title: 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 ED(50) of the N-acetylated disaccharide derived from heparan sulfate (430 Da) is 88 microm compared with 250 microm 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 Ca(2+)-ATPase inhibitor), vanadate (cytoplasmic membrane Ca(2+)-ATPase inhibitor), and nifedipine and verapamil (Ca(2+) 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(+)/Ca(2+) exchanger promoting the extrusion of Ca(2+). This was further substantiated by binding experiments and circular dichroism analysis with the exchanger inhibitor peptide.
authors.affiliation: Departamento de Bioquimica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua 3 de Maio 100, Brazil.
authors.forename: Samuel K
authors.initials: SK
authors.lastname: Shinjo
authors.forename: Ivarne L S
authors.initials: IL
authors.lastname: Tersariol
authors.forename: Vitor
authors.initials: V
authors.lastname: Oliveira
authors.forename: Clóvis R
authors.initials: CR
authors.lastname: Nakaie
authors.forename: Maria E M
authors.initials: ME
authors.lastname: Oshiro
authors.forename: Alice T
authors.initials: AT
authors.lastname: Ferreira
authors.forename: Isabel A
authors.initials: IA
authors.lastname: Santos
authors.forename: Carl P
authors.initials: CP
authors.lastname: Dietrich
authors.forename: Helena B
authors.initials: HB
authors.lastname: Nader
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002942:Circular Dichroism; D003600:Cytosol / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000378:metabolism*; D013552:Swine
chemical_list: D002118:Calcium; D004187:Disaccharides; D006493:Heparin; D006497:Heparan Sulfate; D019831:Sodium-Calcium Exchanger