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Abstract

It has recently been shown that the α-adrenoceptors mediating vasoconstriction of porcine carotid arteriovenous anastomoses resemble both α₁- and α₂-adrenoceptors, but no attempt was made to identify the specific subtypes (α_1A, α_1B and α_1D) involved. Therefore, the present study was designed to elucidate the specific subtype(s) of α₁-adrenoceptors involved in the above response, using the α₁-adrenoceptor agonist phenylephrine and α₁-adrenoceptor antagonists 5-methylurapidil (α_1A), L-765 314 (α_1B) and BMY 7378 (α_1D). Ten-minute intracarotid infusions of phenylephrine (1, 3 and 10 μgkg⁻¹.min⁻¹) induced a dose-dependent decrease in total carotid and arteriovenous anastomotic conductance, accompanied by a small tachycardia. These carotid vascular effects were abolished by L-765 314 (1000 μgkg⁻¹; i.v.), while these responses were only attenuated by 5-methylurapidil (1000 μgkg⁻¹; i.v.), and BMY 7378 (1000 μgkg⁻¹; i.v.). Furthermore, intravenous bolus injections of phenylephrine (3 and 10 μgkg⁻¹) produced a dose-dependent vasopressor response, which was only affected by 1000 μgkg⁻¹ of 5-methylurapidil, while the other antagonists were ineffective. These results, coupled to the binding affinities of the above antagonists at the different α₁-adrenoceptors, suggest that both α_1A- and α_1B-adrenoceptors mediate constriction of carotid arteriovenous anastomoses in anaesthetized pigs. In view of the less ubiquitous nature of α_1B- compared to α_1A-adrenoceptors, the development of potent and selective α_1B-adrenoceptor agonists may prove to be important for the treatment of migraine.

Keywords

arteriovenous anastomoses carotid migraine pig

Introduction

Although the pathophysiology of migraine has not yet been completely unravelled, there is little doubt that dilatation of large cephalic arteries and, possibly, arteriovenous anastomoses is involved in the headache phase of migraine (1–4). Indeed, over the years we have shown that two important groups of drugs that are highly effective in the acute treatment of migraine, i.e. the triptans and ergot alkaloids, potently constrict carotid arteriovenous anastomoses (4–6). While the carotid vasoconstrictor effect of sumatriptan, as well as some other triptans, is mediated by the 5-HT_1B receptor (4, 6, 7), the ergot-induced vasoconstriction involves, in addition to 5-HT_1B receptors (8), also α-adrenoceptors (9).

There are several reasons to believe that α-adrenoceptors may regulate vascular tone of carotid arteriovenous anastomoses, providing a potential avenue for the development of new antimigraine drugs. It is well known that α-adrenoceptors play an important role in the regulation of vascular tone and blood pressure (10) and that stimulation of the receptors results in constriction of the isolated carotid artery (11–15). Furthermore, administration of α-adrenoceptor antagonists to conscious pigs as well as pigs under thiopentone anaesthesia results in an increase in arteriovenous anastomotic blood flow (16, 17). More recently, we have shown that both α₁- and α₂-adrenoceptors mediate the constriction of carotid arteriovenous anastomoses in anaesthetized pigs (18).

The objective of the present study was to elucidate the subtype(s) of α₁-adrenoceptors — α_1A-, α_1B- and α_1D-adrenoceptor subtypes (19–22) — involved in the constriction of carotid arteriovenous anastomoses in anaesthetized pigs. For this purpose, we investigated the effects of 5-methylurapidil, L-765 314 and BMY 7378, which are preferential antagonists, respectively, at α_1A-, α_1B- and α_1D-adrenoceptors (see Table 1 for affinity constants) (23–25), on the carotid vasoconstriction induced by the α₁-adrenoceptor agonist phenylephrine in a well-defined in vivo animal model predictive for antimigraine activity (5, 26). Unfortunately, selective agonists at the α₁-adrenoceptor subtypes are not available.

TABLE 1

Binding affinity constants (pK_i values) for cloned human α₁-adrenoceptor subtypes

Compound	α_1a	α_1b	α_1d
Prazosin	9.4 a	10.2 a	9.8 a
5-Methylurapidil	8.7 a	7.6 a	7.5 a
L-765 314	6.4 b	8.7 b	7.5 b
BMY 7378	6.6 c	7.2 c	9.4 c

Data from

Weinberg et al. (23)

Patane et al. (24)

Goetz et al. (25).

Materials and methods

General

After an overnight fast, 41 domestic pigs (Yorkshire × landrace; female; 10–14 kg) were anaesthetized with azaperone (120 mg, intramuscular, i.m.), midazolam hydrochloride (5 mg, i.m.) and sodium pentobarbital (600 mg, intravenous, i.v.). After tracheal intubation, the animals were connected to a respirator (BEAR 2E, BeMeds AG, Baar, Switzerland) for intermittent positive pressure ventilation with a mixture of room air and oxygen. Respiratory rate, tidal volume and oxygen supply were adjusted to keep arterial blood gas values within physiological limits (pH: 7.35–7.48; pCO₂: 35–48 mmHg; pO₂: 100–120 mmHg). Anaesthesia was maintained with a continuous i.v. infusion of sodium pentobarbital (20 mgkg⁻¹.h⁻¹). It may be pointed out that this anaesthetic regimen, together with bilateral vagosympathectomy (see below), leads to an increase in heart rate and dilatation of arteriovenous anastomoses due to a loss of parasympathetic and sympathetic tone, respectively. Indeed, basal arteriovenous anastomotic blood flow is considerably higher in sodium pentobarbital-anaesthetized pigs (70–80% of carotid blood flow; present results) than in conscious (< 5% of carotid blood flow; 16) or fentanyl/thiopental anaesthetized (∼19% of carotid blood flow; 17) pigs. A high basal carotid arteriovenous anastomotic blood flow is particularly useful for investigating the effects of drugs that constrict these ‘shunt’ vessels.

A catheter was placed in the inferior vena cava via the left femoral vein for the administration of sodium pentobarbital, vehicle (distilled water) or the antagonists. Another catheter was placed in the aortic arch via the left femoral artery for the measurement of arterial blood pressure (Combitrans disposable pressure transducer; Braun, Melsungen, Germany) and arterial blood withdrawal for the measurement of blood gases (ABL-510; Radiometer, Copenhagen, Denmark). Subsequently, bilateral vagosympathectomy was performed in order to prevent the possible influence via baroreceptor reflexes on phenylephrine-induced carotid vascular responses. Two hub-less needles, each connected to a polyethylene tube, used for the administration of radioactive microspheres and phenylephrine, respectively, were inserted into the right common carotid artery against the direction of blood flow for uniform mixing.

Total common carotid blood flow was measured with a flow probe (internal diameter: 2.5 mm) connected to a sine-wave electromagnetic flow meter (Transflow 601-system, Skalar, Delft, The Netherlands), as described elsewhere (5, 27). Heart rate was measured with a tachograph (CRW, Erasmus University, Rotterdam, The Netherlands) triggered by electrocardiogram signals. Arterial blood pressure, heart rate and total carotid blood flow were continuously monitored on a polygraph (CRW, Erasmus University). During the experiment, body temperature was kept at ∼37 °C and the animal was continuously infused with physiological saline to compensate for fluid losses.

The ethical committee of the Erasmus University Rotterdam, dealing with the use of animals in scientific experiments, approved the protocols followed in this investigation.

Distribution of carotid blood flow

As described in detail previously (27–29), the distribution of carotid blood flow was determined with 15.5 ± 0.1 μm (sd) diameter microspheres labelled with ¹⁴¹Ce, ¹¹³Sn, ¹⁰³Ru, ⁹⁵Nb or ⁴⁶Sc (NEN Dupont, Boston, USA). For each measurement, about 200 000 microspheres, labelled with one of the radioisotopes, were mixed and injected into the right common carotid artery. At the end of the experiment, the animal was sacrificed by an overdose of sodium pentobarbital and the heart, lungs, kidneys and all ipsilateral cranial tissues were dissected out, weighed and put in plastic vials. The radioactivity in these vials was counted for 10 min in a γ-scintillation counter (Packard, Minaxi autogamma 5000), using suitable windows to discriminate the different isotopes (¹⁴¹Ce: 120–167 KeV, ¹¹³Sn: 355–435 KeV, ¹⁰³Ru: 450–548 KeV, ⁹⁵Nb: 706–829 KeV and ⁴⁶Sc: 830–965 KeV). All data were processed by a set of specially designed computer programs (27). The fraction of carotid blood flow distributed to the different tissues (CaBF _tis) was calculated by the following equation:

CaB F_{tis} = (I_{tis} / I_{tot}) \times CaBF,

where I_tis and I_tot denote the tissue and total (i.e. the sum of all tissue) radioactivity, respectively, of each radioisotope and CaBF represents the common carotid artery blood flow at the time of microsphere injection. Since little or no radioactivity was detected in the heart and kidneys, all microspheres trapped in the lungs reached this tissue from the venous side after escaping via carotid arteriovenous anastomoses. Therefore, the amount of radioactivity in the lungs was used as an index of the arteriovenous anastomotic fraction of the total carotid blood flow (5). Vascular conductance (ml/min mmHg⁻¹) was calculated by dividing blood flow (ml/min) by mean arterial blood pressure (mmHg).

Experimental protocol

After a stabilization period of at least 60 min, baseline values of heart rate, mean arterial blood pressure, total carotid blood flow and its distribution into arteriovenous anastomotic and capillary fractions, as well as arterial blood gases, were measured. Thereafter, the animals were divided into seven groups, receiving i.v. infusions (0.5 ml/min for 10 min) of either vehicle (distilled water; n = 8), 5-methylurapidil (300 or 1000 μgkg⁻¹; n = 6 each dose), L-765 314 (300 or 1000 μgkg⁻¹; n = 6 and 3, respectively) or BMY 7378 (300 or 1000 μgkg⁻¹; n = 6 each dose). After a waiting period of 15 min, all variables were reassessed. Subsequently, the animals received cumulative doses of phenylephrine (1, 3 and 10 μgkg⁻¹ min⁻¹) infused into the right common carotid artery (0.1 ml/min for 10 min). All variables were collated again 10 min after the start of each agonist infusion.

At least 90 min after the last microsphere injection, the animals received i.v. bolus injections of phenylephrine (3 and 10 μgkg⁻¹) and peak changes in mean arterial blood pressure were noted.

Data presentation and statistical analysis

All data have been expressed as mean±sem. In order to correct for potential baseline differences caused by the antagonists or vehicle, the percentage changes induced by phenylephrine from the values after administration of the different antagonists or vehicle were calculated in each group. The significance of the percentage changes induced by the different doses of phenylephrine within one group was evaluated with Duncan's new multiple range test, once an analysis of variance (randomized block design) had revealed that the samples represented different populations (30). Percentage changes caused by phenylephrine in the different treatment groups were compared with the percentage changes caused by the corresponding phenylephrine dose in the vehicle-treated group using Student's unpaired t-test. Statistical significance was accepted at P < 0.05 (two-tailed).

Drugs

Apart from the anaesthetics azaperone (Stresnil^®; Janssen Pharmaceuticals, Beerse, Belgium), midazolam hydrochloride (Dormicum^®; Hoffmann La Roche b.v. Mijdrecht, The Netherlands) and sodium pentobarbital (Apharmo, Arnhem, The Netherlands), the compounds used in this study were: l-phenylephrine hydrochloride, 5-methylurapidil, BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro [4,5]decane-7,9-dione dihydrochloride) dihydrochloride (all from Sigma-Aldrich Chemie b.v., Zwijndrecht, The Netherlands) and L-765314 (4-amino-2-[4-[1-(benzyloxycarbonyl)-2(S)-[[(1,1-dimethylethyl)amino] carbonyl]-piperazinyl]-6,7-dimethoxyquinazoline hydrochloride) (Merck & Co. Inc., West Point, PA 19486, USA). Finally, heparin sodium (Leo Pharmaceutical Products, Weesp, The Netherlands) was used to prevent clotting of blood in the catheters.

All drugs were dissolved in distilled water. A short period of heating was needed to dissolve 5-methylurapidil (acidified to pH = 6.8–7.0 with 0.1 m HCl) and L-765314. The doses of the drugs refer to their respective salts.

Results

Baseline values and effect of antagonists per se

Baseline values of haemodynamic variables in anaesthetized pigs (n = 41) were: heart rate (100 ± 2 beats/min), mean arterial blood pressure (93 ± 2 mmHg), total carotid blood flow (123 ± 6 ml/min) and conductance (132 ± 6 10⁻² ml/min mmHg⁻¹), arteriovenous anastomotic blood flow (95 ± 6 ml/min) and conductance (102 ± 6 10⁻² ml/min mmHg⁻¹) and capillary blood flow (28 ± 2 ml/min) and conductance (30 ± 2 10⁻² ml/min mmHg⁻¹). There were no major differences between the baseline values in the different groups of animals (data not shown).

No haemodynamic changes were observed with vehicle or BMY 7378 (data not shown). 5-Methylurapidil (1000 μgkg⁻¹) slightly decreased mean arterial blood pressure (−8 ± 4%) and increased heart rate (9 ± 1%) as well as capillary blood flow (35 ± 4%) and conductance (49 ± 9%). L-765 314 (1000 μgkg⁻¹) decreased mean arterial blood pressure (−9 ± 4%) and increased capillary conductance (27 ± 11%).

Systemic haemodynamic responses to intracarotid infusions of phenylephrine

As shown in Table 2, after treatment with vehicle intracarotid infusions of phenylephrine (1, 3 and 10 μgkg⁻¹.min⁻¹) caused a dose-dependent increase in heart rate by up to 28 ± 5%, without affecting mean arterial blood pressure. This phenylephrine-induced tachycardia was slightly less (maximal increase: 11 ± 3%) after 300 μgkg⁻¹ of 5-methylurapidil (probably due to higher initial value), but was not different after the highest dose of 5-methylurapidil (1000 μgkg⁻¹). In animals treated with either 1000 μgkg⁻¹ of L-765314 or BMY7378, a small decrease in mean arterial blood pressure (9 ± 1 or 8 ± 4%, respectively) was observed with phenylephrine; however, when compared with the corresponding blood pressure change in vehicle-treated animals, statistical significance was not reached.

TABLE 2

Changes in heart rate and mean arterial blood pressure induced by 10-min intracarotid infusions of phenylephrine in anaesthetized pigs treated i.v. with either vehicle, 5-methylurapidil, L-765 314 or BMY 7378

Variables and treatment group	Dose (μg/kg)	Values before phenylephrine ∗	Phenylephrine (μg/kg.min⁻¹)
Variables and treatment group	Dose (μg/kg)	Values before phenylephrine ∗	1	3	10
Heart rate (beats/min)
Vehicle	5 ml	95 ± 2	98 ± 2	105 ± 3^a	121 ± 5^a
5-Methylurapidil	300	112 ± 5	111 ± 6^b	112 ± 5^b	123 ± 4^ab
1000	101 ± 3	103 ± 4	106 ± 4	117 ± 3^a
L-765 314	300	98 ± 5	101 ± 5	107 ± 5^a	129 ± 7^a
1000	93 ± 2	93 ± 3	97 ± 4	112 ± 6^a
BMY 7378	300	99 ± 4	101 ± 3	104 ± 3	119 ± 5^a
1000	109 ± 6	112 ± 6	117 ± 6^a	127 ± 6^a
Mean arterial blood pressure (mmHg)
Vehicle	5 ml	97 ± 2	97 ± 3	96 ± 3	99 ± 5
5-Methylurapidil	300	80 ± 5	82 ± 5	79 ± 6	83 ± 6
1000	95 ± 5	89 ± 5^b	87 ± 5	89 ± 6
L-765 314	300	84 ± 6	83 ± 7	84 ± 8	87 ± 8
1000	84 ± 3	80 ± 4^a	78 ± 2^a	79 ± 4^a
BMY 7378	300	85 ± 3	85 ± 4	84 ± 3	90 ± 3
1000	91 ± 5	88 ± 5	86 ± 4	84 ± 4^a

∗

Values after treatment with respective antagonist or vehicle. a, P < 0.05 vs. baseline. b, P < 0.05 vs. response (% response from respective baseline) to the corresponding phenylephrine dose in animals treated with vehicle.

Carotid haemodynamic responses to intracarotid infusions of phenylephrine

Absolute values of total carotid, arteriovenous anastomotic and capillary vascular conductances in the different groups of animals before and after intracarotid infusions of phenylephrine are shown in Fig. 1. In animals treated with vehicle, phenylephrine produced a dose-dependent decrease in total carotid conductance by up to 75 ± 4%. Since phenylephrine did not change the vascular conductance in the capillary fraction, the decrease in total carotid conductance was exclusively caused by a decrease in the arteriovenous anastomotic fraction (maximal response: 92 ± 3%).

Figure 1

Effects of 10-min intracarotid infusions of phenylephrine on total carotid, arteriovenous anastomotic (AVA) and capillary vascular conductances in anaesthetized pigs treated i.v. with either vehicle (Veh; n = 8), 5-methylurapidil (5-MU; 300 or 1000 μgkg⁻¹; n = 6 each dose), L-765314 (L; 300 or 1000 μgkg⁻¹; n = 6 and 3, respectively) or BMY 7378 (BMY; 300 or 1000 μgkg⁻¹; n = 6 each dose). Baseline; Phenylephrine: 1 μg.kg⁻¹.min⁻¹, 3 μg.kg⁻¹.min⁻¹, ▪ 10 μg.kg⁻¹.min⁻¹. ∗P < 0.05 vs. baseline (B; values after treatment); †P < 0.05 vs. response (% response from respective baseline) of the corresponding phenylephrine dose in vehicle-treated animals.

As shown in Figs 1 and 2, the constrictor effect of phenylephrine on carotid arteriovenous anastomoses was not affected by 300 μgkg⁻¹ of either 5-methylurapidil or BMY 7378, but was significantly attenuated by 300 μgkg⁻¹ of L-765314 and 1000 μgkg⁻¹ of 5-methylurapidil and BMY 7378. Furthermore, after the highest dose of L-765 314, the responses to phenylephrine were clearly abolished and the values did not significantly differ from those before phenylephrine infusion.

Figure 2

Percentage changes in arteriovenous anastomotic (AVA) conductance induced by 10-min intracarotid infusions of phenylephrine in anaesthetized pigs treated i.v. with either vehicle (○n = 8), (a) 5-methylurapidil (5-MU; 300 □ or 1000 ▪ μg.kg⁻¹; n = 6 each dose); (b) L-765314 (L; 300 □ or 1000 ▪μg.kg⁻¹; n = 6 and 3, respectively); (c) BMY 7378 (BMY; 300 □ or 1000 ▪μg.kg⁻¹; n = 6 each dose; right graph). ∗P < 0.05 vs. baseline (B; values after treatments); †P < 0.05 vs. response (% response from respective baseline) of the corresponding phenylephrine dose in vehicle-treated animals. Note that the control curves are identical in each graph.

Since mean arterial blood pressure was little affected by the intracarotid infusions of phenylephrine, the responses of vascular conductances were qualitatively and quantitatively similar to those of blood flow (data not shown).

Changes in mean arterial blood pressure by i.v. bolus administration of phenylephrine

In vehicle-treated animals, bolus injections of phenylephrine (3 and 10 μgkg⁻¹, i.v) produced a dose-dependent increase in mean arterial blood pressure, yielding peak responses of 28 ± 3 and 51 ± 3%, respectively (Fig. 3). These phenylephrine-induced vasopressor responses were significantly attenuated by 1000 μgkg⁻¹ of 5-methylurapidil (peak responses: 17 ± 3 and 31 ± 4%, respectively), but were not affected by L-765314 (300 or 1000 μgkg⁻¹), BMY 7378 (300 or 1000 μgkg⁻¹) or 300 μgkg⁻¹ of 5-methylurapidil.

Figure 3

Peak changes in mean arterial blood pressure (MAP) induced by bolus injection of phenylephrine (□ 3 or ▪ 10 μg.kg⁻¹, i.v.) in animals treated i.v. with either vehicle (Veh; n = 8), 5-methylurapidil (5-MU; 300 or 1000 μg.kg⁻¹; n = 6 each dose), L-765 314 (L; 300 or 1000 μg.kg⁻¹; n = 6 and 3, respectively) or BMY 7378 (BMY; 300 or 1000 μg.kg⁻¹; n = 6 each dose). ∗P < 0.05 vs. percentage response of the corresponding phenylephrine dose in vehicle-treated animals.

Discussion

General

We have recently shown that both α₁- and α₂-adrenoceptors mediate constriction of arteriovenous anastomoses within the carotid vascular bed in anaesthetized pigs (18). This conclusion was based on the findings that (i) intracarotid administration of phenylephrine (α₁-adrenoceptor agonist) and BHT933 (α₂-adrenoceptor agonist) decreased total carotid blood flow exclusively confined to the arteriovenous anastomotic fraction, without affecting mean arterial blood pressure; and (ii) these effects of phenylephrine and BHT933 were selectively antagonized by the α₁- and α₂-adrenoceptor antagonists, prazosin and rauwolscine, respectively (18). This is also in agreement with recent findings demonstrated in the external carotid vascular bed of anaesthetized dogs (31).

Based on radioligand binding, molecular biology and isolated tissue experiments, it is known that α₁-adrenoceptors are a heterogeneous group of receptors, currently subdivided into α_1A, α_1B and α_1D subtypes (21, 32). As reviewed by Vargas and Gorman (10), the α_1A-adrenoceptor subtype, which is widely distributed throughout the body and is the major subtype regulating systemic vascular resistance and blood pressure; α_1D-adrenoceptors seem to play only a minor role in blood pressure regulation (33). Whereas there is limited information concerning the vascular effects mediated by α_1B-adrenoceptors, it has been shown that constriction of the isolated carotid artery of the dog and rabbit mainly resembles the cloned α_1B-adrenoceptor (10). However, no information is available on the subtype mediating vasoconstrictor effects within the carotid arterial bed in vivo. Therefore, the objective of the present study was to identify the α₁-adrenoceptor subtype(s) that mediate constriction in the carotid vasculature in anaesthetized pigs, with particular emphasis on the arteriovenous anastomotic fraction, which may be of relevance to migraine therapy (1, 5–7)

In recent years, several selective antagonists at α-adrenoceptor subtypes have been developed (Table 1). In the present study, we made use of 5-methylurapidil and BMY 7378, which have frequently been used to characterize α_1A- and α_1D-adrenoceptors, respectively (Table 1; 22, 25, 32, 34, 35–37). To block α_1B-adrenoceptors, we employed L-765 314, which shows a moderate to high selectivity for cloned α_1b-adrenoceptors over cloned α_1a- or α_1d-adrenoceptors (24). Many studies have used the clonidine derivative, chloroethylclonidine, for this purpose (32, 38). However, it is now evident that chloroethylclonidine alkylates several other receptors as well (32, 39, 40).

Systemic and carotid haemodynamic effects of different antagonists

Whereas the vehicle was devoid of any systemic and carotid haemodynamic effects, administration of the α_1A-adrenoceptor antagonist 5-methylurapidil produced a small hypotension and tachycardia (Table 2). Similar hypotensive effect was observed earlier with 5-methylurapidil (41) and may be related to either blockade of vascular smooth muscle α_1A-adrenoceptors, which play an important role in the maintenance of vascular tone (10) or to its agonist properties at central 5-HT_1A receptors (42). Admittedly, we do not have a clear-cut explanation for the slight hypotension and increase in capillary conductance produced by L-765 314, since it has been shown by Piascik et al. (43) that α_1B-adrenoceptors play only a minor role in the contraction of peripheral blood vessels in vitro.

Changes in heart rate and mean arterial blood pressure by phenylephrine

Intracarotid infusions of phenylephrine produced only minor systemic haemodynamic responses in vehicle-treated animals. The tachycardia (Table 2), which was also observed with i.v. phenylephrine (data not shown), most likely involves an interaction with β-adrenoceptors (18). Furthermore, i.v. administration of phenylephrine (3 and 10 μgkg⁻¹) induced a dose-dependent increase in blood pressure (Fig. 3), which was antagonized by 100 μgkg⁻¹ of prazosin, but not by 300 μgkg⁻¹ of rauwolscine (Willems et al., unpublished observations). In view of the antagonism of this response by 5-methylurapidil, but not by L-765 314 (α_1B-adrenoceptor antagonist) or BMY 7378 (α_1D-adrenoceptor antagonist) (Fig. 3), the pressor response to phenylephrine is likely to be mediated by α_1A-adrenoceptors. In keeping with the approximately 10-fold higher affinity at the cloned α_1a-adrenoceptor displayed by prazosin compared to 5-methylurapidil (Table 1), a 10-fold higher dose of 5-methylurapidil (1000 μgkg⁻¹) was needed to produce antagonism of the phenylephrine-induced vasopressor response. Thus, as previously discussed (10), these results support the role of α_1A-adrenoceptors in the increase in peripheral vascular resistance and concomitant hypertensive effect upon activation.

Carotid haemodynamic responses to intracarotid infusions of phenylephrine

As previously reported (18), phenylephrine caused a pronounced and dose-dependent decrease in total carotid conductance, which was exclusively caused by constriction of carotid arteriovenous anastomoses; nutrient vascular conductance was not modified (Fig. 1). These carotid vasoconstrictor responses were not affected by treatment with 300 μgkg⁻¹ of the α_1D-adrenoceptor antagonist BMY 7378. This dose of BMY 7378 should be sufficient to block α_1D-adrenoceptors in view of comparable affinities of prazosin and BMY 7378 at the cloned human α_1d-adrenoceptor (Table 1) and the fact that 100 μgkg⁻¹ of prazosin abolished this response (18). On this basis, the involvement of α_1D-adrenoceptors in the cranial vasoconstriction induced by phenylephrine in anaesthetized pigs seems highly unlikely. Nevertheless, as shown in Figs 1 and 2, the higher dose of BMY 7378 (1000 μgkg⁻¹) produced a slight, but significant, attenuation in the phenylephrine-induced total carotid and arteriovenous anastomotic vasoconstriction. Since BMY7378 displays a moderate affinity at α_1A- and α_1B-adrenoceptors (pK_i: 6.6 and 7.2, respectively; Table 1), a non-selective blockade of these receptors (which can mediate carotid vasoconstriction; see below) is a likely explanation. Indeed, in two experiments a combination of 5-methylurapidil (1000 μgkg⁻¹) and BMY 7378 (1000 μgkg⁻¹) did not cause any more attenuation of the phenylephrine-induced arteriovenous constriction than 5-methylurapidil (1000 μgkg⁻¹) alone (see below).

The α_1B-adrenoceptor antagonist L-765314 abolished the constriction of carotid arteriovenous anastomoses by phenylephrine, a finding that supports the role of α_1B-adrenoceptors in this effect. Interestingly, the α_1A-adrenoceptor antagonist, 5-methylurapidil was also able to antagonize the cranial vasoconstrictor effects of phenylephrine, but in contrast to L-765 314, the highest dose of phenylephrine still elicited a 50% decrease in arteriovenous anastomotic conductance (Figs 2 and 3). Thus, at similar doses, L-765314 acted as a more potent antagonist when compared with 5-methylurapidil. The above findings lead us to conclude that both α_1A- and α_1B-adrenoceptors mediate the phenylephrine-induced constriction of carotid arteriovenous anastomoses, whereas the α_1D-adrenoceptor plays a minor role, if any.

Admittedly, a critique of the above conclusion may be that, unlike in vitro studies, the exact concentration of α₁-adrenoceptor antagonists at the receptor site can be influenced by pharmacokinetic differences in such an in vivo investigation. However, current techniques do not allow us to study carotid arteriovenous anastomoses in vitro and, to some extent, we have tried to ensure effective antagonist concentration at the receptor site by using as high dose of antagonists as possible. Moreover, we would like to emphasis the fact that selective agonists at the different α₁-adrenoceptor subtypes are currently unavailable. Since porcine α₁-adrenoceptor subtypes have not yet been cloned, the selectivity of the subtype-selective antagonists (see Table 1) is based on their affinity for human cloned receptors.

Possible clinical implications

Lastly, we would like to consider possible clinical implications of the present results showing that α_1A- and α_1B-adrenoceptors mediate the constriction of carotid arteriovenous anastomoses in anaesthetized pigs. To date all acutely acting antimigraine agents, such as the triptans and ergot alkaloids, potently constrict carotid arteriovenous anastomoses (44, 45). Moreover, dilatation of these ‘shunt’ vessels may be involved in the pathophysiology of the headache phase of migraine (1, 5, 6). Since a vasoconstrictor effect in this experimental model seems to be highly predictive for antimigraine efficacy, a α_1A- (such as A61603, see 46) or α_1B-adrenoceptor agonist (which is yet to be developed) should be able to abort migraine headaches. Of the two α₁-adrenoceptor subtypes, the α_1B-adrenoceptor is an interesting target for future antimigraine drugs, especially when considering that this receptor, unlike the α_1A-adrenoceptor, does not seem to be much involved in the constriction of the peripheral blood vessels (10, 43). Indeed, our results suggest that the hypertensive effect produced by intravenous administration of phenylephrine is predominantly mediated via the α_1A-, but not α_1B-adrenoceptor (Fig. 3). An α_1B-adrenoceptor agonist may have a major advantage over the currently available acute antimigraine drugs, which all constrict human isolated coronary artery (47), where, importantly, the α_1B-adrenoceptor is not present (48).

In conclusion, the present study shows that both α_1A- and α_1B-adrenoceptors mediate constriction of porcine carotid arteriovenous anastomoses produced by phenylephrine. Since the α_1B-adrenoceptor subtype is not much involved in constriction of the systemic vasculature, a cranioselective vasoconstriction may be achieved using selective α_1B-adrenoceptor agonists, which may prove effective in migraine.

Footnotes

Acknowledgements

We would like to thank Dr M.A. Patane (Merck & Co. Inc., USA) for generously providing L-765 314.

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α 1 -Adrenoceptor Subtypes Mediating Vasoconstriction in the Carotid Circulation of Anaesthetized Pigs: Possible Avenues for Antimigraine Drug Development

Abstract

Keywords

Introduction

Materials and methods

General

Distribution of carotid blood flow

Experimental protocol

Data presentation and statistical analysis

Drugs

Results

Baseline values and effect of antagonists per se

Systemic haemodynamic responses to intracarotid infusions of phenylephrine

Carotid haemodynamic responses to intracarotid infusions of phenylephrine

Changes in mean arterial blood pressure by i.v. bolus administration of phenylephrine

Discussion

General

Systemic and carotid haemodynamic effects of different antagonists

Changes in heart rate and mean arterial blood pressure by phenylephrine

Carotid haemodynamic responses to intracarotid infusions of phenylephrine

Possible clinical implications

Footnotes

Acknowledgements

References