Introduction

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Donitriptan is a unique high-efficacy agonist at 5-HT_1B/1D receptors; it is currently being evaluated for efficacy in the acute relief of migraine headache in phase II clinical trials (Dukat, 2001; John et al., 1999, 2000). One of the key pharmacological actions of donitriptan is 5-HT_1B receptor-mediated cranioselective vasoconstriction (Tom et al., 2002; Van den Broek et al., 2002a), due mostly to closure of cephalic arteriovenous anastomoses (AVAs; Tom et al., 2002). Immunohistochemical studies in the human trigemino-cerebrovascular system have demonstrated that 5-HT_1B receptors are localized to blood vessels, but not 5-HT_1D receptors (Longmore et al., 1997). Closure of cephalic AVAs is a common feature of tryptamine-derived, selective 5-HT_1B/1D receptor agonists (triptans; De Vries et al., 1999a), which may be associated with increases in systemic arterial-jugular venous oxygen saturation difference (AVOSD; De Vries et al., 1996; Tom et al., 2002). Before the advent of the triptans, the nonselective 5-HT_1B receptor agonist ergotamine (Villalón et al., 1999) was also shown to elicit selective carotid vasoconstriction confined to cephalic AVAs (Johnston and Saxena, 1978). Moreover, increases in AVOSD due to decreases in jugular venous oxygen saturation and oxygen partial pressure (pO₂) without affecting systemic arterial oxygen saturation or pO₂ were also observed in this study (Johnston and Saxena, 1978). However, it is presently unknown whether the recently described triptan 5-HT_1B/1D receptor agonist-induced increases in AVOSD are due to changes in systemic arterial or jugular venous oxygen saturation, or both.

The aim of the present investigation was therefore to elucidate the mechanism of donitriptan-induced increases in AVOSD. The results indicate that donitriptan selectively decreases jugular venous pO₂ and hemoglobin oxygen saturation while increasing jugular venous pCO₂ concomitantly with carotid vasoconstriction. The results are discussed in light of established mechanisms of action of triptan 5-HT_1B/1D receptor agonists in relation to migraine relief.

	Materials and Methods

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Animal Preparation. Twenty-seven male Landrace pigs (18-24 kg; M. Gaec, Sorèze, France) were premedicated with intramuscular administration of azaperone (3 mg/kg) after an overnight fast. General anesthesia was then induced with sodium pentobarbitone (25 mg/kg, i.v.; Sanofi, Montpellier, France). The animals were intubated, ventilated by positive pressure (Alpha 100; Minerve, Esternay, France), and anesthesia was maintained with a continuous infusion via the right saphenous vein of sodium pentobarbitone (6-18 mg/kg/h). Respiratory rate and tidal volume were carefully adjusted for maintaining blood gases within physiological limits (ABL 510; Radiometer, Copenhagen, Denmark). Body temperature was maintained constant between 37° and 38.5°C by a servo-controlled heating blanket.

Experimental Protocol. Experiments were carried out in two separate groups of pigs: vehicle (a mixture of 40% polyethyleneglycol 300 in sterile saline, 0.9%, n = 9) and donitriptan (n = 7). After completion of surgical procedures, a stabilization period of at least 30 min was observed.

Drugs. Azaperone was purchased from Janssen Pharmaceuticals (Antwerp, Belgium). Donitriptan [4-[4-[2-[3-(2 amino-ethyl)-1H-indol-5-yloxy]-acetyl]-piperazin-1-yl]-benzonitrile hydrochloride] and GR 127935 [N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2[-methyl-4(5-methyl-1,2,4)-oxadiazol-3-yl]-(1,1 biphenyl)-4-carboxamide dihydrochloride] were synthesized by the Department of Analytical Chemistry and Division of Medicinal Chemistry IV at the Centre de Recherche Pierre Fabre (Perez and John, 1999). Donitriptan was dissolved in 40% polyethyleneglycol 300 in sterile saline (0.9%), whereas GR 127935 was dissolved in sterile saline (0.9%). Drugs were weighed as base, taking into account the salt-to-base ratio.

Data and Statistical Analysis. Dose-response curves were fitted using an operational sigmoid model (Origin; OriginLab Corp., Northampton, MA) from relative maximal effects induced by agonists. From the results of these analyses, the geometric mean dose of agonist producing 50% of the maximal response (ED₅₀) was calculated with 95% confidence intervals.

	Results

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Effects of Donitriptan on Blood Gas Parameters. The effects of donitriptan on blood gases are presented in Table 1. The initial oxygen partial pressure values in arterial blood (pAO₂) were comparable among both groups (P = N.S.). In vehicle-treated animals, pAO₂ slightly but significantly decreased throughout the experiment reaching 114.2 ± 6.3 mm Hg by the end of experiments from an initial value of 122.5 ± 5.7 mm Hg (P < 0.05; Table 1). In donitriptan-treated animals, a comparably slight decrease in pAO₂ occurred. The oxygen partial pressure in jugular venous blood (pVO₂) was also determined in both groups. In the vehicle group, pVO₂ remained unchanged throughout the experiments (P = N.S.; Table 1). Donitriptan moderately but significantly decreased pVO₂ only at 40 µg/kg compared with vehicle; the maximal decrease reached 31 ± 6% (P < 0.05). The initial values of pACO₂ measured in the donitriptan-treated group were similar to those of the vehicle group. In both groups, pACO₂ values did not significantly change between the beginning and the end of the experiments. Baseline pVCO₂ values did not significantly differ between vehicle and donitriptan groups. Vehicle was devoid of significant effects per se on pVCO₂ as indicated in Table 1. On the other hand, donitriptan from 2.5 µg/kg induced marked and significant increases in pVCO₂ (P < 0.05 compared with vehicle group; Table 1). This constitutes one of the major findings of the present study. It is interesting to note that no significant variations in pH were found in the arterial or venous blood analyses (Table 1).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effects of donitriptan (filled symbols, n = 7) on oxygen saturation in systemic arterial blood (AOS, circles) and oxygen saturation in jugular venous blood (VOS, squares) in the anesthetized pig. Data are mean ± S.E.M. values of percent changes from baseline. *, P < 0.05 versus vehicle-treated animals (open symbols, n = 9) by one-way ANOVA followed by Dunnett's test.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Maximal variations induced by donitriptan (solid bars, n = 7 and 5 with GR 127935) or vehicle (open columns, n = 9 and 6 with GR 127935) in arteriovenous difference in oxygen saturation (AVOSD, panel A) and in total carotid vascular resistance (TCVR, panel B) in the absence (left panel) and in presence of GR 127935 (0.63 mg/kg, right panel). Data are mean ± S.E.M. values of percent changes from baseline. *, P < 0.05 versus vehicle group by one-way ANOVA followed by Dunnett's test; a, P < 0.05 versus group without GR 127935 by one-way ANOVA followed by Dunnett's test.

Effects of Donitriptan on Hemodynamic Parameters. The effects of donitriptan on blood pressure, heart rate (HR), and blood flows are summarized in Table 2. Under baseline conditions, these cardiovascular parameters were not significantly different from those in the vehicle group, with the exception of TCBF, which was significantly higher in donitriptan than in controls (P < 0.05; Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Effects of donitriptan (n = 7) and its vehicle (n = 9) on hemodynamic parameters in the anesthetized pig Mean arterial pressure (MAP), HR, LVP, ABF, TCBF, and left anterior descending coronary artery blood flow (LADBF) were measured. Data are the changes () ± SEM in hemodynamic parameters following vehicle or donitriptan administration.

Effects of GR 127935. In an additional series of experiments, donitriptan was re-examined in animals pretreated by GR 127935 (0.63 mg/kg), a relatively selective 5-HT_1B/1D receptor antagonist (Skingle et al., 1996). Under these conditions, baseline pAO₂, pVO₂, pACO₂, pVCO₂, AOS, VOS, and AVOSD values were similar in vehicle- and donitriptan-treated animals (data not shown). GR 127935 alone did not significantly modify these parameters. The maximal changes in AVOSD induced by GR 127935 alone were 22 ± 11% (P = 0.27 versus vehicle without GR 127935). In the presence of GR 127935, in vehicle- or donitriptan-treated animals a comparable, slight decrease in pAO₂ occurred (maximal variation, 7.1 ± 4.8 mm Hg and 5.1 ± 2.4 mm Hg, P = 0.68, respectively). In the same manner, pVO₂ was moderately decreased but not significantly, and this tendency appeared in both groups. GR 127935 reduced the donitriptan-induced increase in pVCO₂ (P = N.S. versus vehicle, data not shown). AOS remained constant during the experiments in vehicle- and donitriptan-treated animals, maximal variations were 0.3 ± 0.3 and 0.5 ± 0.5%, respectively. The effects of GR 127935 on donitriptan-induced decreases in VOS were also studied. GR 127935 abolished the fall in VOS induced by donitriptan. The maximal changes in vehicle- and donitriptan-treated pigs were 19 ± 10 and 9 ± 3%, respectively (P = 0.38). Thus, in the presence of GR 127935, donitriptan-induced increases in AVOSD were abolished (maximal variation 29 ± 19 versus 22 ± 11% in vehicle group; Fig. 2A).

	Discussion

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The mechanism of donitriptan-induced increases in AVOSD was investigated in the anesthetized farm pig. Donitriptan dose dependently increased AVOSD and decreased jugular venous hemoglobin oxygen saturation from 0.63 µg/kg i.v. without affecting systemic arterial oxygen saturation. Donitriptan-induced increases in AVOSD are therefore mediated by decreased jugular venous oxygen saturation. These effects were accompanied by decreases in jugular venous pO₂ and increases in pCO₂, demonstrating augmented cephalic oxygen consumption and metabolism. Donitriptan also concomitantly and dose dependently reduced carotid blood flow from 0.16 µg/kg. The 5-HT_1B/1D receptor antagonist, GR 127935 (Skingle et al., 1996) significantly reduced both donitriptan-induced increases in AVOSD and carotid vascular resistance, indicating the likely involvement of 5-HT_1B receptors. The present results are in agreement with a previous study (John et al., 1999) and those of a recent study (Tom et al., 2002), which showed dose-dependent 5-HT_1B receptor-mediated decreases in carotid vascular conductance due to a highly selective reduction in cephalic arteriovenous anastomotic (AVA) blood flow. These latter effects were accompanied by an increase in AVOSD (Tom et al., 2002). Indeed, since constriction of cephalic AVAs is a model which has a predictive value for antimigraine activity (Tfelt-Hansen et al., 2000), donitriptan should be effective in the acute treatment of migraine.

Donitriptan-Induced Increases in AVOSD. Donitriptan dose dependently reduced jugular VOS and pVO₂ without significantly affecting systemic AOS or pAO₂. Consequently, the donitriptan-induced increases in AVOSD can be explained by the selective decreases in jugular VOS. The most plausible explanation for the selective decreases in jugular VOS and pVO₂ evoked by donitriptan is closure of cephalic AVAs, which underlies 5-HT_1B receptor-mediated cranial vasoconstriction in the present model (Tom et al., 2002). An unexpected finding in the present study, however, was that donitriptan also dose dependently and significantly increased jugular pVCO₂. Consequently, an additional effect of donitriptan in enhancing cerebral metabolism must also be considered. Augmented pVCO₂ concomitant with decreases in pVO₂ are hallmarks of increased tissue metabolism (Dejours, 1963). Systemic AOS or pO₂ were not modified by donitriptan, thereby allowing effects on hemoglobin affinity for oxygen or on pulmonary blood oxygenation to be ruled out. Metabolic acidosis can also be excluded because no variations in arterial or venous pH or pACO₂ were detected. Donitriptan significantly decreased jugular venous pO₂ and hemoglobin oxygen saturation, indicating enhanced cephalic oxygen extraction. However, one of the main observations of the present study is the marked and dose-dependent increase in jugular venous pCO₂ in the absence of changes in arterial pCO₂, which were consistently and robustly elicited by donitriptan. This strongly suggests that, in addition to promoting cephalic oxygen extraction from arterial blood, as indicated by significant decreases in venous oxygen saturation and pO₂, donitriptan also enhances tissue metabolism, as indicated by significant increases in jugular venous pCO₂. It is noteworthy that physiological arteriovenous differences in pCO₂ are small (around 5-7 mm Hg; compared with a 50-60 mm Hg difference for pO₂), indicating a relatively low sensitivity of this parameter to changes in tissue metabolism. This is due to larger (around 17-fold) tissue reserves of dissolved CO₂ compared with O₂ (Farhi and Rahn, 1960) because of the greater water solubility of the former. A small but statistically significant increase in venous pCO₂ therefore reflects marked increases in tissue CO₂ reserve and metabolism (Dejours, 1963). Consequently, it cannot be excluded that donitriptan stimulates oxygen consumption and cerebral metabolism via 5-HT_1B receptor activation, in addition to 5-HT_1B receptor-mediated vasoconstriction of cranial AVAs. It was previously reported (Johnston and Saxena, 1978) that ergotamine evoked similar changes in blood gases to those presently described for donitriptan, in increasing jugular venous pO₂ and oxygen saturation, and with a notable propensity to increasing, albeit nonstatistically significantly, pVCO₂ (Johnston and Saxena, 1978).

Enhancement of Tissue Metabolism by Donitriptan. The mechanism by which donitriptan enhances cerebral metabolism clearly deserves further study. On the basis of the present results and current knowledge in the field, we tentatively propose the following scheme of events: 5-HT_1B receptors have been demonstrated on endothelial cells of human brain arteries and microvessels (Riad et al., 1998; Nilsson et al., 1999; Van den Broek et al., 2002b), but their precise function is unknown. Interestingly, endothelial cells are regulators of oxygen consumption via the cytochrome c oxidase oxygen sensor (Clementi et al., 1999), and 5-HT_1B receptor activation by sumatriptan and other triptans enhances cellular metabolic rate (Pauwels, 1998). Thus, by activating endothelial 5-HT_1B receptors in the cerebral microcirculation, donitriptan could enhance oxygen consumption and tissue metabolism. This could provide an additional mechanism explaining, at least in part, the observed decreases in venous oxygen saturation and pO₂ and increases in pVCO_2. Measurement of AVOSD in humans during migraine attacks before and after triptan administration could therefore test the clinical validity of the present data and provide further insight into the mechanism of acute antimigraine activity of triptans. The feasibility of such an approach has been demonstrated by Heyck (1969) in studies in which he measured AVOSD during attacks of migraine using the external jugular to obtain venous blood.