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

T. K. Ghosh, P. S. Eis, J. M. Mullaney, C. L. Ebert, D. L. Gill

Research output: Contribution to journalArticle

349 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)11075-11079
Number of pages5
JournalJournal of Biological Chemistry
Volume263
Issue number23
StatePublished - Jan 1 1988

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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