Characterization of specific opioid binding sites in neural membranes from the myenteric plexus of porcine small intestine.

Delta- and kappa-opioid receptors (OPRs), but not micro-OPRs, are expressed in the myenteric plexus of the porcine distal small intestine. In a subpopulation of myenteric neurons, delta- and kappa-OPRs seem to be colocalized and may functionally interact. In this study, radioligand binding was used to characterize myenteric OPR populations in detail. The nonselective OPR antagonist [3H]diprenorphine bound to a single, high-affinity site in myenteric neural membrane homogenates. Naloxone displaced 65 and 59% of [3H]diprenorphine binding from this site in Na(+)-free Tris and Krebs-HEPES buffers, respectively. Naltrexone-derived delta- and kappa-OPR antagonists, including naltriben, 7-benzylidenenaltrexone, nor-binaltorphimine, and 5'-guanidinonaltrindole, displaced [3H]diprenorphine from two distinct binding sites to levels similar to that of naloxone. The selective delta-OPR ligands Tyr-1,2,3,4-tetrahydroisoquinoline-Phe-Phe-OH (TIPP), [D-Pen2,D-Pen5]enkephalin (DPDPE), [D-Ala2, Glu4]deltorphin II, and (+)-4-[(alphaR)-alpha((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl-3-methoxybenzyl)-N,N-diethylbenzamide (SNC-80) and the kappa-OPR agonist (D-(5alpha,7alpha,8beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxoaspiro-(4,5)dec-8-yl) benzeneacetamide (U-69,593) displaced [3H]diprenorphine from three independent binding sites; these included high-affinity delta- and kappa-OPR sites, and a residual binding site. Residual [3H]diprenorphine binding was displaced by the selective kappa-OPR antagonist nor-binaltorphimine after saturation of delta and kappa sites, respectively, with DPDPE and U-69,593. The residual binding site displayed low affinity for delta- and kappa-OPR agonists and TIPP, as well as moderate affinity for naltrexone-derived ligands, properties reminiscent of delta-/kappa-OPR heterodimers.