Regional and subcellular distribution of [125I]endothelin binding sites in rat brain.
The binding of [125I]endothelin-1 (125I-ET-1) to membranes from whole rat brain, from individual brain regions, and derived from subcellular fractionation of whole rat brain was investigated. 125I-ET-1 binding to whole rat brain membranes was rapid, concentration-dependent, saturable, and characterized as irreversible because it was not displaced by unlabeled endothelin-1 (ET-1) and different concentrations of ligand produced, with time, a similar magnitude of binding. The maximum binding site capacity and second-order forward rate association constant of binding were 1,946 +/- 147 fm/mg protein and 5.53 +/- 1.72 x 10(6) M-1 s-1. Removal of either extramembranal calcium or membrane-bound calcium and calcium binding proteins did not affect the binding of 125I-ET-1 to whole rat brain membranes. The brain stem and cerebellum contained the highest levels of 125I-ET-1 binding sites, whereas the cerebral cortex, striatum, and hippocampus contained binding site levels three- to fourfold less. Subcellular fractionation of whole rat brain and subsequent analyses of the distribution of 125I-ET-1 binding demonstrated a twofold enrichment of binding sites in the synaptosomal fraction compared to the homogenate. The myelin fraction contained a similar density of binding sites compared to the homogenate, while the mitochondrial and microsomal fractions contained considerably less binding sites. The ribosomal fraction did not contain any 125I-ET-1 binding sites. The subcellular distribution of 125I-ET-1 binding sites did not correlate with the distribution of 5'-nucleotidase, cytochrome-C oxidase, phosphodiesterase, and alkaline phosphatase. Depletion of extracellular calcium increased 125I-ET-1 binding in the synaptosomal fraction but not in the myelin and mitochondrial fractions.(ABSTRACT TRUNCATED AT 250 WORDS)
Bolger
G T
GT
Department of Pharmacology, Bio-Mega Inc., Laval, Quebec, Canada.
Berry
R
R
Jaramillo
J
J
D000818:Animals; D015415:Biomarkers; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure*; D002118:Calcium / Q000502:physiology; D016232:Endothelins / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017466:Receptors, Endothelin; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism / Q000502:physiology
D015415:Biomarkers; D016232:Endothelins; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D002118:Calcium