Localization of purine metabolizing enzymes in bovine brain microvessel endothelial cells: an enzymatic blood-brain barrier for dideoxynucleosides?

Abstract

Purpose: The specific activities of the purine and pyrimidine metabolizing enzymes, purine nucleoside phosphorylase (PNP), adenosine deaminase (ADA) and cytidine deaminase (CDA) were determined in bovine brain microvessel endothelial cells (BBMECs), whole cerebral tissue and erythrocytes. In addition, the substrate specificities (Km and Vmax) of purified calf spleen PNP for inosine and 2',3'-dideoxyinosine (ddI) and of purified calf intestinal ADA for 2',3'-dideoxyadenosine (ddA), 6-chloro-2',3'-dideoxypurine (6-Cl-ddP), and 2'-beta-fluoro-2', 3'-dideoxyadenosine (F-ddA) have been explored.

Methods: BBMECs were isolated from bovine cerebral cortex by a two step enzymatic dispersion treatment followed by centrifugation over 50% Percoll density gradients. Activities of alkaline phosphatase, gamma-glutamyl transpeptidase, ADA, PNP and CDA were determined in various tissue homogenates (cerebral cortex, BBMECs and erythrocytes). Enzyme kinetic studies were also conducted using commercially available enzymes and several nucleoside analogs of interest.

Results: The activities of ADA and PNP were 42-fold and 247-fold higher in the cerebral microvessels than in the cerebral cortex, respectively, while there was no detectable CDA activity in the microvessel fraction and very little overall activity in the cortex.

Conclusions: ADA and PNP may serve as an enzymatic blood-brain barrier for some of the anti-HIV dideoxynucleosides. Simulations of brain availability for ddI, ddA, 6-Cl-ddP, and F-ddA demonstrated that the quantitative significance of enzyme localization may vary dramatically, however, depending on the membrane permeability of the drug and its bioconversion rate constant within the endothelial cell.