Solubilization of High‐Affinity, Guanine Nucleotide‐Sensitive μ‐Opioid Receptors from Rat Brain Membranes

Abstract

High‐affinity μ‐opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane‐associated receptors with morphine during solubilization in the detergent 3‐[(3‐cholamidopropyl)dimethyl]‐1‐propane sulfonate appeared to stabilize the μ‐opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3‐[(3‐cholamidopropyl)dimethyl]‐1‐propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [3H][d‐Ala2,N‐Me‐Phe4,Gly‐ol5]‐enkephalin ([3H]DAMGO), a μ‐selective opioid agonist, with high affinity (KD = 1.90 ± 0.93 nM; Bmax = 629 ± 162 fmol/mg of protein). Of the membrane‐associated [3H]‐DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific [3H]DAMGO binding was completely abolished in the presence of 10 µM guanosine 5′‐O‐(3‐thiotriphosphate). The solubilized receptor also bound [3H]diprenorphine, a nonselective opioid antagonist, with high affinity (KD = 1.4 ± 0.39 nM, Bmax = 920 ± 154 fmol/mg of protein). Guanosine 5′‐O‐(3‐thiotriphosphate) did not diminish [3H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 µM competed with [3H]diprenorphine for the solubilized binding sites; in contrast, [d‐Pen2,d‐Pen5]‐enkephalin, a δ‐selective opioid agonist, and U50488H, a κ‐selective opioid agonist, failed to compete with [3H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. In the absence of guanine nucleotides, the DAMGO displacement curve for [3H]diprenorphine binding sites better fit a two‐site than a one‐site model with KDhigh = 2.17 ± 1.5 nM, Bmax = 648 ± 110 fmol/mg of protein and KDlow = 468 ± 63 nM, Bmax = 253 ± 84 fmol/mg of protein. In the presence of 10 µM guanosine 5′‐O‐(3‐thiotriphosphate), the DAMGO displacement curve better fit a one‐ than a two‐site model with KD = 815 ± 33 nM, Bmax = 965 ± 124 fmol/mg of protein.

Evidence for MOR on cell membrane, sarcoplasmatic reticulum and mitochondria in left ventricular myocardium in rats

“…Heart tissue was solubilized according to Weems et al [37]. Western blot analyses were performed in duplicate as previously described using specific antisera for MOR and POMC [6,38].…”

Cellular localization and adaptive changes of the cardiac delta opioid receptor system in an experimental model of heart failure in rats

“…Heart tissue (ACF: n = 3; Control: n = 3) was solubilized according to Weems and colleagues [28]. Western blot analyses were performed in duplicate as previously described using specific antisera for DOR and PENK [29].…”

Upregulation of the kappa opioidergic system in left ventricular rat myocardium in response to volume overload

“…Heart tissue was solubilized according to Weems et al [26]. Western blot analyses were performed in duplicate as previously described by using specific antisera for KOR and PDYN [27].…”