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.

Methylglyoxal Inhibits the Binding Step of Collagen Phagocytosis

“…Notably, the spreading processes that occur in early stages of collagen phagocytosis are regulated by intracellular calcium signaling (11,36). Previous studies of endothelial cells have demonstrated an impairment of calcium signaling due to glycation of specific matrix proteins, but the mechanism that mediates this disruption is unknown (37,46). We found that MGO-treated collagen blocked the increases of intracellular calcium concentration seen in cells treated with control collagen beads, consistent with the notion that MGO inhibits collagen recognition and adhesion.…”

The Pathobiology of Diabetic Complications

“…Again, how do we know that this piece of the puzzle is really important? We know that this is important from many animal studies such as several published by George King, showing that inhibition of PKC prevented early changes in the diabetic retina and kidney (27,31,32). Increased hexosamine pathway activity.…”

Dietary hyperglycemia, glycemic index and metabolic retinal diseases

“…(Erickson et al 2007) Conversely, inhibition of PKC decreases breakdown of the iBRB and prevents early changes in the diabetic retina. (Bishara et al 2002; Brownlee 2005; Ishii et al 1996; Koya et al 2000; Saishin et al)…”