Date of Award
Doctor of Philosophy (PhD)
Professor Edwin E. Daniel
Localization and subtype distribution of the receptors for neurotensin and opioids in canine small intestine was studied by the radioligand binding technique.
Extensive dissection procedure was developed to separate a) longitudinal muscle (LM) layer containing myenteric plexus (MP), b) the circular muscle (CM) layer containing the deep muscular plexus (DMP) and c) the submucosa containing the submucous plexus (SMP).
Purified membranes were prepared from the LM, MP, CM, DMP and SMP by differential and the density gradient centrifugations and using the markers 5'-nucleotidase (for smooth muscle plasma membranes), NADPH cytochrome C reductase (for endoplasmaic reticulum), cytochrome C oxidase (for mitochondrial membranes), specific binding of [³H]saxitoxin for the neuronal membranes and the content of the vasoactive intestinal polypeptide immunoreactive material as a measure of intact synaptosomes.
The fractions enriched in the membranes from LM, CM, DMP, MP and SMP were used to study the distribution and properties of neurotensin receptors using [¹²⁵I]Tyr³-neurotensin and of opioid receptors using [³H]diprenorphine, [³H]etorphine and [³H]ethylketocyclazocine.
Neurotensin receptors were confined to the CM, DMP and SMP. These receptors had the similar affinity and recognition properties at their high affinity sites (Kd 0.1 - 0.2 nM). The low affinity receptors on the DMP were of lower affinity than their CM counterparts (Kd 40 nM vs 3 nM). The receptors on the CM differed from those on the DMP in their radiation target size (mw ~ 190,000 da. on the CM and ~120,000 da. on the DMP). Reduced disulfide bridges were required for the binding to both the CM and DMP neurotensin receptors. However, in vitro, the excitation, but not the inhibition, of the circular muscle strips to added neurotensin was abolished on reduction of the disulfide bonds.
Opioid receptors were present on the DMP, MP and SMP but not on any smooth muscle. The receptors on all the three plexuses had similar affinity for the non-selective opioid ligand [³H]diprenorphine (Kd ~0.1 - 0.2 nM). Both the DMP and MP contained ~40-45% of μ- and δ-subtypes of opioid receptors as assessed in competition studies using μ-selective PLO17, and δ-selective DPDPE. SMP contained a higher proportion of μ- over δ-subtype (64% μ and 24% δ). All three plexuses contained similar proportion of k-subtype (10-15%), assessed by competition studies with dynorphin[1-13] and U-50488H, and confirmed by saturation experiments with [³H]ethylketocyclazocine with or without sheilded μ- and δ-receptors. Ionic regulation of these receptors was similar to those observed for the opioid receptors in other systems.
Therefore the action of neurotensin on the motility of the canine small intestine may be the combined result of its action on the smooth muscle and on the modulation of the release of other mediators presynaptically at the DMP level. The action of opioids on the motility is probably through the modulation of the release of other mediatorsat the DMP and MP level: direct action of opioids on the smooth muscle is not supported by the present studies.
Ahmad, Sultan, "Peptide Receptors in Canine Small Intestine" (1989). Open Access Dissertations and Theses. Paper 1923.