Phorbol ester binding sites in human brain: characterization, regional distribution, age-correlation, and alterations...

We have characterized and localized phorbol ester binding sites in human autopsied brains, using [3H]phorbol 12,13-dibutyrate ([3H]PDBu). When the tissue was homogenized in the absence of Ca2+ chelator (10 mM EGTA/2 mM EDTA), Scatchard analysis of the specific [3H]PDBu bindings to both particulate and soluble fractions yielded a single class of high-affinity binding site (Kd = 7.1 and 7.4 nM: Bmax = 45.4 and 3.1 pmol/mg protein, respectively). The particulate fraction retained the majority of [3H]PDBu binding (98% of total binding activity), while the soluble fraction was almost devoid of binding activity (2%). In the presence of Ca2+ chelator, more of the activity was found in the soluble fraction (30%). The binding of [3H]PDBu was potently inhibited by active phorbol esters and related diterpenes with Ki of nanomolar concentration but not by inactive ones. Diolein (OAG), a synthetic diacylglycerol, and polymixin B, an inhibitor of protein kinase C (PKC), inhibited the binding moderately (Ki = 5.8 and 1.3 microM, respectively). H-7, an inhibitor of PKC and cyclic nucleotides-dependent kinase, did not compete with [3H]PDBu for the binding sites (Ki greater than 100,000 nM). The regional distribution of specific [3H]PDBu binding in the human brain was rather uneven and resembled that of [3H]PDBu autoradiograms and PKC-like immunoreactivities in the rat brain. The binding capacities were generally in the order: rhinencephalon greater than basal ganglia greater than cerebral cortex greater than diencephalon greater than cerebellum greater than mesencephalon. Age-related loss of binding sites was observed in the prefrontal cortex of the subjects 33-81 years of age. In Parkinson's disease, the phorbol ester binding showed a significant reduction in the substantia nigra, caudate putamen, and pallidum, whereas it was unchanged in the prefrontal cortex and caudate nucleus of schizophrenics, when compared with the relevant controls.