Ouabain protects rat hearts against ischemia-reperfusion injury via pathway involving src kinase, mitoKATP, and ROS

We showed recently that mitochondrial ATP-dependent K(+) channel (mitoK(ATP)) opening is required for the inotropic response to ouabain. Because mitoK(ATP) opening is also required for most forms of cardioprotection, we investigated whether exposure to ouabain was cardioprotective. We also began to map the signaling pathways linking ouabain binding to Na(+)-K(+)-ATPase with the opening of mitoK(ATP). In Langendorff-perfused rat hearts, 10-80 microM ouabain given before the onset of ischemia resulted in cardioprotection against ischemia-reperfusion injury, as documented by an improved recovery of contractile function and a reduction of infarct size. In skinned cardiac fibers, a ouabain-induced protection of mitochondrial outer membrane integrity, adenine nucleotide compartmentation, and energy transfer efficiency was evidenced by a decreased release of cytochrome c and preserved half-saturation constant of respiration for ADP and adenine nucleotide translocase-mitochondrial creatine kinase coupling, respectively. Ouabain-induced positive inotropy was dose dependent over the range studied, whereas ouabain-induced cardioprotection was maximal at the lowest dose tested. Compared with bradykinin (BK)-induced preconditioning, ouabain was equally efficient. However, the two ligands clearly diverge in the intracellular steps leading to mitoK(ATP) opening from their respective receptors. Thus BK-induced cardioprotection was blocked by inhibitors of cGMP-dependent protein kinase (PKG) or guanylyl cyclase (GC), whereas ouabain-induced protection was not blocked by either agent. Interestingly, however, ouabain-induced inotropy appears to require PKG and GC. Thus 5-hydroxydecanoate (a selective mitoK(ATP) inhibitor), N-(2-mercaptopropionyl)glycine (MPG; a reactive oxygen species scavenger), ODQ (a GC inhibitor), PP2 (a src kinase inhibitor), and KT-5823 (a PKG inhibitor) abolished preconditioning by BK and blocked the inotropic response to ouabain. However, only PP2, 5-HD, and MPG blocked ouabain-induced cardioprotection.     

PDE4 inhibitor, roflumilast protects cardiomyocytes against NO-induced apoptosis via activation of PKA and Epac dual pathways

Myocyte apoptosis plays an important role in myocardial infarction and cAMP is crucial in the regulation of myocyte apoptosis. Phosphodiesterase-4 (PDE4) inhibitor blocks the hydrolysis of cAMP via inhibition of PDE4 and is attractive candidate for novel anti-inflammatory drugs. However, its function in cardiovascular diseases and cardiomyocyte apoptosis is unclear. Therefore, we investigated whether roflumilast, a PDE4 inhibitor, exerts protective effect against NO-induced apoptosis in both of H9c2 cells and neonatal rat cardiomyocytes (NRCMs), focusing on cAMP downstream molecules such as protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). According to our data, intracellular cAMP was increased by roflumilast treatment in H9c2 cells and NRCMs. Roflumilast inhibited SNP-induced apoptosis and this effect was reversed by PKA specific inhibitor H-89 and KT-5720. In addition, PKA specific activator N(6)-benzoyladenosine 3',5-cyclic monophosphate (N(6)Bz-cAMP) mimicked the effects of roflumilast. CREB phosphorylation by roflumilast was also inhibited by H-89, indicating that roflumilast protects SNP-induced apoptosis via PKA-dependent pathway. Roflumilast increased Epac1/GTP-Rap1 and the protective effect was abolished by Epac1 siRNA transfection, demonstrating that Epac signaling was also involved in this protective response. In support, Epac specific activator 8-(4-chlrorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP) protected SNP-induced apoptosis. PI3K/Akt inhibitor LY294002 blocked roflumilast-induced Akt phosphorylation and protective effect. Furthermore, inhibition of Epac1 with siRNA had no effect on roflumilast-induced CREB phosphorylation, whereas inhibited Akt phosphorylation, implicating that Akt phosphorylation was regulated by Epac pathway. In addition, it was also observed that rolipram and cilomilast exert similar effects as roflumilast. In summary, our data indicate that roflumilast protects NO-induced apoptosis via both cAMP-PKA/CREB and Epac/Akt-dependent pathway. Our study suggests a possibility of PDE4 inhibitor roflumilast as a potential therapeutic agent against myocardial ischemia/reperfusion (I/R) injury.     

Bradykinin induces mitochondrial ROS generation via NO,cGMP, PKG,and mitoKATP channel opening and leads to cardioprotection

Bradykinin (BK) mimics ischemic preconditioning by generating reactive oxygen species (ROS). To identify intermediate steps that lead to ROS generation, rabbit cardiomyocytes were incubated in reduced MitoTracker Red stain, which becomes fluorescent after exposure to ROS. Fluorescence intensity in treated cells was expressed as a percentage of that in paired, untreated cells. BK (500 nM) caused a 51 +/- 16% increase in ROS generation (P < 0.001). Coincubation with either the BK B2-receptor blocker HOE-140 (5 microM) or the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) prevented this increase, which confirms that the response was receptor mediated and ROS were actually being measured. Closing mitochondrial ATP-sensitive K+ (mitoKATP) channels with 5-hydroxydecanoate (5-HD, 1 mM) prevented increased ROS generation. BK-induced ROS generation was blocked by Nomega-nitro-m-arginine methyl ester (m-NAME, 200 microM), which implicates nitric oxide as an intermediate. Blockade of guanylyl cyclase with 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10 microM) aborted BK-induced ROS generation but not that from diazoxide, a direct opener of mitoKATP channels. The protein kinase G (PKG) blocker 8-bromoguanosine-3',5'-cyclic monophosphorothioate (25 microM) eliminated the effects of BK. Conversely, direct activation of PKG with 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (100 microM) increased ROS generation (39 +/- 15%; P < 0.004) similar to BK. This increase was blocked by 5-HD. Finally, the nitric oxide donor S-nitroso-N-acetylpenicillamine (1 microM) increased ROS by 34 +/- 6%. This increase was also blocked by 5-HD. In intact rabbit hearts, BK (400 nM) decreased infarction from 30.5 +/- 3.0 of the risk zone in control hearts to 11.9 +/- 1.4% (P < 0.01). This protection was aborted by either 200 microM m-NAME or 2 microM ODQ (35.4 +/- 5.7 and 30.4 +/- 3.0% infarction, respectively; P = not significant vs. control). Hence, BK preconditions through receptor-mediated production of nitric oxide, which activates guanylyl cyclase. The resulting cGMP activates PKG, which opens mitoKATP. Subsequent release of ROS triggers cardioprotection.     

N-oleoyldopamine, a novel endogenous capsaicin-like lipid, protects the heart against ischemia-reperfusion injury via activation of TRPV1

N-oleoyldopamine (OLDA), a bioactive lipid originally found in the mammalian brain, is an endovanilloid that selectively activates the transient receptor potential vanilloid type 1 (TRPV1) channel. This study tests the hypothesis that OLDA protects the heart against ischemia and reperfusion (I/R) injury via activation of the TRPV1 in wild-type (WT) but not in gene-targeted TRPV1-null mutant (TRPV1(-/-)) mice. Hearts of WT or TRPV1(-/-) mice were Langendorffly perfused with OLDA (2 x 10(-9) M) in the presence or absence of CGRP8-37 (1 x 10(-6) M), a selective calcitonin gene-related peptide (CGRP) receptor antagonist; RP-67580 (1 x 10(-6) M), a selective neurokinin-1 receptor antagonist; chelerythrine (5 x 10(-6) M), a selective protein kinase C (PKC) antagonist; or tetrabutylammonium (TBA, 5 x 10(-4) M), a nonselective K(+) channel antagonist, followed by 35 min of global ischemia and 40 min of reperfusion (I/R). Left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), coronary flow (CF), and left ventricular peak positive dP/dt (+dP/dt) were evaluated after I/R. OLDA improved recovery of cardiac function after I/R in WT but not TRPV1(-/-) hearts by increasing LVDP, CF, and +dP/dt and by decreasing LVEDP. CGRP8-37, RP-67580, chelerythrine, or TBA abolished the protective effect of OLDA in WT hearts. Radioimmunoassay showed that the release of substance P (SP) and CGRP after OLDA treatment was higher in WT than in TRPV1(-/-) hearts, which was blocked by chelerythrine or TBA. Thus OLDA exerts a cardiac protective effect during I/R injury in WT hearts via CGRP and SP release, which is abolished by PKC or K(+) channel antagonists. The protective effect of OLDA is void in TRPV1(-/-) hearts, supporting the notion that TRPV1 mediates OLDA-induced protection against cardiac I/R injury.     

ATP-sensitive potassium channel opener iptakalim protects against MPP-induced astrocytic apoptosis via mitochondria and mitogen-activated protein kinase signal pathways

Inhibition of astrocytic apoptosis has been regarded as a novel prospective strategy for treating neurodegenerative disorders such as Parkinson's disease. In the present study, we demonstrated that iptakalim (IPT), an ATP-sensitive potassium channel (K(ATP) channel) opener, exerted protective effect on MPP(+)-induced astrocytic apoptosis, which was reversed by selective mitochondrial K(ATP) channel blocker 5-hydroxydecanoate. Further study revealed that IPT inhibited glutathione (GSH) depletion, mitochondrial membrane potential loss and subsequent release of pro-apoptotic factors (cytochrome c and apoptosis-inducing factor (AIF), and c-Jun NH(2)-terminal kinase/mitogen-activated protein kinases (MAPK) phosphorylation induced by MPP(+). Meanwhile, extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 inhibited the protective effect of IPT on MPP(+)-induced astrocytic apoptosis. Furthermore, IPT could also activate ERK/MAPK and maintain increased phospho-ERK1/2 level after MPP(+) exposure. Taken together, these findings reveal for the first time that IPT protects against MPP(+)-induced astrocytic apoptosis via inhibition of mitochondria apoptotic pathway and regulating the MAPK signal transduction pathways by opening mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels in astrocytes. And targeting K(ATP) channels expressed in astrocytes may provide a novel therapeutic strategy for neurodegenerative disorders.     

Total Ca2+ handling for E-C coupling in the whole heart: an integrative analysis

We assessed total Ca2+ handling (transport, flux) in excitation-contraction (E-C) coupling in a beating left ventricle (LV). We developed a new integrative analysis method that utilizes the internal Ca2+ recirculation fraction (RF), O2 consumption (V(O2)) for Ca2+ handling, and O2 cost of Emax (contractility index) of the LV. We obtained the RF from the beat constant of the exponential decay component of the postextrasystolic potentiation, and the O2 cost of Emax from V(O2) measured at different Emax. Our equation calculated the unknown total Ca2+ handling, futile Ca2+ cycling, and Ca2+ reactivity of Emax from the RF and Ca2+ handling V(O2). The calculated total Ca2+ handling fell between 30 and 110 micromol/kg, depending on Emax and pathological conditions. Our method also allowed an assessment of futile Ca2+ cycling and Ca2+ reactivity of Emax in a beating LV. These data are not available using conventional methods. Our method can be used to better understand the pathophysiology of total Ca2+ handling in a beating heart.     

The delta-opioid receptor agonist DADLE at reperfusion protects the heart through activation of pro-survival kinases via EGF receptor transactivation

The specific delta-opioid receptor agonist [D-Ala(2)-D-Leu(5)]enkephalin (DADLE) protects against infarction in the heart when given before ischemia. In rabbit, this protection leads to phosphorylation of the pro-survival kinases Akt and extracellular signal-regulated kinase (ERK) and is dependent on transactivation of the epidermal growth factor receptor (EGFR). DADLE reportedly protects rat hearts at reperfusion. We therefore tested whether DADLE at reperfusion could protect isolated rabbit hearts subjected to 30 min of regional ischemia and 120 min of reperfusion and whether this protection is dependent on Akt, ERK, and EGFR. DADLE (40 nM) was infused for 1 h starting 5 min before reperfusion and reduced infarct size from 31.0 +/- 2.3% in the control group to 14.6 +/- 1.6% (P = 0.01). This protection was abolished by cotreatment of the metalloproteinase inhibitor (MPI) and the EGFR inhibitor AG1478. In contrast, 20 nM DADLE, although known to be protective before ischemia, failed to protect. Western blotting revealed that DADLE's protection was correlated to increase in phosphorylation of the kinases Akt and ERK1 and -2 in reperfused hearts (2.5 +/- 0.5, 1.6 +/- 0.2, and 2.3 +/- 0.7-fold of baseline levels, P < 0.05 vs. control). The DADLE-dependent increases in Akt and ERK1/2 phosphorylation were abolished by either MPI or AG1478, confirming a signaling through the EGFR pathway. Additionally, DADLE treatment increased phosphorylation of EGFR (1.4 +/- 0.2-fold, P = 0.03 vs. control). Thus the delta-opioid agonist DADLE protects rabbit hearts at reperfusion through activation of the pro-survival kinases Akt and ERK and is dependent on the transactivation of the EGFR.     

Western diet leads to aging-related tumorigenesis via activation of the inflammatory,UPR, and EMT pathways

Among the principal causative factors for the development of complications related to aging is a diet rich in fats and sugars, also known as the Western diet. This diet advocates numerous changes that might increase the susceptibility to initiate cancer and/or to create a tissue microenvironment more conducive to the growth of malignant cells, thus favoring the progression of cancer and metastasis. Hypercaloric diets in general lead to oxidative stress generating reactive oxygen species and induce endoplasmic reticulum stress. Our results demonstrate that mice bearing tumors fed with a Western diet presented bigger tumor mass with increased insulin sensitivity in these tissues. Several markers of insulin signaling, such as AKT phosphorylation and mTOR pathway, are promoted in tumors of Western diet-fed animals. This process is associated with increased macrophage infiltration, activation of unfolded protein response pathway, and initiation of epithelial–mesenchymal transition (EMT) process in these tumor tissues. Summing up, we propose that the Western diet accelerates the aging-related processes favoring tumor development.Subject terms: Melanoma, TOR signalling, Mechanisms of disease, Ageing     

ANG II stimulates PKC-dependent ERK activation,DNA synthesis,and cell division in intestinal epithelial cells

PKC, a major target for the tumor-promoting phorbol esters, has been implicated in the signal transduction pathways that mediate important functions in intestinal epithelial cells, including proliferation and carcinogenesis. With the use of IEC-18 cells arrested in G0/G1, addition of phorbol esters resulted in a modest increase in [3H]thymidine incorporation and a slight shift toward the S and G2/M phases of the cell cycle, whereas the combination of EGF and phorbol 12,13-dibutyrate (PDB) synergistically stimulated DNA synthesis. To investigate the effects of receptor-mediated PKC activation on mitogenesis, we demonstrated that ANG II induced ERK activation, a response completely blocked by pretreatment with mitogen/extracellular signal-regulated kinase inhibitors or specific PKC inhibitors. Furthermore, ANG II stimulated an over threefold increase in [3H]thymidine incorporation that was corroborated by flow cytometric analysis of the cell cycle to levels comparable to that achieved by the combination of EGF and PDB. Taken together, our results indicate that receptor-mediated PKC activation, as induced by ANG II, transduces mitogenic signals leading to DNA synthesis and cell proliferation in IEC-18 cells.     

Variable effects of the mitoKATP channel modulators diazoxide and 5-HD in ATP-depleted renal epithelial cells

The role of mitochondrial K_ATP (mitoK_ATP) channels in renal ischemia-reperfusion injury is controversial with studies showing both protective and deleterious effects. In this study, we compared the effects of the putative mitoK_ATP opener, diazoxide, and the mitoK_ATP blocker, 5-hydroxydecanoate (5-HD) on cytotoxicity and apoptosis in tubular epithelial cells derived from rat (NRK-52E) and pig (LLC-PK1) following in vitro ischemic injury. Following ATP depletion-recovery, there was a significant increase in cytotoxicity in both NRK cells and LLC-PK1 cells although NRK cells were more sensitive to the injury. Diazoxide treatment attenuated cytotoxicity in both cell types and 5-HD treatment-increased cytotoxicity in the sensitive NRK cells in a superoxide-dependant manner. The protective effect of diazoxide was also reversed in the presence of 5-HD in ATP-depleted NRK cells. The ATP depletion-mediated increase in superoxide was enhanced by both diazoxide and 5-HD with the effect being more pronounced in the cells undergoing 5-HD treatment. Further, ATP depletion-induced activation of caspase-3 was decreased by diazoxide in NRK cells. In order to determine the signaling pathways involved in apoptosis, we examined the activation of Erk and JNK in ATP-depleted NRK cells. Diazoxide-activated Erk in ATP-depleted cells, but did not have any effect on JNK activation. In contrast, 5-HD did not impact Erk levels but increased JNK activation even under controlled conditions. Further, the use of a JNK inhibitor with 5-HD reversed the deleterious effects of 5-HD. This study demonstrates that in cells that are sensitive to ATP depletion-recovery, mitoK_ATP channels protect against ATP depletion-mediated cytotoxicity and apoptosis through Erk- and JNK-dependant mechanisms.     

SP600125, a selective JNK inhibitor, protects ischemic renal injury via suppressing the extrinsic pathways of apoptosis

Accumulating evidence suggests that c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in renal ischemia/reperfusion injury. However, the downstream mechanism that accounts for the proapoptotic actions of JNK during renal ischemia/reperfusion has not been elucidated. We report that SP600125, a potent, cell-permeable, selective, and reversible inhibitor of c-Jun N-terminal kinase (JNK), potently decreased renal epithelial tubular cell apoptosis induced by renal ischemia/reperfusion via suppression of the extrinsic pathway. This corresponds to the decrease in JNK phosphorylation at 20 min and c-Jun phosphorylation (Ser63/73) at 3 h after renal ischemia. Additionally, SP600125 attenuated the increased expression of FasL induced by ischemia/reperfusion at 3 h. The administration of SP600125 prior to ischemia was also protective. Thus, our findings imply that SP600125 can inhibit the activation of the JNK-c-Jun-FasL pathway and protect renal tubular epithelial cells against ischemia/reperfusion-induced apoptosis. Taken together, these results indicate that targeting the JNK pathway provides a promising therapeutic approach for renal ischemia/reperfusion injury.     

Pseudomonas aeruginosa alkaline protease blocks complement activation via the classical and lectin pathways

The complement system rapidly detects and kills Gram-negative bacteria and supports bacterial killing by phagocytes. However, bacterial pathogens exploit several strategies to evade detection by the complement system. The alkaline protease (AprA) of Pseudomonas aeruginosa has been associated with bacterial virulence and is known to interfere with complement-mediated lysis of erythrocytes, but its exact role in bacterial complement escape is unknown. In this study, we analyzed how AprA interferes with complement activation and whether it could block complement-dependent neutrophil functions. We found that AprA potently blocked phagocytosis and killing of Pseudomonas by human neutrophils. Furthermore, AprA inhibited opsonization of bacteria with C3b and the formation of the chemotactic agent C5a. AprA specifically blocked C3b deposition via the classical and lectin pathways, whereas the alternative pathway was not affected. Serum degradation assays revealed that AprA degrades both human C1s and C2. However, repletion assays demonstrated that the mechanism of action for complement inhibition is cleavage of C2. In summary, we showed that P. aeruginosa AprA interferes with classical and lectin pathway-mediated complement activation via cleavage of C2.     

The functional microdomain in transmembrane helices 2 and 7 regulates expression, activation, and coupling pathways of the gonadotropin-releasing hormone receptor.

Structural microdomains of G protein-coupled receptors (GPCRs) consist of spatially related side chains that mediate discrete functions. The conserved helix 2/helix 7 microdomain was identified because the gonadotropin-releasing hormone (GnRH) receptor appears to have interchanged the Asp(2.50) and Asn(7.49) residues which are conserved in transmembrane helices 2 and 7 of rhodopsin-like GPCRs. We now demonstrate that different side chains of this microdomain contribute specifically to receptor expression, heterotrimeric G protein-, and small G protein-mediated signaling. An Asn residue is required in position 2.50(87) for expression of the GnRH receptor at the cell surface, most likely through an interaction with the conserved Asn(1.50(53)) residue, which we also find is required for receptor expression. Most GPCRs require an Asp side chain at either the helix 2 or helix 7 locus of the microdomain for coupling to heterotrimeric G proteins, but the GnRH receptor has transferred the requirement for an acidic residue from helix 2 to 7. However, the presence of Asp at the helix 7 locus precludes small G protein-dependent coupling to phospholipase D. These results implicate specific components of the helix 2/helix 7 microdomain in receptor expression and in determining the ability of the receptor to adopt distinct activated conformations that are optimal for interaction with heterotrimeric and small G proteins.     

MHC-based fiber type and E-C coupling characteristics in mechanically skinned muscle fibers of the rat

In this study, we investigated whether the previously established differences between fast- and slow-twitch single skeletal muscle fibers of the rat, in terms of myosin heavy chain (MHC) isoform composition and contractile function, are also detectable in excitation-contraction (E-C) coupling. We compared the contractile responsiveness of electrophoretically typed, mechanically skinned single fibers from the soleus (Sol), the extensor digitorum longus (EDL), and the white region of the sternomastoid (SM) muscle to t-system depolarization-induced activation. The quantitative parameters assessed were the amplitude of the maximum depolarization-induced force response (DIFR(max); normalized to the maximum Ca(2+)-activated force in that fiber) and the number of responses elicited until the force declined by 75% of DIFR(max) (R-D(75%)). The mean DIFR(max) values for type IIB EDL and type IIB SM fibers were not statistically different, and both were greater than the mean DIFR(max) for type I Sol fibers. The mean R-D(75%) for type IIB EDL fibers was greater than that for type I Sol fibers as well as type IIB SM fibers. These data suggest that E-C coupling characteristics of mechanically skinned rat single muscle fibers are related to MHC-based fiber type and the muscle of origin.     

Interactions between adenosine and K+ channel-related pathways in the coupling of somatosensory activation and pial arteriolar dilation

Multiple, perhaps interactive, mechanisms participate in the linkage between increased neural activity and cerebral vasodilation. In the present study, we assessed whether neural activation-related pial arteriolar dilation (PAD) involved interactions among adenosine (Ado) A(2) receptors (A(2)Rs), large-conductance Ca(2+)-operated K(+) (BK(Ca)) channels, and inward rectifier K(+) (K(ir)) channels. In rats with closed cranial windows, we monitored sciatic nerve stimulation (SNS)-induced PAD in the absence or presence of pharmacological blockade of A(2)Rs (ZM-241385), ecto-5'-nucleotidase (α,β-methylene-adenosine diphosphate), BK(Ca) channels (paxilline), and K(ir) channels (BaCl(2)). Individually, these interventions led to 53-66% reductions in SNS-induced PADs. Combined applications of these blockers led to little or no further repression of SNS-induced PADs, suggesting interactions among A(2)Rs and K(+) channels. In the absence of SNS, BaCl(2) blockade of K(ir) channels produced 52-80% reductions in Ado and NS-1619 (BK(Ca) channel activator)-induced PADs. In contrast, paxilline blockade of BK(Ca) channels was without effect on dilations elicited by KCl (K(ir) channel activator) and Ado suffusions, indicating that Ado- and NS-1619-associated PADs involved K(ir) channels. In addition, targeted ablation of the superficial glia limitans was associated with a selective 60-80% loss of NS-1619 responses, suggesting that the BK(Ca) channel participation (and paxilline sensitivity) derived largely from channels within the glia limitans. Additionally, blockade of either PKA or adenylyl cyclase caused markedly attenuated pial arteriolar responses to SNS and, in the absence of SNS, responses to Ado, KCl, and NS-1619. These findings suggested a key, possibly permissive, role for A(2)R-linked cAMP generation and PKA-induced K(+) channel phosphorylation in somatosensory activation-evoked PAD.     

Role of charged residues in coupling ligand binding and channel activation in the extracellular domain of the glycine receptor

The glycine receptor is a member of the ligand-gated ion channel receptor superfamily that mediates fast synaptic transmission in the brainstem and spinal cord. Following ligand binding, the receptor undergoes a conformational change that is conveyed to the transmembrane regions of the receptor resulting in the opening of the channel pore. Using the acetylcholine-binding protein structure as a template, we modeled the extracellular domain of the glycine receptor alpha1-subunit and identified the location of charged residues within loops 2 and 7 (the conserved Cys-loop). These loops have been postulated to interact with the M2-M3 linker region between the transmembrane domains 2 and 3 as part of the receptor activation mechanism. Charged residues were substituted with cysteine, resulting in a shift in the concentration-response curves to the right in each case. Covalent modification with 2-(trimethylammonium) ethyl methanethiosulfonate was demonstrated only for K143C, which was more accessible in the open state than the closed state, and resulted in a shift in the EC50 toward wild-type values. Charge reversal mutations (E53K, D57K, and D148K) also impaired channel activation, as inferred from increases in EC50 values and the conversion of taurine from an agonist to an antagonist in E53K and D57K. Thus, each of the residues Glu-53, Asp-57, Lys-143, and Asp-148 are implicated in channel gating. However, the double reverse charge mutations E53K:K276E, D57K:K276E, and D148K:K276E did not restore glycine receptor function. These results indicate that loops 2 and 7 in the extracellular domain play an important role in the mechanism of activation of the glycine receptor although not by a direct electrostatic mechanism.     

Schisandrol B promotes liver enlargement via activation of PXR and YAP pathways in mice

BackgroundSchisandrol B (SolB) is one of the bioactive components from a traditional Chinese medicine Schisandra chinensis or Schisandra sphenanthera. It has been demonstrated that SolB exerts hepatoprotective effects against drug-induced liver injury and promotes liver regeneration. It was found that SolB can induce hepatomegaly but the involved mechanisms remain unknown.PurposeThis study aimed to explore the mechanisms involved in SolB-induced hepatomegaly.MethodsMale C57BL/6 mice were injected intraperitoneally with SolB (100 mg/kg) for 5 days. Serum and liver samples were collected for biochemical and histological analyses. The mechanisms of SolB were investigated by qRT-PCR and western blot analyses, luciferase reporter gene assays and immunofluorescence.ResultsSolB significantly increased hepatocyte size and proliferation, and then promoted liver enlargement without liver injury and inflammation. SolB transactivated human PXR, activated PXR in mice and upregulated hepatic expression of its downstream proteins, such as CYP3A11, CYP2B10 and UGT1A1. SolB also significantly enhanced nuclear translocation of PXR and YAP in human cell lines. YAP signal pathway was activated by SolB in mice.ConclusionThese findings demonstrated that SolB can significantly induce liver enlargement, which is associated with the activation of PXR and YAP pathways.     

Apelin-13 protects the heart against ischemia-reperfusion injury through inhibition of ER-dependent apoptotic pathways in a time-dependent fashion

Endoplasmic reticulum (ER) stress is activated during and contributes to ischemia-reperfusion (I/R) injury. Attenuation of ER stress-induced apoptosis protects the heart against I/R injury. Using apelin, a ligand used to activate the apelin APJ receptor, which is known to be cardioprotective, this study was designed to investigate 1) the time course of changes in I/R injury after ER stress; 2) whether apelin infusion protects the heart against I/R injury via modulation of ER stress-dependent apoptosis signaling pathways; and 3) how phosphatidylinositol 3-kinase (PI3K)/Akt, endothelial nitric oxide synthase (eNOS), AMP-activated protein kinase (AMPK), and ERK activation are involved in the protection offered by apelin treatment. The results showed that, using an in vivo rat I/R model induced by 30 min of ischemia followed by reperfusion, infarct size (IS) increased from 2 h of reperfusion (34.85 ± 2.14%) to 12 h of reperfusion (48.98 ± 3.35, P < 0.05), which was associated with an abrupt increase in ER stress-dependent apoptosis activation, as evidenced by increased CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, and JNK activation (CHOP: 2.49-fold increase, caspase-12: 2.09-fold increase, and JNK: 3.38-fold increase, P < 0.05, respectively). Administration of apelin at 1 μg/kg not only completely abolished the activation of ER stress-induced apoptosis signaling pathways at 2 h of reperfusion but also significantly attenuated time-related changes at 24 h of reperfusion. Using pharmacological inhibition, we also demonstrated that PI3K/Akt, AMPK, and ERK activation were involved in the protection against I/R injury via inhibition of ER stress-dependent apoptosis activation. In contrast, although eNOS activation played a role in decreasing IS at 2 h of reperfusion, it failed to modify either IS or ER stress-induced apoptosis signaling pathways at 24 h after reperfusion.