Fibromyalgia syndrome (FMs) is a chronic pain syndrome characterized by widespread pain and stiffness, multiple tender points, and fatigue [¹]. This pain syndrome has an incidence of 2% in the general population and occurs with higher frequency among women in middle age [²]. FM is often associated with increased prevalence of depressive symptoms, major depression and anxiety [³]. The cause and pathophysiology of FMs is unclear; pathophysiological hypotheses include impairment in the functioning of the hypothalamic-pituitary axis and alterations in specific neurotransmitters such as substance P, N-methyl-D-aspartate, noradrenaline (norepinephrine) and serotonin (5-HT). However, interest has been growing in a possible involvement of 5-HT in FM. Indeed, strong evidence has accumulated to support the hypothesis that a deficiency in serotonergic neuronal functioning might be related to the pathophysiology of FM [^5-8]. Patients with FM have been found to have decreased concentrations of 5-HT and tryptophan (5-HT precursor) in serum and cerebrospinal fluid. 5-HT, in particular, is theorized to have a function in stage 4 sleep and in the pain threshold [⁹]. This neurotransmitter is implicated in psychiatric disorders such as depression, anxiety, and obsessive compulsive disorder. Stressful experiences lead to depression in some people who are already genetically predisposed, and increase the probability of FM exordium. The 5-HT gene could moderate the serotonergic response to stress [⁴].

As a mediator, 5-HT exerts its actions by means of interaction with distinct receptors, which are differentiated on the basis of structures, molecular mechanisms and pharmacological profiles [⁹]. On this basis, drugs acting on 5-HT receptors, in particular on 5-HT₂ and 5-HT₃, are being used or investigated for the clinical management of FM [¹⁰,¹¹]. Among drugs targeting 5-HT receptors, ketanserin is a selective 5-HT₂ antagonist that can reduce the hyperalgesia, spontaneous pain, sleep disorders and other symptoms of FM [¹²], and granisetron and tropisetron are selective 5-HT₃ receptor antagonists that show clinical efficacy in FM [¹¹].

In an analogous manner to other transmitters, the endogenous activity of 5-HT is controlled by a specific 5-HT transporter (SERT), which mediates the intracellular reuptake of 5-HT and can be specifically blocked by selective 5-HT reuptake inhibitors (SSRIs) such as paroxetine and fluoxetine. SERT is widely expressed in intestinal epithelial cells, in central or peripheral serotonergic neurons and in platelets; it shares common molecular and physiological features in these locations [^13-15]. Clinical studies have demonstrated the efficacy of SSRI in FM [¹⁶], but the data are not unequivocal. Nociception refers to the physiological process of transmitting a painful stimulus from the periphery through afferent neurons to the cerebral cortex. It has been postulated that serotonergic neurotransmission has a significant function in nociception [¹⁷,¹⁸]; alterations in 5-HT metabolism and transmission might therefore be important in the pathogenesis of FM. These findings support the proposal that aberrant pain perception in FM also results from an instability of the 5-HT system in FM. There is also evidence that changes in the expression of SERT are due to a polymorphism in the transcription region in patients with FM [¹⁹].

In the present study, both the expression and functionality of SERT were determined in platelets collected from patients with FM, with the following aims: first, to perform a comparison with the pharmacological profile of platelet SERT in healthy volunteers, and second, to examine putative correlations of SERT characteristics with the severity of symptoms such as tiredness, Fibromyalgia Impact Questionnaire (FIQ) score or tender point index (TPi).

[³H]Paroxetine binding sites on the SERT of platelet membranes were used as a first marker of serotonergic function. Table 1 shows the mean B_max values for the density, and K_d values for the affinity, of [³H]paroxetine binding on platelets in the patients and in the healthy controls. A statistically significant difference was found between the two groups for B_max (1,048 ? 30 versus 1,260 ? 34 fmol/mg for control; mean ? SEM) but not for K_d (0.086 ? 0.021 nM versus 0.077 ? 0.012 nM for control; mean ? S.E.M.).

The evaluation of symptom severity in patients with FM (Table 2) yielded tiredness scores from 2 to 10 with a mean of 7.4 ? 2.2 (SD), TPi values from 1 to 3 with a mean of 2.2 ? 0.5 (SD), and FIQ scores from 8 to 94 with a mean of 57.1 ? 21.3 (SD). The severity of symptoms was shown to be related to the maximal binding capacity of platelet SERT, in that the statistical analysis showed a negative correlation between symptom scores and the respective B_max values (p < 0.0001 for tiredness; p < 0.0001 for TPi and p < 0.0001 for FIQ). By contrast, no significant correlation was found when comparing symptom severity with K_d values.

Age did not seem to influence the binding parameters of platelet SERT in either patients with FM or healthy volunteers. [³H]Serotonin uptake on platelets was used as marker of the functionality of SERT. V_max and K_m values of [³H]serotonin uptake in patients with FM accounted for 90 ? 4 pmol/10⁹ platelets per minute and 96 ? 15 nM, respectively (means ? SEM; Table 3). A comparison of these parameters with those in healthy volunteers indicated significant differences for V_max values but not for K_m values: whereas the mean V_max in the patients with FM was significantly lower (90 ? 4 versus 114 ? 6 pmol/10⁹ platelets per minute for controls; p < 0.001), the mean K_m in the patients with FM was not significantly different (96 ? 15 versus 114 ? 15 nM for controls; Table 2).

The severity of the symptoms was related to V_max and K_m in that the statistical analysis showed a negative correlation between the symptom scores and the respective V_max values (p < 0.0001 for tiredness; p < 0.05 for TPi and p < 0.001 for FIQ). We also found a positive correlation between K_m values and tiredness (p < 0.001), TPi (p < 0.001) and FIQ (p < 0.001).

The covariate age was shown not to have an effect on the variability of the V_max or K_m values in either patients with FM or healthy volunteers.

The present study is the first examination of the relationships between SERT expression and kinetic parameters of 5-HT uptake. We have found statistically significant differences in B_max and V_max values between patients with FM and controls. Moreover, we found a negative correlation between symptom scores and the respective B_max and V_max values (B_max and V_max versus tiredness, TPi and FIQ score). The platelet is considered to be a peripheral model of neuronal activity with respect to 5-HT function. In fact, previous studies have demonstrated that the same SERT is expressed in the central nervous system and platelets [¹⁴]. Moreover, the identity between the two structures, as confirmed by sequence homologies through cloning studies [¹⁵], has provoked a surge of different studies in neuropsychiatric disorders, given the possibility of exploring peripherally a mechanism of the central nervous system [²⁶]. Recently it has been proposed that altered serotonergic neuronal function might be related to the pathophysiology of FM [⁵,⁸,²⁷]. These findings prompted us to investigate the characteristics of SERT in the platelets of patients with FM. B_max and K_d values of [³H]Paroxetine binding were assumed to represent SERT density and ligand binding affinity, respectively, whereas V_max and K_m values of [³H]Serotonin uptake were taken as estimates of SERT rate and affinity, respectively. A very interesting observation was that both binding and uptake parameters differed significantly from those of healthy volunteers.

The patients with FM have fewer SERTs expressed on the cellular membrane than healthy subjects (a decreased B_max, perhaps because the SERTs are less transcribed). Besides having fewer SERTs, patients with FM have a deficit in functionality (demonstrated by a decrease in transport rate).

Such combined changes in B_max and V_max values allow the inference that the efficiency of 5-HT uptake by platelet SERT is altered. Our previous studies demonstrated an alteration of SERT density and of the uptake rate of SERT in psychiatric patients [²⁸,²⁹]. Consistent with this suggestion was the correlation analysis in the present study: the lower the density and rate of SERT on platelet membranes, the higher the severity of FM symptoms. Moreover, the K_m values were also positively correlated with tiredness, TPi and FIQ.

A reduced density and rate of SERT are consistent with previous observations indicating that levels of 5-HT are altered in patients with FM [³⁰,³¹]. The biophysiological mechanism of FM has been proposed to be similar to that in depression, and it has been suggested that this is likely to result from a neuroendocrine/neurotransmitter dysregulation [³²]. However, we suppose that the alterations in B_max and V_max values are not related to the pathophysiology of FM but are a consequence of FM. Our hypothesis is that a decrease in B_max and V_max of SERT is due to a pain stimulus [³³,³⁴].

It has been shown that the decreased pain perception threshold during depression is likely to result from a dysfunction in several neurotransmitter systems, especially the serotoninergic one, which is also involved in the pathophysiology of depression [³⁵]. In addition, an excessive stimulation of peripheral 5-HT receptors would account for pain and might explain why the clinical use of 5-HT₃ receptor antagonists such as tropisetron or granisetron can promote the relief of disturbance associated with FM [¹¹,³⁶].

SERT has been investigated previously in patients with FM, with discordant results. Russell and colleagues [³⁷] found a higher B_max in patients with FM than in healthy controls, whereas other authors found normal B_max values [²⁸,³⁵] using [³H]Paroxetine or [³H]Imipramine [³⁸]. In our experiments we used [³H]Paroxetine, which binds with high affinity to a specific population of binding sites located on human platelets and neuronal membranes, associated with 5-HT uptake mechanisms [³⁹]. The present results indicate a decrease in the density and rate of platelet SERT in patients with FM, and allow us to propose a specific role for SERT in the pathogenesis of FM. In fact, we avoided the inclusion of patients with FM who had psychiatric components because it is known that in psychiatric disorders such as depression, the expression of SERT is altered [^40-42] and it is very difficult to identify the role of the two components in patients with FM who have comorbid psychiatric disorders. Thus, the changes in B_max and V_max demonstrated in our study may be due to FM only.

There is also a possible contribution from 5-HT to the aetiology of FM because of the efficacy of SSRIs in the management of chronic pain in idiopathic pain disorders [⁴³]. Thus, in view of the decreased B_max and V_max values found in our subset of mentally healthy patients, who were SSRI free, we propose that there is a compensatory mechanism in the central nervous system to relieve the pain. This may clarify the improvement in the therapeutic effectiveness of SSRI in the patients with FM.

This is to our knowledge the first observation that, apart from a decrease in expression, there is also an alteration in the rate of SERT which seems to depend not only on the SERT number. In fact, B_max is not correlated with V_max (data not shown).

In the patients with FM, the decrease in 5-HT levels, which had already been observed, together with the impaired SERT functionality, might contribute to the pathogenesis of the disease, both in quantity and rate. In fact, these two factors are important because they are correlated with the level of disease severity.

Thus, the results of the present study are in agreement with the hypothesis that a deficit in the 5-HT transporter site could be of pathogenetic significance in FM syndrome.

5-HT = serotonin; B_max = maximal membrane binding capacity; FIQ = Fibromyalgia Impact Questionnaire; FM = fibromyalgia; K_d = dissociation constant; K_m = Michaelis constant; SERT = specific serotonin transporter; SSRI = selective 5-HT reuptake inhibitor; TPi = tender point index; V_max = maximal uptake rate of SERT.

The authors declare that they have no competing interests.

L Bazzichi was responsible for the design of the study and patient recruitment. GG was responsible for the design of the study and was the supervisor of the laboratory analysis. LB drafted the manuscript and was the coordinator of the laboratory activity. GM performed the statistical analysis. LF and PI conducted the binding assays. FDF and TG were responsible for the uptake assays. CG drafted the manuscript and performed the statistical analysis. AR was responsible for the preparation of the samples and for their storage. AL and SB were general supervisors of the research group. All authors read and approved the final manuscript.