Substance P evokes a rise in intracellular calcium concentration in cultured myenteric neurons via an inositol trisphosphate pathway

activates similar signaling pathways, has no trophic effects on the gastrointestinal tract. In order to elucidate the cause of the difference, we stably transfected Chinese hamster ovary cells with the cloned human CCKB receptor (CHO-CCK) or m3 receptor (CHO-M3). We found that the activation of the CCK receptor by CCK-8 (0.1 10 riM), but not the m3 receptor by carbachol (up to 0.1 mM), increased the DNA synthesis monitored by 3H-thymidine uptake. We considered these cells were the good models reflecting the physiological phenomena. A transient activation of p42/p44 MAP kinases by carbachol was observed in CHO-M3 ceils as the same extent as that by CCK-8 in the CHO-CCK cells, indicating that the initial signaling is common between these two receptors. When the expression of c-fos gene was quantified after the stimulation of these cells, m3-mediated response was only evident at the highest concentration, whereas that by CCK-8 was not attenuated, suggesting some attenuation beyond MAP kinases in the cholinergic pathway. Ratiometric imaging analysis of the cells using a Ca 2+ indicator, Fura-2, revealed that the maximal ligand occupation of either receptor induced a rapid, transient increase in [Ca2+]i followed by a sustained plateau phase. After 2 hours of stimulation, the [Ca2+]i response to the m3 receptor disappeared, whereas the response to CCKB receptor activation remained as a repetitive [Ca2+]i oscillation. Removal of extracellular Ca 2+, which abolished the [Ca2+]i oscillation, completely inhibited the DNA synthesis in the CHO-CCK cells. When the intracellular C-terminal part of CCKB receptor was truncated or replaced with the corresponding part of m3 receptor, the trophic effect disappeared. We also made chimera receptor of CCKB posessing the third intracellular loop of m3-type, but it could not be permanently expressed in the cell. These results suggest that the desensitization mechanisms might be principal determinants of cell proliferation via Gq-protein coupled receptors, and the persistence of [Ca2+]i increase as [Ca2+]i oscillation could be essential for this type of signal transduction. This work was supported in part by a grant from the Japanese Ministry of Education, Sience, Sports, and Culture.