Biomedical subjects

Tadeusz Pawelczyk

Publications and source records attributed to Tadeusz Pawelczyk.

16 recordsLinked to original sources

PubMed2006

Expression of adenosine receptors in cardiac fibroblasts as a function of insulin and glucose level.

Adenosine among other factors is known to regulate the growth and function of cardiac fibroblasts (CFs). Its action is mediated by cell-surface receptors linked to a variety of signaling systems. The goal of present work was to examine the effects of glucose and insulin on adenosine receptors (ARs) mRNA and protein level in primary culture of rat CFs by means of real-time PCR and Western blot. Elevated glucose level increased the expression of A(1)-AR, A(2A)-AR, decreased the expression of A(3)-AR, and had no effect on A(2B)-AR expression. On the other hand insulin suppressed the expression of A(1)-AR, and A(2B)-AR, and had no effect on A(2A)-AR and A(3)-AR expression. Our measurements showed that accumulation of cAMP in response to ARs agonists correlated well with the changes in receptors expression level. These results indicate that changes in glucose and insulin level independently and differentially regulate the ARs expression and functional state in CFs.

Adenosine↗

PubMed2006

Reduced ability to release adenosine by diabetic rat cardiac fibroblasts due to altered expression of nucleoside transporters.

Adenosine produced by cardiac cells is known to attenuate the proliferation of cardiac fibroblasts (CFs), inhibit collagen synthesis, and protect the myocardium against ischaemic and reperfusion injury. Diabetic patients' hearts exhibit ventricular hypertrophy and demonstrate reduced tolerance to hypoxia or ischaemia. In this study, we characterize the effects of glucose and insulin on processes that determine the release of adenosine from CFs. We showed that during ATP depletion, rat CFs cultured in the absence of insulin release significantly less adenosine compared to cells grown in the presence of insulin. Moreover, under both conditions the quantity of released adenosine depends on glucose concentration. We demonstrate that this is due to altered expression of nucleoside transporters. High glucose (25 mm) induced 85% decrease in nucleoside transporter ENT1 mRNA levels. Decrease of the insulin level below 10(-11) m resulted in over 3-fold increase in the nucleoside transporter CNT2 mRNA content. Measurements of adenosine transport in CFs cultured in the presence of 5 mm glucose and 10 nm insulin showed that the bidirectional equilibrative adenosine transport accounted for 70% of the overall adenosine uptake. However, cells grown in the presence of high glucose (25 mm) demonstrated 65% decrease of the bidirectional equilibrative adenosine transport. Experiments on CFs cultured in the absence of insulin showed that the unidirectional Na(+)-dependent adenosine uptake rose in these cells more than 4-fold. These results indicate that the development of diabetes may result in an increased uptake of interstitial adenosine by CFs, and reduction of the ability of these cells to release adenosine during ATP deprivation.

Adenosine↗

PubMed2006

Cell cycle inhibitory protein p27 in human middle ear cholesteatoma.

AIM: To evaluate the immunohistochemical and molecular presentation of protein p27 in cholesteatoma. METHODS: 42 cholesteatoma samples and 6 external ear canal skin (EECS) specimens were investigated and analyzed taking into consideration congenital, acquired, recurrent cholesteatoma, and EECS. RESULTS: The expression of p27 was found in 16 (38.1%) out of 42 specimens of cholesteatoma and in 5 (83.3%) out of 6 specimens of EECS. There was a significant difference in p27-positive staining rate between EECS and cholesteatoma epithelium (p < 0.008). The presence of p27 was detected in 10 cases of acquired cholesteatoma, 2 cases of congenital and 3 cases of recurrent cholesteatoma. There was no significant difference between the presence of p27 in cholesteatoma and EECS (p = 0.01). CONCLUSION: The down-regulation of p27 is a key player in cell cycle control and plays an undefined role in the pathogenesis of all types of cholesteatoma.

Adolescent↗

PubMed2006

RS-alpha-lipoic acid protects cholinergic cells against sodium nitroprusside and amyloid-beta neurotoxicity through restoration of acetyl-CoA level.

The work presented here verifies the hypothesis that RS-alpha-lipoic acid may exert its cholinoprotective and cholinotrophic activities through the maintenance of appropriate levels of acetyl-CoA in mitochondrial and cytoplasmic compartments of cholinergic neurons. Sodium nitroprusside (SNP) and amyloid-beta decreased pyruvate dehydrogenase, choline acetyltransferase activities, acetyl-CoA content in mitochondria and cytoplasm, as well as increased fraction of non-viable, trypan blue positive cells in cultured differentiated cholinergic SN56 neuroblastoma cells. Lipoic acid totally reversed toxin-evoked suppression of choline acetyltrasferase and pyruvate dehydrogenase activities, as well as mitochondrial and cytoplasmic acetyl-CoA levels, and partially attenuated increase of cell mortality. Significant negative correlations were found between enzyme activities, acetyl-CoA levels and cell mortality in different neurotoxic and neuroprotective conditions employed here. The level of cytoplamic acetyl-CoA correlated with mitochondrial acetyl-CoA, whereas choline acetyltransferase activity followed shifts in cytoplasmic acetyl-CoA. Thus, we conclude that, in cholinergic neurons, particular elements of the pyruvate-acetyl-CoA-acetylcholine pathway form a functional unit responding uniformly to nerotoxic and neuroprotectory conditions.

Acetyl Coenzyme A↗

PubMed2006

Diabetes-induced decrease of adenosine kinase expression impairs the proliferation potential of diabetic rat T lymphocytes.

The proliferative response of T lymphocytes is a crucial step in cell-mediated immunity. This study was undertaken to investigate the mechanisms leading to the impaired proliferative response of diabetic T lymphocytes. T cells that had been isolated from the spleen of normal rats and cultured in medium containing 20 mm glucose and no insulin displayed the same degree of proliferative impairment as cells isolated from diabetic rats. The rate of T-cell proliferation, when induced with concanavalin A or anti-CD3 and anti-CD28 antibodies, was not affected by the inhibition of nucleoside transporters. T cells cultured at high glucose concentrations in the absence of insulin displayed decreased expression of adenosine kinase, and released measurable extracellular quantities of adenosine. Under resting conditions, the level of cAMP was 5.9-fold higher in these cells compared to cells grown in low glucose and in the presence of insulin. Experiments with specific adenosine receptor agonists and antagonists showed that adenosine-induced suppression of diabetic T cell proliferation was mediated by the A2A adenosine receptor, but not by the A2B receptor. Treatment of diabetic T cells with 10 microm H-89, a specific protein kinase A inhibitor, restored T-cell proliferation. These results show that suppressed proliferation of diabetic T lymphocytes is evoked by the decreased expression of adenosine kinase, leading to the outflow of adenosine from the cell. Extracellular adenosine then stimulates the A2A receptor and induces cAMP production, leading to the activation of protein kinase A, and suppression of T-cell proliferation.

Adenosine Kinase↗

PubMed2005

Differential effect of insulin and elevated glucose level on adenosine handling in rat T lymphocytes.

Reduced proliferation potential is among other T cell functional defects long known feature of diabetes. However, the mechanism responsible for this impairment is still unknown. Our study was undertaken to investigate the effect of changes in glucose and insulin concentrations on adenosine metabolism, transport and receptor-mediated action in rat T lymphocytes. Presented results indicate that vulnerability of T cells to metabolic stress is determined by insulin but not by glucose concentration. However, glucose and insulin differentially affected the activities of adenosine metabolizing enzymes in resting and proliferating T cells. The Con A-induced proliferation of cultured T lymphocytes did not depended on expression level and functional state of nucleoside transporters. Inhibition of adenosine kinase (AK) with 5-iodotubercidin lowers the proliferation potential of T cells to the level observed for insulin-deprived cells. Moreover, insulin-deprived T lymphocytes but not cells cultured in the presence of insulin released significant quantities of adenosine. Under resting conditions, the cAMP level was fivefold higher in cells deprived of insulin comparing to cells cultured in the presence of insulin. Exposition of insulin-deprived T lymphocytes to specific antagonist (ZM241385) of A2a receptor but not to specific antagonist (Alloxazine) of A2b receptor suppressed cAMP elevation and completely restored the proliferation potential of T cells. Concluding, adenosine released by insulin-deprived T cells due to suppressed AK activity by acting on A2a receptors leads to increases in cAMP level and suppression of T cell proliferation. We assume that this mechanism may significantly contribute to immune impairment observed in diabetes.

Adenosine↗

PubMed2005

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

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.

Animals↗

PubMed2005

Effect of L-carnitine on acetyl-CoA content and activity of blood platelets in healthy and diabetic persons.

BACKGROUND: Excessive blood platelet activity contributes to vascular complications in diabetic persons. Increased acetyl-CoA in platelets from diabetic persons has been suggested to be a cause of this hyperactivity. We therefore investigated whether L-carnitine, which up-regulates metabolism of acetyl-CoA in muscles and brain, may affect platelet function in healthy and diabetic individuals. METHODS: We obtained platelets from healthy and diabetic persons and measured acetyl-CoA concentrations, malonyl dialdehyde (MDA) synthesis, and platelet aggregation in the absence and presence of L-carnitine. Activities of selected enzymes involved in glucose and acetyl-CoA metabolism were also assessed. RESULTS: Fasting glucose, fructosamine, and hemoglobin A1c were present in significantly higher amounts in the blood of diabetic patients than in healthy individuals. Activities of carnitine acetyltransferase, glucose-6-phosphate dehydrogenase, oxoglutarate dehydrogenase, and fatty acid synthase were 17%-62% higher in platelets from diabetic patients. Mitochondrial acetyl-CoA was increased by 98% in platelets from diabetic patients, MDA synthesis was increased by 73%, and platelet aggregation by 60%. L-Carnitine had no or only a slight effect on these indices in platelets from healthy individuals, but in platelets from diabetic patients, L-carnitine caused a 99% increase in acetyl-CoA in the cytoplasmic compartment along with increases in MDA synthesis and platelet aggregation. CONCLUSIONS: Excessive platelet activity in persons with diabetes may result from increased acetyl-CoA, which apparently increases synthesis of lipid activators of platelet function. L-Carnitine may aggravate platelet hyperactivity in diabetic persons by increasing the provision of surplus acetyl-CoA to the cytoplasmic compartment.

Acetyl Coenzyme A↗

PubMed2005

Region-specific alterations of adenosine receptors expression level in kidney of diabetic rat.

Pathological alterations of renal function in insulin-dependent diabetes have been attributed to numerous factors, including adenosine. This study examined the expression levels of adenosine receptors (ARs) in the kidney of the streptozotocin-induced diabetic rat. In the diabetic kidney A1-AR mRNA levels increased 1.7- and 2.8-fold in cortex and medulla, respectively. This was accompanied by increased A1-AR protein levels in membranes of kidney cortex (1.5-fold) and medulla (threefold). A1-AR immunoreactivity increased strongly along medullar tubules especially in the collecting duct. The levels of A2a-AR mRNA increased twofold in diabetic kidney cortex but remained unchanged in medulla; however, A2a-AR protein levels increased more than threefold in cortex. Immunohistochemistry showed increased A2a-AR immunoreactivity in luminal membranes of cortical collecting ducts and in epithelial cells of preglomerular vessels. There were no significant changes in A2b-AR expression in diabetic kidney except in medullar membranes, where the receptor protein content decreased by 60%. A3-AR mRNA levels in diabetic kidney remained unchanged, but membrane-associated A3-AR protein levels increased by 70% in diabetic kidney cortex and decreased by 80% in medulla. These changes in ARs genes expression, receptor protein content, and cellular and tissue distribution, correspond to abnormalities characteristic of the diabetic kidney, suggesting involvement in pathogenesis of diabetic nephropathy.

Animals↗

PubMed2004

Differential effect of insulin and elevated glucose level on adenosine transport in rat B lymphocytes.

Impaired lymphocyte function is a common feature of human diabetes. Nucleoside transport across the plasma membrane is an essential step during lymphocyte growth and activation. In our study, we evaluated the impact of diabetic conditions on nucleoside transport system in B lymphocytes. Examination of the nucleoside transporters expression level in B lymphocytes isolated from diabetic rats revealed significant changes in their mRNA levels. Experiments performed on B cells cultured in medium containing defined concentrations of glucose and insulin showed that the rENT1 mRNA level was sensitive to extracellular glucose concentration and was not affected by insulin. Increase of glucose concentration from 5 to 20 mM caused a decrease of rENT1 mRNA by 80% and was associated with decreased adenosine uptake by the B cells. The effect of glucose was blocked by PD98059, a MAPK kinase inhibitor. The mRNA levels of rENT2 and rCNT2 were highly dependent on insulin but not on glucose concentration. Exposure of lymphocytes to 10 nM insulin resulted in a 2-fold increase in rENT2 mRNA and a 50% decrease in the rCNT2 mRNA level. Alterations in mRNA levels of rENT2 and rCNT2 were associated with changes in adenosine transport. Insulin-induced changes in expression level of rENT2 were blocked by wortmannin, an inhibitor of phosphatidylinositide 3-kinase, whereas the effect of insulin on rCNT2 was inhibited by PD98059 and to a lesser extend by wortmannin. In summary, impaired nucleoside transport in diabetic B lymphocytes results from alterations in the expression of nucleoside transporters, which are independently and differentially regulated by glucose and insulin.

Adenosine↗

PubMed2004

Insulin and glucose induced changes in expression level of nucleoside transporters and adenosine transport in rat T lymphocytes.

Adenosine is an endogenous agent exerting potent action on the immune system including regulation of lymphocyte functioning. Impaired T lymphocyte functioning is a common feature of diabetes. The aims of this study were to examine the effects of glucose and insulin on nucleoside transporters (NT) expression level and adenosine (Ado) transport in rat T lymphocytes cultured under the defined concentrations of glucose and insulin. Performed experiments revealed that rat T lymphocytes expressed the equilibrative nucleoside transporter type 1 and 2 (rENT1, rENT2) and concentrative nucleoside transporter type 2 (rCNT2). The mRNA levels of rENT2 and rCNT2 were highly dependent on insulin but were not affected by changes in extracellular glucose concentration. Exposition of T cells to 10nM insulin resulted in 73% increase in rENT2 mRNA and 50% decrease in the rCNT2 mRNA level. The level of rENT1 mRNA was sensitive to extracellular glucose concentration but not to insulin. The highest differences among cells cultured in high (20mM) and low (5mM) glucose were observed in equilibrative nitrobenzylthioinosine sensitive adenosine transport, which was lowered by 65% in cells cultured at high glucose. Alterations in adenosine transport were accompanied by changes in adenosine accumulation in the cell. These results indicate that adenosine transport in rat T lymphocytes is independently and differentially regulated by glucose and insulin by means of changes in the nucleoside transporters expression level. Altered adenosine transport has a great impact on its intracellular level. This suggests that under diabetic conditions adenosine action on T lymphocytes might be altered.

Adenosine↗

PubMed2004

Ubc9-induced inhibition of diadenosine triphosphate hydrolase activity of the putative tumor suppressor protein Fhit.

Fhit protein is the product of the putative tumor suppressor fragile histidine triad (FHIT) gene. The way by which Fhit exerts its antitumor activity remains largely unknown, although the Fhit-Ap3A complex is believed to be the native signaling form of Fhit. Here, we have shown that Fhit protein interacts with hUbc9, a recombinant human SUMO-1 conjugating enzyme, in an adenosine(5')triphospho(5')nucleoside (Ap3N)-dependent manner. Our experiments showed that the dinucleoside polyphosphate hydrolase activity of Fhit is suppressed by interacting with hUbc9 protein. In the presence of equimolar hUbc9 the Vmax and Km activity of Fhit was decreased by 35%. Analysis of Fhit kinetics in the presence of different fixed concentrations of Ubc9 showed that Ubc9 is an uncompetitive inhibitor. Including SUMO-1 protein in the assay neither affected the Fhit activity nor modified the effect of Ubc9 on Fhit kinetics. Our data suggest that hUbc9-induced inhibition of Fhit may result in an elongation of the Fhit-Ap3A signaling complex lifetime leading to alteration of its antitumor activity.

Acid Anhydride Hydrolases↗

PubMed2003

Insulin induces expression of adenosine kinase gene in rat lymphocytes by signaling through the mitogen-activated protein kinase pathway.

The activity of adenosine kinase (AK) was significantly impaired in splenocytes isolated from diabetic rats. Administration of insulin to diabetic animals restored AK activity, protein, and mRNA levels in diabetic splenocytes. Experiments performed on cultured rat lymphocytes demonstrated that insulin did not change the stability of AK mRNA. Insulin induced AK gene expression in a dose- and time-dependent manner. Maximal increases in AK mRNA (3.9-fold) and activity level (3.7-fold) were observed at the fourth and fifth hours of cell incubation with 10 nM insulin, respectively. The insulin effect on AK expression was not influenced by dibutyryl cAMP (dcAMP). On the other hand dcAMP weakly increased (1.7-fold) basal expression of AK. Exposure of rat lymphocytes to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), or rapamycin, an inhibitor of mTOR, did not affect the ability of insulin to stimulate expression of AK. Prior treatment of the cells with 10 microM PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase (MEK) completely blocked insulin-stimulated expression of AK gene. Insulin produced a significant transient increase in the tyrosine phosphorylation of ERK1/2, and PD98059 inhibited this phosphorylation. Furthermore exposure of cells to insulin has resulted in transient phosphorylation of Elk-1 on Ser-383 and sustained elevation of c-Jun and c-Fos protein. The maximal phosphorylation of Elk-1 was observed at 15 min, and was blocked by PD98059. We concluded that insulin stimulates AK gene expression through a series of events occurring sequentially. This includes activation of the MAPK cascade and subsequent phosphorylation of Elk-1 followed by increased expression of c-fos and c-jun genes.

Adenosine Kinase↗

PubMed2003

The effect of insulin on expression level of nucleoside transporters in diabetic rats.

Evidence that the time course of insulin-induced changes in adenosine level in diabetic rats is different from that observed for expression of adenosine kinase prompted us to study the insulin effect on expression of nucleoside transporters in tissues of diabetic rats. RNase protection assay demonstrated that mRNA levels of equilibrative (rENT) and Na+-dependent nucleoside transporters (rCNT) were altered in diabetic tissues. The rENT1 mRNA level with respect to values obtained in age- and sex-matched nondiabetic rats was decreased by 45, 32, and 10% in diabetic heart, liver, and kidney, respectively. The level of rENT2 mRNA was lowered by 40% in diabetic kidney and heart, and by 24% in diabetic liver. Changes in the expression pattern of rCNT1 and rCNT2 in diabetic tissues differed significantly from that observed for rENT. The levels of rCNT1 and rCNT2 mRNA did not change significantly in diabetic kidney. In diabetic heart, the mRNA levels of rCNT1 and rCNT2 increased 1.7- and 2-fold, respectively. Changes in expression of nucleoside transporters were accompanied by alterations in adenosine content. Administration of insulin to diabetic rats resulted in a drop in adenosine concentration in examined tissues and return of the rCNT1, rCNT2, and rENT2 but not rENT1 mRNA levels to values observed in nondiabetic rats. In summary, these data demonstrate that insulin affects expression of nucleoside transporters in a cell-specific manner. We conclude that change in the expression level of the nucleoside transporters occurring in tissues of diabetic rat is an important factor influencing adenosine levels in the cell.

Adenosine↗

PubMed2003

Isozymes delta of phosphoinositide-specific phospholipase C and their role in signal transduction in the cell.

Phospholipase C (PLC, EC 3.1.4.11) is an enzyme crucial for the phosphoinositol pathway and whose activity is involved in eukaryotic signal transduction as it generates two second messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). There are four major types of phospholipase C named: beta, gamma, delta and the recently discovered epsilon, but this review will focus only on the recent advances for the delta isozymes of PLC. So far, four delta isozymes (named delta1-4) have been discovered and examined. They differ with regard to cellular distribution, activities, biochemical features and involvement in human ailments.

Active Transport, Cell Nucleus↗

PubMed2002

Insulin restores expression of adenosine kinase in streptozotocin-induced diabetes mellitus rats.

The activity of adenosine kinase is significantly impaired in tissues of diabetic rat. Changes in the activity of adenosine kinase were accompanied by alterations in its mRNA and protein level. These changes depended on insulin level and were not related to glucose level. During the first 7 h after insulin treatment the level of adenosine kinase mRNA, protein and enzymatic activity in kidneys, liver and heart returned to normal values. The observed relation between insulin and adenosine kinase expression level may suggest that insulin increases the rate of adenosine kinase gene transcription. Decreased activity of adenosine kinase was associated with elevated level of adenosine in diabetic tissues. On the 10th day after the STZ treatment there was a 3.5 and 2-fold increase in adenosine content in heart and liver, respectively. On the other hand, in diabetic kidney adenosine level was elevated only by 20%. Administration of insulin to diabetic rats resulted in a drop of adenosine to the level seen in normal heart and liver whereas, in kidneys the adenosine content was 50% lower than that observed under normal conditions. The time-dependent coarse of changes in adenosine level was different from that observed for adenosine kinase activity, what may suggest that other factors, possibly nucleoside transporters are also important for controlling adenosine level in the cell.

Adenosine↗