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Abstract

The 5-HT_1B/1D receptor agonist sumatriptan is effective in aborting acute attacks of migraine and is known to cause constriction of cranial arteries as well as some peripheral blood vessels. The present study set out to investigate whether 5-HT_1B and/or 5-HT_1D receptors mediate contractions of the human isolated middle meningeal and temporal arteries (models for anti-migraine efficacy) and coronary artery and saphenous vein (models for side-effect potential). Concentration-response curves were made with sumatriptan (1 nM-100 μM) in blood vessels in the absence or presence of selective antagonists at 5-HT_1B (SB224289) and 5-HT_1D (BRL15572) receptors. SB224289 antagonized sumatriptan-induced contractions in all blood vessels, although the antagonism profile was different amongst these blood vessels. In the temporal artery, SB224289 abolished contraction to sumatriptan, whereas in the middle meningeal artery and saphenous vein sumatriptan-induced contractions were blocked in an insurmountable fashion. Moreover, SB224289 acted as a weak surmountable antagonist in the coronary artery (_pK_B: 6.4 ± 0.2). In contrast, BRL15572 had little or no effect on sumatriptan-induced contractions in the four blood vessels investigated. In situ hybridization revealed the expression of 5-HT_1B receptor mRNA in the smooth muscle as well as endothelial cells of the blood vessels, whereas the mRNA for the 5-HT_1D receptor was only very weakly expressed. These results show that the 5-HT_1B receptor is primarily involved in sumatriptan-induced contractions of human cranial as well as peripheral blood vessels.

Keywords

5-HT receptors BRL15572 coronary artery cranial arteries human migraine saphenous vein SB224289 sumatriptan

Introduction

Sumatriptan, the first of the 5-HT_1B/1D receptor agonists, is highly effective in aborting migraine headaches (1, 2). It is believed that migraine headache results from dilation of extracerebral cranial blood vessels and that sumatriptan, as well as other triptans, constricts these dilated vessels (2, 3). Although the triptans are known to be cranioselective, they all have the propensity to constrict the coronary artery (4–7). Constriction of the coronary artery may lead to cardiovascular adverse events, including myocardial ischaemia and infarction in predisposed individuals (8).

The triptans are high affinity agonists at 5-HT_1B and 5-HT_1D receptors, but their vasoconstrictor effect seems to be mediated via the 5-HT_1B receptor (2). Using RT-PCR techniques, it has also been shown that the 5-HT_1B receptor mRNA is predominant over 5-HT_1D receptor mRNA in the human middle cerebral (9, 10), middle meningeal (11), temporal (12) and coronary (13, 14) arteries. The 5-HT_1B receptor protein has also been localized in the smooth muscle layer as well as endothelium of the human middle cerebral (15), middle meningeal (4, 16) and coronary (4, 13, 16) arteries, where the 5-HT_1D receptor protein is not, or poorly, expressed. Functional pharmacological in vitro studies suggest that sumatriptan behaves as a full agonist in blood vessels (17–21), which do not contract in response to selective 5-HT_1D receptor agonists (14, 22).

A more direct evidence for a 5-HT_1B receptor-mediated vasoconstriction to sumatriptan can be obtained from antagonist studies. Until now, studies in the human isolated middle meningeal (7, 18, 23) and coronary (7, 17, 24, 25) arteries and saphenous vein (21) have used non-selective 5-HT_1B/1D receptor antagonists (GR127935, GR55562 and GR125743). Recently, SB224289 and BRL15572 have been introduced as selective 5-HT_1B and 5-HT_1D receptor antagonists, respectively (26, 27). The use of these compounds revealed that sumatriptan-induced contractions in human isolated temporal (12) and small pulmonary (28) arteries as well as canine (29) and porcine (30) carotid vascular beds are mediated via the 5-HT_1B receptor. Using both functional in vitro and in situ hybridization techniques, we investigated the role of 5-HT_1B and 5-HT_1D receptors in mediating contractions of the human isolated middle meningeal and temporal arteries (models for therapeutic efficacy in migraine) and coronary artery and saphenous vein (models for peripheral side-effect potential) (6, 7, 31).

Material and methods

Tissue collection

The middle meningeal (four male, five female; age 30–72 years) and temporal (three female; age 45–59 years) arteries and saphenous vein (eight male, one female; age 45–78 years) were obtained postoperatively from patients undergoing craniotomy (middle meningeal and temporal artery: eight aneurysms; four meningiomas) or coronary artery bypass grafting (saphenous vein) at the Erasmus University Medical Centre, Rotterdam, The Netherlands. The blood vessels were placed in a propylene tube filled with ice-cold (0–4°C) physiological saline, transported immediately to the laboratory and used within 2 h of surgery.

The right epicardial coronary artery was obtained from nine heart-beating organ donors (three male, six female; 37–64 years) who died of non-cardiac disorders (six cerebrovascular accident, two cerebral infarction, one head trauma). The Rotterdam Heart Valve Bank, Rotterdam, The Netherlands provided the hearts, after donor mediation by Bio Implant Services Foundation/Eurostransplant Foundation, Leiden, The Netherlands. The vessel was stored overnight in a modified Krebs bicarbonate solution (see below) and used the next day.

Organ bath experiments

Measurement of vascular contractions

The methods used were similar to those described in detail earlier (6, 7). Briefly, approximately 4-mm segments, obtained from pieces of the middle meningeal (n=6), temporal (n=3) and coronary (n=6) arteries and saphenous vein (n=6), were mounted on metal prongs in organ baths, containing a modified Krebs bicarbonate solution (pH 7.4; 37°C), aerated with 95% O₂ and 5% CO₂. The composition (mM) of the Krebs bicarbonate solution was NaCl 119, KCl 4.7, CaCl₂ 1.25 (or 2.5 for coronary artery and saphenous vein), MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25 and glucose 11.1 (or 8.3 for coronary artery and saphenous vein). The cyclo-oxygenase inhibitor indomethacin (0.1 µm) was added to the Krebs solution to prevent prostaglandin synthesis. In addition, the Krebs solution was enriched with the muscarinic receptor antagonist atropine, the histamine H₁ receptor antagonist mepyramine, the mixed 5-HT_2/7 receptor antagonist mesulergine, the α₁-adrenoceptor antagonist prazosin, the inhibitor of neuronal uptake₁ imipramine (all 1 µm) and the inhibitor of extra-neuronal uptake₂ corticosterone (10 µm), to exclude the putative involvement of these receptors/mechanisms.

Changes in isometric tension were registered on recording set-ups from either EMKA Technology (Paris, France) for the middle meningeal and temporal arteries or Harvard Apparatus (South Natick, Massachusetts, USA) for the coronary artery and saphenous vein. The segments were allowed to equilibrate for at least 30 min, stretched to a passive tension of 4 mN (middle meningeal and temporal arteries), 15 mN (coronary artery) or 10 mN (saphenous vein). Since sumatriptan is metabolized by monoamine oxidase (32), we then treated the segments with pargyline (100 µm) for 15 min to prevent its possible breakdown. After washing, all segments were exposed two to three times to either 0.1 µm prostaglandin F _2α (PGF _2α; middle meningeal and temporal arteries) or 30 mm KCl (coronary artery and saphenous vein) to demonstrate the reproducibility of the evoked contractions. Subsequently, the relaxation response to 10 nm substance P (1 nm in the case of coronary artery or 0.1 µm bradykinin in the case of saphenous vein) in vessel segments pre-contracted with PGF _2α (1 µm) was used to verify the functional integrity of the endothelium.

After washing, the segments were allowed to equilibrate for 60 min, with replacement of the Krebs solution every 15 min. The segments were then studied in a paired parallel experimental set-up (6, 7), where a single concentration response curve to sumatriptan (1 nm-100 µm) was constructed in each segment incubated for 60 min with either the vehicle, SB224289, BRL15572 or both antagonists together. As only three segments could be obtained from the human temporal artery, addition of both antagonists together was omitted. In view of the affinity of SB224289 and BRL15572 at recombinant h5-HT_1B (pK_i: 8.0 and 6.1, respectively) and h5-HT_1D receptors (pK_i: 6.2 and 7.9, respectively) (26, 27), we initially employed the two antagonists in a concentration range of 100–300 nm in the coronary artery (without the cocktail of inhibitors as mentioned above) and saphenous vein. As in these concentrations neither antagonist affected the concentration response curves of sumatriptan, all experiments were performed with 1 µm of SB224289 and BRL15572.

Data presentation

Contractile responses were expressed as percentage of the contractile response to 1 µm PGF _2α (temporal and middle meningeal arteries) or 100 mm KCl (coronary artery and saphenous vein). The occasional spontaneous phasic contractions, observed in some coronary artery and saphenous vein segments, were ignored when measuring contractions. When the concentration contraction curve to sumatriptan did not attain a plateau, the contraction with its highest concentration (100 µm) was considered as the apparent maximum contraction (E_max). Initially, the mean value of E_max of sumatriptan observed in individual experiments was calculated. The mean concentration response curves were analysed with a non-linear regression fitting technique for sigmoidal functions with variable slope using Graphpad Prism 3.0 (Graphpad Software Inc., San Diego, California, USA) to calculate potency (pEC₅₀) and Hill slopes for the agonists in the absence or presence of antagonists. When mean (apparent) E_max and Hill slopes were not significantly different in control and antagonist experiments, a surmountable antagonism was assumed. The whole data set was then transformed using SPSS 7.5 non-linear regression statistics (SPSS Inc., Chicago, Illinois, USA) into a dependent fitting model, where the mean maximal-induced contraction was set to the respective agonist control value. In case of a parallel rightward shift, a Schild regression analysis was performed with a slope set to unity to calculate the pK_B value. Due to the insurmountable behaviour of SB224289 in the middle meningeal artery and saphenous vein, pK_B values could not be calculated. Instead, we calculated the negative logarithm of the mean concentration of sumatriptan eliciting a contraction equivalent to 25% of E_max in the individual control experiments (pEC_25%) in the absence or presence of antagonists. This parameter enabled us to compare the properties of the antagonists in these vascular preparations against low concentrations of sumatriptan.

Statistical analysis

Differences between the (apparent) E_max of sumatriptan in the absence or presence of antagonists were analysed with a paired t-test, using Graphpad Prism 3.0 (Graphpad Software Inc., San Diego, California, USA). In case the E_max values in the absence of antagonists were similar to those in the presence of antagonist, the differences in Hill slopes and pEC₅₀ values of mean concentration response curves between vehicle and antagonist groups were analysed with a one-way analysis of variance (anova) followed by Dunnett's multiple comparison t-test. In case E_max values were different between the respective groups, we performed a two-way repeated measurement analysis of variance (RM-anova) followed by Bonferroni's multiple comparison t-test to evaluate the effect of treatments (i.e. control or antagonist) at repetitive concentrations of sumatriptan. Differences between the pEC_25% values of sumatriptan in the absence or presence of antagonists were calculated according to paired t-test. In all cases statistical significance was assumed when P < 0.05. Data are presented as mean±SE mean.

Molecular biological experiments

In situ hybridization

5-HT_1B and 5-HT_1D receptor mRNAs were localized employing non-radioactive in situ hybridization on the human middle meningeal artery, coronary artery and saphenous vein (n=3 each). After cleaning the surrounding tissue the blood vessels were fixed for 24 h in 4% paraformaldehyde dissolved in phosphate buffered saline. Chinese hamster ovary (CHO) cells expressing either the human recombinant 5-HT_1B or 5-HT_1D receptor served as positive controls. After dehydration with increasing percentage of ethanol in phosphate buffered saline, the blood vessels were embedded in paraffin and 5-µm thick sections were cut with a microtome (model HM325, Microm GmbH, Walldorf, Germany). The sections were mounted on superfrost plus® glass slides (Menzel-Glaser, Braunschweig, Germany) and dried at 37°C for 48 h.

Recombinant plasmid DNAs encoding the human 5-HT_1B (Genbank accession number D10995, nucleotide 40–390, length 350 bp) or 5-HT_1D (Genbank accession number M81589; nucleotide 1–400, length 400 bp) receptor were employed for the preparation of non-radioactive cRNA probes. Linearized DNA templates were transcribed to synthesize the sense and antisense cRNA probes, using T₇ or SP₆ RNA polymerase as per protocol described for the DIG-RNA labelling kit. DIG-labelled cRNA probes were quantified by dot blotting and using serial dilutions of standard DIG-labelled control RNA supplied in the kit. Treatment of tissue sections and cells and subsequent hybridization was performed as described earlier (33). The tissues were hybridized with 25 ng of cRNA probe per slide for 16 h at 55°C and the DIG-labelled hybrids were detected by incubation with antidigoxigenin antibody (1 : 2000 dilution) conjugated to alkaline phosphatase for 2.5 h at room temperature. The immunodetection of DIG-labelled hybrids was done using 4-nitroblue tetrazolium chloride (NBT) as chromogen and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) as coupling agent. Slides were counter-stained with nuclear red solution, dehydrated with ethanol gradients and mounted with Euparal (Chroma-Gesellschaft, Schmid GmbH, Köngen, Germany). Cells and tissue sections were visualized under a light microscope (model Leica DM RBE, Leica NL, BV, Rijswijk, The Netherlands) and photographed using a CCD video camera (Sony DXC-950, Sony Corporation, Tokyo, Japan). The sense riboprobes were included as negative controls, which showed no or little staining compared with antisense riboprobes.

Endothelial staining

Serial sections of 5-µm thickness were processed for immunohistochemical localization of endothelial cell marker, CD31. Sections were deparaffinized, rehydrated and incubated with pronase (1 mg/ml) at 37°C for 10 min prior to incubation with specific purified mouse monoclonal antibodies raised against human CD31 (Neomarkers, Union City, California, USA). To block non-specific binding, sections were incubated with 10% normal goat serum diluted in 5% bovine serum albumin in phosphate buffered saline (pH=7.4). Subsequently, sections were incubated overnight at 4°C with primary antibodies, CD31 (1 : 75 v/v). Incubation for 30 min with secondary biotinylated anti-immunoglobulins (Multilink®, 1 : 75 v/v, Biogenex, San Ramon, USA) and tertiary complex of streptavidin conjugated to alkaline phosphatase (Label® 1 : 50 v/v, Biogenex) were used to enhance the detection sensitivity. Colour was developed using New Fuchsine as chromogen, while endogenous alkaline phosphatase activity was inhibited by 0.01 m levamisole (Sigma Chemical Co., St Louis, USA). Slides were counterstained with Mayer's haematoxylin. Positive controls consisted of human cardiac tissue where intense staining was seen in the endothelium of all blood vessels. The optimal dilutions of primary antibody were identified by examining the intensity of staining obtained with a series of dilutions, which gave specific and easily visible signal on paraffin sections of control tissues. Slides were mounted and visualized using light-microscopy. Negative controls consisted of omission of the primary antibody.

Ethical approval

The Medical Ethics Committee of the Erasmus University Medical Centre Rotterdam, dealing with the use of human material for scientific experiments, approved the protocols for this investigation.

Compounds and kits

For pharmacological study, the following compounds were used: atropine sulphate, bradykinin acetate, corticosterone, 5-hydroxytryptamine creatinine sulphate (serotonin; 5-HT), imipramine hydrochloride, indomethacin hydrochloride, mepyramine maleate, pargyline hydrochloride, prostaglandin F _2α tris salt (PGF _2α) and substance P acetate (all purchased from Sigma Chemical Co.), mesulergine hydrochloride (gift: Novartis AG, Basel, Switzerland), prazosin hydrochloride (gift: Pfizer, Sandwich, UK), sumatriptan succinate, SB224289 (2,3,6,7-tetrahydro-1′-methyl-5-[2′-methyl-4′(5-methyl-1,2,4-oxadiazo l-3-yl) biphenyl-4-carbonyl] furo [2,3f]indole-3-spiro-4′-piperidine hydrochloride) and BRL15572 (1-3-chlorophenyl-4-[3,3-diphenyl(2-(S,R) hydroxypropanyl) piperazine] hydrochloride), both from GlaxoSmithKline, Harlow, Essex, UK, courtesy of Dr A. A. Parsons. Stock solutions of corticosterone (100 mm), indomethacin, mesulergine, prazosin, SB224289 and BRL15572 (all 10 mm) were dissolved in 100% v/v dimethyl sulphoxide and further diluted in distilled water. All other compounds were dissolved in distilled water.

For molecular biology experiments, the materials used were: acetic anhydride, diethyl pyrocarbonate, levamisol, maleic acid, triethanolamine and xylene (Sigma Chemical Co.), antidigoxigenin-AP Fab fragments, blocking reagent for nucleic hybridization, DIG-RNA labelling kit, glycine and RNase T₁ (Boehringer Mannheim, Almere, The Netherlands), bovine serum albumin, dextran sulphate, ethylene diaminetetraacetic acid, ficoll, hering sperm DNA, phenol and Tris-HCl (Amersham Pharmacia Biotech Benelux, Roosendaal, The Netherlands), BCIP, formamide, NBT, proteinase K, sodium citrate and yeast tRNA (Life Technologies, Breda, The Netherlands), DNase, RNase inhibitors and Triton-X-100 (Promega Benelux, Leiden, The Netherlands) and RNase away solution (Molecular Bio-products, San Diego, CA, USA).

Results

Relaxation responses to substance P and bradykinin

The relaxation to substance P (10 nm) amounted to 29% (range: 17–44%, n=6) and 42% (range: 9–86%, n=3) of pre-contraction with 1 µm PGF _2α in the middle meningeal and temporal arteries, respectively. In the coronary artery, relaxation to substance P (1 nm) was 46% (range: 30–66%, n=6) of pre-contraction to 1 µm PGF _2α. In the saphenous vein, relaxation to bradykinin (1 µm) was 54% (range: 26–82%, n=6) of pre-contraction to 1 µm PGF _2α.

Effects of sumatriptan on cranial arteries

Concentration response curves to sumatriptan in the middle meningeal and temporal arteries are depicted in Fig. 1. In both blood vessels, sumatriptan elicited a concentration-dependent contraction. The E_max values (efficacy) of sumatriptan in the middle meningeal and temporal arteries were 83 ± 15% and 68 ± 28% of the contraction to 1 µm PGF _2α, respectively, and the pEC₅₀ values (potency) amounted to 6.7 ± 0.2 and 6.7 ± 0.3, respectively (Table 1).

Table 1

Apparent E_max and pEC₅₀ values of sumatriptan in contracting human isolated blood vessels in the absence or presence of antagonists

	Middle meningeal artery		Temporal artery		Coronary artery		Saphenous vein
Antagonist	E_max (%)	pEC₅₀	E_max (%)	pEC₅₀	E_max (%)	pEC₅₀	E_max (%)	pEC₅₀
None	83 ± 15	6.7 ± 0.2	68 ± 28	6.7 ± 0.3	13 ± 2	5.7 ± 0.1	62 ± 4	6.1 ± 0.1
SB224289 (1 µm)	40 ± 11∗	ND	2 ± 1∗	ND	11 ± 3	5.0 ± 0.1∗	35 ± 7∗	ND
BRL15572 (1 µm)	72 ± 10	6.8 ± 0.2	47 ± 24	6.5 ± 0.2	14 ± 2	5.7 ± 0.1	44 ± 7∗	6.1 ± 0.1
SB224289 (1 µm) + BRL15572 (1 µm)	53 ± 14	ND	–	–	10 ± 2	5.0 ± 0.2∗	32 ± 4∗	ND

Data are means± SEM (n = 3–6). Apparent E_max is presented as percentage of contraction elicited by either 1 µm PGF _2α (middle meningeal and temporal artery) or 100 mm KCl (coronary artery and saphenous vein). ND = not determined because of insurmountable nature of the antagonism; – = not investigated.

^∗Significantly different from sumatriptan control (P < 0.05).

Figure 1

Cumulative concentration response curves to sumatriptan in the human isolated middle meningeal (n = 5–6) and temporal (n = 3) arteries in the absence (control; ○) or presence of SB224289 (1 µm; •), BRL15572 (1 µm; ▴) or both antagonists (1 µm each; ▪). Symbols and vertical bars represent the means± SEM. ∗Significant difference from control contraction elicited by respective concentrations of sumatriptan (Bonferroni multiple comparison t-test, P < 0.05).

The responses to sumatriptan in both cranial arteries were antagonized by the 5-HT_1B receptor antagonist SB224289 (1 µm) and the magnitude of antagonism did not correlate with the functional integrity of the endothelium (substance P response). As SB224289 decreased the E_max of sumatriptan, the antagonism was apparently insurmountable and pEC₅₀ values of sumatriptan were not determined (Fig. 1, Table 1). The 5-HT_1D receptor antagonist BRL15572 (1 µm) had no effect on either the E_max or pEC₅₀ values of sumatriptan. Also, no additional antagonism was observed with the combination of the two antagonists (Fig. 1, Table 1).

Due to the insurmountable antagonism of sumatriptan by SB224289, pK_B values could not be estimated. Alternatively, we calculated the negative logarithm of sumatriptan concentration eliciting a response equivalent to 25% of sumatriptan control E_max (pEC_25%) in the absence or presence of the antagonists in the middle meningeal artery; the temporal artery data was not further processed as SB224289 virtually abolished sumatriptan-induced contractions (Table 2). While BRL15572 was ineffective, the pEC_25% of sumatriptan was significantly decreased by SB224289. No additional antagonism was observed with the combination of the two antagonists (Table 2).

Table 2

pEC_25% of sumatriptan in contracting the middle meningeal artery and saphenous vein in the absence or presence of antagonists

Antagonist	Middle meningeal artery	Coronary artery	Saphenous vein
None	6.8 ± 0.2	6.3 ± 0.1	6.4 ± 0.1
SB224289 (1 µm)	5.6 ± 0.4∗	5.7 ± 0.3	5.3 ± 0.2∗
BRL15572 (1 µm)	7.0 ± 0.5	6.1 ± 0.1	6.3 ± 0.1
SB224289 (1 µm) + BRL15572 (1 µm)	6.0 ± 0.5	5.7 ± 0.2∗	5.7 ± 0.3∗

Data are mean± SEΜ (n = 4–6). pEC_25% represents the negative logarithm of the concentrations of sumatriptan eliciting a contraction equivalent to 25% of individual control E_max (middle meningeal artery, 20.8 ± 3.8% of the response to 1 µm PGF _2α; coronary artery, 3.3 ± 0.4% of the response to 100 mm KCl; saphenous vein, 15.2 ± 0.9% of the response to 100 mm KCl).

^∗Significantly different from sumatriptan control (P < 0.05).

Effect of sumatriptan on peripheral vessels

Sumatriptan also contracted the human coronary artery and saphenous vein in a concentration-dependent manner. The pEC₅₀ and (apparent) E_max of sumatriptan were, respectively, 5.7 ± 0.1 and 13 ± 2% of the response to 100 mm KCl in the coronary artery and 6.1 ± 0.1 and 62 ± 4% of the response to 100 mm KCl in the saphenous vein (Fig. 2, Table 1).

Figure 2

Cumulative concentration response curves to sumatriptan in the human isolated coronary artery (n = 5–6) and saphenous vein (n = 4–6) in the absence (control; ○) or presence of SB224289 (1 µm; •), BRL15572 (1 µm; ▴) or both antagonists (1 µm each; ▪). Symbols and vertical bars represent the means± SEM. ∗Significant difference from control contraction elicited by respective concentrations of sumatriptan (Bonferroni multiple comparison t-test, P < 0.05).

In the coronary artery, SB224289 caused a small parallel rightward shift in the concentration response curve to sumatriptan, yielding a significant decrease in the pEC₅₀ (5.0 ± 0.1) with no change in the apparent E_max (11 ± 3% of the response to 100 mm KCl) of sumatriptan. Schild regression analysis revealed a pK_B value of 6.4 ± 0.2 for SB224289 against sumatriptan. The shift in the concentration response curve of sumatriptan by SB224289 was independent of the functional integrity of the endothelium. BRL15572 did not have any effect on sumatriptan-induced contraction. Incubation with both antagonists together resulted in a small parallel rightward shift as was noticed with SB224289 alone (Fig. 2, Table 1).

In the saphenous vein, the response to sumatriptan was antagonized by SB224289 (1 µm). Although the contraction to sumatriptan increased with concentration, the response did not reach a plateau with the highest concentration used and therefore pEC₅₀ and pK_B values after SB224289 were not determined. As observed in the other blood vessels, the antagonism of SB224289 was independent of the functional integrity of the endothelium. Incubation with BRL15572 (1 µm) resulted in a slight attenuation of the response to 10 µm sumatriptan as well as its E_max, but no change was observed in the pEC₅₀ value of sumatriptan. Also, no additional antagonism was observed with the combination of the two antagonists (Fig. 2, Table 1). The pEC_25% of sumatriptan was not affected by BRL15572, but was reduced after SB224289 alone or in combination with BRL15572 (Table 2).

In situ hybridization

The specificity of mRNA signals and standardization of the in situ hybridization conditions were first established by the use of specific sense and antisense probes in CHO cells expressing either the human 5-HT_1B or 5-HT_1D receptor. Strong cytoplasmic and nuclear mRNA expression as a dark purple/blue colour for respective antisense riboprobes was observed in these cells, whereas sense riboprobes depicted no staining. Furthermore, the sense riboprobes did not show any specific mRNA expression assessed as purple/blue staining in the tissue samples included in this study. If any staining with sense riboprobes was detected, it was always far less than the staining with antisense probes (data not shown).

Cellular localization of mRNAs for the 5-HT_1B and 5-HT_1D receptors in the human middle meningeal and coronary arteries and saphenous vein is depicted in Fig. 3. In the middle meningeal artery, hybridization with antisense riboprobe showed 5-HT_1B receptor mRNA signals in the medial smooth muscle layer as well as in the endothelial cells, whereas adventitial cells showed only a faint staining. Although not so intense, the coronary artery and saphenous vein also expressed the 5-HT_1B receptor mRNA in the vascular smooth muscle cells. Specific expression for the 5-HT_1B receptor was seen in the luminal endothelial cells in the case of coronary artery. In order to verify the in situ hybridization data for the endothelial cell expression of 5-HT_1B receptor mRNA, CD31 immunohistochemistry was performed and it confirmed a cytoplasmic endothelial cell staining (Fig. 3). In contrast to the 5-HT_1B receptor mRNA, no signals were detected for the 5-HT_1D receptor mRNA in both smooth muscle and endothelial cells in the middle meningeal artery and saphenous vein. However, a weak staining for the 5-HT_1D receptor mRNA was noticed in some endothelial cells in the coronary artery and in adventitial cells in the case of middle meningeal as well as coronary arteries (Fig. 3).

Figure 3

Photographs of immunohistochemical staining with CD31 endothelial marker (top panels) and in situ hybridization with DIG-labelled cRNA probes for h5-HT_1B (middle panels) and h5-HT_1D (lower panels) receptor mRNAs in the human middle meningeal and coronary arteries and saphenous vein. The reddish colour in the top panels denotes endothelial cells, while the purple precipitates in the middle panels correspond to 5-HT_1B receptor mRNA signals. The 5-HT_1D receptor mRNA (lower panels) as well as the sense riboprobes, as exemplified by the left middle panel inset, did not show signals in the three blood vessels. Scale bar = 50 µm.

Discussion

Craniovascular selectivity of sumatriptan

Sumatriptan contracted in a concentration-dependent manner both cranial and peripheral blood vessels, used as models for anti-migraine activity (middle meningeal and temporal artery) and possible peripheral side-effect potential (coronary artery and saphenous vein), respectively. The potency of sumatriptan was higher at the middle meningeal and temporal arteries (pEC₅₀: 6.7 each) than at the coronary artery (pEC₅₀: 5.7) and saphenous vein (pEC₅₀: 6.1). Compared with the other vessels, the efficacy (E_max) of sumatriptan in the coronary artery was clearly lower (see Table 1). These data, which confirm the cranioselectivity of sumatriptan, are in accordance with other studies dealing with middle meningeal (4, 6, 18, 23), temporal (12, 18) and coronary (6, 7, 13, 17, 31, 34) arteries and the saphenous vein (6, 7, 21).

Receptors mediating sumatriptan-induced contractions

The data obtained in in vitro functional studies show that the 5-HT_1B receptor antagonist SB224289 clearly attenuated sumatriptan-induced contractions, although the 5-HT_1D receptor antagonist BRL15572 had little effect on the concentration response curves to sumatriptan; the combination of SB224289 and BRL15572 did not reveal any additional antagonism. These results establish that the 5-HT_1B receptor mediates the contractile effects of sumatriptan in both cranial and peripheral blood vessels, while the 5-HT_1D receptor does not seem to play any role. The latter conclusion is also supported by the fact that selective 5-HT_1D receptor agonists (PNU-109291 and L775 606) did not contract isolated blood vessels (14, 22, 35) and, interestingly, one such compound (PNU-142633) was also found ineffective in migraine (36). Based on the high affinity of sumatriptan for the 5-HT_1F receptor (pKi: 7.9) (37), it has been argued that this receptor might play a role in the therapeutic action of sumatriptan (38). Although we cannot rule this out, the 5-HT_1F receptor is not involved in vasoconstriction, as selective 5-HT_1F receptor agonists (LY344864 and LY334370) show no vasoconstrictor effect (14, 39, 40). In addition, Verheggen et al. (12) have suggested that in the presence of SB224289 high concentrations of sumatriptan can elicit contractions of the human isolated temporal artery via the 5-HT_2A receptor. This was not the case in the present studies, probably because we included the mixed 5-HT_2/7 receptor antagonist mesulergine (pKB: 9.1 and 8.2, respectively) (41), in the Krebs solution. However, the 5-HT₂ receptor antagonist ketanserin was unable to block sumatriptan-induced contractions in the human middle meningeal (18) and coronary (17, 34) arteries as well as the saphenous vein (21).

It may be noted that SB224289 antagonized the responses to sumatriptan in an insurmountable manner in the middle meningeal artery, whereas in the temporal artery the contractions were virtually abolished. In the coronary artery, there was a weak competitive (surmountable) antagonism, whilst in the saphenous vein an intermediate antagonistic response was observed. The nature of the difference in the antagonistic behaviour of SB224289 in these blood vessels is not clear. A possible explanation (42) could be that SB224289, which acts as an inverse agonist in cells expressing recombinant 5-HT_1B receptors (43), may also do so at constitutive 5-HT_1B receptors. Another explanation for the observed differences could be the influence of receptor density and receptor reserve, but, in view of considerably higher 5-HT_1B receptor density in the meningeal artery as compared with the coronary artery (16), we should have observed a surmountable antagonism in the middle meningeal artery and insurmountable antagonism in the coronary artery. Finally, in view of the localization of the 5-HT_1B receptor on the endothelium (see below), the effect of SB224289 may be influenced to a different degree by endothelial factors (either contractile or relaxing) released upon activation of the 5-HT_1B receptor. Whatever the mechanism, differences in the nature of antagonism have also been observed between sumatriptan and the non-selective 5-HT_1B/1D receptor antagonist GR127935 (14, 23).

It is surprising that SB224289 proved to be a weak antagonist in our experiments as its pK_B (6.4 ± 0.2) against sumatriptan in the coronary artery resembled more its pK_i (6.2) at the 5-HT_1D receptor and was far less than its pK_i at the 5-HT_1B receptor (8.2, 26, 27). However, if 5-HT_1D receptors were involved, we would have found BRL15572 to be an even more potent antagonist than SB224289; this was obviously not the case. The involvement of 5-HT_1B receptor in the sumatriptan-induced coronary artery contraction is also supported by previous investigations using non-selective 5-HT_1B/1D receptor antagonists (6, 7, 13, 24, 34). However, the weaker antagonism by SB224289 than these non-selective antagonists suggests that an additional mechanism is partly responsible for the coronary contraction induced by sumatriptan. The nature of this additional mechanism is not known, but it cannot be related to 5-HT₂ receptors in view of the presence of mesulergine in the organ bath. Certainly, this additional mechanism is of interest with regard to the sumatriptan-related cardiac side-effects and may prove relevant in future anti-migraine drug development.

Vascular localization of 5-HT_1B and 5-HT_1D receptor mRNA

Although RT-PCR studies have shown the presence of 5-HT_1B receptor mRNA in the human middle meningeal (11) and coronary (13, 14) arteries, the cellular localization of 5-HT receptor subtypes at mRNA level has not been demonstrated. Our results of in situ hybridization showed the mRNA expression of 5-HT_1B receptor in both smooth muscle and endothelium, whereas the expression of 5-HT_1D receptor mRNA was weak, if any, in the three human blood vessels investigated (Fig. 3). These results are in agreement with the 5-HT_1B and 5-HT_1D receptor protein localization in coronary and middle meningeal arteries (4, 13, 16). The localization of 5-HT_1B receptor mRNA within the smooth muscle cells of these blood vessels, and the lack of 5-HT_1D receptor mRNA, strongly suggest that contraction to sumatriptan is mediated via the 5-HT_1B receptor rather than the 5-HT_1D receptor. Although the exact role of the endothelial 5-HT_1B receptor in these vessels is not well understood, we cannot rule out the release of relaxing and/or contractile substances upon stimulation.

Conclusion

In conclusion, our data show that sumatriptan contracts the human middle meningeal, temporal and coronary arteries and saphenous vein via the 5-HT_1B receptor, but not 5-HT_1D receptor. In the human coronary artery, the contraction may also be mediated by an unknown, SB224289-resistant, mechanism.

Footnotes

Acknowledgements

The authors wish to express their gratitude to the staff of the Neurosurgery (Head: Professor C.J.J. Avezaat) and Cardiothoracic Surgery (Head: Professor A.J.J.C. Bogers) Units, Erasmus University Medical Centre, Rotterdam, as well as the Rotterdam Heart Valve Bank (Dr A.P.A. Stegmann) for providing human blood vessels. We thank Dr Mike Mosley (Pfizer Ltd, Sandwich, UK) for kindly providing us with recombinant plasmid DNAs encoding human 5-HT_1B and 5-HT_1D receptors and transfected CHO cells. Dr H. Motulsky (Graphpad Software Inc., San Diego, California, USA) and Dr P.G.H. Mulder (Department of Epidemiology and Biostatistics, Erasmus University Medical Centre, Rotterdam) helped us with statistical analyses of the data.

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Characterization of Sumatriptan-Induced Contractions in Human Isolated Blood Vessels Using Selective 5-HT 1B and 5-HT 1D Receptor Antagonists and In Situ Hybridization

Abstract

Keywords

Introduction

Material and methods

Tissue collection

Organ bath experiments

Measurement of vascular contractions

Data presentation

Statistical analysis

Molecular biological experiments

In situ hybridization

Endothelial staining

Ethical approval

Compounds and kits

Results

Relaxation responses to substance P and bradykinin

Effects of sumatriptan on cranial arteries

Effect of sumatriptan on peripheral vessels

In situ hybridization

Discussion

Craniovascular selectivity of sumatriptan

Receptors mediating sumatriptan-induced contractions

Vascular localization of 5-HT1B and 5-HT1D receptor mRNA

Conclusion

Footnotes

Acknowledgements

References

Vascular localization of 5-HT_1B and 5-HT_1D receptor mRNA