Dietary palmitate and oleate differently modulate insulin sensitivity in human skeletal muscle.

AIMS/HYPOTHESIS: Energy-dense nutrition generally induces insulin resistance, but dietary composition may differently affect glucose metabolism. This study investigated initial effects of monounsaturated vs saturated lipid meals on basal and insulin-stimulated myocellular glucose metabolism and insulin signalling. METHODS: In a randomised crossover study, 16 lean metabolically healthy volunteers received single meals containing safflower oil (SAF), palm oil (PAL) or vehicle (VCL). Whole-body glucose metabolism was assessed from glucose disposal (Rd) before and during hyperinsulinaemic-euglycaemic clamps with D-[6,6-2H2]glucose. In serial skeletal muscle biopsies, subcellular lipid metabolites and insulin signalling were measured before and after meals. RESULTS: SAF and PAL raised plasma oleate, but only PAL significantly increased plasma palmitate concentrations. SAF and PAL increased myocellular diacylglycerol and activated protein kinase C (PKC) isoform θ (p < 0.05) but only PAL activated PKCɛ. Moreover, PAL led to increased myocellular ceramides along with stimulated PKCζ translocation (p < 0.05 vs SAF). During clamp, SAF and PAL both decreased insulin-stimulated Rd (p < 0.05 vs VCL), but non-oxidative glucose disposal was lower after PAL compared with SAF (p < 0.05). Muscle serine1101-phosphorylation of IRS-1 was increased upon SAF and PAL consumption (p < 0.05), whereas PAL decreased serine473-phosphorylation of Akt more than SAF (p < 0.05). CONCLUSIONS/INTERPRETATION: Lipid-induced myocellular insulin resistance is likely more pronounced with palmitate than with oleate and is associated with PKC isoforms activation and inhibitory insulin signalling. TRIAL REGISTRATION: ClinicalTrials.gov .NCT01736202. FUNDING: German Federal Ministry of Health, Ministry of Culture and Science of the State North Rhine-Westphalia, German Federal Ministry of Education and Research, European Regional Development Fund, German Research Foundation, German Center for Diabetes Research.

Yoda1 and phosphatidylserine exposure in red cells from patients with sickle cell anaemia.

Phosphatidylserine (PS) exposure is increased in red cells from sickle cell anaemia (SCA) patients. Externalised PS is prothrombotic and attractive to phagocytes and activated endothelial cells and thus contributes to the anaemic and ischaemic complications of SCA. The mechanism of PS exposure remains uncertain but it can follow increased intracellular Ca2+ concentration ([Ca2+]i). Normally, [Ca2+]i is maintained at very low levels but in sickle cells, Ca2+ permeability is increased, especially following deoxygenation and sickling, mediated by a pathway sometimes called Psickle. The molecular identity of Psickle is also unclear but recent work has implicated the mechanosensitive channel, PIEZO1. We used Yoda1, an PIEZO1 agonist, to investigate its role in sickle cells. Yoda1 caused an increase in [Ca2+]i and PS exposure, which was inhibited by its antagonist Dooku1 and the PIEZO1 inhibitor GsMTx4, consistent with functional PIEZO1. However, PS exposure did not necessitate an increase in [Ca2+]i. Two PKC inhibitors were also tested, chelerytherine chloride and calphostin C. Both reduced PS exposure whilst chelerytherine chloride also reduced Yoda1-induced increases in [Ca2+]i. Findings are therefore consistent with the presence of PIEZO1 in sickle cells, able to mediate Ca2+ entry but that PKC was also involved in both Ca2+ entry and PS exposure.

Circular RNA PRKCI and microRNA-545 relate to sepsis risk, disease severity and 28-day mortality.

This study aimed to explore the clinical values of circular RNA protein kinase C iota (circ-PRKCI) and its target microRNA-545 (miR-545) in sepsis patients. Plasma samples of 121 sepsis patients and 60 healthy controls (HCs) were collected, then circ-PRKCI and miR-545 expressions were detected using RT-qPCR. Sepsis patients' demographics, biochemical indexes, medical histories, infection information were recorded. Besides, comprehensive disease scores (APACHE II score and SOFA score) were assessed within 24 h after admission. According to the survival status, 28-day mortality was calculated. Decreased circ-PRKCI expression and increased miR-545 expression were observed in sepsis patients compared to HCs, both of which had close correlations with sepsis risk. Besides, circ-PRKCI was negatively correlated with miR-545 in sepsis patients and HCs, respectively. Circ-PRKCI was negatively correlated with serum creatinine, white blood cell, C-reactive protein, APACHE II score, SOFA score, but positively correlated with albumin, which also related to blood stream infection (as primary infection site) and anaerobes infection in sepsis patients. Whereas the miR-545 showed a roughly opposite tendency. Decreased circ-PRKCI and increased miR-545 expressions were discovered in deaths compared to survivors, and both of them had values for predicting 28-day mortality risk in sepsis patients, which were slightly lower than the predictive values of APACHE II score and SOFA score for predicting 28-day mortality risk. Multivariate logistic analyses displayed circ-PRKCI as an independent factor predicting decreased 28-day mortality risk. In conclusion, circ-PRCKI insufficiency and miR-545 sufficiency were related to sepsis risk, clinical disease severity and 28-day mortality risk.

CircPRKCI relieves lipopolysaccharide-induced HK2 cell injury by upregulating the expression of miR-545 target gene ZEB2.

The aim of this study was to investigate the possible influences of circPRKCI abnormal expression on lipopolysaccharide (LPS)-induced HK2 cell injury and its mechanism. The circPRKCI level was identified in serum samples from patients with urosepsis and healthy subjects, as well as LPS-treated HK2 cells by qRT-PCR. Cell viability, apoptosis, expression of proteins associated with apoptosis, and expression of pro-inflammatory cytokines in LPS-treated HK2 cells were measured. Effects of circPRKCI abnormal expression on LPS-induced HK2 cell injury were then evaluated. Afterward, the binding miRNA of circPRKCI and target gene of miRNA were identified, and the involvements of NF-kB pathway signaling pathway with the effects of circPRKCI were finally studied. CircPRKCI was significantly down-regulated in serum samples from patients with urosepsis and LPS-treated HK2 cells. LPS-induced decrease of cell viability, increase of cell apoptosis, as well as elevated productions of tumor necrosis factor (TNF)-α, interleukins (IL)-1ß, IL-6, and IL-8 in HK2 cells were attenuated by overexpressed circPRKCI. In addition, circPRKCI negatively regulated the expression of miR-545, and miR-545 up-regulation reversed the inhibiting effects of circPRKCI overexpression on LPS-induced HK2 cell injury. Moreover, zinc finger E-box-binding homeobox 2 (ZEB2) was identified as a target gene of miR-545, and ZEB2 overexpression partly reversed the effects of miR-545 up-regulation on LPS-induced HK2 cell injury. Furthermore, NF-kB pathway was revealed to be associated to the effects of circPRKCI on LPS-induced HK2 cell injury. This research indicated that the highly expressed circPRKCI relieved inflammatory injury induced by LPS in HK2 cells by suppressing miR-545/ZEBs and depressing the briskness of NF-kB pathway.

Iguratimod represses B cell terminal differentiation linked with the inhibition of PKC/EGR1 axis.

BACKGROUND: This study aimed to explore the molecular mechanism and clinical relevance of iguratimod in the regulation of human B cell terminal differentiation. METHODS: An in vitro human antibody-secreting cell (ASC) differentiation system was established to test the effect of iguratimod. B cell phenotype and key transcription factors (TFs) relevant to ASC differentiation were analyzed through flow cytometry and qPCR. The COX-2 activity was measured by enzyme immunoassay (EIA). RNA sequencing was used to identify potential targets of iguratimod. We enrolled six treatment-naive rheumatoid arthritis (RA) patients whose blood samples were collected for phenotypic and molecular studies along with 12-week iguratimod monotherapy. RESULTS: Iguratimod inhibited human ASC generation without affecting B cell activation and proliferation. Iguratimod showed only weak COX-2 activity. Gene set enrichment analysis (GSEA) identified that protein kinase C (PKC) pathway was targeted by iguratimod which was confirmed by PKC activity detection. Furthermore, early growth response 1 (EGR1), a target of PKC and a non-redundant TF for ASC differentiation, was found to be the most downregulated gene in iguratimod-treated B cells. Lastly, iguratimod monotherapy decreased peripheral ASCs and was associated with improved disease activity. The expression of major ASC-related TFs, including EGR1, was similarly downregulated in patient blood samples. CONCLUSIONS: Iguratimod inhibits ASC differentiation both in vitro and in RA patients. Our study suggests that PKC/EGR1 axis, rather than COX-2, is critically involved in the inhibitory effect by iguratimod on human ASC differentiation. Iguratimod could have a broader application to treat B cell-related autoimmune diseases in clinics.

Season Dependent Changes in the Expression of Protein Kinase C Isoenzymes in a Female Patient with Systemic Lupus Erythematosus.

We aimed to answer the question whether the decreased expression of protein kinase C (PKC) isoenzymes in the peripheral blood mononuclear cells (PBMC) of patients with systemic lupus erythematosus (SLE) is inherited or not. For this reason we examined the expression of PKC isoenzymes in a European white girl with acute SLE and in her healthy mother and father simultaneously in summer and winter during one year using western blotting and densitometry. We found that in the father the expression of PKC isoenzymes did not differ from that of eight healthy controls included women and men. However, in the "SLE-free" mother and in the patient arrived in July with acute symptoms of lupus, the expression of PKC isoenzymes showed a season dependent undulation in parallel. Namely, in summer the expression values were significantly lower, in winter they were significantly higher than those in the controls. Thus, the decreased expression of PKC isoenzymes in the PBMC of SLE patient is not a disease specific marker; it appears also in her lupus free mother. This phenomenon may be due to a season dependent female genetic background. However, the low PKC levels in summer can still decrease further the low production of IL-2 in T cells of lupus patients augmenting the existing AP-1 defects. This is the first report on the season and female dependent inherited changing of PKC expression in a European white patient with SLE and her mother. Further studies are needed to confirm these findings in other populations.

Cross talk between serine/threonine and tyrosine kinases regulates ADP-induced thromboxane generation in platelets.

ADP-induced thromboxane generation depends on Src family kinases (SFKs) and is enhanced with pan-protein kinase C (PKC) inhibitors, but it is not clear how these two events are linked. The aim of the current study is to investigate the role of Y311 phosphorylated PKCδ in regulating ADP-induced platelet activation. In the current study, we employed various inhibitors and murine platelets from mice deficient in specific molecules to evaluate the role of PKCδ in ADP-induced platelet responses. We show that, upon stimulation of platelets with 2MeSADP, Y311 on PKCδ is phosphorylated in a P2Y1/Gq and Lyn-dependent manner. By using PKCδ and Lyn knockout murine platelets, we also show that tyrosine phosphorylated PKCδ plays a functional role in mediating 2MeSADP-induced thromboxane generation. 2MeSADP-induced PKCδ Y311 phosphorylation and thromboxane generation were potentiated in human platelets pre-treated with either a pan-PKC inhibitor, GF109203X or a PKC α/ß inhibitor and in PKC α or ß knockout murine platelets compared to controls. Furthermore, we show that PKC α/ß inhibition potentiates the activity of SFK, which further hyper-phosphorylates PKCδ and potentiates thromboxane generation. These results show for the first time that tyrosine phosphorylated PKCδ regulates ADP-induced thromboxane generation independent of its catalytic activity and that classical PKC isoforms α/ß regulate the tyrosine phosphorylation on PKCδ and subsequent thromboxane generation through tyrosine kinase, Lyn, in platelets.

Protein kinase Cι is a new prognostic factor in gastric cancer.

PURPOSES: Protein kinase Cι (PKCι) is an important oncogenic K-ras effector, and its expression is correlated with tumor angiogenesis. The role of PKCι in gastric cancer remains unclear. The aim of this study was to clarify the role of PKCι in gastric cancer. METHODS: Twenty-eight patients with gastric cancer who underwent gastrectomy were enrolled in this study. The expression of PKCι mRNA was determined, as were the clinicopathological factors. The patients were divided into PKCι high and low expression groups. The 5-year survival rate, ERK mRNA level and VEGF mRNA level were compared between the two groups. The prognostic factors were investigated by a multivariate analysis. RESULTS: High expression of PKCι was observed to be associated with a lack of differentiation, tumor invasion ≥muscularis propria≤, stage III and IV disease and peritoneal dissemination. The 5-year survival rate in the PKCι high group was lower than that in the PKCι low group. The multivariate analysis revealed that a high expression level of PKCι was an independent prognostic factor. The expression levels of ERK and VEGF in the PKCι high group were higher than those in the PKCι low group. CONCLUSION: Our results indicate that PKCι is correlated with tumor progression and angiogenesis. PKCι may be a new prognostic factor for gastric cancer.

Mechanism of race-dependent platelet activation through the protease-activated receptor-4 and Gq signaling axis.

OBJECTIVE: Black individuals are at an increased risk of myocardial infarction and stroke, 2 vascular diseases with strong thrombotic components. Platelet activation is a key step in platelet clot formation leading to myocardial infarction and stroke, and recent work supports a racial difference in platelet aggregation through the thrombin protease-activated receptors (PARs). The underlying mechanism for this racial difference, however, has not been established. Determining where in the signaling cascade these racial differences emerge will aid in understanding why individuals of differing racial ancestry may possess an inherent difference in their responsiveness to antiplatelet therapies. APPROACH AND RESULTS: Washed human platelets from black volunteers were hyperaggregable in response to PAR4-mediated platelet stimulation compared with whites. Interestingly, the racial difference in PAR4-mediated platelet aggregation persisted in platelets treated ex vivo with aspirin and 2MeSAMP (2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate), suggesting that the racial difference is independent of secondary feedback. Furthermore, stimulation of platelets from black donors with PAR4-activating peptide showed a potentiated level of activation through the Gq pathway compared with platelets from white donors. Differences in signaling included increased Ca(2+) mobilization, Rap1 (Ras-related protein 1) activation, and integrin αIIbß3 activation with no observed difference in platelet protein expression between the groups tested. CONCLUSIONS: Our study is the first to demonstrate that the Gq pathway is differentially regulated by race after PAR4 stimulation in human platelets. Furthermore, the racial difference in PAR4-mediated platelet aggregation persisted in the presence of cyclooxygenase and P2Y12 receptor dual inhibition, suggesting that current antiplatelet therapy may provide less protection to blacks than whites.

Role of a Janus kinase 2-dependent signaling pathway in platelet activation.

INTRODUCTION: Janus kinases (JAKs) are intracellular non-receptor tyrosine kinases that transduce cytokine-mediated signals through a pathway mediated by JAK and the signal transducer and activator of transcription (STAT) proteins. The JAK-STAT pathway is involved in immune response, inflammation, and tumorigenesis. Platelets are anuclear blood cells that play a central role in hemostasis. METHODS: The aggregometry, immunoblotting, and platelet functional analysis used in this study. RESULTS: We found that the JAK2 inhibitor AG490 (25 and 50µM) attenuated collagen-induced platelet aggregation and calcium mobilization in a concentration-dependent manner. In the presence of AG490, the phosphorylation of PLCγ2, protein kinase C (PKC), Akt or JNK in collagen-activated aggregation of human platelets was also inhibited. In addition, we found that various inhibitors, such as the PLCγ2 inhibitor U73122, the PKC inhibitor Ro318220, the phospoinositide 3-kinase inhibitor LY294002, the p38 mitogen-activated protein kinase inhibitor SB203580, the ERK inhibitor PD98059, and the JNK inhibitor SP600125, had no effects on collagen-induced JAK2 activity. However, U73122, Ro318220 and SP600125 significantly diminished collagen-induced STAT3 phosphorylation. These findings suggest that PLCγ2-PKC and JNK are involved in JAK2-STAT3 signaling in collagen-activated platelets. CONCLUSION: Our results demonstrate that the JAK2-STAT3 pathway is involved in collagen-induced platelet activation through the activation of JAK2-JNK/PKC-STAT3 signaling. The inhibition of JAK2 may represent a potential therapeutic strategy for the preventing or treating thromboembolic disorders.

Protective effect of short-term genistein supplementation on the early stage in diabetes-induced renal damage.

Hyperglycemia-induced oxidative stress has been concerned in the development of diabetic nephropathy (DN), which may cause kidney damage associated with inflammation and fibrosis. This study has been conducted to investigate the role of genistein supplementation in an acute DN state. Mice with FBG levels more than 250 mg/dL after alloxan injection (single i.p., 150 mg/kg) were considered as diabetic. Diabetic mice (DM) were further subdivided according to their FBG levels, medium-high FBG (DMMH < 450 mg/dL) and high FBG (DMH; 450 mg/dL) and were administrated by an AIG-93G diet supplemented with different doses of genistein (0, 0.025 or 0.1%). After 2 weeks' treatment, the levels of kidney malondialdehyde (MDA), blood urea nitrogen (BUN), and plasma creatinine and lipid profiles, as well as oxidative stress and inflammation-related markers, were measured (P < 0.05). Genistein supplementation improved levels of FBG in the DMMH groups, but not in the DMH group, regardless of the treatment dose. Moreover, the supplementation attenuated kidney oxidative stress indicated by MDA, BUN, and plasma creatinine. In addition, genistein treatment decreased inflammatory markers such as nuclear factor kappa B (p65), phosphorylated inhibitory kappa B alpha, C-reactive protein, monocyte chemotactic protein-1, cyclooxygenase-2, and tumor necrosis factor-alpha and improved oxidative stress markers (nuclear-related factor E2, heme oxygenase-1, glutathione peroxidase, and superoxide dismutase isoforms) in treatment groups, regardless of the genistein treatment dose. Furthermore, genistein supplementation inhibited the fibrosis-related markers (protein kinase C, protein kinase C-beta II, and transforming growth factor-beta I) in the DN state. However, 0.1% genistein supplementation in diabetes with high FBG levels selectively showed a preventive effect on kidney damage. These results suggest that genistein might be a good protective substance for DN through regulation of oxidative stress and inflammation. In particular, genistein is more efficient in diabetes patients with medium-high blood glucose levels. Finally, it is required to establish the beneficial dosage of genistein according to blood glucose levels.

A functional interaction between TRPC/NCKX induced by DAG plays a role in determining calcium influx independently from PKC activation.

Ca(2+)influx might occur through K(+)-dependent Na(+)/Ca(2+) exchanger operating in reverse mode (rNCKX). In a cellular model different from platelets, an interaction between canonical transient receptor potential cation (TRPC) channels and NCX has been found. The aim of this study was to verify whether the TRPC/NCKX interaction operates in human platelets. Our results showed that the diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced rNCKX-mediated Ca(2+) influx through TRPC-mediated Na(+) influx. DAG-induced activation of TRPC/NCKX occurs independently of protein kinase C (PKC) activation, as PKC inhibitor did not modify OAG-mediated Ca(2+) influx. Moreover, as both rNCKX and TRPC inhibitors reduced OAG-induced platelet aggregation which, conversely, was increased by flufenamic acid, known to develop TRPC activity, it could be suggested that the TRPC/NCKX interaction has a role in OAG-dependent platelet aggregation.

Effects of mild-warming moxibustion on Bcl-2 and PKC expressions of peripheral blood in elderly people.

OBJECTIVE: To explore the anti-aging effects of mild-warming moxibustion on Bcl-2 and PKC expression in peripheral blood and general symptoms in elderly people. METHODS: A total of 61 elderly people and 30 non-elderly people were enrolled. The total effective rate of mild-warming moxibustion was assessed by symptom scores, and Bcl-2 and PKC expression in peripheral blood was detected by flow cytometry. RESULTS: The total effective rate in the mild-warming moxibustion group was significantly higher than in the blank control group (P < 0.01). Bcl-2 and PKC expression rates in peripheral blood in the blank control group were lower than in the normal control group (< 0.01), but higher after mild-warming moxibustion (P < 0.01). CONCLUSION: The anti-aging effects of mild-warming moxibustion may be due to increased Bcl-2 and PKC expression in peripheral blood in aged people.

Platelet protein kinase C and brain-derived neurotrophic factor levels in borderline personality disorder patients.

Borderline personality disorder (BPD) is a prevalent and difficult to treat psychiatric condition characterized by abrupt mood swings, intense anger and depression, unstable interpersonal relationships, impulsive self-destructive behavior and a suicide rate of approximately 10%. Possible underlying molecular dysregulations in BPD have not been well explored. Protein kinase C (PKC) and brain-derived neurotrophic factor (BDNF) have both been implicated in affective disorders, but their role in BPD has not been examined. Platelets were isolated from blood obtained from 24 medication-free BPD patients and 18 healthy control subjects. PKC-α, phosphorylated-PKC-α (p-PKCα), PKC-ßII, and BDNF were measured in platelet homogenates by immunoblotting. In the males, platelet BDNF and PKC-α levels were lower in patients than controls. p-PKC-α and PKC-ßII were lower at trend levels. In the entire sample, platelet p-PKCα and PKC-α activity were lower, at a trend level, in patients compared to controls. This is the first report to our knowledge of PKC and BDNF activity in BPD and calls for replication. These findings are consistent with altered PKC and BDNF activity in a range of neuropsychiatric conditions including bipolar disorder, depression and suicide.

Glucose and collagen regulate human platelet activity through aldose reductase induction of thromboxane.

Diabetes mellitus is associated with platelet hyperactivity, which leads to increased morbidity and mortality from cardiovascular disease. This is coupled with enhanced levels of thromboxane (TX), an eicosanoid that facilitates platelet aggregation. Although intensely studied, the mechanism underlying the relationship among hyperglycemia, TX generation, and platelet hyperactivity remains unclear. We sought to identify key signaling components that connect high levels of glucose to TX generation and to examine their clinical relevance. In human platelets, aldose reductase synergistically modulated platelet response to both hyperglycemia and collagen exposure through a pathway involving ROS/PLCγ2/PKC/p38α MAPK. In clinical patients with platelet activation (deep vein thrombosis; saphenous vein graft occlusion after coronary bypass surgery), and particularly those with diabetes, urinary levels of a major enzymatic metabolite of TX (11-dehydro-TXB2 [TX-M]) were substantially increased. Elevated TX-M persisted in diabetic patients taking low-dose aspirin (acetylsalicylic acid, ASA), suggesting that such patients may have underlying endothelial damage, collagen exposure, and thrombovascular disease. Thus, our study has identified multiple potential signaling targets for designing combination chemotherapies that could inhibit the synergistic activation of platelets by hyperglycemia and collagen exposure.

Platelet protein kinase C-theta deficiency with human RUNX1 mutation: PRKCQ is a transcriptional target of RUNX1.

OBJECTIVE: Mutations in the hematopoietic transcription factor RUNX1 cause thrombocytopenia and impaired platelet function. In a patient with a heterozygous mutation in RUNX1, we have described decreased platelet pleckstrin phosphorylation and protein kinase C- (PKC-, gene PRKCQ) associated with thrombocytopenia, impaired platelet aggregation, and dense granule secretion. Little is known regarding regulation of PKC- in megakaryocytes and platelets. We have addressed the hypothesis that PRKCQ is a direct transcriptional target of RUNX1. METHODS AND RESULTS: In a chromatin immunoprecipitation assay using megakaryocytic cells, there was RUNX1 binding in vivo to PRKCQ promoter region -1225 to -1056 bp containing a RUNX1 consensus site ACCGCA at -1088 to -1069 bp; an electrophoretic mobility shift assay showed RUNX1 binding to the specific site. In RUNX1 overexpression studies, PKC- protein expression and promoter activity were enhanced; mutation of RUNX1 site showed decreased activity even with RUNX1 overexpression. Lastly, PRKCQ promoter activity and PKC- protein were decreased by short interfering RNA knockdown of RUNX1. CONCLUSIONS: Our results provide the first evidence that PRKCQ is regulated at the transcriptional level by RUNX1 in megakaryocytic cells and a mechanism for PKC- deficiency associated with RUNX1 haplodeficiency.

SSRI augmentation of antipsychotic alters expression of GABA(A) receptor and related genes in PMC of schizophrenia patients.

Clinical studies have shown that negative symptoms of schizophrenia unresponsive to antipsychotic given alone can improve after augmentation with SSRI antidepressant. Laboratory investigations into the mechanism of this synergism showed that co-administration of SSRI and antipsychotic produces changes in GABA(A) receptor and related systems, which differ from the effects of each drug alone. To examine the clinical relevance of these findings, the current study examined the effects of SSRI augmentation treatment on GABA(A) receptor and related systems in schizophrenia patients. Schizophrenia patients with high levels of negative symptoms unresponsive to antipsychotic treatment received add-on fluvoxamine (100 mg/d). Blood was taken before and 1, 3 and 6 wk after adding fluvoxamine and peripheral mononuclear cells (PMC) isolated. RNA encoding for GABA(A)ß3, 5-HT2A, and 5-HT7 receptors, PKCß2, and brain-derived neurotrophic factor (BDNF) was assayed with real-time RT-PCR. Plasma BDNF protein was assayed using ELISA. Clinical symptoms were assessed with validated rating scales. We found significant increase in mRNA encoding for GABA(A)ß3 and 5-HT2A, 5-HT7 receptors and BDNF and a reduction in PKCß2 mRNA. Plasma BDNF protein concentrations were increased. There were significant correlations among the genes. Clinical symptoms improved significantly. mRNA expression of PKCß2, 5-HT2A and 5-HT7 showed significant associations with clinical symptoms. Combined SSRI+antipsychotic treatment is associated with changes in GABA(A) receptor and in related signalling systems in patients. These changes may be part of the mechanism of clinically effective drug action and may prove to be biomarkers of pharmacological response.

Inhibitory effects of resveratrol on platelet activation induced by thromboxane a(2) receptor agonist in human platelets.

Resveratrol (RSVL), a polyphenolic compound found in red wine is believed to be a contributor in decreasing the incidence of coronary heart disease. Although its primary target is unknown, it blocks platelet aggregation by an ill-defined mechanism. Protein kinase C (PKC), which would redistribute from the cytosol to the platelet membrane upon platelet stimulation, plays a key role in the signal transduction system of platelets in human. In this study, we investigated the effect of RSVL and a PKC inhibitor (DL-erythro-1,3-Dihydroxy-2-aminooctadecane, PKCI) on platelet aggregation induced by a thromboxane A(2) receptor agonist (U46619, 9,11-Dideoxy-11α, 9α-epoxymethanoprostaglandin F(2α)) using a platelet aggregometer. We also studied the platelet membranebound fibrinogen (PFig) content and the activity of protein kinase C (PKC) in platelets from healthy volunteers using flow cytometry, and a phosphorimaging system, respectively. Our results showed that RSVL blocked platelet aggregation and PFig content induced by U46619 in a concentration-dependent manner. PKCI and RSVL had an additive effect in inhibiting platelet aggregation and PFig content. Furthermore, RSVL (final concentration 50 µM) remarkably depressed the activity of PKC in the membrane of platelets and the percentage of membrane PKC activity in total PKC activity. Taken together, these results suggested that RSVL suppressed U46619-induced platelet aggregation and PFig content partially through the inhibition of the activity of PKC in platelets.

Inhibition of ATP release from erythrocytes: a role for EPACs and PKC.

OBJECTIVE: Here we demonstrate that, in human erythrocytes, increases in cAMP that are not localized to a specific receptor-mediated signaling pathway for ATP release can activate effector proteins resulting in inhibition of ATP release. Specifically we sought to establish that exchange proteins activated by cAMP (EPACs) inhibit ATP release via activation of protein kinase C (PKC). METHODS: ATP release stimulated by iloprost (ILO), or isoproterenol (ISO), was determined in the absence and presence of selective phosphodiesterase inhibitors and/or the EPAC activator, 8CPT2OMecAMP (8CPT). To determine whether EPACs inhibit ATP release via activation of PKC, erythrocytes were incubated with phorbol 12-myristate 13-acetate (PMA) prior to either forskolin or ILO in the absence and presence of a PKC inhibitor, calphostin C (CALC). RESULTS: Selective inhibition of PDEs in one pathway inhibited ATP release in response to activation of the other cAMP-dependent pathway. 8CPT and PMA inhibited both ILO- and ISO-induced ATP release. Inhibition of ATP release with 8CPT was rescued by CALC. CONCLUSION: These results support the hypothesis that cAMP not localized to a specific signaling pathway can activate EPACs which inhibit ATP release via activation of PKC and suggest a novel role for EPACs in erythrocytes.