Noncompetitive antagonism of BIBN4096BS on CGRP-induced responses in human subcutaneous arteries

Abstract

We investigated the antagonistic effect of 1-piperidinecarboxamide, N-[2-[[5amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS) on the calcitonin gene-related peptide (CGRP)-induced responses by using isometric myograph and FURA-2 technique in human subcutaneous arteries removed in association with abdominal surgery. BIBN4096BS, at the concentration of 1 pm, had no significant effect on the CGRP-induced relaxation in these vessels. At the concentration of 10 pM, BIBN4096BS had a competitive antagonistic-like behaviour characterized by parallel rightward shift in the log CGRP concentration-tension curve with no depression of the E(max). At the higher concentrations (0.1 and 1 nM), BIBN4096BS had a concentration-dependent noncompetitive antagonistic effect on the CGRP-induced responses. The efficacy and potency of CGRP was significantly greater in the smaller (lumen diameter approximately 200 microM) human subcutaneous arteries compared to the larger ones. The apparent agonist equilibrium dissociation constant, K(A), for CGRP(1) receptors in the human subcutaneous arteries was approximately 1 nM. Analysis of the relationship between receptor occupancy and response to CGRP indicates that the receptor reserve is relatively small. Using reverse transcriptase-polymerase chain reaction (RT-PCR), the presence of mRNA sequences encoding the calcitonin receptor-like receptor, receptor activity modifying protein (RAMP1, RAMP2, RAMP3) and receptor component protein were demonstrated in human subcutaneous arteries, indicating the presence of CGRP(1)-like receptor and the necessary component for the receptor activation. In conclusion, the inhibitory action of BIBN4096BS at the low concentration (10 pM) on the CGRP-tension curve (but not intracellular calcium concentration ([Ca(2+)](i)) resembles what is seen with a reversible competitive antagonist. However, at the higher concentrations (0.1 and 1 nM), BIBN4096BS acts as a selective noncompetitive inhibitor at CGRP(1) receptors in human subcutaneous arteries.

Figure 1

Effect of increasing concentrations of BIBN4096BS (10 pM, 0.1 nM, 1 nM) on the CGRP-induced reduction in the tension (a) and [Ca²⁺]_i (b) of human subcutaneous arteries. The control CGRP concentration–response curves (CRCs) are based on the pooled data. Points represent mean values and vertical bars indicate±s.e.m. Relative tension and [Ca²⁺]_i are given as percentages of the initial steady-state levels induced by 300 nM U46619. A paired two-tailed t-test was used to compare the mean values between the first and second curves (^*P<0.05, ^**P<0.01, ^***P<0.001).

Figure 2

The relationship between the size of lumen diameter of human subcutaneous arteries and (a) the maximal CGRP-induced relaxation in these vessels (linear regression analysis: r=0.91; P<0.0001; n=24) and (b) the sensitivity (pD₂) of human subcutaneous arteries to CGRP (linear regression analysis: r=0.62; P=0.0012; n=24).

Figure 3

Regression line for the plot of reciprocals of equieffective concentrations of CGRP without (A[M] and with 0.1 nM BIBN4096BS (A′[M]).

Figure 4

Demonstration of mRNA encoding the CRLR (size: 497 bp), RAMP1 (size: 445 bp), RAMP2 (size: 283 bp), RAMP3 (size: 159 bp) and RCP (size: 392 bp) in the human subcutaneous areteries by RT-PCR. Ladder: 100-base pair ladder. Blind 1 and 2 are negative controls using primers for CRLR and RAMP1, respectively, without the reverse transcriptase enzyme. Negative controls using primers for RAMP2, RAMP3 and RCP were also performed (not shown).