Demonstration of a functional motilin receptor in TE671 cells from human cerebellum

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 Ca2+ 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 I-125-[Nle(1,3)]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 Ca2+-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 Ca2+-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 IP3-sensitive Ca2+ stores. These cells may serve as a model of the central motilin receptor. (C) 2001 Elsevier Science B.V. All rights reserved.