For inhibition experiments with various phospholipase inhibitors, after a 24-hour incubation with [3H]AA (as performed and described previously), the cells were washed twice with DMEM/BSA. The washed cells were treated with 1 ��M concentrations of the various inhibitors for Tipifarnib purchase 15 minutes before the addition of peptide #1 (0.1 ��M) and were incubated for an additional 30 minutes, after which the release of [3H]AA was measured by counting the supernatant in a ��-counter. Statistical Analysis. All results are expressed as mean �� S.E.M from at least four experiments. P < 0.05 was considered statistically significant, using analysis of variance (ANOVA, nonparametric) or the paired t test when performed with a specifically matched paired control. Prism GraphPad 4.0 was used for all statistical analyses.

In addition, binding data and monophasic dose-response curves of cellular activation were curve-fitted using Prism (nonlinear curve-fitting). Results Affinities for Human Bn Receptors. In this study, we compared the ability of the naturally occurring Bn-related peptides GRP and NMB and the universal Bn-receptor agonist ligand peptide #1 (Mantey et al., 1997; Pradhan et al., 1998) to interact with and activate the three human Bn-receptor subtypes��hGRP-R, hNMB-R, and hBRS-3 (Jensen et al., 2008)��with that of MK-5046, a recently reported (Guan et al., 2011; Sebhat et al., 2011) BRS-3-receptor selective nonpeptide agonist, and Bantag-1, a BRS-3 selective peptide antagonist (Guan et al., 2010; Feng et al., 2011).

To be certain the results were reflective of the pharmacology of these peptides, two different Bn-receptor�Ccontaining cells were used to assess the results with each human Bn-receptor subtype. In the cells containing hBRS-3, hBRS-3 transfected Balb 3T3 cells (Benya et al., 1995; Mantey et al., 1997) and NCI-N417 lung cancer cells, which contain a native hBRS-3�Creceptor (Ryan et al., 1998b; Uehara et al., 2011), the universal ligand peptide #1 had a high affinity for the hBRS-3-receptor (2.5�C2.7 nM; Fig. 1, A and B; Table 1), similar to what was reported by others (Gonzalez et al., 2008; Sancho et al., 2010; Uehara et al., 2011); this affinity was 2-fold lower than the affinity observed with the antagonist Bantag-1 (1.0�C1.6 nM; Fig. 1; Table 1). However it was 8-fold higher than that for the nonpeptide agonist MK-5046 (17�C18 nM; Fig.

1; Table 1), and >10,000-fold higher than the affinity of GRP and NMB for each hBRS-3 cell line Batimastat (Fig. 1; Table 1). TABLE 1 Binding affinities of different ligands for human BRS-3, GRP, and NMB receptors in various cells Fig. 1. Comparison of the affinities of the peptide agonists GRP, NMB, and peptide #1, nonpeptide agonist MK-5046, and the putative peptide antagonist Bantag-1 for the hBRS-3�Creceptor in (A) hBRS-3 Balb 3T3 cells and (B) NCI-N417 cells. The peptides were … In the two cell lines containing hGRP-R, hGRP-R-transfected Balb 3T3 cells (Benya et al.