title: Matrix protein glycation impairs agonist-induced intracellular Ca2+ signaling in endothelial cells.
abstract: Studies have shown diabetes to be associated with alterations in composition of extracellular matrix and that such proteins modulate signal transduction. The present studies examined if non-enzymatic glycation of fibronectin or a mixed matrix preparation (EHS) alters endothelial cell Ca(2+) signaling following agonist stimulation. Endothelial cells were cultured from bovine aorta and rat heart. To glycate proteins, fibronectin (10 microg/ml), or EHS (2.5 mg/ml) were incubated (37 degrees C, 30 days) with 0.5 M glucose-6-phosphate. Matrix proteins were coated onto cover slips after which cells (10(5) cells/ml) were plated and allowed to adhere for 16 h. For measurement of intracellular Ca(2+), cells were loaded with fura 2 (2 microM) and fluorescence intensity monitored. Bovine cells on glycated EHS showed decreased ability for either ATP (10(-6) M) or bradykinin (10(-7) M) to increase Ca(2+) (i). In contrast, glycated fibronectin did not impair agonist-induced increases in Ca(2+) (i). In the absence of extracellular Ca(2+), ATP elicited a transient increase in Ca(2+) (i) consistent with intracellular release. Re-addition of Ca(2+) resulted in a secondary rise in Ca(2+) (i) indicative of store depletion-mediated Ca(2+) entry. Both phases of Ca(2+) mobilization were reduced in cells on glycated mixed matrix; however, as the ratio of the two components was similar in all cells, glycation appeared to selectively impair Ca(2+) release from intracellular stores. Thapsigargin treatment demonstrated an impaired ability of cells on glycated EHS to increase cytoplasmic Ca(2+) consistent with decreased endoplasmic reticulum Ca(2+) stores. Further support for Ca(2+) mobilization was provided by increased baseline IP(3) levels in cells plated on glycated EHS. Impaired ATP-induced Ca(2+) release could be induced by treating native EHS with laminin antibody or exposing cells to H(2)O(2) (20-200 microM). Glycated EHS impaired Ca(2+) signaling was attenuated by treatment with aminoguanidine or the antioxidant alpha-lipoic acid. The results demonstrate that matrix glycation impairs agonist-induced Ca(2+) (i) increases which may impact on regulatory functions of the endothelium and implicate possible involvement of oxidative stress.
authors.affiliation: Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia.
authors.forename: Nour B
authors.initials: NB
authors.lastname: Bishara
authors.forename: Marjorie E
authors.initials: ME
authors.lastname: Dunlop
authors.forename: Timothy V
authors.initials: TV
authors.lastname: Murphy
authors.forename: Ian A
authors.initials: IA
authors.lastname: Darby
authors.forename: M A
authors.initials: MA
authors.lastname: Sharmini Rajanayagam
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hill
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002417:Cattle; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000187:drug effects / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000378:metabolism* / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism*; D007797:Laminin / Q000037:antagonists & inhibitors; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000502:physiology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000906:Antibodies; D000975:Antioxidants; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D017127:Glycation End Products, Advanced; D007295:Inositol Phosphates; D007797:Laminin; D016877:Oxidants; D017382:Reactive Oxygen Species; D000255:Adenosine Triphosphate; D001920:Bradykinin; D002118:Calcium