sr-146131 and Neuroblastoma

The effect of ¿2-[4-(4-chloro-2, 5-dimethoxy-phenyl)-5-[2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoy l]-5, 7-dimethyl-indol-1-yl¿-acetic acid (SR146131), a novel non-peptide agonist of cholecystokinin (CCK) CCK(1) receptors, was compared to the effect of sulphated cholecystokinin octapeptide (CCK-8-S) on CCK(1) receptors of the human neuroblastoma cell line IMR-32. SR146131 inhibited [125I]CCK-8-S binding to IMR-32 cells at nanomolar concentrations. SR146131 and CCK-8-S increased intracellular free Ca(2+) levels ([Ca(2+)](i)) in the same concentration range (EC(50)=6+/-2.3 and 1.3+/-0.14 nM, respectively). Although the shape of the [Ca(2+)](i) increase induced by CCK-8-S and SR146131 was slightly different, extracellular Ca(2+) removal affected the response of both compounds to a similar degree, and the response of both compounds was essentially due to Ca(2+) release from intracellular stores. This was also confirmed by measuring the [Ca(2+)](i) response of single cells: both compounds induced [Ca(2+)](i) oscillations at subnanomolar concentrations and elicited a large peak increase in [Ca(2+)](i) at higher concentrations (EC(50)=0.5+/-0.04 and 5.7+/-1.9 nM for CCK-8-S and SR146131, respectively). Both CCK-8-S and SR146131 induced a sustained increase of phosphoinositide turnover in these cells, and acted at similar concentrations (EC(50)=2.7+/-0.7 and 6+/-3.1 nM, respectively), although the maximal effect of SR146131 was somewhat lower than the effect of CCK-8-S. These data show that SR146131 activates human CCK(1) receptors on IMR-32 cells in a manner and with a potency similar to that of CCK-8-S.

Topics: Benzodiazepinones; Binding, Competitive; Calcium; Devazepide; Dose-Response Relationship, Drug; Humans; Indoles; Iodine Radioisotopes; Neuroblastoma; Phenylurea Compounds; Phosphatidylinositols; Radioligand Assay; Receptors, Cholecystokinin; Sincalide; Thiazoles; Tumor Cells, Cultured

SR146131 inhibited the binding of [125I]-Bolton Hunter (BH)-sulfated cholecystokinin octapeptide (CCK-8S) for the human recombinant cholecystokinin subtype 1 (CCK1) receptor (IC50 = 0.56 nM) with high (300-fold) selectivity to the CCK2 receptor. The biological activity of SR146131 was characterized in vitro in a NIH-3T3 cell line expressing the human recombinant CCK1 receptor (3T3-hCCK1). Measuring intracellular calcium release, SR146131 behaved as a full agonist with an efficacy comparable with that of CCK-8S (EC50 = 1.38 +/- 0.06 nM). On individual cells, SR146131 induced, like CCK-8S, Ca2+ oscillations at subnanomolar concentrations and sustained responses at higher concentrations. Like CCK-8S, SR146131 also fully stimulated inositol monophosphate formation (EC50 = 18 +/- 4 nM). SR146131 partially activated mitogen-activated protein kinase and enhanced the expression of the immediate early gene krox 24. In the human CHP212 and IMR32 neuroblastoma cell lines, which constitutively express the CCK1 receptor, SR146131 behaved as a partial agonist on intracellular calcium release and inositol monophosphate formation. All of these effects of SR146131 were inhibited by the CCK1 receptor antagonists SR27897B and devazepide, suggesting that the effects of SR146131 were entirely mediated by the CCK1 receptor. In contrast, high concentrations (>1 microM) of SR146131 had only minimal effects on CCK-8S-stimulated and unstimulated Chinese hamster ovary (CHO) cells expressing the human CCK2 receptor, indicating that SR146131 is functionally inactive on the CCK2 receptor. In conclusion, these in vitro experiments show that SR146131 is a highly potent and selective agonist of the CCK1 receptor.

Topics: 3T3 Cells; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; CHO Cells; Cricetinae; Devazepide; DNA-Binding Proteins; Early Growth Response Protein 1; Genes, Immediate-Early; Hormone Antagonists; Humans; Immediate-Early Proteins; Indoleacetic Acids; Indoles; Inosine Monophosphate; Mice; Neuroblastoma; Receptor, Cholecystokinin A; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; Recombinant Proteins; Sincalide; Thiazoles; Transcription Factors; Tumor Cells, Cultured