REGIONAL AND SUBCELLULAR-DISTRIBUTION OF [I-125] ENDOTHELIN BINDING-SITES IN RAT-BRAIN

THE binding of [I-125]endothelin-1 (I-125-ET-1) to membranes from whole rat brain, from individual brain regions, and derived from subcellular fractionation of whole rat brain was investigated. I-125-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 I-125-ET-1 to whole rat brain membranes. The brain stem and cerebellum contained the highest levels of I-125-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 I-125-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 I-125-ET-1 binding sites. The subcellular distribution of I-125-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 I-125-ET-1 binding in the synaptosomal fraction but not in the myelin and mitochondrial fractions. The second-order association rate constant for I-125-ET-1 binding in the mitochondrial fraction was significantly different from that calculated for the synaptosomal and myelin fractions. These results demonstrate the presence of substantial densities of high-affinity binding sites for ET-1 in rat brain and suggest that both plasmalemmal and intracellular I-125-ET-1 binding sites with different properties may exist in the brain.