Tadeusz Pawelczyk

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.

Amplification of c-myc gene and overexpression of c-Myc protein in breast cancer and adjacent non-neoplastic tissue.

BACKGROUND Deregulated c-Myc expression and alterations of c-myc oncogene have been reported to play an important role in breast cancer tumorigenesis. We examined the relationship between c-Myc protein level, amplification of c-myc oncogene and commonly used clinical and pathologic factors. METHODS The studies were conducted on 94 ductal and lobular cancers. Amplification of c-Myc was assessed by the semiquantitative multiplex PCR assay. The amount of c-Myc protein was estimated by the densitometry analysis of Western blots. RESULTS Amplification of c-Myc was found in 21% of examined cancers. There was no association of c-myc amplification with established risk factors. Overexpression of c-Myc protein without c-myc amplification was associated with negative status of axillary lymph node. The size of lobular carcinoma displaying overexpression of c-Myc and the normal copy number of c-myc gene was significantly smaller than the size of tumor with elevated c-Myc and amplification of c-myc gene (p < 0.01). Within tumors displaying overexpression of c-Myc protein and c-myc gene amplification the size of ductal carcinoma was smaller than the size of lobular carcinoma (p < 0.007). CONCLUSION Data presented in this study suggest that alterations of c-myc gene and c-Myc protein level might be related to breast cancer progression. The prognostic utility of elevated level of c-Myc protein associated with normal status of c-myc gene for patients with lobular carcinoma requires further studies.

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 μm 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.

Acetyl-CoA the key factor for survival or death of cholinergic neurons in course of neurodegenerative diseases.

Glucose-derived pyruvate is a principal source of acetyl-CoA in all brain cells, through pyruvate dehydogenase complex (PDHC) reaction. Cholinergic neurons like neurons of other transmitter systems and glial cells, utilize acetyl-CoA for energy production in mitochondria and diverse synthetic pathways in their extramitochondrial compartments. However, cholinergic neurons require additional amounts of acetyl-CoA for acetylcholine synthesis in their cytoplasmic compartment to maintain their transmitter functions. Characteristic feature of several neurodegenerating diseases including Alzheimer’s disease and thiamine diphosphate deficiency encephalopathy is the decrease of PDHC activity correlating with cholinergic deficits and losses of cognitive functions. Such conditions generate acetyl-CoA deficits that are deeper in cholinergic neurons than in noncholinergic neuronal and glial cells, due to its additional consumption in the transmitter synthesis. Therefore, any neuropathologic conditions are likely to be more harmful for the cholinergic neurons than for noncholinergic ones. For this reason attempts preserving proper supply of acetyl-CoA in the diseased brain, should attenuate high susceptibility of cholinergic neurons to diverse neurodegenerative conditions. This review describes how common neurodegenerative signals could induce deficts in cholinergic neurotransmission through suppression of acetyl-CoA metabolism in the cholinergic neurons.

Alterations of Adenine Nucleotide Metabolism and Function of Blood Platelets in Patients With Diabetes

Increased activity of blood platelets contributes to vascular complications in patients with diabetes. The aim of this work was to investigate whether persisting hyperglycemia in diabetic patients generates excessive accumulation of ATP/ADP, which may underlie platelet hyperactivity. Platelet ATP and ADP levels, thiobarbituric acid–reactive species synthesis, and aggregation of platelets from patients with diabetes were 18–82% higher than in platelets from healthy participants. In patients with diabetes, platelet stimulation with thrombin caused about two times greater release of ATP and ADP than in the healthy group while decreasing intraplatelet nucleotide content to similar levels in both groups. This indicates that the increased content of adenylate nucleotides in the releasable pool in the platelets of diabetic patients does not affect their level in metabolic cytoplasmic/mitochondrial compartments. Significant correlations between platelet ATP levels and plasma fructosamine, as well as between platelet ATP/ADP and platelet activities, have been found in diabetic patients. In conclusion, chronic hyperglycemia-evoked elevations of ATP/ADP levels and release from blood platelets of patients with diabetes may be important factors underlying platelet hyperactivity in the course of the disease.

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.

Effects of zinc on SN56 cholinergic neuroblastoma cells

Zinc is a trace element necessary for proper development and function of brain cells. However, excessive accumulation of zinc exerts several cytotoxic effects in the brain. The aim of this work was to see whether cytotoxic effects of zinc are quantitatively correlated with changes in acetyl‐CoA metabolism. The zinc levels up to 0.20 mmol/L caused concentration‐dependent inhibition of pyruvate dehydrogenase (PDH) activity that correlated with the increase in trypan blue‐positive fraction and the decrease in cultured cell number (r = 0.96, p = 0.0001). Chronic exposure of cells to 0.15 mmol/L zinc decreased choline acetyltransferase and aconitase activities, cytoplasmic acetyl‐CoA and whole cell ATP level by 38%, 57%, 35%, and 62%, respectively but caused no change in mitochondrial acetyl‐CoA level and activities of other enzymes of glycolytic and tricarboxylic acid cycle. dl‐α‐lipoamide when added simultaneously with zinc to cultured cells or their homogenates attenuated its chronic or acute suppressive effects. In homogenates of chronically Zn‐treated cells, lipoamide overcame PDH but not aconitase inhibition. Presented data indicate that acute‐transient elevation of zinc caused reversible inhibition of PDH, aconitase activities and acetyl‐CoA metabolism, which when prolonged could lead to irreversible enzyme inactivation yielding decrease in cell viability and secondary suppression of their cholinergic phenotype.

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.

RS-α-lipoic acid protects cholinergic cells against sodium nitroprusside and amyloid-β neurotoxicity through restoration of acetyl-CoA level

The work presented here verifies the hypothesis that RS‐α‐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‐β 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.

Relationships between cholinergic phenotype and acetyl-CoA level in hybrid murine neuroblastoma cells of septal origin

High susceptibility of cholinergic neurons to neurotoxic signals may result from their utilization of acetyl‐CoA for both energy production and acetylcholine synthesis. SN56 cholinergic cells were transfected stably with cDNA for choline acetyltransferase. Transfected cells (SN56ChAT2) expressed choline acetyltransferase activity and acetylcholine content, 17 times and 2 times higher, respectively, than did nontransfected cells. Transfection did not change pyruvate dehydrogenase but decreased the acetyl‐CoA level by 62%. Differentiation by cAMP and retinoic acid caused an increase of choline acetyltransferase activity and decrease of acetyl‐CoA levels in both cell lines. Negative correlation was found between choline acetyltransferase activity and acetyl‐CoA level in these cells. SN56ChAT2 cells were more susceptible to excess NO than were native SN56 cells, as evidenced by the thiazolyl blue reduction assay. Thus, the sensitivity of cholinergic neurons to pathologic conditions may depend on the cholinergic phenotype‐dependent availability of acetyl‐CoA.