title: Effect of intracerebroventricular beta-funaltrexamine on mu opioid receptors in the rat brain: consideration of binding condition.
abstract: 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 [3H][[cap]dAla2,MePhe4,Gly-ol5]enkephalin (DAMGO)[3H][D-Pen2, D-Pen5]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-HCI buffer containing EGTA and leupeptin for 1.5 to 3 h; ii) binding in Tris-HCI buffer containing bacitracin, leupeptin, chymostatin and bestatin for 3 to 4 h; iii) binding in Tris-HCI 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 [3H]DAMGO binding in the whole brain with i but not with ii. With iii, 20 nmol beta-FNA reduced [3H]DAMGO binding, but not 2 or 6 nmol. Saturation experiments with i showed that the reduction in [3H]DAMGO binding after 6 or 20 nmol beta-FNA was due to a decrease in Bmax and an increase in KD. For delta binding, there was no significant change in [3H]DPDPE (2 nM) binding with i after 2, 6 or 20 nmol beta-FNA. Thus, under i, icv beta-FNA reduced [3H]DAMGO binding significantly without affecting [3H]DPDPE binding. In addition, mu binding was also conducted with 1 nM [3H]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 [3H]naloxone binding under each of the three conditions. Competitive inhibition of 1 nM [3H]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 icv beta-FNA caused a reduction in Bmax without affecting the KD 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 [3H]naloxone, which always displayed high affinity regardless of agonist affinity states, under each of the three conditions (iv, v and vi).
authors.affiliation: Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: L Y
authors.initials: LY
authors.lastname: Liu-Chen
authors.forename: H H
authors.initials: HH
authors.lastname: Yang
authors.forename: S
authors.initials: S
authors.lastname: Li
authors.forename: J U
authors.initials: JU
authors.lastname: Adams
mesh_terms: D000700:Analgesics / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D020881:Enkephalin, D-Penicillamine (2,5)-; D004745:Enkephalins / Q000378:metabolism; D007276:Injections, Intraventricular; D008297:Male; D009270:Naloxone / Q000378:metabolism; D009271:Naltrexone / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D017450:Receptors, Opioid, mu / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014316:Tritium
chemical_list: D000700:Analgesics; D004745:Enkephalins; D009292:Narcotic Antagonists; D017450:Receptors, Opioid, mu; D014316:Tritium; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009270:Naloxone; D009271:Naltrexone; C024524:beta-funaltrexamine; D020881:Enkephalin, D-Penicillamine (2,5)-