Henri B. Weems’s research while affiliated with Uniformed Services University of the Health Sciences and other places

Antibodies directed against the C-terminal and the N-terminal regions of the μ-opioid receptor were generated to identify the G proteins that coimmunoprecipitate with the μ receptor. Two fusion proteins were constructed: One contained the 50 C-terminal amino acids of the μ receptor, and the other contained 61 amino acids near the N terminus of the receptor. Antisera directed against both fusion proteins were capable of immunoprecipitating ∼70% of solubilized rat brain μ receptors as determined by [3H][D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin ([3H]DAMGO) saturation binding. The material immunoprecipitated with both of the antisera was recognized as a broad band with a molecular mass between 60 and 75 kDa when screened in a western blot. Guanosine 5′-O-(3-thiotriphosphate) (GTPγS) had an EC50 of 0.4 nM in diminishing [3H]DAMGO binding to the immunoprecipitated pellet. The ratio of G proteins to μ receptors in the immunoprecipitated material was 1:1. When the material immunoprecipitated with affinity-purified antibody was screened for the presence of G protein α subunits, it was determined that Gαo, Gαi1, Gαi3, and to a lesser extent Gαi2, but not Gαs or Gαq/11, were coimmunoprecipitated with the μ receptor. Inclusion of GTPγS during the immunoprecipitation process abolished the coimmunoprecipitation of G proteins.

Antibodies directed against the C-terminal and the N-terminal regions of the mu-opioid receptor were generated to identify the G proteins that coimmunoprecipitate with the mu receptor. Two fusion proteins were constructed: One contained the 50 C-terminal amino acids of the mu receptor, and the other contained 61 amino acids near the N terminus of the receptor. Antisera directed against both fusion proteins were capable of immunoprecipitating approximately 70% of solubilized rat brain mu receptors as determined by [3H][D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin ([3H]DAMGO) saturation binding. The material immunoprecipitated with both of the antisera was recognized as a broad band with a molecular mass between 60 and 75 kDa when screened in a western blot. Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) had an EC50 of 0.4 nM in diminishing [3H]DAMGO binding to the immunoprecipitated pellet. The ratio of G proteins to mu receptors in the immunoprecipitated material was 1:1. When the material immunoprecipitated with affinity-purified antibody was screened for the presence of G protein a subunits, it was determined that G(alpha)o, G(alpha)i1, G(alpha)i3, and to a lesser extent G(alpha)i2, but not G(alpha)s or G(alpha)q11, were coimmunoprecipitated with the mu receptor. Inclusion of GTPgammaS during the immunoprecipitation process abolished the coimmunoprecipitation of G proteins.

High-affinity mu-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 mu-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-Dl5]-enkephalin([3H]DAMGO), a mu-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 microM 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 microM competed with [3H]diprenorphine for the solubilized binding sites; in contrast, [D-Pen2, D-Pen5]-enkephalin, a delta-selective opioid agonist, and U50488H, a kappa-selective opioid agonist, failed to compete with [3H]diprenorphine for the solubilized binding sites at concentrations of < 1 microM. 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 microM 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.

The effect of chronic naltrexone administration on the expression of mu opioid receptors on 7315c tumor cells was examined. Osmotic minipumps containing either saline or naltrexone were subcutaneously implanted into Buffalo rats that had been injected intraperitoneally with 7315c cells. Fourteen days after the pumps were implanted, 7315c tissue and brain tissue were removed and examined for their ability to bind [3H]DAMGO and to respond to morphine (or DAMGO) and guanosine 5'-O-(3-thiotriphosphate) in an adenylyl cyclase assay. Naltrexone treatment caused a doubling in the density of [3H]DAMGO binding sites in both whole brain membranes and the 7315c cell membranes. Naltrexone treatment may have slightly diminished the affinity of mu opioid receptors for [3H]DAMGO (by 1.5- to 2-fold), but the precision of the assay was inadequate to determine whether this difference was significant. Naltrexone treatment also had no effect on the potency or efficacy of guanosine 5'-O-(3-thiotriphosphate) in diminishing [3H]DAMGO binding to either whole brain or 7315c cell membranes. The influence of naltrexone treatment on opioid inhibition of adenylyl cyclase activity was also investigated in both tissues. In 7315c membranes, naltrexone treatment caused a 40% increase in the efficacy (maximal effect) of morphine but had no effect on the potency (IC50) of morphine in inhibiting forskolin-stimulated adenylyl cyclase activity. In whole brain membranes from control rats, DAMGO failed to affect significantly forskolin-stimulated adenylyl cyclase. However, in whole brain membranes from naltrexone-treated rats, DAMGO caused a 30% inhibition of forskolin-stimulated adenylyl cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

High-affinity mu-opioid receptors have been solubilized from 7315c cell membranes. Occupancy of the membrane-associated receptors with morphine before their solubilization in the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate was critical for stabilization of the receptor. The solubilized opioid receptor bound [3H]-etorphine with high affinity (KD = 0.304 +/- 0.06 nM; Bmax = 154 +/- 33 fmol/mg of protein). Of the membrane-associated [3H]etorphine binding sites, 40 +/- 5% were recovered in the solubilized fraction. Both mu-selective and non-selective enkephalins competed with [3H]etorphine for the solubilized binding sites; in contrast, delta- and kappa-opioid enkephalins failed to compete with [3H]etorphine for the solubilized binding sites at concentrations of < 1 microM. The mu-selective ligand [3H][D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin also bound with high affinity (KD = 0.79 nM; Bmax = 108 +/- 17 fmol/mg of protein) to the solubilized material. Of the membrane-associated [3H][D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin binding sites, 43 +/- 3% were recovered in the solubilized material. Guanosine 5'-O-(3-thiotriphosphate), GTP, and guanosine 5'-O-(2-thiodiphosphate), but not adenylylimidodiphosphate, diminished [3H][D-Ala2,N-Me-Phe4,Gly5-ol] enkephalin binding in a concentration-dependent manner. Finally, mu-opioid receptors from rat brain membranes were also solubilized in a high-affinity, guanine nucleotide-sensitive state if membrane-associated receptors were occupied with morphine before and during their solubilization with the detergent 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate.

: High-affinity μ-opioid receptors have been solubilized from 7315c cell membranes. Occupancy of the membrane-associated receptors with morphine before their solubilization in the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate was critical for stabilization of the receptor. The solubilized opioid receptor bound [3H]-etorphine with high affinity (KD= 0.304 ± 0.06 nM; Bmax= 154 ± 33 fmol/mg of protein). Of the membrane-associated [3H]etorphine binding sites, 40 ± 5% were recovered in the solubilized fraction. Both μ-selective and non-selective enkephalins competed with [3H]etorphine for the solubilized binding sites; in contrast, 5- and K-opioid enkephalins failed to compete with [3H]etorphine for the solubilized binding sites at concentrations of

Enantiomers of terfenadine were resolved by high-performance liquid chromatography (HPLC) using a chiral stationary phase (CSP) column packed with beta-cyclodextrin (beta-CD) covalently bound to silica. Separation was achieved in both the reverse phase and normal phase modes. Resolution of enantiomers was confirmed by ultraviolet-visible absorption, circular dichroism, and mass spectral analysis.

Enantiomeric pairs of the antihistaminic drug terfenadine and its carboxylic acid derivative were directly separated by HPLC using an ovomucoid protein column. Absolute configurations of terfenadine enantiomers were assigned by comparing their circular dichroism spectra with those of 1-phenyl-1-butanol enantiomers of known absolute stereochemistry. Terfenadine and its major carboxylic acid metabolite extracted from blood plasma following an oral administration of a racemic terfenadine to rats were found to be enriched in the (S)- and (R)-enantiomers, respectively. The results indicated that the (R)-enantiomer of an orally administered racemic terfenadine was preferentially oxidized in rats to form a carboxylic acid metabolite enriched in the (R)-enantiomer.