Endothelin-1 levels and albuminuria in patients with type 2 diabetes mellitus.

AIM: To evaluate the relationship of plasma endothelin-1 (ET-1) levels, a marker of endothelial dysfunction, and urinary albumin excretion in patients with type 2 diabetes mellitus (DM). METHODS: Cross-sectional study was conducted in 279 patients (132 males, mean age: 58.7+/-11.0 years, mean DM duration: 11.3+/-8.1 years). Urinary albumin excretion, ET-1, and insulin were measured. Insulin sensitivity was estimated by homeostasis model assessment (HOMA-ir) index. RESULTS: ET-1 was associated with urinary albumin excretion after controlling for age, gender, body mass index, blood pressure, HbA1c test, and total cholesterol (R=0.436; adjusted R(2)=0.190, P<0.01). Furthermore, there was a progressive increase in plasma ET-1 levels from patients with normoalbuminuria (n=187, 0.92+/-0.50pg/ml), microalbuminuria (n=68, 1.13+/-0.52pg/ml) to macroalbuminuria (n=24, 1.93+/-1.10pg/ml, P<0.01). CONCLUSION: There is an independent association of plasma ET-1 levels with urinary albumin excretion. In addition, plasma ET-1 levels started to increase in the normal values of urinary albumin excretion suggesting that in patients with type 2 DM endothelial dysfunction is already present, in urinary albumin excretion values considered normal.

Indomethacin stimulates activity and expression of ecto-5'-nucleotidase/CD73 in glioma cell lines.

Gliomas are the most common and devastating primary tumors of the central nervous system. Ecto-NTPDases and ecto-5'-nucleotidase/CD73 can control extracellular ATP/adenosine levels, which have been described as proliferation factors. Here, we investigate the influence of indomethacin on the enzyme cascade that catalyses the interconversion of purine nucleotides in U138-MG and C6 glioma cell lines. Exposure of glioma cells to 100 microM indomethacin for 48 h caused increases of 52% (P < 0.05) and 62% (P < 0.05) in the AMP hydrolysis rate in C6 and U138-MG cell lines, respectively. Indomethacin treatments also increased ATP hydrolysis. Significant increase in ecto-5'-nucleotidase/CD73 mRNA and protein levels were observed after treatment with indomethacin. Pretreatment of glioma cells with a specific antagonist of the adenosine A(3) receptor, MRS1220 (1 microM; 9-Chloro-2-(2-furanyl)-5-((phenylacetyl)amino)-[1,2,4]triazolo[1,5-c]quinazoline), significantly reduced the inhibition of cell proliferation induced by indomethacin. In addition, a significant increase in mRNA levels of the adenosine A(3) receptor was observed after treatment with indomethacin. In conclusion, our data indicate that adenosine A(3) receptors and the enzyme, ecto-5'-nucleotidase/CD73, are involved in the anti-proliferative effect of indomethacin in glioma cells.

Nonsteroidal anti-inflammatory drugs inhibit the growth of C6 and U138-MG glioma cell lines.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used drugs for the treatment of inflammatory disease and have a chemopreventive effect in a variety of tumors. Several studies have demonstrated unequivocally that certain NSAIDs cause antiproliferative effects independent of cyclooxygenase (COX) activity. In this study, we investigated the effect of chemically unrelated NSAIDs in the proliferation of glioma cell lines and the possible mechanisms involved in indomethacin-mediated inhibition of proliferation in glioma cells lines. The glioma cell lines were treated with NSAIDs and proliferation was measured by cell counting. Indomethacin, acetaminophen, sulindac sulfide and NS-398 (N-[2-cyclohexyloxy)-4-nitrophenyl]methane-sulfonamide) induced a time- and concentration-dependent inhibition of C6 rat glioma cell proliferation. The inhibition of COX by chemically unrelated NSAIDs leads to inhibition of rat and human glioma cell proliferation. The tetrazolium reduction assay (MTT) indicated a reduction in cell viability induced by indomethacin. None of the NSAIDs tested induced caspase-3/7 activation, assayed with a fluorigenic substrate. The indomethacin-induced inhibition of C6 cells proliferation was abrogated by the use of the c-Src inhibitor, PP2 and the MEK inhibitor, PD 098059, suggesting COX-independent mechanisms. Indomethacin decreased the percentage of cells in the S phase, with relative increases in the G0/G1 and/or the G2/M phase. NSAIDs may be clinically important for pharmacological intervention in gliomas.

P2X7 receptors stimulate AKT phosphorylation in astrocytes.

1. Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X(7) subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. 2. P2Y and P2X receptor agonists ATP, uridine 5'-triphosphate (UTP) and 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration- and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X(7) receptor. Activation was maximal at 5 - 10 min and was sustained for 60 min; the EC(50) for BzATP was approximately 50 microM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. 3. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X(7) receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. 4. Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. 5. In conclusion, our data indicate that stimulation of astrocytic P2X(7) receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism.

ERK, PKC and PI3K/Akt pathways mediate extracellular ATP and adenosine-induced proliferation of U138-MG human glioma cell line.

OBJECTIVE: Extracellular nucleotides and nucleosides induce proliferation in a set of human glioma cell lines. In this study we investigate the signal transduction pathways involved in ATP and adenosine-mediated proliferation in U138-MG human glioma cells. METHODS: Cell proliferation was accessed through [(3)H]thymidine incorporation, cell counting and flow cytometry. Protein phosphorylation was detected through Western blotting. RESULTS: ATP or adenosine (100 microM) induced extracellular signal-regulated protein kinase (ERK), Akt and GSK3beta phosphorylation. The increase in [(3)H]thymidine incorporation induced by ATP or adenosine was decreased when cells were incubated with LY 294002 (by +/-90%), GF 109203X (by +/-76%) or PD 098059 (by +/-63%). The increase in cell numbers with ATP or adenosine was less after a 48-hour treatment of cells with ATP or adenosine plus GF 109203X (by +/-66%) or LY 294002 (by +/-83%). Percentage of cells in S phase was decreased in cells treated with LY 294002 plus ATP when compared to ATP- treated cells. CONCLUSION: Stimulation of purinergic receptors in U138-MG cells leads to cell proliferation mediated by PI3K/Akt, ERK and PKC signaling. It may be clinically important for pharmacological intervention in gliomas to associate purinergic receptor antagonists and signal transduction pathways blockers.

Extracellular nucleotides and nucleosides induce proliferation and increase nucleoside transport in human glioma cell lines.

Extracellular purines (adenosine triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine) and pyrimidines (uridine 5'-triphosphate (UTP) and UDP) are important signaling molecules that mediate diverse biological effects via P1 and P2 purinergic receptors. The human glioma cell lines U87 MG, U251 MG and U138 MG were treated with purines and pyrimidines for 24 or 48 h and proliferation was measured by [3H]-thymidine incorporation, flow cytometry and cell counting. The studies showed that extracellular nucleotides and nucleosides induce proliferation of the studied glioma cells. Incorporation of [3H]-thymidine followed the order of ATP approximately equal to guanosine approximately equal to inosine approximately equal to adenosine > UTP > ADP while ATPgammaS and 2MeSATP had no effect. The effect of ATP was partially inhibited by suramin and by reactive blue 2 (RB2). Co-treatment with the following antagonists of P1 purinoreceptors DPCPX, CPT or 8PT did not block the effect of adenosine while a specific antagonist of the A3 receptor, MRS1220, totally blocked the effect of adenosine. ATP and adenosine also increased the overall uptake of [3H]-thymidine into the cell, producing a positive effect on the [3H]-thymidine incorporation measurements. These data indicate that the uptake of thymidine and proliferation of gliomas can be induced by purines and pyrimidines via both P1 and P2 purinoceptors.