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Changes of muscarinic cholinergic binding by lymphocytes in parkinson's disease with and without dementia.

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Changes of Muscarinic
Cholinergic Binding by
Lymphocytes in Parhnson’s
Disease with and
without Dementia
Jose Martin Rabey, MD,* Elisheva Grynberg, BSc,? and
Eran Graff, P h D t
Following the report of animal studies {I21 performed on rat lymphocytes, we demonstrated that peripheral lymphocytes from patients with “probable”
AD exhibited a marked reduction in cholinergic binding capacity, as measured by [3H]quinuclidinyl benzilate (QNB), a muscarinic receptor blocker 113, 141. In
this study, we examine if the binding characteristics of
lymphocytes may be altered in patients with PD with
and without dementia.
Patients and Methods
A population of 44 out-patients with idiopathic PD (age
We compared the muscarinic cholinergic binding in
lymphocytes of 44 patients with idiopathic Parkinson’s
disease with 23 age-matched normal volunteers, using
{3H)quinuclidinyl benzilate. In 24 patients with Parkinson’s disease without dementia, binding was normal in
12, below control values in 6, whereas the remaining
6 (all on anticholinergic medication) showed very high
binding. I n all 20 patients with Parkinson’s disease and
with dementia, the binding was below control levels,
indicating that in these patients, as in patients with Alzheimer’s dementia, the cholinergic muscarinic binding
by lymphocytes is reduced.
Rabey JM, Grynberg E, Graff E. Changes of
muscarinic cholinergic binding by lymphocytes in
Parkinson’s disease with and without dementia.
Ann Neurol 1991;30:847-850
The underlying cause of the mental deficits observed in
Parkinson’s disease (PD)is controversial. Some authors
11-31 suggested that patients with P D exhibit a subcortical dementia attributable to dysfunction of basal
ganglia and frontal subcortical connections. Alternatively, identification of Alzheimer-type histopathological changes in the brain of some patients with P D led
others C4, 51 to suggest that the dementia of P D was
due to concomitant Alzheimer’s disease (AD). This hypothesis was supported by the discovery that both patients with AD and PD have atrophy of the nucleus
basalis of Meynert, the source of cholinergic innervation of the cerebral cortex 16-81. In addition, decreased choline acetyltransferase activity (indicative of
cholinergic innervation) has been found in the cortical
areas and hippocampus of patients with PD 191. This
cholinergic deficiency has been related to the degree
of intellectual deterioration observed in these patients
[9-117.
range, 56-82 yr) were included in the study. Criteria for the
diagnosis included the presence of at least three of the four
major motor manifestations: tremor, postural instability, bradykinesia, and muscular rigidity. Twenty of the patients also
suffered from dementia according to the Diagnostic and Statistical Manual of Mental Disorders-IIIR criteria [ 151. Patients with any metabolic or cardiovascular disorders and
those suffering from brain infarcts (as ascertained by computed tomography) were excluded. Patients with mental impairment considered to be drug-related were also excluded.
The scoring of dementia was performed using the Short
Mental Test (SMT) [ 161. Our previous analysis demonstrated
that a score of ?9% or above indicated “normal” mental
status, whereas a cut-point of 70% was used to separate
definitely demented patients [ 161.
We included 24 patients with PD who achieved more than
79% in the SMT (nondemented) and 20 who scored less than
70% (demented). Motor features were graded according to
the Hoehn-Yahr scale. All of them were on levodopa therapy
alone or combined with bromocriptine, except 6 patients
who suffered from P D without dementia and also took anticholinergics (trhexyphenidyl hydrochloride). The patients
with PD were compared with 23 age-matched healthy volunteers (age range, 55-83 yr).
Blood Collection
Blood was collected by venipuncture using heparin as an
anticoagulant.
Isolation of Lymphocytes
Human lymphocytes were isolated on a Ficoll Hypaque gradient. Following centrifugation for 45 minutes at 1,000 g,
the lymphocyte layer was separated and then washed twice in
RPMI 1640 nutrient-medium (GIBCO, Grand Island, NY).
Washed lymphocytes were resuspended in RPMI, counted
with a hemocytometer, and their viability determined by trypan blue exclusion test.
QNB Bindinghsay
From the ‘Department of Neurology and the ?Department of
Chemical Pathology, Tel-Aviv Medical Center, Sackler School of
Medicine, Tel-Aviv University, Israel.
Received Jan 22, 1991, and in revised form Jun 6. Accepted for
publication Jufl 11, 1991.
Address correspondence to Dr Rabey, Department of Neurology,
Ichilov Hospital, 6 Weizman St, Tel-Aviv, Israel 64239.
One million lymphocytes were incubated at room temperature for 45 minutes in 0.5 ml RPMI medium containing
different concentrationsofQNB ( 5 , 10,25,50,75, 100, 150,
200, 300 nM). Specific binding was routinely determined by
the preincubation of parallel tubes with 100 pM atropine
sulfate for 15 minutes before adding QNB. The reaction was
stopped by adding 3 ml of ice-cold saline, followed by rapid
filtration on prewet Whatman GFiB filters. After being
Copyright 0 1771 by the American Neurological Association
847
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50
5c
60
70
Age
80
90
in Y e a r s
Fig I . Maximum binding capacity (Bm.j values determined by
Scatchard analysis of3{H}quinuclidinylbenzilate (QNB) binding to (ymphoqtesfrom normal volunteers of various ages (black
circles) and from patients with PaTkin.ron:r disease without dementia (black squares). Parkinsonian patients treated with
antimuscarinic drugs (empty squares with dots) showed
higher binding. The solid line represents a regression line of the
a k a from normal subjects. B,, values demonstrated a high positive correlation with age (r = 0.963).The broken line represents the 95% conjdence interval. Note that 6 patients with
Parkinson's disease without dementia s h w low binding.
washed with 6 ml saline, the radioactivity on the filters was
measured by liquid scintillation spectrometry.
Specific binding, expressed as the difference in QNB
bound to lymphocytes in the presence or absence of atropine,
constitutes 50 to 70% of total binding. One hundred bound
femtomoles corresponds to a difference of approximately
2,000 counts per minute. The protein content in the lymphocyte samples was determined by the method of Lowry and
colleagues [17].
Analysis
of Radioligand Binding
Analysis of the results was performed according to Scatchard
1181. Binding at the low concentration (up to 10 mM) could
not be fitted and thus the maximum binding capacity (B-)
and the dissociation constant (Kd) values were calculated
from the linear regression lines of the higher QNB concentrations (25-300 nM). It has been suggested that retention
of QNB binding by viable lymphocytes may be due to a
process of uptake internalization or nonspecific trapping
1131. We found this suggestion unlikely because washing the
lymphocytes after incubation with ice-cold fresh media (a
5-min process) resulted in an 80 to 85%) loss of radioactivity.
Statisticdl Analysis
Regression analysis of B, and Kd values as related to age
was performed in normal control subjects. In addition, the
prediction interval (95% confidence level) of the regression
line was calculated and illustrated to indicate possible significant deviations from normal values.
848 Annals of Neurology
AAA
A
I
60
70
80
Age in Y e a r s
90
Fig 2. Maximum binding capacity ( B d values determined by
Scatchard analysis of i{H)quinuclidinyl benzilate (QNB) binding to (ymphocytesfrom normal volunteers of various ages (circles) and from patients with Parkinson's disease with dementia
(triangles). The solid line is a regression line of the data from
normal subjects. B,, values demonstrated a high positive correlation with age (r = 0.9631. Note the lower B,, values of patients with Parkinson? disease with dementia. The broken
line represents the prediction interval.
Results
In the PD group without dementia (n = 24) (Fig l),
6 showed binding below control levels and 12 showed
normal binding; 6 o n anticholinergic therapy showed
very high binding (50-100% higher than control levels). The B, values of patients with PD with dementia
fell below the prediction interval (95% confidence
level) (Fig 2); 15 showed very low binding compared
to the control subjects, whereas in 5 patients B, values were only slightly lower. The Kd values did not
change significantly among the groups studied (Kd of
control group, 62 nM; PD group, 58 nM; PD with
dementia group, 67 nM). The low binding capacity observed in patients with PD with dementia had no clear
relation to the severity of the symptoms according to
the SMT. There was also no correlation between the
binding and the motor status of the patients, according
to the Hoehn-Yahr grading.
Discussion
In spite of the fact that alterations in cortical cholinergic innervation in AD and PD are well established,
results regarding brain cholinergic receptors are inconclusive. When compared with age-matched controls,
cortical muscarinic receptors in AD were found unchanged [20, 217, decreased 122-24}, or increased
[25, 267, whereas in PD, one group reported an increase [9, 111 and suggested that it was due to anticholinergic treatment or denervation hypersensitivity, or
both.
It was recently reported that muscarinic receptors in
the brain can be divided into at least two major sub-
Vol 30 No 6 December 1991
types: MI, labeled by [3H)pirenzepine 127, 28) and
M,, labeled by [3H)oxotremorine [29) or C3H}AFDX116 [30). In light of this new finding, it has been shown
that in AD there is a selective cortical loss of the presynaptic M, receptors, which are located on cholinergic
cells 122, 31).
The existence of cholinergic muscarinic receptors
was demonstrated and characterized in intact peripheral lymphocytes [32-361, as well as in lysed lymphocyte membranes 1351. We have suggested the existence
of a functional analogy between muscarinic binding in
the brain and in circulating lymphocytes of rats 112).
Moreover, we have also shown, as have other researchers, that lymphocytes from patients with “probable”
AD have lower binding than age-matched control subjects [13, 14, 37, 381. This report demonstrates that
lymphocytes from patients with PD with dementia also
show lower muscarinic binding. It is also of interest to
note that some patients with PD without dementia
show low binding. It might be useful to follow these
patients to determine if they later develop dementia.
The biological significance of cholinergic receptor
changes in the membrane of lymphocytes exceeds the
scope of this study and deserves further consideration,
and a detailed characterization of this binding site
should be addressed. Alternatively, a correlation between brain and lymphocyte muscarinic receptors may
not exist at all; both systems are affected concomitantly
but separately during the development of the disease.
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