apyrase and Hyperglycemia

Chronic activation of proinflammatory caspase-1 in the retinas of diabetic animals and patients in vivo and retinal Müller cells in vitro is well documented. In this study we characterized how elevated glucose and extracellular purines contribute to the activation of caspase-1 in a cultured rat Müller cell (rMC-1) model. The ability of high glucose (25 mM, 24 h) to activate caspase-1 was attenuated by either apyrase, which metabolizes extracellular ATP to AMP, or adenosine deaminase (ADA), which metabolizes extracellular adenosine to inosine. This suggested that autocrine stimulation of ATP-sensing P2 receptors and adenosine-sensing P1 receptors may in part mediate the response to high glucose. Exogenous ATP, 5'-N-ethylcarboxamido-adenosine (NECA), a nonselective P1 receptor agonist, or forskolin (FSK) increased caspase-1 activity in rMC-1 cells cultured in control glucose (5 mM) medium. Accumulation of active caspase-1 was also increased by dipyridamole, which suppresses adenosine reuptake. High-glucose stimulation of caspase-1 was attenuated by suramin, a nonselective P2 antagonist, or A2 adenosine receptor antagonists, but not by antagonism of P2X7 ATP-gated ion channel receptors. Although high glucose increased P2X7 mRNA, neither P2X7 protein nor function was detected in rMC-1 cells. The increased caspase-1 activity stimulated by high glucose, FSK, NECA, or ATP was correlated with increased gene expression of caspase-1 and thioredoxin-interacting-protein (TXNIP). These findings support a novel role for autocrine P1 and P2 purinergic receptors coupled to cAMP signaling cascades and transcriptional induction of caspase-1 in mediating the high-glucose-induced activation of caspase-1 and secretion of IL-1β in a cell culture model of nonhematopoietic retinal Müller cells.

Topics: Adenosine A2 Receptor Antagonists; Adenosine Deaminase; Adenosine-5'-(N-ethylcarboxamide); Animals; Apyrase; Caspase 1; Cell Line; Colforsin; Dipyridamole; Enzyme Inhibitors; Glucose; Hyperglycemia; Purinergic P2X Receptor Antagonists; Rats; Receptors, Purinergic P1; Receptors, Purinergic P2; Retina; Suramin

Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications.. An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. The calcineurin inhibitor cyclosporin A or the novel NFAT blocker A-285222 prevented glucose-induced OPN expression. Furthermore, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A-285222 for 4 weeks or prevented in arteries from NFATc3(-/-) mice.. These results identify a glucose-sensitive transcription pathway in vivo, revealing a novel molecular mechanism that may underlie vascular complications of diabetes.

Topics: Animals; Apyrase; Arteries; Binding Sites; Blood Glucose; Calcineurin; Calcineurin Inhibitors; Cyclosporine; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Disease Models, Animal; Enzyme Inhibitors; Female; Glucose Tolerance Test; Humans; Hyperglycemia; Jurkat Cells; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; NFATC Transcription Factors; Osteopontin; Promoter Regions, Genetic; Pyrazoles; RNA, Messenger; Signal Transduction; Time Factors; Transcriptional Activation; Transfection; Uridine Triphosphate