Proteomic study of amyloid beta (25-35) peptide exposure to neuronal cells: Impact on APE1/Ref-1's protein-protein interaction.

The genotoxic, extracellular accumulation of amyloid ß (Aß) protein and subsequent neuronal cell death are associated with Alzheimer's disease (AD). APE1/Ref-1, the predominant apurinic/apyrimidinic (AP) endonuclease and essential in eukaryotic cells, plays a central role in the base excision repair (BER) pathway for repairing oxidized and alkylated bases and single-strand breaks (SSBs) in DNA. APE1/Ref-1 is also involved in the redox activation of several trans-acting factors (TFs) in various cell types, but little is known about its role in neuronal functions. There is emerging evidence for APE1/Ref-1's role in neuronal cells vulnerable in AD and other neurodegenerative disorders, as reflected in its nuclear accumulation in AD brains. An increase in APE1/Ref-1 has been shown to enhance neuronal survival after oxidative stress. To address whether APE1/Ref-1 level or its association with other proteins is responsible for this protective effect, we used 2-D proteomic analyses and identified cytoskeleton elements (i.e., tropomodulin 3, tropomyosin alpha-3 chain), enzymes involved in energy metabolism (i.e., pyruvate kinase M2, N-acetyl transferase, sulfotransferase 1c), proteins involved in stress response (i.e., leucine-rich and death domain, anti-NGF30), and heterogeneous nuclear ribonucleoprotien-H (hnRNP-H) as being associated with APE1/Ref-1 in Aß(25-35)-treated rat pheochromocytoma PC12 and human neuroblastoma SH-SY5Y cell lines, two common neuronal precursor lines used in Aß neurotoxicity studies. Because the levels of some of these proteins are affected in the brains of AD patients, our study suggests a neuroprotective role for APE1/Ref-1 via its association with those proteins and modulating their cellular functions during Aß-mediated neurotoxicity.

The role of eNOS, iNOS, and NF-kappaB in upregulation and activation of cyclooxygenase-2 and infarct size reduction by atorvastatin.

Pretreatment with atorvastatin (ATV) reduces infarct size (IS) and increases myocardial expression of phosphorylated endothelial nitric oxide synthase (p-eNOS), inducible NOS (iNOS), and cyclooxygenase-2 (COX2) in the rat. Inhibiting COX2 abolished the ATV-induced IS limitation without affecting p-eNOS and iNOS expression. We investigated 1) whether 3-day ATV pretreatment limits IS in eNOS(-/-) and iNOS(-/-) mice and 2) whether COX2 expression and/or activation by ATV is eNOS, iNOS, and/or NF-kappaB dependent. Male C57BL/6 wild-type (WT), University of North Carolina eNOS(-/-) and iNOS(-/-) mice received ATV (10 mg.kg(-1).day(-1); ATV(+)) or water alone (ATV(-)) for 3 days. Mice underwent 30 min of coronary artery occlusion and 4 h of reperfusion, or hearts were harvested and subjected to ELISA, immunoblotting, biotin switch, and electrophoretic mobility shift assay. As a result, ATV reduced IS only in the WT mice. ATV increased eNOS, p-eNOS, iNOS, and COX2 levels and activated NF-kappaB in WT mice. It also increased myocardial COX2 activity. In eNOS(-/-) mice, ATV increased COX2 expression but not COX2 activity or iNOS expression. NF-kappaB was not activated by ATV in the eNOS(-/-) mice. In the iNOS(-/-) mice, eNOS and p-eNOS levels were increased but not iNOS and COX2 levels; however, NF-kappaB was activated. In conclusion, both eNOS and iNOS are essential for the IS-limiting effect of ATV. The expression of COX2 by ATV is iNOS, but not eNOS or NF-kappaB, dependent. Activation of COX2 is dependent on iNOS.

Caffeinated coffee blunts the myocardial protective effects of statins against ischemia-reperfusion injury in the rat.

PURPOSE: We asked whether caffeinated coffee (CC) blunts the infarct size (IS)-limiting effects of atorvastatin (ATV). BACKGROUND: Adenosine receptor activation is essential for mediating the IS-limiting effects of statins. Caffeine is a nonspecific adenosine receptor blocker, and thus drinking CC may block the myocardial protective effects of statins. METHODS: Rat received 3-day ATV (10 mg/kg/day) or water by oral gavage once daily. Drinking water was replaced by water + sugar (7.5 g/100 ml), CC with sugar, or decaffeinated coffee (DC) with sugar. On the 4th day, rats were anesthetized and underwent 30 min of coronary artery occlusion and 4 h reperfusion. Area at risk was assessed by blue dye and infarct size by TTC. RESULTS: Body weight and area at risk was comparable among groups. IS was 25.1 +/- 3.9% of the area at risk in the control group. In rats not receiving ATV, CC (25.5 +/- 3.1%) and DC (34.0 +/- 2.8%) did not affect IS. IS was significantly reduced by ATV in the water + sugar (11.7 +/- 0.7%, p = 0.015) and DC (11.5 +/- 1.0%; p < 0.001) groups, but not in the CC group (32.3 +/- 3.0%; p = 0.719). ATV increased myocardial levels of Ser-473 phosphorylated Akt in the water + sugar and DC groups, but not in the CC group. CONCLUSIONS: CC, but not DC, abrogated the IS-limiting effects of ATV by blocking the adenosine receptors and preventing the phosphorylation of Akt. CC did not affect IS in rats not receiving ATV.

The cardioprotective effect of a statin and cilostazol combination: relationship to Akt and endothelial nitric oxide synthase activation.

BACKGROUND: Atorvastatin (ATV) protects against ischemia-reperfusion by upregulating Akt and subsequently, endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177. However, when given orally, high doses of ATV (10 mg/kg/d) are needed to achieve maximal protective effect in the rat. Protein kinase A (PKA) also phosphorylates eNOS at Ser-1177. As PKA activity depends on cAMP, cilostazol (CIL), a phosphodiesterase type III inhibitor, may stimulate NO production by activating PKA. HYPOTHESIS: CIL and ATV may have synergistic effects on eNOS phosphorylation and myocardial infarct size (IS) reduction. METHODS: Sprague-Dawley rats received 3-day oral pretreatment with: (1) water; (2) low dose ATV (2 mg/kg/d); (3) CIL (20 mg/kg/d): (4) ATV+CIL. Rats underwent 30 min coronary artery occlusion and 4 h reperfusion, or hearts explanted for immunoblotting without being subjected to ischemia. Area at risk (AR) was assessed by blue dye and IS by triphenyl-tetrazolium-chloride. RESULTS: Body weight and the size of AR were comparable among groups. There were no significant differences among groups in mean blood pressure and heart rate. CIL, but not ATV, reduced IS. IS in the ATV+CIL group was significantly smaller than the other three groups (P < 0.001 for each comparison). ATV, CIL and their combination did not affect total eNOS expression. ATV at 2 mg/kg/d did not affect Ser-1177 P-eNOS levels, whereas CIL increased it (258 +/- 15%). The level of myocardial P-eNOS levels was highest in the ATV+CIL group (406 +/- 7%). CONCLUSIONS: ATV and CIL have synergistic effect on eNOS phosphorylation and IS reduction. By increased activation of eNOS, CIL may augment the pleiotropic effects of statins.

The central role of adenosine in statin-induced ERK1/2, Akt, and eNOS phosphorylation.

Statins activate phosphatidylinositol-3-kinase, which activates ecto-5'-nucleotidase and phosphorylates 3-phosphoinositide-dependent kinase-1 (PDK-1). Phosphorylated (P-)PDK-1 phosphorylates Akt, which phosphorylates endothelial nitric oxide synthase (eNOS). We asked if the blockade of adenosine receptors (A(1), A(2A), A(2B), or A(3) receptors) could attenuate the induction of Akt and eNOS by atorvastatin (ATV) and whether ERK1/2 is involved in the ATV regulation of Akt and eNOS. In protocol 1, mice received intraperitoneal ATV, theophylline (TH), ATV + TH, or vehicle. In protocol 2, mice received intraperitoneal injections of ATV, U0126 (an ERK1/2 inhibitor), ATV + U0126, or vehicle; 8 h later, hearts were assessed by immunoblot analysis. In protocol 3, mice received intraperitoneal ATV alone or with 8-sulfophenyltheophylline (SPT); 1, 3, and 6 h after injection, hearts were assessed by immunoblot analysis. In protocol 4, mice received intraperitoneal ATV alone or with SPT, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC), alloxazine, or MRS-1523; 3 h after injection, hearts were assessed by immunoblot analysis. ATV increased P-ERK, P-PDK-1, Ser(473) P-Akt, Thr(308) P-Akt, and P-eNOS levels. TH blocked ATV-induced increases in P-ERK, Ser(473) P-Akt, Thr(308) P-Akt, and P-eNOS levels without affecting the induction of P-PDK-1 by ATV. U0126 blocked the ATV induction of Ser(473) P-Akt and Thr(308) P-Akt while attenuating the induction of P-eNOS. A detectable increase in P-ERK, Ser(473) P-Akt and P-eNOS was seen 3 and 6 h after injection but not at 1 h. DPCPX, CSC, and alloxazine partially blocked the ATV induction of P-ERK, Ser(473) P-Akt, and P-eNOS. In conclusion, blockade of adenosine A(1), A(2A), and A(2B) receptors but not A(3) receptors inhibited the induction of Akt and eNOS by statins. Adenosine was required for ERK1/2 activation by statins, which resulted in Akt and eNOS phosphorylation.