca2+. The loss of cell membrane integrity further increases Ca2+ influx, which may elicit a self-amplifying process of cell membrane leakage.

Role of Na+,K+ pumps in restoring contractility following loss of cell membrane integrity in rat skeletal muscle

In skeletal muscles, electrical shocks may elicit acute loss of force, possibly related to increased plasma membrane permeability, induced by electroporation (EP). We explore the role of the Na(+),K(+) pumps in force recovery after EP. Isolated rat soleus or extensor digitorum longus (EDL) muscles were exposed to EP paradigms in the range 100-800 V cm(-1), and changes in tetanic force, Na(+),K(+) contents, membrane potential, (14)C-sucrose space and the release of the intracellular enzyme lactic acid dehydrogenase (LDH) were characterized. The effects of Na(+),K(+) pump stimulation or inhibition were followed. Electroporation caused voltage-dependent loss of force, followed by varying rates and degrees of recovery. EP induced a reversible loss of K(+) and gain of Na(+), which was not suppressed by tetrodotoxin, but associated with increased (14)C-sucrose space and release of LDH. In soleus, EP at 500 V cm(-1) induced complete loss of force, followed by a spontaneous, partial recovery. Stimulation of active Na(+),K(+) transport by adrenaline, the beta(2)-agonist salbutamol, calcitonin gene-related peptide (CGRP) and dibutyryl cyclic AMP increased initial rate of force recovery by 183-433% and steady-state force level by 104-143%. These effects were blocked by ouabain (10(-3) m), which also completely suppressed spontaneous force recovery. EP caused rapid and marked depolarization, followed by a repolarization, which was accelerated by salbutamol. Also in EDL, EP caused complete loss of force, followed by a spontaneous partial recovery, which was markedly stimulated by salbutamol. Electroporation induces reversible depolarization, partial rundown of Na(+),K(+) gradients, cell membrane leakage and loss of force. This may explain the paralysis elicited by electrical shocks. Na(+),K(+) pump stimulation promotes restoration of contractility, possibly via its electrogenic action. The major new information is that the Na(+),K(+) pumps are sufficient to compensate a simple mechanical leakage. This may be important for force recovery in leaky muscle fibres.