Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2017 May 2;174(13):2001–2014. doi: 10.1111/bph.13799

Olcegepant blocks neurogenic and non‐neurogenic CGRPergic vasodepressor responses and facilitates noradrenergic vasopressor responses in pithed rats

V H Avilés‐Rosas 1, E Rivera‐Mancilla 1, B A Marichal‐Cancino 1, G Manrique‐Maldonado 1, A H Altamirano‐Espinoza 1, A Maassen Van Den Brink 2, C M Villalón 1,✉
PMCID: PMC6398519  PMID: 28369749

Abstract

Background and Purpose

Olcegepant (BIBN4096BS) is a selective non‐peptide CGRP receptor antagonist with acute antimigraine properties. Since systemic vascular tone is modulated by perivascular (primary sensory CGRPergic and sympathetic) nerves, this randomized study investigated in pithed rats the effect of acute i.v. treatment with olcegepant on the neurogenic and non‐neurogenic: (i) CGRPergic vasodepressor responses; and (ii) noradrenergic vasopressor responses. The pithed rat is an experimental model predictive of systemic (cardio) vascular side effects.

Experimental Approach

Seventy‐five male Wistar rats (divided into 15 groups, n = 5 each) were pithed, artificially ventilated and prepared for: (i) spinal stimulation (T9–T12; 0.56–5.6 Hz) of the sensory CGRPergic vasodepressor outflow or i.v. bolus injections (0.1–1 μg·kg−1) of α‐CGRP, substance P or acetylcholine, which induced frequency‐dependent or dose‐dependent vasodepressor responses; or (ii) spinal stimulation (T7–T9; 0.03–3 Hz) of the sympathetic vasopressor outflow or i.v. bolus injections (0.03–3 μg·kg−1) of noradrenaline, which produced frequency‐dependent or dose‐dependent vasopressor responses.

Key Results

Olcegepant (1000 and 3000 μg·kg−1, i.v.) dose‐dependently blocked the vasodepressor responses to sensory nerve stimulation or i.v. α‐CGRP, without affecting those to substance P or acetylcholine. Whereas it potentiated the vasopressor responses to sympathetic nerve stimulation or i.v. noradrenaline.

Conclusions and Implications

Olcegepant (i.v.) selectively blocked the neurogenic and non‐neurogenic CGRPergic vasodepressor responses. This blockade by olcegepant potentiated the neurogenic and non‐neurogenic noradrenergic vasopressor responses in pithed rats, an effect that might result in an increased vascular resistance and, consequently, in a prohypertensive action.

Abbreviations

D‐R curve

dose–response curve

S‐R curve

stimulus–response curve

Introduction

CGRP and its receptors play a role in the modulation of central and peripheral physiological functions, including pain transmission and cardiovascular homeostasis as well as cardiovascular disease (Russell et al., 2014; MaassenVanDenBrink et al., 2016). For example, an excessive increase in plasma levels of CGRP, released from capsaicin‐sensitive sensory nerves, is associated with several diseases, including pulmonary hypertension, heart failure, ischaemia and migraine (Russell et al., 2014; MaassenVanDenBrink et al., 2016). With the development of potent non‐peptide CGRP receptor antagonists (e.g. olcegepant, telcagepant, etc.), the importance of CGRP in the pathophysiology of migraine has been established (Villalón and Olesen, 2009; Edvinsson, 2015).

Other findings reveal that systemic vascular resistance is modulated by two classes of perivascular nerves, namely, sympathetic vasoconstrictor (via noradrenaline release; Hoffman, 2001) and primary sensory vasodilator (mainly via CGRP release; Smillie and Brain, 2011) nerves. Indeed, Kawasaki et al. (1988) showed that CGRP is a vasodilator neurotransmitter in rat mesenteric resistance vessels, which decreases vascular resistance. In keeping with this, Taguchi et al. (1992) demonstrated that electrical spinal (T9–T12) stimulation of the perivascular sensory outflow in pithed rats results in vasodepressor responses mediated by vasodilatation of the mesenteric vascular bed, which are blocked by the CGRP receptor antagonist CGRP8‐37. However, CGRP8‐37 had to be given as an i.v. continuous infusion to produce an effective blockade, which disappeared when this infusion was interrupted (Taguchi et al., 1992).

Although olcegepant (BIBN4096BS) was the first CGRP receptor antagonist effective in acute migraine treatment when given i.v. (Olesen et al., 2004), no study has yet reported whether olcegepant affects the neurogenic and non‐neurogenic systemic vascular tone in the pithed rat model, which is predictive of systemic (cardio) vascular side effects (Valdivia et al., 2004). The pithed rat model, which lacks a functional central nervous system (see Methods section), is devoid of: (i) the influence of the autonomic and sensory nervous systems; (ii) compensatory baroreflex mechanisms that take place in intact or anaesthetized animals; and (iii) the ‘buffering capacity’ of the baroreceptor reflexes that allow a quick recovery after changes in blood pressure. Clearly, these are not physiological conditions, but this model allows us to selectively stimulate the sensory CGRPergic vasodepressor outflow and the sympathetic vasopressor outflow (see below). Accordingly, as a first approach, in this study in pithed rats we investigated the effect of i.v. bolus injections of olcegepant on: (i) the vasodepressor responses induced by either selective spinal (T9–T12) stimulation of the sensory CGRPergic vasodepressor outflow (Taguchi et al., 1992) or i.v bolus injections of rat α‐CGRP; (ii) the vasodepressor responses to substance P http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2098 or acetylcholine that are unrelated to CGRPergic mechanisms; and (iii) the vasopressor responses induced by either selective spinal (T7–T9) stimulation of the sympathetic vasopressor outflow (Gillespie et al., 1970) or i.v bolus injections of noradrenaline.

We hypothesized that olcegepant will block the CGRPergic vasodepressor responses and, consequently, will boost the noradrenergic vasopressor responses. A preliminary account of this investigation was presented at the 2014 British Pharmacological Society Winter Meeting (Villalón et al., 2014).

Methods

Compliance with ARRIVE, legal and ethical requirements as well as with the new guidance on design and statistical analysis requirements

A total of 75 male normotensive Wistar rats (provided by our institutional animalarium) were used in the present study. The animals were maintained on a 12/12 h light–dark cycle (lights on at 07 h) and housed on wood‐based bedding (provided by our institutional animalarium) in plastic cages (1580 cm2 by 18 cm depth) in a special room at a constant temperature (22 ± 2°C) and humidity (50%), with food and water freely available in their home cages (five rats per cage).

The animals were initially divided into three main sets: set 1 (n = 40); set 2 (n = 20); and set 3 (n = 15) for the different treatments (see succeeding texts and Figure 1). All experimental protocols were approved by our institutional ethics committee (CICUAL‐Cinvestav; permission protocol number 507‐12) and followed the regulations established by the Mexican Official Norm (NOM‐062‐ZOO‐1999), in full compliance with: (i) the guide for the Care and Use of Laboratory Animals in USA; (ii) the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath & Lilley, 2015); and (iii) the new guidance on experimental design and analysis for the British Journal of Pharmacology (Curtis et al., 2015), with randomization throughout. For more specific details on design and statistical analysis, see the sections entitled: (i) Sample size calculation, randomization and blinding ; and (ii) Data presentation and statistical evaluation .

Figure 1.

Figure 1

Open in a new tab

Experimental protocols showing the number of animals used in the three main sets of pithed rats as well as their corresponding division into the different subsets and groups used in the present study. Note that: (i) in set 1 and set 3 (for the induction of vasodepressor responses), methoxamine was continuously infused during and until the end of the experiments in order to maintain diastolic blood pressure around 165 mmHg; and (ii) in all treatment subgroups n = 5 each (with no exception). For further specific details on these experimental protocols, see the corresponding sections.

General methods

All animals where initially anaesthetised with sodium pentobarbital (60 mg·kg−1, i.p.). The adequacy of anaesthesia before pithing was judged by the absence of ocular reflexes, a negative tail flick test and corporal relaxation, amongst others. After cannulation of the trachea, the rats were pithed by inserting a stainless steel rod through the orbit and foramen magnum into the vertebral foramen (Shipley and Tilden, 1947). Immediately afterwards, the animals were artificially ventilated with room air by an Ugo Basile pump (56 strokes min−1; stroke volume: 20 mL·kg−1), as previously established (Kleinman and Radford, 1964). Subsequently, in 20 rats of set 1 and 10 rats of set 2, the pithing rod was replaced by an electrode: (i) enamelled except for a 1.5 cm length 9 cm from the tip, so that the uncovered segment was situated at T9–T12 of the spinal cord (n = 20; Figure 1) to stimulate the sensory CGRPergic nerves supplying the systemic vasculature to produce vasodepressor responses (Taguchi et al., 1992; Villalón et al., 2008); and (ii) enamelled except for a 1 cm length 9 cm from the tip, so that the uncovered segment was situated at T7–T9 of the spinal cord (n = 10; Figure 1) to stimulate the thoracic sympathetic nerves supplying the systemic vasculature to produce vasopressor responses (Gillespie et al., 1970).

After cervical bilateral vagotomy, the three sets of rats were treated as follows:

  1. Set 1 (animal weight: 300–350 g; age range: 16–20 weeks; n = 40) for the analysis of the vasodepressor responses induced by either electrical stimulation of the perivascular sensory CGRPergic outflow or i.v. bolus injections of exogenous α‐CGRP. This weight range is optimal for eliciting vasodepressor responses under our experimental conditions, and the cardiovascular model in pithed rats has been in use for several years (Villalón et al., 2008; Marichal‐Cancino et al., 2013). Catheters were placed in: (a) the left femoral vein for the continuous infusion of methoxamine; (b) the right femoral vein for the bolus injection of gallamine or exogenous α‐CGRP; (c) the left jugular vein for the continuous infusion of hexamethonium; and (d) the right jugular vein for the bolus injections of olcegepant or its corresponding vehicle. Then, this set was divided into two subsets (n = 20 each; see Figure 1) to analyse the vasodepressor responses induced by, respectively: (i) spinal (T9–T12) electrical stimulation of the vasodepressor sensory outflow (0.56, 1, 1.8, 3 and 5.6 Hz); and (ii) i.v. bolus injections of exogenous α‐CGRP (0.1, 0.18, 0.3, 0.56 and 1 μg·kg−1). Each response was obtained under unaltered baseline values ​​of blood pressure and heart rate, as reported previously (Villalón et al., 2008; Marichal‐Cancino et al., 2013).

  2. Set 2 (animal weight: 250–280 g; age range: 12–16 weeks; n = 20) for the analysis of the vasopressor responses induced by either electrical stimulation of the perivascular sympathetic outflow or i.v. bolus injections of exogenous noradrenaline. This weight range is optimal for eliciting vasopressor responses under our experimental conditions, and the cardiovascular model in pithed rats has been in use for several years (Villalón et al., 1995a,b, 1998). Catheters were placed in: (a) the left and right femoral veins for the bolus injections of, respectively, gallamine and exogenous noradrenaline; and (b) the left and right jugular veins for the bolus injections of, respectively, olcegepant and its corresponding vehicle. Subsequently, this set was divided into two subsets (n = 10 each; Figure 1) to analyse the vasopressor responses induced by, respectively: (i) electrical stimulation of the preganglionic spinal (T7–T9) vasopressor sympathetic outflow, as previously established by Gillespie et al. (1970), using stimulation frequencies of 0.03, 0.1, 0.3, 1 and 3 Hz; and (ii) i.v. bolus injections of exogenous noradrenaline (0.03, 0.1, 0.3, 1 and 3 μg·kg−1). Each response was obtained under unaltered baseline values ​​of blood pressure and heart rate, as previously described (Villalón et al., 1995a,b, 1998).

  3. Set 3 (animal weight: 300–350 g; age range: 16–20 weeks; n = 15) for the analysis of the vasodepressor responses induced by i.v. bolus injections of exogenous substance P followed by acetylcholine. This weight range is optimal for eliciting vasodepressor responses to i.v. bolus injections of compounds under our experimental conditions. The dose–response curve (D‐R curve) for substance P was constructed and 20 min later, when diastolic blood pressure had returned to baseline values, the D‐R curve for acetylcholine was constructed. Catheters were placed in: (a) the left femoral vein for the continuous infusion of methoxamine; (b) the right femoral vein for the sequential bolus injections of exogenous substance P and acetylcholine; (c) the left and right jugular veins for the bolus injections of, respectively, olcegepant and its corresponding vehicle. Afterwards, this set was divided into three groups (n = 5 each) to analyse the vasodepressor responses induced by consecutive i.v. bolus injections of substance P and acetylcholine in animals pretreated with: (i) nothing (for control responses); (ii) vehicle of olcegepant (1 mL·kg−1, i.v.); and (iii) olcegepant (3000 μg·kg−1, i.v.; Figure 1). The i.v. bolus injections of substance P followed by those of acetylcholine (both at 0.1, 0.18, 0.3, 0.56 and 1 μg·kg−1) were given using a sequential schedule.

In the above three sets, an additional catheter (connected to a Grass pressure transducer; P23 XL) was placed in the left carotid artery for recording arterial blood pressure. Heart rate was measured with a tachograph (7P4F, Grass Instrument Co., Quincy, MA, USA) triggered from the blood pressure signal. Furthermore, both blood pressure and heart rate were recorded simultaneously by a model 7D Grass polygraph (Grass Instrument Co., Quincy, MA, USA).

The vasodepressor/vasopressor stimulus–response curves (S‐R curves) and D‐R curves elicited by, respectively, electrical stimulation and exogenous agonists (α‐CGRP/noradrenaline/substance P/acetylcholine) were completed in 50–60 min, and each response was elicited under unaltered values of resting blood pressure. The electrical stimuli (Hz) and the i.v. dosing with the exogenous agonists (μg·kg−1) were given at 5–15 min intervals (see below). The body temperature of each pithed rat was maintained at 37°C by a lamp and monitored with a rectal thermometer.

Sample size calculation, randomization and blinding

Sample size calculation

On the basis of studies previously published by our group (e.g. Villalón et al., 1995a,b; 1998; 2008; Marichal‐Cancino et al., 2013), sample size was calculated by using SigmaPlot (v 12.0) as follows: (i) minimum detectable difference in mean = 25 mmHg, (ii) standard deviation = 11; (iii) groups = 3; (iv) desired power ≥ 0.8; (v) α (two‐tailed) = 0.05. Accordingly, n = 5 per group was established (see Figure 1 for specific treatment details).

Randomization

The animals, initially divided into three main sets (n = 40, 20 and 15 as described previously), were randomized by our animalarium staff by marking each rat and then allocated to a group by using a table of random numbers. Once in our lab, the animals were further randomized for each treatment assignment (randomized block design) with the use of a free randomizer (https://www.randomizer.org). The order of treatment was also randomized for each animal.

Blinding

All of our experimental values (i.e. the changes in diastolic blood pressure and heart rate) in each group of animals were simultaneously obtained by at least two different operators (coauthors in this study), of whom only one knew the treatment assignment for each group. Admittedly, our experimental protocols and data analysis were not blinded as required by the new guidance for publication in the British Journal of Pharmacology (Curtis et al., 2015), as our experiments were performed between 2013 and 2014.

Experimental protocols

Protocol 1. Vasodepressor responses induced by either electrical stimulation of the perivascular sensory CGRPergic outflow or i.v. bolus injections of exogenous α‐CGRP

In the first subset (n = 20) of set 1, the pithing rod was replaced by an electrode enamelled except for 1.5 cm length 9 cm from the tip, so that the uncovered segment was situated at T9–T12 of the spinal cord to allow selective stimulation of the sensory CGRPergic vasodepressor outflow; a similar electrode was placed dorsally (Villalón et al., 2008). Before electrical stimulation, the animals received (i.v.): (i) gallamine (25 mg·kg−1), to avoid electrically‐induced muscular twitching; and (ii) 10 min later, a continuous infusion of hexamethonium (2 mg·kg−1·min−1) to block the electrically‐induced vasopressor responses that result from stimulation of the preganglionic sympathetic vasopressor outflow (Villalón et al., 1995a,b, 1998). After 10 min, baseline values of diastolic blood pressure (an indicator of peripheral vascular resistance) and heart rate were determined.

Then, diastolic blood pressure was increased and maintained at approximately 165 mmHg by an i.v. continuous infusion of methoxamine (10–15 μg·kg−1·min−1), as reported previously (Villalón et al., 2008; Marichal‐Cancino et al., 2013), to allow the subsequent induction of vasodepressor responses. After a stable haemodynamic condition for at least 30 min, baseline values of diastolic blood pressure and heart rate were determined again. Then, this subset was divided into four groups (n = 5 each, as previously calculated) that were pretreated with i.v. injections of 300, 1000 or 3000 μg·kg−1 olcegepant or its corresponding vehicle (1 mL·kg−1 bidistilled water, pH: 6.5–7.0; see Figure 1 for further details). Ten minutes later, the sensory CGRPergic outflow was electrically stimulated by applying trains of 10 s (monophasic rectangular pulses of 2 ms duration and 50 V), at increasing stimulation frequencies (0.56, 1, 1.8, 3 and 5.6 Hz) during the methoxamine infusion.

The second subset (n = 20) of set 1 was divided into four groups n = 5 each; (Figure 1) that were pretreated with i.v. injections of 300, 1000 or 3000 μg·kg−1 olcegepant, or its vehicle. In these groups: (i) the pithing rod was left throughout the experiments; (ii) the administration of both gallamine and hexamethonium was omitted as there was no electrical stimulation; and (iii) instead of electrical stimulation, the animals received consecutive i.v. bolus injections of exogenous α‐CGRP (0.1, 0.18, 0.3, 0.56 and 1 μg·kg−1) for inducing vasodepressor responses (Figure 1). It must be emphasized that only one S‐R curve or D‐R curve was carried out per animal (pretreated with olcegepant or its vehicle) since tachyphylaxis of the CGRPergic vasodepressor responses was observed when eliciting a second S‐R curve or D‐R curve, as previously shown by Villalón et al. (2008). This means that the second S‐R curve or D‐R curve was not reproducible in the same animal as it was markedly attenuated.

Protocol 2. Vasopressor responses induced by either electrical stimulation of the perivascular sympathetic outflow or i.v. bolus injections of exogenous noradrenaline

In the first subset (n = 10) of set 2, the pithing rod was replaced by an electrode enamelled except for a 1 cm length 9 cm from the tip, so that the uncovered segment was situated at T7–T9 of the spinal cord for selective preganglionic stimulation of the sympathetic vasopressor outflow (Villalón et al., 1995 a,b, 1998). Prior to electrical stimulation, the animals received gallamine (25 mg·kg−1, i.v.) to avoid electrically‐induced muscular twitching. After a stable haemodynamic condition for at least 30 min, baseline values of diastolic blood pressure and heart rate were determined. Under these conditions, the sympathetic vasopressor outflow was stimulated by applying trains of 10 s (monophasic rectangular pulses of 2 ms duration and 50 V, at 0.03, 0.1, 0.3, 1 and 3 Hz). Subsequently, this subset was subdivided into 2 treatment groups (n = 5 each, Figure 1) comprising consecutive i.v. bolus injections of, respectively: (i) vehicle of olcegepant (1 mL·kg−1 bidistilled water, given twice); and (ii) olcegepant (1000 and 3000 μg·kg−1). Then 10 min after each dose of vehicle or olcegepant, a S‐R curve was elicited again.

The second subset (n = 10) of set 2 (also divided into two groups, n = 5 each; Figure 1) was prepared as described previously for the first subset, except that: (i) the pithing rod was left in throughout the experiments; (ii) the administration of gallamine was omitted as there was no electrical stimulation; and (iii) instead of electrical stimulation, the animals received i.v. bolus injections of exogenous noradrenaline (0.03, 0.1, 0.3, 1 and 3 μg·kg−1) for inducing vasopressor responses (Figure 1).

It is noteworthy that, unlike the induction of vasodepressor responses (see above), the noradrenergic S‐R curves and D‐R curves are highly reproducible when constructing a second (and even a third) S‐R curve and D‐R curve, as previously reported (Villalón et al., 1995 a,b; 1998). That is why these curves were analysed in the same groups of animals before and after treatment with olcegepant or its vehicle (Figure 1).

Protocol 3. Vasodepressor responses induced by consecutive i.v. bolus of substance P and acetylcholine in control rats and in rats pretreated i.v. with olcegepant or its vehicle

The third set of rats (n = 15) was prepared as previously described in Protocol 1 for eliciting the vasodepressor responses to exogenous α‐CGRP. The only difference was that, instead of i.v. α‐CGRP, the animals received consecutive i.v. bolus injections of exogenous substance P and acetylcholine (both at 0.1, 0.18, 0.3, 0.56 and 1 μg·kg−1; Figure 1). Accordingly, this set of rats was divided into three groups (n = 5 each), namely, control group (no pretreatment), group pretreated with vehicle (1 mL·kg−1) and group pretreated with olcegepant (3000 μg·kg−1). Then 10 min later, the D‐R curves to substance P and acetylcholine were recorded.

Other procedures applying to protocols 1, 2 and 3

The doses of hexamethonium and/or methoxamine were infused at a rate of 0.02 mL·min−1 by a WPI model sp100i pump (World Precision Instruments Inc., Sarasota, FL, USA). The intervals between the different stimulation frequencies or doses of α‐CGRP, substance P, acetylcholine and noradrenaline depended on the duration of their effects on diastolic blood pressure (5–10 min). In each case, we waited until diastolic blood pressure had completely returned to baseline values. At the end of the experiment, the respiration pump was switched off.

Data presentation and statistical evaluation

All data in the text and figures are presented as the mean ± SEM. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). The peak changes in diastolic blood pressure produced by the different treatments were determined and expressed as follows: (i) for electrical stimulation of the sensory CGRPergic vasodepressor outflow as well as for the i.v. bolus injections of exogenous α‐CGRP, substance P and acetylcholine, the resulting vasodepressor responses are expressed as % changes from baseline diastolic blood pressure (as previously reported by Villalón et al., 2008; Marichal‐Cancino et al., 2013); and (ii) for electrical stimulation of the sympathetic vasopressor outflow and i.v. bolus of exogenous noradrenaline, the resulting vasopressor responses are expressed as changes from baseline diastolic blood pressure (as previously reported by Villalón et al., 1995a,b; 1998). Moreover, the difference between the changes in diastolic blood pressure within one subgroup of animals was evaluated with Student–Newman–Keuls test, once a two‐way repeated measures ANOVA (randomized block design) had revealed that the samples represented different populations (Steel and Torrie, 1980). Statistical significance was accepted at P < 0.05.

Compounds

Apart from the anaesthetic (sodium pentobarbital), the compounds used in the present study (obtained from the sources indicated) were: gallamine triethiodide, hexamethonium chloride, methoxamine hydrochloride, rat α‐CGRP, substance P, acetylcholine chloride, noradrenaline hydrochloride, hydrochloric acid and sodium hydroxide (Sigma Chemical Co., St. Louis, MO, USA); and olcegepant (BIBN4096BS; Tocris Bioscience Co., Park Ellisville, MO, USA). All compounds were dissolved in physiological saline, except: (i) olcegepant, which was initially dissolved in 0.5 mL of 1 N HCl, then diluted with 4 mL of bidistilled water and then adjusted to pH 6.5–7.0 with 1 N NaOH (Arulmani et al., 2004); and (ii) hydrochloric acid and sodium hydroxide, which were dissolved in bidistilled water. These vehicles had no effect on the baseline values of diastolic blood pressure or heart rate (not shown). Fresh solutions were prepared for each experiment. The doses mentioned in this text refer to the free base of substances, except in the case of gallamine and noradrenaline where they refer to the corresponding salts.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).

Results

Systemic haemodynamic variables

The baseline values of diastolic blood pressure and heart rate in the 75 rats were 66 ± 8 mmHg and 303 ± 9 beats min−1 respectively. These variables remained unchanged after the i.v. bolus injection of gallamine or during the i.v. infusion of hexamethonium (not shown). Moreover, in all cases, diastolic blood pressure (but not heart rate) was significantly increased 20 min after the infusion of methoxamine had commenced (Table 1 and Figure 2A–C), as reported in previous studies from our group (e.g. Villalón et al., 2008; González‐Hernández et al., 2010, 2011; Marichal‐Cancino et al., 2013). Except when constructing S‐R curves or D‐R curves (see below), the methoxamine‐induced increases in diastolic blood pressure were sustained throughout the experiments (Figure 2A–C), as reported earlier (e.g. Villalón et al., 2008; González‐Hernández et al., 2010; 2011; Marichal‐Cancino et al., 2013). In addition, the effects produced by vehicle or the different doses of olcegepant on diastolic blood pressure and heart rate in the different groups are shown in Table 1. It is noteworthy that in the animals receiving methoxamine infusions (for eliciting vasodepressor responses), after i.v. administration of: (i) 300 or 1000 μg·kg−1 olcegepant (or the corresponding volumes of vehicle or nothing) the values of diastolic blood pressure and heart rate remained with no significant changes (P > 0.05); and (ii) 3000 μg·kg−1 olcegepant the values of diastolic blood pressure markedly decreased (P < 0.05) (Table 1). In contrast, in the absence of the methoxamine infusions (for eliciting noradrenergic vasopressor responses), the values of diastolic blood pressure remained without significant changes (P > 0.05) after i.v. administration of vehicle or all doses of olcegepant (Table 1). Moreover, none of the above i.v. treatments significantly affected (P > 0.05) the values of heart rate (Table 1).

Table 1.

Effects of i.v. continuous infusions of methoxamine (10–15 μg·kg−1·min−1) and/or the subsequent i.v. administration of vehicle (1 mL·kg−1) or olcegepant (300, 1000 and/or 3000 μg·kg−1) on the baseline values of diastolic blood pressure and heart rate in pithed rats

Treatments Diastolic blood pressure (mmHg) Heart rate (beats min−1)
n per group Before During methoxamine infusion After vehicle or nothing After olcegepant 300 or 1000 μg·kg−1 After olcegepant 3000 μg·kg−1 Before During methoxamine infusion After vehicle or nothing After olcegepant 300 or 1000 μg·kg−1 After olcegepant 3000 μg·kg−1
Vasodepressor S‐R curve 20 58 ± 2 166 ± 10* 170±7δ 165±8δ 96 ± 2** 317 ± 4 305 ± 6 315±8δ 310±5δ 300 ± 4
D‐R curve to α‐CGRP 20 62 ± 5 175 ± 6* 175±9δ 175±8δ 100 ± 5** 313 ± 2 315 ± 4 310±2δ 320±7δ 308 ± 4
D‐R curves to substance P
and acetylcholine		 15 65 ± 3 170 ± 5* 167±5δ ‐ 95 ± 8** 300 ± 5 310 ± 7 310±7δ ‐ 308 ± 5
Vasopressor S‐R curve 10 71 ± 5 ‐ 70±8δ 69±6δ 72 ± 7 290 ± 4 ‐ 300±8δ 299±10δ 304 ± 7
D‐R curve to noradrenaline 10 75 ± 2 ‐ 72±2δ 73±4δ 70 ± 3 300 ± 4 ‐ 302±9δ 303±6δ 298 ± 9
Open in a new tab
‐

Indicates that this (sub)group did not receive the treatment described.

*

Significantly different (P < 0.05) from the corresponding value obtained before the methoxamine infusion for producing vasodepressor responses.

**

Significantly different (P < 0.05) from the corresponding value obtained after 1000 μg·kg−1 olcegepant or 1 mL·kg−1 vehicle.

δ

These values remained practically unchanged 10 min after the i.v. administration of: (i) 1 mL·kg−1 vehicle (or nothing); or (ii) 300 or 1000 μg·kg−1 olcegepant. Accordingly, only one set of data (i.e. after 1 mL·kg−1 vehicle or after 1000 μg·kg−1 olcegepant) is shown for the sake of clarity and for avoiding a table unnecessarily exceeding the page dimensions. For further details, see the description of experimental protocols.

Figure 2.

Figure 2

Open in a new tab

Original tracings illustrating: (i) The vasodepressor responses to (A) electrical stimulation of the perivascular sensory CGRPergic outflow, (B) i.v. bolus injections of exogenous α‐CGRP and (C) i.v. bolus injections of exogenous substance P and acetylcholine after i.v. administration of vehicle (1 mL·kg−1). In all cases, the vasodepressor responses were not accompanied by changes in heart rate. Similar protocols were followed after 300, 1000 and/or 3000 μg·kg−1 olcegepant (not shown). (ii) The vasopressor responses to: (D) electrical stimulation of the sympathetic vasopressor outflow; and (E) i.v. injections of exogenous noradrenaline after i.v. administration of vehicle (1 mL·kg−1); in both cases the vasopressor responses were accompanied by small changes in heart rate. Similar protocols were followed after 1000 and 3000 μg·kg−1 olcegepant (not shown).

Vasodepressor responses produced by electrical stimulation of the perivascular sensory CGRPergic outflow or by i.v. administration of α‐CGRP, substance P and acetylcholine

The onset of the responses induced by stimulation (0.56, 1, 1.8, 3 and 5.6 Hz) of the perivascular CGRPergic sensory outflow (T9–T12) or by sequential i.v. bolus injections of α‐CGRP, substance P and acetylcholine during methoxamine infusions resulted in frequency‐dependent and dose‐dependent vasodepressor responses (Figure 2). The electrically‐induced vasodepressor responses were a result of selective stimulation of the sensory CGRPergic vasodepressor outflow since heart rate was not altered (Figure 2A), as previously reported (Villalón et al., 2008). Moreover, the vasodepressor responses to α‐CGRP (Figure 2B) as well as substance P and acetylcholine (Figure 2C) were not accompanied by changes in heart rate. In all cases, the resulting S‐R curves and D‐R curves were significant (P < 0.05) when compared with their corresponding baseline values.

Effect of olcegepant or its vehicle on the vasodepressor responses induced by electrical stimulation or exogenous α‐CGRP

Figure 3 shows the vasodepressor responses induced by electrical stimulation (S‐R curves; upper panel) and sequential i.v. bolus of exogenous α‐CGRP (D‐R curves; lower panel) in animals pretreated with vehicle (1 mL·kg−1) or olcegepant (300, 1000 and 3000 μg·kg−1). A two‐way analysis of variance comparing between the treatment groups and the effects of varying the nerve stimulation frequency or dose of α‐CGRP demonstrated the main effects as differences between groups [F(4,64) = 16.764, P < 0.05] and interactions [F(4,64) = 8.115, P < 0.05] in the resulting S‐R curves and also in the D‐R curves [F(4,64) = 49.959, P < 0.05] and [F(4,64) = 15.877, P < 0.05] respectively. In rats pretreated with vehicle both the S‐R curve (Figure 3A) and D‐R curve (Figure 3E) did not significantly differ (P > 0.05) from the corresponding control curve (i.e. resulting from the animals that did not receive vehicle or olcegepant). In contrast: (i) 300 μg·kg−1 olcegepant tended to attenuate the neurogenic vasodepressor response at 5.6 Hz, although this attenuation was not significant (Figure 3B), while it produced a significant blockade of the vasodepressor responses induced by the two highest doses of CGRP (Figure 3F); (ii) 1000 μg·kg−1 olcegepant produced a significant blockade of only the vasodepressor response produced by 5.6 Hz (Figure 3C), while it produced a significant blockade of the vasodepressor responses induced by the two highest doses of CGRP (Figure 3G); and (iii) 3000 μg·kg−1 olcegepant abolished the vasodepressor responses to both sensory electrical stimulation (Figure 3D) and exogenous α‐CGRP (Figure 3H).

Figure 3.

Figure 3

Open in a new tab

Olcegepant blocks the vasodepressor responses to electrical stimulation and exogenous α‐CGRP. Vasodepressor responses (during methoxamine infusions) induced by selective stimulation of the perivascular sensory CGRPergic outflow (upper panels) and i.v. bolus injections of exogenous α‐CGRP (lower panels) in pithed rats pretreated i.v. with vehicle (A and E: 1 mL·kg−1; n = 5 each) or olcegepant (B and F: 300 μg·kg−1; C and G: 1000 μg·kg−1; and D and H: 3000 μg·kg−1; n = 5 each). *P < 0.05 versus the corresponding control response. Note that either the vehicle S‐R curve or the vehicle D‐R curve shown for comparing with the effects of every olcegepant dose in the corresponding upper and lower panels was obtained from the same subgroup of animals, but it is shown for the sake of clarity when making comparisons. Moreover, 10 min after 3000 μg·kg−1 olcegepant a significant decrease in diastolic blood pressure (P < 0.05) was produced.

Effect of olcegepant or its vehicle on the vasopressor responses produced by electrical sympathetic stimulation or exogenous noradrenaline

Electrical stimulation of the sympathetic vasopressor outflow and sequential i.v. bolus injections of exogenous noradrenaline resulted in, respectively, frequency‐dependent and dose‐dependent increases diastolic blood pressure (see Figure 4), which were transient in nature (Figure 2D,E). The electrically‐induced vasopressor responses were due to selective sympathetic stimulation, since only inconsistent increases in heart rate were observed, as reported earlier (Villalón et al., 1995a,b, 1998). Exogenous noradrenaline also produced, as expected, dose‐dependent increases in heart rate (Figure 2E); these effects were not evaluated further. Main differences between groups [F(2,32) = 4.338, P < 0.05] and interactions [F(8,32) = 5.501, P < 0.05] were detected in the resulting S‐R curves; whereas only main differences between groups [F(4,74) = 7.109, P < 0.05] were detected in the D‐R curves. After i.v. administration of vehicle, both the sympathetically‐induced (Figure 4A) and noradrenaline‐induced (Figure 4C) vasopressor responses remained unchanged (P > 0.05). In contrast, i.v. administration of 1000 and 3000 μg·kg−1 olcegepant increased (P < 0.05): (i) the sympathetically induced vasopressor responses at high stimulation frequencies (1 and 3 Hz; Figure 4B); and (ii) the noradrenaline‐ induced vasopressor responses at lower doses (0.03, 0.1 and 0.3 μg·kg−1; Figure 4D) and tended to increase (not significantly) the vasopressor responses at 1 and 3 μg·kg−1 noradrenaline.

Figure 4.

Figure 4

Open in a new tab

Olcegepant increases the vasopressor responses to electrical stimulation and exogenous noradrenaline. Vasopressor responses (in the absence of methoxamine infusions) induced by electrical stimulation of the perivascular sympathetic outflow (upper panels) and i.v. bolus injections of exogenous noradrenaline (lower panels) in pithed rats before and after i.v. administration of either vehicle (A and C: 1 mL·kg−1 distilled water, pH: 6.5–7.0; n = 5) or olcegepant (B and D: 1000 and 3000 μg·kg−1; n = 5). *P < 0.05 (responses after 1000 and 3000 μg·kg−1 olcegepant) versus the corresponding control response. It is noteworthy that 10 min after 1 mL·kg−1 vehicle as well as after 1000 and 3000 μg·kg−1 olcegepant no significant changes in baseline diastolic blood pressure and heart rate (P > 0.05) were produced.

Effect of olcegepant on the vasodepressor responses induced by substance P and acetylcholine

As shown in Figure 5, sequential i.v. bolus injections of substance P followed by acetylcholine during the continuous infusion of methoxamine resulted in dose‐dependent decreases in diastolic blood pressure in control, vehicle‐pretreated and olcegepant‐pretreated animals, which were significant (P < 0.05) when compared with their corresponding baseline values. These vasodepressor responses to substance P and acetylcholine remained unchanged (P > 0.05) after i.v. administration of vehicle (Figure 5A,B) or 3000 μg·kg−1 olcegepant (Figure 5C,D).

Figure 5.

Figure 5

Open in a new tab

Olcegepant does not affect the vasodepressor responses to substance P and acetylcholine (during methoxamine infusions) in pithed rats. (i) Upper panel: animals pretreated with nothing (control responses) and compared with those pretreated with vehicle (A and B: 1 mL·kg−1 distilled water, pH: 6.5–7.0, n = 5 each); and (ii) lower panel: animals pretreated with vehicle (1 mL·kg−1, n = 5 each; same group described above) and compared with those pretreated with olcegepant (C and D: 3000 μg·kg−1; n = 5 each). The control responses to substance P and acetylcholine and those induced after vehicle did not significantly differ from those induced after olcegepant (P > 0.05 in all cases). Moreover, 10 min after 3000 μg·kg−1 olcegepant a significant decrease in diastolic blood pressure (P < 0.05) was produced.

Discussion and conclusions

General

In addition to the implications discussed below, our study shows that: (i) 3000 μg·kg−1 olcegepant abolished the vasodepressor responses induced by electrical sensory stimulation (1.8, 3.1 and 5.6 Hz) and α‐CGRP (all doses); (ii) 1000–3000 μg·kg−1 olcegepant increased the vasopressor responses to electrical sympathetic stimulation (1 and 3 Hz) and noradrenaline (0.03, 0.1 and 3 μg·kg−1); and (iii) these effects were selective, as the responses to substance P and acetylcholine remained unaltered after 3000 μg·kg−1 olcegepant. Hence, our study in pithed rats (which lack compensatory baroreflex mechanisms) suggests that olcegepant might induce prohypertensive mechanisms by boosting the vasoconstrictor (and consequently the vasopressor) responses evoked by sympathetic stimulation and noradrenaline. Consistent with this suggestion, Mai et al. (2014) showed in α‐CGRP knockout mice an increase in blood pressure associated with an increase in vascular sympathetic tone. Admittedly, Smillie et al. (2014) found that basal values of blood pressure in wild‐type and α‐CGRP knockout mice were similar, but some experimental differences between these contrasting studies include: (i) Mai et al. (2014) employed global knockout α‐CGRP/calcitonin mice and used telemetry to continuously record blood pressure; and (ii) Smillie et al. (2014) employed selective α‐CGRP knockout mice and used the tail cuff method to monitor blood pressure (this allows a single measurement each day). However, as Smillie et al. (2014) reported an exaggerated increase in blood pressure when the α‐CGRP knockout mice were used in the angiotensin II hypertension model, both studies support the notion that sensory CGRPergic nerves could be playing a protective role against hypertension. However, the fact that CGRP receptor antagonists (given as a single administration or during a short treatment period) do not affect basal blood pressure in healthy humans (Olesen et al., 2004; Petersen et al., 2005), or in humans with migraine and coronary artery disease (Ho et al., 2012), implies that CGRP does not seem to play a primary role in the regulation of basal blood pressure. Additional clinical studies analysing the cardiovascular effects produced by a long‐term blockade of CGRP receptors are required to shed further light on this issue (González‐Hernández et al., 2016).

Systemic haemodynamic changes

The i.v. infusions of hexamethonium (to block the vasopressor autonomic outflow) and methoxamine (for a sustained increase in blood pressure) represent a conditio sine qua non to induce vasodepressor responses by application of electrical stimulation, CGRP, substance P or acetylcholine (Taguchi et al., 1992). Under these conditions, when blood pressure is raised, 3000 μg·kg−1 olcegepant decreased diastolic blood pressure. This olcegepant‐induced vasodepressor effect was not observed in anaesthetised rats (Arulmani et al., 2004) or in pithed rats with a diastolic blood pressure of around 65 mm Hg (i.e. without methoxamine infusion; Table 1). This difference is probably due to opposing levels of vascular tone between the two conditions, with the elevated level of vascular tone induced by methoxamine revealing the vasodilator effect of the high dose of olcegepant. The mechanism mediating this effect of olcegepant was not further investigated.

Blockade by olcegepant of the sensory vasodepressor outflow and exogenous α‐CGRP

Taguchi et al. (1992) showed that an infusion of CGRP8‐37, a CGRP receptor antagonist, blocked the vasodepressor responses evoked by electrical stimulation (T9–T12) and exogenous CGRP. However, this blockade disappeared when this infusion was stopped. In contrast to CGRP8‐37, single i.v. injections of olcegepant dose‐dependently blocked the vasodepressor responses induced by electrical stimulation or rat α‐CGRP (Figure 3). These results are similar to those reported by Arulmani et al. (2004) and Petersen et al. (2004) in anaesthetised rats, although the former authors used human α‐CGRP (instead of rat α‐CGRP) and olcegepant; these experimental differences may help explain why 3000 μg·kg−1 olcegepant was less effective to block the vasodepressor responses to human α‐CGRP in anaesthetised rats (Arulmani et al., 2004).

Interestingly, there are some differences in the profile of blockade produced by 300, 1000 and 3000 μg·kg−1 olcegepant on the vasodepressor responses induced by electrical stimulation(S‐R curves) and rat α‐CGRP (D‐R curves) (Figure 3). Although we have no clear‐cut explanation for (nor a direct experimental proof to definitely support) these differences, four possibilities could be proposed, namely:

  1. Spinal stimulation of the T9–T12 or T7–T9 segments induces local (mesenteric) haemodynamic changes (Gillespie et al., 1970; Kawasaki et al., 1988; Taguchi et al., 1992), while i.v. bolus injections of noradrenaline and CGRP produce systemic (generalized) haemodynamic changes.

  2. The potentially high concentrations of neurotransmitters released from perivascular nerves after electrical stimulation.

  3. Prejunctional CGRP autoreceptors inhibit the sensory release of CGRP in the rat mesenteric bed (Nuki et al., 1994). Thus, blockade of these receptors by olcegepant may result in facilitation of CGRP release; this would explain why the sensory nerve stimulation responses require higher doses of olcegepant (as compared to the responses to α‐CGRP) to be blocked. Admittedly, further experiments falling beyond the scope of this study will be required to validate this possibility.

  4. Electrical stimulation of the sensory fibres may induce, in addition to CGRP, release of substance P and neurokinin A (Brain and Cambridge, 1996). Hence, the potential contribution of the latter neuropeptides to the electrically‐induced vasodepressor responses, although small and predominantly mediated by endothelial mechanisms (Taguchi et al., 1992), would be masked by the release of CGRP. This hypothesis may explain, at least in part, why the vasodepressor responses to nerve stimulation (unaffected after 300 μg·kg−1 olcegepant) were abolished after 3000 μg·kg−1 olcegepant. In keeping with this hypothesis, the vasodepressor responses to exogenous α‐CGRP (mediated exclusively by vascular CGRP receptors) were dose‐dependently blocked (starting with 300 μg·kg−1) by olcegepant (Figure 3).

Further evidence supporting olcegepant's selectivity to block CGRP receptors

Apart from considering that substance P is co‐localized with CGRP in sensory nerves (Price and Flores, 2007), endothelial mechanisms are also involved in the vasodilator responses to substance P (via the NK1 receptor; Lerner and Persson, 2008) and acetylcholine (via the M3 receptor; Hoffman and Taylor, 2001). Hence, the fact that the vasodepressor responses to substance P and acetylcholine were not modified by 3000 μg·kg−1 olcegepant (Figure 5C,D) supports olcegepant's selectivity to block CGRP receptors. Consistent with these findings, Doods et al. (2000) have shown in binding studies that olcegepant: (i) is a selective CGRP antagonist as it displayed no significant affinity for a set of 75 different receptors or enzyme systems (IC50> > 1000 nM); and (ii) exhibits an about 200 fold higher affinity for human CGRP receptors (pKi: 10.84) compared to rat CGRP receptors (pKi: 8.46). These binding data may also help explain why higher blocking doses of olcegepant were required in rats (present results) as compared to humans (Olesen et al., 2004).

Potentiation by olcegepant of the vasopressor responses produced by electrical sympathetic stimulation and exogenous noradrenaline: potential clinical implications

The vascular tone is mainly modulated by the sympathetic and sensory nervous systems (Hoffman, 2001; Smillie and Brain, 2011). Thus, the fact that 1000–3000 μg·kg−1 olcegepant potentiated the vasopressor responses produced by electrical sympathetic stimulation and exogenous noradrenaline (Figure 4) suggests that olcegepant may have potential prohypertensive properties when used frequently in acute antimigraine therapy, particularly in patients with cardiovascular diseases. These potential prohypertensive properties may be mediated by changing the balance between vasodilator/vasoconstrictor mechanisms. Indeed, under our experimental conditions, the electrical stimulation of spinal T9, which is shared in our sympathetic vasopressor (T7–T 9) and sensory vasodepressor (T 9–T12) protocols of electrical stimulation, may actually result in a sensory CGRP release. Thus, the resulting vasopressor responses induced by this procedure may have been, at least partly, overshadowed by the sensory CGRP release from electrical stimulation of the sensory vasodepressor CGRPergic outflow.

In addition to the above neurogenic release of CGRP, a basal circulating CGRP concentration in the pg/pM range has been reported in several species (see Villalón and Olesen, 2009). Thus, once the systemic vasodilatation (vasodepressor responses) produced by CGRP has been blocked by olcegepant, it allows to detect the increase of the vasopressor responses produced by electrical sympathetic stimulation and exogenous noradrenaline in pithed rats (devoid of compensatory baroreflex mechanisms which, admittedly, is not a physiological condition). On this basis, it is not unreasonable to suggest that this vascular noradrenergic facilitation might result in a potential prohypertensive action if olcegepant were taken frequently. Notwithstanding, we have to admit that 3000 μg·kg−1 olcegepant (i.v.) produced no increase in blood pressure in anaesthetised rats (Arulmani et al., 2004). This apparent discrepancy may be explained by activation of baroreflex mechanisms opposing any vasopressor effects, since those anaesthetised rats (unlike our experimental protocols in pithed rats) had an intact central nervous system.

Furthermore, the fact that 1000 and 3000 μg·kg−1 olcegepant significantly potentiated the vasopressor responses produced by: (i) 1 and 3 Hz (but not by lower stimulation frequencies); and (ii) 0.03, 0.1 and 0.3 μg·kg−1 noradrenaline (but not by higher doses) (Figure 4) deserves further considerations. In this respect, Bulloch and McGrath (1988) have suggested that a substantial component of the vasopressor response produced by 3 Hz in pithed rats, being resistant to blockade of α1 adrenoceptors, may involve a prominent role of purinergic co‐transmitters. Accordingly, it is tempting to propose that the above difference in olcegepant's profile of potentiation (Figure 4) may involve, at least in part, additional actions on the sympathetic release of co‐transmitters. Consistent with this view, Donoso et al. (2012) demonstrated in vitro that CGRP inhibits the sympathetic release of noradrenaline, ATP and neuropeptide Y. Since this effect was prevented with CGRP8‐37 or H‐89, they suggested the role of prejunctional CGRP receptors. These findings may also explain the differences observed in the above potentiation by olcegepant (Figure 4). Thus, we cannot categorically exclude the possibility that both pre‐ and post‐junctional CGRPergic mechanisms may modulate the sympathetic vasopressor outflow. Admittedly, further experiments that fall beyond the scope of the present study will be required to validate this possibility.

Lastly, it is worth considering the possible clinical relevance of our findings, particularly within the context of the potential cardiovascular risks associated with blocking acutely CGRP receptors. Indeed, our results show that 1000–3000 μg·kg−1 olcegepant facilitates the noradrenergic vasopressor responses in pithed rats. Accordingly, one may suggest that in patients with frequent migraine attacks and cardiovascular pathologies, the acute systemic blockade of CGRP receptors could represent a risk for worsening their cardiovascular conditions (e.g. a prohypertensive action). Evidently, it remains to be investigated whether a similar facilitation of the noradrenergic vasopressor responses (and a resulting prohypertensive action) is also produced after i.v. administration of other gepants or monoclonal antibodies towards CGRP and the CGRP receptor.

In conclusion, systemic blockade of CGRP receptors by 1000 and 3000 μg·kg−1 olcegepant: (i) dose‐dependently and selectively blocked the vasodepressor responses induced by electrical sensory stimulation and exogenous rat α‐CGRP; and (ii) facilitated the vasopressor responses induced by electrical sympathetic stimulation (1 and 3 Hz) or exogenous noradrenaline (0.03, 0.1 and 0.3 μg·kg−1). Both effects may result in a prohypertensive action.

Author contributions

V.H.A.R., E.R.M., B.A.M.C., G.M.M. and A.H.A.E. performed the research. C.M.V., V.H.A.R., G.M.M. and A.M.V.D.B. designed the research study. V.H.A.R., E.R.M., B.A.M.C., G.M.M., and A.H.A.E. analysed the data. V.H.A.R., B.A.M.C., A.M.V.D.B. and C.M.V. wrote the paper.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Acknowledgements

The authors would like to thank Mr. Mauricio Villasana and Mrs. Belinda Villanueva (B. Pharm.) for their skilful technical/administrative assistance. The present study was financially supported by Consejo Nacional de Ciencia y Tecnología (CONACyT; grant no. 219707; México D.F.).

Avilés‐Rosas, V. H. , Rivera‐Mancilla, E. , Marichal‐Cancino, B. A. , Manrique‐Maldonado, G. , Altamirano‐Espinoza, A. H. , Maassen Van Den Brink, A. , and Villalón, C. M. (2017) Olcegepant blocks neurogenic and non‐neurogenic CGRPergic vasodepressor responses and facilitates noradrenergic vasopressor responses in pithed rats. British Journal of Pharmacology, 174: 2001–2014. doi: 10.1111/bph.13799.

References

  1. Alexander SPH, Davenport AP, Kelly E, Marrion N, Peters JA, Benson HE et al. (2015). The concise guide to PHARMACOLOGY 2015/16: G protein‐coupled receptors. Br J Pharmacol 172: 5744–5869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arulmani U, Schuijt MP, Heiligers JPC, Willems EW, Villalón CM, Saxena PR (2004). Effects of the calcitonin gene‐related peptide (CGRP) receptor antagonist BIBN4096BS on α‐CGRP‐induced regional haemodynamic changes in anaesthetised rats. Basic Clin Pharmacol Toxicol 94: 291–297. [DOI] [PubMed] [Google Scholar]
  3. Brain SD, Cambridge H (1996). Calcitonin gene‐related peptide: vasoactive effects and potential therapeutic role. Gen Pharmacol 27: 607–611. [DOI] [PubMed] [Google Scholar]
  4. Bulloch JM, McGrath JC (1988). Selective blockade by nifedipine of ‘purinergic’ rather than adrenergic nerve‐mediated vasopressor responses in the pithed rat. Br J Pharmacol 95: 695–700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Curtis MJ, Bond RA, Spina D, Ahluwalia A, Alexander SP, Giembycz MA et al. (2015). Experimental design and analysis and their reporting: new guidance for publication in BJP. Br J Pharmacol 172: 3461–3471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Donoso MV, Hermosilla D, Navarrete C, Álvarez P, Lillo JG, Huidobro‐Toro JP (2012). Reciprocal sympatho‐nensory control: functional role of nucleotides and calcitonin gene related peptide in peripheral neuroeffector junction. Neuroscience 203: 216–229. [DOI] [PubMed] [Google Scholar]
  7. Doods H, Hallermayer G, Wu D, Entzeroth M, Rudolf K, Engel W et al. (2000). Pharmacological profile of BIBN4096BS, the first selective small molecule CGRP antagonist. Br J Pharmacol 129: 420–423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Edvinsson L (2015). CGRP receptor antagonists and antibodies against CGRP and its receptor in migraine treatment. Br J Clin Pharmacol 80: 193–199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gillespie JS, MacLaren A, Pollock D (1970). A method of stimulating different segments of the autonomic outflow from the spinal column to various organs in the pithed cat and rat. Br J Pharmacol 40: 257–267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. González‐Hernández A, Manrique‐Maldonado G, Lozano‐Cuenca J, Muñoz‐Islas E, Centurión D, MaassenVanDenBrink A et al. (2011). The 5‐HT1 receptors inhibiting the rat vasodepressor sensory CGRPergic outflow: further involvement of 5‐HT1F, but not 5‐HT1A or 5‐HT1D, subtypes. Eur J Pharmacol 659: 233–243. [DOI] [PubMed] [Google Scholar]
  11. González‐Hernández A, Marichal‐Cancino BA, Lozano‐Cuenca J, López‐Canales JS, Muñoz‐Islas E, Ramírez‐Rosas MB et al. (2016). Heteroreceptors modulating CGRP release at neurovascular junction: potential therapeutic implications on some vascular‐related diseases. Biomed Res Int 2016, Article ID 2056786, 17 pages, 10.1155/2016/2056786 (Open Access). [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. González‐Hernández A, Muñoz‐Islas E, Lozano‐Cuenca J, Ramírez‐Rosas MB, Sánchez‐López A, Centurión D et al. (2010). Activation of 5‐HT1B receptors inhibits the vasodepressor sensory CGRPergic outflow in pithed rats. Eur J Pharmacol 637: 131–137. [DOI] [PubMed] [Google Scholar]
  13. Ho TW, Ho AP, Chaitman BR, Johnson C, Mathew NT, Kost J et al. (2012). Randomized, controlled study of telcagepant in patients with migraine and coronary artery disease. Headache 52: 224–235. [DOI] [PubMed] [Google Scholar]
  14. Hoffman BB (2001). Catecolamines, sympathomimetic drugs, and adrenergic receptor antagonists In Hardman JG, Limbird LE, man AG (eds). Goodman & Gilman's. The Pharmacological Basis of Therapeutics (10th Edition) McGraw‐Hill: New york, 215–268. [Google Scholar]
  15. Hoffman BB, Taylor P (2001). Neurotransmission: the autonomic and somatic motor nervous systems In: Hardman JG, Limbird LE, Gilman AG. (eds). Goodman & Gilman's The Pharmacological Basis of Therapeutics, 10th edn. McGraw‐Hill: New york, pp. 115–154. [Google Scholar]
  16. Kawasaki H, Takasaki K, Saito A, Goto K (1988). Calcitonin gene‐related peptide acts as a novel vasodilator neurotransmitter in mesenteric resistance vessels of the rat. Nature 335: 164–167. [DOI] [PubMed] [Google Scholar]
  17. Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG (2010). Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 160: 1577–1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kleinman L, Radford E (1964). Ventilation standards for small mammals. J Appl Physiol 19: 360–362. [DOI] [PubMed] [Google Scholar]
  19. Lerner UH, Persson E (2008). Osteotropic effects by the neuropeptides calcitonin gene‐related peptide, substance P and vasoactive intestinal peptide. J Musculoskelet Neuronal Interact 8: 154–165. [PubMed] [Google Scholar]
  20. MaassenVanDenBrink A, Meijer J, Villalón CM, Ferrari MD (2016). Wiping out CGRP: potential cardiovascular risks. Trends Pharmacol Sci 37: 779–788. [DOI] [PubMed] [Google Scholar]
  21. Mai TH, Wu J, Diedrich A, Garland EM, Robertson D (2014). Calcitonin gene‐related peptide (CGRP) in autonomic cardiovascular regulation and vascular structure. J Am Soc Hypertens 8: 286–296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Marichal‐Cancino BA, Altamirano‐Espinoza AH, Manrique‐Maldonado G, MaassenVanDenBrink A, Villalón CM (2013). Role of pre‐junctional CB1, but not CB2, TRPV1 or GPR55 receptors in anandamide‐induced inhibition of the vasodepressor sensory CGRPergic outflow in pithed rats. Basic Clin Pharmacol Toxicol 114: 240–247. [DOI] [PubMed] [Google Scholar]
  23. McGrath JC, Lilley E (2015). Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP. Br J Pharmacol 172: 3189–3193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nuki C, Kawasaki H, Takasaki K, Wada A (1994). Pharmacological characterization of presynaptic calcitonin gene‐related peptide (CGRP) receptors on CGRP‐containing vasodilator nerves in rat mesenteric resistance vessels. J Pharmacol Exp Ther 268: 59–64. [PubMed] [Google Scholar]
  25. Olesen J, Diener HC, Husstedt IW, Goadsby PJ, Hall D, Meier U et al. (2004). Calcitonin gene‐related peptide receptor antagonist BIBN 4096 BS for the acute treatment of migraine. N Engl J Med 350: 1104–1110. [DOI] [PubMed] [Google Scholar]
  26. Petersen KA, Birk S, Doods H, Edvinsson L, Olesen J (2004). Inhibitory effect of BIBN4096BS on cephalic vasodilatation induced by CGRP or transcranial electrical stimulation in the rat. Br J Pharmacol 143: 697–704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Petersen KA, Birk S, Lassen LH, Kruuse C, Jonassen O, Lesko L et al. (2005). The CGRP antagonist, BIBN4096BS does not affect cerebral or systemic haemodynamics in healthy volunteers. Cephalalgia 25: 139–147. [DOI] [PubMed] [Google Scholar]
  28. Price TJ, Flores CM (2007). Critical evaluation of the colocalization between calcitonin gene‐related peptide, substance P, transient receptor potential vanilloid subfamily type 1 immunoreactivities, and isolectin B4 binding in primary afferent neurons of the rat and mouse. J Pain 8: 263–272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Russell FA, King R, Smillie SJ, Kodji X, Brain SD (2014). Calcitonin gene‐related peptide: physiology and pathophysiology. Physiol Rev 94: 1099–1142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shipley RE, Tilden JH (1947). A pithed rat preparation suitable for assaying pressor substances. Proc Soc Exp Biol Med 64: 453–455. [DOI] [PubMed] [Google Scholar]
  31. Smillie SJ, Brain S (2011). Calcitonin gene‐related peptide (CGRP) and its role in hypertension. Neuropeptides 45: 93–104. [DOI] [PubMed] [Google Scholar]
  32. Smillie SJ, King R, Kodji X, Outzen E, Pozsgai G, Fernandes E et al. (2014). An ongoing role of α‐calcitonin gene–related peptide as part of a protective network against hypertension, vascular hypertrophy, and oxidative stress. Hypertension 63: 1056–1062. [DOI] [PubMed] [Google Scholar]
  33. Southan C, Sharman JL, Benson HE, Faccenda E, Pawson AJ, Alexander SP et al. (2016). The IUPHAR/BPS guide to PHARMACOLOGY in 2016: towards curated quantitative interactions between 1300 protein targets and 6000 ligands. Nucl Acids Res 44: D1054–D1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Steel RGD, Torrie JH (1980). Principles and Procedures of Statistics, a Biomedical Approach, 2nd edn. McGraw‐Hill, Kogakusha Ltd: Tokyo. [Google Scholar]
  35. Taguchi T, Kawasaki H, Imamura T, Takasaki K (1992). Endogenous calcitonin gene‐related peptide mediates nonadrenergic noncholinergic depressor response to spinal cord stimulation in the pithed rat. Circ Res 71: 357–364. [DOI] [PubMed] [Google Scholar]
  36. Valdivia LF, Centurión D, Perusquía M, Arulmani U, Saxena PR, Villalón CM (2004). Pharmacological analysis of the mechanisms involved in the tachycardic and vasopressor responses to the antimigraine agent, isometheptene, in pithed rats. Life Sci 74: 3223–3234. [DOI] [PubMed] [Google Scholar]
  37. Villalón CM, Albarrán JA, Lozano J, Pertz HH, Görnemann T, Centurión D (2008). Pharmacological profile of the clonidine‐induced inhibition of vasodepressor sensory outflow in pithed rats: correlation with alpha2A/2C‐adrenoceptors. Br J Pharmacol 154: 51–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Villalón CM, Avilés‐Rosas VH, Rivera‐Mancilla E, Altamirano‐Espinoza AH, Manrique‐Maldonado G, Villanueva‐Castillo B (2014). Intravenous olcegepant blocks the sensory vasodepressor CGRPergic outflow and facilitates the sympathetic vasopressor outflow in pithed rats. Proceedings of the 2014 British Pharmacological Society Winter Meeting (Pharmacology 2014) at http://www.pA2online.org/abstracts/Vol12Issue3abst005P.pdf
  39. Villalón CM, Centurión D, Rabelo G, de Vries P, Saxena PR, Sánchez A (1998). The 5‐HT1‐like receptors mediating inhibition of sympathetic vasopressor outflow in the pithed rat: operational correlation with the 5‐HT1A, 5‐HT1B and 5‐HT1D subtypes. Br J Pharmacol 124: 1001–1011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Villalón CM, Contreras J, Ramírez E, Castillo C, Perusquía M, López FJ et al. (1995a). 5‐hydroxytryptamine inhibits pressor responses to preganglionic sympathetic nerve stimulation in pithed rats. Life Sci 7: 803–812. [DOI] [PubMed] [Google Scholar]
  41. Villalón CM, Contreras J, Ramírez E, Castillo C, Perusquía M, Terrón JA (1995b). Characterization of prejunctional 5‐HT receptors mediating inhibition of sympathetic vasopressor responses in the pithed rat. Br J Pharmacol 116: 3330–3336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Villalón CM, Olesen J (2009). The role of CGRP in the pathophysiology of migraine and efficacy of CGRP receptor antagonists as acute antimigraine drugs. Pharmacol Ther 124: 309–323. [DOI] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES