Modification of the peripheral-type benzodiazepine receptor by arachidonate, diethylpyrocarbonate and thiol reagents

Abstract

Peripheral type benzodiazepine receptors are differentially modified by arachidonate, diethylpyrocarbonate and thiol reagents, as evidenced by the finding that binding of a proposed agonist ([3H]R05-4864) and a proposed antagonist ([3H]PK 11195) to rat kidney mitochondrial membranes can be modified separately. (1) Arachidonate significantly lowered the affinity of the peripheral-type benzodiazepine receptor for R05-4864 and diazepam but did not alter the affinity for PK 11195 and only slightly altered for dipyridamole. In contrast, diethylpyrocarbonate inhibited [3H]PK 11195 binding by 43% while reducing [3H]R05-4864 binding only 16%. (2) Diethylpyrocarbonate treatment causes a reduction in affinity for both dipyridamole and PK 11195, did not change the affinities for R05-4864 and diazepam but increased the affinities for diuretics. PK 11195, R05-4864 and dipyridamole totally protected peripheral benzodiazepine receptors against inactivation by diethylpyrocarbonate. In contrast, diethylpyrocarbonate inactivation of [3H]PK 11195 binding was increased after preincubation of peripheral benzodiazepine receptors with metolazone. Arachidonate both lowers the affinity for [3H]R05-4864 and eliminated the ability of R05-4864 to protect peripheral benzodiazepine receptors against inactivation by diethylpyrocarbonate. (3) Among different thiol reagents, only dithiothreitol treatment resulted in 26 and 43% inhibition of [3H]PK 11195 and [3H]R05-4864 binding, respectively. These results indicate that the peripheral-type benzodiazepine receptor molecule possesses at least two different conformations or separate mutually exclusive sites for agonists and antagonists. Additionally, the interaction of the thiazide class of diuretics with peripheral-type benzodiazepine receptors seems to require binding sites on the receptor molecule in addition to those for specific agonist and antagonist, whereas dipyridamole appears to bind to the antagonist site or conformation.