title: Competitive, reversible, and potent antagonism of inositol 1,4,5-trisphosphate-activated calcium release by heparin.
abstract: The action of inositol 1,4,5-trisphosphate (InsP3) in releasing intracellular Ca2+ is shown to be competitively and potently antagonized by the glycosaminoglycan, heparin. Using either permeabilized cells of the DDT1MF-2 smooth muscle cell line, or an isolated microsomal membrane fraction derived from intact cells, heparin (4-6 kDa) at 10 micrograms/ml was observed to completely block the action of InsP3 in releasing Ca2+ accumulated via the ATP-dependent Ca2+ pump. In permeabilized cells, heparin had no effect on Ca2+ pump activity or on passive Ca2+ fluxes contributing to equilibrium Ca2+ accumulation. Heparin up to 100 micrograms/ml had no effect on the GTP-activated Ca2+ translocation process previously characterized in this cell line. Half-maximal inhibition of Ca2+ release activated by 10 microM InsP3 occurred with heparin at approximately 0.6 and 0.2 microgram/ml in permeabilized cells and isolated microsomes, respectively. Using microsomes, InsP3 dose-response curves in the presence and absence of 0.2 microgram/ml heparin (approximately 40 nM) revealed a 10-fold increase in apparent Km for InsP3 (0.31 microM in the absence of heparin) with no change in Vmax, indicating a competitive action of heparin. The results revealed a very high apparent affinity of heparin for the InsP3 active site, with a calculated Ki value of 2.7 nM. Heparin was shown to rapidly (within 20 s) reverse prior full activation of InsP3-mediated Ca2+ release returning the Ca2+ equilibrium back to that observed without InsP3. This reversal occurs even after prolonged (6 min) InsP3 activation. These results indicate a specific, high affinity, and competitive antagonism of the InsP3 active site by heparin. The rapidly induced reversal of InsP3-activated Ca2+ release by heparin strongly suggests that InsP3 directly activates a channel which remains open only while InsP3 is associated and closes immediately upon InsP3 dissociation.
mesh_terms: D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006493:Heparin / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000037:antagonists & inhibitors*; D007700:Kinetics; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism; D010539:Permeability; D013403:Sugar Phosphates / Q000037:antagonists & inhibitors*
chemical_list: D007295:Inositol Phosphates; D013403:Sugar Phosphates; D000001:Calcimycin; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D002118:Calcium