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Pharmacological characterization of calcitonin gene-related peptide receptors
and BIBN4096BS -- a novel CRPG receptor antagonist
Wu, D.
Publication date
1999
Link to publication
Citation for published version (APA):
Wu, D. (1999). Pharmacological characterization of calcitonin gene-related peptide receptors
and BIBN4096BS -- a novel CRPG receptor antagonist.
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CHAPTER 5
Characterisation of the hemodynamic effects of CGRP in anesthetized rats.
The influence of CGRP-antagonists.
1. Introduction
Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide that is expressed by the same gene as the endocrine hormone calcitonin (1). CGRP is expressed in a- and ß- forms that vary by one and three amino acids in rats and humans (2-4), respectively. CGRP is the most potent vasodilating substance so far known. Its vasodilation has been shown in a variety of isolated vessels and in vivo in different species, including humans (5-9). Immunochemical studies have shown that CGRP is distributed broadly within the cardiovascular system as a dense peripheral sensory network that innervates the arteries, veins, heart and viscera (10-15). In animal studies, positive inotropic and chronotropic effects of CGRP have been observed in the isolated spontaneously beating right atrium. This activity is not modified by metoprolol, mepyramine, Cimetidine, or indomethacin, suggesting a direct cardiac site of action (16). The hypothesis that there may exist specific receptors in the heart and that inotropic CGRP activity occurs in vivo still needs further investigation.
Two different CGRP receptor subtypes (CGRPi and CGRP2) have been proposed to occur on the basis of differential antagonist affinities and agonist potencies in peripheral tissues
(17-18). The c-terminal h-aCGRP sequence h-aCGRP(s-37) is considered to be selective for CGRPi receptors found in the guinea pig atrium, whereas the agonist [cys(ACM)2,7]hCGRPcc
is considered to be selective for the CGRP2 receptor originally described in the rat vas deferens. A new potent analogue [cys(Et)2,7]hCGRPa displayed a high potency to inhibit the
rat vas deferens twitch response, whereas in the guinea pig atrium this analogue induced only a slight inotropic effect at very high concentration (19). These findings suggested that [cys(Et)2,7]hCGRPa may be of value to investigate the functional relevance of the CGRP2
receptor.
Adrenomedullin (ADM) is a 52 amino acid peptide present in human plasma (20). It has been found to display a few biological activities in common with CGRP (21). Adrenomedullin shows some structural similarity to the calcitonin, to the pancreatic 13-cell peptide, amylin (22)
and to CGRP. This prompted us to investigate whether these compounds may have similar hemodynamic effects and may act through CGRP receptors, or via a different receptor population.
The first aim of the present study was to investigate the hemodynamic effects of CGRP related peptides in vivo in rats. The second aim was to pharmacologically characterize the CGRP receptors by using various CGRP related peptide agonists and three CGRP antagonists, including BEBN4096BS, a modified dipeptide known to be a potent CGRP antagonist (23).
2. Materials and methods
Male Wistar rats (Chbb: Thorn 350-400g) which had been fasted for 1 day were anaesthetised with sodium pentobarbitone (induction with 60mg/kg , i.p. and maintained with a infusion of 30mg/kg/h , s.c. in the abdominal skin through a 23G needle using a solution of 10mg/ml). The trachea was cannulated and the animals were artificially respired (80 strokes/min) with room air supplemented by oxygen, to maintain arterial blood gases within physiological limits. The body temperature was maintained at 37°C with a heating pad. The right femoral artery and vein were cannulated for the continuous measurement of arterial blood pressure and intravenous administration of agonists, respectively. The left femoral vein was cannulated for the intravenous administration of antagonists. Heart rate was derived from the blood pressure signal. After administration of heparin (1000 IU/kg), the right common carotid artery was cannulated and a catheter with a microtip pressure transducer was inserted via the right carotid artery into the lumen of the left ventricle to monitor left ventricular pressure (LVP) and its first derivative (LV ± dp/dpmax).All parameters were recorded on a 8 channel polygraph. Following surgery , the animals were allowed to equilibrate for 20min to achieve stable resting levels.
2.1. Experimental protocol
2.1.1. Dose-response studies of agonists:
The dose-response curves with respect to the effect of peptide agonists were constructed by applying intravenous injections of increasing doses of each peptide in a volume of 0.5ml/kg,
at 15min intervals. Each bolus dose was followed by 0.2ml saline to ensure the delivery of the peptide. As a control, 0.4ml vehicle (saline) was tested prior to drug administration. Only one dose-response curve was made in each animal. The same protocol was followed for examining the antagonists alone.
2.1.2. Effects of antagonists:
To investigate the antagonist potency of h-aCGRP(8-37), h-ßCGRP(8-37) and BIBN4096BS
against CGRP related peptide agonists induced hemodynamic responses, the reproducibility of CGRP agonists induced hemodynamic responses was examined first by repeating 7 times the application of a fixed dose of each agonist. The choice of dose was based on a comparison of the potency of CGRP agonists induced hemodynamic effects from the prior study. The effects of the antagonists were determined after having recorded two reproducible responses to the CGRP agonists. Subsequently, this procedure was repeated with increasing concentrations of the antagonists (h-aCGRP(8-37> h-ßCGRP(8-37) and BIBN4096BS ).Each
dose of antagonist was injected 5min prior to the agonist. Only one dose-response curve was made in each animal. In order to establish the responses of h-ADM in the presence of an antagonist (h-aCGRP(8.37) , h-ßCGRP(8-37) and BIBN4096BS, respectively), the antagonist
was injected 5min prior to the second administration of h-ADM. Only one dose of antagonist was used in each animal.
2.2. Statistical analysis
Drug-induced hemodynamic effects were calculated as peak percentage changes from resting levels, measured immediately prior to each dose. Relative potencies of the agonists were determinied by comparing ED30 values, (i.e. the dose required to produce a 30% decrease in diastolic blood pressure [DBP] ). Relative potencies of the antagonists were determined by comparing ID50 values, (i.e. the dose required to produce a 50% inhibition of the agonist response of the first application). All values were expressed as mean ± S.E.M.. Student's t-test was used for statistical evaluation. P values <0.05 were considered to be significant.
2.3. Drugs used
The following drugs and chemicals were used: human adrenomedullin, human calcitonin and human amylin were purchased from Bachern, California, U.S.A.; r-ßCGRP and h-ßCGRP(8-37) were purchased from Neosystem, Strasbourg, France; h-aCGRP(g.37), h-aCGRP, h-BCGRP
and r-aCGRP were purchased from Saxon Biochemicals GmbH, Hannover, Germany; [Cys(Et)2,7]hCGRPa was purchased from Wherl GmbH Wolfenbüttel, Germany; rat amylin
was purchased from Polypeptide, Wolfenbüttel, Germany; BEBN4096BS was synthesized by Boehringer Ingelheim Pharma KG, Biberach /Riss, Germany. All peptides were dissolved in distilled water. BIBN4096BS was dissolved in a small volume (20nl) IN HCl, further diluted with saline, then pH was adjusted to 6.5-7.0 by IN NaOH. Stock solutions (2xl0'3 M) of each
compound were stored in aliquots at -20CC until needed. Solutions were diluted to the final
concentration with saline.
3. Results
In table 1 the initial values of 5 parameters (diastolic blood pressure, heart rate, left ventricular pressure, LV +dp/dtmax, and LV -dp/dtmax ) after 20 min of stabilisation in anesthetized rats are shown.
Table 1. Initial values ( mean+ S.E.M., n=138 ) of the diastolic blood pressure PBP), heart rate (HR), left ventricular pressure (LVP), and its first derivative (+ dp/dtmax) in anesthetized rats.
parameter Initial value
DBP HR LVP (+)dp/dtmax (-)dp/dtmax 109.12+/-0.27 429.53+/-0.75 135.47+/-0.30 8721.16+/2.21 7845.31+/-2.61 ( mmHg ) ( beats/min ) (mmHg) ( mmHg/s ) ( mmHg/s )
For abbreviations see text.
Table 2. Decrease in diastolic blood pressure (DBP), left ventricular pressure (LVP), and its first derivative^(+ dp/dtmax) caused by treatment of anesthetized rats with CGRP agonists. The effects of CGRP agonists are expressed as ED30 values (nmol/kg).
DBP (mmHg) LVP (mmHg) (+)dp/dtmax (mmHg/s) (-)dp/dtmax (mmHg/s) h-«CGRP 0.074 +/-0.0038 0.20 +/-0.023 0.14 +/-0.039 0.068 +/-0.011 h-ßCGRP 0.068 +/-0.0057 0.20 +/-0.032 0.38 +/-0.14 0.057 +/-0.0065 r-aCGRP 0.059 +/-0.0074 0.21 +/-0.046 0.21 +/-0.042 0.063 +/-O.016 r-ßCGRP 0.079 +/-0.025 0.16 +/-0.020 0.22 +/-0.049 0.067 +/-0.015 ET-hCGRPa 0.51 +/-0.078 1.04 +MI.17 1.74 +/-0.44 0.44 +/-0.084 h-ADM 0.46 +/-0.037 0.96 +/-0.36 0.88 +/-0.16 0.50 +/-0.045 r-amylin 3.11 +/-0.59 7.18 +/-0.72 9.41 +/-2.36 3.72 +/-0.85
For abbreviations see text.
1.5 HR 5 0 , 40 _ » _ h - a C G R P ? , h-nr.PiRP — 3 0 , a ~~ 1 « _ * _ r - a C G R P S 20 u c ~ 10 . 0 . _ o _ r - ß C G R P S 20 u c ~ 10 . 0 . - * ^ ^ = " X " —I _ a _ E t - h C G R P _ o _ h-ADM S 20 u c ~ 10 . 0 . 0 0.01 0.03 ^x—i—x^r*"^—, , 0.1 0.3 1 3 10 30 _;,-—r-amylin nmol/kg
3.1. Hemodynamic effects of CGRP agonists
The peptides h-aCGRP, h-ßCGRP, r-aCGRP, r-ßCGRP, [Cys(Et)2'7]hCGRPa, h-ADM, and
h-amylin decreased DBP, LVP, +dp/dtmax, -dp/dtmax and increased HR in a dose-related manner ( Fig. 1 ). Relative potencies of the agonists were determined by comparing ED30 values ( table 2 ). h-aCGRP, h-ßCGRP, r-aCGRP, r-ßCGRP were equipotent; human [Cys(Et)2,7]hCGRPa as well as h-ADM proved 10 times less potent than 4 CGRP; r-amylin
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^ ra O) « IT Ewas 100 times less potent than the 4 forms of CGRP; h-calcitonin and h-amylin were inactive at the highest dose used ( 10'8 mol/kg ). Reproducible responses for a fixed dose of h-ocCGRP
(lu"10 mol/kg), h-ßCGRP (ÏO-10 mol/kg), r-aCGRP (10'10 mol/kg), r-ßCGRP (10'10 mol/kg),
[Cys(Et)2,7]hCGRPa (6xl0"'° mol/kg) and r-amylin(6xl0'9 mol/kg) were obtained 7 times in
the same animal with no significant difference compared with the first application ( paired t-test, p>0.05). However, for h-ADM(3xl0'10 mol/kg), this response was only obtained twice in
the same animal. The receptor desentization as effected by a diminished response was observed from the third application of h-ADM onwards.
Table 3. Antagonistic effects of h-aCGRP(8.37), h-BCGRP(8.37) and BIBN4096BS on CGRP-induced
hemodynamic actions (DBP, LVP.+ dp/dtmax) in anesthetized rats. The antagonistic effects are expressed as ID50 values (nmol/kg).
agonist
antagonist
agonist h-aCGRP8-37 h-BCGRP8-37 BIBN4096BN DBP h-aCGRP 23.2 +/-4.2 31.3 +/-3.5 33.1 +/-4.6 DBP h-ßCGRP 172 +/-29 121 +/-39 81.7 +/-7.6 DBP r-aCGRP 234 +/-27 45.6 +/-12.5 57.2 +/-9.3 DBP r-ßCGRP 82.1 +/-29.9 98.4 +/-24.8 35.3 +/-8.6 DBP Et-hCGRPa 18.2 +/-4.6 24.9 +/-5.8 8.46 +/-1.69 DBP r-amylin 54.1 +/-8.0 63.0 +/-15.2 141 +/-27 LVP h-aCGRP 18.5 +/-4.7 32.1 +/7.4 30.7 +/-6.7 LVP h-ßCGRP 195 +/-23 127 +/-38 75.6 +/-14.1 LVP r-aCGRP 240 +/-38 53.0 +/-16.2 52.4 +/-4.9 LVP r-ßCGRP 87.8 +/-33.9 130 +/-17 31.4 +/-9.7 LVP Et-hCGRPa 35.9 +/-12.7 30.8 +/-7.9 11.5 +/-1.8 LVP r-amylin 60.1 +/-2.3 43.2 +/-8.8 116 +/-40 (+)dp/dtmax h-aCGRP 10.7 +/-2.3 24.1 +/-7.4 27.7 +/-6.9 (+)dp/dtmax h-ßCGRP 232 +/-14 107 +/-29 78.3 +/-16.8 (+)dp/dtmax r-aCGRP 282 +/-46 28.6 +/-10.4 49.7 +/-13.3 (+)dp/dtmax r-ßCGRP 50.2 +/-14.4 97.2 +/-14.4 54.7 +/-25.2 (+)dp/dtmax Et-hCGRPa 7.9 +/-2.14 40.5 +/-16.1 3.39 +/-0.79 (+)dp/dtmax r-amylin 58.9 +/-17.1 37.9 +/-16.5 80.3 +/-38.4 (-)dp/dtmax h-aCGRP 11.8 +/-2.1 15.3 +/-5.6 28.6 +/-17.5 (-)dp/dtmax h-ßCGRP 148 +/-26 93.9 +/-27.5 76.1 +/-22.0 (-)dp/dtmax r-aCGRP 182 +/-43 29.1 +/-7.8 49.5 +/-11.0 (-)dp/dtmax r-ßCGRP 72.8 +/-18.4 69.3 +/-21.7 26.1 +/-8.7 (-)dp/dtmax Et-hCGRPa 17.2 +/-6 18.4 +/-3.5 4.92 +/-1.58 (-)dp/dtmax r-amylin 45.5 +/-7.1 40.1 +/-16.2 95.3 +/-42.2 96
3.2. Effect of the CGRP antagonists
Systemic administration alone, h-aCGRP(8-37) was inactive up to a dose of 3xl0"7 mol/kg, but
in a dose of KT6 mol/kg, it decreased DBP ( 59.94+A2.85 % ), LVP ( 38.52+/-4.07 % ),
+dp/dtmax (51.49+/-5.7 % ), -dp/dtmax (61.94+/-4.61 % ) and also reduced HR ( 18.5+/-2.95 % ). Doses of 3xl0"7 mol/kg and 10"6 mol/kg, BD3N4096BS did not block this effect of
h-aCGRP(8.37).
The three CGRP antagonists h-aCGRP(8.37), h-ßCGRP(8-37) and BIBN4096BS
dose-dependently antagonized the hemodynamic effects of h-ocCGRP, h-ßCGRP, aCGRP, r-ßCGRP, [Cys(Et)2,7]hCGRPa, r-amylin in anesthetized rats (Fig 2,3,4 ). However, the three
antagonists did not block the effects of h-ADM. The relative potencies of the antagonists were determined by comparing BD50 values ( table 3 ). The order of potency of h-ocCGRP(8-37) concerning the reduction of the CGRP agonists induced hemodynamic effects was [Cys(Et)2l7]hCGRPa >h-aCGRP>r-amylin >r-ßCGRP>h-ßCGRP>r-ocCGRP. The order of
potency of h-ßCGRP(g.37) was [Cys(Et)2,7]hCGRPa
>h-aCGRP>r-aCGRP>r-amylin>r-ßCGRP>h-ßCGRP. The order of potency of BIBN4096BS was [Cys(Et)2,7]hCGRPa
>h-aCGRP>r-ßCGRP>r-aCGRP>h-ßCGRP>r-amylin.
4. Discussion
CGRP is one of the most potent vasodilators known (24). The hypotensive and tachycardie effects of systemic administration of CGRP have been described in several species including humans (25-29). The present study in anesthetized rats has shown that CGRP receptor agonists induced a reduction in diastolic blood pressure, left ventricular pressure and ±dp/dtmax, wheseas heart rate is increased. The 4 forms of the CGRP agonists ( aCGRP, h-ßCGRP, r-aCGRP, r-ßCGRP) were approximately equipotent, whereas h-ADM was 10 times less potent and r-amylin was 100 times less potent in producing hemodynamic effects than the 4 forms of CGRP. This finding is consistent with previous reports (21). [Cys(Et)2'7]hCGRPa
has been proposed as a potent CGRP2 receptor selective analogue (19), it was 10 times less potent than 4 forms of CGRP in producing hemodynamic effects in vivo, calcitonin and
amylin were inactive ( ÏO"8 mol/kg ). CGRP has been shown to exert positive inotropic and
chronotropic effects on guinea pig isolated atrium (30-31). In near-term fetal sheep, the injections of CGRP into left pulmonary artery increased heart rate stepwise, without causing changes in pulmonary or carotid arterial pressure (32). Therefore, CGRP receptor agonists-induced increases in heart rate are not only secondary to decreased systemic blood pressure but also reflect a positive chronotropic effect in the rats. The first derivative of LVP (dp/dtmax) is a sensitive parameter for the assessment of cardiac contractility, and it is both load- and HR-dependent (33). In the present study, no change on preload was observed (data not shown). Therefore, the CGRP receptor agonist-induced decreases in maximal dp/dt were due to decreases on afterload (DBP). Upon systemic administration h-aCGRP(8.37) alone
proved inactive up to a dose of 3xl0'7 mol/kg, but in a dose of 10"6 mol/kg, it showed
nonspecific effects, decreased DBP, LVP, +dp/dtmax, -dp/dtmax, and decreased HR.
Upon comparison of the vasorelaxant effects of repeated exposure of pig small-diameter LAD coronary arterial rings to CGRP and nitrates, Foulkes et al. (34-35) found that, unlike nitrates, tolerance to CGRP did not develop. In the present study , reproducible responses for a fixed dose of 5 forms of CGRP ( h-aCGRP, h-ßCGRP, r-aCGRP, r-ßCGRP and [Cys(Et)2'7]hCGRPa ) and r-amylin were obtained 7 times in the same animal, and tolerance
was not observed. However, for h-ADM, reproducible responses were only obtained twice in the same animal, the receptor desensitization was observed from the third application of h-ADM onwards. This may be caused by a long lasting hypotensive effect of h-ADM.
The three CGRP antagonists h-aCGRP(8-37), h-ßCGRP(8.37) and BIBN4096BS produced
dose-related effects in antagonizing hemodynamic effects of h-aCGRP, h-ßCGRP, aCGRP, r-ßCGRP, [Cys(Et)2,7]hCGRPa and r-amylin in anesthetized rats (Fig 2, 3, 4. ). However, the
three antagonists did not counteract the h-ADM induced effects. h-ßCGRP as well as r-aCGRP were 10 times less sensitive to blockade by h-r-aCGRP(8-37). [Cys(Et)2,7]-hCGRPa
effects were the most sensitive to a reduction by the three CGRP antagonists. Different ID50 values of CGRP antagonists in antagonizing the CGRP agonists induced hemodynamic effects in vivo, may indicate the existence of CGRP receptor subtypes.
Amylin has been reported to interact both with CGRP receptors (36-39), and with amylin receptors (38, 40). In the present study , the hemodynamic effects of r-amylin were blocked
by three different CGRP antagonists. At high doses, h-aCGRP(8-37) and h-ßCGRP(8-37)
completely blocked the r-amylin-induced hemodynamic effects in vivo, although BIBN4096 did not, indicating that in vivo r-amylin may act through both CGRP receptors and amylin receptors, whereas BEBN4096BS could be sensitive to CGRP receptors but not to amylin receptors.
The ADM-induced increase in cyclic AMP levels in rat cultured vascular smooth muscle cells was reported to be blocked by h-aCGRP(8-37) (41-42). h-aCGRP(8-37) also attenuated the
vasodilator response to ADM in the perfused mesenteric artery of the rats (43). Accordingly, ADM seems to be able to activate CGRP receptors. However, the binding of [125I]-ADM in
vascular smooth muscle cells was inhibited by ADM but not by CGRP, suggesting that special ADM receptors may exist (42). In the present study, the hemodynamic effects of h-ADM in anesthetized rats remained uninfluenced by three different CGRP antagonists, indicating that these effects are not mediated via the activation of CGRP receptors.
In conclusion, CGRP-related peptide agonists produced dose-related hemodynamic effects in anesthetized rats.The 4 forms of CGRP (h-ctCGRP, h-ßCGRP, r-ctCGRP and r-ßCGRP )were approximately equipotent. [Cys(Et)2,7]hCGRPa as well as h-ADM were 10 times less potent
and r-amylin was 100 times less active, whereas h-calcitonin and h-amylin were inactive. Tolerance towards CGRP and r-amylin did not develop, but it was observed for h-ADM. The present has study demonstrated that BIBN4096BS is a potent CGRP antagonist. At a dose of 10"6 mol/Kg, hccCGRP(837) showed nonspecific effects, decreased DBP, LVP , +dp/dtmax,
-dp/dtmax, but it reduced HR, but BIBN4096BS did not counteract these effects. Different ID50 values of CGRP antagonists with respect to CGRP receptor agonists-induced hemodynamic effects in vivo suggest the existence of different CGRP receptor subtypes. The hemodynamic effect of r-amylin in vivo was mediated via the activation of CGRP receptors, but not the effects of h-ADM.
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