Evidence for distinct phencyclidine and SKF10047 receptors following detergent treatment of rat brain membranes.

In order to compare characteristics of benzomorphan and phencyclidine receptors, binding of the ligands [3H]SKF10047 and [3H]phencyclidine (PCP) was measured in intact rat synaptosomal membranes and in membranes treated with a detergent (CHAPS, a twitterionic derivative of cholic acid). Ligand binding was quantified in the particle containing fractions and in particle-free supernatants. About 20% of the SKF binding sites could be solubilized from the membranes by CHAPS under the conditions used here, while all PCP binding sites remained associated to particles. This observation and the inhibition patterns found for the two ligands indicate that the PCP receptors and the SKF receptors as delineated in this paper are indeed separate.

Effect of opioid agonists selective for three subclasses of opioid receptors and one sigma receptor agonist on dopamine stimulated adenylate cyclase in rat caudate nucleus.

The adenylate cyclase in rat caudate nucleus homogenate could be stimulated by dopamine and less potently by the dopamine D1 receptor specific agonist SKF38393. Agonists selective for mu[D-Ala2, MePhe4Gly(ol)5]enkephalin (DAGO) and delta opioid receptors [D-Pen2, D-Pen5]enkephalin (dPen-dPen), inhibited the dopamine but not the dopamine D1 stimulated adenylate cyclase. The kappa opioid agonist, U69593, had no effect, probably due to low kappa receptor contents in rat caudate nucleus. 10(-4) M of the sigma receptor specific agonist, 1,3-di-o-tolylguanidine (DTG), potentiated the dopamine as well as the dopamine D1 stimulated adenylate cyclase while lower concentrations of DTG had no effect.

The effect of two different buffers on the high affinity 3H-ethylketocyclazocine and 3H-SKF10047 binding to guinea pig brain membranes.

In vitro mu and delta opioid receptor binding is known to be influenced by ions. High affinity 3H-SKF10047 and 3H-ethylketocyclazocine binding sites are found in brain membranes and postulated to be similar to mu opioid receptor binding. To investigate this postulate, we have studied how the high affinity binding of 3H-SKF10047, 3H-ethylketocyclazocine, a tritiated mu agonist, mu antagonist and delta agonist is altered when the radioreceptor binding assay incubation buffer is changed. The binding of 3H-ethylketocyclazocine and the mu antagonist (3H-naloxone) is highest in isotonic HEPES buffer, while the binding of the mu (3H-dihydromorphine) and delta (3H-D-ala-D-leu-enkephalin) agonist is highest in hypotonic Tris-HCl buffer. 3H-SKF10047 binding is similar in the two buffers. The inhibition of 3H-ethylketocyclazocine, 3H-SKF10047 and tritiated mu and delta opioid ligands by seven unlabeled ligands is then compared in the two buffers. Morphine chloride is a more potent inhibitor of 3H-ethylketocyclazocine binding and tritiated mu ligand in hypotonic Tris-HCl buffer than in isotonic HEPES buffer. The potency of naloxone, nalorphine, SKF10047, D-ala-D-leu-enkephalin, cyclazocine and phencyclidine in inhibiting 3H-ethylketocyclazocine binding is independent of the buffer system. None of the seven unlabelled substances change potency with buffer change when inhibiting the 1.2 nM 3H-SKF10047 binding. In sum our results show that 1 nM 3H-ethylketocyclazocine binding is influenced by buffer change in a manner very similar to mu ligand binding, while the 1.2 nM 3H-SKF10047 binding is only slightly influenced by buffer change and therefore different from mu ligand binding.

Detergent extraction from rat synaptosomal plasma membranes reveals difference in mu and delta opioid receptor binding.

Rat synaptosomal plasma membranes were extracted with a detergent (CHAPS, a zwitterionic derivative of cholic acid). mu and delta opioid receptor binding and adenylate cyclase activities were tested in the intact membranes and in the supernatants from detergent treated membranes. The 6000 X g/8 min. supernatant contained mu receptor binding equal to 33% of the mu receptor binding measured in the untreated membranes. When the detergent treated membranes were sedimented at (50,000 X g/10 min.), 23% of the mu receptor binding was recovered in the supernatant. After a 100,000 X g/30 min. centrifugation the supernatant contained 10% of the mu receptor binding when compared to untreated membranes. Of the delta receptor binding found in intact membranes, 10% or less was recovered in the 3 supernatants described above. Furthermore, the mu and delta receptor binding were distributed differently among particles in the supernatants. This indicates differences in the chemical properties of the mu and delta opioid receptors. Adenylate cyclase assays showed that the G/F site of this enzyme complex was inactivated in the supernatants from detergent treated membranes parallel to the delta receptor binding decrease. However, the catalytic part of adenylate cyclase was present in the supernatants and seemed resistant to the detergent.