Michał Komoszyński

Adenosine A2A receptors in Parkinson’s disease treatment

Latest results on the action of adenosine A2A receptor antagonists indicate their potential therapeutic usefulness in the treatment of Parkinson’s disease. Basal ganglia possess high levels of adenosine A2A receptors, mainly on the external surfaces of neurons located at the indirect tracts between the striatum, globus pallidus, and substantia nigra. Experiments with animal models of Parkinson’s disease indicate that adenosine A2A receptors are strongly involved in the regulation of the central nervous system. Co-localization of adenosine A2A and dopaminergic D2 receptors in striatum creates a milieu for antagonistic interaction between adenosine and dopamine. The experimental data prove that the best improvement of mobility in patients with Parkinson’s disease could be achieved with simultaneous activation of dopaminergic D2 receptors and inhibition of adenosine A2A receptors. In animal models of Parkinson’s disease, the use of selective antagonists of adenosine A2A receptors, such as istradefylline, led to the reversibility of movement dysfunction. These compounds might improve mobility during both monotherapy and co-administration with L-DOPA and dopamine receptor agonists. The use of adenosine A2A receptor antagonists in combination therapy enables the reduction of the L-DOPA doses, as well as a reduction of side effects. In combination therapy, the adenosine A2A receptor antagonists might be used in both moderate and advanced stages of Parkinson’s disease. The long-lasting administration of adenosine A2A receptor antagonists does not decrease the patient response and does not cause side effects typical of L-DOPA therapy. It was demonstrated in various animal models that inhibition of adenosine A2A receptors not only decreases the movement disturbance, but also reveals a neuroprotective activity, which might impede or stop the progression of the disease. Recently, clinical trials were completed on the use of istradefylline (KW-6002), an inhibitor of adenosine A2A receptors, as an anti-Parkinson drug.

Emerging role of extracellular nucleotides and adenosine in multiple sclerosis

Extracellular nucleotides and adenosine play important roles in inflammation. These signaling molecules interact with the cell-surface-located P2 and P1 receptors, respectively, that are widely distributed in the central nervous system and generally exert opposite effects on immune responses. Indeed, extracellular ATP, ADP, UTP, and UDP serve as alarmins or damage-associated molecular patterns that activate mainly proinflammatory mechanisms, whereas adenosine has potent anti-inflammatory and immunosuppressive effects. This review discusses the actual and potential role of extracellular nucleotides and adenosine in multiple sclerosis (MS).

Extracellular purine metabolism in blood vessels (Part II): Activity of ecto-enzymes in blood vessels of patients with abdominal aortic aneurysm.

Both platelet aggregation and high blood pressure are associated with development of atherosclerosis. Among other factors that modulate platelet aggregation and blood pressure, extracellular purines (e-purines) influence these processes via purinoceptors P1 and P2 for which they are natural ligands. We hypothesized that ecto-enzymes such as nucleoside triphosphate diphosphohydrolases (NTPDases), adenylate kinase, 5′-nucleotidase, and adenosine deaminase that regulate the level of e-purines may be involved in the development of atherosclerosis. The enzymatic assays were performed either on the fragments of human abdominal aortas obtained after death or on abdominal aneurysm samples collected during surgery. The substrates and products such as adenine nucleosides and nucleotides were analyzed using reverse phase high-performance liquid chromatography (HPLC) method. Here, we estimated and demonstrated the activities of these ecto-enzymes in the patients with atherosclerosis or atherosclerosis-like diseases such as abdominal aneurysm, myocardial infarction, or Leriche syndrome (LS) with worse thrombosis of extremities. In particular, we noticed reduction in activity of NTPDase1app, NTPDase2app, ecto-adenylate kinase app, and ecto-adenosine deaminaseapp; however, ecto-5 ′-nucleotidaseapp that hydrolyzed e-adenosine monophosphate (e-AMP) into e-adenosine did not show any significant changes. This led us to suggest that alteration of the activity of examined ecto-enzymes is responsible for the development of atherosclerosis or atherosclerosis-like diseases.

The role of purinergic signaling in the etiology of migraine and novel antimigraine treatment

Etiopathogenesis of migraine involves different structures of the central nervous system: the trigeminal nerve with nuclei located in the brain stem, vascular system, and the cerebral cortex as well as diverse mechanisms and pathological processes. The multidirectional action of purines in different cell types (blood vessels, neurons, and satellite glial cells) and through different types of purinergic receptors contributes to the etiopathogenesis of migraine pain. Adenosine triphosphate (ATP) and its derivatives are involved in initiation and propagation of migrenogenic signals in several ways: they participate in vasomotor mechanism, cortical spreading depression, and in fast transmission or cross-excitation based on the satellite glial cells in trigeminal ganglion. Contribution of purinergic signaling in the conduction of pain is realized through the activation of P1 and P2 receptors expressed widely in the central nervous system: on the neurons and glial cells as well as on the smooth muscles and endothelium in the vascular system. Therefore, the purinergic receptors can be an excellent target for pharmacologists constructing new antimigraine therapeutics. Moreover, the mechanisms facilitating ATP and adenosine degradation may prevent vasodilatation and thus avoid a secondary central sensitization during a migraine attack. Thus, agonists and antagonists of P receptors as well as ecto-enzymes metabolizing nucleotides/nucleosides could gain the growing attention as therapeutic agents.

Subcellular and surface localization of the membrane-bound apyrase (atp diphosphohydrolase ec 3.6.1.5) from wheat seedlings

Abstract It has been demonstrated by differential centrifugation methods and separation of membranes on a sucrose gradient that wheat membrane apyrase is bound

Purification and properties of adenosinetriphosphatase from Zea mays seedling microsomes

Abstract A simple method was developed for selective solubilization of membrane ATPase from etiolated corn seedlings using 0.01% Triton X100 and 0.01% deoxycholate containing 200 mM KI. An 81-fold enriched enzyme preparation, with specific activity of 133 μmol Pi/mg protein/hr, was obtained. The enzyme stored in 25 mM Tris-HCl buffer (pH 7.5) at 4° showed rapid loss of activity. The enzyme was stabilized by 1 mM EDTA with addition of 1.2 mM Mg 2+ °. Mg 2+ and Ca 2+ (1.2 mM) increased enzymatic activity by 12 and 10.8% respectively, whereas Na + and K + brought about a 20% increase in ATP-hydrolysis. The effect of combined mono- and di-valent ions was neither synergistic nor additive. Ouabain exerted no effect on enzyme activity. The enzyme showed two pH optima (6.0 and 7.5) in the presence of Na + and K + , and one optimum at pH 6.5 in the absence of these ions. On polyacrylamide gel the enzyme was resolved into two protein bands, both exhibiting ATPase activity. It is suggested that the soluble enzyme from the microsomal fraction of corn seedlings contains two ATP-hydrolyzing enzymes, one of them being stimulated by Na + and K + ions.

Rola ektopuryn w procesie od zapalenia do demielinizacji – perspektywy powstania nowych metod leczenia stwardnienia rozsianego

Nucleotides released from activated and/or injured cells activate P2 receptors. Extracellular nucleotides serve as danger signals or damage-associated molecular patterns (DAMPs) that trigger various immune responses. Indeed, P2 receptors are highly expressed in the astrocytes, microglia and other immune cells such as T and B lymphocytes that migrate to the central nervous system. The activation of P2 receptors triggers the secretion of proinflammatory cytokines and chemokines as well as immune cell migration and proliferation that contribute to demyelination and axonal damage. The activation of P2 receptors is controlled by the ectonucleotidases which hydrolyze extracellular nucleotides. Ecto-NTPDases and ecto-5′-nucleotidase are expressed in the astrocytes, oligodendrocytes, microglia, endothelial cells and activated T cells. The hydrolysis of extracellular ATP and ADP by enzymes results in the generation of extracellular adenosine. This nucleoside interacts with P1 receptors and activates anti-inflammatory and immuno-suppressive responses in the cells involved in MS.

Isolation and bioinformatic analysis of seven genes encoding potato apyrase. Bacterial overexpresssion, refolding and initial kinetic studies on some recombinant potato apyrases.

Here we have isolated seven apyrase encoding cDNA sequences (StAPY4-StAPY10) from the potato variety Saturna tuber cDNA library by affecting necessary modifications in the screening protocol. The cDNA sequences were identified with a pair of primers complementary to the most conserved sequences identified in potato variety Desiree apyrase genes. Our data strongly suggest the multigenic nature of potato apyrase. All deduced amino acid sequences contain a putative signal sequence, one transmembrane region at the amino terminus and five apyrase conserved regions (ACRs) (except StAPY6). Phylogenetic analysis revealed that encoded proteins shared high level of DNA sequence identity among themselves, representing a family of proteins markedly distinct from other eukaryotic as well as prokaryotic apyrases. Two cDNA sequences (StAPY4 and StAPY6) were overexpressed in bacteria and recombinant proteins were found accumulated in inclusion bodies, even thought they were fused with thioredoxin-tag. Additionally, we present the first successful in vitro attempt at reactivation and purification of recombinant potato apyrase StAPY6. The ratio of ATPase/ADPase hydrolysis of recombinant StAPY6 was determined as 1.5:1. Unlike other apyrases the enzyme lacked ACR5 and was endowed with lower molecular weight, high specificity for purine nucleotides and very low specificity for pyrimidine, suggesting that StAPY6 is a potato apyrase, not described so far.

In vitro renaturation of proteins from inclusion bodies

Recombinant proteins and enzymes are commonly used in many areas of our life, such as diagnostics, industry and medicine, due to heterologous synthesis in prokaryotic expression systems. However, a high expression level of foreign protein in bacteria cells results in formation of inactive and insoluble aggregates--inclusion bodies. Reactivation of aggregated proteins is a complex and time-consuming process. Every protein requires experimental optimization of the process conditions. The choice of the refolding method depends on the type of recombinant protein and its physical, chemical and biological properties. Recovery of the activity of proteins accumulated in inclusion bodies can be divided into 4 steps: 1) inclusion bodies isolation, 2) solubilization of aggregates, 3) renaturation, 4) purification of catalytically active molecules. Efficiency of the refolding process depends on many physical factors and chemical and biological agents. The above parameters determine the time of the folding and prevent protein aggregation. They also assist the folding and have an influence on the solubility and stability of native molecules. To date, dilution, dialysis and chromatography are the most often used methods for protein refolding.

Extracellular-Purine Metabolism in Blood Vessels (Part I). Extracellular-Purine Level in Blood of Patients with Abdominal Aortic Aneurysm

Adenosine and adenosine derivatives are the main regulators of purinoceptors (P1 and P2) mediated hemostasis and blood pressure. Since impaired hemostasis and high blood pressure lead to atherosclerosis and to the development of aneurysm, in this study we tested and compared the concentration of extracellular purines (e-purines) in the blood in of patients having abdominal aortic aneurysm with that from healthy volunteers. Whereas adenine nucleosides and nucleotides level in human blood plasma was analysed using reverse phase high performance liquid chromatography (HPLC), cholesterol concentration was estimated by an enzymatic assay. We did not find any correlation between e-purines concentration and the age of healthy volunteers. Furthermore, the sum level of e-purines (ATP, ADP, AMP, adenosine, and inosine) in the control group did not exceed 70 μM, while it was nearly two-fold higher in the blood of patients having abdominal aortic aneurysm, (123 μM). In a special case of people with Leriche Syndrome, a disease characterized by deep atherosclerotic changes, the e-purines level had further increased. Additionally, we also report typical atherosclerotic changes in the aorta using histological assays as well as total cholesterol rise. The significant rise in cholesterol concentration in the blood of the patients with abdominal aortas aneurysm, compared with the control groups, was not unique since 23% of the healthy people also exceeded the normal level of cholesterol. Therefore, our results strongly indicate that the estimation of e-purines concentration in the blood may serve as another indicator of atherosclerosis and warrant further consideration as a futuristic diagnostic tool.