[D-Arg(1),D-Trp(5,7,9),Leu(11)]substance P coordinately and reversibly inhibits bombesin- and vasopressin-induced signal transduction pathways in Swiss 3T3 cells

The novel substance P (SP) analogue, [D-Arg(1),D-Trp(5,7,9),Leu(11)]SP like [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]SP inhibited DNA synthesis induced by bombesin, vasopressin, and bradykinin, but did not interfere with the mitogenic response induced by other growth factors or pharmacological agents in Swiss 3T3 cells. [D-Arg(1),D-Trp(5,7,9),Leu(11)]SP reversibly inhibited bombesin-induced DNA synthesis, causing a 6-fold greater rightward shift in the bombesin dose response than [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]SP at identical concentrations (10 mu M). We found that the new, more potent, SP analogue coordinately and reversibly inhibited bombesin-induced Ca2+ mobilization and protein kinase C (PKC) and mitogen-activated protein (MAP) kinase activation. The dose-response curves for bombesin-induced Ca2+ mobilization and MAP kinase activation were similarly displaced (51- and 40-fold, respectively) by [D-Arg(1),D-Trp(5,7,9),Leu(11)]SP. In addition, [D-Arg(1),D-Trp(5,7,9),Leu(11)]SP reversibly inhibited bombesin-induced tyrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 bands as well. as p125 focal adhesion kinase. [D-Arg(1),D-Trp(5,7,9),Leu(11)]SP also reversibly and coordinately inhibited vasopressin-induced Ca2+ mobilization, PKC stimulation, MAP kinase activation, tyrosine phosphorylation, and DNA synthesis in Swiss 3T3 cells. Surprisingly, deletion of the terminal Leu of [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]SP to yield [D-Arg(1),D-Phe(5),D-Trp(7,9)]SP1-10 resulted in a selective loss of inhibitory activity of this analogue against bombesin but not vasopressin-stimulated DNA synthesis, Ca2+ mobilization, and MAP kinase activation. Collectively, these results suggest that SP analogues act at the receptor level to coordinately and reversibly antagonize bombesin- or vasopressin-induced signal transduction in Swiss 3T3 cells.