Molecular and Cellular Biochemistry

Protection of the ex vivo rat heart from ischemia/reperfusion injury can be provided by ischemic preconditioning (IPC). Previous studies revealed that a complex of pannexin-1 with the P2X7 receptor forms a channel during IPC that results in the release of cardioprotectants such as adenosine and sphingosine 1-phosphate (S1P) that bind to G-protein-coupled cell surface receptors triggering cardioprotective cell signaling pathways. Antagonists of both pannexin-1 (carbenoxolone and mefloquine) and P2X7 receptors (brilliant blue G) are known to block IPC when administered at the time of preconditioning (Vessey et al. J Cardiovasc Pharmacol Ther 15:190, 2010). We now demonstrate that these same antagonists also block the cardioprotective effects of IPC when added after the index ischemia during full reperfusion. Likewise, addition at full reperfusion of binding antagonists to the endogenous cardioprotectants S1P (VPC) or adenosine (8-SPT) reduced the effectiveness of IPC. These data suggest that IPC has a component that requires the release of cardioprotectants via pannexin-1/P2X7 channels not only during preconditioning phase but again during the early stages of reperfusion following the index ischemia. It was found that the level of cardioprotectant release required at reperfusion to achieve cardioprotection was lower when hearts had been preconditioned. Further, pharmacologic preconditioning with S1P or adenosine was also blocked at reperfusion by antagonists of the pannexin-1/P2X7 channels indicating that pharmacologic preconditioning also requires opening of the channel at full reperfusion. In untreated hearts, key components of the PI3 kinase/Akt signaling pathway were revealed by western analysis to be lost during ischemia. This correlates with an inability to generate phospho-Akt at reperfusion. IPC prevents this loss and thereby primes the cell for response to cardioprotectants released at full reperfusion.

Oxidative metabolism of blood-borne fuels provides myocardium the energy required to sustain its contractile performance. Recent research has revealed that, in addition to supplying energy, certain fuels are able to detoxify harmful oxidants and bolster the myocardium's endogenous antioxidant defenses. These antioxidant capabilities could potentially protect the myocardium from the ravages of reactive oxygen and nitrogen intermediates generated upon reperfusion of ischemic myocardium. This article reviews experimental evidence that two fuels, pyruvate and acetoacetate, provide such antioxidant protection. Pyruvate's antioxidant properties stem in part from its α-keto carboxylate structure, which enables it to directly, non-enzymatically neutralize peroxides and peroxynitrite. Also, citrate, which accumulates in pyruvate-perfused myocardium following anaplerotic pyruvate carboxylation, supports NADPH production to maintain glutathione:glutathione disulfide (GSH/GSSG) redox potential, the central component of the myocardial antioxidant system. Like pyruvate, acetoacetate restores GSH/GSSG and increases contractile function of post-ischemic stunned myocardium, although some of its antioxidant mechanisms may differ from pyruvate's. Both compounds restore β-adrenergic signaling and inotropism, which are compromised in stunned myocardium. N-acetylcysteine, a pharmacological antioxidant that does not provide energy, duplicated the salutary effects of pyruvate and acetoacetate on post-ischemic β-adrenergic signaling and GSH/GSSG. These findings reveal novel, energy-independent mechanisms for enhancement of post-ischemic cardiac performance by metabolic fuels.

As exposure to polycyclic aromatic hydrocarbons (PAHs; a family of environmental toxicants) have been implicated in cardiovascular diseases, the ability of the aortic tissue to process these toxicants is important from the standpoint of abdominal aortic aneurysms and atherosclerosis. Benzo(a)pyrene (B(a)P), a representative PAH compound is released into the environment from automobile exhausts, industrial emissions, and considerable intake of B(a)P is also expected in people who are smokers and barbecued red meat eaters. Therefore, knowledge of B(a)P metabolism in the cardiovascular system will be of importance in the management of vascular disorders. Toward this end, subcellular fractions (nuclear, cytosolic, mitochondrial, and microsomal) were isolated from the aortic tissues of Apo E mice that received a 5 mg/kg/week of B(a)P for 42 days and 0.71 mg/kg/day for 60 days. The fractions were incubated with 1 and 3 μM B(a)P. Post incubation, samples were extracted with ethyl acetate and analyzed by reverse-phase HPLC. Microsomal B(a)P metabolism was greater than the rest of the fractions. The B(a)P metabolite levels generated by all the subcellular fractions showed a B(a)P exposure concentration-dependent increase for both the weekly and daily B(a)P treatment categories. The preponderance of B(a)P metabolites such as 7,8-dihydrodiol, 3,6-, and 6,12-dione metabolites are interesting due to their reported involvement in B(a)P-induced toxicity through oxidative stress.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily and appear to have beneficial effects in the cardiovascular system. PPARβ/δ has been shown previously to exert an inhibitory effect on cardiac myocyte hypertrophy in vivo and in vitro although the exact mechanism is not fully clear yet. The principal signaling pathways that have been involved in triggering cardiac hypertrophic response are mitogen-activated protein kinases (MAPKs) and PI3K/Akt cascades. In this study, we sought to evaluate the potential effects evoked by PPARβ/δ activation on signaling pathways that are implicated in cardiac myocyte growth responses. The selective PPARβ/δ agonist GW0742 attenuated ERK1/2 and Akt phosphorylation that was stimulated by growth promoting agonists (phenylephrine, insulin or IGF-1). This effect was not reversed by the specific PPARβ/δ antagonist, GSK0660, but was inhibited by vanadate, a potent protein tyrosine phosphatase inhibitor. In addition, GW0742 prevented the oxidation and inactivation of PTEN supporting further the notion that its inhibitory action on the agonist-induced kinase phosphorylation is mediated by the modulation of phosphatase activity. Furthermore, GW0742 abolished the agonist-induced intracellular generation of reactive oxygen species, independently of PPARβ/δ activation. Our data reveals a new non-genomic mechanism of GW0742, which ameliorates the generation of reactive oxygen species and attenuates ERK1/2 and PI3K/Akt signaling, with implications in the regulation of cardiac hypertrophic response.

The backbone dynamics of bovine heart fatty acid binding protein (H-FABP) and porcine ileal lipid binding protein (ILBP) were studied by 15N NMR relaxation (T1 and T2) and steady state heteronuclear 15N{1H} NOE measurements. The microdynamic parameters characterizing the backbone mobility were determined using the ‘model-free’ approach. For H-FABP, the non-terminal backbone amide groups display a rather compact protein structure of low flexibility. In contrast, for ILBP an increased number of backbone amide groups display unusually high internal mobility. Furthermore, the data indicate a higher degree of conformational exchange processes in the μsec-msec time range for ILBP compared to H-FABP. These results suggest significant differences in the conformational stability for these two structurally highly homologous members of the fatty acid binding protein family.

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl peptidase type-1 (PAP1). The cloned nucleotide sequence has an ORF coding for a primary sequence of 209 amino acid residues, which displays 98% identity with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary between the two sequences. The recombinant bovine PAP1 with a C-terminal His6 tag (rBtaPAP16H) was expressed in Escherichia coli XL10-Gold cells and purified by immobilised nickel ion affinity chromatography resulting in a yield of 2.6 mg of PAP1 per litre of culture. Purified rBtaPAP16H had a specific activity of 3633 units mg−1. SDS-PAGE revealed a band for bovine PAP1 with a molecular weight of ∼24 kDa, which is in good agreement with previously reported data on PAP1. The K m and k cat values obtained for rBtaPAP16H were 59 μM and 3.5 s−1, respectively. The optimum pH for activity was 9.0–9.5 and the optimum temperature was 37 °C. rBtaPAP16H was found to have an absolute requirement for the thiol-reducing agent DTT, consistent with the expected property of a cysteine protease. Kinetic studies using the peptides pGlu-His-Pro-NH2 (TRH), pGlu-Ala and pGlu-Val revealed K i values of 44.1, 141 and 652.17 μM, respectively. The lowest K i, observed for Thyrotropin-releasing Hormone (TRH), indicates that rBtaPAP16H has a higher affinity for tripeptides over dipeptides.

In plants, protein kinase CK2 is involved in different processes that control many aspects of metabolism and development. In mammals and yeast the enzyme is a heterotretamer composed of two types of subunits. During years the subunit composition of the maize protein kinase CK2 enzyme has been a source of controversy. We have recently characterized the maize holoenzyme subunits. Our results show that multiple catalytic and regulatory subunits are expressed in maize and are able to specifically interact with other α and β subunits suggesting a high level of heterogeneity in the typical heterotetrameric structure. Here, we summarize data available on plant CK2 enzymes, in order to clarify the distinctive features and functions of plant protein kinase CK2.