Heparin and heparan sulfate disaccharides bind to the exchanger inhibitor peptide region of Na+/Ca2+exchanger and reduce the cytosolic calcium of smooth muscle cell lines - Requirement of C4-C5 unsaturation and 1 -> 4 glycosidic linkage for activity

Abstract

Heparin and heparan sulfate fragments, obtained by bacterial heparinase and heparitinases, bearing an unsaturation at C4-C5 of the uronic acid moiety, are able to produce up to 80% reduction of the cytosolic calcium of smooth muscle cell lines. Unsaturated disaccharides from chondroitin sulfate, dermatan sulfate, and hyaluronic acid are inactive, indicating that, besides the unsaturation of the uronic acid, a vicinal 1 --> 4 glycosidic linkage is needed. An inverse correlation between the molecular weight and activity is observed. Thus, the ED50 of the N-acetylated disaccharide derived from heparan sulfate (430 Da) is 88 muMm compared with 250 muMm of the trisulfated disaccharide (650 Da) derived from heparin. Except for enoxaparin (which contains an unsaturation at the non-reducing end and 1 --> 4 glycosidic linkage), other low molecular weight heparins and native heparin are practically inactive in reducing the cytosolic calcium levels. Thapsigargin (sarcoplasmic reticulum. Ca2+-ATPase inhibitor), vanadate (cytoplasmic membrane Ca2+-ATPase inhibitor), and nifedipine and verapamil (Ca2+ channel antagonists) do not interfere with the effect of the trisulfated disaccharide upon the decrease of the intracellular calcium. A significant decrease of the activity of the trisulfated disaccharide is observed by reducing extracellular sodium, suggesting that the fragments might act upon the Na+/Ca2+ exchanger promoting the extrusion of Ca2+. This was further substantiated by binding experiments and circular dichroism analysis with the exchanger inhibitor peptide.