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1

Recent advances in studies on biochemical and structural properties of equilibrative and concentrative nucleoside transporters

100%
Podgorska M., Kocbuch K., Pawelczyk T.
Acta Biochimica Polonica
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2005
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tom 52
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nr 4
Nucleoside transporters (NT) facilitate the movement of nucleosides and nucleobases across cell membranes. NT-mediated transport is vital for the synthesis of nucleic acids in cells that lack de novo purine synthesis. Some nucleosides display biological activity and act as signalling molecules. For example, adenosine exerts a potent action on many physiological processes including vasodilatation, hormone and neurotransmitter release, platelet aggregation, and lipolysis. Therefore, carrier-mediated transport of this nucleoside plays an important role in modulating cell function, because the efficiency of the transport processes determines adenosine availability to its receptors or to metabolizing enzymes. Nucleoside transporters are also key elements in anticancer and antiviral therapy with the use of nucleoside analogues. Mammalian cells possess two major nucleoside transporter families: equilibrative (ENT) and concentrative (CNT) Na+-dependent ones. This review characterizes gene loci, substrate specificity, tissue distribution, membrane topology and structure of ENT and CNT proteins. Regulation of nucleoside transporters by various factors is also presented.
2
Content available remote

Protein kinase C mediated high glucose effect on adenosine receptors expression in rat B lymphocytes

88%
Sakowicz-Burkiewicz M., Kocbuch K., Grden M., Szutowicz A., Pawelczyk T.
Journal of Physiology and Pharmacology
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2009
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tom 60
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nr 3
145-153
Hyperglycemia-induced alterations of adenosine receptors (ARs) expression are implicated in the pathomechanism leading to impaired function of the lymphocytes in diabetes. However, the signaling pathways utilized by glucose to regulate ARs expression are unknown. This work was undertaken to investigate the impact of high glucose level on the ARs expression in rat B lymphocytes. The results presented in this report demonstrate that rat B lymphocytes express all four types of ARs at the mRNA and protein level. Exposing B cells to high glucose (25 mM) suppressed the expression of A1-AR, A2B-AR, and A3-AR, but had no effect on the expression of A2A-AR. A selective inhibitor of Ca2+-dependent protein kinase C (PKC) isoforms suppressed the high glucose effect on A3-AR expression. Inhibition of PKC- with rottlerin blocked the high glucose effect on A1-AR mRNA level. An inhibitor of Raf-1 kinase completely blocked the high glucose effect on A2B-AR expression. The suppression of A1-AR and A2B-AR mRNA expression induced by high glucose was blocked by an inhibitor (PD98059) of MAPK kinase (MEK). In conclusion, high glucose utilizes a signaling pathway involving some elements of the MAPK pathway and different PKC isoforms to suppress the expression of A1-AR, A2B-AR, and A3-AR in rat B lymphocytes.
3
Content available remote

Adenosine 5'-triphosphate is the predominant source of peripheral adenosine in human B lymphoblasts

88%
Sakowicz-Burkiewicz M., Kocbuch K., Grden M., Szutowicz A., Pawelczyk T.
Journal of Physiology and Pharmacology
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2010
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tom 61
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nr 4
491-499
Adenosine 5'-triphosphate (ATP) and adenosine are the crucial endogenous signaling molecules in immunity and inflammation. In this study we identified the source of extracellular adenosine in human B lymphoblasts, and evaluate the ATP release and metabolism. We observed that the B cells continuously released substantial quantities of ATP (35 pmol/106 cells) when subjected to slow motion in the incubation medium. The adenosine level in the B cell incubation medium was very low, and increased (5-fold) upon inhibition of adenosine deaminase activity with 10 µM of 2-deoxycoformycin (DCF). Inclusion of an inhibitor of equilibrate nucleoside transport (nitrobenzylthioinosine) in the incubation medium in the presence of DCF resulted in the elevation of adenosine level by 9-fold. Inhibition of ecto-ATPase activity with 100 µM of ARL67156 was associated with a 2-fold increase of the extracellular ATP level and a 3-fold decrease of adenosine concentration in the cell culture media. Inclusion of ,ß-methyleneadenosine 5'-diphosphate, a selective inhibitor of ecto-5'-nucleotidase in the incubation medium resulted in a significant decrease (7-fold) the adenosine concentration. In conclusion, our results indicate that ATP released from the B cell is the primary source of peripheral adenosine, and that the activities of ecto enzymes and the efficiency of Ado uptake through the nucleoside transporters determine the Ado level on the B cell surface.
4

Effect of insulin and glucose on adenosine metabolizing enzymes in human B lymphocytes

88%
Kocbuch K., Sakowicz-Burkiewicz M., Grden M., Szutowicz A., Pawelczyk T.
Acta Biochimica Polonica
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2009
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tom 56
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nr 3
439-446
In diabetes several aspects of immunity are altered, including the immunomodulatory action of adenosine. Our study was undertaken to investigate the effect of different glucose and insulin concentrations on activities of adenosine metabolizing enzymes in human B lymphocytes line SKW 6.4. The activity of adenosine deaminase in the cytosolic fraction was very low and was not affected by different glucose concentration, but in the membrane fraction of cells cultured with 25 mM glucose it was decreased by about 35% comparing to the activity in cells maintained in 5 mM glucose, irrespective of insulin concentration. The activities of 5'-nucleotidase (5'-NT) and ecto-5'-NT in SKW 6.4 cells depended on insulin concentration, but not on glucose. Cells cultured with 10-8 M insulin displayed an about 60% lower activity of cytosolic 5'-NT comparing to cells maintained at 10-11 M insulin. The activity of ecto-5'-NT was decreased by about 70% in cells cultured with 10-8 M insulin comparing to cells grown in 10-11 M insulin. Neither insulin nor glucose had an effect on adenosine kinase (AK) activity in SKW 6.4 cells or in human B cells isolated from peripheral blood. The extracellular level of adenosine and inosine during accelerated catabolism of cellular ATP depended on glucose, but not on insulin concentration. Concluding, our study demonstrates that glucose and insulin differentially affect the activities of adenosine metabolizing enzymes in human B lymphocytes, but changes in those activities do not correlate with the adenosine level in cell media during accelerated ATP catabolism, implying that nucleoside transport is the primary factor determining the extracellular level of adenosine.
5

Analysis of OPN gene expression in the colorectal cancer

76%
Lubkowska M., Szulc A., Kocbuch K., Strzelczyk B., Kruszewski W., Pawelczyk T.
Acta Biochimica Polonica
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2006
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tom 53
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nr Supplement 1
6

Zinc as early neurotoxic signal in cholinergic SN56 neuroblastoma cells

76%
Dys A., Ronowska A., Gul-Hinc A. S., Klimaszewska-Lata J., Kocbuch K., Szutowicz A., Bielarczyk H.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
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nr 1
Preferential loss of septal cholinergic neurons is a main cause of cognitive deficits in various encephalopathies. Zinc excess is one of multiple pathologic signals contributing to mechanisms of Alzheimer’s and other neurodegenerative diseases. We suggest that zinc may be involved in early excitotoxic phase of neuronal injury. In homogenates of SN56 cholinergic neuroblastoma cells, Zn caused instant inhibition of pyruvate dehydrogenase (PDH), aconitase, isocitrate dehydrogenase (IDH) and ketoglutarate dehydrogenase (KDH) activities with Ki values equal to 0.08, 0.008, 0.005 and 0.005 mM, respectively. Short term, 30 minute exposition to Zn caused a concentration dependent increase in mortality of cAMP/retinoic acid differentiated SN56 cholinergic cells (DC) that was two times higher than that of differentiated ones (NC). Zn also decreased cytoplasmic acetyl-CoA as well as ACh content and inhibited its release. Exposition of DC and NC to increasing concentrations of Zn yielded concave up non saturable accumulation plots that reached level of 60 nmol/mg protein at 0,15 mM extracellular concentration of a cation. In these conditions no change in whole cell Ca level was observed. However the level of intramitochondrial Ca was decreased by 30%, at 100 % increase of cytoplasmic Ca. Significant, direct correlation between Zn accumulation and cytoplasmic Ca concentration (r=0.97, p=0.028) and the inverse one with mitochondrial Ca (r=- 0.96, p=0.028) were found, respectively. On the other hand, 24 h cell exposition to 0,15 mM Zn increased its intracellular content from 1.4 to about 6 nmol/mg protein at simultaneous 40% decrease of whole cell Ca level. Zn caused no significant changes in the density of ZnT1 and ZnT4 transporter proteins in the cells. Presented data indicate the coexistence in SN56 cell plasma membranes low density - high-affinity and high density - low affinity Zn-transporting sites. Inhibition of mitochondrial Na-Ca exchanger by accumulated Zn might cause depletion of Ca in mitochondria. In addition chronic exposition to Zn apparently induced adaptative mechanisms eliminating excess of the metal from the cells. These changes may directly inhibit intramitochondrial acetyl-CoA synthesis and its transport to cytoplasmic compartment, yielding impairment of cell viability and suppression their transmitter functions. Supported by MWiSW NN401 0299 37, St57 and W-109 MUG projects.
7
Content available remote

Impact of adenosine receptors on immunoglobulin production by human peripheral blood B lymphocytes

76%
Sakowicz-Burkiewicz M., Kocbuch K., Grden M., Maciejewska I., Szutowicz A., Pawelczyk T.
Journal of Physiology and Pharmacology
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2012
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tom 63
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nr 6
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