Characterization of inositol 1,4,5-trisphosphate receptors and calcium mobilization in a hepatic plasma membrane fraction.
The distribution of hepatic binding sites for the calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (IP3), was analyzed in subcellular fractions of the rat liver by binding studies with [32P]IP3 and compared with the Ca2+ release elicited by IP3 in each fraction. Three major subcellular fractions enriched in plasma membrane, mitochondria, and endoplasmic reticulum were characterized for their 5'-nucleotidase, glucose-6-phosphatase, succinate reductase, and angiotensin II binding activities. The fraction enriched in plasma membrane showed 7- and 20-fold increases in IP3 binding capacity over those enriched in endoplasmic reticulum and mitochondria, respectively, and contained a single class of high-affinity binding sites with Kd of 1.7 +/- 1.0 nM and concentration of 239 +/- 91 fmol/mg protein. IP3 binding reached equilibrium in 30 min at 0 degrees C, and the half-time of dissociation was about 15 min. The specificity of the IP3 binding sites was indicated by their markedly lower affinities for inositol 1-phosphate, phytic acid, fructose 1,6-bisphosphate, 2,3-bisphosphoglycerate, and inositol 1,3,4,5-tetrakisphosphate. The Ca2+-releasing activity of IP3 in the subcellular fractions was monitored with the fluorescent indicator, Fura-2. All three fractions showed ATP-dependent Ca2+ uptake and rapidly released Ca2+ in response in IP3. The fraction enriched in plasma membrane was the most active in this regard, releasing 174 +/- 67 pmol Ca2+/mg of protein compared to 45 +/- 10 and 48 +/- 7 pmol/mg protein for the fractions enriched in endoplasmic reticulum and mitochondria, respectively. These data suggest that the [32P]IP3 binding sites represent specific intracellular receptors through which IP3 mobilizes Ca2+ from a storage site associated (or co-purifying) with the plasma membrane of the rat liver. It is likely that a specialized vesicular system (to which IP3 can bind and trigger the release of Ca2+) is located in close proximity with the plasma membrane and is thus adjacent to the site at which IP3 is produced during stimulation of the hepatocyte by Ca2+-mobilizing hormones.
Guillemette
G
G
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Balla
T
T
Baukal
A J
AJ
Catt
K J
KJ
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D002462:Cell Membrane / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D013403:Sugar Phosphates / Q000378:metabolism*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D013403:Sugar Phosphates; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D002118:Calcium