Acetylcholine mediated vasodilatation in the microcirculation of patients with chronic fatigue syndrome — a short review
Authors: Spence VA, Khan F, Kennedy G, Abbot NC & Belch JJF.
Institution: Vascular Diseases Research Unit, University Department of Medicine, Ninewells Hospital & Medical School, Dundee DD1 9SY, UK
Support: Studies reported here were funded by the charity ME Research Group for Education and support (MERGE — charity number 1080201), Perth, UK. Laser Doppler equipment was purchased by a grant from the Disability Aid Foundation. Further support was also received from the Sir John Fisher Foundation (Educational Grant).
The etiology of chronic fatigue syndrome (CFS) remains controversial and a number of hypotheses have been put forward to explain it. Research into the condition is hindered by the considerable heterogeneity seen across patients but several reports have highlighted disturbances to cholinergic mechanisms in terms of central nervous system activity, neuromuscular function and autoantibodies to muscarinic cholinergic receptors. This paper examines an altogether separate function for acetylcholine and that is its role as an important and generalized vasodilator.
Most diseases are accompanied by a blunted response to acetylcholine but the opposite is true for CFS. Such sensitivity is normally associated with physical training so the finding in CFS is anomalous and may well be relevant to vascular symptoms that characterize many patients. There are several mechanisms that might lead to ACh endothelial sensitivity in CFS patients and various experiments have been designed to unravel the enigma. These are reported here.
Publication: Prostaglandins, Leukotrienes and Essential Fatty Acids 2004; 70: 403–7
Comment by MERGE:
CFS is currently defined exclusively by a number of non-specific symptoms that are common to many conditions, but there has been speculation that many of the neurological symptoms might be cholinergically mediated (1). This conjecture is supported by findings of increased levels of free choline in the central nervous system of CFS patients (2–4). As well as these neurological findings, however, there has been a recent report of autoantibodies specifically to muscarinic receptors in many CFS patients, suggesting that there might well be subgroups within the CFS construct that are associated with autoimmune abnormalities of cholinergic, muscarinic receptors (5).
Apart from its neurotransmitter functions, acetylcholine is, of course, a well-established and prominent vasodilator whose action is dependent upon an intact layer of functioning endothelial cells that line the lumen of all blood vessels. This mini-review brings together a number of novel studies, examining blood vessel endothelial biology in CFS/ME patients, which have now been published by the research group at the University of Dundee, UK. This group has found clear abnormalities associated with a specific endothelium-dependent vasodilator — acetylcholine — and the review sets out to explain the biology and significance of the acetylcholine pathway as it affects endothelial cells, and what experiments are needed to unravel the mystery of the sensitivity seen in CFS/ME patients. The review is divided into 5 sections and a conclusion, and an overview of each is given in brief below.
Acetylcholine but not nitric oxide sensitivity in CFS patients
A common test of endothelial integrity is the response of blood vessels to both endothelium-dependent vasodilators like acetylcholine and endothelium-independent vasodilators like nitric oxide (NO) via an NO donor like sodium nitroprusside.
In most medical conditions associated with cardiovascular disease there is a blunted response to acetylcholine. However, the research group have reported increased responses to a cumulative dose regime of acetylcholine delivered by iontophoresis into the cutaneous microcirculation of 22 CFS patients when compared with 22 age and gender-matched control subjects (6). In the same patients the response to iontophoretic application of sodium nitroprusside, a donor of NO, was normal compared with controls (6).
There are many possible explanations for endothelial sensitivity to acetylcholine, ranging from sensitivity of the G-protein muscarinic (m3) receptor on the surface of the endothelial cell, low levels of the enzyme acetylcholinesterase (AChE) as expressed on endothelial cells and up-regulation of one of many of the post-receptor signaling mechanisms. Since the response to NO was normal, the group has assumed that smooth muscle cell biology was not implicated in the sensitivity to acetylcholine.
Testing the sensitivity of the endothelial acetylcholine muscarinic and other receptors
In a further set of experiments, single doses of 1% acetylcholine, 0.06% bradykinin (BK), 0.15% substance P (SP) (all endothelium-dependent vasodilators) and 1% sodium nitroprusside (SNP) (endothelium-independent) were delivered by iontophoresis for 80 s using a 0.1 mA current into the forearm skin of 30 well-defined CFS/ME patients and 30 well-matched control subjects. The experiments were carried out under controlled experimental conditions as previously reported for each of these vasodilators (7). The results showed that resting skin perfusion was normal and the peak responses to the single doses of acetylcholine, BK and NO were also normal.
There was a significantly increased response to SP in CFS patients and this was often accompanied by a spreading flare and localized oedema: a finding not observed in control subjects. It is possible that the increased response to SP is a consequence of a heightened sensitivity to SP in terms of its histamine releasing properties (8).
Indeed, sensitivity to histamine has been implicated in CFS pathogenesis, and the group proposes, therefore, that there is no widespread upregulation of endothelial G protein receptor function in CFS patients.
Prolongation of the acetylcholine-mediated blood flow response and the relation to blood cholinergic enzymes
A further study demonstrated that acetylcholine sensitivity in CFS patients might be explained by prolonged action of the vascular response to acetylcholine (9). The group tested this by recording the dynamics of the acetylcholine-stimulated blood flow response for up to 30 min to allow the rate of decay of the response to be calculated.
Two points were determined, t75 and t50, corresponding to the times taken for the blood flow response to return to 75% and 50%, respectively, of the peak response to acetylcholine minus the baseline flow. Also, in the same study, the group determined levels of red blood cell acetylcholinesterase and plasma butrylcholinesterase to check any relationship between blood cholinesterase measurements and the endothelial responses to acetylcholine. The data demonstrated that the dynamics of the acetylcholine-stimulated blood flow response is significantly different in CFS patients compared with control subjects, in that the action of acetylcholine is prolonged in the CFS patients.
The group has postulated that this prolonged acetylcholine response might be related to inhibition of endothelial expression of the enzyme acetylcholinesterase (10), possibly via a viral mechanism, since herpes virus is known to inhibit acetylcholinesterase within cholinergically sensitive cells and lymphocytic choriomeningitis virus can inhibit acetylcholinesterase within cholinergically-sensitive cells for years after infection.
Acetylcholine sensitivity is specific to a sub-group of patients within the CFS construct
The group has also recently completed a study looking at acetylcholine and methacholine responses in three groups of patients, all of whom fulfill criteria for CFS (11). They had hypothesized that farm-workers who developed a CFS-like syndrome when exposed to organophosphate cholinesterase inhibitors present in sheep dip might also be acetylcholine-sensitive.
Also, they had speculated that those with Gulf War syndrome who had taken the cholinesterase inhibitor pyridostigmine bromide as a nerve protection agent and who had been exposed to organophosphate de-lousing powder might be acetylcholine-sensitive. To test this, the group studied acetylcholine responses in 46 CFS patients described as patients with CFS/ME, 24 farmer workers (CFS/OP) and 25 Gulf War soldiers (CFS/GWS). All of these patients fulfilled the CDC criteria for CFS (12). The results demonstrated that only the CFS/ME patients were sensitive to acetylcholine (11).
The second aim was to test further the hypothesis that vascular hypersensitivity in CFS was due to a reduction in endothelial cholinesterase activity, and this hypothesis was tested by assessing the blood flow responses to methacholine, a vasodilator that is almost identical to acetylcholine, but which is much less influenced by the action of cholinesterase.
The prediction was that if these acetylcholine results were a consequence of reduced cholinesterase activity, there would be no difference between the vascular responses to acetylcholine and methacholine. In control subjects, methacholine responses were indeed significantly greater than those for acetylcholine and this was also true for those with CFS/OP and CFS/GWS conditions. In the CFS/ME group there were no significant differences between acetylcholine and methacholine responses. These results point to a problem with acetylcholinesterase under-expression on the vascular endothelium of CFS patients.
The fatty acid hypothesis and the acetylcholine vasodilator pathway
While the current data might point to under-expression of acetylcholinesterase on the endothelium of CFS patients, there are, in fact, several possible pathways for the blood vessel to dilate following stimulation of the acetylcholine muscarinic receptor through to the vascular smooth muscle.
Under normal circumstances with an intact endothelium it is thought that the calcium-activated NO pathway is responsible for the major component of acetylcholine-mediated vasodilatation. Phospholipase A2 activated prostacyclin (PGI2) also contributes to vasodilatation, and the contribution of this pathway to total relaxation can be determined by blocking with cyclo-oxygenase inhibitors such as aspirin.
Under pathological conditions in which the NO pathway is disrupted, however, endothelium-derived hyperpolarizing factor (EDHF) may also be a prominent vasodilator (13,14), and this is activated by a cytochrome P450 epoxygenase such as epoxyeicosotrienoic (EET) acid which is a metabolic derivative of arachadonic acid (AA). What contribution each of these pathways makes to the enhanced sensitivity to acetylcholine in CFS patients is intriguing and needs to be determined. Such research is entirely feasible and urgently required.
In a number of separate studies, this group has demonstrated abnormalities of the acetylcholine endothelium-dependent vasodilator pathway in CFS patients. Sensitivity to acetylcholine seems to be restricted to those patients within the CFS construct who fit descriptions for "sporadic" ME/CFS/PVFS but not those with Gulf War syndrome, those exposed to organophosphate compounds or, indeed, those with fibromyalgia (15).
The vascular endothelium is a dynamic organ which is influenced by many biological factors such as age, hormonal status, blood pressure, hyperlipidaemia and cardiovascular disease progression, so it is, at best, a blunt instrument for the assessment of specific disturbances to cholinergic mechanisms.
Nevertheless, the model used in the studies above has been well tested by this research group, and also by many other international groups of researchers working in vascular medicine (16–18). The research group is therefore confident that the findings of increased sensitivity to acetylcholine in CFS patients are robust and unusual.
Increased sensitivity to acetylcholine is normally associated with trained athletes, while CFS patients are characterized (according to the Fukuda 1994 definition) as having "a substantial reduction in previous levels of occupational, educational, social or personal activities". These results are important in terms of vascular control mechanisms in this patient group and may be relevant to the problem of orthostatic instability that is so evident in most CFS patients.
The findings reported in this review are specific to endothelial cholinergic activity and may or may not be applicable to other more widespread neurotransmitter functions of acetylcholine. Recent evidence in a very small group of patients suggested that CFS might be the consequence of a cholinergic dysautonomia, and that treatment with cholinesterase inhibiting agents might well be therapeutic (19).
Such a hypothesis is in direct contrast with the findings reported by this group at the University of Dundee, and so great caution is still needed in treating an illness with such obvious heterogeneity. It is important that the mechanisms underlying the pattern of abnormal peripheral endothelial cholinergic activity that has been described in this series of papers is unraveled, and that the significance of altered cholinesterase activity, the prostanoid pathway and the role of endothelium-derived hyperpolarizing factor (EDHF) is determined. MERGE is currently funding a follow-up study to continue these important investigations.
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Source: © 2003-2004 MERGE, online at http://www.meresearch.org.uk