TY - JOUR
T1 - Competitive, reversible, and potent antagonism of inositol 1,4,5-trisphosphate-activated calcium release by heparin
AU - Ghosh, T. K.
AU - Eis, P. S.
AU - Mullaney, J. M.
AU - Ebert, C. L.
AU - Gill, D. L.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1988
Y1 - 1988
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0023690141&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0023690141&partnerID=8YFLogxK
M3 - Article
C2 - 3136153
AN - SCOPUS:0023690141
VL - 263
SP - 11075
EP - 11079
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 23
ER -