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Abstract

Both calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are potent vasodilators that have been shown to induce headache in migraine patients. Their antagonists are effective in the treatment of migraine attacks. In the present study, we hypothesize that vasodilation induced by the NO donor glyceryltrinitrate (GTN) or by CGRP is partially mediated via large conductance calcium-activated potassium (BK_Ca) channels. The effects of the BK_Ca channel selective inhibitor iberiotoxin on dural and pial vasodilation induced by CGRP, GTN and endogenously released CGRP by transcranial electrical stimulation (TES) were examined. Iberiotoxin significantly attenuated GTN-induced dural and pial artery dilation in vivo and in vitro, but had no effect on vasodilation induced by CGRP and TES. Our results show that GTN- but not CGRP-induced dural and pial vasodilation involves opening of BK_Ca channels in rat.

Keywords

CGRP dural and pial arteries GTN iberiotoxin transcranial electrical stimulation

Introduction

Our previous human studies have demonstrated that nitric oxide (NO) and calcitonin gene-related peptide (CGRP) are likely to be involved in migraine pathogenesis (1–4). It has been suggested that the interaction of NO and CGRP is implicated in the development of vascular headaches (5, 6). Both substances are endogenous vasodilators that act via different mechanisms. CGRP is known to cause increased levels of intracellular cAMP, whereas NO stimulates guanylyl cyclase with a subsequent increase in cGMP levels. Inherited dysfunction of voltage gated Ca²⁺ and Na⁺ channels and Na⁺/K⁺ pump subunits, as well as mutation in neuronal voltage gated Na⁺ channels, can cause familial hemiplegic migraine (7), and it has been suggested that the common types of migraine may also be caused by channel dysfunction (8).

Whether channels other than Ca²⁺ and Na⁺ are involved in mechanisms responsible for migraine is still unknown. Potassium (K⁺) channels are widely distributed and participate in the regulation of cerebral vascular tone (9). Furthermore, ATP-sensitive K⁺ channel (K_ATP) openers are potent vasodilators (10) and may induce headache in humans (11, 12). Large conductance calcium-activated K⁺ (BK_Ca) channels are thought to play a critical role in the regulation of cerebral artery diameter (13). These data indicate that K⁺ channels may be relevant targets in headache research.

It has been suggested that BK_Ca channels may be activated by protein kinase A (PKA) and protein kinase G (PKG) via the respective cAMP and cGMP pathways in patch-clamp experiments (14, 15). Furthermore, BK_Ca channels modulate the vasodilator response to both exogenous nitrovasodilators and endogenous receptor-mediated NO release in isolated arteries (16, 17).

We focused on the effect of BK_Ca channels on NO- and CGRP-induced vasodilation within the dural and pial vessels, because these molecules play a causal role in migraine (1–4).

In order to study the interaction of BK_Ca channels with CGRP and NO and to compare responses in pial and dural arteries, a rat genuine closed cranial window model was used in combination with studies of isolated middle meningeal (MMA) and middle cerebral (MCA) arteries. The genuine closed cranial model allows a direct measurement of dural and pial artery diameter and local cortical cerebral blood flow by laser Doppler flowmetry (LCBF_Flux) in anaesthetized animals. Furthermore, it provides the opportunity to investigate the craniovascular effects of infused vasoactive substances and endogenous CGRP after electrical stimulation (18, 19).

The aim was to: (i) confirm the vasodilator effects in vivo of CGRP, glyceryltrinitrate (GTN) and transcranial electrical stimulation (TES) on dural and pial artery diameter, LCBF_Flux(20–22) and mean arterial blood pressure (MABP); (ii) determine whether the selective BK_Ca channel inhibitor iberiotoxin is able to attenuate effects of exogenous CGRP, of CGRP released by TES and of GTN; and (iii) confirm effects of CGRP and GTN and their inhibition by iberiotoxin in isolated MCAs and MMAs in vitro.

Materials and methods

In vivo studies

Surgical preparation

This study complied with The Danish Animal Experimentation Inspectorate (file: 2001/561-390) on the care and use of animals. All experiments were conducted using the closed cranial window technique as previously described by Williamson (18). Male Sprague—Dawley rats (300–400 g) were anaesthetized throughout the experiments with pentobarbital (Mebumal^®; The Danish Hospital Pharmacy, Glostrup, Denmark). Anaesthesia was induced by 60 mg/kg i.p. and throughout the experiments anaesthesia was maintained by i.v. infusion of 20 mg/kg per hour pentobarbital. The depth of anaesthesia was judged by suppression of the hind paw withdrawal reflex. The animals were mechanically ventilated. The respirator delivered 3–3.5 ml/stroke of O₂ 30% and N₂O 70%, at a ventilation rate of 60–65 strokes/min (Rodent Ventilator; UGO BASILE, Comerio, Italy). In the genuine closed cranial window many combinations of anaesthetics were tried by Petersen and colleagues, including inhalation anaesthetics, before deciding on the combination of Mebumal (pentobarbital) and N₂O. It has been concluded that this combination had the least effect on the cerebral blood flow and the cerebral arteries during long-term observation (19). The femoral arteries and veins were cannulated bilaterally by catheters (0.40 mm i.d.) (Portex^®; Astratech, Taastrup, Denmark) for administration of test substances, blood sampling and measurement of MABP and arterial blood gases (ABG) (Transducer TCM4-7; World Precision Instruments, Sarasota, FL, USA). The animals were placed in a stereotaxic frame, the skull exposed and the parietal bone thinned by drilling with a saline-cooled dental drill until the dural and pial arteries were clearly visible through the intact skull. Before placing the video camera, warm (37 °C) mineral oil was applied.

Intravital microscopy

The vessels were imaged by a Sony^® video camera with microscope (Sony^® DSP digital camera, MS 50 objective; Tokyo, Japan) and the real-time image was displayed on a television screen. Dural and pial artery diameters were continuously measured using a Video Dimension Analyser (VDA; Living Systems Instrumentation^®, Burlington VT, USA). Data were collected and analysed by data acquisition and analysis software (Perisoft version 1; Perimed Systems^®, Järfälla, Sweden). LCBF_Flux (Perimed Periflux^® 4001; Perimed AB) was measured by a probe 90° bent (Perimed 410, fibre separation 0.25 mm; Perimed AB) held by a micromanipulator. The probe was placed free of the bone but in touch with mineral oil, which enhances the accuracy of the LCBF_Flux measurement (20). The probe was placed, using a micromanipulator, under visual control on the surface of the cranial window with no or few observable vessels.

Blood pressure was measured continuously by connecting the catheter inserted in the femoral artery to a transducer (Transducer TCM4-7; World Precision Instruments). Blood gases were measured at the beginning and end of each experiment (ABL520; Radiometer, Br⊘nsh⊘j, Denmark). Because changes in P_aCO₂, P_aO₂ and pH can affect the diameter of pial and dural artery as well as LCBF_Flux, these values were kept within normal limits (19). Body temperature was controlled by a heating blanket and kept at 37 ± 0.5 °C. (Letica^® Scientific Instruments, Barcelona, Spain).

Experimental protocols

Electrical stimulation parameters. TES was used to evoke dural and pial vessel dilation with a bipolar stimulating electrode (NE-200x; Harvard Instruments, Edenbridge, UK) that was placed on the surface of the cranial window approximately 200 µm from the vessels chosen for measurement. The surface of the cranial window was stimulated at 5 Hz, 1 ms for 10 s (Grass Stimulator S48; Grass Instruments, Quincy, MA, USA). The stimulation was done with increasing voltage until maximal dilation was observed. The same voltage was used for the subsequent TES in the same animal (23).

The effect of CGRP, GTN, TES and inhibition by iberiotoxin. It has been shown that CGRP at a dose of 0.3 µg/kg causes maximal dilation of dural and pial vessels, providing a fall in MABP of < 25 mmHg in anaesthetized rats (24). The reproducible response for CGRP (0.3 µg/kg) has been shown previously on all measured parameters in the genuine closed cranial window model, when an i.v. bolus of CGRP was administered seven times at 15-min intervals (24). For TES it has also been shown that after an initial voltage determination, it is possible to evoke the same dilatory response for both dural and pial arteries seven times at 25-min intervals without any significant differences (24). LCBF_Flux was not measured in the experiments with TES for technical reasons. It has been shown that 0.1 mg/kg of iberiotoxin infusion over 5 min could be used without haemodynamic effects per se (25).

In GTN dose–effective studies we have found that a bolus dose of 20 µg/kg caused maximal dilation of dural and pial arteries with a fall in MABP of up to 35 mmHg in anaesthetized rats (not published).

After a stabilization period of about 1–1.5 h, baseline values were recorded and a blood sample taken to determine ABG. First, bolus CGRP (0.3 µg/kg), GTN (20 µg/kg) or TES was given. After a wash-out period of 10–12 min, iberiotoxin infusion 0.1 mg kg⁻¹ was given in a volume of 1 ml over 5 min followed by bolus CGRP (0.3 µg/kg), GTN (20 µg/kg) or TES.

In vitro studies

Male Sprague–Dawley rats (300–380 g; Denmark) were exanguinated during CO₂ anaesthesia. The brain was removed and the first branch of the MMAs and MCAs were dissected out using an operation microscope. Each vessel was cut into 1–2 mm long circular segments and placed in an ice-cold buffer solution gassed with 5% CO₂ in O_2. The composition of the buffer was (mm): NaCl 119, NaHCO₃ 15, KCl 4.6, CaCl₂ 1.5, NaH₂PO₄ 1.2, MgCl₂ 1.27, and glucose 5.5, pH 7.4. Throughout the experiment the buffer was continuously aerated with oxygen enriched with 5% CO₂, resulting in a pH of 7.4. In order to determine vessel tension, each segment was mounted on two metal wires 40 µm in diameter in a myograph (Model 610m: Danish Myo Technology, Aarhus, Denmark). The artery segments were allowed to equilibrate for approximately 30 min. The vessels were stretched to the internal circumference the vessel would have if relaxed and exposed to a passive transmural pressure of 45 mmHg (6.0 kPa) for the dural artery and 52 mmHg (7.0 kPa) for the MCA. This was in order to achieve maximal active force development (26). Following a second 30-min equilibration period, the vessels were constricted twice by 63 mm KCl in a modified buffer solution in which NaCl was substituted for KCl on an equimolar basis. The contraction amounted to 0.21 ± 0.03 mN (n = 19) in the MMA and 0.79 ± 0.07 mN (n = 26) in MCAs. In order to study the relaxant effect of GTN and CGRP, the cerebral arteries were precontracted with 3 × 10⁻⁶ m prostaglandin F_2α (PGF_2α). This resulted in a stable tension of 0.12 ± 0.02 mN (n = 19) in the MMAs and 0.70 ± 0.07 mN (n = 26) in the MCAs, to which the agonist was added in a single concentration. The tension lasted for at least 20–30 min without a significant fall in tone. In blockade experiments, 10⁻⁷ m iberiotoxin was added to the tissue bath 15 min before the addition of GTN 10⁻⁶ m or CGRP 10⁻⁸ m. Of four tissue segments, two served as controls (i.e. GTN or CGRP without blocker) and the others were treated with iberiotoxin.

Data analysis

For in vivo studies the maximal effect (peak) of GTN, TES or CGRP on dural, pial and LCBF_Flux was calculated as the percentage increase or decrease from baseline. The prestimulation baseline was defined as the average of the 60 s preceding the stimulation. The diameters of the dural and pial arteries and LCBF_Flux were measured in arbitrary units, MABP in mmHg, and presented as percentage change from baseline.

All data are presented as mean ± SEM. Statistical significance was set at P < 0.05. Non-parametric Wilcoxon test was used for in vivo studies. SPSS version 10.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analysis, GraphPrism^® (version 3; San Diego, CA, USA) for graphical evaluation.

For in vitro studies all data are presented as mean ± SEM. Number of rats = n. The Mann–Whitney U-test was used to determine statistical significance, assumed when P < 0.05.

Drugs

Iberiotoxin were obtained from Tocris Cookson Inc. (Bristol, UK). Final concentrations were made using normal saline. Rat αCGRP was obtained from Neosystem (Strasbourg, France). GTN was obtained from The Danish Hospital Pharmacy, Glostrup, Denmark (Glycerylnitrat^®). Stock solutions of the drugs were kept frozen in small aliquots and diluted in isotonic saline (0.9%) immediately before use.

Results

The effect of NO donor GTN and inhibition by iberiotoxin in the genuine closed cranial window model

An infusion of GTN (20 µg/kg) increased dural artery diameter by 100 ± 12% and pial artery diameter by 43 ± 5% (n = 6) (Table 1, Figs 1 and 2a,b).

Figure 1

Trace showing the effect of glyceryltrinitrate (GTN) (20 µg/kg) and inhibition by iberiotoxin (0.1 mg/kg) on mean arterial blood pressure, dural and pial artery diameter and local cortical cerebral blood flow using the genuine closed cranial window model. A, GTN (20 µg/kg) bolus; B, pause; C, iberiotoxin (0.1 mg/kg) infusion over 5 min; D, pause; E, GTN (20 µg/kg) bolus; F, pause.

Figure 2

Effect of glyceryltrinitrate (GTN) (20 µg/kg) on (a) dural, (b) pial artery diameter, (c) local cortical cerebral blood flow (LCBF_Flux) and (d) mean arterial blood pressure (MABP) in the absence and presence of iberiotoxin (0.1 mg/kg). Values given as mean ± SEM, n = 6. Statistical analysis comparing responses in the absence and presence of iberiotoxin performed by non-parametric Wilcoxon test: NS, P > 0.05; ∗P < 0.05.

Table 1

Effect of CGRP (0.3 µg/kg), GTN (20 µg/kg) and TES on dural/pial artery diameter, LCBF_Flux and MABP

Infusion or TES	Percent change in artery diameter
	Dura			Pia
	Control	n	Iberiotoxin	Control	n	Iberiotoxin	n
GTN 20 μg/kg	+100 ± 12	6	+12 ± 6∗	+43 ± 5	6	+19 ± 6∗	6
CGRP 0.3 μg/kg	+89 ± 17	6	+92 ± 19, NS	+12 ± 3	6	+16 ± 5, NS	6
TES	+124 ± 19	6	+123 ± 20, NS	+71 ± 36	6	+73 ± 36, NS	6

Infusion or TES	Percent change in LCBF_Flux			Percent change in MABP
Infusion or TES	Control	n	Iberiotoxin	Control	n	Iberiotoxin	n
GTN 20 μg/kg	+29 ± 4	6	+9 ± 2∗	−39 ± 5	6	−15 ± 2∗	6
CGRP 0.3 μg/kg	+22 ± 4	6	+21 ± 3, NS	−17 ± 3	6	−17 ± 3, NS	6
TES	–	–	–	−3 ± 1	6	−2 ± 1, NS	6

Values given as mean ± SEM. Statistical analysis comparing dura vs. pia responses in the absence and presence of iberiotoxin to calcitonin gene-related peptide (CGRP), glyceryltrinitrate (GTN) and transcranial electrical stimulation (TES) performed by non-parametric Wilcoxon test: NS, P > 0.05;

∗

P < 0.05. LCBF_Flux, Local cortical cerebral blood flow; MABP, mean arterial blood pressure;

An infusion of iberiotoxin (0.1 mg/kg) over 5 min had no effect on MABP, LCBF_Flux or dural/pial artery diameter (n = 6) (P > 0.05). Iberiotoxin (0.1 mg/kg) attenuated GTN-induced dural artery dilation to 12 ± 6% (P = 0.031, n = 6) and pial artery dilation to 19 ± 6% (P = 0.031, n = 6) (Table 1, Fig. 2a,b). The same dose of GTN caused an increase in LCBF_Flux of 29 ± 4% (n = 6) and a decrease in MABP of 39 ± 5 (n = 6) (Table 1). These effects were significantly attenuated by iberiotoxin (Table 1, Fig. 2c,d).

The effect of CGRP and inhibition by iberiotoxin in the genuine closed cranial window model

An infusion of CGRP (0.3 µg/kg) increased dural artery diameter by 81 ± 16% and pial artery diameter by 12 ± 3% (n = 6) (Table 1). A CGRP infusion (0.3 µg/kg) caused an increase in LCBF_Flux of 22 ± 4% (n = 6) and a decrease in MABP of 17 ± 3% (Table 1). Iberiotoxin (0.1 mg/kg) did not significantly attenuate the CGRP-induced effects in the dural and pial arteries, LCBF_Flux or MABP (Table 1).

The effect of TES and inhibition by iberiotoxin in the genuine closed cranial window model

TES increased dural artery diameter by 124 ± 19% and pial artery diameter by 71 ± 36% (n = 6) (Table 1). Iberiotoxin (0.1 mg/kg) did not significantly attenuate dural or pial artery dilation induced by TES (Table 1).

The effect of iberiotoxin on CGRP- and GTN-induced relaxation in isolated dural and cerebral arteries in vitro

When given alone, 10⁻⁷ m iberiotoxin induced weak, slowly developing contraction of the MMAs and MCAs. The amount of contraction was not measured because PGF_2α was added before the contraction had reached a steady level of tension. A single concentration of CGRP (10⁻⁸ m) resulted in a relaxation amounting to 46 ± 11% (n = 5) and 29 ± 7% (n = 5), respectively, in the rat MMA and MCA. In the presence of 10⁻⁷ m iberiotoxin the respective relaxations were 34 ± 13% (n = 4; P > 0.05) and 31 ± 8% (n = 5; P > 0.05). GTN (10⁻⁶ m) resulted in a relaxation amounting to 64 ± 8% (n = 6) and 70 ± 7% (n = 7), respectively, of rat MMAs and MCAs (Fig. 3a,b). In the presence of 10⁻⁷ m iberiotoxin the relaxations were 34 ± 9% (n = 6; P < 0.05) and 47 ± 6% (n = 7; P < 0.05), respectively, in rat MMAs and MCAs (Fig. 3a,b).

Figure 3

In vitro relaxation induced by a single concentration of glyceryltrinitrate (GTN) in the absence and presence of 10⁻⁷ m iberiotoxin in rat (a) dural and (b) middle cerebral arteries. The relaxation of each segment tested was calculated as a percentage of the precontraction induced by 3 × 10⁻⁶ m prostaglandin F_2α. Each bar represents the mean values with SEM shown by vertical bars, n = 6–7. Statistical analysis comparing responses in the absence and presence of iberiotoxin (10⁻⁷ m) performed by Mann–Whitney U-test: NS, P > 0.05; ∗P < 0.05.

Discussion

The main finding of the present study is that the BK_Ca channel selective blocker iberiotoxin can attenuate GTN- but not CGRP-induced dural and pial artery dilation in vivo and in vitro, suggesting a role of BK_Ca channels in GTN-induced cranial vasodilation in the rat.

Effect of GTN and inhibition by iberiotoxin

This study has shown that bolus GTN decreases systemic blood pressure and dilates dural and pial arteries in vivo in the closed cranial window model. The responses in dural and pial arteries were verified on isolated rat MMAs and MCAs in vitro. In previous studies we have suggested that the effect on dural arteries is direct, whereas the dilation of pial arteries is mostly or completely autoregulatory and secondary to a drop in systemic blood pressure (24). However, in isolated arteries, where there was no autoregulatory effect and no blood–brain barrier, GTN was equipotent in inducing dilation of the two types of vessel. The GTN-induced dilation in both arterial beds was attenuated in vivo and in vitro by iberiotoxin. In our in vivo experiments GTN caused an increase in LCBF_Flux and a fall in MABP that were attenuated by iberiotoxin. Several animal studies have shown that topical and systemic GTN dilates pial arteries (27, 28). In the anaesthetized cat, infusion of nitroglycerin caused dilation of the suprasylvian pial artery (29). In addition, it has been shown that topical application of the NO donors cause an increase in meningeal blood flow (5). In the rat, activation of BK_Ca, but not K_ATP channels has been observed to contribute to NO-induced vasodilation both in vivo and in vitro (25). Thus, it seems unlikely that K_ATP channels are involved in NO-induced vasodilation in the rat.

The effect of CGRP and inhibition by iberiotoxin

In line with several other investigations, we have shown that i.v. administration of CGRP decreases systemic blood pressure and dilates dural and pial arteries in vivo in the closed cranial window model (24, 30–31). From our previous studies we have concluded that the effect on dural arteries is direct, whereas the dilation of pial arteries is mostly or completely autoregulatory and secondary to a drop in systemic blood pressure (19). However, this difference is probably due to poor penetration of CGRP through the blood–brain barrier, as CGRP administered to isolated arteries in myograph baths was equipotent on the MMA and MCA.

Our results are in agreement with several studies that have examined the effect of CGRP on cerebral arteries in vitro (32), pial arterioles in vivo (33) and in the genuine closed cranial window model (24). The present study has shown that CGRP in vivo and in vitro causes dilation of dural and pial vessels not attenuated by iberiotoxin. In another study, we have shown that the K_ATP channel blocker glibenclamide inhibits CGRP-induced dilation of dural and pial vessels in vivo. This result also confirms that the dose and concentration of CGRP used in the present study was not too high to be inhibited by a potassium channel blocker. Thus, it seems that GTN and CGRP in rat dural and pial circulation act via two different types of K⁺ channels.

The effect of electrical stimulation and inhibition by iberiotoxin

TES caused dilation of dural and pial vessels without affecting MABP. In our experiments, iberiotoxin could not attenuate these effects. It has been suggested that activation of the trigeminal ganglion by TES primarily releases CGRP (34, 35). This is further supported by the inhibitory effect of CGRP antagonists on TES-mediated dilation of dural and pial arteries (24). Thus, iberiotoxin failed to attenuate dural/pial vasodilation induced by exogenous CGRP and CGRP evoked by TES.

Possible role of BK_Ca channels in migraine pathogenesis

Within the cranium only a few structures, such as nociceptive afferents at meningeal blood vessels and at the large cerebral arteries, are able to generate nociceptive signals (36). When these vessels are stimulated experimentally during brain surgery, patients experience a throbbing unilateral migraine-like pain (36). Experimental and clinical data suggest that the trigeminovascular system of large dural vessels, rather than pial vessels, is involved in the pathogenesis of headache (36–40). CGRP plays a pivotal role in migraine, as infusion of CGRP can trigger a migraine attack in migraineurs (3) and the CGRP receptor antagonist BIBN 4096 BS is effective in the treatment of acute attacks of migraine (4). In a similar way, NO also seems to be involved in the generation of severe vascular headaches, because administration of the NO donor GTN causes migraine headaches in migraineurs (1) and the nitric oxide synthase inhibitor L-NMMA is effective in the treatment of the acute migraine attack (2). It has been suggested that the activation of the trigeminocerebrovascular system by NO involves interaction with CGRP, thereby causing the dural blood vessel dilation during migraine (6). Our results are in agreement with a previous study, which suggested that activation of BK_Ca channels may contribute to NO-induced vasodilation in the heart (25).

In summary, our experiments confirm that NO produces marked dilation of meningeal blood vessels, which is believed to produce migraine headache in the clinic. Thus, BK_Ca channels may be involved in migraine pathogenesis, since inhibition of the BK_Ca channels within cranial vessels significantly reduces NO (GTN)-induced dural vasodilation.

Acknowledgement

This study was supported by The Else Torps Legat, The Novo Nordisk foundation and The Lundbeck foundation.

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Role of BK Ca Channels in Cephalic Vasodilation Induced by CGRP,NO and Transcranial Electrical Stimulation In The Rat

Abstract

Keywords

Introduction

Materials and methods

In vivo studies

Surgical preparation

Intravital microscopy

Experimental protocols

In vitro studies

Data analysis

Drugs

Results

The effect of NO donor GTN and inhibition by iberiotoxin in the genuine closed cranial window model

The effect of CGRP and inhibition by iberiotoxin in the genuine closed cranial window model

The effect of TES and inhibition by iberiotoxin in the genuine closed cranial window model

The effect of iberiotoxin on CGRP- and GTN-induced relaxation in isolated dural and cerebral arteries in vitro

Discussion

Effect of GTN and inhibition by iberiotoxin

The effect of CGRP and inhibition by iberiotoxin

The effect of electrical stimulation and inhibition by iberiotoxin

Possible role of BKCa channels in migraine pathogenesis

Acknowledgement

References

Possible role of BK_Ca channels in migraine pathogenesis