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 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 p K 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.