Abstract
Glutamate may play an important role in the pathogenesis of migraine: glutamate release in the brain may be involved in the development of spreading depression and increased concentrations of this amino acid have been reported in plasma and platelets from migraine patients. Here we assessed platelet glutamate uptake and release in 25 patients affected by migraine with aura (MA) and 25 patients affected by migraine without aura (MoA), comparing the results with a group of 20 healthy matched controls. Both glutamate release from stimulated platelets and plasma concentrations of the amino acid were assessed by high-performance liquid chromatography, and were increased in both types of migraine, although more markedly in MA. Platelet glutamate uptake, assessed as 3H-glutamate intake, was increased in MA, while it was reduced in MoA with respect to the control group. These results support the view that MA might involve different pathophysiological mechanisms from MoA and, specifically, up-regulation of the glutamatergic metabolism. Understanding these dysfunctional pathways could lead to new, possibly more successful therapeutic approaches to the management of migraine.
Introduction
Glutamate is the most important excitatory neurotransmitter in the central nervous system (CNS), playing an important function also in the control of the cerebral energetic metabolism, and becoming excitotoxic when present at excessive extracellular concentrations. Excessive neuronal stimulation by glutamate, due to defective removal of extracellular glutamate from the synaptic cleft by neuronal and glial glutamate transporters, may trigger an enzymatic cascade of events leading to cell death, a mechanism called excitotoxicity (1).
Glutamate is known to determine neuronal damage following cerebral ischaemia (2) and also to play a major role in the pathophysiology of epilepsy (3). Considerable evidence indicates that glutamate may play a role in the pathogenesis of migraine also. Glutamate release in the brain, acting on N-methyl-D-aspartate (NMDA) receptors, may be involved in the initiation, propagation and duration of spreading depression, a phenomenon implicated in the pathophysiology of migraine attacks (4); the cerebral concentration of magnesium decreases during a migraine attack (5), causing enhancement of the sensitivity of NMDA receptors to glutamate. In susceptible individuals, excessive oral intake of glutamate may induce migraine-like features (6), revealing a possible contribution of peripheral glutamate to this pathology. Indeed, increased concentrations of glutamate have been reported in plasma in migraine patients with respect to controls (7, 8) and in migraine patients during attacks compared with the resting state (8); while platelet levels of glutamate have been shown to be raised in patients with migraine with aura (MA) (9). Higher concentrations of glutamate have also been found in the cerebrospinal fluid of migraine patients as opposed to controls (10).
Platelet activation and release of granule constituents are known to play an important role in the pathophysiology of migraine (11) and it is interesting that these cells can be considered as a useful peripheral model when studying glutamatergic homeostasis. In fact, platelets express glutamate receptors and transporters, and re-uptake glutamate from the blood with an energy-dependent mechanism similar to that described in the central nervous system (12).
Therefore, in order to determine whether or not peripheral glutamatergic dysfunction might be operative in the pathogenesis of migraine, we assessed glutamate plasma concentrations, its release from platelets following collagen-induced aggregation and platelet re-uptake of this amino acid in patients affected by either MA or migraine without aura (MoA) and compared the results with those obtained from healthy matched controls.
Materials and methods
Twenty-five subjects affected by MA and 25 affected by MoA were recruited during the headache-free period and compared with 20 age- and sex-matched controls without a history of migraine (CTRLS). The clinical diagnosis was made according to the 2004 International Headache Society Classification (13). All subjects had been free of any medication for at least 8 days when studied, in particular of those drugs affecting platelet function; they were not on any particular diet, and were not known to suffer from psychiatric diseases, gout, hypertension, diabetes, epilepsy, neurodegenerative diseases, nephropathy, cardiovascular diseases or coagulation disorders. The patients were sequentially recruited from those attending the Headache Centre of S. Gerardo Hospital, Monza, Italy, and from those attending the Headache Centre of the Neurological Institute C. Besta, Milan, Italy. Demographic data are shown in Table 1. Informed consent was obtained from all subjects.
Clinical and demographic characteristics of the enrolled subjects
MA, Migraine with aura; MoA, migraine without aura; CTRLS, controls.
Platelet preparation
Since food intake may affect plasma glutamate concentrations, blood samples (25 ml) were collected after overnight fasting from the antecubital vein. Platelet-rich plasma (PRP) was obtained after centrifugation at 210 g for 20 min. One aliquot of PRP was then removed and centrifuged at 3600 g for 15 min in order to obtain platelet-poor plasma (PPP) and a pellet of platelets, which was resuspended in 1 ml of 0.39 M sucrose containing 5% dimethylsulphoxide (Sigma-Aldrich, St Louis, MO, USA) and frozen at −80°C until the uptake assay.
Platelet glutamate uptake
Sodium-dependent glutamate uptake in platelets was assessed according to the original method of Mangano and Schwarcz (12) with minor modifications, as previously described (14). Platelet aliquots of 50 µl were added to 425 µl Tris-citrate buffer (pH 7.0) and samples were preincubated for 10 min at 37°C. To investigate the sodium-dependenthigh-affinity uptake, blank samples were preincubated into a similar Tris buffer that had sodium chloride substituted by equimolar choline chloride, and sodium citrate by equimolar potassium citrate. The uptake was started by the addition of 25 µl 3H-glutamate (specific activity = 44 Ci/mmol; NEN Life Science Products, Milan, Italy), 60 µM final concentration, and stopped after 30 min by the addition of 3 ml ice-cold Tris buffer containing 1 mM cold glutamate. Platelets were separated by centrifugation and washed pellets were dissolved in Formule-989 (Du Pont, Boston, MA, USA) and counted in a β-counter with 60% efficiency. All assays were performed in triplicate. Net high-affinity uptake was determined by subtracting no-sodium blanks from the uptake in the presence of sodium and expressed as pmol glutamate/mg proteins per 30 min. Sodium-independent, low-affinity uptake was also calculated as the difference between uptake at 37°C and at 4°C, in the absence of sodium. Protein concentrations were determined by a spectrophotometer using the Bradford reagent.
Glutamate high-performance liquid chromatography assay
An aliquot of 900 µl of PPP was immediately treated with 100 µl of 4 M HClO4 and then neutralized with 50 µl of 7 M K2CO3, in order to avoid enzymatic glutamate degradation (15). The assessment of glutamate levels was performed by reverse-phase high-performance liquid chromatography (HPLC). After precolumn derivatization with o-phthaldialdehyde, samples were run through a C18 reverse-phase column (Beckman Instruments, Fullerton, CA, USA; 250 × 4.6 mm) by a multistep gradient of two solvents (A, 0.1 mol/l sodium acetate buffer, pH 7.2; B, methanol/tetrahydrofuran, 97 : 3 v/v) at a flow rate of 1.5 ml/min. Amino acids were detected by fluorimetry, as previously reported (15).
Platelet glutamate release
Platelet glutamate release was investigated in vitro following collagen-induced platelet aggregation. A blood sample of 10 ml was collected in tubes containing 1 ml of 3.8% sodium citrate and centrifuged to obtain PRP as described above. The PRP was then divided into two aliquots of 500 µl each. The platelet count in the PRP was standardized between 150 000 and 200 000/ml by dilution with PPP obtained from the same patient. One of the two aliquots was incubated for 30 s at 37°C with 3.5 µl of collagen 0.4 µM and the rate of aggregation was evaluated by an Aggrego-Meter (Chrono-Log, Havertown, PA, USA), while the second aliquot was kept untreated at 37°C. The two aliquots of PRP were then centrifuged to obtain the PPP, which was then inactivated for glutamate HPLC assay, as described above. The release of glutamate was calculated as the difference between glutamate concentrations in the presence and absence of collagen stimulation.
Statistical analysis
Results
Intercritical and fasting plasma glutamate levels were significantly increased in patients affected by both types of migraine with respect to healthy matched controls, although this increase was more marked (P < 0.001) in MA patients (∼95% and ∼45% increase, respectively, for MA and MoA vs. controls; P < 0.001; Fig. 1a).
(a) Glutamate plasma concentrations were assessed in the intercritical period in 25 patients affected by migraine with aura (MA), 25 patients affected by migraine without aura (MoA) and 20 healthy matched controls (CTRLS).
Platelet glutamate release following collagen-induced aggregation was assessed in the same blood samples and was increased in MA patients when compared with both controls (∼75%; P < 0.001) and MoA patients (∼30%; P < 0.01; see Fig. 1b). Glutamate release was also increased in MoA with respect to matched controls (∼35%; P < 0.01; Fig. 1b).
Finally, intercritical platelet glutamate uptake was significantly higher (∼45%) in MA patients with respect to controls (P < 0.001; Fig. 1c). In contrast, platelet glutamate uptake was reduced in MoA when compared with both controls (∼45% less; P < 0.001) and, especially, MA patients (about 2.5-fold less; P < 0.001).
Discussion
Much evidence now supports the hypothesis that migraine is associated with an impairment of the glutamatergic metabolism in the brain: glutamate release from the brain might be involved in the genesis and propagation of migraine spreading depression acting on CNS NMDA receptors (4). Cortical spreading depression was blocked by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid in human neocortical slices excised for treatment of intractable epilepsy (16). Moreover, NMDA-mediated transmission is likely to be involved in nociceptive transmission within the trigeminovascular complex (17), the neuronal system responsible for the transmission of pain in migraine. Central sensitization of the trigeminal system may also be involved in the pathogenesis of migraine, and a recent study has shown that cerebrospinal fluid glutamate levels are elevated in patients affected by chronic migraine, especially in those with concomitant fibromyalgia (18).
A role for platelet- or blood-derived glutamate in migraine has been suggested by several studies which reported increased plasma concentrations of glutamate in migraine patients compared with controls (7, 8), particularly in patients affected by MA (8); on the other hand, some studies have reported increased plasma concentrations of glutamate only in MoA (19). These inconsistencies might be ascribed to methodology, since food intake and the lack of the correct procedures for inactivation during sample processing might cause major differences in subsequent assessment of the concentrations of the amino acid (15). Moreover, plasma glutamate is likely to be linked to platelet storage function. In fact, platelets possess high-affinity glutamate transporters (20) which re-uptake glutamate from the blood (12) and express vesicular glutamate transporters also, which pack glutamate into specific secretory vesicles (21). After aggregating stimuli, platelets release glutamate, although the significance of this process is not well understood. Furthermore, platelets express functional glutamate NMDA receptors proposed to modulate platelet aggregation (22).
In MA we found elevated glutamate plasma concentrations, platelet glutamate release and platelet glutamate uptake. This suggests that up-regulation of the glutamatergic metabolism is operative in this condition, possibly due to a putative genetic trait. An interesting possibility is that the increased platelet glutamate re-uptake may be a compensatory mechanism linked to the increased plasma concentration of this amino acid. We recently reported that glutamate can stimulate its own transport in human platelets (23), analogously to what has been previously described for the CNS. Interestingly, glutamate concentrations have been consistently reported as increased in platelets obtained from patients affected by MA, while MoA platelets displayed a glutamate platelet content similar to that of controls (9, 19). Similar mechanisms may be operative in the CNS also. In a recent study, platelet glutamate uptake in patients affected by temporal lobe epilepsy and hippocampal sclerosis has been reported to be increased (24), and the authors proposed that up-regulation of the uptake might be a compensatory phenomenon for the increased glutamate release caused by seizure activity, in order to prevent glutamate excitotoxicity. If we speculate that a similar alteration is present in migraine, we could hypothesize that an abnormal release of glutamate in the intersynaptic space, leading to an increased excitability of the cerebral cortex with development of the spreading depression, may induce an increase of plasma concentrations of the amino acid, and consequently of platelet re-uptake. Strong support for this hypothesis is provided by the fact that lamotrigine, which inhibits glutamate release, reduces the frequency of MA attacks (25). This evidence, coupled with our data, may allow one to hypothesize that glutamate released from platelets plays an important role in the pathophysiology of MA and, based on this assumption, that glutamate release from activated platelets is also blocked by lamotrigine.
MA is also a risk factor, albeit small, for the development of strokes (26). Both hypoxic and ischaemic states produce, in fact, increased release of glutamate in the extracellular space (27) and we recently reported that platelet glutamate release is increased in acute stroke patients (28).
In MoA we also found elevated glutamate plasma concentrations and glutamate platelet release, compared with controls, albeit lower than in MA; on the other hand, platelet glutamate uptake was reduced with respect to the control group. The latter phenomenon could be linked to an impairment of the energetic metabolism in platelets, with alteration of energy-dependent glutamate uptake. However, previous studies have reported several abnormalities of platelet function in both types of migraine (29). Therefore, it is theoretically possible that, against this background of decreased energy metabolism, the smaller increase in plasma glutamate observed in MoA, compared with MA patients, is not able to stimulate the compensatory increase in platelet re-uptake. Moreover, Mallolas and colleagues have recently reported a highly prevalent polymorphism in the promoter of the glutamate transporter EAAT2 gene that abolishes a putative regulatory site for activator protein-2 (AP-2) and creates a new consensus binding site for the repressor transcription factor GC-binding factor 2 (GCF2) (30). Interestingly, since the glutamate transporter EAAT2 is expressed by human platelets (20), a putative genetic predisposition in these patients may help to explain the observed functional down-regulation.
In conclusion, our data further support the idea that MA and MoA have different pathogenic mechanisms and that glutamate stored and released from platelets might play a role in the genesis of migraine symptoms, at least in the case of MA. Studies of larger patient populations and focused on various mechanisms affecting glutamate uptake and the homeostasis of this amino acid are needed to confirm these hypotheses, which may usher in the development of novel therapeutic strategies.