Neuroscience Letters 365 (2004) 19–22

Cholinergic modulation of baker’s yeast cell

phagocytosis by rat astrocytes
Ricardo M. Gómez a , Marı́a I. Berrı́a b , Leonor Sterin-Borda a,∗
a Department of Pharmacology, School of Dentistry, University of Buenos Aires, MT Alvear 2146, 4 piso sector B, 1221 Buenos Aires, Argentina
b Department of Microbiology, Faculty of Medicine, University of Buenos Aires, Buenos Aires, Argentina

Received 4 December 2003; received in revised form 24 March 2004; accepted 6 April 2004

Abstract

Cholinergic regulation of baker’s yeast cell phagocytosis in rat cultured astrocytes was studied. Phagocytic activity was reduced by 1×10−5 M
of atropine or pirenzepine, but not by AF-DX116 or 4-DAMP. In addition, carbachol stimulated phagocytosis in a dose-dependent manner.
Furthermore, only 1×10−5 M of atropine, pirenzepine and 4-DAMP significantly reduced enhanced activity induced by 1×10−7 M carbachol.
It was also observed that l-NMMA, staurosporine, or U-73122, reduced phagocytosis activity while TFP failed to do so. Nitrite levels in
astrocyte supernatants increased after baker’s yeast cells were incorporated to astrocyte cultures, correlating with enhanced phagocytosis
induced by carbachol stimulation, and were reduced by 1 × 10−5 M of atropine, pirenzepine or aminopiridine, but not by AF-DX116 or
4-DAMP. Enhanced NO production triggered by astrocyte phagocytosis may have pathological consequences.
© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Astrocytes; Baker’s yeast cells; Carbachol; Nitric oxide; iNOS

Studies on induced central nervous system (CNS) lesions
in laboratory animals have clearly shown the capacity of
astrocytes to perform phagocytosis [1,5]. This function may
be particularly relevant in the course of autoimmune inflam-
matory response in the rodent or human CNS by helping the
microglia to carry out more efficient clearance of apoptotic
immune cells and thus protecting the CNS from leakage
of potentially harmful substances from secondary necrotic
cells [14].
Astrocytes display muscarinic receptors both in vivo [18]
and in vitro [11] and their specific activation in this cell
type is known to modulate a number of pathophysiolog-
ical states, including proliferation [10], apolipoprotein E
secretion [8], and calcium responses [7], but their role in
phagocytic activity remains unknown. To address this issue,
we used baker’s yeast cells together with a triple staining
procedure on astrocyte cultures [12] in order to study the
muscarinic regulation of astrocyte phagocytic activity.
Astroglial cell cultures were obtained from newborn rat
brain, on the basis of previous description [6]. Briefly, cor-
∗ Corresponding author. Tel.: +54-11-4964-1276;
fax: +54-11-4963-2767.
E-mail address: leo@farmaco.odon.uba.ar (L. Sterin-Borda).
tical hemispheres were harvested, neural tissues digested
by 0.25% trypsin, and 600,000/800,000 cells/ml of growth
medium (d-MEM plus 10% fetal calf serum) seeded in plas-
tic flasks. By changing the supernatant within 24 h, neurons
were eliminated by means of differential attachment, leav-
ing an almost homogenous population of glial cells. After
3 weeks at 37 ◦ C, the already confluent primary cell culture
was shaken for 2 h at 37 ◦ C, and supernatant discarded in
order to detach contaminating oligodendrocytes grown on
top of the remaining adherent astroglial cell monolayer,
which in turn was trypsinized. Resulting cells were then
resuspended (300,000 cells/ml of growth medium), and
seeded as first subculture in 12 (6 × 105 cells/3.8 cm2 ) or
24 (3 × 105 cells/1.88 cm2 ) well plates which included a
glass coverslip. Immunoperoxidase GFAP labeling showed
a high homogeneous population of astrocytes (> 94%, data
not shown).
When 2 days old, first subcultures of astroglial mono-
layers were treated during 1 h at 37 ◦ C with a number
of pharmacological agents and exposed to an autoclaved
baker’s yeast suspension at a multiplicity of infection of 1
during 48 h in a d-MEM plus 5% fetal calf serum at 37 ◦ C.
Supernatants were harvested, clarified and stored at −70 ◦ C
until used and cells were washed several times and fixed

0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2004.04.034
20 R.M. Gómez et al. / Neuroscience Letters 365 (2004) 19–22
Fig. 1. (A–C) Phagocytic activity in cultured astrocytes after different pharmacological treatments. (A) Basal (without carbachol); (B) dose–response
phagocytic curve with carbachol; (C) action of muscarinic cholinergic antagonists upon carbachol effect.
with chilled acid methanol for 10 min. For cell staining, we
used a previously described procedure [12].
Nitrite/nitrate levels were measured with a commercial
kit (Calbiochem). Briefly, nitrate was converted to nitrite
by incubation with nitrate reductase in the presence of
NADPH. Lactate dehydrogenase was then used to destroy
excess NADPH. Equal volumes of sample and Griess
reagent were incubated at room temperature. After 10 min,
absorbance was read at 550 nm. Nitrite concentration was
determined by using sodium nitrate as a standard.
The muscarinic agonist carbachol, the muscarinic blocker
atropine, specific M1 blocker pirenzepine, the inducible
nitric oxide synthase (iNOS) inhibitor 2-amino-4-methyl-
pyridine (AP), and the generic nitric oxide synthase (NOS)
inhibitor l-NMMA were purchased from Sigma (MO,
USA). Specific M2 blocker AF-DX116 is from Boehringer
Ingelheim (CT, USA). Specific M3 blocker 4-DAMP is from
RBI (MA, USA). Staurosporine, specific inhibitor of PKC,
is from Tocris (MO, USA). U-73122, specific inhibitor of
phospholipase C (PLC), is from ICN (OH, USA). Stock so-
lutions were freshly prepared in the corresponding buffers.
We used one-way analysis of variance (ANOVA) followed
by a Newman–Keuls multiple comparison procedure to an-
alyze data. Values of P lower than 0.05 were considered sta-
tistically significant. Data was expressed as means ± S.E.M.
Phagocytic activity was measured by counting yeast cells
in a field (y/f) using a grid in the 40× objective in order to
quantify in a blind manner. Fig. 1A shows that basal phago-
cytic activity (28 ± 2 y/f) is reduced when 1 × 10−6 M of at-
ropine (16.4±2.2 y/f) or pirenzepine (20.8±1.6 y/f) blockers
were used. No changes were observed with the same concen-
tration of AF-DX116 (26.2 ± 1.4 y/f) or 4-DAMP (25 ± 1.6
Fig. 2. (A and B) Triple staining procedure in untreated (A) and treated
carbachol (1 × 10−7 M) astrocytes (B) 250×.
y/f) (n = 5). As depicted in Figs. 1B and 2A, B, carbachol
stimulated phagocytic activity in a dose-dependent manner
when used at concentrations ranging from 1 × 10−9 M to
1 × 10−6 M (% of basal: 1 × 10−11 M, 105 ± 9; 1 × 10−10 M,
110 ± 10; 1 × 10−9 M, 161 ± 11; 1 × 10−8 M, 187 ± 8;
1 × 10−7 M, 196 ± 7; 1 × 10−6 M, 194 ± 8) (n = 5). In order
to confirm these results, the antagonists mentioned above
were used in astrocytes treated with 1 × 10−7 M carbachol
(Fig. 1C). Again, atropine and pirenzepine reduced (22 ± 2
y/f and 26.2 ± 1.5 y/f) the enhanced activity induced by car-
bachol (49 ± 4 y/f) (P < 0.01). No significant changes were
observed when the same concentration of AF-DX116 (46±3
y/f) was used, and, interestingly, the use of 4-DAMP signif-
icantly reduced phagocytic activity to 38.1 ± 3 y/f (n = 5).
We observed similar values (450 ± 55) hematoxylin stained
nucleous/field in treated and untreated monolayers (data not
showed).
In order to determine whether an endogenous nitric ox-
ide signaling system is triggered in carbachol stimulation
of phagocytic activity, a number of additional drugs that in-
hibited enzymatic pathways in NO production were used.
As shown in Fig. 3A, the inhibitor of nitric oxide synthase
l-NMMA (1×10−4 M), the inhibitor of PKC (staurosporine,
1 × 10−9 M), or the inhibitor of PLC (U-73122, 5 × 10−6 M)
significantly reduced the phagocytosis of baker’s yeast cells
Fig. 3. (A and B) Phagocytic activity in cultured astrocytes with different
pharmacological treatments. (A) Open bars: basal (without carbachol);
black bars: action of several enzymatic antagonists upon carbachol ef-
fect. (B) Nitrite levels in supernatants of cultured astrocytes. Open bars:
without baker cells; black bars: undergoing phagocytosis after different
pharmacological treatments.
 R.M. Gómez et al. / Neuroscience Letters 365 (2004) 19–22
(basal, 25±3 and 49±2; l-NMMA, 17±2 and 30±1; stau-
rosporine, 18 ± 1.5 and 29 ± 2; U-73122, 17 ± 5 and 32 ± 2;
n = 5). In contrast, the inhibitor of calcium/calmodulin TFP
(5 × 10−6 M) had no effect (26 ± 2 and 47 ± 3; untreated
and carbachol treated, respectively). Since l-NMMA ex-
erted a significant effect, it was of interest to study nitric
oxide (NO) production measured as nitrate/nitrite in the su-
pernatant. As Fig. 3B shows, NO (2.2 ± 0.5 ␮M) increased
dramatically after baker’s yeast cells were incorporated to
astrocyte cultures (9 ± 1 ␮M). In addition, NO production
closely followed phagocytosis activity, increasing after car-
bachol stimulation (18 ± 2 ␮M) was blocked with atropine
(4.5 ± 0.5 ␮M); pirenzepine (3 ± 0.7 ␮M) and 4-DAMP
(7 ± 0.7 ␮M), but not with AF-DX116 (15 ± 1 ␮M). Lastly,
we used the specific inhibitor of the inducible nitric oxide
synthase (iNOS) AP (5 × 10−5 M) and observed significant
inhibition (2.5 ± 0.4 ␮M), suggesting the involvement of
iNOS during the phagocytic process. As expected, l-NMMA
reduced NO levels as when AP was used and yeast alone
showed undetectable levels after treatment with the respec-
tive drugs (data not shown).
The finding that atropine and pirenzepine reduced basal
phagocytic activity of astrocytes suggests firstly that this
function is modulated by the cholinergic system, and sec-
ondly that such function is preferentially exerted by the M1
receptor. To further study this function, we performed a
dose–response curve with carbachol and, as expected, car-
bachol increased phagocytic uptake of baker’s yeast cells
by rat astrocytes in a concentration dependent manner. The
specificity of this result was strengthened when the enhanced
phagocytic activity induced by carbachol was significantly
reduced using atropine and pirenzepine, less with 4-DAMP
but not with AF-DX116. These findings suggest that in basal
cholinergic phagocyte activity stimulation the subtype M1
receptor plays a major role, but after exogenous stimulation
the subtype M3 is also activated perhaps because subtype M1
receptors are saturated. Given the reduced astrocyte phago-
cytosis activity observed with U-73122 and staurosporine
in contrast to TFP, which had no effect, it seems that such
activity is mediated mainly by the DAG-PKC rather than
the IP3-Ca/Calmomulin pathway. Similar results have been
found in other cell types. In this regard, it has been shown
that in human neutrophils phagocytic ability is inhibited by
forskolin, a potent activator of adenylate cyclase, and re-
versed by either phorbol myristate acetate or diacylglyc-
erol, which are both activators of PKC [3]. Interestingly, it
has been shown that carbachol stimulation of rat astrocytes
elicits a calcium response mediated by the M3 subtype of
muscarinic receptors with PKC appearing to be involved in
maintaining raised calcium concentration [7]. Further stud-
ies will determine whether this mechanism is involved in
PKC stimulation of astrocyte phagocytosis.
According to our findings, NO production appears to oc-
cur as a consequence of either muscarinic stimulation or
enhanced phagocytic activity. Either pathway may prove
relevant considering the proposed roles for NO in CNS
21
pathophysiology, which range from intercellular signaling,
through necrosis of cells and invading pathogens, to the in-
volvement of NO in apoptosis and tissue remodeling [15].
Raised NO levels agree with previous studies suggesting
that NO synthesis occurring in astrocyte phagocytosis is
the result of iNOS induction [13]. However, at least in
macrophages, it has been shown that phagocytosis per se
is not sufficient to induce NOS activity [9]. This could be
explained because astrocyte phagocytosis may be associ-
ated with the secretion of certain cytokines, including tu-
mor necrosis factor-alpha [4], which in turn induces iNOS
expression [2]. Interestingly, the production of NO subse-
quent to phagocytosis seems essential not only for astrocytes
but also for microglia since astrocyte co-cultures as well as
astrocyte-conditioned medium increased microglial phago-
cytic activity by poorly understood mechanisms [16]. On the
other hand, it has recently been shown that carbachol can
stimulate iNOS activity and gene expression via M1 mAchR
activation in cerebral frontal cortex [17].
Be as it may, present results help to clarify the mecha-
nisms involved in the phagocytic process of astrocytes, thus
interpreting their underlying regulatory and signaling roles.
Acknowledgements
This work was partly supported by grants from the Na-
tional Research Council (CONICET), and the University of
Buenos Aires, Argentina.
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