Matrix protein glycation impairs agonist-induced intracellular Ca2+ signaling in endothelial cells

J Cell Physiol. 2002 Oct;193(1):80-92. doi: 10.1002/jcp.10153.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / pharmacology
  • Animals
  • Antibodies / pharmacology
  • Antioxidants / pharmacology
  • Bradykinin / pharmacology
  • Calcium / metabolism
  • Calcium Signaling / drug effects
  • Calcium Signaling / physiology*
  • Cattle
  • Cell Adhesion / drug effects
  • Cells, Cultured
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / metabolism*
  • Extracellular Matrix Proteins / drug effects
  • Extracellular Matrix Proteins / metabolism*
  • Fibronectins / metabolism
  • Glycation End Products, Advanced / metabolism*
  • Glycation End Products, Advanced / pharmacology
  • Inositol Phosphates / metabolism
  • Intracellular Fluid / metabolism*
  • Laminin / antagonists & inhibitors
  • Oxidants / pharmacology
  • Oxidative Stress / physiology
  • Rats
  • Reactive Oxygen Species / metabolism


  • Antibodies
  • Antioxidants
  • Extracellular Matrix Proteins
  • Fibronectins
  • Glycation End Products, Advanced
  • Inositol Phosphates
  • Laminin
  • Oxidants
  • Reactive Oxygen Species
  • Adenosine Triphosphate
  • Bradykinin
  • Calcium