Available online at www.sciencedirect.com

sclENcE @ D I R E c T e

European Journal of Pharmacology 530 (2006) 107- 116

Characterisation of CGRP receptors in human and porcine isolated coronary arteries: Evidence for CGRP receptor heterogeneity

Saurabh Gupta a , Suneet Mehrotra a, Carlos M. Villalón b, Ingrid M. Garrelds a, René de Vries a,

Jorge P. van Kats ', Hari S. Sharma ", Pramod R. Saxena ", Antoinette MaassenVanDenBrink a'*

" Department of Pharmacology, University Medical Center Rotterdam, PO. Box 1738, 3000 DR Rotterdam, The Netherlands Deparlamenlo de Farmacobiologia, Cinvestav-Coapa, Czda. de los Tenorios 235, Col. Granjas-Coapa. Deleg. Tlalpan, C.? 14330, Mé.>~ico D I , México

Thoracic Surgeiy and Heart Valve Bank, Erasmus MC. Universio. Medical Center Rotterdam, PO. Box 1738, 3000 DR Rotterdam, The Netherlands

Received 12 May 2005; received in revised form 1 November 2005; accepted 8 November 2005

Abstract

This study sets out to charactense calcitonin gene-related peptide (CGRP) receptors in human and porcine isolated proximal and distal coronary artenes using BIBN4096BS. Human (h)-aCGRP induced relaxations that were blocked by BIBN4096BS in al1 artenes studied. In contrast to the other vessels, the Schild plot slope in the human distal coronary artery segments (0.68*0.07) was significantly less than unity and BIBN4096BS potently blocked these responses (pKb (10 nM): 9.29*0.34, n=5). In the same preparation, h-aCGF¿F'8.37 behaved as a weak antagonist of h-aCGRP-induced relaxations (pKb (3 pM): 6.28*0.17, n=4), with also a Schild plot slope smaller than unity. The linear agonists, [ e t h y l a m i d e - ~ y s 2 ~ 7 ] - h - a ~ ~ R P ( [ C ~ S ( E ~ ) ' ~ ~ ] - ~ - ~ C G W ) and [ a c e t i m i d o m e t h y l - ~ y s 2 ~ 7 ] - h - ~ ~ ~ ~ ([C~S(AC~)'~']-~-~CGRP), had a high potency (pECSo: 8.21 *0.25 and 7.25*0.14, respectively), suggesting the presence of CGRP2 receptors, while the potent blockade by BIBN4096BS (pKb (10 nM): 10.13*0.29 and 9.95*0.11, respectively) points to the presence of CGRPI receptors. Using RT-PCR, mRNAs encoding for the essential components for functional CGRPI receptors were demonstrated in both human proximal and distal coronary artery. Further, h-aCGRP (100 nM) increased cAMP levels, and this was attenuated by BIBN4096BS (1 pM). The above results demonstrate the presence of CGRP, receptors in al1 coronary artery segments investigated, but the human distal coronary artery segments seem to have an additional population of CGRP receptors not complying with the currently classified C G W l or CGW2 receptors. O 2005 Elsevier B.V. Al1 nghts reserved.

Kqwords: BIBN4096BS; h-acalcitonin gene-related peptide (h-aCGRP); h-aCGRPx.37; [ C ~ S ( A C ~ ) ' . ~ ] - ~ - ~ C G R P ; [ C ~ S ( E ~ ) ~ - ~ ] - ~ - ~ C G R P ; CGRP receptor; Human coronary artery

1. Introduction

The human calcitonin gene-related peptide (h-aCGRP), a 37-amino acid peptide, is one of the most potent endogenous vasodilators known. It exists in two forms, h-aCGRP and h- BCGRP, which differ from each other by three amino acids. In humans, these forms have almost similar biological actions (Quirion et al., 1992; Van Rossum et al., 1997), but are encoded by separate genes. Although CGRP was fírst described in 1982 (Amara et al., 1982) the classification of CGRP receptors has

* Corresponding author. Tel.: +31 10 408 75 37/47; fax: +31 10 408 94 58. E-mail address: a.vanharen-maassenvandenbrink@erasmusmc.nl

(A. MaassenVanDenBrink). URL: http:llwww.erasmusmc.nVpharmacology/ (A. MaassenVanDenBrink).

been painstakingly slow because of the lack of selective and potent ligands.

Presently, CGRP receptors are functionally classified into CGRPl and CGRP2 types. The CGRPl receptor has been cloned and consists of at least three main entities, namely, the calcitonin receptor like receptor (CLR), receptor activity modifying protein- 1 (RAMP-1) and receptor component protein (RCP) (Luebke et al., 1996; Poyner et al., 2002); in contrast, the CGRP2 receptor, described in some animal tissues (Dumont et al., 1997; Quirion et al., 1992), has not yet been reported in humans or deciphered molecularly. Therefore, the classification and characterisation of CGRP receptors are primarily based on different functional pharmacological responses. In this respect, the C-terminal fragment ~ - ( Y C G R P ~ - ~ , has a higher antagonist potency at the prototypic CGRP, receptor described in guinea-

0014-29991s - see front matter O 2005 Elsevier B.V. Al1 rights reserved. doi:10.1016/j.ejphar.2005.11.020

108 S. Gupla e! al. / Euro~>eun Jor~rnal of Pharmacology 530 (2006) 107-11 6

pig atrium (pKb: 7-8) than at the CGRP2 receptor described in rat vas deferens (pKb: 5.5-6.5) (Dennis et al., 1989; Quirion et al., 1992). However, the range of antagonist affinities reported for h-aCGRPx.37 within and between different species (Poyner et al., 2002) is too wide to be explained by the existence ofjust two receptors. Further, the CGRP2 receptor seems to be more sensitive to the linear agonists [ e t h y l a m i d e - ~ y s 2 ~ 7 ] - h - a ~ ~ R P ( [ c ~ ~ ( E ~ ) ~ ' ~ ] - ~ - ~ c G R P ) and [ a c e t i m i d ~ m e t h ~ l - ~ ~ s ~ ~ ~ ] - h - aCGRP ( [ C ~ S ( A C ~ ) ~ . ~ ] - ~ - ~ C G W ) (pECSO: =7) (Dumont et al., 1997) than the CGRPl receptor (Dennis et al., 1990; Mimeault et al.. 1991). It should be noted that the selectivity of these linear agonists is ambiguous; in porcine large coronary artery [ ~ ~ s ( ~ c r n ) ~ . ~ ] - h - a ~ ~ R P acts like a partial agonist (Waugh et al., 1999), while [ ~ ~ s ( ~ t ) ~ . ~ ] - h - a c ~ R P is known to activate the CGRP, receptor in cell lines (Choksi et al., 2002). However, these linear agonists still are important for the study of CGRP receptors in the absence of more selective CGRP receptor agonists and antagonists.

Interestingly, the responses to CGRP receptor agonists vary depending on the location and the size of blood vessels (Foulkes et al., 1991; Sheykhzade and Nyborg, 1998). Moreover, the relaxation induced by CGRP is endothelium-dependent in the rat aorta (Wisskirchen et al., 1999), while it is endothelium- independent in human and porcine artenes (Luu et al., 1995; Wisskirchen et al., 1999). Therefore, the extrapolation of affinities froin one species to another to classify CGRP receptors should be done with caution.

BIBN4096BS, a lys-tyr dipeptide denvative, (l-pipendine- carboxamide, N-[2-[[5-amino- 1 -[[4-(4-pyndiny1)- l -piperazinyl] carbonyl] pentyl] amino]-1-[(3,5-dibromo-4-hydroxyphenyl) methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazoli- nyl), displays a high antagonist potency and selectivity for human CGRP receptors (Doods et al., 2000; Durham, 2004). Moreover, this antagonist is reported to have a 10-fold higher affinity for the CGRP, receptor (Wu et al., 2000) as compared to the CGRP, receptor. These properties of BIBN4096BS provide the possibility for an in-depth characterisation of CGRP receptors. Although the antagonist potency of BIBN4096BS in the human coronary artery has been studied earlier (Edvinsson et al., 2002), a detailed investigation of CGRP receptors in human coronary arteries has not yet been performed. The fact that BIBN4096BS is effective in the acute treatment of migraine (Olesen et al., 2004) underlines the need for a detailed investigation of CGRP receptors in human blood vessels. Therefore, we used BIBN4096BS and other available conventional CGRP receptor ligands to charactense CGRP receptors in human and porcine isolated coronary arteries. A part of this study has been published as an abstract (Gupta et al., 2004).

2. Materials and methods

2.1. Functional studies

Huinan hearts were obtained from 'heart-beating' donors (22 male, 28 feinale: 48*2 years, range 3-66 years) who died due to non-cardiac causes. The hearts were provided by the Heart

Valve Bank, Rotterdam after donor mediation by Bio Implant Services Foundation/Euro Transplant Foundation (Leiden, The Netherlands). Porcine hearts (pigs of either sex; 6- 12 months of age) were obtained from a local slaughterhouse. In both cases, proximal (intemal diameter: 2-3 mm) and distal (interna1 diameter: 250-600 pm) portions of the right coronary artery were dissected, placed in oxygenated Krebs bicarbonate solution (NaCl 118, KC1 4.7, CaCl? 2.5, MgS04 1.2, KH2P04 1.2, NaHC03 25 and glucose 11.1 mM; pH 7.4) and stored ovemight at 4 "C. Vessel segments containing distinct, macro- scopically visible atherosclerotic lesions were not used.

Proxiinal coronary artery segments (3-4 inm length) were suspended with the help of stainless-steel hooks in 15-m1 organ baths with 15 mN pretension (optimal tension shown in earlier experiments). Dista1 artery segments (1-2 mm length) were placed in Mulvany myographs between two parallel titaniurn wires with a tension normalised to 90% of l loo (distance when transmural pressure equals 100 mm Hg), thus achieving optimal conditions for active force development (Nyborg et al., 1987). The organ baths and myograph chambers, containing Krebs bicar- bonate solution (for composition, see above) at 37 "C, were continuously bubbled with 95% 0 2 and 5% CO:. Since spontaneous contractions frequently occurred in hurnan distal coronary arteries, these experiments were performed in the presence of the cyclo-oxygenase inhibitor indomethacin (0.1 pM) Unless mentioned othenvise, no attempt was made to remove the endothelium.

After an initial equilibration period of 30 min, al1 segments were challenged twice with 30 mM KCl at 30-min intervals to v e n 6 reproducibility of the responses. The integnty of the endothelium was assessed by observing relaxations to substance P (1-10 nM) after precontraction with prostaglandin F2a (1 yM, proximal coronary artenes) or U466 19 (9,l l -dideoxy- 1 l a , 9a- epoxymethano-prostaglandin F2a, 10- 100 nM, distal coronary artenes). In 4 human and 5 porcine distal coronary artery segments, the endothelium was denuded using a human hair. After 30 min, 100 mM KC1 was added to determine the reference contractile response in each seginent. Then, a stable contraction plateau of around 60% of the inaximal contraction was obtained with KCl (30 mM) and, subsequently, the CGRP receptor agonists (i.e. h-aCGRP, h-[XGRP, [ ~ ~ s ( ~ c m ) ~ , ' ] - h - aCGRP and [ C ~ S ( E ~ ) ~ ' ~ ] - ~ - ~ C G R P ; range: 0.1 nM-3 pM) were added in a cumulative manner in 0.5 log steps every 5 min or when the inaxiinum effect of a given concentration was reached. In huinan proximal coronary artery, it was difficult to obtain a stable precontraction with 30 mM KCI. In fact, other compounds, such as U46619, showed even more unstable precontraction in pilot experiments, and the responses to concentrations of CGRP <30 nM were small and difficult to discnminate from the spontaneous decline in the baseline tension. Therefore, we chose to start the concentration- response curves to h-aCGRP at a higher concentration, so that quantification was less sensitive to artefacts. Furthermore, in three human proximal vessels, but none of the distal vessels, the control segment did not respond to h-aCGRP and, consequently, al1 segments from these non-responding vessels were excluded from the study. Only one concentration-

S. Gupra et al. / European Journal of Pharmacology 530 (2006) 107-116 1 09

response curve was constructed in the arterial segments, either in the absence or presence of the antagonists (BIBN4096BS, 0.1 nM-10 pM or ~ - C ~ C G R P ~ . ~ ~ , 0.1-10 pM) that were incubated for a period of 30 min, unless mentioned otherwise.

2.2. Isolation o f total RNA and reverse transcriptase- polyrnerase chain reaction (RT-PCR) studies

After isolation from the heart, the segments of proximal and distal human coronary arteries were snap frozen in liquid nitrogen and stored at -80 "C until use. The tissues were transferred to guanidium thiocyanate buffer, homogenised (Ultra-Turrax homogeniser, model T8; Janke and Kunkel Gmbh, Staufen, Germany) and the total RNA was extracted (Chomczynski and Sacchi, 1987; Sharma et al., 1996). The RNA concentration was measured by UV absorbance at a wavelength of 260 nm using a Gene Quant RNAIDNA calculator (Pharmacia-LKB, Uppsala, Sweden) and the quality of RNA was assessed by formaldehyde agarose gel electropho- resis and a DNAlprotein ratio (OD260/OD280) of >1.8. Total RNA was denatured at 65 "C and the first strand of cDNA was synthesised in a reaction volume of 20 p1 by adding the following reagents: reverse transcription buffer (25 rnM Tris-- HC1; pH 8.3, 50 rnM KCl; 5 mM MgCl,, 2 mM dithiothreitol), 1 mM deoxy nucleotide triphosphate (dNTPs), ribonuclease inhibitor (1 Ulpl), random hexamer (150 nglpg total RNA) and finally moloney-murine leukaemia virus-reverse transcriptase (MMLV-RT) (Pro-omega, Benelux b.v., Leiden, The Nether- lands). A control reaction with al1 of the above ingredients, except MMLV-RT, was prepared. The reactions were carried out for 90 min at 42 "C, extended for another 10 min at 75 "C and then cooled at 4 "C. The cDNA thus synthesised was stored at -20 "C until used as a PCR template.

The quality of cDNA was verified by PCR amplification of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) using specific oligonucleotide primers (see Results section). For PCR amplification, a 20 p1 reaction mixture containing the following components was prepared: 1.25 mM of each dNTP, 3 mM MgCl?, PCR buffer (1 x PCR buffer: 10 mM Tris-HC1. pH 8.3,50 mM KCl) Ampli Taq GoldTM enzyme (0.5 U), 0.5 pM of each of the forward and reverse primers and 2 p1 of the cDNA template. After a brief centrifugation the enzyme was first activated for 5 min at 94 "C in a PCR thermocycler (model PTC- 100TM, MJ Research Inc., Watertown, MA, U.S.A.). PCR was carried out as: 45 s at 94 "C, 45 s at 55 "C and 90 s at 72 "C with a total of 40 cycles. Finally, the reaction was extended for an additional 10 min. For PCR amplification of RAMP-3 cDNA template obtained from the proximal coronary artery, we reduced the concentration of MgCll from 3 mM to 2 mM and performed 35 instead of 40 cycles to eliminate an unspecific band, while the remaining part of the conditions remained unchanged. An aliquot of PCR reaction product was analysed on 2% agarose gel, visualised under UV light and digitally photographed. The different CGRP receptor components were semi-quantitatively assessed using ID-analysis software (Kodak Digital Science 1D Image Analysis, Version 1.6, Scientific Imaging System, Rochester, NY, U.S.A.).

2.3. cAMP rneasurernents

Human proximal and distal, as well as porcine distal coronary artery segrnents were incubated in a medium contain- ing isobutylmethylxanthine (IBMX, 0.5 rnM) for 30 min in the absence or presence of BIBN4096BS (1 pM). The arterial segments were exposed to KC1 (30 mM), challenged with h- aCGRP (100 nM) or forskolin (10 pM) for 5 min and then snap frozen. Forskolin, which increases intracellular cAMP concen- trations by activating adenylyl cyclase, was used to assess the specificity of BIBN4096BS for CGRP-mediated increases in cAMP concentrations. The samples were stored at - 80 "C until cAMP assay using the ELISA kit and manual (R and D Systems Europe Ltd., Abingdom, U.K.).

2.4. Data presentation and statistical analysis

The relaxant responses elicited by agonists are expressed as percentage relaxation of the tone induced by 30 mM KCl. Al1 data are presented as means5S.E.M and n represents the number of blood vessels obtained from different donors. The concentration-response curves for al1 agonists were analysed using non-linear regression analysis and the potency of agonists was expressed as pECso using Graph Pad Prism 3.01 (Graph Pad Software Inc., San Diego, CA, U.S.A.). The blocking potency of the antagonists was estimated by calculating EC50 ratios and plotting a Schild-plot (Arunlakshana and Schild, 1959) using linear regression to get the slope value. Only the ECSo ratios > 2 were used for Schild plot analysis and for calculation of respective pKb values. In certain cases, the slope value was significantly different from one, thus prohibiting us to calculate pA2 values. Therefore, to enable a uniform comparison between groups, "apparent pKb" values were calculated for the antagonists at each given concentration, constraining the slope to unity. Correlation analyses were carried out using Pearson's coefficient of correlation using SPSS 11.01 (SPSS Inc., Chicago, Illinois, U.S.A.). Statistical significance was deter- mined by unpaired Student t-test, with differences considered significant at P 5 0.05. The ethical committee of Erasmus MC, Rotterdam, approved the study.

2.5. Cornpounds

The compounds used in the present study (obtained from the sources indicated) were: h-aCGRP, h-PCGRP, [ C ~ S ( A C ~ ) ~ . ~ ] - h-aCGRP, [Cys ( E ~ ) ~ ~ ~ ] - ~ - ~ c G R P and ~ - ( Y C G R P ~ . ~ ~ (Poly- peptide, Wolfenbüttel Germany); BIBN4096BS (gift from Dr. Henn Doods, Boehringer Ingelheim Pharma K.G., Biberach, Germany); U46619, isobutylmethylxanthine (IBMX), forsko- lin and substance P (Sigma Chemicals Co., Steinheim, Germany); and KC1 (Merck, Darmstad, Germany). Al1 com- pounds were dissolved in distilled water except for forskolin, which was dissolved in dimethylsulfoxide and BIBN4096BS, which was dissolved in 4% HCl(1 N) to obtain a 0.01 M stock solution; the latter was then diluted with distilled water. Al1 peptides and antagonists were stored in aliquots at - 80 "C.

110 S. Gupta et al. / European Jou~nal of Pharmaco1og)i 530 (2006) 107-116

3. Results

3.1. Functionul studies

3.1.1. Human coronary arteries The contraction induced by 100 mM KCl was significantly

higher in the proximal (56*3 mN) than in the distal ( 8 5 1 mN) coronary arteries. In contrast, the endothelium-dependent relaxant response to substance P (10 nM) was less in the proximal than in the distal coronary segments (265 13 and 76k 4%, respectively).

h-cxCGRP induced a concentration-dependent relaxation in precontracted human coronary arteries (Fig. 1). The maximal response to h-cxCGRP was significantly less in the proximal (E,,,: 43 * 7% of contraction to 30 rnM KCI) than in the distal (E,,,,: 92*4%) segments. In distal segments where no attempt was made to remove the endothelium. we assessed the cor- relation between the pECSO and E,,,,, of h-cxCGRP and the relaxation to substance P, as determined in each experiment. These correlations were not significant (Pearson's r=0.066 and 0.088, respectively; P=0.76 and 0.69, respectively, n=23-27), indicating that the endothelium is not involved in relaxations to h-aCGRF'. Accordingly, in distal coronary segments, we also performed an additional series of ex- penments where the endothelium was denuded (relaxation to substance P: 1 .O 5 1.4% and 64 522% of the precontraction to

U466 19 in endothelium-denuded and -intact segments; respectively). In these segments, both the E,,, (96&2.3% and 96*2.1%, respectively) and pECjo (9.23*0.44 and 9.18*0.86, respectively, n=4) of h-cxCGRP were similar. BIBN4096BS did not change the basa1 tone or the contmctionto KCl (30 mM) in either the proximal or distal human coronary artery. In proximal segments, the antagonism seemed to be non-competitive (Fig. 1A) as demonstrated by a significant suppression of the E,,, of h-aCGRP (43 *7%), even at low concentrations of BIBN4096BS (E,,,: 2455% and 18*8% and in the presence of 0.1 and 1 nM of BIBN4096BS, respectively). However, in view of the large variability inherent to this preparation, as described in Materials and methods section, these data should be interpreted with caution.

In distal segments BIBN4096BS concentration-dependent- ly antagonised the responses to h-aCGRP, but the Schild plot slope was significantly less than one (0.6850.07; Table 1). Further experiments were carried out in the human distal coronary artery segments to investigate the anomalous nature of the slope. To exclude that this finding was due to the inability of BIBN4096BS to reach equilibrium conditions afier 30 min, we performed experiments in distal coronary arteries with BIBN4096BS using a longer incubation period (2 h; Fig. 1 B). In addition, we conducted experiments using a wider range of concentrations of BIBN4096BS (Fig. 1C) to obtain an estimate of the Schild slope based on more data

e Control +- 3 nM BIBN4096BS 0 3 0 nM BIBN4096BS

+ 0.1 nM BIBN4096BS +- 10 nM BIBN4096BS + 100 nM BIBN4096BS -C 1 nM BIBN4096BS

A Hurnan proxirnal artery

+ 1 jtM BIBN4096BS

B Human distal artery

1001 , u I

-7.5 -7.0 -6.5 -6.0 -5.5 Log [h-a CGRP] (M)

C Human distal artery

-10 -9 -8 -7 -6 -5 Log [h-a CGRP] (M)

-10 -9 -8 -7 -6 -5 Log [h-a CGRP] (M)

o 1 I I 1 1

-10 -9 -8 -7 -6

Log [BIBN4096BS] (M)

Fig. 1. Effect of increasing concentrations of BIBN4096BS on the relaxant responses to h-aCGRP in proximal (panel A) and distal (panels B and C) segments ofthi human right coronary artery (n =6-11) and average Schild plot slope (panel D) obtained from panel C. BIBN4096BS (0.1 nM-1 pM) was incubated for 0.5 h (panel A, C and D) or 2 h (panel B).

S. Gupta et al. / Eiri-apean Journal of'Phai-macology 530 (2006) 107-116

Table 1 Effect of BIBN4096BS on relaxations to CGRP receptor agonists in human distal coronary artery segments

Agonists E m a x pECii> Slope Apparent pKh BIBN4096BS

0.1 nM I nM 10 nM 0.1 yM 1 m h-(YCGRP 92h4 (10) 8.05h0.23 (10) 0.68h0.07 (5)" 9.29h0.34 (5) 8.66hO.25 (10) 8.41h0.26 (10) h-(YCGRP' 9013 (6) 8.27h0.43 (6) 0.64h0.05 (6)" 9.56h0.22 (5) 9.33h0.25 (6) 9.13h0.17 (4) h-aCG~p' 8 2 i 4 (1 1) 8.6010.22 ( 1 1) 0.6510.09 (9)" 9.7210.18 (4) 10.1210.18 (9) 9.76*0.16 (10) 9.29h0.20 (6) [ ~ y s ( ~ c m ) ~ . ' ] 93*2 (8) 7.25h0.14 (8) 0.4910.13 (5)" 11.0310.42 (4) 10.2410.26 (7) 9.9550.11 (9) [~ys(~ t )" ' ] 9013 (9) 8.21h0.25 (9) 1.13h0.25 (6) 10.67h0.37 (3)' 10.29í0.18 (8) 10.13h0.29 (9) 10.2610.36 (3) h-PCGRP 86h4 (8) 8.80*0.21 (8) 0.7010.08 (8)" 9.88+0.40 (2) 9.51 10 .20 (8) 9.19h0.30 (8) 8.86h0.22 (8)

BIBN4096RS (0.1 nM-1 pM) was incubated for 0.5 h with concentrations increasing in one-log steps, except in the case of the human coronary artery and h-NCGRP where two additional groups (-more concentrations or '2-h incubation) were used. E,,,,, was expressed as ?0 of the response to 30 mM KC1. Al1 data are meanshS.E.M ( 1 1 ) . "Slopes of the Schild regression significantly different ( P 5 0.05) frorn unity. [ ~ y s ( ~ c m ) ' . ~ ] , [C~S(AC~) ' . ' ]~-NCGRP; [C~S(E~)'.'], [C~S(E~)'-']~-NCGRP. "pk',, at 0.3 nM BIBN4096BS.

points than in our initial experiments (Fig. ID). In both cases, the slope remained significantly less than one (0.65 50.09 and 0.6450.05, respectively), but the longer incubation period did increase the potency of BlBN4096BS (see apparent pKh values in Table 1).

~ - C X C G R P ~ . ~ ~ also antagonised the responses to h-aCGRP in the human distal coronary artery (Fig. 2), but with a low potency (pKb at 3 pM: 6.28 h0 . 17 n =4), and again the Schild plot slope was significantly less than one (0.3650.05, n=4).

Both linear agonists induced concentration-dependent relaxations and BIBN4096BS. similarly as with h-aCGRP, antagonised the responses to [ ~ ~ s ( ~ c m ) ' ~ ~ ] - h - a ~ ~ in a seemingly cornpetitive manner with a slope significantly less than one (0.49h0.13; Fig. 3). Although the antagonism of BIBN4096BS appeared competitive in nature, it should be kept in mind that in view of the low potency of [Cys ( ~ c m ) ~ . ~ l - h - a ~ ~ R P the values had to be extrapolated for calculation of the pharmacological parameters. In contrast, the slope obtained with [ C ~ S ( E ~ ) ~ . ~ ] - ~ - ~ C G R P ( l . 13 1-0.25) was not different from unity. h-PCGRP relaxed the distal coronary artery with a potency not different from that of h-aCGRP (Fig. 3; Table l), and this response was potently antagonised by BIBN4096BS with a Schild plot slope (0.7010.08) significantly less than one.

Human distal artery

+ Control

-10 -9 -8 -7 -6 -5

Log [h-a CGRP] (M)

Fig. 2. Effect of increasing concentrations of ~ - N C G R P ~ . ~ ~ on the relaxant responses to h-aCGRP in distal segrnents of the human right coronary artery (n=4-12). ~ - N C G R P ~ - ~ ~ (100 nM-10 pM) was incubated for 0 3 h.

3.1.2. Porcine coronary arteries The contractile response to 100 mM KCl in proximal

coronary arteries was 6956 mN as compared to 1454 mN in the distal segments. h-aCGRP induced concentration-depen- dent relaxations in both proximal and distal coronary artery segments (Fig. 4). Endothelium denudation in the porcine distal coronary segments resulted in significantly decreased relaxa- tions to substance P (7h4% and 7 8 5 7% of the precontraction to U466 19 for endothelium-denuded and -intact artery segments, respectively). In contrast, the relaxations to h-(CGRP in endo- thelium-denuded segments (E,,,,: 97 12%; pECso: 8.80h0.17) were not different from those in control segments (E,,,,,: 95h2%; pECS0: 8.7710.16, ti=5). Similar to the human coronary artery, the E,,,, of h-aCGRP was significantly less in porcine proximal segments (7213%) than in distal segments (95 h 2%). Moreover, h-aCGRP-induced relaxations were blocked with equal potency by BIBN4096BS (1 pM) in the proximal and distal segments (pKb: 7.44h0.10 and 7.6310.14, respectively), while the Schild plot slope obtained in both cases were not different from unity (0.8050.18 and 0.8750.10, respectively).

3.2. RT-PCR studies iti humati cororiaty arteries

Using human specific forward and reverse primers, designed on the basis of nucleotide sequences of several components of the CGRP receptor family reported in the NCBI Genebank (Table 2), PCR products corresponding in size to CLR, RAMP-1, RAMP-2, RAMP-3, RDC and RCP were consistently amplified in both proximal and distal human coronary arteries (Fig. 5). Semi-quantitative analysis revealed no difference in the expression levels of these CGRP receptor components, except for a two-fold higher expression of RAMP-2 in the distal as compared to the proximal segments.

3.3. cAMP measurernents iti humati atid porcine coronary arteries

h-aCGRP (100 nM) increased cAMP concentrations significantly in the human proximal and distal artery segments as compared to the segments incubated with

S Gupfa el al. / European Jounrril of'Phurmacologv 530 (7006) 107-116

I I I I 1 100 -10 -9 -8 -7 -6 -5

Log [ [ ~ y s ( ~ c m ) ~ ' ~ ] - h - a CGRP] (M) L0g [ [ ~ ~ c ( ~ t ) ~ ' ~ ] - h - a CGRP] (M)

-@ Control

+ 0.1 nM BlBN4096BS

-+ 0.3 nM BlBN4096BS

-)- 1 nM BlBN4096BS

+ 10 nM BlBN4096BS

+ 100 nM BIBN4096BS

+ 1 pM BlBN4096BS

1 0 0 ' 1 1 I 1 1 1

-10 -9 -8 -7 -6 -5

LOg [-h-b CGRP] (M)

Fig. 3. Effect of increasing concentrations of BIBN4096BS on the relaxant responses to [ C ~ S ( A C ~ ) * . ' ] - ~ - ~ C G R P , [Cys (E~)'- ']-~-~cGRP and h-PCGRP in distal segments of the human right coronaiy aitery (n=5-9). BIBN4096BS (0.1 nM-1 pM) was incubated for 0.5 h.

vehicle (42+ 7 versus 107 1 2 1 and 14*9 versus 106169 pM mg protein-' rng tissue ' , respectively). This increase was significantly blocked by BIBN4096BS in both the human artery segments (Fig. 6). Siinilarly in porcine distal coronary artenes also there was a significant increase in cAMP levels in response to 11-(wCGRP and this effect was antagonised by BlBN4096BS (Fig. 6). BIBN4096BS did not significantly inliibit the forskolin-induced increase in the cAMP levels, either in human (389* 134 and 292* 1 10 pM mg protein ' mg t issue ' in the absence or presence of antagonist, respectively, P=0.47, n=5) or porcine (200*40 and 141 *72 pM mg protein- ' mg tissue- ', respectively, P=O.61, n=4) distal coronary artenes.

4. Discussion

4.1. filnctional interaction hetween CGRP receptor rigonists and antagowists

In both human and porcine proximal (internal diameter: 2-3 mm) as well as distal (internal diameter: 250-600 pm) segments of the right coronary artery, h-(wCGRP elicited a concentration-dependent relaxation that was antagonised by BIBN4096BS, which proved more potent in the humans than in the pig. Moreover, our results demonstrate that these relaxations were endothelium-independent in both human and porcine distal coronary artery. Our findings, showing that the

42- Control U 300 nM BlBN4096BS U 3 yM BIBN4096BS + 100 nM BIBN4096BS t l pM BIBN4096BS t 10 yM BIBN4096BS

Porcine proximal artery Porcine dista1 artery

, -1 O -9 -8 -7 -6 -5

Log [h-a CGRP] (M) -1 0 -9 -8 -7 -6 -5

Log [h-n CGRP] (M)

Fig. 4. Effect of increasing concentrations of BIBN4096BS on the relaxant responses to h-nCCiRP in proximal and distal segments of the porcine right coronaiy artery (n=6-8). BIBN4096BS (0.1 nM- 1 pM) was incubated for 0.5 h.

S. Gupta et al. / Europeati Joiirnd o/ Pk~irni(ico1ogy 530 (2006) 107-116 11 3

Table 2 Detection of molecular components relevant for CGRP receptors

Components NCBT genebank accession no. Fonvard primer (5'-3') Reverse primer (5'-3') Amplified sequence Size (bp)

h-CLR AY389506 TCAAGAGCCTAAGTTGCCAAA IL4TCAGCACAAATTCAATGCC 497-1057 560 h-RAMP-1 NM005855 CTGCCAGGAGGCTAACTACG GACCACGATGAAGGGGTAGA 78-376 298 h-RAMP-2 BC040107 GGGGGACGGTGAAGAACTAT GTTGGCAAAGTGGATCTGGT 164-391 227 h-RAMP-3 BC05385 AAGGTCTTCGCAGACATGAT GCAGTTGGAGAAGAACTGCC 123-312 189 h-RCP U51 134 AACTGATCTGAAAGAGCAGCG TCTTCTTCTGCTCAGCCTCTG 12 1-465 344 h-RDC- 1 AF030297 ACGTGGTGGTCTTCCTTGTC AAGGCCTTCATCAGCTCGTA 770-990 220 GAPDH BC023632 TGACTTCAACAGCACCC TACATGACAAGGTGCGGCTC 906-1254 348

Each set of fonvard and reverse primers was designed from the nucleotide sequences reported in the NCBT genebank. CLR, Calcitonin receptor like receptor; RAMP, receptor activity modifiing protein: RCP, receptor component protein; RDC-I glyceraldehyde-3-phosphate dehydrogenase.

the orphan receptor (originally cloned from canine thyroid cDNA); GAPDH,

resppnses to h-aCGRP in al1 four segment types are mediated by the CGRP receptor, are consistent with those reported recently in the human (Edvinsson et al., 2002; Hasbak et al., 2003) and porcine (Wu et al., 2002) left anterior descending coronary artery, where the CGRP-induced relaxation was also antag- onised by BIBN4096BS with similar potencies.

As reported earlier (Sheykhzade and Nyborg, 1998, Foulkes et al., 1991), we also found that the efficacy of CGRP inversely related to the vessel diameter, E,,,, values in both human and porcine arteries were lower in the proximal (43*7% and 72*3%, respectively) than in the distal (92*4% and 95*2%, respectively) coronary segments. The antagonism in the case of human proximal arteries seems to be non-competitive, as demonstrated by ttie suppression of the E,,,,. Although these data should be interpreted witti caution, it is worth noting that a similar non-competitive betiaviour of BIBN4096BS has been reported in human subcutaneous arteries at similar concentra- tions (0.1 and 1 nM) (Sheyktizade et al., 2004).

The Schild plot slope in the tiuman distal coronary artery (0.68 50.07) was significantly lower ttian unity and, therefore,

Human proximal artery M 1 2 3 4 5 6 C

additional experiments were undertaken with human distal coronary arteries. To rule out that the Schild plot slope was less than unity because BIBN4096BS had not yet reached the equilibrium conditions in the distal segments. we employed a longer incubation period (2 h) with the antagonist. Notwith- standing, the slope (0.65*0.09) remained significantly less than one, although the potency of the antagonist increased with longer incubation period. Within this framework, if there were a non-equilibrium state between an antagonist and its re- ceptor, the Schild plot slope would tend to be greater than unity (Kenakin, 1993) instead of less than one. as was the case in our experiments. The fact that a longer incubation period increased the potency of BIBN4096BS (apparent pKb at 10 nM: 10.12*0.18) is in line with previous observations on slow on and off kinetics of BIBN4096BS (Schindler and Doods, 2002). Similarly, as with the experiments employing a longer incubation period for BIBN5096BS, the slope of the Schild plot obtained with more concentrations of BIRN4096BS re- mained less than one (0.64*0.05).

In subsequent studies, we used the conventional antagonist h-aCGRPx.37 as a tool to distinguish between CGRPl (pKb: 7- 8) and CGRP2 (pKb: 5.5-6.5) receptors (Dennis et al., 1989; Quirion et al., 1992; Wisskirchen et al., 1998). The fact that h-

Human distal artery M 1 2 3 4 M 5 6

Fig. 5. Agarose gel electrophoresis of PCR amplified products derived from cDNA obtained from proximal (upper panel) and distal (lower panel) segments of the human nght coronary artery. The different lanes marked on top of the panels denote: + x 174 DNAMae 111 marker (M), RAMP-1 (298 bp; l), RAMP-2 (227 bp; 2), RAMP-3 (189 bp; 3), CLR (560 bp; 4), RCP (344 bp; 5) and RDC (220 bp; 6). The size of 3 marker bands is indicated in the left margins.

0 Vehicle m h-a CGRP

F"1 h-a CGRP + BIBN4096BS

Hurnan coronary Porcine coronary

i t

Proximal Distal Distal artery ariery ariery

Fig. 6. Effect of BTBN4096BS (1 FM) on the increases in cAMP concentration by h-nCGRP (1 00 nM) in the human (proximal and distal segments) and porcine (distal segments) right coronary artery (n=5-17). *Significantly (P<0.05) different from vehicle-treated segments.

S Gupta et al. /Europea~t Jourr7al of Phat.tri(rco10~~ 530 (2006) 107-116

< Y C G R P ~ . ~ ~ antagonised h-aCGRP with a low potency (pKb: 6.28i0.17) supports the possible involvement of CGRPz receptors (Juaneda et al., 2000). Again, the Schild plot slope was less than unity, demonstrating that the smaller Schild slope values should most likely be attributed to a heterogeneous receptor population in the human distal coronary artery and is not due to a BIBN4096BS-specific phenomenon. Further, our finding showing that the relatively selective CGRP2 receptor agonists, [ ~ ~ s ( A c m ) ~ ~ ~ l - h - a ~ ~ R P and [ C ~ S ( E ~ ) ' . ~ ] - ~ - ~ C G R P (Dennis et al., 1989; Juaneda et al., 2000; Quirion et al., 1992), also induced concentration-dependent relaxations indicate the presence of putative CGRP2 receptors. However, in similar experiments performed in left anterior descending coronary artery segments (1-2 mrn extemal diameter) obtained from patients with cardiac myopathy, Hasbak et al. (2003) observed only a very weak relaxant effect of [ C ~ S ( A C ~ ) ' ~ ~ ] - ~ - ~ C G R P (1 pM) amounting to less than 5% of the effect we observed. This difference may be due to the fact that we used donors without cardiac pathology and, moreover, used smaller size segments (0.25-0.6 mm intemal diameter) of the right coronary artery, which may contain different subtypes of CGRP-sensitive receptors. The selectivity of the linear agonists is not unequivocal; Waugh et al. (1999), who observed that with increasing concentrations of KCl (8-15 mM) used as precontraction, the relaxation induced by [ ~ ~ s ( ~ c m ) ~ ' ~ ] - h - aCGRP in pig coronary arteries decreased from 90% to almost zero, demonstrating that [ C ~ ~ ( A C ~ ) ~ . ~ ] - ~ - ~ C G R P may act as a partial agonist. However, in our preparation this was not the case, since even with a precontraction induced by 30 mM KC1 the E,,,,, of [ C ~ S ( A C ~ ) ~ ~ ~ ] - ~ - ~ C G R P was as high as 93 i2%. Further, the other linear agonist [ c ~ ~ ( E ~ ) " ~ ] - ~ - ~ c G R P acti- vated CGRP1-like receptor in SK-N-MC and Col-29 cells, while the same agonist acted on CGRPz-like receptors in rat vas deferens with a pECSO "8 (WU et al., 2000). In the present study, [ c ~ ~ ( E ~ ) ~ ~ ~ ] - ~ - ~ c G R P also relaxed arteries with a similar potency (pEC5,,: 8.2 1 i0.25). According to Kenakin (1993), different slopes obtained with Schild plots for different agonists with the same antagonist indicate a heterogeneous receptor population. Indeed, in contrast to our experiments with h-aCGRP and [ C ~ S ( A C ~ ) ' . ~ ] - ~ - ~ C G R P , the Schild slopes obtained with BIBN4096BS antagonising [ ~ ~ s ( E t ) ~ , ' ] - h-aCGRP was equal to unity, suggesting that these agonists do not stimulate the same receptor population or activate same receptor but with different potencies. Further, the observation that the apparent pKb values decrease with increasing concentrations of antagonist in case the slope was less than one again points to a heterogeneous CGRP receptor population (Table 1).

Intriguingly, the fact that the apparent pKb of BIBN4096BS was around 10 for both linear agonists points to the presence of CGRPl receptors as BIBN4096BS is supposed to be at least 10 times less potent for CGRP2 receptors (Wu et al., 2000). Hence, it is reasonable to assume that the receptors in the human distal coronary artery do not completely comply with the pharmaco- logical profile of the presently accepted CGRP receptor classification (Juaneda et al., 2000); notwithstanding, it would be prudent to consider severa1 lines of arguments within this

framework. Firstly, Sheykhzade et al. (2004) have recently suggested a non-competitive nature of antagonism of BIBN4096BS at concentrations higher than 10 pM against h- aCGRP-induced relaxations in human subcutaneous arteria Although, as mentioned above, this might be the case in our results with the proximal coronary artery, it does not apply to the distal coronary artery, where even at high concentrations of BIBN4096BS the E,,,, to h-aCGRP or other agonist was not suppressed showing antagonism is competitive in nature. Secondly, the interaction between BIBN4096BS and the adrenomedullin receptor might be responsible for the slope of the Schild plot being less than one. However, the affinity of BIBN4096BS for adrenomedullin receptors is too low (ICSo: 10.3 pM) for such an interaction (Doods et al., 2000). Thirdly, metabolism of h-aCGRP or CGRP receptor ligands by tissue peptidases can affect the data obtained. Nevertheless, inhibition of these peptidases has shown that the contribution of metabolism in determining the affinity of CGRP ligands is negligible (Wu et al., 2000). Fourthly, the linear agonists might not be selective enough to fonn the basis for the classification of CGRP receptors. In view of our findings, we also have our reservation towards the purported selectivity of linear agonist towards CGRP2 receptors, as both agonists under similar experimental conditions yielded significantly different Schild plot slopes with BIBN4096BS, suggesting that these agonists do not activate the same receptor subtypes. BIBN4096BS is supposed to be selective for the CGRP receptor, but it showeda similar or even higher antagonist potency with the linear agonists in human distal coronary artery as compared to h- aCGRP.Finally, the slope being different from one cannot be attributed only to BIBN4096BS as in the case of porcine proximal and distal coronary arteries the slope was not significantly different from one, and on the other hand h- C X C G R P ~ . ~ ~ with h-aCGRP also produced a slope less than one, thus underlining a heterogeneous receptor population in the human distal coronary artery.

4.2. Molecular components of the CGRP receptor family

Our RT-PCR results showed the expression of vanous CGRP receptor components like CLR, RCP, RDC- 1, RAMP-1, RAMP- 2 and RAMP-3 in both proximal and distal human coronary arteries; these molecular studies confinned the presence of al1 the essential components (CLR + RAMP- 1 + RCP) required for functional CGRP receptors in the above coronary segments. In this context, CLR is a G-protein coupled receptor and the presence of RAMP-1 ensures intracellular trafficking and maturation of the receptor (McLatchie et al., 1998). The third entity, RCP, is required for the formation of a high-affinity G- protein-coupled receptor, there by ensuring the signal transduc- tion of CLR (Evans et al., 2000). Unfortunately, the molecular counterpart for the CGRP2 receptor is yet to be determined.

4.3. Transduction mechanisms of the CGRP receptors

Both in human and porcine coronary arteries cAMP levels increased after a challenge with h-aCGRP. The fact that this

S. Gupta et al. /European Journal ofPharmacology 530 (2006) 107-116 115

increase in cAMP was abolished after incubation with BIBN4096BS clearly demonstrates that the relaxations to h- uCGW in the above blood vessels are, at least partly, mediated via this second messenger system.

In conclusion, the relaxation to h-aCGW in the human distal coronary arteries is mediated by C G W l receptors as demonstrated by the high apparent pKb values ( ~ 9 - 1 0 ) obtained with BIBN4096BS. In addition, another C G W receptor subtype, possibly also acting via an increase in C M , seems to be present in the human distal coronary artery. Although the nature of this receptor is not entirely clear, it shows some functional characteristics of C G W 2 receptors, such as the low antagonist potency of h-aCGRPx.37 and the high potency of the linear agonists, whereas the high apparent pKb values of BIBN4096BS point to the presence of C G W , receptors. A prerequisite to further characterise these receptors would be the advent of more selective C G W receptor agonists and antagonists along with the molecular characterisation of the putative C G W 2 receptor.

Acknowledgements

We would like to acknowledge Dr. H. Doods (Boehringer Ingelheim Pharma, Biberach, Germany) for the generous gift of BIBN4096BS and other C G W receptor-ligands and Dr. P.G.H. Mulder (Department of Epidemiology and Biostatistics, Erasmus MC, Rotterdam, The Netherlands) for his kind help in the statistical analyses. This study was partly supported from funds obtained from Boehringer Ingelheim Pharma KG (Biberach, Germany) for a contract research project with Erasmus Pharma B.V.

References

Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S., Evans, R.M., 1982. Altemative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 298, 240-244.

Amlakshana, O., Schild, H.O., 1959. Some quantitative uses of drug antagonists. Br. J. Pharmacol. 14, 48-58.

Choksi: T., Hay, D.L., Legon, S., Poyner, D.R., Hagner, S., Bloom. S.R., Smith, D.M., 2002. Comparison of the expression of calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) with CGFP and adrenomedullin binding in cell lines. Br. J. Pharmacol. 136, 784-792.

Chornczynski, P., Sacchi, N,, 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Bio- chem. 162, 156-159.

Dennis, T., Foumier, A,, St. Pierre, S., Quirion, R., 1989. Structure-activity profile of calcitonin gene-related peptide in peripheral and brain tissues. Evidence for receptor multiplicity. J. Pharmacol. Exp. Ther. 25 1, 7 18-725.

Dennis, T., Foumier, A,, Cadieux, A,, Pomerleau, F., Jolicoeur, F.B., St. Pierre, S., Quirion, R., 1990. h-CGW,-,,: a calcitonin gene-related peptide antagonist revealing calcitonin gene-related peptide receptor heterogeneity in brain and periphery. J. Pharmacol. Exp. Ther. 254, 123-128.

Doods, H., Hallermayer, G., Wu, D., Entzeroth, M., Rudolf, K.: Engel, W., Eberlein, W., 2000. Pharmacological profile of BIBN4096BS, the first selective small molecule CGFP antagonist. Br. J. Pharmacol. 129,420-423.

Dumont, Y., Foumier, A,, St.-Pierre, S., Quirion, R., 1997. A potent and selective CGRP2 agonist, [ C ~ S ( E ~ ) ~ , ~ ] ~ - C G R P alpha: comparison in prototypical CGRP, and CGRP' in vitro bioassays. Can. J. Physiol. Pharmacol. 75, 671-676.

Durharn, P.L.. 2004. CGFP-receptor antagonists-a ffesh approach to migraine therapy? N. Engl. J. Med. 350, 1073-1075.

Edvinsson, L., Alm, R.; Shaw, D., Rutledge, R.Z., Koblan, K.S., Longmore, J., Kane, S.A., 2002. Effect of the CGFP receptor antagonist BIBN4096BS in human cerebral, coronaiy and omental arteries and in SK-N-MC cells. Eur. J. Pharmacol. 434, 49-53.

Evans, B.N., Rosenblatt, M.I., Mnayer, L.O., Oliver, K.R., Dickerson, I.M., 2000. CGFP-RCP, a novel protein required for signal transduction at calcitonin gene-related peptide and adrenomedullin receptors. J. Biol. Chem. 275, 31438-31443.

Foulkes, R., Shaw, N., Bose. C.. Hughes, B., 1991. Differential vasodilator profile of calcitonin gene-related peptide in porcine large and small diameter coronaiy artery rings. Eur. J. Pharmacol. 201, 143-149.

Gupta, S., Mehrotra, S.. Garrelds, I.M., Sharma, H.S., Saxena, P.R., MaassenVanDenBrink, A., 2004. Evidence for a heterogeneous CGFP receptor population in the human coronary artery. Neuropeptides 38, 122-123.

Hasbak, P., Saetrum Opgaard, O., Eskesen, K., Schifter, S., Arendrup, H., Longmore, J., Edvinsson, L., 2003. Investigation of CGFP receptors and peptide pharmacology in human coronary arteries. Characterization with a nonpeptide antagonist. J. Pharmacol. Exp. Ther. 304, 326-333.

Juaneda, C., Dumont, Y., Quirion, R., 2000. The molecular pharmacology of CGRP and related peptide receptor subtypes. Trends Pharmacol. Sci. 2 1, 432-438.

Kenakin, T., 1993. Competitive antagonism. Pharmcologic Analysis of Drug- receptor Interaction. Raven Press, Ltd, New York, pp. 278-322.

Luebke, A.E., Dahl, G.P., Roos, B.A., Dickerson, I.M., 1996. Identification of a protein that confers calcitonin gene-related peptide responsiveness to oocytes by using a cystic fibrosis transmembrane conductance regulator assay. Proc. Natl. Acad. Sci. U. S. A. 93, 3455-3460.

Luu, T.N., Dashwood, M.R., Chester, A.H., Muddle, J.R., Yacoub, M.H., 1995. Calcitonin gene-related peptide in healthy and atheromatous human epicardial coronary arteries. Function and receptor characteriza- tion. J. Vasc. Res. 32, 93-99.

McLatchie, L.M., Fraser, N.J., Main, M.J., Wise, A,, Brown, J., Thompson, N,, Solari, R., Lee, M.G., Foord, S.M., 1998. RAMPs regulate the transpon and ligand specificity of the calcitonin-receptor-like receptor. Nature 393, 333-339.

Mimeault, M., Foumier, A.: Dumont, Y., St-Pierre, S., Quirion, R., 1991. Comparative affinities and antagonistic potencies of various human calcitonin gene-related peptide fragments on calcitonin gene-related peptide receptors in brain and periphery. J. Pharmacol. Exp. Ther. 258, 1084- 1090.

Nyborg, N.C., Baandrup, U., Mikkelsen, E.O., Mulvany. M.J., 1987. Active, passive and myogenic characteristics of isolated rat intramural coronary resistance arteries. Pflügers Arch. 410, 664-670.

Olesen, J.. Diener, H.C., Husstedt, I.W., Goadsby, P.J., Hall, D., Meier, U., Pollentier, S., Lesko, L.M., 2004. Calcitonin gene-related peptide receptor antagonist BIBN 4096 BS for the acute treatment of migraine. N. Engl. J. Med. 350, 1104-1 110.

Poyner, D.R., Sexton, P.M., Marshall, I., Smith, D.M., Quirion, R., Bom, W., Muff, R., Fischer, J.A., Foord, S.M., 2002. Intemational Union of Pharmacology: XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. Pharmacol. Rev. 54, 233-246.

Quirion, R., Van Rossum, D., Dumont, Y., St-Pierre, S., Foumier, A., 1992. Characterization of CGFP, and CGRP2 receptor subtypes. Ann. N. Y. Acad. Sci. 657, 88-105.

Schindler, M., Doods, H.N., 2002. Binding properties of the novel, non-peptide CGRP receptor antagonist radioligand, [ 3 ~ ] ~ ~ ~ ~ 4 0 9 6 ~ ~ . Eur. J. Pharma- col. 442, 187-193.

Sharma, H.S., Maulik, N,, Gho, B.C., Das, D.K., Verdouw, P.D., 1996. Coordinated expression of heme oxygenase-1 and ubiquitin in the porcine heart subjected to ischemia and reperfusion. Mol. Cell. Biochem. 157, 111-116.

Sheykhzade, M., Nyborg, N.C., 1998. Caliber dependent calcitonin gene-related peptide-induced relaxation in rat coronary arteries: effect of K- on the tachyphylaxis. Eur. J. Pharmacol. 351, 53-59.

116 S. Gupta et al. /European Journal of'Pharmacologv 530 (2006) 107-116

Sheykhzade, M., Lind, H., Edvinsson, L., 2004. Noncompetitive antagonism of BIBN4096BS on CGW-induced responses in human subcutaneous arteries. Br. J. Pharmacol. 143, 1066-1073.

Van Rossum, D., Hanisch, U.K., Quirion, R., 1997. Neuroanatomical localization, pharmacological charactenzation and functions of CGW, related peptides and their receptors. Neurosci. Biobehav. Rev. 2 1, 649-678.

Waugh, D.J., Bockman, C.S., Smith, D.D., Abel, P.W., 1999. Limitations in using peptide drugs to characterize calcitonin gene-related peptide receptors. J. Pharmacol. Exp. Ther. 289, 1419-1426.

Wisskirchen, F.M., Buri, R.P., Marshall, I., 1998. Pharmacological character- ization of C G W receptors mediating relaxation of the rat pulmonary artery and inhibition of twitch responses of the rat vas deferens. Br. J. Pharmacol. 123. 1673-1683.

Wisskirchen, F.M., Gray, D.W., Marshall, I., 1999. Receptors mediating CGRP- induced relaxation in the rat isolated thoracic aorta and porcine isolated coronary ariery differentiated by h-alpha-CGRP8.37. Br. J. Pharmacol. 128, 283-292.

Wu, D., Eberlein, W., Rudolf, K., Engel, W., Hallermayer, G., Doods, H., 2000. Characterisation of calcitonin gene-related peptide receptors in rat ahiurn and vas deferens: evidence for a [ ~ ~ s ( ~ t ) ~ . ~ ] h - ~ ~ ~ ~ - ~ r e f e r r i n ~ receptor. Eur. J. Pharmacol. 400, 3 13-3 19.

Wu, D., Doods, H., Amdt, K., Schindler, M., 2002. Development and potential of non-peptide antagonists for calcitonin-gene-related peptide (CGRP) receptors: evidence for C G W receptor heterogeneiiy. Biochem. Soc. Trans. 30,468-473.