01 nM) and a half-maximal effect at 0.70�C1.0 nM (Fig. 4, A and B; Table 2). Peptide #1 was 3- to 12-fold more potent at activating PLC in hNMB-R cells than was the natural ligand NMB (1�C12 blog post nM; Fig. 4, A and B; Table 2), and 38- to 700-fold more potent at stimulating the production of [3H]IP than was GRP. Neither Bantag-1 nor MK-5046 activated hGRP-R, even at concentrations of 1000 nM (Fig. 4, A and B; Table 2). To further explore the ability of Bantag-1 to function as a possible hBRS-3�Creceptor antagonist, we assessed its ability to inhibit a maximally effective concentration of either peptide #1 (Fig. 5, A and B) or the nonpeptide agonist MK-5046 (Fig. 5, C and D). The antagonist Bantag-1 causes a concentration-dependent inhibition of PLC stimulation caused by both agonists (i.e.

, peptide #1, MK-5046) in the two hBRS-3 cell types (Fig. 5, A�CD). With peptide #1, Bantag-1 caused detectable inhibition at 1 nM, half-maximal inhibition at 52.3 �� 2.7 nM, and complete inhibition at 10 ��M in hBRS-3 Balb 3T3 cells (Fig. 5A). In NCI-N417 cells, Bantag-1 caused detectable inhibition of stimulation by peptide #1 at 30 nM, half-maximal inhibition at 137 �� 15 nM, and complete inhibition at 10 ��M (Fig. 5B). With MK-5046 (0.01 ��M) in hBRS-3 Balb 3T3 cells, Bantag-1 caused half-maximal inhibition at 27.5 �� 2.5 nM (Fig. 5C), whereas in NCI-N417 cells half-maximal inhibition occurred at 3.2 �� 0.56 nM (Fig. 5D). A 100 nM concentration of Bantag-1 caused a parallel rightward shift in the peptide #1 (Fig. 6, A and C) and MK-5046 (Fig. 6, B and D) dose-response curves for generation of [3H]IP.

A lesser concentration of Bantag-1 caused a proportional shift in the peptide #1 dose-response curve (data not shown), and when this rightward shift was analyzed by determining the Schild coefficient, it was 1.04 �� 0.09 for peptide #1 in hBRS-3 Balb 3T3 cells (not significantly different than unity), compatible with it functioning as a competitive antagonist with an affinity of 0.57 �� 0.15 nM. Fig. 5. Bantag-1 inhibits [3H]IP production in hBRS-3 Balb 3T3 (A and C) and NCI-N417 (B and D) cells. (A and B) Antagonist Bantag-1 alters [3H]IP production stimulated by 0.1 ��M peptide #1. The results are expressed as the percentage of stimulation caused … Fig. 6. Bantag-1 alters the dose-response curves of peptide #1 and MK-5046 stimulated [3H]IP in cells containing hBRS-3.

(A and C) Effect of the antagonist Bantag-1 (100 nM) on the dose-response curve of peptide #1 for stimulating [3H] production. The results … To gain possible insight into the biphasic nature of the MK-5046 dose-response curve for stimulating [3H]IP generation, Cilengitide we examined the effect of adding increasing concentrations of MK-5046 to a maximally effective concentration of peptide #1 (Fig. 7, A and B) or of adding increasing concentrations of the antagonist Bantag-1 to a supramaximal concentration of MK-5046 (Fig. 7, C and D).