Pharmacological characterization of inositol-1,4,5,-trisphosphate binding to membranes from retina and retinal cultures.
Light and excitatory amino acids (EAA) stimulate the phosphoinositide cycle in the vertebrate retina. The regulation of Ca2+ release from intracellular stores by inositol-1,4, 5-trisphosphate (IP3) involves an interaction of this compound with specific receptors. By means of [3H]IP3-specific binding, we studied the kinetic and pharmacological properties of IP3 receptors in the chick retina as well as in primary cultures of neurons and glia from this tissue. The equilibrium time for the binding reaction was 15 min and was optimal at alkaline pH (8.3). IP3 receptor displayed high affinity (K(B) approximately 40 nM) and selectivity for D-IP3, compared to D-IP4 > L-IP3 > D-IP2 > D-IP1. These characteristics were the same in subcellular fractions from outer (P1) and thinner (P2) plexiform layers, binding sites being more abundant in P2 (2.65 pmol/mg protein). IP3 receptors were present in both neuronal and glial cultures, but were concentrated in neuronal cultures. Binding was not affected by ryanodine, or caffeine, related to calcium-induced calcium release (CICR) channels, nor by the endoplasmic reticulum Ca2+ ATPase inhibitor thapsigargin, while heparin affectively inhibited IP3 binding. GSSG and thimerosal increased the affinity of [3H]IP3 binding from IC50 approximately 80 nM to IC50 approximately 40 nM; this effect was reversed by DTT. Binding in zero Ca2+ was decreased by low concentrations of Ca2+ (350 nM). These results suggest that actions of IP3 in the retina are regulated by physiological changes in intracellular pH and Ca2+ concentrations, as well as by the oxidation state of the receptor. Additionally, the presence of IP3 receptors in Müller glia opens the possibility of IP3 participation in nonsynaptic signalling through Ca2+ waves in glial cells.
López-Colomé
A M
AM
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México.
Lee
I
I
D000704:Analysis of Variance; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002645:Chickens; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000494:pharmacology; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D009457:Neuroglia / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D012160:Retina / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D013849:Thimerosal / Q000494:pharmacology
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D018160:Receptors, Cytoplasmic and Nuclear; D013849:Thimerosal; D015544:Inositol 1,4,5-Trisphosphate; D005978:Glutathione; D004229:Dithiothreitol; D019803:Glutathione Disulfide