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For a reappraisal of the current perplexing notion that the small intestine of the rat lacks in vitro opiate receptor binding, crude microsomal fractions were prepared from longitudinal muscle with attached myenteric plexus, isolated from rat small intestine, carefully dissected free from the mesentery. These preparations, when incubated with the potent opiate etorphine, tritium labelled at high specific activity (31 Ci/mmole), exhibited high affinity (K D = 1.2 nM) saturable binding. This in vitro binding of 3 H-etorphine (2×10 −9 M) was stereospecific, since it was completely prevented by 1×10 −6 M levorphanol and unaffected by the same concentration of dextrorphan. When 10 mM NaCl was added, considerably less naloxone was required than in control medium for inhibiting the binding of tritiated etorphine to the rat small intestine longitudinal muscle microsomal preparation. Our present evidence for substantial in vitro opiate receptor binding in the rat small intestine agrees with previous results of pharmacological and in vivo binding studies supporting the existence of specific sites of action for narcotics therein.

Scatchard analysis of 3H-dihydromorphine (3H-DHM) binding to rat brain subcellular fractions gave a curvilinear plot in the microsomal (P3) and a linear plot in the mitochondrial (P2) fraction, respectively. In contrast, plots of 3H-ethylketocyclazocine (3H-EK) binding were linear in the P3 and curvilinear in the P2 fraction. No differences were seen in the potency of morphine and ketazocine in displacing either 3H-DHM or 3H-EK in the P2 fraction. In the P3 fraction, however, morphine was more potent than ketazocine in displacing 3H-DHM, but the reverse was true when 3H-EK was used as the ligand. Isolation of the P3 fraction may facilitate the demonstration of a kappa receptor in the rat brain.

The rat mesentery was found to contain an interfering material responsible for failure to detect in vitro opiate receptor binding in preparations from rat small intestine longitudinal muscle. A crude microsomal fraction obtained from the mesentery, after dilution so as to deliver 8 μg protein per sample, was still capable of producing substantial inhibition (50%) of 0.4 nM 3H-etorphine binding to 0.4 mg protein from brain microsomes. Scatchard analysis indicated a decrease in the number of binding sites without relevant changes in their affinity. The inhibitory potency of rat mesentery preparations on tritiated etorphine binding was : reduced but not abolished in the presence of soybean trypsin inhibitor; unaffected by EGTA 2.5 × 10−3M, which completely preserved binding to brain microsomes incubated with 50 ng/ml phospholipase A2 from porcine pancreas; increased by preincubation at 37°C with brain microsomes; variably affected - depending on the separately prepared batches of mesentery tested - by boiling, which however revealed a heat-resistent component consistently accounting to a large extent for the observed binding inhibition. Interference by an unidentified factor occurring in the mesentery - perhaps an opioid peptide - and possible contamination with pancreatic tissue containing proteases, presumably explain why previous studies failed to demonstrate in vitro opiate receptor binding in rat small intestine.