(Leu13)-Motilin (human, porcine)

Pharmacological studies indicate that in man and in rabbit, but not in dog, motilin has a direct influence upon gastrointestinal smooth muscle. In accordance with this hypothesis we have presented direct biochemical evidence for the presence of motilin receptors on rabbit smooth muscle tissue. We have now extended our studies to human and canine tissue. Tissue homogenates were studied in binding experiments with iodinated porcine [Leu13]motilin and iodinated canine motilin. It was ascertained that the iodination procedure had little effect on the biological activity of the porcine analogue. In the human antrum specific binding of the iodinated porcine analogue was only found in the smooth muscle layer. It was absent in mucosal or serosal preparations. At 30 degrees C and pH 8.0, binding was maximal after 60 min of incubation, and was reversed by the addition of unlabeled porcine motilin. Binding was enhanced in the presence of calcium and magnesium ions. At a concentration of 10 mM MgCl2, binding was 220% of the binding observed in its absence. Displacement studies with synthetic porcine [Leu13]motilin or synthetic natural porcine motilin indicated a dissociation constant (Kd) of 3.6 +/- 1.6 nM and a maximal binding capacity (Bmax) of 77 +/- 9 fmol per mg protein. Canine motilin displaced iodinated porcine motilin with an apparent Kd of 2.2 +/- 0.9 nM. Compared to antral binding, receptor density decreased aborally and orally, and was absent in jejunum and ileum. In dog specific binding could not be demonstrated in antral and duodenal tissue, neither with labeled porcine nor with labeled canine motilin. However, labeled canine motilin was equipotent to labeled porcine motilin in binding studies with human tissue: the dissociation constant was 0.9 +/- 0.6 nM. The present studies therefore demonstrate the existence of a specific motilin receptor in the antroduodenal region of the human gut. Apparently, such receptors are not present in the canine gut. Our data support the hypothesis that in the human gastrointestinal tract, the gastroduodenal area is motilin's target region.

Background:Our laboratory has described the presence of motilin receptors in the rabbit cerebellum. We discovered its presence in the human TE671 cell line, which is of cerebellar origin.Methods:Cytosolic Ca(2+) fluxes were monitored on a confocal microscope in cells loaded with Indo-1 and stimulated with motilin under various conditions. Binding studies were performed with 125I-[Nle(13)]porcine motilin. Using primers, PCR for the motilin receptor was performed.Results:Cells responded to motilin after 45+/-20 s. At different concentrations of motilin (10(-8), 10(-7), 10(-6.5), 10(-6) and 10(-5) M) the percentage of responding cells was 0+/-0, 0.6+/-1.5, 4.9+/-4.7, 21.7+/-15 and 35.7+/-12, respectively. The response was blocked by the motilin antagonists [Phe(3), Nle(13)]po-motilin (0.8+/-1.8%) and GM-109 (0.0+/-0.0%) and mimicked by the agonist ABT-229 (23.6+/-15%). After stimulation with motilin, ABT-229 or [Phe(3),Leu(13)]po-motilin, but not with the antagonist GM-109, cells were desensitized. The response to motilin persisted in Ca(2+)-free solution (22.8+/-14.7%), was not affected by nifedipine (44+/-11%) but was abolished by incubation with thapsigargin (0+/-0%). Neither ryanodine, nor a previous stimulation with caffeine (0+/-0%) in Ca(2+)-free Krebs, nor both could block the response to motilin (28, 32.0+/-5.7, 41.3+/-6.1%, respectively). Binding studies revealed two binding sites for motilin, with a pK(d) of 8.9+/-0.05 and 6.11+/-0.61 (n=4). There were 100 times more low than high affinity receptors per cell. The presence of receptor mRNA was confirmed by PCR.Conclusion:Functional motilin receptors are present in TE671 cells. The response requires intracellular IP(3)-sensitive Ca(2+) stores. These cells may serve as a model of the central motilin receptor.

We describe the antagonistic properties due to the replacement of Pro3 by phenylalanine in porcine motilin. The analogue, [Phe3,Leu13] porcine motilin (OHM-11526), displaces iodinated [Nle13]porcine motilin bound to a homogenate of rabbit antral smooth muscle tissue. The dissociation constant (pKd) was 9.26 +/- 0.04, versus 9.11 +/- 0.01 for motilin and 8.24 +/- 0.06 for ANQ-11125, the (1-14) fragment of OHM-11526. The Hill coefficient was close to one and Schild plot analysis confirmed the competitive nature of the interaction. In the tissue bath OHM-11526 was unable to induce contractions of segments of rabbit duodenum. At a concentration of 10(-6) M, OHM-11526 was unable to induce contractions of segments of rabbit duodenum. At a concentration of 10(-6) M, OHM-11526 inhibited the effect of maximally effective doses of porcine motilin and of the erythromycin derivative, EM-523, but was without effect on contractions induced by acetylcholine, substance P and serotonin. Increasing doses of OHM-11526 shifted the dose-response curves of motilin and EM-523 to the right, but caused a depression of the maximal response as well. From the motilin curves, and assuming a dual competitive and non-competitive interaction, the pA2 was 7.79 +/- 0.08, the pD'2 6.91 +/- 0.08. The EM-523 curves yielded comparable data (pA2 = 8.10 +/- 0.12 and pD'2 = 7.06 +/- 0.13). OHM-11526 also blocked the motilin responses observed with smooth muscle strips from the rabbit and human antrum. However, in a preparation of the chicken small intestine, OHM-11526 was a full agonist with a potency (pD2 = 6.84) comparable to that of porcine motilin (pD2 = 6.71). Our data confirm the interaction of motilides with the motilin receptor. Due to its increased affinity for the motilin receptor, OHM-11526 will be a valuable took for studying the physiology of motilin and the pharmacology of motilin and motilides.