Inositol 1,4,5-trisphosphate binds to a specific receptor and releases microsomal calcium in the anterior pituitary gland.
The properties of inositol 1,4,5-trisphosphate (InsP3) receptor sites in the anterior pituitary were evaluated by binding studies with InsP3 labeled with 32P to high specific radioactivity. Specific binding of Ins[32P]P3 was demonstrable in pituitary membrane preparations and was linearly proportional to the amount of membrane added over the range 0.5-2 mg of protein. Kinetic studies showed that specific InsP3 binding was half-maximal in about 40 sec and reached a plateau after 15 min at 0 degree C. Addition of 1 microM unlabeled InsP3 was followed by rapid dissociation of the bound ligand, according to a single exponential function with half-time of about 1 min. Scatchard analysis of the binding data was consistent with a single set of high-affinity sites with Kd of 1.1 +/- 0.4 nM and maximal binding capacity of 28 +/- 15 fmol/mg of protein (n = 6). The specificity of Ins[32P]P3 binding to these sites was illustrated by the much weaker affinity for structural analogs such as inositol 1-phosphate, phytic acid, 2,3-bisphosphoglycerate, and fructose 1,6-bisphosphate. To assess the functional relevance of the InsP3 binding sites, the Ca2+-releasing activity of InsP3 was measured in pituitary membrane preparations. In the presence of oligomycin (2.5 micrograms/ml), Ca2+ movements were monitored with the fluorescent indicator fura-2 (free acid). Under these conditions, 1 mM ATP caused rapid uptake of Ca2+ by a vesicular component of the membrane fraction. Addition of InsP3 (50-2000 nM) caused a dose-dependent release of Ca2+ with a half-maximal effect at 240 nM. In the presence of 3% polyethylene glycol, GTP also stimulated calcium release. However, a nonhydrolyzable GTP analog, guanosine 5'-[gamma-thio]triphosphate, did not release calcium and completely blocked the effect of GTP. Under physiological conditions within the cytosol, the high-affinity InsP3 binding sites characterized in pituitary membranes could serve as the putative receptors through which InsP3 triggers Ca2+ mobilization in the anterior pituitary gland.
Guillemette
G
G
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Bethesda, MD 20892.
Balla
T
T
Baukal
A J
AJ
Catt
K J
KJ
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D006160:Guanosine Triphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013403:Sugar Phosphates / Q000378:metabolism*
D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D013403:Sugar Phosphates; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D002118:Calcium