[D-Arg1,D-Phe5,D-Trp7,9,Leu11]-Substance P

Ghrelin is a GH-releasing peptide that also has an important role as an orexigenic hormone-stimulating food intake. By measuring inositol phosphate turnover or by using a reporter assay for transcriptional activity controlled by cAMP-responsive elements, the ghrelin receptor showed strong, ligand-independent signaling in transfected COS-7 or human embryonic kidney 293 cells. Ghrelin and a number of the known nonpeptide GH secretagogues acted as agonists stimulating inositol phosphate turnover further. In contrast, the low potency ghrelin antagonist, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P was surprisingly found to be a high potency (EC50 = 5.2 nm) full inverse agonist as it decreased the constitutive signaling of the ghrelin receptor down to that observed in untransfected cells. The homologous motilin receptor functioned as a negative control as it did not display any sign of constitutive activity; however, upon agonist stimulation the motilin receptor signaled as strongly as the unstimulated ghrelin receptor. It is concluded that the ghrelin receptor is highly constitutively active and that this activity could be of physiological importance in its role as a regulator of both GH secretion and appetite control.

Substance P derivatives are potential therapeutic compounds for the treatment of small cell lung cancer and can cause apoptosis in small cell lung cancer cells in culture. These peptides act as broad spectrum neuropeptide antagonists, blocking calcium mobilization induced by gastrin-releasing peptide, bradykinin, cholecystokinin, and other neuropeptides. We show that [D-Arg1,D-Phe5,D-Trp7,9, Leu11]substance P has unique agonist activities in addition to this described antagonist function. At doses that block calcium mobilization by neuropeptides, this peptide causes activation of c-Jun N-terminal kinase and cytoskeletal changes in Swiss 3T3 fibroblasts and stimulates migration and calcium flux in human neutrophils. Activation of c-Jun N-terminal kinase is dependent on the expression of the gastrin-releasing peptide receptor in rat 1A fibroblasts, demonstrating that the responses to the peptide are receptor-mediated. We hypothesize that [D-Arg1,D-Phe5,D-Trp7,9, Leu11]substance P acts as a biased agonist on neuropeptide and related receptors, activating certain guanine nucleotide-binding proteins through the receptor, but not others.

BALB/c 3T3 cells do not normally express receptors for bombesin-like peptides [bombesin (Bn), gastrin-releasing peptide (GRP), and neuromedin B (NmB)]. Transfection of BALB/c 3T3 cells with complementary DNA-encoding GRP receptors or NmB receptors leads to stable expression of functional GRP receptors (GRP Rt) or NmB receptors (NmB Rt), respectively, which are coupled to cell membrane ion channels. Whole cell current analysis using patch electrodes shows that the activation of these newly expressed receptors induces cation conductance increases, most frequently a Ca(2+)-activated plasma membrane K+ conductance. The dose-response (peak-current) relations of both transfected receptor subtypes were sigmoidal and exhibited threshold activation concentration in the picomole range and the saturation of responses to higher concentrations than 10(-8) M. The GRP Rt responded about equally to GRP, NmB, and Bn when compared at equimolar levels, despite their known difference in binding affinity for the three peptides (GRP, Bn > NmB). In contrast, for the NmB Rt, the NmB was more potent than GRP or Bn. Among four GRP/Bn-receptor antagonists tested, the [D-Phe6]Bn(6-13) ethyl ester suppressed GRP Rt responses at low concentrations (10(-7) M).