Substance P-related antagonists inhibit vasopressin and bombesin but not 5'-3-O-(thio)triphosphate-stimulated inositol phosphate production in Swiss 3T3 cells.

The substance P (SP) analogues [DArg1, DPhe5, DTrp7,9, Leu11] SP (AntD) and [Arg6, DTrp7,9, MePhe8] SP (6-11) (AntG) inhibit the action of many different neuropeptides including SP. These analogues might be useful in the treatment of small cell lung cancer but their mechanism of action is unclear. Here, we analyzed the effect of AntD and AntG on neuropeptide vs. guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)-stimulated inositol phosphate generation in permeabilized Swiss 3T3 cells. AntD inhibited vasopressin and bombesin stimulated inositol phosphate formation (IC50 of 0.75 microM and 2 microM, respectively). Similarly, AntG inhibited vasopressin-stimulated inositol phosphate generation with an IC50 of 1 microM. Strikingly, neither AntD up to 10 microM nor AntG up to 20 microM was able to inhibit GTP gamma S-stimulated inositol phosphate generation. Dose-response curves of neuropeptide-induced inositol phosphate generation were dramatically displaced to the right by either 10 microM AntD or 20 microM AntG. However, neither antagonist affected the dose response of GTP gamma S-stimulated inositol phosphate generation. Furthermore, 20 microM AntD had no effect on AIF-4-induced inositol phosphates in COS-1 cells transfected with G alpha q. AntD inhibited [3H]vasopressin binding competitively in intact Swiss 3T3 cells and both AntD and AntG inhibited [3H]vasopressin binding in Swiss 3T3 and rat liver membranes. Scatchard analysis revealed that AntD inhibited vasopressin binding by reducing receptor affinity without affecting receptor number in both intact and membrane preparations of Swiss 3T3 cells. The results strongly suggest that SP analogues AntD and AntG block the action of the Ca2+ mobilizing neuropeptides at the receptor level, rather than inhibiting G protein-stimulated inositol phosphate production.