Migraine Patients Show an Increased Density of Dopamine D3 and D4 Receptors on Lymphocytes

Abstract

Recent studies have revealed peculiar functional and genetic features of dopamine receptors in migraine. As peripheral blood lymphocytes (PBL) may represent a tool for peripheral detection of neuroreceptors, we compared the expression of dopamine D3 (DRD3) and D4 (DRD4) receptors on PBL in migraine patients and in healthy controls using radioligand binding assay techniques in the presence of antidopamine D2-like receptor antibodies. The dopamine D2-like receptor agonist [³H]7-OH-DPAT was used as a radioligand. An increased density of both DRD3 (P = 0.0006) and DRD4 (P = 0.002) on PBL was observed in migraineurs compared with controls. This up-regulation might reflect central and/or peripheral dopamine receptor hypersensitivity due to hypofunction of the dopaminergic system. These findings support the view that dopamine D2-like receptors are involved in the determination of the so-called migraine trait, which may help to elucidate several clinical features of the disease.

Introduction

Following the first clinical and pharmacological suggestions of altered dopaminergic neurotransmission in migraineurs, recent studies on dopamine receptors have further emphasized the dopamine and migraine link (1, 2). It is becoming increasingly clear that dopamine receptors in migraineurs have peculiar functional and genetic features. In fact, they have a lower activation threshold, as is demonstrated by the fact that very low doses (10 µg/kg) of apomorphine, which at most activate prejunctional dopamine receptors in healthy subjects, are able to activate both pre- and post-junctional receptors in migraine patients (3). Moreover, a recent genetic study has shown a positive association between allele 1 of the dopamine D2 receptor (DRD2) and the subgroup of migraineurs presenting both nausea and yawning immediately before or during the pain phase of migraine (4). In addition, there is a debate on the possibility that a specific DRD2 polymorphism, the NcoI C/C genotype, might increase the susceptibility to migraine with aura (5, 6).

Peripheral blood lymphocytes (PBL) express dopamine D3 (DRD3), D4 (DRD4) and D5 (DRD5) receptor subtypes, as well as several other neurotransmitter and neuropeptide receptors. The assay of PBL neurotransmitter and neuropeptide receptors seems to represent a promising tool for the peripheral detection of these receptors as their expression might reflect the status of homologous brain receptors (7–19).

We have previously demonstrated an increased density of DRD5 on PBL in migraineurs (20). In this study, we compared the expression of DRD3 and DRD4 on PBL in migraine patients and in healthy controls using a combined radioligand binding assay and immunochemical protocol recently developed for discriminating DRD3 and DRD4 in PBL (21).

Methods

Patients

We studied 25 patients suffering from migraine with (n = 5) and without (n = 20) aura according to the IHS criteria (22) (females = 18, males = 7; mean age = 31.3 years, range 21–47; illness duration = 18.6 years, range 3–37; attacks frequency=3.9/month, range 1–7), and 20 healthy control subjects (females = 15, males = 5; mean age=32.8 years, range 23–50). Migraineurs had either never been treated with any prophylactic drug or had discontinued treatment at least 2 months prior to enrolment. In two patients the interval between the last attack and the test was 3 days. In the remaining patients the mean interval was 9.9 days (range 5–19). Medications taken by the patients to treat their last attack were indometacin (n = 7), ibuprofen (n = 5), ASA (n = 5), paracetamol (n = 4), sumatriptan (n = 4). No patient used anti-emetics or ergotamine.

All subjects gave their informed consent and the study was approved by the local ethical committee.

PBL preparation and radioligand binding assay

The PBL separation was performed as detailed elsewhere (9).

A PBL suspension (250 µl) containing 2 × 10⁶ cells was incubated in triplicate for 60 min at 25°C with increasing concentrations (0.05–2 nM) of [³H]7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7OH-DPAT) alone or plus 10 µM dopamine to define non-specific binding. The incubation buffer used was 50 mm Tris HCl (pH 7.4) containing 1 mm EDTA, 50 µM quinoline, 0.1% bovine serum albumin and 0.005% ascorbic acid. These experiments were performed to determine the optimal radioligand concentration to use in subsequent standard assay and receptor affinity studies in migraineurs and controls. In another session of experiments, the above suspension of PBL was incubated for 1–2 h at 25°C with 0.05–2 µg/ml anti-DRD3 or anti-DRD4 receptor immunoglobulins IgGs according to the procedure detailed elsewhere (21). At the end of incubation PBL were centrifuged, pelletted and incubated with 0.3 nM [³H]7OH-DPAT alone or plus 10 µM dopamine to define non-specific binding. In a series of preliminary experiments, PBL suspension was incubated with IgGs preadsorbed with synthetic peptides used to raise antibodies. At the end of incubation with [³H]7OH-DPAT, PBL were isolated onto Whatman GF-B glass fibre filters with a manifold apparatus. Filters were then rapidly washed twice with ice-cold incubation buffer and transferred into scintillation spectrometry at an efficiency of 40%. The assays for migraineurs and controls were performed at the same time to avoid batch variation in the assay.

Data and statistical analysis

Data from binding and competition experiments were calculated with the Radlig programme (23). Dissociation constant (Kd) and maximum density of binding site (Bmax) values were derived from Scatchard plots of saturation isotherms. [³H]7-OH-DPAT binding in the presence of preadsorbed antibodies was considered as total binding. Binding values obtained in the presence of concentrations of antibodies against DRD3 and DRD4 causing maximal inhibition of radioligand binding to PBL (0.5–1 µg/ml) were considered to represent non-specific retention of radioligand to PBL preparation or to be the result of labelling sites other than DRD3 and DRD4. The difference between total binding and non-specific radioligand retention was considered as immunochemically defined specific binding.

The statistical significance of differences in the density of [³H]7-OH-DPAT binding between the groups studied was assessed by t-test for unpaired data. Pearson's correlation coefficient was used to check for any correlation of DRD3 and DRD4 Bmax values with age, disease duration and attack duration. Spearman's correlation coefficient was used to compare dopamine receptor Bmax values with attack frequency and severity of accompanying symptoms. Multiple regression analysis was used in migraineurs to study the effects of age, sex, illness duration, attack frequency and duration and accompanying symptoms on DRD3 and DRD4 Bmax values.

Chemicals

[³H]7-OH-DPAT (specific activity 98 Ci/mmol) was purchased from the Amersham Radiochemical Centre (Amersham, Buckinghamshire, United Kingdom). Rabbit antidopamine D3 (cat. no. 324402) and D4 (cat. no. 324405) IgGs and corresponding receptor blocking peptides were purchased from Calbiochem-Nova-Biochem (San Diego, California, USA). Other chemicals were purchased from RBI Biochemicals, Inc. (Natick, Massachusetts, USA) or Sigma Chemical Co. (St. Louis, Missouri, USA).

Results

[³H]7-OH-DPAT binding to PBL was saturable, concentration-dependent and of high affinity in migraine patients and healthy control subjects. Migraine patients showed a significant increase in Bmax values of [³H]7-OH-DPAT binding sites to PBL (P = 0.006) compared with healthy controls (39.0 ± 20.3 fmol/10⁶ cells and 15.1 ± 2.4 fmol/10⁶, respectively), whereas no difference was found in Kd values (0.3 ± 0.02 and 0.3 ± 0.03, respectively) (Fig. 1). Bmax values of [³H]7-OH-DPAT binding did not correlate with either the age and sex of patients and controls, with the type of migraine, or with the clinical features of the patients (illness duration, attack frequency and duration, quality or severity of accompanying symptoms).

Figure 1

Scatchard plots of specific [³H]7-OH-DPAT binding to peripheral blood lymphocytes from migraine patients (filled circles) and healthy controls (open circles). Values of [³H]7-OH-DPAT specifically bound to peripheral blood lymphocytes are expressed in fmol/10⁶ cells. Points are the mean of triplicate determinations. S. E. was less than 10%.

Using anti-DRD3 and DRD4 antibodies, the maximal inhibition of [³H]7-OH-DPAT binding to PBL was obtained with a concentration of 0.5 µg/ml of each antibody. In both migraine patients and healthy controls, the DRD3 was the most represented D2-like receptor subtype (Fig. 2). The use of the anti-DRD3 and DRD4 IgGs for the discrimination of the dopamine D2-like receptor subtypes labelled by [³H]7-OH-DPAT confirmed a statistically significant increase in Bmax values of both DRD3 (P = 0.0006) and DRD4 (P = 0.002) on PBL in migraine patients compared with healthy controls (24.8 ± 9.9 fmol/10⁶ cells vs. 9.5 ± 2.7 fmol/10⁶ cells and 19.8 ± 7.2 fmol/10⁶ cells vs. 7.0 ± 1.0 fmol/10⁶ cells, respectively) (Fig. 2).

Figure 2

[³H]7-OH-DPAT binding to peripheral blood lymphocytes in the absence (total binding, first column) or in the presence of 0.5 µg/ml concentration of antidopamine D3 (second column), D4 (third column) receptor IgGs, or of both IgGs (fourth column). Data are the mean ± S.E. HC = healthy controls; M = migraine patients. *P = 0.006; **P = 0.0006; ***P = 0.002.

Discussion

Human PBL express DRD3, DRD4 and DRD5 subtypes, whereas no evidence exists of the expression of DRD1 and DRD2 (7–12). DRD1 and DRD5 belong to the D1-like receptor superfamily, whereas DRD2, DRD3 and DRD4 belong to the D2-like receptor superfamily (24–26). Extending our previous study in which an up-regulation of DRD5 on PBL was reported (20), we describe an increased density of dopamine D2-like receptors, namely of DRD3 and DRD4 subtypes, on PBL in migraine patients.

The characterization of dopamine D2-like receptors on PBL by radioligand binding assay techniques using tritiated neuroleptics as radioligands has long been debated (27). The use of the preferential DRD3 agonist [³H]7-OH-DPAT as radioligand has provided consistent results (10), even though it labels not only DRD3 but also DRD4 and sigma receptors (28, 29). The cloning of dopamine receptor subtypes (25) and the subsequent development of antibodies against dopamine D1-like and D2-like receptor subtypes has led to a better characterization of different dopamine receptor subtypes. Therefore, in the present study we assayed dopamine D2-like receptor subtypes on PBL using a combined radioligand binding assay protocol with [³H]7-OH-DPAT as radioligand in association with anti-DRD3 and DRD4 IgGs (21). The fact that the binding of [³H]7-OH-DPAT to PBL was sensitive to anti-DRD3 and DRD4 IgGs is in agreement with the labelling of DRD3 and DRD4 (21), whose presence on PBL has also been demonstrated by molecular biology studies. DRD3 was the most represented subtype both in migraineurs and healthy controls. We documented a greater density of both DRD3 (P = 0.0006) and DRD4 (P = 0.002) on PBL in migraineurs than in healthy controls.

PBL synthesize dopamine and other cathecolamines. Dopamine, cathecolamines and their precursors suppress PBL proliferation and cytokine production by acting as auto and/or paracrine regulators of PBL activity through induction of apoptosis (30). However, the role of dopamine receptors on PBL remains to be determined. Nevertheless, it has been reported in different neurological diseases that the expression pattern of neuroreceptors on PBL seems to be related to that of corresponding brain receptors, as is suggested by the decreased muscarinic cholinergic binding on PBL in Alzheimer's disease (31) and in Parkinson's disease with dementia (32), by the increased expression of dopamine receptors in de novo Parkinsonian patients (33), as well as by the increased expression of β-adrenoreceptors in patients with pure autonomic failure (34).

The mechanism responsible for the increased expression of DRD3 and DRD4 on PBL in migraine is unclear. It could be hypothesized that this receptor up-regulation represents a peripheral adaptative response to central dopaminergic alterations. A growing body of data indicate an involvement of dopaminergic neurotransmission in migraine, although no unequivocal evidence has yet emerged demonstrating a precise role in the pathophysiology of the attack (1, 2). A central and peripheral hyperactivity of the dopaminergic system, probably associated with receptor hypersensitivity, has been demonstrated in migraine by pharmacological challenges with dopamine agonists. Consequently, the increased density of DRD3 and DRD4 on PBL might be seen as a peripheral expression of this dopamine receptor supersensitivity.

Our study adds new elements to the debate on the involvement of dopamine D2-like receptors in migraine. Considerable interest has recently been focused on the possibility that specific alleles of DRD2 are involved in the susceptibility, clinical expression and psychiatric comorbidity of migraine (4–6, 35). Interestingly, DRD2, DRD3 and DRD4 are located in the intermediate and medial subnuclei of the nucleus of the solitary tract and in the dorsal motor vagal nucleus, which are brainstem areas of marked interest in migraine, whereas DRD3 has been detected in the area postrema (36). Furthermore, D2-like pre-junctional receptors located on noradrenergic sympathetic endings (together with D1-like receptors located on vascular smooth muscles) may influence cerebral haemodynamic responses (37).

We conclude that the increased expression of DRD3 and DRD4 on PBL in migraine might reflect central and/or peripheral dopamine receptor hypersensitivity due to dopaminergic hypofunction. Even when considering the limitation that research findings do not inequivocably indicate as yet a precise role of dopamine in the pathophysiology of migraine attack, the present data support the view that dopamine D2-like receptors are at least involved in the determination of the so-called migraine trait, which may help to shed light on several clinical features of the disease.