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Cholinergic muscarinic binding by human lymphocytes Changes with aging antagonist treatment and senile dementia of the Alzheimer type.

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Cholinergc Muscarinic Binding by Human
Lymphocytes: Changes with Aging,
Antagonist Treatment, and Sende Dementia
of the Alzheimer Type
-
Jose M. Rabey, MD, Louis Shenkman, MD, and Gad M. Gilad, PhD”
In peripheral blood lymphocytes (mixed lymphocytes isolated on a Ficoll-Hypaque density gradient) derived from
normal human subjects, cholinergic muscarinic binding capacity was found to increase with age. In contrast, lymphocytes derived from patients with “probable” senile dementia of the Alzheimer type (SDAT) exhibited a marked
reduction in binding capacity. Treatment of these patients with antimuscarinic drugs was associated with increased
muscarinic binding by lymphocytes. These results indicate that cholinergic muscarinic binding by peripheral blood
lymphocytes may be useful in the study of alterations associated with aging and SDAT, as well as in evaluating changes
induced by certain cholinergic drug treatments.
Rabey JM, Shenkman L, Gilad GM: Cholinergic muscarinic binding by human lymphocytes: changes with
aging, antagonist treatment, and senile dementia of the Alzheimer type. Ann Neurol 20:628-631, 1986
Lymphocytes respond to cholinergic agonists with
increased cyclic-guanosine monophosphate (GMP)
levels, enhanced ribonucleic acid and protein synthesis, and altered immune function 116, 28, 301. These
induced alterations are diminished or blocked by the
specific muscarinic antagonist atropine, which implies
the presence of muscarinic cholinergic receptor sites
on lymphocytes. Indeed, muscarinic receptors recently
were demonstrated and characterized in murine lymphocytes {2, 14, 191 and in peripheral circulating human blood lymphocytes 1343. As diminished function
of central cholinergic systems has been implicated in
the deterioration of mental faculties during aging 14, 9,
127 and as an etiological factor in senile dementia of
the Alzheimer type (SDAT) [8, 10, 13,23, 25, 27, 29,
32 J, a peripheral marker for central muscarinic cholinergic function would be very useful E27, 291. It was,
therefore, of interest to determine whether any
changes in muscarinic binding by peripheral lymphocytes occur during normal aging and in senile dementia. Furthermore, it was important to establish whether
treatment with muscarinic agonists or antagonists
evokes changes in lymphocyte muscarinic receptors,
similar to the changes such agents evoke in brain muscarinic receptors {5, 20, 311.
Methods
Patients
From the Center for Neuroscience and Behavioral Research,
Isotope Department, The Weizrnann Institute of Science, Rehovot,
Israel; and the Department of Neurology, Ichilov Hospital, Sackler
School of Medicine, Tel Aviv University, Tel Aviv, Israel.
Received Dec 24, 1985, and in revised form Apr 14, 1986. Accepted for publication Apr 16, 1986.
Address reprint requests to Dr Gilad, Isotope Department, The
Weizmann Institute of Science, 76100 Rehovot, Israel.
62 8
Studies were carried out on human volunteers of both sexes.
Six age-matched healthy volunteers were compared with 5
patients suffering from myasthenia gravis treated with muscarinic agonists (neostigmine [Prostigmin) or pyridostigmine)
for 4 to 8 months, and with 12 patients suffering from senile
dementia with extrapyramidal features treated with muscarinic antagonists (trihexyphenidyl or biperiden) for 4 to 12
months. Twenty-two age-matched healthy volunteers served
as control subjects for 27 patients with “probable” SDAT
(accordingto DSM-111 criteria [ 11)) who were diagnosed on
the basis of their mental status as suffering from marked
confusion, disorientation, inability to concentrate,and memory loss. Patients with any metabolic or cardiovascular disorders and those suffering from multiinfarct dementia (as ascertained by computed tomographic scans) were excluded.
Blood Collections
Blood was collected by venipuncture using heparin as an
anticoagulant.
Isolation of Lymphocytes
Human lymphocytes were isolated on a Ficoll-Hypaque gradient [7]. Following centrifugation for 45 minutes at 1,000g,
the lymphocyte layer was separated and then washed twice in
RPMI 1640 nutrient medium (GIBCO, Grand Island, NY).
*present address: ~~i~ ~
Saba, Israel.
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uar
Eflect of Various Drugs on Lymphocyte QNB Bindin?
1
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200
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9
0
’
Drug
/
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B r n y 8 5 . 4 ~ l / l X l OCell*
I2 6x105 binding sites/cell)
Kd
-,
D
:62 2nM
.
44
Free,[%] Q N B ( n M )
Fi g 1 . Specz’jic {’H}quinuclidinyl benzilate ([ ?H}QNB) binding
to human lymphocytes (from a 34-year-old man) as a function of
concentration. Each point represents the mean value of duplicate
measurements. The inset shows a Scatchard plot of the results,
the derived dissociation constant (Kd) and maximum binding
capacity (BmU)values, and the number of binding sites per cell.
Washed lymphocytes were resuspended in RPMI, counted
with a hemocytometer, and their viability determined by trypan blue exclusion.
{’ H)Quinzlclidinyl Benzilate Binding Assay
One million lymphocytes were incubated at room temperature for 45 minutes in 0.5 ml of RPMI medium containing different concentrations of [3H]quinuclidinyl benzilate
(QNB; 5-200 nM). Specific binding was routinely determined by the preincubation of parallel tubes with 100 p~
atropine for 15 minutes before adding QNB. The reaction
was stopped by adding 3 ml of ice-cold saline, followed by a
rapid filtration on prewetted Whatman GF/B filters. After
washing with 6 ml of saline, the radioactivity on the filters
was measured by liquid scintillation spectrometry. Figure 1
illustrates a characteristic saturation curve of Q N B binding
by human lymphocytes. Specific binding, expressed as the
difference in Q N B bound to lymphocytes in the presence of
atropine, constitutes 50 to 70% of the total binding measured in the absence of atropine. One hundred femtomoles
bound corresponds to a difference of about 2,000 cpm.
Analyses of radioligand binding experiments were performed according to Scatchard 1261.Binding at the lower
concentrations (up to 10 nM) could not be fitted, and thus
the maximum binding capacity (Bmm;425.4 fmol/l X lo6
cells) and the dissociation constant (Kd; 62.2 nM) values were
calculated from the linear regression line of the higher Q N B
concentrations (25-150 nM) (see Fig 1).
The binding characteristics of several cholinergic drugs
were examined (Table). The maximal displacement of lymphocyte Q N B binding was highest for dexetimide (which
was also the most potent), followed by atropine. A more
potent displacement was observed with dexetimide than with
levetimide, demonstrating stereospecificity of the binding
site (the dexetimide and levetimide were a generous gift
from Dr Pierre Laduron, Janssen Pharmaceutica, Beerse,
Belgium) [lS]. Nicotine had no practical effect on Q N B
Dexetimide
Levetimide
Atropine
Scopolamine
Nicotine
Oxotremorine
Acetylcholine
Maximal
Displacement (c/c)
IC50 (CLM)
70
5
60
15
12
20
50
30
40
70
60
10
50
40
“Results are expressed as the mean value of three separate determinations, with a range of concentrations between 1 and 500 (*M.
QNB
= [iH]quinuclidinyl benzilate; IC50 = concentration at 50%
displacement.
binding, further indicating the muscarinic character of the
binding site.
It has been suggested that retention of Q N B by viable
lymphocytes may be due to a process of uptake, internalization, or nonspecific trapping [IS]. We found this unlikely
because washing the lymphocytes after the incubation with
ice-cold fresh media (a 5-minute process) resulted in an 80 to
85% loss of radioactivity.
Protein Determination
The content of proteins in the lymphocyte samples was determined by the method of Lowry and colleagues { 171.
Results
lncreased QNB Binding in Hzlman Lymphocytes
after Antagonist Treatment
Treatment of patients with the antimuscarinic drugs
trihexyphenidyl or biperiden resulted in an increased
QNB binding capacity by lymphocytes. The increased
binding capacity after cholinergic antagonist treatment
was due to higher B,
values, while I(d values did not
change significantly (Fig 2).
No change in QNB binding capacity was observed
in lymphocytes from 5 patients suffering from myasthenia gravis who were treated with the cholinergic
agonists neostigmine and pyridostygmine (see Fig 2).
Changes in Lymphocytes with Age and SDAT
The binding capacity of QNB increased with patient
age (Fig 3). The age-dependent increase in binding
capacity was highly correlated with increased B,
values, indicating an age-dependent increase in the
number of binding sites per lymphocyte. The values
of Kd decreased slightly in patients over 50 years of
age. Thus, a moderate age-dependent decrease in Kd
values, indicating an increased affinity of the ligand to
its receptor, also contributed to the observed increase
in QNB binding capacity.
Lymphocytes from a group of 27 patients with probable SDAT had significantly lower B,
values as comRabey et al: Cholinergic Binding by Lymphocytes
629
Fig 2. Differences in maximum binding capacity (Bmu)(lefi)
and KJ (right) values after Scatchard analyses of {~'Hjquinuclidinyl benzilate binding to lymphocytesfrom 7 patients with
senile dementia (SD) of the Alzheimer type. 5 patients with myasthenia gravis treated with neostigmine methysulfate (Prostigmin) and pyridostigmine (cholinergic agonists), and 12 patients
with SD exhibiting extrapyramidalfeatures treated with trihexyphenidyl and biperiden (cholinergicantagonists). The mean results ( t standzrd error of the mean) are expressed as the percent.tge of control values (shaded area) obtained from age-matched
healthy volunteers.
I
I
I
I
I
20
40
60
80
I
1
I
100
20
Age (year)
I
40
I
60
I
80
l
l
100
Fig 3. Scatter diagrams of maximum binding capacity (Bmx)
(Left)and Kd (right) values determined by Scatchard analyses of
{3H}quinuclidinylbenzilate ([3H]QNB) binding to lymphocytes
from normaZfemales and males of various ages (solid circles)
and from patients with senile dementia of the Alzheimer type
(open circles). The broken lines are linear regression lines o f the
dzta from nomzal subjects. B,, values demonstrated a high positive correlation with age (r = 0.62).Note the lower B,, values
of senile dementia patients. K d values demonstrated only a moderate negatioe correlation with age (r = 0.45) and were unchanged in patients with senile dementia.
pared with age-matched control subjects, while Kd
values did not differ significantly (see Figs 2 and 3).
Discussion
The present study demonstrates that (1)peripheral circulating blood lymphocytes possess cholinergic muscarinic binding sites, which are responsive to phar-
macological treatment with antimuscarinic drugs; (2)
there is an age-dependent increase in muscarinic binding capacity by adult lymphocytes; and (3) patients
with SDAT exhibit a marked reduction in muscarinic
binding capacity by lymphocytes.
This, study supports previous findings that muscarinic binding sites are present on human lymphocytes
134). However, the kinetics of QNB binding to human lymphocytes are quite different from those reported for muscarinic receptors in rat brain tissue [33J
This discrepancy holds true even when the lymphocyte
findings are compared to measurements done in rat
brain slices [lS}, indicating a much lower affinity
(about 100-fold) for the ligand to muscarinic receptors
on lymphocytes as compared to those in rat brain. Caution must be taken, however, in the interpretation of
the binding experiments. Assumptions made in analyses of binding experiments with membrane preparations may not prove valid for intact cells under physiological conditions [22]. Therefore, the Kd or 50%
displacement values in our study probably reflect the
contribution of many physiological processes rather
than simply representing the affinity of the binding site
to the ligand 122). For this reason we chose to deal
only with the linear part of the Scatchard plots (see Fig
1) and avoid further interpretation of the data. Yet,
in spite of these kinetic differences, the lymphocyte
response to chronic treatment with antimuscarinic
drugs-increased muscarinic binding-is similar to the
response observed in rodent brain after similar treatment 15, 20, 311, suggesting a functional homology.
The lack of change in lymphocyte muscarinic binding sites following agonist treatment was expected. It
was highly improbable that treatment with the indirect
agonists (acetylcholinesterase inhibitors) used in this
study would result in any significant elevation of blood
acetylcholine levels that would affect the binding sites.
Treatment with direct muscarinic agonists might have
had such1 an effect.
The qge-dependent increase in muscarinic binding
capacity by lymphocytes is intriguing. This may be a
result of specific alterations in cholinergic reception by
the lymphocyte or, alternatively, a result of changes in
other membrane constituents, which may in turn lead
to alterations in the general physical properties of the
lymphocyte membrane. Such enhanced binding was
not observed in the brain; rather, no alteration o r reduced receptor numbers was observed [8, 24, 271.
Since neuronal cell death is a general characteristic of
the aging brain, a probable reason for such observations is the death of different numbers of neurons
bearing rnuscarinic receptors. Cell counts were not
performed in previous studies [8, 24, 271. Thus it appears that comparisons between brain muscarinic binding and lymphocyte muscarinic binding will have to be
done on a\ per-cell basis.
630 Annals of Neurology Vol 20 No 5 November 1986
The reduction in binding sites associated with
SDAT is of great interest for the following reasons: (1)
it indicates that the hypofunction of the cholinergic
system that is most often associated with SDAT apparently is not confined to the brain but is expressed in
the periphery as well; and (2) it implies that the immune system is also affected, primarily or secondarily
C3, 6, 21). A recent report that confirms the present
finding demonstrated a reduction in Q N B binding by
lymphocytes from patients with Alzheimer’s disease
111. The changes observed in this study could, in principle, originate from different populations of B and T
lymphocytes 13, b}. In fact, a recent report suggests
that Q N B binding is greater in B-cell than in T-cell
murine lymphocytes [ 2 } . This will have to be determined in humans as well.
Our results suggest that cholinergic muscarinic binding by circulating blood lymphocytes may serve as a
useful peripheral marker reflecting drug-induced
changes and alterations associated with aging and
SDAT.
Dr Gilad is an incumbent of the Paul and Gabriella Rosenbaum
Career Development Chair, in perpetuity, established by the Paul
and Gabriella Rosenbaum Foundation, Chicago, IL.
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Rabey et al: Cholinergic Binding by Lymphocytes
631
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