EFFECT OF INTRACEREBROVENTRICULAR BETA-FUNALTREXAMINE ON MU-OPIOID RECEPTORS IN THE RAT-BRAIN - CONSIDERATION OF BINDING CONDITION

Effects of 24 h pretreatment with intracerebroventricular (icv) beta-funaltrexamine (beta-FNA) on brain opioid receptor binding in rats were examined under various conditions. Agonist binding to mu and delta opioid receptors (with [H-3][[cap]dAla(2),MePhe(4), Gly-ol(5)]enkephalin (DAMGO)[H-3][D-Pen(2), D-Pen(5)]enkephalin (DPDPE), respectively) was performed under three different conditions: i) pretreatment of membranes with GDP and Na+ and binding in the presence of Mg++ in Tris-HCl buffer containing EGTA and leupeptin for 1.5 to 3 h; ii) binding in Tris-HCl buffer containing bacitracin, leupeptin, chymostatin and bestatin for 3 to 4 h; iii) binding in Tris-HCl buffer containing EGTA and leupeptin for 45 min. Condition i was shown to convert opioid receptors to a high affinity state for agonists, beta-FNA (2, 6 or 20 nmol) significantly reduced 1 nM [H-3]DAMGO binding in the whole brain with i but not with ii. With iii, 20 nmol beta-FNA reduced [H-3]DAMGO binding, but not 2 or 6 nmol. Saturation experiments with i showed that the reduction in [H-3]DAMGO binding after 6 or 20 nmol beta-FNA was due to a decrease in B-max and an increase in K-D. For delta binding, there was no significant change in [H-3]DPDPE (2 nM) binding with i after 2, 6 or 20 nmol beta-FNA. Thus, under i, icv beta-FNA reduced [H-3]DAMGO binding significantly without affecting [H-3]DPDPE binding. In addition, mu binding was also conducted with 1 nM [H-3]naloxone under three different conditions: iv) in the presence of Na+ and GDP; v) in the presence of Na+, Gpp(NH)p and Mg++; vi) in the presence of Na+. Both iv and v were shown to shift opioid receptors to a low affinity state for agonists. beta-FNA (20 nmol) significantly decreased 1 nM [H-3]naloxone binding under each of the three conditions. Competitive inhibition of 1 nM [H-3]naloxone binding by DAMGO in the presence of Na+ and GDP showed that receptors existed in a single low affinity state for DAMGO, and that icy beta-FNA caused a reduction in B-max without affecting the K-D of DAMGO. In summary, when all the receptors were converted to a high agonist affinity state i or a low agonist affinity state iv, the changes in mu binding induced by beta-FNA could be revealed with agonist binding. Additionally, changes in mu binding induced by beta-FNA could be detected with [H-3]naloxone, which always displayed high affinity regardless of agonist affinity states, under each of the three conditions (iv, v and vi).