Competitive, reversible, and potent antagonism of inositol 1,4,5-trisphosphate-activated calcium release by heparin

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 μg/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 μg/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 μM InsP₃ occurred with heparin at approximately 0.6 and 0.2 μg/ml in permeabilized cells and isolated microsomes, respectively. Using microsomes, InsP₃ does-response curves in the presence and absence of 0.2 μg/ml heparin (approximately 40 nM) revealed a 10-fold increase in apparent K(m) for InsP₃ (0.31 μM in the absence of heparin) with no change in V(max), indicating a competitive action of heparin. The results revealed a very high apparent affinity of heparin for the InsP₃ active site, with a calculated K(i) 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.