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 Ca²⁺ signaling following agonist stimulation. Endothelial cells were cultured from bovine aorta and rat heart. To glycate proteins, fibronectin (10 μg/ml), or EHS (2.5 mg/ml) were incubated (37°C, 30 days) with 0.5 M glucose-6-phosphate. Matrix proteins were coated onto cover slips after which cells (10⁵ cells/ml) were plated and allowed to adhere for 16 h. For measurement of intracellular Ca²⁺, cells were loaded with fura 2 (2 μM) 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 Ca²⁺_i. In contrast, glycated fibronectin did not impair agonist-induced increases in Ca²⁺_i. In the absence of extracellular Ca²⁺, ATP elicited a transient increase in Ca²⁺_i consistent with intracellular release. Re-addition of Ca²⁺ resulted in a secondary rise in Ca²⁺_i indicative of store depletion-mediated Ca²⁺ entry. Both phases of Ca²⁺ 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 Ca²⁺ release from intracellular stores. Thapsigargin treatment demonstrated an impaired ability of cells on glycated EHS to increase cytoplasmic Ca²⁺ consistent with decreased endoplasmic reticulum Ca²⁺ stores. Further support for Ca²⁺ mobilization was provided by increased baseline IP₃ levels in cells plated on glycated EHS. Impaired ATP-induced Ca²⁺ release could be induced by treating native EHS with laminin antibody or exposing cells to H₂O₂ (20–200 μM). Glycated EHS impaired Ca²⁺ signaling was attenuated by treatment with aminoguanidine or the antioxidant α-lipoic acid. The results demonstrate that matrix glycation impairs agonist-induced Ca²⁺_i increases which may impact on regulatory functions of the endothelium and implicate possible involvement of oxidative stress. J. Cell. Physiol. 193: 80–92, 2002.