The effect of ghrelin on the regulation of antioxidantenzymes and inducible nitric oxide synthase in rat liver

U poslednjih nekoliko godina, objavljeni naučni podaci sve više ukazuju na antioksidantne i antiinflamatorne efekte grelina. U ovoj doktorskoj disertaciji su izučavani efekti grelina na ekspresiju enzima jetre: superoksid dismutaze (engl. Superoxide Dismutase, SOD), katalaze (engl. Catalase, CAT), glutation peroksidaze (engl. Glutathione peroxidase, GPx) i glutation reduktaze (engl. Glutathione reductase, GR), kao i ekspresiju njihovog uzvodnog regulatora, nuklearnog faktora-kB (engl. Nuclear factor- kappa B, NFκB). Takođe su izučavani efekti grelina na regulaciju enzima inducibilne azot-monoksid-sintaze (engl. Inducible nitric oxide synthase, iNOS; NOS2) u jetri pacova. Jedan od ciljeva istraživanja u okviru ove doktorske disertacije je bio i utvrđivanje uloge kinaze regulisane ekstraćelijskim signalima (engl. Extracellular regulated protein kinase, ERK1/2) i protein kinaze B (engl. Protein kinase B, Akt) u grelinom regulisonoj aktivnosti i ekspresiji antioksidatnivnih enzima i iNOS u jetri pacova. Kontrolni, normalno hranjeni mužjaci pacova, Wistar soja (mase 200-250g) kao i mužjaci pacova koji su bili na dijeti bogatoj mastima (engl. High fat diet, HF), tretirani su grelinom (0,3nmol/5μl) intracerebroventrikularnim (ICV) injeciranjem, u bočne cerebralne komore svaka 24 h, u trajanju od 5 dana. Dva sata nakon poslednjeg tretmana, životinje su žrtvovane i izolovane su jetre. Za utvrđivanje nivoa ekspresije proteina: iNOS, antioksidativnih enzima, fosforilacije Akt na Ser473, ERK 1/2 i NFκB, subjedinica p50 i p65, primenjena je Western blot metoda. Dobijeni rezultati pokazuju da dolazi do povećanja ekspresije proteina za enzime antioksidativne zaštite: Cu/Zn-SOD (p<0,05), Mn-SOD (p<0,05), CAT (p<0,01), GPx, (p<0,001), kao i GR (p<0,01) u grupi normalno hranjenih životinja tretiranih grelinom u poređenju sa kontrolnim životinjama. Grelin značajno smanjuje ekspresiju iNOS (p<0,001) kao i fosforilaciju NFκB subjedinica p65 (p<0,001) i p50 (p<0,05) u grupi normalno hranjenih životinja u poređenju sa kontrolnom grupom pacova. Nivo fosforilacije Akt na Ser473 značajno je veći u grelinom tretiranoj grupi nego kod kontrolnih životinja (p<0,01)...In recent years, there have been prominent findings suggesting that ghrelin has antioxidant and anti-inflammatory effects. In this study we investigated the effects of ghrelin on protein expression of liver antioxidant enzymes superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) as well as protein expression of their upstream regulator, nuclear factor kappa B (NFκB). We have also studied the effects of ghrelin on liver inducible nitric oxide synthase (iNOS) protein expression. Furthermore, we aimed to investigate whether extracellular regulated protein kinase (ERK 1/2) and protein kinase B (Akt) are involved in ghrelin regulated liver antioxidant enzymes and iNOS protein expression. Male Wistar rats (200-250g) were treated with ghrelin (0,3nmol/5μl) injected into the lateral cerebral ventricle every 24 h for 5 day, 2 h after the last treatment the animals were sacrificed and liver excised. For protein expression of antioxidant enzymes, iNOS, phosphorylation of Akt at Ser473, ERK1/2 and nuclear factor B (NFκB) subunits 50 and 65, Western blot method was used. Results show significantly higher protein expression of Cu/Zn SOD (p<0,05), Mn-SOD (p<0,05), CAT (p<0,01), GPx, (p<0,001), and GR (p<0,01) in the liver isolated from ghrelin treated animals compared with control animals. On the contrary, ghrelin significantly (p<0,001) reduced protein expression of iNOS. In addition, phosphorylation of NFκB subunits p65 and p50 were significantly (p<0,05) decreased by ghrelin when compared with controls. Phosphorylation of ERK1/2 and phosphorylation of Akt at Ser473 were significantly higher in ghrelin treated than in control animals (p<0,05 for ERK1/2; p<0,01 for Akt). In conclusion, results from this study show that activation of Akt and ERK1/2 are involved in ghrelin-mediated regulation of protein expression of antioxidant enzymes and iNOS in the rat liver

Nitric Oxide and its Role in Cardiovascular Diseases

Nitric oxide synthases (NOS) are the enzymes responsible for nitric oxide (NO) generation. NO is a free radical which reacts with various molecules to cause multiple biological effects. It is clear that the generation and actions of NO under physiological and pathophysiological conditions are exquisitely regulated and extend to almost every cell type and function within the circulation. While the molecule mediates many physiological functions, an excessive presence of NO is toxic to cells. The enzyme NOS, constitutively or inductively, catalyses the production of NO in several biological systems. NO is derived not only from NOS isoforms but also from NOS-independent sources. In mammals, to date, three distinct NOS isoforms have been identified: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). The molecular structure, enzymology and pharmacology of these enzymes have been well defined, and reveal critical roles for the NOS system in a variety of important physiological processes. This review focuses on recent advances in the understanding of the interactions between NOS enzymes and pathophysiology of cardiovascular diseases (CVD) and the role of NO agonists as potential therapeutic agents in treatment of CVD.The Open Nitric oxide Journal has been discontinued

Regulation of Inducible Nitric Oxide Synthase (iNOS) and its Potential Role in Insulin Resistance, Diabetes and Heart Failure

Nitric oxide synthases (NOS) are the enzymes responsible for nitric oxide (NO) generation. NO is a reactive oxygen species as well as a reactive nitrogen species. It is a free radical which mediates several biological effects. It is clear that the generation and actions of NO under physiological and pathophysiological conditions are regulated and extend to almost every cell type and function within the circulation. In mammals 3 distinct isoforms of NOS have been identified: neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). The important isoform in the regulation of insulin resistance (IR) is iNOS. Understanding the molecular mechanisms regulating the iNOS pathway in normal and hyperglycemic conditions would help to explain some of vascular abnormalities observed in type 2 diabetes mellitus (T2DM). Previous studies have reported increased myocardial iNOS activity and expression in heart failure (HF). This review considers the recent animal studies which focus on the understanding of regulation of iNOS activity/expression and the role of iNOS agonists as potential therapeutic agents in treatment of IR, T2DM and HF

DETERMINATION OF TOTAL ANTIOXIDANT STATUS (TAS) AS A BIOCHEMICAL PARAMETER IN CONTROL OF WORKERS ’ HEALTH

Abstract — Despite its importance for human health, the capacity of the antioxidative defense system (AOS) is still not included among regular biochemical analyses. In this study, we estimated the blood plasma level of the total antioxidant status (TAS) as an appropriate parameter for AOS monitoring with the aim of including it among other biochemical parameters in the regular control of health of the working population. We have applied a method developed by Randox Laboratories (England) that can be routinely performed on automatic chemical analyzers. The TAS level, in correlation with corresponding biochemical parameters, could contribute to better evaluation of the health of individuals and the working population

Efekti karvedilola na aktivnost/ekspresiju inducibilne azot oksid sintetaze (iNOS) kod bolesnika sa hroničnom srčanom insuficijencijom (HSI)

Previous studies have reported increased myocardial inducible nitric oxide synthase (iNOs) expression and activity in chronic heart failure (ChF). Nitric oxide (NO) is a free radical which reacts with various molecules to cause multiple biological effects. Carvedilol is a α1-, β1-, and β2-adrenergic antagonist, used in therapy of hypertension and ChF. recently, it has been shown that carvedilol has an effect as NO quenching agent. Carvedilol presents several other mechanisms of action that converge to improve the symptomatology of hypertension and CHF. In addition to the adrenergic antagonism, carvedilol is also an antioxidant and endothelin suppressor. The molecular mechanism for the NO quenching potency of carvedilol remains to be determined. In cardiac cells the major pathway responsible for the regulation of iNOS activity/expression involves the activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt). Thus, in our current ongoing and future studies, we will test the hypothesis that in disease states such as CHF where the PI3K/Akt pathway is interrupted, the regulation of iNOS activity/expression will be abolished. as a corollary of this primary hypothesis, we postulate that in CHF carvedilol stimulates cardiovascular iNOS via a mechanism involving activation of PI3K/akt cascade and further amplifies iNOS activity/expression. Moreover, since the PI3K/akt pathway have been acknowledged as a critically important mediator of cardiac iNOS regulation, PI3K/akt are identified as one of key targets for novel therapeutic interventions to minimize irreversible tissue damage associated with CHF and hypertension. A safer technology to regulate in vivo synthesis of PI3K/akt by generic manipulation would be a welcome work.Azot oksid (NO) je slobodni radikal čije reakcije dovode do različitih bioloških dejstava. Karvedilol je antagonist α1-, β1-,i β2 adrenergičkih receptora koji se koristi u terapiji hipertenzije i nedavno je pokazano da hemijski reaguje sa NO. Pored adrenergičke blokade karvedilol doprinosi poboljšanju hipertenzije i HSI drugim mehanizmima. Takođe, pokazano je da karvedilol ima ulogu antioksidansa i ulogu u supresiji endotelina. Precizan mehanizam interakcije karvedilola i NO do sada nije u potpunosti razjašnjen. aktivnost/ekspresija inducibilne azot oksid sintetaze (iNOS) u kardiomiocitima dominantno je regulisana aktivacijom fosfatidilinozitol 3-kinaze (PI3K) i protein kinaze B (akt). U našim tekućim i planiranim istraživanjima, polazimo od hipoteze da je u HSI poremećen PI3K/akt signalni put, a time konsekventno i smanjena aktivnost/ekspresija iNOS-a. Takođe, naša hipoteza se bazira na pretpostavci da karvedilol u HSI stimuliše iNOS u kardiovaskularnom sistemu preko aktivacije PI3K/Akt kaskade. U zaključku, regulacija PI3K/Akt signalnog puta ima veoma bitnu ulogu u regulaciji iNOS-a u stanjima HSI. S obzirom da PI3K/Akt signalni put ima biološku ulogu kao kritično-važni signalni put u delovanju adrenergičkih lekova, kao što je karvedilol, to ga čini ključnim za terapeutske intervencije u cilju minimalizacije ireverzibilnih tkivnih i ćelijskih oštećenja koja su asocirana sa HSI i hipertenzijom

Thrombin and vascular inflammation

Vascular endothelium is a key regulator of homeostasis. In physiological conditions it mediates vascular dilatation, prevents platelet adhesion, and inhibits thrombin generation. However, endothelial dysfunction caused by physical injury of the vascular wall, for example during balloon angioplasty, acute or chronic inflammation, such as in atherothrombosis, creates a proinflammatory environment which supports leukocyte transmigration toward inflammatory sites. At the same time, the dysfunction promotes thrombin generation, fibrin deposition, and coagulation. The serine protease thrombin plays a pivotal role in the coagulation cascade. However, thrombin is not only the key effector of coagulation cascade; it also plays a significant role in inflammatory diseases. It shows an array of effects on endothelial cells, vascular smooth muscle cells, monocytes, and platelets, all of which participate in the vascular pathophysiology such as atherothrombosis. Therefore, thrombin can be considered as an important modulatory molecule of vascular homeostasis. This review summarizes the existing evidence on the role of thrombin in vascular inflammation

Thrombin stimulates VSMC proliferation through an EGFR-dependent pathway: involvement of MMP-2

In this study, the role of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK1/2), heparin-binding EGF-like growth factor (HB-EGF), general metalloproteinases, matrix metalloproteinases-2 (MMP-2) in mediating the mitogenic action of thrombin in rat vascular smooth muscle cells (VSMC) was investigated. The incubation of rat VSMC with thrombin (1 U/ml) for 5 min resulted in significant (p lt 0.001) increase of ERK1/2 phosphorylation by 8.7 +/- A 0.9-fold, EGFR phosphorylation by 8.5 +/- A 1.3-fold (p lt 0.001) and DNA synthesis by 3.6 +/- A 0.4-fold (p lt 0.001). Separate 30-min pretreatments with EGFR tyrosine kinase irreversible inhibitor, 10 A mu M PD169540 (PD), and 20 A mu M anti-HB-EGF antibody significantly reduced thrombin-stimulated EGFR and ERK1/2 phosphorylation by 81, 72 % and by 48 and 61 %, respectively. Furthermore, the same pretreatments with PD or anti-HB-EGF antibody reduced thrombin-induced VSMC proliferation by 44 and 45 %, respectively. In addition, 30-min pretreatments with 10 A mu M specific MMP-2 inhibitor significantly reduced thrombin-stimulated phosphorylation of both EGFR and ERK1/2 by 25 %. Moreover, the same pretreatment with MMP-2 inhibitor reduced thrombin-induced VSMC proliferation by 45 %. These results show that the thrombin-induced DNA synthesis correlates with the level of ERK1/2 activation rather than EGFR activation. These results further suggest that thrombin acts through EGFR and ERK 1/2 signaling pathways involving MMP-2 to upregulate proliferation of VSMC

Retraction Note: Thrombin stimulates VSMC proliferation through an EGFR-dependent pathway: involvement of MMP-2

The Editors-in-Chief have retracted this article. After publication, concerns were raised regarding image irregularities in the Figures. Specifically, In Figs. 1a and 6a, the p-EGFR lanes 1 and 2 appear highly similar, but the t-EGFR blots are different. In Figs. 2a and 3a, the t-EGFR panels appear highly similar. Fig. 2a and 3a t-EGFR lanes 3 and 4 appear highly similar to Fig. 4a t-EGFR lanes 1 and 2 (flipped horizontally). Fig. 4b p-ERK lanes 1, 2 and 4 appear highly similar to Fig. 6b p-ERK lanes 3, 2 and 4, respectively (flipped horizontally). In Figs. 5b and 7b, the t-EGFR panels appear highly similar. The Editors-in-Chief therefore no longer have confidence in the presented data. Katarina Smiljanic and Esma R. Isenovic do not agree to this retraction. Pierre Marche is deceased. Milan Obradovic, Aleksandra Jovanovic, Jelena Djordjevic, Branislava Dobutovic and Danimir Jevremovic have not responded to any correspondence from the publisher about this retraction.Retraction note to: [http://dx.doi.org/10.1007/s11010-014-2151-y

A novel hypothesis regarding the possible involvement of cytosolic phospholipase 2 in insulin-stimulated proliferation of vascular smooth muscle cells

Insulin (INS) via INS receptor acts as a mitogen in vascular smooth muscle cells (VSMCs) through stimulation of multiple signaling mechanisms, including p42/44 mitogen-activated protein kinase (ERK1/2) and phosphatidyl inositol-3 kinase (PI3K). In addition, cytosolic phospholipase 2 (cPLA(2)) is linked to VSMCs proliferation. However, the upstream mechanisms responsible for activation of cPLA(2) are not well defined. Therefore, this investigation used primary cultured rat VSMCs to examine the role of PI3K and ERK1/2 in the INS-dependent phosphorylation of cPLA(2) and proliferation induced by INS. Exposure of VSMCs to INS (100 nM) for 10 min increased the phosphorylation of cPLA(2) by 1.5-fold (p lt 0.01), which was blocked by the cPLA(2) inhibitor MAFP (10 mu M; 15 min). Similarly, the PI3K inhibitor LY294002 (10 mu M; 15 min) and ERK1/2 inhibitor PD98059 (20 mu M; 15 min) abolished the INS-mediated increase in cPLA(2) phosphorylation by 59% (p lt 0.001), and by 75% (p lt 0.001), respectively. Further, inhibition of cPLA2 with cPLA2 inhibitor MAFP abolished the INS-stimulated ERK1/2 phosphorylation by 65% (p lt 0.01). Incubation of rat VSMCs with INS resulted in an increase of VSMCs proliferation by 85% (p lt 0.001). The effect of INS on VSMCs proliferation was significantly (p lt 0.01) reduced by pretreatment with MAFP. Thus, we hypothesized that INS stimulates VSMCs proliferation via a mechanism involving the PI3K-dependent activation of cPLA(2) and release of arachidonic acid (AA), which activates ERK1/2 and further amplifies cPLA(2) activity. (C) 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved

The role of antioxidative treatment in diabetes mellitus

Oksidativni stres (OxS) je skup poremećaja dinamičke ravnoteže prooksidanasa i antioksidanasa koji nastaje usled povećane produkcije slobodnih radikala (SR) ili usled smanjene aktivnosti antioksidativnog zaštitnog sistema (AOS). Bolest koja se, takođe, povezuje sa OxS je i diabetes mellitus (DM). DM karakteriše povećani nivo glukoze u cirkulaciji, kao i drugi biohemijski poremećaji koji nastaju kao posledica neadekvatne produkcije ili neadekvatnog dejstva insulina. Dijabetes tipa 1 (DMT1) je prototip autoimune bolesti sa intenzivnim OxS. Antioksidativni enzim katalaza (CAT) smanjuje produkciju vodonik-peroksida koji može biti veoma toksičan za ćelije pankreasa. Povećanje aktivnosti CAT u svim fazama DMT1 indirektno potvrđuje značaj OxS u nastanku ove bolesti. Visoko reaktivni SR, nastali uglavnom zbog hiperglikemije, izazivaju OxS što dodatno ubrzava razvoj i napredovanje DM kao i njegovih komplikacija koje su uzrokovane redukovanom aktivnošću AOS. Uprkos brojnim dokazima o štetnim posledicama OxS u DM, veliki broj kliničkih ispitivanja sa klasičnim antioksidansima, nažalost, nije pokazao efikasnost njihove primene u lečenju DM. Novi terapijski pristup, koji uključuje primenu kako standardnih tako i novih antioksidanasa, se predlaže u algoritmima lečenja DM. Važno je ukazati na značaj doze primenjenih antioksidanasa, obzirom da je dokazano da pojedinačne visoke doze mogu delovati prooksidaciono. Takođe, važna je i dozna uravnoteženost primenjenih antioksidanasa i antidijabetika, u cilju efikasnijeg lečenja obolelih od DM. U okviru ovog rada fokusirali smo se na ulogu antioksidansa u lečenju DM.Oxidative stress (OxS) represents an imbalance in dynamic equilibrium between prooxidants and antioxidants caused by increased free radical (FR) production or decreased activity of antioxidative system (AOS). OxS is involvedin many diseases such is diabetes mellitus (DM). DM presents with elevated blood glucose level and other biochemical disorders related to the inappropriate insulin secretion or improper insulin action. DM type 1 (DMT1) is an autoimmune disease with excessive OxS. The activity of antioxidative enzyme catalase (CAT) diminishes the production of hydrogen peroxide which is highly toxic for pancreatic cells. The increased activity of catalase (CAT) found in DMT1 patients signifies the importance of OxS in the pathogenesis of this disease. The generation of highly reactive FR, induced by hyperglycaemia, triggers the development of oxidative stress. Furthermore, OxS accelerates the development of DM and its complications which are related to the decreased activity of AOS. However, the standard antioxidant drugs failed in clinical trials for DM. New antioxidant agents combined with standard drugs are now proposed in therapy for DM. It is important to acknowledge that high doses of antioxidant agents could paradoxically have prooxidant effect. In addition, it is important to establish the dosage balance between antioxidant and antidiabetic drugs. In this article, we discuss the antioxidative agents and their significance in treatment of DM