Platanosides, a Potential Botanical Drug Combination, Decrease Liver Injury Caused by Acetaminophen Overdose in Mice.

Oxidative stress plays an important role in acetaminophen (APAP)-induced hepatotoxicity. Platanosides (PTSs) isolated from the American sycamore tree (Platanus occidentalis) represent a potential new four-molecule botanical drug class of antibiotics active against drug-resistant infectious disease. Preliminary studies have suggested that PTSs are safe and well tolerated and have antioxidant properties. The potential utility of PTSs in decreasing APAP hepatotoxicity in mice in addition to an assessment of their potential with APAP for the control of infectious diseases along with pain and pyrexia associated with a bacterial infection was investigated. On PTS treatment in mice, serum alanine aminotransferase (ALT) release, hepatic centrilobular necrosis, and 4-hydroxynonenal (4-HNE) were markedly decreased. In addition, inducible nitric oxide synthase (iNOS) expression and c-Jun-N-terminal kinase (JNK) activation decreased when mice overdosed with APAP were treated with PTSs. Computational studies suggested that PTSs may act as JNK-1/2 and Keap1-Nrf2 inhibitors and that the isomeric mixture could provide greater efficacy than the individual molecules. Overall, PTSs represent promising botanical drugs for hepatoprotection and drug-resistant bacterial infections and are effective in protecting against APAP-related hepatotoxicity, which decreases liver necrosis and inflammation, iNOS expression, and oxidative and nitrative stresses, possibly by preventing persistent JNK activation.

Combination therapy of lovastatin and AMP-activated protein kinase activator improves mitochondrial and peroxisomal functions and clinical disease in experimental autoimmune encephalomyelitis model.

Recent studies report that loss and dysfunction of mitochondria and peroxisomes contribute to the myelin and axonal damage in multiple sclerosis (MS). In this study, we investigated the efficacy of a combination of lovastatin and AMP-activated protein kinase (AMPK) activator (AICAR) on the loss and dysfunction of mitochondria and peroxisomes and myelin and axonal damage in spinal cords, relative to the clinical disease symptoms, using a mouse model of experimental autoimmune encephalomyelitis (EAE, a model for MS). We observed that lovastatin and AICAR treatments individually provided partial protection of mitochondria/peroxisomes and myelin/axons, and therefore partial attenuation of clinical disease in EAE mice. However, treatment of EAE mice with the lovastatin and AICAR combination provided greater protection of mitochondria/peroxisomes and myelin/axons, and greater improvement in clinical disease compared with individual drug treatments. In spinal cords of EAE mice, lovastatin-mediated inhibition of RhoA and AICAR-mediated activation of AMPK cooperatively enhanced the expression of the transcription factors and regulators (e.g. PPARα/ß, SIRT-1, NRF-1, and TFAM) required for biogenesis and the functions of mitochondria (e.g. OXPHOS, MnSOD) and peroxisomes (e.g. PMP70 and catalase). In summary, these studies document that oral medication with a combination of lovastatin and AICAR, which are individually known to have immunomodulatory effects, provides potent protection and repair of inflammation-induced loss and dysfunction of mitochondria and peroxisomes as well as myelin and axonal abnormalities in EAE. As statins are known to provide protection in progressive MS (Phase II study), these studies support that supplementation statin treatment with an AMPK activator may provide greater efficacy against MS.

LP340, a novel histone deacetylase inhibitor, decreases liver injury and fibrosis in mice: role of oxidative stress and microRNA-23a.

Effective therapy for liver fibrosis is lacking. Here, we examined whether LP340, the lead candidate of a new-generation of hydrazide-based HDAC1,2,3 inhibitors (HDACi), decreases liver fibrosis. Liver fibrosis was induced by CCl4 treatment and bile duct ligation (BDL) in mice. At 6 weeks after CCl4, serum alanine aminotransferase increased, and necrotic cell death and leukocyte infiltration occurred in the liver. Tumor necrosis factor-α and myeloperoxidase markedly increased, indicating inflammation. After 6 weeks, α-smooth muscle actin (αSMA) and collagen-1 expression increased by 80% and 575%, respectively, indicating hepatic stellate cell (HSC) activation and fibrogenesis. Fibrosis detected by trichrome and Sirius-red staining occurred primarily in pericentral regions with some bridging fibrosis in liver sections. 4-Hydroxynonenal adducts (indicator of oxidative stress), profibrotic cytokine transforming growth factor-ß (TGFß), and TGFß downstream signaling molecules phospho-Smad2/3 also markedly increased. LP340 attenuated indices of liver injury, inflammation, and fibrosis markedly. Moreover, Ski-related novel protein-N (SnoN), an endogenous inhibitor of TGFß signaling, decreased, whereas SnoN expression suppressor microRNA-23a (miR23a) increased markedly. LP340 (0.05 mg/kg, ig., daily during the last 2 weeks of CCl4 treatment) decreased 4-hydroxynonenal adducts and miR23a production, blunted SnoN decreases, and inhibited the TGFß/Smad signaling. By contrast, LP340 had no effect on matrix metalloproteinase-9 expression. LP340 increased histone-3 acetylation but not tubulin acetylation, indicating that LP340 inhibited Class-I but not Class-II HDAC in vivo. After BDL, focal necrosis, inflammation, ductular reactions, and portal and bridging fibrosis occurred at 2 weeks, and αSMA and collagen-1 expression increased by 256% and 560%, respectively. LP340 attenuated liver injury, ductular reactions, inflammation, and liver fibrosis. LP340 also decreased 4-hydroxynonenal adducts and miR23a production, prevented SnoN decreases, and inhibited the TGFß/Smad signaling after BDL. In vitro, LP340 inhibited immortal human hepatic stellate cells (hTERT-HSC) activation in culture (αSMA and collagen-1 expression) as well as miR23a production, demonstrating its direct inhibitory effects on HSC. In conclusions, LP340 is a promising therapy for both portal and pericentral liver fibrosis, and it works by inhibiting oxidative stress and decreasing miR23a.

MD-2 is involved in the stimulation of matrix metalloproteinase-1 expression by interferon-γ and high glucose in mononuclear cells - a potential role of MD-2 in Toll-like receptor 4-independent signalling.

We reported recently that treatment of diabetic apolipoprotein E-deficient mice with the Toll-like receptor 4 (TLR4) antagonist Rs-LPS, a lipopolysaccharide isolated from Rhodobacter sphaeroides, inhibited atherosclerosis. Since it is known that Rs-LPS antagonizes TLR4 by targeting TLR4 co-receptor MD-2, this finding indicates that MD-2 is a potential target for the treatment of atherosclerosis. In this study, we determined if MD-2 is involved in the gene expression regulated by signalling pathways independent of TLR4. Given that interferon-γ (IFNγ) and hyperglycaemia play key roles in atherosclerosis, we determined if MD-2 is involved in IFN-γ and high-glucose-regulated gene expression in mononuclear cells. Results showed that IFN-γ and high glucose synergistically stimulated matrix metalloproteinase 1 (MMP-1), a proteinase essential for vascular tissue remodelling and atherosclerosis, in U937 mononuclear cells, but Rs-LPS inhibited the MMP-1 stimulation. To provide more evidence for a role of MD-2 in IFN-γ-stimulated MMP-1, studies using antibodies and small interfering RNA demonstrated that MD-2 blockade or knockdown attenuated the effect of IFN-γ on MMP-1. Furthermore, studies using PCR arrays showed that MD-2 blockade had a similar effect as IFN-γ receptor blockade on the inhibition of IFN-γ-stimulated pro-inflammatory molecules. Although these findings indicate the involvement of MD-2 in IFN-γ signalling, we also observed that MD-2 was up-regulated by IFN-γ and high glucose. We found that MD-2 up-regulation by IFN-γ played an essential role in the synergistic effect of IFN-γ and LPS on MMP-1 expression. Taken together, these findings indicate that MD-2 is involved in IFN-γ signalling and IFN-γ-augmented MMP-1 up-regulation by LPS.

LP342, a novel histone deacetylase inhibitor, decreases nitro-oxidative stress, mitochondrial dysfunction and hepatic ischaemia/reperfusion injury in mice.

Objectives: Some histone deacetylase (HDAC) isoforms contribute to ischaemia/reperfusion (IR) injury (IRI). Here, we examined whether LP342, the lead candidate of a new generation of hydrazide-based HDAC inhibitors (HDACi), decreases hepatic IRI. Methods: IR was induced by clamping blood vessels to ~70% of the livers of mice for 1 h. Key findings: At 6 h after reperfusion, ALT markedly increased, and wide-spread necrosis, leukocyte infiltration, and apoptosis occurred. LP342 treatment (1 mg/kg, ip) at 20 h or 1 h before ischaemia markedly decreased IRI whereas LP342 treatment upon reperfusion was marginally protective. Nitro-oxidative stress, c-Jun-N-terminal kinase (JNK) activation, and mitochondrial dysfunction contribute to IRI. 4-Hydroxynonenal, 3-nitrotyrosine, inducible nitric oxide synthase (iNOS), JNK activation and Sab binding increased markedly after IR, which LP342 blunted. LP342 also induced thioredoxin-1 expression before and after IR. LP342 also decreased mitochondrial depolarisation as detected by intravital microscopy at 2 h after IR. Lastly, LP342 increased acetylation of both histone-3 (class I HDAC substrate) and NFκB p65 but not tubulin (class II HDAC substrate) before and after IR. Conclusions: This novel HDACi protects against IRI most likely by epigenetic upregulation of antioxidant proteins and post-translational modifications of NFκB thus inhibiting iNOS expression and inflammatory responses.

Different signaling mechanisms regulating IL-6 expression by LPS between gingival fibroblasts and mononuclear cells: seeking the common target.

To reduce connective tissue IL-6 level stimulated by LPS, it is essential to control IL-6 expression in both mononuclear cells and fibroblasts. However, it is unclear whether the regulatory mechanisms for both cells are similar or not. In this study, we found that signaling pathways mediating LPS-stimulated IL-6 in mononuclear U937 cells and fibroblasts were different. Furthermore, our studies showed that while LPS activated AP-1 and NFκB in U937 cells, it only activated NFκB in fibroblasts. Analysis of nuclear AP-1 subunits showed that LPS stimulated c-Fos, Fra-1 and Jun D activities in U937 cells, but not fibroblasts. The lack of ERK involvement in LPS-stimulated IL-6 in fibroblasts was further supported by the observations that simvastatin, which is known to target ERK-AP-1, failed to inhibit LPS-stimulated IL-6 by fibroblasts. Finally, we showed that targeting NFκB pathway was highly effective in inhibition of LPS-stimulated IL-6 in coculture of U937 cells and fibroblasts.

Mitochondrial depolarization after acute ethanol treatment drives mitophagy in living mice.

Ethanol increases hepatic mitophagy driven by unknown mechanisms. Type 1 mitophagy sequesters polarized mitochondria for nutrient recovery and cytoplasmic remodeling. In Type 2, mitochondrial depolarization (mtDepo) initiates mitophagy to remove the damaged organelles. Previously, we showed that acute ethanol administration produces reversible hepatic mtDepo. Here, we tested the hypothesis that ethanol-induced mtDepo initiates Type 2 mitophagy. GFP-LC3 transgenic mice were gavaged with ethanol (2-6 g/kg) with and without pre-treatment with agents that decrease or increase mtDepo-Alda-1, tacrolimus, or disulfiram. Without ethanol, virtually all hepatocytes contained polarized mitochondria with infrequent autophagic GFP-LC3 puncta visualized by intravital microscopy. At ~4 h after ethanol treatment, mtDepo occurred in an all-or-none fashion within individual hepatocytes, which increased dose dependently. GFP-LC3 puncta increased in parallel, predominantly in hepatocytes with mtDepo. Mitochondrial PINK1 and PRKN/parkin also increased. After covalent labeling of mitochondria with MitoTracker Red (MTR), GFP-LC3 puncta encircled MTR-labeled mitochondria after ethanol treatment, directly demonstrating mitophagy. GFP-LC3 puncta did not associate with fat droplets visualized with BODIPY558/568, indicating that increased autophagy was not due to lipophagy. Before ethanol administration, rhodamine-dextran (RhDex)-labeled lysosomes showed little association with GFP-LC3. After ethanol treatment, TFEB (transcription factor EB) translocated to nuclei, and lysosomal mass increased. Many GFP-LC3 puncta merged with RhDex-labeled lysosomes, showing autophagosomal processing into lysosomes. After ethanol treatment, disulfiram increased, whereas Alda-1 and tacrolimus decreased mtDepo, and mitophagy changed proportionately. In conclusion, mtDepo after acute ethanol treatment induces mitophagic sequestration and subsequent lysosomal processing.Abbreviations : AcAld, acetaldehyde; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ALD, alcoholic liver disease; Alda-1, N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; LMNB1, lamin B1; MAA, malondialdehyde-acetaldehyde adducts; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MPT, mitochondrial permeability transition; mtDAMPS, mitochondrial damage-associated molecular patterns; mtDepo, mitochondrial depolarization; mtDNA, mitochondrial DNA; MTR, MitoTracker Red; PI, propidium iodide; PINK1, PTEN induced putative kinase 1; PRKN, parkin; RhDex, rhodamine dextran; TFEB, transcription factor EB; Tg, transgenic; TMRM, tetramethylrhodamine methylester; TOMM20, translocase of outer mitochondrial membrane 20; VDAC, voltage-dependent anion channel.

Adipocyte-mononuclear cell interaction, Toll-like receptor 4 activation, and high glucose synergistically up-regulate osteopontin expression via an interleukin 6-mediated mechanism.

Although it has been reported that osteopontin, a matrix glycoprotein and proinflammatory cytokine, mediates obesity-induced adipose tissue macrophage infiltration and insulin resistance, it remains unclear how osteopontin is up-regulated in adipose tissue in obese humans and animals. In this study, we incubated U937 mononuclear cells with adipocytes in a transwell system and studied how cell interaction regulated osteopontin expression. Results showed that coculture of U937 cells with adipocytes led to a marked increase in osteopontin production when compared with that released by independent cultures of U937 cells. Moreover, lipopolysaccharide or palmitic acid-induced TLR4 activation and high glucose further augmented the coculture-stimulated osteopontin secretion. Similar observations were made in the coculture of human primary monocytes and adipocytes. Real time PCR studies showed that coculture of U937 cells and adipocytes increased osteopontin mRNA in U937 cells, but not adipocytes, suggesting that adipocyte-derived soluble factor may stimulate osteopontin expression by U937 cells. In our studies to explore the underlying mechanism, we found that the neutralizing antibodies against interleukin (IL)-6 or IL-6 small interfering RNA transfection in adipocytes effectively inhibited coculture-stimulated osteopontin expression, suggesting that IL-6 released by adipocytes plays an essential role in the coculture-stimulated osteopontin expression by U937 cells. In conclusion, this study has demonstrated that cell interaction, TLR4 activation, and high glucose up-regulate osteopontin expression, and adipocyte-derived IL-6 played a major role in the up-regulation.

Lactate boosts TLR4 signaling and NF-kappaB pathway-mediated gene transcription in macrophages via monocarboxylate transporters and MD-2 up-regulation.

It has been shown that lactate induces insulin resistance. However, the underlying mechanisms have not been well understood. Based on our observation that lactate augments LPS-stimulated inflammatory gene expression, we proposed that lactate may enhance TLR4 signaling in macrophages, which has been shown to play an important role in insulin resistance in adipocytes. In this study, we demonstrated that lactate stimulated MD-2, a coreceptor for TLR4 signaling activation, NF-kappaB transcriptional activity, and the expression of inflammatory genes in human U937 histiocytes (resident macrophages). Similar enhancement of the inflammatory gene expression by lactate was also observed in human monocyte-derived macrophages. The essential role of MD-2 in lactate-augmented TLR4 signaling was confirmed by observation that the suppression of MD-2 expression by small interfering RNA led to significant inhibition of inflammatory gene expression. To further elucidate how lactate treatment enhances TLR4 activation, we showed that the augmentation of inflammatory gene expression by lactate was abrogated by antioxidant treatment, suggesting a critical role of reactive oxygen species in the enhancement of TLR4 activation by lactate. Finally, we showed that alpha-cyano-4-hydroxycinnamic acid, a classic inhibitor for monocarboxylate transporters, blocked lactate-augmented inflammatory gene expression and nuclear NF-kappaB activity, indicating that lactate transport through monocarboxylate transporters is required for lactate-enhanced TLR4 activation. Collectively, this study documents that lactate boosts TLR4 activation and NF-kappaB-dependent inflammatory gene expression via monocarboxylate transporters and MD-2 up-regulation.

IL-6 and high glucose synergistically upregulate MMP-1 expression by U937 mononuclear phagocytes via ERK1/2 and JNK pathways and c-Jun.

Matrix metalloproteinases (MMPs) play a pivotal role in tissue remodeling and destruction in inflammation-associated diseases such as cardiovascular disease and periodontal disease. Although it is known that interleukin (IL)-6 is a key proinflamatory cytokine, it remains unclear how IL-6 regulates MMP expression by mononuclear phagocytes. Furthermore, it remains undetermined how IL-6 in combination with hyperglycemia affects MMP expression. In the present study, we investigated the regulatory effect of IL-6 alone or in combination with high glucose on MMP-1 expression by U937 mononuclear phagocytes. We found that IL-6 is a powerful stimulator for MMP-1 expression and high glucose further augmented IL-6-stimulated MMP-1 expression. We also found that high glucose, IL-6, and lipopolysaccharide act in concert to stimulate MMP-1 expression. In the studies to elucidate underlying mechanisms, the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways were found to be required for stimulation of MMP-1 by IL-6 and high glucose. We also observed that IL-6 and high glucose stimulated the expression of c-Jun, a key subunit of AP-1 known to be essential for MMP-1 transcription. The role of c-Jun in MMP-1 expression was confirmed by the finding that suppression of c-Jun expression by RNA interference significantly inhibited MMP-1 expression. Finally, we demonstrated that similarly to U937 mononuclear phagocytes, IL-6 and high glucose also stimulated MMP-1 secretion from human primary monocytes. In conclusion, this study demonstrated that IL-6 and high glucose synergistically stimulated MMP-1 expression in mononuclear phagocytes via ERK and JNK cascades and c-Jun upregulation.

Altered dopamine transporter function and phosphorylation following chronic cocaine self-administration and extinction in rats.

Cocaine binds with the dopamine transporter (DAT), an effect that has been extensively implicated in its reinforcing effects. However, persisting adaptations in DAT regulation after cocaine self-administration have not been extensively investigated. Here, we determined the changes in molecular mechanisms of DAT regulation in the caudate-putamen (CPu) and nucleus accumbens (NAcc) of rats with a history of cocaine self-administration, followed by 3weeks of withdrawal under extinction conditions (i.e., no cocaine available). DA uptake was significantly higher in the CPu of cocaine-experienced animals as compared to saline-yoked controls. DAT V(max) was elevated in the CPu without changes in apparent affinity for DA. In spite of elevated CPu DAT activity, total and surface DAT density and DAT-PP2Ac (protein phosphatase 2A catalytic subunit) interaction remained unaltered, although p-Ser- DAT phosphorylation was elevated. In contrast to the CPu, there were no differences between cocaine and saline rats in the levels of DA uptake, DAT V(max) and K(m) values, total and surface DAT, p-Ser-DAT phosphorylation, or DAT-PP2Ac interactions in the NAcc. These results show that chronic cocaine self-administration leads to lasting, regionally specific alterations in striatal DA uptake and DAT-Ser phosphorylation. Such changes may be related to habitual patterns of cocaine-seeking observed during relapse.

Simvastatin suppresses LPS-induced MMP-1 expression in U937 mononuclear cells by inhibiting protein isoprenylation-mediated ERK activation.

Matrix metalloproteinase (MMP) plays a crucial role in periodontal disease and is up-regulated by oral Gram-negative, pathogen-derived LPS. In this study, we reported that simvastatin, a 3-hydroxyl-3-methylglutaryl-CoA reductase inhibitor, effectively inhibited LPS-stimulated MMP-1 as well as MMP-8 and MMP-9 expression by U937 mononuclear cells. Our studies showed that the geranylgeranyl transferase inhibitor inhibited LPS-stimulated MMP-1 expression, and addition of isoprenoid intermediate geranylgeranyl pyrophosphate (GGPP) reduced the inhibitory effect of simvastatin on LPS-stimulated MMP-1 expression. We also demonstrated that simvastatin inhibited the activation of Ras and Rac, and the inhibition was abolished by addition of GGPP. The above results indicate that protein isoprenylation is involved in the regulation of MMP-1 expression by LPS and simvastatin. Moreover, we showed that simvastatin inhibited LPS-stimulated nuclear AP-1, but not NF-kappaB activity, and the inhibition was reversed by addition of GGPP. Simvastatin also inhibited LPS-stimulated ERK but not p38 MAPK and JNK. Finally, we showed that the inhibition of LPS-stimulated ERK activation by simvastatin was reversed by GGPP. Taken together, this study showed that simvastatin suppresses LPS-induced MMP-1 expression in U937 mononuclear cells by targeting protein isoprenylation-mediated ERK activation.

Race-related differences in functional antibody response to pneumococcal vaccination in HIV-infected individuals.

BACKGROUND: Both HIV positivity and African American (AA) ethnicity are associated with increased incidence of invasive pneumococcal disease (IPD). Poor immune response to pneumococcal polysaccharide-based vaccines may contribute to the race related increased frequency of IPD in African American HIV positive individuals. METHODS: Caucasian and AA HIV-infected (HIV+) individuals 40-65 years old with CD4+ T cells/µl (CD4) >200 on antiretroviral therapy (ART) received either the 13-valent pneumococcal conjugate vaccine (PCV) followed by the 23-valent pneumococcal polysaccharide vaccine (PPV) or PPV only. Serum IgG, IgM and opsonophagocytic antibody responses to serotypes 14 and 23F as well as serum IgG and opsonophagocytic antibody responses to serotype 19A were measured pre- and post-vaccination. We measured serum markers of inflammation in all participants and performed single cell gene expression profiling at the baseline by HD Biomark in Caucasians and African Americans. RESULTS: There were no significant differences in pre-immunization inflammatory markers or post-vaccination IgG and IgM concentrations between Caucasian and African American participants. However, we found significantly lower opsonophagocytic activity in response to serotypes 14 and 19A in the AA group compared to the Caucasian group. There was no association between inflammatory markers and immune response to vaccination, however we found extensive biomodal variation in gene expression levels in single IgM+ memory B cells. Differentially expressed genes may be related to differences in the immune response between ethnic groups. CONCLUSIONS: Distinct racial differences were found in the functional immune response following either PPV and/or PCV/PPV immunization in HIV-positive adults, although these differences were serotype dependent. Decreased ability to respond to vaccination may in part explain racial disparities in pneumococcal disease epidemiology. ClinicalTrials.gov ID: NCT03039491.

Eomes partners with PU.1 and MITF to Regulate Transcription Factors Critical for osteoclast differentiation.

Bone-resorbing osteoclasts (OCs) are derived from myeloid precursors (MPs). Several transcription factors are implicated in OC differentiation and function; however, their hierarchical architecture and interplay are not well known. Analysis for enriched motifs in PU.1 and MITF chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) data from differentiating OCs identified eomesodermin (EOMES) as a potential novel binding partner of PU.1 and MITF at genes critical for OC differentiation and function. We were able to demonstrate using co-immunoprecipitation and sequential ChIP analysis that PU.1, MITF, and EOMES are in the same complex and present as a complex at OC genomic loci. Furthermore, EOMES knockdown in MPs led to osteopetrosis associated with decreased OC differentiation and function both in vitro and in vivo. Although EOMES is associated with embryonic development and other hematopoietic lineages, this is the first study demonstrating the requirement of EOMES in the myeloid compartment.

Dysregulation of dopamine transporter trafficking and function after abstinence from cocaine self-administration in rats: evidence for differential regulation in caudate putamen and nucleus accumbens.

The profound alterations produced by cocaine on dopamine (DA) neurotransmission raise the possibility that dopamine transporter (DAT)-expressing neurons may modify DA transport in response to repeated cocaine exposure to maintain the appropriate efficiency of DA clearance. In this study, we determined the changes in molecular mechanisms of DAT regulation in rats with a history of repeated cocaine self-administration followed by 3 weeks of abstinence. Using ex vivo caudate putamen (CPu) and nucleus accumbens (NAcc) synaptosomal preparations, we found that DA uptake was significantly higher in the CPu and NAcc of cocaine-experienced animals compared with yoked saline animals. Surface distribution, p-Ser phosphorylation, and protein phosphatase 2A catalytic subunit (PP2Ac) interaction of DAT were all altered in the CPu. Maximal velocity (V(max)) values were elevated both in the CPu and NAcc of cocaine-experienced rats compared with saline controls. Although there was no change in the apparent affinity for DA in the CPu, increased DA affinity was evident in the NAcc. Consistent with elevated DAT activity in cocaine-experienced animals, a higher level of surface DAT, DAT-PP2Ac association, and decreased serine phosphorylation of DAT were observed in the CPu, but not in the NAcc. These results, for the first time, suggest that chronic cocaine self-administration followed by abstinence leads to persisting alterations in normal DAT trafficking and catalytic regulatory cascades in the CPu and NAcc in a brain region-specific manner.

Modulation of serotonin transporter function by kappa-opioid receptor ligands.

Kappa opioid receptor (KOR) agonists produce dysphoria and psychotomimesis. While KOR agonists produce pro-depressant-like effects, KOR antagonists produce anti-depressant-like effects in rodent models. The cellular mechanisms and downstream effector(s) by which KOR ligands produce these effects are not clear. KOR agonists modulate serotonin (5-HT) transmission in the brain regions implicated in mood and motivation regulation. Presynaptic serotonin transporter (SERT) activity is critical in the modulation of synaptic 5-HT and, subsequently, in mood disorders. Detailing the molecular events of KOR-linked SERT regulation is important for examining the postulated role of this protein in mood disorders. In this study, we used heterologous expression systems and native tissue preparations to determine the cellular signaling cascades linked to KOR-mediated SERT regulation. KOR agonists U69,593 and U50,488 produced a time and concentration dependent KOR antagonist-reversible decrease in SERT function. KOR-mediated functional down-regulation of SERT is sensitive to CaMKII and Akt inhibition. The U69,593-evoked decrease in SERT activity is associated with a decreased transport Vmax, reduced SERT cell surface expression, and increased SERT phosphorylation. Furthermore, KOR activation enhanced SERT internalization and decreased SERT delivery to the membrane. These data demonstrate that KOR activation decreases 5-HT uptake by altering SERT trafficking mechanisms and phosphorylation status to reduce the functional availability of surface SERT.

A role for p38 mitogen-activated protein kinase in the regulation of the serotonin transporter: evidence for distinct cellular mechanisms involved in transporter surface expression.

The serotonin transporter (SERT) is regulated by various signaling mechanisms that may operate to maintain appropriate levels of synaptic serotonin (5-HT). We demonstrate that one of the mitogen-activated protein kinases (MAPKs), p38 MAPK, regulates SERT. Treatment of rat midbrain synaptosomes with p38 MAPK-specific inhibitors, PD169316 [4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole] or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole], reduced 5-HT uptake. An additive SERT inhibition by PD169316 and beta-phorbol 12-myristate 13-acetate (beta-PMA) indicated the involvement of a protein kinase C (PKC)-independent MAPK pathway. Kinetic studies indicated a significant decrease in the transport capacity (V(max)) after PD169316 treatment of synaptosomes. Biotinylation studies showed reduced SERT proteins in the plasma membrane of synaptosomes after p38 MAPK inhibition and PKC activation. Phosphorylation studies using synaptosomes revealed decreased SERT phosphorylation by PD169316 but increased phosphorylation by beta-PMA. d-Amphetamine enhanced SERT basal phosphorylation and PD169316 blocked this effect. SERT interaction with protein phosphatase 2A catalytic subunit and syntaxin 1A decreased after PD169316 or beta-PMA treatment of synaptosomes. In synaptosomes, PKC activation but not p38 MAPK inhibition resulted in SERT redistribution from cholesterolrich lipid raft fractions to nonlipid raft fractions. The presence of phospho-p38 MAPK in synaptosomes and human embryonic kidney 293 (HEK-293) cells suggested the presence of constitutively active p38 MAPK in these preparations. Cotransfection of HEK-293 cells with SERT and a constitutively active form of MAP kinase kinase 3b(E) [MKK3b(E)] increased 5-HT transport, and RNA interference targeted to p38 MAPK inhibited 5-HT uptake, confirming the involvement of active p38 MAPK in SERT expression. Although PD169316 inhibited SERT insertion to the plasma membrane, beta-PMA increased SERT internalization in HEK-293 cells. Together, these results indicate a distinct role of p38 MAPK in SERT regulation.

S-Nitrosoglutathione ameliorates acute renal dysfunction in a rat model of lipopolysaccharide-induced sepsis.

OBJECTIVE: Sepsis induces an inflammatory response that results in acute renal failure (ARF). The current study is to evaluate the role of S-Nitrosoglutathione (GSNO) in renoprotection from lipopolysaccharide (LPS)-induced sepsis. METHODS: Rats were divided to three groups. First group received LPS (5 mg/kg body weight), second group was treated with LPS + GSNO (50 µg/kg body weight), and third group was administered with vehicle (saline). They were sacrificed on day 1 and 3 post-LPS injection. Serum levels of nitric oxide (NO), creatinine and blood urea nitrogen (BUN) were analysed. Tissue morphology, T lymphocyte infiltrations, and the expression of inflammatory (TNF-α, iNOS) and anti-inflammatory (IL-10) mediators as well as glutathione (GSH) levels were determined. KEY FINDING: Lipopolysaccharide significantly decreased body weight and increased cellular T lymphocyte infiltration, caspase-3 and iNOS and decreased PPAR-γ in renal tissue. NO, creatinine and BUN were significantly elevated after LPS challenge, and they significantly decreased after GSNO treatment. TNF-α level was found significantly increased in LPS-treated serum and kidney. GSNO treatment of LPS-challenged rats decreased caspase-3, iNOS, TNF-α, T lymphocyte infiltration and remarkably increased levels of IL-10, PPAR-γ and GSH. CONCLUSION: GSNO can be used as a renoprotective agent for the treatment of sepsis-induced acute kidney injury.

GABA transmission in the nucleus accumbens is altered after withdrawal from repeated cocaine.

Repeated cocaine causes enduring changes in dopamine and glutamate transmission in the nucleus accumbens, and dopamine and glutamate terminals synapse on GABAergic accumbens neurons. The present study demonstrates that there are changes in GABA transmission in the accumbens at 3 weeks after discontinuing daily cocaine injections. No-net flux microdialysis revealed a significant increase in the basal levels of extracellular GABA in the accumbens of cocaine-treated rats. The elevated extracellular GABA was normalized by blocking voltage-dependent Na+ channels and provided increased tone on GABA(B) presynaptic autoreceptors and heteroreceptors because blocking GABA(B) receptors produced a greater elevation in extracellular GABA, dopamine, and glutamate in cocaine-treated compared with control subjects. For many G-protein-coupled receptors, increased agonist can cause receptor desensitization. Consistent with GABA(B) receptor desensitization, baclofen-stimulated GTPgammaS binding was reduced, and the reduction in G-protein coupling was accompanied by reduced Ser phosphorylation of the GABA(B2) receptor subunit. No effect by repeated cocaine was found in the levels of total GABA(B1) or GABA(B2) protein. Together, these data demonstrate that withdrawal from repeated cocaine treatment produces an increase in the basal levels of extracellular GABA in the accumbens that depends on neuronal activity. The increase may be mediated in part by functional desensitization of GABA(B) receptors, likely the result of diminished Ser phosphorylation of the GABA(B2) receptor.

AKP-11 - A Novel S1P1 Agonist with Favorable Safety Profile Attenuates Experimental Autoimmune Encephalomyelitis in Rat Model of Multiple Sclerosis.

Sphingosine-1-phosphate receptor 1 (S1P1) mediated regulation of lymphocyte egress from lymphoid organs is recognized as the mechanism of FTY720 (Fingolimod, Gilenya) efficacy in relapsing-remitting forms of multiple sclerosis (RRMS). In this study we describe a novel S1P1 agonist AKP-11, next generation of S1P1 agonist, with immunomodulatory activities in cell culture model and for therapeutic efficacy against an animal model of MS, i.e. experimental autoimmune encephalomyelitis (EAE) but without the adverse effects observed with FTY720. Like FTY720, AKP-11 bound to S1P1 is internalized and activates intracellular AKT and ERKs cellular signaling pathways. In contrast to FTY720, AKP-11 mediated S1P1 downregulation is independent of sphingosine kinase activity indicating it to be a direct agonist of S1P1. The S1P1 loss and inhibition of lymphocyte egress by FTY720 leads to lymphopenia. In comparison with FTY720, oral administration of AKP-11 caused milder and reversible lymphopenia while providing a similar degree of therapeutic efficacy in the EAE animal model. Consistent with the observed reversible lymphopenia with AKP-11, the S1P1 recycled back to cell membrane in AKP-11 treated cells following its withdrawal, but not with withdrawal of FTY720. Accordingly, a smaller degree of ubiquitination and proteolysis of S1P1 was observed in AKP-11 treated cells as compared to FTY720. Consistent with previous observations, FTY720 treatment is associated with adverse effects of bradycardia and lung vascular leaks in rodents, whereas AKP-11 treatment had undetectable effects on bradycardia and reduced lung vascular leaks as compared to FTY720. Taken together, the data documents that AKP-11 treatment cause milder and reversible lymphopenia with milder adverse effects while maintaining therapeutic efficacy similar to that observed with FTY720, thus indicating therapeutic potential of AKP-11 for treatment of MS and related autoimmune disorders.