Effect of BIBP3226 on inositol phosphate accumulation and cytosolic calcium level in control and NPY Y1 receptor expressing CHO-K1 cells.

BIBP3226 was developed as a potent, selective and competitive antagonist for NPY Y1 receptors by mimicking the C-terminal part of NPY. In agreement with previous studies, NPY mediated a pertussis toxin sensitive elevation of intracellular calcium concentration in CHO-K1 cells that express recombinant human NPY Y1 receptors which can be inhibited by BIBP3226. Surprisingly micromolar concentrations of BIBP3226 were found to induce by itself a fast increase of intracellular calcium concentration followed by a sustained elevated level of this ion. These responses of BIBP3226 are not mediated by NPY receptor activation since (1) they are still present after NPY receptor activation and desensitization, (2) they are also evoked by the receptor inactive enantiomer BIBP3435, (3) they are not affected by pretreatment of the cells with pertussis toxin, (4) they also occur in non-transfected CHO-K1 cells. Preincubation of the cells with EGTA abolished only the sustained increase calcium concentration elicited by BIBP3226 suggesting that the fast increase of intracellular calcium concentration reflects the mobilization of intracellular calcium pools. The ability of thapsigargin to completely inhibit BIBP3226 mediated responses, in the presence or absence of extracellular calcium indeed indicated that BIBP3226 mobilizes intracellular Ins(1,2,3)P3 sensitive calcium stores. In agreement, BIBP3226 was found to activate phospholipase C since the responses were completely inhibited by U73122. Furthermore, when measured in the presence of 10 mM LiCl, BIBP3226 caused an increased accumulation of inositol phosphates. This effect of BIBP3226 is likely to be mediated by activation of an until now unknown receptor or cellular target that is endogeneously expressed in CHO-K1 cells.