Abstract

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) and vasoactive intestinal polypeptide are structurally and functionally closely related but show differences in migraine-inducing properties. Mechanisms responsible for the difference in migraine induction are unknown. Here, for the first time, we present a head-to-head comparison study of the immediate and long-lasting observations of the migraine-inducing, arterial, physiological and biochemical responses comparing PACAP38 and vasoactive intestinal polypeptide. In a double-blind crossover study 24 female migraine patients without aura were randomly allocated to intravenous infusion of PACAP38 (10 pmol/kg/min) or vasoactive intestinal polypeptide (8 pmol/kg/min) over 20 min. We recorded incidence of migraine during and after infusion (0–24 h). Magnetic resonance angiography of selected extra- and intracranial arteries, blood samples (plasma PACAP38 and vasoactive intestinal polypeptide and serum tryptase), and vital signs (blood pressure, heart rate, respiratory frequency, and end-tidal pressure of CO2) was recorded before and up to 5 h after infusion. Twenty-two patients [mean age 24 years (range 19–36)] completed the study on both days. Sixteen patients (73%) reported migraine-like attacks after PACAP38 and four after vasoactive intestinal polypeptide (18%) infusion (P = 0.002). Three of four patients, who reported migraine-like attacks after vasoactive intestinal polypeptide, also reported attacks after PACAP38. Both peptides induced marked dilatation of the extracranial (P < 0.05), but not intracranial arteries (P > 0.05). PACAP38-induced vasodilatation was longer lasting (>2 h), whereas vasoactive intestinal polypeptide-induced dilatation was normalized after 2 h. We recorded elevated plasma PACAP38 at 1 h after the start of PACAP38 infusion only in those patients who later reported migraine attacks. Blood levels of vasoactive intestinal polypeptide and tryptase were unchanged after PACAP38 infusion. In conclusion, PACAP38-induced migraine was associated with sustained dilatation of extracranial arteries and elevated plasma PACAP38 before onset of migraine-like attacks. PACAP38 has a much higher affinity for the PAC1 receptor and we therefore suggest that migraine induction by PACAP38 may be because of activation of the PAC1 receptor, which may be a future anti-migraine drug target.

Introduction

In recent years there has been considerable interest in the role of pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) in migraine (Schytz et al., 2010). Intravenous infusion of PACAP38 induces migraine attacks in migraine sufferers and anti-migraine drugs are effective in the treatment of PACAP38-induced migraine (Schytz et al., 2009). In contrast, vasoactive intestinal polypeptide (VIP), a structurally-related neuropeptide, does not seem to induce migraine attacks (Rahmann, 2008). Both neuropeptides dilate cephalic arteries in healthy subjects (Hansen et al., 2006; Birk et al., 2007; Amin et al., 2012) and in migraineurs (Rahmann, 2008; Schytz et al., 2009). Interestingly, PACAP38 induces longer lasting vasodilatation than VIP, and we have recently shown that this effect lasted up to 5 h (Amin et al., 2012). PACAP38 and VIP (VPAC) bind with equal affinity to VPAC1 and VPAC2 receptors (Harmar et al., 2012). Given that PACAP38 has a much higher affinity for the PAC1 receptor (Harmar et al., 2012), migraine induction by PACAP38 may be because of activation of the PAC1 receptor (Schytz et al., 2010). Studies in migraine sufferers examined the physiological effects of PACAP38 (Schytz et al., 2009) and VIP (Rahmann, 2008) over a relatively short period (90 min), and patients reported migraine attacks 4–5 h after the start of PACAP38 infusion (Schytz et al., 2009), but not after VIP (Rahmann, 2008). The pathophysiological mechanisms of PACAP38-induced migraine are still unknown. Possible mechanisms may include prolonged dilatation of cranial arteries, increased endogenous production of PACAP38, and mast cell degranulation. In support, prolonged dilatation may activate the perivascular afferents leading to initiation of an inflammatory response (Wolff, 1963). To date, no studies investigated physiological changes during pre-ictal and ictal periods in human provocation studies (i.e. at least 5 h). For definitive demonstration of a difference between PACAP38 and VIP, a head-to-head double-blind crossover study is necessary. We present such a study with long-lasting observations of the migraine-inducing, arterial, physiological, and biochemical responses comparing PACAP38 and VIP. We hypothesized that PACAP38 compared to VIP would induce more migraine and a longer lasting dilatation of extracranial arteries. We also hypothesized no effect of either neuropeptide on the circumference of cerebral arteries and that PACAP38-induced migraine attacks would be preceded by changes in plasma levels of PACAP38 and mast cell degranulation marker tryptase.

Materials and methods

We recruited 24 female migraine patients without aura according to the International Classification of Headache Disorders (Headache Classification Committee of the International Headache Society, 2004) through a Danish website for recruitment of participants for biomedical research projects (www.forsoegsperson.dk). Exclusion criteria were: a history of other types of headaches (except episodic tension-type headache <5 days per month), any daily medication intake (except oral contraceptives), serious somatic or psychiatric disease, hypertension (systolic blood pressure >150 mmHg and/or diastolic blood pressure >100 mmHg), pregnant or breastfeeding females, and any contraindications for MRI scans. All participants used safe contraceptive methods.

The study was approved by the Ethical Committee of the Capital (Copenhagen) Region (H-1-2011-023) and conducted in accordance with the Declaration of Helsinki. In addition, information regarding the study was registered in www.clinicaltrials.gov (NCT01471990). All participants gave their written consent to participate in the study after receiving written and oral study information.

Experimental design

In a double-blinded crossover design the patients received 10 pmol/kg/min PACAP38 or 8 pmol/kg/min VIP (Calbiochem®) in random order over 20 min on 2 days separated by at least 1 week. These concentrations of PACAP38 and VIP showed equipotent initial vasodilator effect on the superficial temporal artery in previous studies (Rahmann, 2008; Schytz et al., 2009). Before the first study day, each participant underwent a general physical examination. All patients reported to the laboratory headache-free and without having a migraine attack within 5 days or other types of headache 48 h before the start of the study. The intake of coffee, tea, cocoa or other methylxanthine-containing foods and beverages was not allowed for 8 h before the study start. All participants fasted completely from 4 h before the experiment start.

On the study day, the patients were placed in the supine position in the magnetic resonance scanner room and a venous catheter (Venflon, Becton Dickinson Infusion Therapy) was inserted into the left antecubital vein for drug infusion. We collected blood samples to determine the baseline haematocrit, haemoglobin, PACAP38, VIP, and tryptase levels. We performed a baseline magnetic resonance angiography (MRA) on a 3.0 T Philips Achieva Scanner (Philips Medical Systems) using an eight-element phased-array receiver head coil. The patients were monitored with ECG, heart rate (by ECG monitoring function attached to the magnetic resonance scanner), end-tidal partial pressure of CO2 (PetCO2), respiratory frequency (Capnograph, Datex), and blood pressure (Veris monitor, Medrad). Intravenous infusion of either PACAP38 or VIP was started immediately after the baseline scan. The study medication was randomized, blinded and packed by a pharmacist (Centralapoteket). Every package consisted of two 10 ml containers labelled, for example Patient 1, Day 1 and Patient 1, Day 2. The packages were allocated successively to the patients as they came to the first study day. The patients were not removed from the scanner between the baseline scan and the subsequent MRA session at 20 min after the start of the infusion. This subsequent MRA scan session lasted for ∼20 min, after which the patients were allowed to leave the scanner and walk around or lie in a bed. Between 60 to 90 min after the start of the infusion, patients were allowed to eat and drink, and were then placed into the magnetic resonance scanner again. The second post-infusion MRA scan session was started at time point 120 min, and the last scan session at time point 5 h. The patients were removed from the scanner between the last two scan sessions. If participants reported symptoms meeting criteria for migraine-like attacks (Schytz et al., 2009) within 5 h after the start of the infusion, they were scanned immediately and if possible, once more 30 min after treatment with 6 mg sumatriptan by subcutaneous injection (Fig. 1).

Figure 1

Flow chart of the methodological procedures during the hospital phase. MRA, blood pressure, heart rate, PetCO2, respiratory frequency, and blood levels of PACAP38, VIP, tryptase, haemoglobin, and haematocrit were recorded at baseline. Subsequently, infusion of either PACAP38 or VIP was started for 20 min. The MRA recordings were repeated at 20 min, 2 h and if no migraine-like attack, at 5 h. If the patient reported a migraine-like attack, an immediate MRA was done followed by treatment with 6 mg subcutaneous sumatriptan injection, and another MRA 30 min after the injection. Blood levels of PACAP38, VIP and tryptase were taken at time points 60 min and 300 min. Headache characteristics, blood pressure, heart rate, PetCO2, and respiratory frequency were recorded every 5 to 10 min until 90 min and every 30 min until 5 h.

Figure 1

Flow chart of the methodological procedures during the hospital phase. MRA, blood pressure, heart rate, PetCO2, respiratory frequency, and blood levels of PACAP38, VIP, tryptase, haemoglobin, and haematocrit were recorded at baseline. Subsequently, infusion of either PACAP38 or VIP was started for 20 min. The MRA recordings were repeated at 20 min, 2 h and if no migraine-like attack, at 5 h. If the patient reported a migraine-like attack, an immediate MRA was done followed by treatment with 6 mg subcutaneous sumatriptan injection, and another MRA 30 min after the injection. Blood levels of PACAP38, VIP and tryptase were taken at time points 60 min and 300 min. Headache characteristics, blood pressure, heart rate, PetCO2, and respiratory frequency were recorded every 5 to 10 min until 90 min and every 30 min until 5 h.

Recording of variables

We recorded MRA, heart rate, PetCO2, respiratory frequency, mean arterial blood pressure, headache intensity, and other symptoms at fixed time points before and after intravenous infusion. MRA was recorded as described above. The heart rate, PetCO2, and respiratory frequency were recorded every fifth min until 90 min after the start of infusion. The blood pressure, headache intensity, and other symptoms were recorded every 10 min until 90 min after the start of infusion. Between 90 min and 5 h, all variables were recorded every 30 min (i.e. at 120, 150, 180, 210, 240, 270 and 300 min). Headache intensity was rated on a verbal rating scale from 0 to 10 [0 = no headache; 1 = a very mild headache (including a feeling of pressing or throbbing); 10 = worst imaginable headache] (Iversen et al., 1989). Blood samples for quantification of PACAP38, VIP, and tryptase were collected at baseline, at 60 min, and 300 min after the start of infusion.

Plasma concentrations of PACAP38 and vasoactive intestinal polypeptide

Blood samples for PACAP38 and VIP measurements were collected in ice-chilled tubes containing 50 IU of heparin and 500 kIU aprotinin (Trasylol) per ml blood. Plasma was separated by centrifugation at 1851g for 10 min at 4°C within 30 min and then frozen at −25°C. The radioimmunoassays were performed as previously described for PACAP38 (Birk et al., 2007) and VIP (Rahmann, 2008).

Serum concentration of tryptase

Venous blood was collected into vacutainers containing clot activator and gel for serum separation. After 30 min of coagulation, samples were centrifuged at 2500g for 10 min at room temperature. The serum samples were stored at −80°C until analysis. Serum tryptase (including all forms of α-tryptase and β-tryptase) was measured by a fluorescence immunoassay (Phadia) for the Phadia 250 analyser.

Imaging protocol

For vessel imaging, a 3D inflow gradient echo sequence was used. First, a scout MRA was performed using field of view (180 × 180 × 120 mm3); acquired matrix size (MxP) of 120 × 120; acquired voxel resolution 1.5 × 1.5 × 2.4 mm3; reconstructed resolution 0.70 × 0.70 × 1.20 mm3; repetition time 23 ms; echo time 3.9 ms; flip angle 20°; parallel imaging (sense) factor 3; two chunks; total scan duration of 73 s. This scout MRA was used to plan the subsequent high-resolution scans. The first high-resolution MRA scan was planned to cover the large arteries and used field of view 200 × 200 × 74 mm3; acquired matrix size 800 × 406; acquired voxel resolution 0.25 × 0.49 × 1.00 mm3; reconstructed resolution 0.20 × 0.20 × 0.50 mm3; repetition time 25 ms; echo time 3.5; flip angle 20°; sense factor 2; four chunks; total scan duration of 9 min 3 s. If the participant was not removed from the magnetic resonance scanner between the baseline and the first post-infusion scan, we used same resolution but a shorter field of view (200 × 200 × 37) with total scan duration time 4 min 31 s. Based on the first high-resolution MRA, we planned another MRA scan with even higher spatial resolution to visualize smaller arteries, using the following parameters: field of view 200 × 200 × 16 mm3; acquired matrix size 800 × 571; acquired voxel resolution 0.25 × 0.35 × 0.70 mm3; reconstructed resolution 0.20 × 0.20 × 0.35 mm3; repetition time 25 ms; echo time 3.5; flip angle 20°; sense factor 3; two chunks; total scan duration of 5 min 29 s.

Magnetic resonance angiography data analysis

The acquired MRAs were analysed by LKEB-MRA vessel wall analysis software program (version 6.2007) (de Koning et al., 2003), which has been used in previous migraine studies (Schoonman et al., 2008; Asghar et al., 2011; Amin et al., 2013a). The software provides automated contour detection and quantification of the luminal boundaries every 0.2 mm perpendicular to the centreline in the chosen vessel segments. Investigators who performed the analysis (F.M.A., E.L.) were masked to the experimental day and scan session. We obtained an average of 26 values (i.e. 5 mm long vessel segment) for each arterial measurement. We differentiated between intracranial and extracranial arteries. The intracranial arteries were further differentiated as cerebral and extracerebral. The cerebral arteries included the middle cerebral artery, the cerebral segment of the internal carotid artery (ICAcerebral), and the basilar artery. The extracerebral artery was the cavernous segment of the internal carotid artery (ICAcavernous), which was defined as the initial portion of the ICA, when it enters the cranial cavity through the carotid canal, but before the siphon in the cavernous sinus. The extracranial vessel segments included the superficial temporal artery, the middle meningeal artery, the external carotid artery and the cervical segment of the internal carotid artery (ICAcervical). The middle cerebral artery was identified by marking the start of the branch from the main trunk of the ICA. The ICAcerebral was identified just before the ICA became middle cerebral artery. The basilar artery segment was marked using the point where it divides into the posterior cerebral arteries as reference. The ICAcavernous start was selected where the ICA exits the skull bone and enters the intracranial cavity (i.e. at the point it appears as a circle on the axial MRA images). The middle meningeal artery segment was identified by marking the branch from the main trunk of the maxillary artery, and the ICAcervical and superficial temporal artery segments were identified using the ipsilateral start of the middle meningeal artery as reference point.

Statistical analysis

We calculated our sample size based on detection of at least 45% difference in migraine induction between PACAP38 (65%) and VIP (20%) at 5% significance level with 80% power [standard deviation (SD) 0.9]. We calculated that 20 patients should be included in the crossover study, and then increased the sample size to 24 to increase confidence in our findings.

All absolute values are presented as mean, except headache scores, which are presented as median values. Peak mean vascular variables and per cent changes are presented with 95% confidence intervals (CI). Measurements before the start of infusion of each dose were defined as baseline. We calculated median peak headache score and median time to peak headache and to onset of attack after PACAP38 and VIP.

The mean plasma half-life of PACAP38 is 3.5 min (Birk et al., 2007) and that of VIP 1.0 min (Domschke et al., 1978). In addition, the vasoactive effect of PACAP38 lasts for at least 5 h (Amin et al., 2012). We therefore defined an early phase as 0 to 2 h, a delayed phase from 2 to 5 h and a post-hospital phase from 5 to 24 h after start of infusion.

The primary end-points of the study were: the difference in incidence of migraine-like attacks between PACAP38 and VIP; the difference in incidence of headache between PACAP38 and VIP; the difference in area under the curve (AUC) for headache score during the early phase; and the delayed phase between PACAP38 and VIP. The secondary end-points were difference in AUC0-2h for arterial circumferences (middle cerebral artery, superficial temporal artery, and middle meningeal artery), mean arterial blood pressure, heart rate, respiratory frequency, and PetCO2 between PACAP38 and VIP; difference in arterial circumferences (external carotid artery, ICAcervical, ICAcavernous, ICAcerebral, and basilar artery) between baseline and 2 h after infusion; the difference in arterial circumference (external carotid artery, ICAcervical, ICAcavernous, ICAcerebral, and basilar artery) between baseline and migraine-like attack, and before versus after treatment with sumatriptan; the difference in blood levels of PACAP38, VIP and tryptase at 60 min, between the two experimental days.

Headache incidence, migraine-like attacks, and adverse events were analysed as binary categorical data with the McNemar test. We calculated the AUC according to the trapezium rule (Matthews et al., 1990) to obtain summary measures and to analyse the differences between PACAP38 and VIP. Baseline was subtracted before calculating AUC to reduce variation between sessions. Analysis of AUC values, circumferences, and blood concentrations was performed with a paired t-test. Headache scores were analysed using the Wilcoxon signed rank test. We tested for period and carry-over effect using independent samples t-tests according to Altman (1999).

The analyses were performed using IBM® SPSS® Statistics 20.0 for MAC. Level of significance at 0.05 was accepted for all tests and no correction for multiple comparisons was made.

Results

Twenty-four female migraineurs without aura participated in the study. Detailed headache and migraine history is shown in Table 1. Twenty-two patients [mean age 24 years (range 19–36); mean height 169 cm (range 157–183); mean weight 62 kg (range 51–77)], completed the study on both experimental days, as two participants dropped out after the first study day (PACAP38 day in both cases). Contact was lost with one of these patients, whereas the second patient declined to participate because of an unpleasant feeling after PACAP38. These two participants were therefore not included in the comparison between the two experimental days, but only in the comparison within the PACAP38 day. There were no day-to-day differences of any variables at baseline, except the heart rate, which was slightly higher on the VIP day (Table 2). We further found no period or carry-over effect for circumference changes of middle cerebral artery, middle meningeal artery, and superficial temporal artery (baseline to 20 min) or blood level changes of PACAP38, VIP and tryptase (baseline to 60 min) (P ≥ 0.119). There were also no right-to-left differences or differences between arterial circumferences at baseline on the two experimental days (Table 3). We have therefore presented the mean circumferences of right and left sided arteries.

Table 1

Clinical characterization of all 24 migraine without aura patients

Subject Age (years) Disease duration (years) Migraine frequency (attacks/month) Triptan use (days/month) Analgesic use (days/month) Tension-type headache (days/month) Alcohol induced migraine/headache 
VP01 22 0–1 No/yes 
VP02 24 1–2 No/yes 
VP03 23 11 1–2 No/yes 
VP04 22 1–2 No/yes 
VP05 36 11 1–2 No/yes 
VP06 19 1–2 No/no 
VP07 21 >3 No/yes 
VP08 31 15 >3 Yes/yes 
VP09 19 14 >3 No/yes 
VP10 28 22 1–2 No/yes 
VP11 20 17 >3 No/yes 
VP12 23 15 2–3 No/yes 
VP13 21 10 2–3 Yes/yes 
VP14 31 13 >3 Yes/yes 
VP15 26 15 >3 <5 NA 
VP16 20 >3 No/yes 
VP17 21 1–2 No/yes 
VP18 22 1–2 Yes/yes 
VP19 27 >3 Yes/yes 
VP20 34 0–1 Yes/yes 
VP21 22 0–1 Yes/yes 
VP22 22 10 >3 Yes/yes 
VP23 22 1–2 No/yes 
VP24 19 >3 No/yes 
Subject Age (years) Disease duration (years) Migraine frequency (attacks/month) Triptan use (days/month) Analgesic use (days/month) Tension-type headache (days/month) Alcohol induced migraine/headache 
VP01 22 0–1 No/yes 
VP02 24 1–2 No/yes 
VP03 23 11 1–2 No/yes 
VP04 22 1–2 No/yes 
VP05 36 11 1–2 No/yes 
VP06 19 1–2 No/no 
VP07 21 >3 No/yes 
VP08 31 15 >3 Yes/yes 
VP09 19 14 >3 No/yes 
VP10 28 22 1–2 No/yes 
VP11 20 17 >3 No/yes 
VP12 23 15 2–3 No/yes 
VP13 21 10 2–3 Yes/yes 
VP14 31 13 >3 Yes/yes 
VP15 26 15 >3 <5 NA 
VP16 20 >3 No/yes 
VP17 21 1–2 No/yes 
VP18 22 1–2 Yes/yes 
VP19 27 >3 Yes/yes 
VP20 34 0–1 Yes/yes 
VP21 22 0–1 Yes/yes 
VP22 22 10 >3 Yes/yes 
VP23 22 1–2 No/yes 
VP24 19 >3 No/yes 
Table 2

Comparison of mean values (± SD) of variables at baseline between the PACAP38 and VIP days (n = 22)

Variable PACAP38 day (± SD) VIP day (± SD) P-value 
PetCO2 (kPa) 4.69 (0.32) 4.73 (0.31) 0.317 
Respiratory frequency 15 (4) 14 (3) 0.063 
Systolic blood pressure (mmHg) 116 (11) 114 (8) 0.145 
Diastolic blood pressure (mmHg) 67 (6) 65 (7) 0.425 
Mean arterial blood pressure (mmHg) 83 (7) 81 (6) 0.184 
Heart rate (beats/min) 62 (8) 65 (8) 0.026 
Haemoglobin 8.01 (0.42) 8.09 (0.52) 0.445 
Hematocrit 0.38 (0.02) 0.38 (0.03) 0.404 
VIP (pmol/l) 6.03 (1.77) 6.73 (3.81) 0.104 
Tryptase (µg/l) 4.14 (2.62) 4.07 (2.43) 0.581 
Variable PACAP38 day (± SD) VIP day (± SD) P-value 
PetCO2 (kPa) 4.69 (0.32) 4.73 (0.31) 0.317 
Respiratory frequency 15 (4) 14 (3) 0.063 
Systolic blood pressure (mmHg) 116 (11) 114 (8) 0.145 
Diastolic blood pressure (mmHg) 67 (6) 65 (7) 0.425 
Mean arterial blood pressure (mmHg) 83 (7) 81 (6) 0.184 
Heart rate (beats/min) 62 (8) 65 (8) 0.026 
Haemoglobin 8.01 (0.42) 8.09 (0.52) 0.445 
Hematocrit 0.38 (0.02) 0.38 (0.03) 0.404 
VIP (pmol/l) 6.03 (1.77) 6.73 (3.81) 0.104 
Tryptase (µg/l) 4.14 (2.62) 4.07 (2.43) 0.581 

PetCO2 = end-tidal pressure of CO2.

P-values: the paired samples t-test.

Table 3

Comparison of the mean circumference (mm) of selected extracranial and intracranial arteries at baseline between PACAP38 and VIP days

Artery PACAP38 VIP P-value 
ECA (mm) 9.09 8.88 0.361 
STA (mm) 5.05 5.14 0.514 
MMA (mm) 4.54 4.43 0.315 
ICAcervical (mm) 14.48 14.52 0.825 
ICAcavernous (mm) 13.54 13.41 0.640 
ICAcerebral (mm) 11.24 11.02 0.381 
MCA (mm) 9.14 8.91 0.167 
BA (mm) 9.38 9.18 0.077 
Artery PACAP38 VIP P-value 
ECA (mm) 9.09 8.88 0.361 
STA (mm) 5.05 5.14 0.514 
MMA (mm) 4.54 4.43 0.315 
ICAcervical (mm) 14.48 14.52 0.825 
ICAcavernous (mm) 13.54 13.41 0.640 
ICAcerebral (mm) 11.24 11.02 0.381 
MCA (mm) 9.14 8.91 0.167 
BA (mm) 9.38 9.18 0.077 

ECA = external carotid artery; STA = superficial temporal artery; MMA = middle meningeal artery; MCA = middle cerebral artery; BA = basilar artery.

P-values: the paired samples t-test.

Incidence of migraine-like attacks and headache

After PACAP38 infusion, 16 of 22 patients (73%) reported migraine-like attacks (0–24 h) compared with 4 of 22 (18%) after VIP (P = 0.002) (Tables 4 and 5). The median time to onset of attack was 4 h 15 min (range 20 min to 9 h) after start of PACAP38 infusion, and 4 h 30 min (range 70 min to 24 h) after start of VIP infusion. The median intensity of immediate headache (verbal rating scale 1) after VIP infusion in four patients, who later developed migraine-like attacks, was similar to median intensity (verbal rating scale 1) of 18 patients, who did not report migraine-like attacks after VIP.

Table 4

Headache characteristics after intravenous PACAP38 infusion in 24 patients

Subject MLA or PH (onset time) Headache characteristicsa Associated symptomsb Treatment (time) /efficacy 
VP01 PH (4 h 30 min) Bilat/pres/6/– –/–/– – 
PH (6 h) Bilat/pres/6/– –/–/– Ibuprofen (6 h) /yes 
VP02 – – Yes/–/– – 
– – –/–/– – 
VP03 PH (10 min) Bilat/pres/1/– –/–/– – 
PH (6 h) Bilat/pres/1/yes –/–/– Acetylsalicylic acid + caffeine (7 h) /yes 
VP04 MLA (3 h 30 min) Right/throb/7/yes Yes/yes/yes Sumatriptan (4 h 40 min) /yes 
PH (6 h) Right/pres/1/– Yes/–/yes – 
VP05 PH (20 min) Left/pres/2/– Yes/yes/– – 
MLA (7 h) Left/throb/3/yes Yes/–/– Eletriptan (7 h) /yes 
VP06 PH (20 min) Bilat/pres/1/– Yes/–/– – 
PH (15 h) Right/throb/1/– –/–/– – 
VP07 MLA (2 h) Right/throb/4/yes Yes/yes/– – 
PH (6 h) Bilat/pres/3/– Yes/–/– Paracetamol (6 h) /yes, metoclopramide (6 h) /yes 
VP08 PH (10 min) Left/throb/1/– –/–/– – 
MLA (8 h) Left/throb/3/yes Yes/–/– Zolmitriptan (8 h) /yes 
VP09 MLA (4 h 30 min) Bilat/throb/4/yes –/yes/yes – 
MLA (6 h) Bilat/throb/9/yes –/yes/yes Sumatriptan (6 h) /yes 
VP10 MLA (20 min) Left/throb/6/yes Yes/yes/yes Sumatriptan (75 min) /– 
MLA (6 h) Bilat/pres/5/yes Yes/yes/yes Sumatriptan (18 h) /– 
VP11 MLA (2 h) Right/pres/2/yes Yes/–/– – 
MLA (13 h) Right/throb/9/yes Yes/yes/yes Sumatriptan (13 h) /yes 
VP12 PH (30 min) Bilat/pres/5/– –/–/– – 
PH (6 h) Bilat/pres/2/– –/–/– – 
VP13 MLA (5 h) Left/throb/4/yes –/yes/yes Sumatriptan (5 h 30 min) /yes 
PH (24 h) Bilat/pres/6/yes –/yes/– – 
VP14 MLA (4 h) Right/throb/4/yes Yes/yes/yes Sumatriptan (5 h 10 min) /– 
MLA (6 h) Bilat/throb/2/yes Yes/yes/yes – 
VP15 MLA (4 h 30 min) Right/throb/2/– –/yes/yes – 
MLA (6 h) Right/throb/3/– –/yes/yes Almotriptan (6 h) /yes 
VP16 MLA (2 h) Bilat/throb/3/yes Yes/yes/yes Sumatriptan (2 h 45 min) /–, metoclopramide (3 h 25 min) /– 
Bc – – –/–/– – 
VP17 – – –/–/– – 
– – –/–/– – 
VP18 MLA (50 min) Bilat/throb/4/yes Yes/yes/yes Sumatriptan (3 h 20 min) /yes 
PH (14 h) Bilat/pres/1/– –/–/– Acetylsalicylic acid + codeine (13 h)/yes 
VP19 PH (5 h) Bilat/pres/3/– –/–/– Nimesolide (5 h) /yes 
MLA (9 h) Left/pres/3/– –/–/– Eletriptan (9 h) /yes 
VP20 PH (60 min) Bilat/throb/1/yes –/–/– – 
MLA (7 h) Bilat/throb/1/yes Yes/–/– – 
VP21 MLA (20 min) Right/throb/6/yes Yes/yes/yes Sumatriptan (3 h) /yes 
PH (6 h) Bilat/pres/1/yes Yes/–/– – 
VP22 PH (20 min) Bilat/pres/2/– –/yes/– – 
MLA (7 h) Bilat/pres/2/– –/yes/yes Sumatriptan (7 h) /yes 
VP23 MLA (4 h 30 min) Bilat/pres/7/yes Yes/–/– – 
– – – Cetirizine (12 h) /yesd 
VP24 MLA (5 h) Right/throb/4/yes Yes/–/– – 
MLA (6 h) Right/throb/5/yes Yes/–/– Sumatriptan (6 h) /yes, clemastine (8 h) /–, paracetamol (8 h) /–, methylprednisolonsuccinate i.v. (8 h 30 min) / yes 
Subject MLA or PH (onset time) Headache characteristicsa Associated symptomsb Treatment (time) /efficacy 
VP01 PH (4 h 30 min) Bilat/pres/6/– –/–/– – 
PH (6 h) Bilat/pres/6/– –/–/– Ibuprofen (6 h) /yes 
VP02 – – Yes/–/– – 
– – –/–/– – 
VP03 PH (10 min) Bilat/pres/1/– –/–/– – 
PH (6 h) Bilat/pres/1/yes –/–/– Acetylsalicylic acid + caffeine (7 h) /yes 
VP04 MLA (3 h 30 min) Right/throb/7/yes Yes/yes/yes Sumatriptan (4 h 40 min) /yes 
PH (6 h) Right/pres/1/– Yes/–/yes – 
VP05 PH (20 min) Left/pres/2/– Yes/yes/– – 
MLA (7 h) Left/throb/3/yes Yes/–/– Eletriptan (7 h) /yes 
VP06 PH (20 min) Bilat/pres/1/– Yes/–/– – 
PH (15 h) Right/throb/1/– –/–/– – 
VP07 MLA (2 h) Right/throb/4/yes Yes/yes/– – 
PH (6 h) Bilat/pres/3/– Yes/–/– Paracetamol (6 h) /yes, metoclopramide (6 h) /yes 
VP08 PH (10 min) Left/throb/1/– –/–/– – 
MLA (8 h) Left/throb/3/yes Yes/–/– Zolmitriptan (8 h) /yes 
VP09 MLA (4 h 30 min) Bilat/throb/4/yes –/yes/yes – 
MLA (6 h) Bilat/throb/9/yes –/yes/yes Sumatriptan (6 h) /yes 
VP10 MLA (20 min) Left/throb/6/yes Yes/yes/yes Sumatriptan (75 min) /– 
MLA (6 h) Bilat/pres/5/yes Yes/yes/yes Sumatriptan (18 h) /– 
VP11 MLA (2 h) Right/pres/2/yes Yes/–/– – 
MLA (13 h) Right/throb/9/yes Yes/yes/yes Sumatriptan (13 h) /yes 
VP12 PH (30 min) Bilat/pres/5/– –/–/– – 
PH (6 h) Bilat/pres/2/– –/–/– – 
VP13 MLA (5 h) Left/throb/4/yes –/yes/yes Sumatriptan (5 h 30 min) /yes 
PH (24 h) Bilat/pres/6/yes –/yes/– – 
VP14 MLA (4 h) Right/throb/4/yes Yes/yes/yes Sumatriptan (5 h 10 min) /– 
MLA (6 h) Bilat/throb/2/yes Yes/yes/yes – 
VP15 MLA (4 h 30 min) Right/throb/2/– –/yes/yes – 
MLA (6 h) Right/throb/3/– –/yes/yes Almotriptan (6 h) /yes 
VP16 MLA (2 h) Bilat/throb/3/yes Yes/yes/yes Sumatriptan (2 h 45 min) /–, metoclopramide (3 h 25 min) /– 
Bc – – –/–/– – 
VP17 – – –/–/– – 
– – –/–/– – 
VP18 MLA (50 min) Bilat/throb/4/yes Yes/yes/yes Sumatriptan (3 h 20 min) /yes 
PH (14 h) Bilat/pres/1/– –/–/– Acetylsalicylic acid + codeine (13 h)/yes 
VP19 PH (5 h) Bilat/pres/3/– –/–/– Nimesolide (5 h) /yes 
MLA (9 h) Left/pres/3/– –/–/– Eletriptan (9 h) /yes 
VP20 PH (60 min) Bilat/throb/1/yes –/–/– – 
MLA (7 h) Bilat/throb/1/yes Yes/–/– – 
VP21 MLA (20 min) Right/throb/6/yes Yes/yes/yes Sumatriptan (3 h) /yes 
PH (6 h) Bilat/pres/1/yes Yes/–/– – 
VP22 PH (20 min) Bilat/pres/2/– –/yes/– – 
MLA (7 h) Bilat/pres/2/– –/yes/yes Sumatriptan (7 h) /yes 
VP23 MLA (4 h 30 min) Bilat/pres/7/yes Yes/–/– – 
– – – Cetirizine (12 h) /yesd 
VP24 MLA (5 h) Right/throb/4/yes Yes/–/– – 
MLA (6 h) Right/throb/5/yes Yes/–/– Sumatriptan (6 h) /yes, clemastine (8 h) /–, paracetamol (8 h) /–, methylprednisolonsuccinate i.v. (8 h 30 min) / yes 

A = in-hospital phase (0–5 h);

B = post-hospital phase (6–24 h);

MLA = migraine-like attack; PH = peak headache (if no MLA was reported); N/a = .

aHeadache characteristics: localization/quality/intensity/aggravation by movement (throb = throbbing; pres = pressing).

bAssociated symptoms: nausea/photophobia/phonophobia.

cThis information obtained by telephone because no diary was received.

dMedication taken against facial puffing.

Table 5

Headache characteristics after intravenous infusion of VIP in 24 patients

Subject MLA or PH (onset time) Headache characteristicsa Associated symptomsb Treatment (time)/efficacy 
VP01 – – –/–/– – 
PH (19 h) Bilat/pres/4/yes –/–/– – 
VP02 PH (2 h) Left/throb/3/yes –/–/– – 
PH (6 h) Left/throb/3/yes –/–/– – 
VP03 MLA (70 min) Bilat/throb/8/yes Yes/yes/yes Sumatriptan (2 h 45 min) /yes, metoclopramide (3 h 25 min) /yes 
PH (24 h) Bilat/pres/1/– Yes/–/– Paracetamol (24 h) /yes 
VP04 PH (30 min) Bilat/pres/3/– –/–/– – 
PH (13 h) Bilat/pres/2/yes Yes/yes/– – 
VP05 PH (20 min) Left/pres/1/– –/–/– – 
– – –/–/– – 
VP06 PH (40 min) Bilat/pres/1/– –/–/– – 
– – –/–/– – 
VP07 MLA (3 h) Right/throb/6/yes –/yes/yes Sumatriptan (4 h 30 min) /yes 
MLA (14 h) Right/throb/6/yes –/yes/yes Acetylsalicylic acid + caffeine (17 h) /– 
Acetylsalicylic acid + caffeine (21 h) /– 
VP08 PH (20 min) Bilat/throb/2/– –/–/– – 
MLA (6 h) Left/throb/3/– Yes/–/– Zolmitriptan (6 h) /yes 
VP09 PH (20 min) Bilat/pres/1/– –/–/– – 
– – –/–/– – 
VP10 PH (20 min) Left/throb/2/yes –/–/yes – 
PH (18 h) Left/throb/2/yes –/–/– – 
VP11 – – –/–/– – 
– – –/–/– – 
VP12 PH (5 h) Bilat/pres/2/– –/–/– – 
PH (6 h) Bilat/pres/3/– –/–/– Paracetamol (6 h) /yes 
VP13 PH (10 min) Bilat/pres/1/– –/–/– – 
PH (17 h) Bilat/pres/1/– –/–/– – 
VP14 – – –/–/yes – 
PH (17 h) Right/throb/1/yes –/–/– – 
VP15 PH (5 h) Bilat/pres/3/yes –/–/– – 
MLA (6 h) Right/throb/5/yes Yes/–/– Almotriptan (7 h) /– 
VP16 PH (10 min) Right/pres/2/– –/–/– – 
Bc – – –/–/– – 
VP17 – – –/–/– – 
– – –/–/– – 
VP18 PH (10 min) Bilat/pres/1/– –/–/– – 
PH (9 h) Bilat/throb/2/– –/–/– – 
VP19 – – –/–/– – 
– – –/–/– – 
VP20 Ad N/a N/a N/a N/a 
Bd N/a N/a N/a N/a 
VP21 – – –/–/– – 
– – –/–/– – 
VP22 PH (30 min) Bilat/pres/2/– –/–/– – 
– – –/–/– – 
VP23 PH (20 min) Bilat/pres/1/– Yes/–/– – 
PH (16 h) Bilat/pres/1/– –/–/– – 
VP24 Ad N/a N/a N/a N/a 
Bd N/a N/a N/a N/a 
Subject MLA or PH (onset time) Headache characteristicsa Associated symptomsb Treatment (time)/efficacy 
VP01 – – –/–/– – 
PH (19 h) Bilat/pres/4/yes –/–/– – 
VP02 PH (2 h) Left/throb/3/yes –/–/– – 
PH (6 h) Left/throb/3/yes –/–/– – 
VP03 MLA (70 min) Bilat/throb/8/yes Yes/yes/yes Sumatriptan (2 h 45 min) /yes, metoclopramide (3 h 25 min) /yes 
PH (24 h) Bilat/pres/1/– Yes/–/– Paracetamol (24 h) /yes 
VP04 PH (30 min) Bilat/pres/3/– –/–/– – 
PH (13 h) Bilat/pres/2/yes Yes/yes/– – 
VP05 PH (20 min) Left/pres/1/– –/–/– – 
– – –/–/– – 
VP06 PH (40 min) Bilat/pres/1/– –/–/– – 
– – –/–/– – 
VP07 MLA (3 h) Right/throb/6/yes –/yes/yes Sumatriptan (4 h 30 min) /yes 
MLA (14 h) Right/throb/6/yes –/yes/yes Acetylsalicylic acid + caffeine (17 h) /– 
Acetylsalicylic acid + caffeine (21 h) /– 
VP08 PH (20 min) Bilat/throb/2/– –/–/– – 
MLA (6 h) Left/throb/3/– Yes/–/– Zolmitriptan (6 h) /yes 
VP09 PH (20 min) Bilat/pres/1/– –/–/– – 
– – –/–/– – 
VP10 PH (20 min) Left/throb/2/yes –/–/yes – 
PH (18 h) Left/throb/2/yes –/–/– – 
VP11 – – –/–/– – 
– – –/–/– – 
VP12 PH (5 h) Bilat/pres/2/– –/–/– – 
PH (6 h) Bilat/pres/3/– –/–/– Paracetamol (6 h) /yes 
VP13 PH (10 min) Bilat/pres/1/– –/–/– – 
PH (17 h) Bilat/pres/1/– –/–/– – 
VP14 – – –/–/yes – 
PH (17 h) Right/throb/1/yes –/–/– – 
VP15 PH (5 h) Bilat/pres/3/yes –/–/– – 
MLA (6 h) Right/throb/5/yes Yes/–/– Almotriptan (7 h) /– 
VP16 PH (10 min) Right/pres/2/– –/–/– – 
Bc – – –/–/– – 
VP17 – – –/–/– – 
– – –/–/– – 
VP18 PH (10 min) Bilat/pres/1/– –/–/– – 
PH (9 h) Bilat/throb/2/– –/–/– – 
VP19 – – –/–/– – 
– – –/–/– – 
VP20 Ad N/a N/a N/a N/a 
Bd N/a N/a N/a N/a 
VP21 – – –/–/– – 
– – –/–/– – 
VP22 PH (30 min) Bilat/pres/2/– –/–/– – 
– – –/–/– – 
VP23 PH (20 min) Bilat/pres/1/– Yes/–/– – 
PH (16 h) Bilat/pres/1/– –/–/– – 
VP24 Ad N/a N/a N/a N/a 
Bd N/a N/a N/a N/a 

A = in-hospital phase (0–5 h);

B = post-hospital phase (6–24 h);

MLA = migraine-like attack; PH = peak headache (if no MLA was reported); N/a = .

aHeadache characteristics: localization/quality/intensity/aggravation by movement (throb = throbbing; pres = pressing).

bAssociated symptoms: nausea/ photophobia/ phonophobia.

cThis information obtained by telephone because no diary was received.

dDid not attend for Day 2.

In total 20 patients (91%) experienced headache (0–24 h) after PACAP38 infusion compared with 18 patients (82%) after VIP infusion (P = 0.625). The median peak headache intensity was 4 (range 0–9) on the verbal rating scale after PACAP38 and 1.5 (range 0–8) after VIP. The median time to peak headache occurred 2 h 45 min (range 10 min to 6 h) after PACAP38 and 25 min (range 10 min to 11 h) after VIP.

The AUC0-2 h for PACAP38-induced headache during the early phase (126) was larger than after VIP (47) (P = 0.03). Furthermore, the AUC2-5 h for PACAP38-induced headache was also larger in the delayed phase (297) compared with after VIP (102), (P = 0.003), (Fig. 2).

Eighteen patients used rescue medication after PACAP38 [median time 6 h (range 1 h 15 min to 13 h)] versus five patients after VIP [median time 6 h (range 2 h 45 min to 7 h)] (P = 0.002). Thirteen patients took triptans. The headache response at 1 and 2 h after treatment is shown in Fig. 3.

Figure 2

Individual and median (thick line) headache intensity scores on a verbal rating scale from 0–5 h after PACAP38 (A) and VIP (B) infusion in 22 patients with migraine. The AUC for PACAP38-induced headache during the early phase (AUC0-2 h: 126) was larger than after VIP (AUC0-2 h: 46.8) (P = 0.03). The AUC for PACAP38-induced headache was also larger in the delayed phase (AUC2-5 h: 297) compared to after VIP (AUC2-5 h: 102) (P = 0.003).

Figure 2

Individual and median (thick line) headache intensity scores on a verbal rating scale from 0–5 h after PACAP38 (A) and VIP (B) infusion in 22 patients with migraine. The AUC for PACAP38-induced headache during the early phase (AUC0-2 h: 126) was larger than after VIP (AUC0-2 h: 46.8) (P = 0.03). The AUC for PACAP38-induced headache was also larger in the delayed phase (AUC2-5 h: 297) compared to after VIP (AUC2-5 h: 102) (P = 0.003).

The effect of PACAP38 and vasoactive intestinal polypeptide on the cranial arteries

AUC0-2 h for the superficial temporal artery (n = 9) and middle meningeal artery (n = 10) circumference was significantly larger after PACAP38 than after VIP (P = 0.002; P = 0.028). We found no difference in the AUC0-2 h for the middle cerebral artery (P = 0.348, n = 12) (Figs 4 and 5).

Figure 3

Headache intensity on the verbal rating scale (0–10) before and after treatment with a triptan in 12 patients with PACAP38-induced head pain. Median time to treatment was 5 h 10 min (range 1 h 15 min to 13 h). Median headache intensity was 4 (range 2 to 9) before treatment, 1 (range 0 to 9) at 1 h after treatment (P = 0.001), and 0.5 (range 0 to 7) at 2 h after treatment (P = 0.0001). The post-treatment data are not available for one patient (Patient VP16).

Figure 3

Headache intensity on the verbal rating scale (0–10) before and after treatment with a triptan in 12 patients with PACAP38-induced head pain. Median time to treatment was 5 h 10 min (range 1 h 15 min to 13 h). Median headache intensity was 4 (range 2 to 9) before treatment, 1 (range 0 to 9) at 1 h after treatment (P = 0.001), and 0.5 (range 0 to 7) at 2 h after treatment (P = 0.0001). The post-treatment data are not available for one patient (Patient VP16).

Figure 4

Per cent circumference changes of the superficial temporal artery (STA), middle meningeal artery (MMA), and middle cerebral artery (MCA) after PACAP38 and VIP infusion. The superficial temporal artery dilated 49.5% (95% CI 30.5 to 68.6) at 20 min and 43.9% (95% CI 34.1 to 53.7) at 2 h after PACAP38 (filled triangle). The changes after VIP were 49.1% (95% CI 42.0 to 56.1) at 20 min and −1.3% (95% CI −5.4 to 2.7) at 120 min (open triangle). The per cent circumference changes of the middle meningeal artery were 31.6% (95% CI 23.7 to 39.5) and 17.0% (95% CI 8.4 to 25.6) at 20 and 120 min after PACAP38 (filled circle). The changes after VIP were 31.4% (95% CI 16.3 to 46.5) at 20 min and −1.6% (95% CI −11.2 to 8.0) at 120 min (open circle). The middle cerebral artery changed 0.9% (95% CI −2.6 to 4.4) and −0.4% (95% CI −3.4 to 2.6) at 20 and 120 min after PACAP38 (filled square). The changes after VIP were 0.6% (95% CI −5.3 to 6.4) at 20 min and 2.1% (95% CI −3.0 to 7.1) at 120 min (open square). AUC for the superficial temporal artery was larger (230) after PACAP38, than after VIP (126), (P = 0.002). AUC was also larger (111) for the middle meningeal artery after PACAP38, than after VIP (66) (P = 0.028). There was no difference in the AUC for the middle cerebral artery (PACAP38: 2 and VIP: 46) (P = 0.348).

Figure 4

Per cent circumference changes of the superficial temporal artery (STA), middle meningeal artery (MMA), and middle cerebral artery (MCA) after PACAP38 and VIP infusion. The superficial temporal artery dilated 49.5% (95% CI 30.5 to 68.6) at 20 min and 43.9% (95% CI 34.1 to 53.7) at 2 h after PACAP38 (filled triangle). The changes after VIP were 49.1% (95% CI 42.0 to 56.1) at 20 min and −1.3% (95% CI −5.4 to 2.7) at 120 min (open triangle). The per cent circumference changes of the middle meningeal artery were 31.6% (95% CI 23.7 to 39.5) and 17.0% (95% CI 8.4 to 25.6) at 20 and 120 min after PACAP38 (filled circle). The changes after VIP were 31.4% (95% CI 16.3 to 46.5) at 20 min and −1.6% (95% CI −11.2 to 8.0) at 120 min (open circle). The middle cerebral artery changed 0.9% (95% CI −2.6 to 4.4) and −0.4% (95% CI −3.4 to 2.6) at 20 and 120 min after PACAP38 (filled square). The changes after VIP were 0.6% (95% CI −5.3 to 6.4) at 20 min and 2.1% (95% CI −3.0 to 7.1) at 120 min (open square). AUC for the superficial temporal artery was larger (230) after PACAP38, than after VIP (126), (P = 0.002). AUC was also larger (111) for the middle meningeal artery after PACAP38, than after VIP (66) (P = 0.028). There was no difference in the AUC for the middle cerebral artery (PACAP38: 2 and VIP: 46) (P = 0.348).

Figure 5

MRA before (A) and 2 h after (B) the start of PACAP38 infusion. PACAP38 (10 pmol/kg) was infused intravenously over 20 min.

Figure 5

MRA before (A) and 2 h after (B) the start of PACAP38 infusion. PACAP38 (10 pmol/kg) was infused intravenously over 20 min.

Figure 6

Mean changes in plasma concentration of PACAP38 (A) and VIP (B) after PACAP38 and VIP infusions in 20 patients. Comparison of the change in plasma PACAP38 after PACAP38 (8.7 pmol/l) and after VIP (−1.1 pmol/l) infusions showed significant difference (P = 0.00005) (A). There was no difference in plasma VIP between the PACAP38 (0.7 pmol/l) and VIP (0.2 pmol/l) days (P = 0.506) (B).

Figure 6

Mean changes in plasma concentration of PACAP38 (A) and VIP (B) after PACAP38 and VIP infusions in 20 patients. Comparison of the change in plasma PACAP38 after PACAP38 (8.7 pmol/l) and after VIP (−1.1 pmol/l) infusions showed significant difference (P = 0.00005) (A). There was no difference in plasma VIP between the PACAP38 (0.7 pmol/l) and VIP (0.2 pmol/l) days (P = 0.506) (B).

Explorative analyses of the circumference changes between baseline and 20 min, and baseline and 2 h after PACAP38 compared to after VIP are shown in Tables 6.

Blood levels of PACAP38, vasoactive intestinal polypeptide and tryptase after intravenous infusion of PACAP38

We found a significant increase in plasma PACAP38 concentration (8.7 pmol/l) at 60 min after infusion. The VIP concentration at 60 min was unchanged (0.6 pmol/l) (Fig. 6).

Explorative analysis revealed that the PACAP38 concentration at 60 min was increased significantly in the group of patients (n = 18) who reported delayed (median onset 4.5 h) migraine attacks (10.2 ± 9.1 pmol/l) than in the group of patients (n = 6) who did not report a delayed attack (4.3 ± 2.4 pmol/l) (P = 0.013).

The serum tryptase concentrations did not change between baseline (3.95 µg/l) and 60 min (3.90 µg/l) after the start of infusion, P = 0.328 (n = 24) (Fig. 7), or 5 h after infusion start [baseline: 3.91 µg/l, and 5 h: 4.05 µg/l (P = 0.287, n = 11)]. In addition, there was no difference in tryptase concentrations between baseline (4.35 µg/l) and PACAP38-induced migraine attacks (4.30 µg/l), P = 0.715 (n = 8).

Plasma PACAP38, vasoactive intestinal polypeptide and tryptase levels after intravenous infusion of vasoactive intestinal polypeptide

The PACAP38 (−1.1 pmol/l) and VIP (0.2 pmol/l) concentration at 60 min after VIP infusion was unchanged (Fig. 6). We found a significant decrease in the tryptase concentration 1 h (3.92 µg/l) after VIP infusion compared to baseline (4.09 µg/l), P = 0.007 (n = 21) (Fig. 7), and no decrease 5 h after VIP [baseline: 4.15 µg/l, and 5 h: 4.01 µg/l (P = 0.172, n = 16)].

Arterial responses during PACAP38-induced attacks

We recorded circumferences of extracranial arteries in nine patients who reported unilateral head pain during PACAP38-induced migraine attacks [median time of onset 3 h (range 20 min to 5 h); median headache intensity 3 (range 1 to 7)]. We found that the external carotid artery, superficial temporal artery, and middle meningeal artery were dilated compared to baseline (Table 7), but there was no difference in circumferences comparing pain to non-pain side (external carotid artery, P = 0.258; superficial temporal artery, P = 0.569; middle meningeal artery, P = 0.561). Compared to baseline we found no difference in the circumference of the intracranial arteries on the pain or non-pain sides during attack (P ≥ 0.099) (Table 7).

Table 6

Comparison of per cent circumference changes (95% CI) from baseline to 20 min and to 2 h between the PACAP38 and VIP days

Artery Time n PACAP38 VIP P-value 
ECA 0–20 min – No data No data – 
0–2 h 11 32.6% (24.0 to 41.2) −0.7% (−3.8 to 2.4) 0.000002 
STA 0–20 min 17 46.5% (35.1 to 58.0) 43.6% (35.2 to 52.0) 0.682 
0–2 h 13 45.3% (37.5 to 53.1) −3.2% (−7.1 to 0.6) <0.000001 
MMA 0–20 min 18 27.3% (21.3 to 33.2) 29.0% (20.6 to 37.3) 0.682 
0–2 h 13 19.3% (12.4 to 26.1) −1.3% (−9.2 to 6.5) 0.001 
MCA 0–20 min 19 −0.4% (−2.8 to 1.9) 1.9% (−2.1 to 5.9) 0.259 
0–2 h 14 −0.6% (−3.1 to 1.9) 1.7% (−2.6 to 6.0) 0.373 
ICAcerebral 0–20 min 14 0.03% (−2.2 to 2.3) 1.4% (−2.7 to 5.5) 0.267 
0–2 h 11 −0.5% (−3.0 to 2.1) 0.2% (−3.3 to 3.8) 0.520 
ICAcavernous 0–20 min 5.3% (−47.2 to 57.9) 11.0% (−86.8 to 108.7) 0.716 
0–2 h 14 −2.9% (−5.5 to −0.3) −0.3% (−3.6 to 2.9) 0.174 
ICAcervical 0–20 min 18 −2.3% (−4.1 to −0.5) 2.3% (0.1 to 4.5) 0.000286 
0–2 h 13 −3.4% (−5.3 to −1.4) −1.6% (−3.6 to 0.4) 0.081 
BA 0–20 min 19 −0.2% (−2.4 to 1.9) 1.1% (−0.1 to 2.4) 0.209 
0–2 h 14 −0.6% (−2.5 to 1.3) 0.5% (−0.6 to 1.6) 0.365 
Artery Time n PACAP38 VIP P-value 
ECA 0–20 min – No data No data – 
0–2 h 11 32.6% (24.0 to 41.2) −0.7% (−3.8 to 2.4) 0.000002 
STA 0–20 min 17 46.5% (35.1 to 58.0) 43.6% (35.2 to 52.0) 0.682 
0–2 h 13 45.3% (37.5 to 53.1) −3.2% (−7.1 to 0.6) <0.000001 
MMA 0–20 min 18 27.3% (21.3 to 33.2) 29.0% (20.6 to 37.3) 0.682 
0–2 h 13 19.3% (12.4 to 26.1) −1.3% (−9.2 to 6.5) 0.001 
MCA 0–20 min 19 −0.4% (−2.8 to 1.9) 1.9% (−2.1 to 5.9) 0.259 
0–2 h 14 −0.6% (−3.1 to 1.9) 1.7% (−2.6 to 6.0) 0.373 
ICAcerebral 0–20 min 14 0.03% (−2.2 to 2.3) 1.4% (−2.7 to 5.5) 0.267 
0–2 h 11 −0.5% (−3.0 to 2.1) 0.2% (−3.3 to 3.8) 0.520 
ICAcavernous 0–20 min 5.3% (−47.2 to 57.9) 11.0% (−86.8 to 108.7) 0.716 
0–2 h 14 −2.9% (−5.5 to −0.3) −0.3% (−3.6 to 2.9) 0.174 
ICAcervical 0–20 min 18 −2.3% (−4.1 to −0.5) 2.3% (0.1 to 4.5) 0.000286 
0–2 h 13 −3.4% (−5.3 to −1.4) −1.6% (−3.6 to 0.4) 0.081 
BA 0–20 min 19 −0.2% (−2.4 to 1.9) 1.1% (−0.1 to 2.4) 0.209 
0–2 h 14 −0.6% (−2.5 to 1.3) 0.5% (−0.6 to 1.6) 0.365 

ECA = external carotid artery; STA = superficial temporal artery; MMA = middle meningeal artery; MCA = middle cerebral artery; BA = the basilar artery; n = number of participants. P-values: the paired t-test comparing relative circumference changes between the PACAP38 and VIP days.

Table 7

Mean circumferences (mm) and the percent change (95% CI) of selected arteries during PACAP38–induced unilateral migraine attacks and at baseline

Artery Side n Baseline Attack Change (%) P-value 
MCA Pain 8.76 8.97 2.7% (−2.7 to 8.0) 0.317 
Non-pain 9.53 9.83 3.2% (−1.8 to 8.2) 0.170 
ICAcerebral Pain 10.92 11.25 3.2% (−1.2 to 7.6) 0.143 
Non-pain 11.81 11.74 −0.6% (−5.0 to 3.8) 0.787 
ICAcavernous Pain 13.16 13.39 1.9% (−0.1 to 4.0) 0.099 
Non-pain 13.12 13.09 −0.2% (−3.6 to 3.3) 0.885 
ICAcervical Pain 14.51 13.91 −4.0% (−7.4 to −0.5) 0.026* 
Non-pain 14.14 13.91 −1.6% (−5.1 to 1.8) 0.316 
MMA Pain 4.64 5.54 19.1% (9.2 to 29.1) 0.004* 
Non-pain 4.36 5.41 23.6% (12.7 to 34.5) 0.003* 
STA Pain 4.97 7.27 47.6% (35.0 to 60.2) 0.00002*** 
Non-pain 5.05 7.68 52.2% (30.7 to 73.8) 0.001** 
ECA Pain 9.84 12.81 31.0% (20.9 to 41.2) 0.00004*** 
Non-pain 9.01 12.23 36.9% (27.4 to 46.4) 0.00001*** 
BA  9.47 9.54 0.9% (−1.6 to 3.3) 0.541 
Artery Side n Baseline Attack Change (%) P-value 
MCA Pain 8.76 8.97 2.7% (−2.7 to 8.0) 0.317 
Non-pain 9.53 9.83 3.2% (−1.8 to 8.2) 0.170 
ICAcerebral Pain 10.92 11.25 3.2% (−1.2 to 7.6) 0.143 
Non-pain 11.81 11.74 −0.6% (−5.0 to 3.8) 0.787 
ICAcavernous Pain 13.16 13.39 1.9% (−0.1 to 4.0) 0.099 
Non-pain 13.12 13.09 −0.2% (−3.6 to 3.3) 0.885 
ICAcervical Pain 14.51 13.91 −4.0% (−7.4 to −0.5) 0.026* 
Non-pain 14.14 13.91 −1.6% (−5.1 to 1.8) 0.316 
MMA Pain 4.64 5.54 19.1% (9.2 to 29.1) 0.004* 
Non-pain 4.36 5.41 23.6% (12.7 to 34.5) 0.003* 
STA Pain 4.97 7.27 47.6% (35.0 to 60.2) 0.00002*** 
Non-pain 5.05 7.68 52.2% (30.7 to 73.8) 0.001** 
ECA Pain 9.84 12.81 31.0% (20.9 to 41.2) 0.00004*** 
Non-pain 9.01 12.23 36.9% (27.4 to 46.4) 0.00001*** 
BA  9.47 9.54 0.9% (−1.6 to 3.3) 0.541 

ECA = external carotid artery; STA = superficial temporal artery; MMA = middle meningeal artery; MCA = middle cerebral artery; BA = basilar artery; n = number of participants.

P-values: the paired t test comparing absolute circumference values during attack and on attack-free days. ***P < 0.001; **P < 0.01; *P < 0.05.

We recorded arterial circumferences in six patients before and after subcutaneous injection of sumatriptan. Sumatriptan significantly constricted all extracranial arteries (external carotid artery, P = 0.034; superficial temporal artery, P = 0.049; middle meningeal artery, P = 0.035; ICAcervical, P = 0.002) and the cavernous part of the ICA (P = 0.000053), but not the cerebral arteries (middle cerebral artery, P = 0.093; ICAcerebral, P = 0.613; basilar artery, P = 0.084) (Fig. 8).

Figure 7

Mean changes in serum concentration of tryptase at 60 min after PACAP38 and VIP infusions in 21 migraine patients. The serum tryptase levels did not differ after infusion of PACAP38 (−0.05 µg/l) and after VIP (−0.16 µg/l) (P = 0.128).

Figure 7

Mean changes in serum concentration of tryptase at 60 min after PACAP38 and VIP infusions in 21 migraine patients. The serum tryptase levels did not differ after infusion of PACAP38 (−0.05 µg/l) and after VIP (−0.16 µg/l) (P = 0.128).

Figure 8

Mean per cent circumference change of extracranial (grey) and intracranial (white) arteries between before and 30 to 60 min after treatment with subcutaneous injection of 6 mg sumatriptan in six patients with PACAP38-induced migraine-like attacks. The circumference change of the ICAx (cervical part) was −14.1% (95% CI −19.5 to −8.6); ICAi (cavernous) −13.8% (95% CI −16.6 to −11.1); middle meningeal artery −12.7% (95% CI −23.6 to −1.8); superficial temporal artery −12.6% (95% CI −26.0 to 0.7); external carotid artery −9.9% (95% CI −19.9 to 0.1); middle cerebral artery −7.1% (95% CI −16.0 to 1.8); basilar artery −2.0% (95% CI −4.7 to 0.8); and ICAc (cerebral) −1.1% (95% CI −6.3 to 4.2).

ICAx = cervical part of the internal carorid artery; ICAi = cavernous part of the internal carotid artery; MMA = middle meningeal artery; STA = superficial temporal artery; ECA = external carotid artery; MCA = middle cerebral artery; BA = basilar artery; ICAc = cerebral part of the internal carotid artery. Paired samples t-test: ***P < 0.0001; **P < 0.005; *P < 0.05.

Figure 8

Mean per cent circumference change of extracranial (grey) and intracranial (white) arteries between before and 30 to 60 min after treatment with subcutaneous injection of 6 mg sumatriptan in six patients with PACAP38-induced migraine-like attacks. The circumference change of the ICAx (cervical part) was −14.1% (95% CI −19.5 to −8.6); ICAi (cavernous) −13.8% (95% CI −16.6 to −11.1); middle meningeal artery −12.7% (95% CI −23.6 to −1.8); superficial temporal artery −12.6% (95% CI −26.0 to 0.7); external carotid artery −9.9% (95% CI −19.9 to 0.1); middle cerebral artery −7.1% (95% CI −16.0 to 1.8); basilar artery −2.0% (95% CI −4.7 to 0.8); and ICAc (cerebral) −1.1% (95% CI −6.3 to 4.2).

ICAx = cervical part of the internal carorid artery; ICAi = cavernous part of the internal carotid artery; MMA = middle meningeal artery; STA = superficial temporal artery; ECA = external carotid artery; MCA = middle cerebral artery; BA = basilar artery; ICAc = cerebral part of the internal carotid artery. Paired samples t-test: ***P < 0.0001; **P < 0.005; *P < 0.05.

Vital signs

We found an increase in the heart rate on the PACAP38 day compared to the VIP day during the early (P = 0.00009) and the delayed phases (P = 0.007) (Fig. 9A). Explorative analysis showed no correlation between heart rate or heart rate increase and the headache intensity reported by the patients (data not shown). The AUC for mean arterial blood pressure was larger after VIP compared to after PACAP38 during the early (P = 0.001) and the delayed phases (P = 0.018) (Fig. 9B). The AUC0-2h for respiratory frequency and PetCO2 was larger in the early phase on the VIP day compared to the PACAP38 day (P = 0.020 and P = 0.039). There was no difference in the AUC2-5h for respiratory frequency and PetCO2 between VIP and PACAP38 in the delayed phase (P = 0.079 and P = 0.294) (Fig. 9C and D) (Table 8).

Figure 9

The effect of PACAP38 and VIP on heart rate (A), mean arterial blood pressure (B), respiratory frequency (C), and the PetCO2 (D) in early (0–2 h, n = 22) and delayed (2–5 h, n = 17) phases after infusion. The AUC for heart rate was larger in the early and delayed phases after PACAP38 compared to VIP (P = 0.00009 and P = 0.007). The AUC for mean arterial blood pressure was larger in the early and delayed phases after VIP compared to PACAP38 (P = 0.001 and P = 0.018). The AUC for respiratory frequency and PetCO2 was larger in the early, but not delayed phases after VIP compared to PACAP38 (respiratory frequency, P = 0.020 and P = 0.079; PetCO2, P = 0.039 and 0.294).

Figure 9

The effect of PACAP38 and VIP on heart rate (A), mean arterial blood pressure (B), respiratory frequency (C), and the PetCO2 (D) in early (0–2 h, n = 22) and delayed (2–5 h, n = 17) phases after infusion. The AUC for heart rate was larger in the early and delayed phases after PACAP38 compared to VIP (P = 0.00009 and P = 0.007). The AUC for mean arterial blood pressure was larger in the early and delayed phases after VIP compared to PACAP38 (P = 0.001 and P = 0.018). The AUC for respiratory frequency and PetCO2 was larger in the early, but not delayed phases after VIP compared to PACAP38 (respiratory frequency, P = 0.020 and P = 0.079; PetCO2, P = 0.039 and 0.294).

Table 8

Comparison of AUC ± SD for the early (0–2 h) and delayed (2–5 h) phases between the PACAP38 and VIP days

Artery Time n PACAP38 VIP P-value 
HR 0–2 h 22 3074 ± 1225 1740 ± 642 0.00009 
2–5 h 17 3121 ± 1893 1644 ± 883 0.007 
MABP 0–2 h 22 204 ± 558 820 ± 477 0.001 
2–5 h 17 63 ± 630 791 ± 1198 0.018 
RF 0–2 h 22 −134 ± 343 34 ± 231 0.020 
2–5 h 17 −114 ± 637 143 ± 327 0.079 
PetCO2 0–2 h 22 −43 ± 32 −27 ± 22 0.039 
2–5 h 17 −27 ± 41 −17 ± 28 0.294 
Artery Time n PACAP38 VIP P-value 
HR 0–2 h 22 3074 ± 1225 1740 ± 642 0.00009 
2–5 h 17 3121 ± 1893 1644 ± 883 0.007 
MABP 0–2 h 22 204 ± 558 820 ± 477 0.001 
2–5 h 17 63 ± 630 791 ± 1198 0.018 
RF 0–2 h 22 −134 ± 343 34 ± 231 0.020 
2–5 h 17 −114 ± 637 143 ± 327 0.079 
PetCO2 0–2 h 22 −43 ± 32 −27 ± 22 0.039 
2–5 h 17 −27 ± 41 −17 ± 28 0.294 

HR = heart rate; MABP = mean arterial blood pressure; RF = respiratory frequency; PetCO2 = end-tidal pressure of CO2.

P-values: the paired samples t-test.

Adverse events

Adverse events were recorded and are shown in Table 9. One patient (Patient VP23) reported puffiness of the face after discharge. Oral antihistamine (cetirizine, 10 mg) was prescribed and the symptoms disappeared. Another patient (Patient VP24) reported severe pain in the jaw and puffiness on the face after 6 h. This patient was treated with antihistamine (clemastine, 2 mg) combined with intravenous methylprednisolone succinate (37.5 mg) in the emergency department. All symptoms gradually disappeared and the patient was discharged after 10 h observation.

Table 9

Adverse events in the hospital phase (0–5 h) after PACAP38 and VIP infusion (n = 22)

Symptom PACAP38 VIP P-value 
Flushing 24/24 (100%) 21/22 (95%) 1.000a 
Heat sensation 23/24 (96%) 22/22 (100%) 1.000a 
Palpitation 23/24 (96%) 21/22 (95%) 1.000a 
Nausea 15/24 (63%) 6/22 (27%) 0.057a 
Photophobia 13/24 (54%) 6/22 (27%) 0.070a 
Phonophobia 10/24 (42%) 4/22 (18%) 0.227a 
Shivering 9/24 (38%) 8/22 (36%) 1.000a 
Nasal congestion 4/24 (17%) 0/22 0.250a 
Facial puffing 4/24 (17%) 0/22 0.250a 
Dizziness 1/24 (4%) 3/22 (14%) 0.500a 
Feeling of numbness of both arms 1/24 (4%) 2/22 (9%) 1.000a 
Tingling in the chest 1/24 (4%) 2/22 (9%) 1.000a 
Vomiting 1/24 (4%) 1/22 (5%) 1.000a 
Feeling of tightening all over the body 1/24 (4%) 1/22 (5%) 1.000a 
Dry mouth 1/24 (4%) 0/22 1.000a 
Increased sensitivity to touch in the face 1/24 (4%) 0/22 1.000a 
Feeling of swollen throat 1/24 (4%) 0/22 1.000a 
Yawning 1/24 (4%) 0/22 1.000a 
Pain in the jaw 1/24 (4%) 0/22 1.000a 
Feeling of gas in the stomach 1/24 (4%) 0/22 1.000a 
Swollen hands 1/24 (4%) 0/22 1.000a 
Visual disturbances (shade on moving objects) 0/24 1/22 (5%) 1.000a 
Tingling in a finger 0/24 1/22 (5%) 1.000a 
Symptom PACAP38 VIP P-value 
Flushing 24/24 (100%) 21/22 (95%) 1.000a 
Heat sensation 23/24 (96%) 22/22 (100%) 1.000a 
Palpitation 23/24 (96%) 21/22 (95%) 1.000a 
Nausea 15/24 (63%) 6/22 (27%) 0.057a 
Photophobia 13/24 (54%) 6/22 (27%) 0.070a 
Phonophobia 10/24 (42%) 4/22 (18%) 0.227a 
Shivering 9/24 (38%) 8/22 (36%) 1.000a 
Nasal congestion 4/24 (17%) 0/22 0.250a 
Facial puffing 4/24 (17%) 0/22 0.250a 
Dizziness 1/24 (4%) 3/22 (14%) 0.500a 
Feeling of numbness of both arms 1/24 (4%) 2/22 (9%) 1.000a 
Tingling in the chest 1/24 (4%) 2/22 (9%) 1.000a 
Vomiting 1/24 (4%) 1/22 (5%) 1.000a 
Feeling of tightening all over the body 1/24 (4%) 1/22 (5%) 1.000a 
Dry mouth 1/24 (4%) 0/22 1.000a 
Increased sensitivity to touch in the face 1/24 (4%) 0/22 1.000a 
Feeling of swollen throat 1/24 (4%) 0/22 1.000a 
Yawning 1/24 (4%) 0/22 1.000a 
Pain in the jaw 1/24 (4%) 0/22 1.000a 
Feeling of gas in the stomach 1/24 (4%) 0/22 1.000a 
Swollen hands 1/24 (4%) 0/22 1.000a 
Visual disturbances (shade on moving objects) 0/24 1/22 (5%) 1.000a 
Tingling in a finger 0/24 1/22 (5%) 1.000a 

P-values: McNemar test: Exact sig. (two-tailed).

aBinomial distribution used.

Discussion

The main findings in the present head-to-head comparison study are that PACAP38 induced more migraine-like attacks and a much longer lasting extracranial arterial dilatation than VIP. Furthermore, PACAP38-induced migraine attacks were preceded by elevated plasma levels of PACAP38, indicating de novo synthesis or release of PACAP38.

Possible vascular and inflammatory mechanisms of PACAP-induced migraine

The present study clearly demonstrates that in spite of many similarities, only PACAP38, but not VIP induces migraine attacks. PACAP38 and VIP receptors are present in cerebral and extracranial arteries (Knutsson and Edvinsson, 2002; Chan et al., 2011), but neither exogenously administered PACAP38 nor VIP caused dilatation of cerebral arteries, suggesting no passage of the peptides across the blood–brain barrier during the experiment. Previous in vitro studies of rat and human cerebral arteries showed that abluminal, but not luminal application of PACAP38 and VIP caused relaxation of smooth muscle cells of the rat middle cerebral artery (Grände et al., 2012). Collectively, these data suggest that central effects of PACAP38 and VIP after systemic administration are unlikely in the human being.

The question is how we can explain the differences between peptides in extracranial arterial responses and to what extent prolonged extracranial dilatation contributes to PACAP38-induced migraine attacks. The messenger RNA of VPAC1, VPAC2 and PAC1 receptors is expressed in smooth muscle cells of human meningeal (Chan et al., 2011) and cerebral (Knutsson and Edvinsson, 2002) arteries. Both peptides bind with equal affinity to the VPAC1 and VPAC2 receptors (Harmar et al., 2012). It seems that the VPAC1 receptor is the main receptor responsible for vasodilatation elicited by PACAP38 and VIP in different vascular beds in the rat (Fahrenkrug et al., 2000; Boni et al., 2009), and that the vascular involvement of VPAC2 and PAC1 receptors in cranial arteries is limited in the rat (Baun et al., 2011). Interspecies differences are unlikely because of a close structural homology between rat and human VPAC1, VPAC2 and PAC1 receptors (Dickinson and Fleetwood-Walker, 1999). At present, we cannot definitively explain the differences in vascular effect between PACAP38 and VIP in humans based on the distribution or receptor profile.

The contribution of immediate or prolonged dilatation of cranial arteries by peptides to the initiation of migraine attacks is uncertain. The fact that all migraine-provoking substances are vasoactive and act outside of the blood–brain barrier (except glyceryl trinitrate) suggests an important role of extracranial arteries, in particular their perivascular innervation in the initiation of migraine attacks. Simple dilatation is unlikely because VIP (Rahmann, 2008), adenosine (Birk et al., 2005), and adrenomedullin (Petersen et al., 2009) dilate extracranial arteries, but do not induce migraine attacks. However, these substances caused dilatation and facial flushing of much shorter duration than PACAP38. On the other hand, glyceryl trinitrate, another reliable migraine trigger induces only a short-lasting dilatation of arteries (Schoonman et al., 2008), suggesting that PACAP-induced migraine may include other mechanisms than long-lasting dilatation per se. In support, patients who reported unilateral migraine-like pain had bilateral arterial dilatations. The trigeminal ganglion (Tajti et al., 1999) and the trigeminal nucleus caudalis (Uddman et al., 2002) contain PACAP38, but not VIP. Furthermore, the messenger RNA of VPAC1, VPAC2 and PAC1 receptors are present in the cranial ganglia with perivascular nerve fibre projections (Knutsson and Edvinsson, 2002). Based on these data, it would be plausible to suggest that PACAP38 can induce prolonged extracranial dilatation as well as neurogenic inflammation. This may lead to sensitization of trigeminal perivascular afferents and thereby to migraine attacks in migraine patients. Sumatriptan blocks neurogenic inflammation (Buzzi and Moskowitz, 1990) and peripheral sensitization (Levy et al., 2004). In the present study, sumatriptan reversed extracranial arterial dilatation induced by PACAP38 and reduced the intensity of migraine headache. These data are consistent with our previous studies (Asghar et al., 2011; Amin et al., 2012, 2013a, b). Together they suggest that sumatriptan exerts its anti-migraine effect by blocking sensory input from the extracranial perivascular sensory nerves and perhaps by constricting extracerebral arteries.

Inflammatory mediators released from mast cells may cause sensitization of meningeal afferents (Levy et al., 2007). PACAP38 has a much stronger degranulatory effect on rat dural mast cells than VIP, but a selective PAC1 receptor agonist was unable to degranulate mast cells (Baun et al., 2012). In the present study we measured tryptase as a marker of mast cell degranulation (Hogan and Schwartz, 1997) and found no increase in serum concentration after PACAP38. This suggests that mast cell degranulation is not involved in migraine generation, which is also supported by a lack of effects of antihistamine drugs mast cell stabilizers (Brandes et al., 2004) in migraine treatment. However, it is possible that systemic tryptase is not an ideal marker for dural mast cell degranulation. We cannot, therefore, completely rule out mast cell degranulation during migraine attacks.

Possible systemic effect of PACAP and migraine induction

PACAP is also found in the gastrointestinal, endocrine, immune and respiratory systems (Vaudry et al., 2009). Among many other mechanisms, PACAP is also involved in energy homeostasis through behavioural and metabolic mechanisms (Resch et al., 2013). Furthermore, it has been described as a master regulator in central and peripheral stress responses through modulation of the hypothalamic–pituitary–adrenal axis (Amir-Zilberstein et al., 2012; Mustafa, 2013). It also regulates gonadotropin expression alone and through modulation of gonadotropin-releasing hormone through intracellular signalling pathways (Halvorson, 2013; Thomas et al., 2013). A recent study by Taylor et al., (2014) suggested that PACAP inhibits calcium-dependent potassium currents in pyramidal neurons by increasing cyclic adenosine monophosphate and activation of protein kinase A-dependent pathways. It further blocks the effects of cocaine- and amphetamine-regulated transcript peptides on feeding and short-term weight gain (Burgos et al., 2013). Maejima et al. (2013) reported that PACAP regulated feeding mechanisms, stress response or autonomic response through activation of increase of calcium-ions in the hypothalamic paraventricular nucleus cells. Thus, PACAP is a multifunctional peptide involved in various cellular and physiological responses, and therefore non-vascular mechanisms of PACAP38 cannot be ruled out as a migraine triggering effect.

Elevated PACAP38 levels after PACAP38 infusion

Birk et al. (2007) examined plasma PACAP38 concentration after intravenous infusion of PACAP38 in healthy subjects. Compared to baseline levels, they found elevated plasma PACAP38 levels (∼3.7 pmol/l) at 60 min after the start of an infusion lasting 20 min. In the present study we found that PACAP38 increased more in patients, who later developed a migraine attack (10.2 pmol/l) than in patients who did not report a delayed migraine attack (3.2 pmol/l) after intravenous PACAP38 infusion. Interestingly, plasma PACAP38 in the non-migraine group (n = 6) seems to be similar to previously reported plasma levels in healthy subjects (Birk et al., 2007). These studies are comparable because both used the same dose, rate of infusion (10 pmol/kg/min for 20 min), and samples were analysed in the same laboratory (J.F.). Given that plasma half-life of PACAP38 is 3.5 min (Birk et al., 2007), exogenously administrated PACAP38 should be completely eliminated 40 min after infusion. Elevated PACAP38 levels thus extend well beyond the clearance of PACAP38. Possible mechanisms for this are (i) impaired elimination; (ii) endogenous release; and (iii) de novo synthesis. The exact source of PACAP38 in plasma is unknown, but activation of cranial parasympathetic and sensory ganglia has been suggested (Edvinsson, 2013). It is possible that PACAP38 in the systemic circulation in our study represents neuronal overflow, i.e. it is released from PACAP38-containing nerve terminals. Elevated PACAP38 levels in patients, who later developed migraine attacks may thus be due to endogenous release from PACAP-containing nerves. Interestingly, a study by Tuka et al. (2013) reported increased PACAP38 levels during migraine attacks compared to outside of attacks. The peripheral PACAP38 concentration (10.2 pmol/l) in patients who later developed migraine is, however, unlikely to explain the sustained dilatation of arteries. The concentration of PACAP that is needed to elicit half of the maximal vasodilatory effect is close to 4 nmol/l. In addition, long-lasting vascular effect is not always related to plasma levels of the substance (e.g. ergots, Tfelt-Hansen et al., 2013). However, this mechanism seems unlikely for PACAP38 as it rapidly dissociates by in vitro washout methods in the laboratory (personal communication, Dr Michael Baun).

PACAP38 and the nociceptive system

The present head-to-head study strongly suggests a pro-nociceptive role of the PAC1 receptor. The PAC1 receptor is expressed in the dorsal horn of the spinal cord (Jongsma et al., 2000), where nociceptive information is processed, and spinal administration of PACAP38 in the rat, decreased dynorphin release by 26%, whereas the dynorphin release was unchanged after VIP (Liu et al., 2011). Moreover, intrathecal application of the selective PAC1 receptor antagonist, PACAP6-38, increased dynorphin release by 38% (Liu et al., 2011) emphasizing the importance of the PAC1 receptor in the regulation of dynorphin. In another study, blockade of the PAC1 receptor by intrathecally administered PACAP6–38 effectively attenuated thermal hyperalgaesia and mechanical allodynia associated with preclinical pain models (Davis-Taber et al., 2008). It was concluded that PAC1 receptor activation at spinal cord level is pro-nociceptive. Reduced nociceptive response to chemical, thermal and mechanical stimuli was, however, reported in the PAC1 receptor knockout mouse (Jongsma et al., 2001). Whether the difference between PACAP38 and VIP is due to the PAC1 receptor or caused by other factors, therefore, remains unclear. However, development of a specific PAC1 receptor antagonist for human use may contribute to a better understanding of migraine pathophysiology, and could possibly become a major breakthrough in migraine treatment.

Conclusion

PACAP38 induces more migraine-like headaches than VIP after intravenous infusion and a long-lasting dilatation of extracranial arteries. PACAP38-induced migraine-like attacks were preceded by elevated levels of plasma PACAP38. Sumatriptan reversed PACA38-induced attacks in parallel with constriction of the dilated extracranial arteries. These data further substantiate the suggestion that PAC1 receptor antagonists may be effective anti-migraine agents.

Acknowledgements

We thank Professor Lene Theil Skovgaard, Department of Biostatistics, University of Copenhagen for statistical advices. We also thank radiologists Dr Vibeke Andrée Larsen and Dr Hayder Obaid Ghani for assessment of the anatomic scans; Dr Song Guo, Peter Frederiksen Svane, Dr Lærke Smedegaard, Julie Ravneberg, Jaychandran Raghava, radiographers Bente S Møller, Marjut Lindhardt, and Helle J Simonsen for help with scans; laboratory technician Liselotte Pflug for help with blood samples.

Funding

The study was supported by grants from the University of Copenhagen, the Lundbeck Foundation through the Centre for Neurovascular Signalling (LUCENS), the Research Foundation of the Capital Region of Denmark, Danish Council for Independent Research-Medical Sciences, the Novo Nordisk Foundation, IMK Almene Foundation, and the Cool Sorption Foundation.

Conflicts of interest

J.O. has received grant or research support from, has been a consultant or scientific adviser for, and has been in the speakers’ bureau of Allergan Inc., AstraZeneca Pharmaceuticals LP, Boehringer Ingelheim, Eli Lilly, GlaxoSmithKline, Janssen Pharmaceutical Products, Lundbeck, Merck, and Pzifer. M.A. is a consultant or scientific adviser for Allergan, Amgen, Alder, and ATI, and primary investigator for an M-1 ATI trial. F.M.A. has received honoraria for lecturing from Allergan and travel grants from MSD. All other authors declare that they have no conflicts of interest.

Abbreviations

    Abbreviations
  • AUC

    area under the curve

  • ICA

    internal carotid artery

  • MRA

    magnetic resonance angiography

  • PACAP

    pituitary adenylate cyclase-activating polypeptide

  • VIP

    vasoactive intestinal polypeptide

  • VPAC

    VIP and PACAP

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