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INTRODUCTION |
Ca2+-regulated exocytosis has been traditionally
regarded as a specialized process present in only a few cell types.
Recently, however, evidence has been accumulating indicating that it is a ubiquitous process observed in a variety of animal cells (1-5). A
previous study from our laboratory showed that between 10 and 20% of
conventional lysosomes in fibroblasts and epithelial cells fuse with
the plasma membrane and release their contents upon elevation of the
intracellular free Ca2+ concentration
([Ca2+]i) (6). Lysosome exocytosis is similar to
other well characterized forms of Ca2+-regulated exocytosis
in specialized cells, in the sense that it is ATP and
temperature-dependent and requires micromolar levels of
[Ca2+]i (6). These findings are consistent with
recent capacitance measurements in Chinese hamster ovary cells, which showed that vesicles in the size range of lysosomes (0.4-1.5
µM) fuse with the plasma membrane in response to 6-20
µM [Ca2+]i (3).
Although the physiological function of regulated lysosome exocytosis is
unclear, an intriguing possibility is that it may participate in plasma
membrane resealing, a process that requires Ca2+ and
exocytosis (5). Several studies describe accumulation and fusion of
intracellular vesicles at the sites of membrane injury (7-9). Although
proposed to belong to the endosomal/lysosomal compartment (10), the
exact identity of these vesicles has remained obscure. The evidence
presented here and in our previous work (6) strongly suggests that the
vesicle population mobilized by Ca2+ and postulated to be
involved in membrane repair may, at least in fibroblasts, correspond to
conventional lysosomes.
Another physiological process known to require mobilization and fusion
of lysosomes with the plasma membrane is cell invasion by the protozoan
parasite Trypanosoma cruzi. Several lines of evidence
indicate that host cell lysosomes are required for cell invasion by
this pathogen. The process involves a gradual recruitment of lysosomes
to the parasite entry site, followed by lysosome fusion and formation
of an intracellular vacuole, through which T. cruzi enters
the cell (11, 12). Cell invasion by T. cruzi is
Ca2+-dependent, because it can be effectively
blocked by loading the host cells with membrane-permeant
Ca2+ chelators or by previously depleting intracellular
Ca2+ stores. Trypomastigotes, the T. cruzi
infective stages, are capable of triggering intracellular free
Ca2+ transients in a variety of mammalian cell types (13,
14).
Although the mechanism responsible is incompletely understood, there
have been several reports of potentiation of exocytosis by agents that
elevate cytosolic cAMP in mammalian cells (15-19). In this study, we
investigated in parallel the effect of modulation in intracellular cAMP
levels on Ca2+-dependent exocytosis of
lysosomes and on cell invasion by T. cruzi. We demonstrate
that cAMP levels regulate Ca2+-evoked lysosome exocytosis
in NRK fibroblasts and that the stimulatory effect of cAMP on lysosome
exocytosis can be further potentiated by microfilament disassembly,
consistent with previous observations in other systems implicating the
cortical actin cytoskeleton as an obstacle for exocytosis (20-22). The
same pattern of regulation is observed on the levels of cell invasion
by T. cruzi trypomastigotes, when the cells are stimulated
by agonists that elevate or reduce intracellular cAMP. Exposure of NRK
cells to the infective trypomastigote stages of T. cruzi
results in elevated intracellular cAMP levels, an effect not observed
with the noninfective epimastigote stages of the parasite. These
findings reinforce previous observations indicating that similar
mechanisms regulate lysosome exocytosis and T. cruzi
invasion of mammalian cells.
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EXPERIMENTAL PROCEDURES |
Materials--
Forskolin, dideoxyforskolin,
3-isobutyl-1-methylxantine
(IBMX),1 8-Br-cAMP (sodium
salt), isoproterenol, phalloidin, cytochalasin D, and protein
G-Sepharose were obtained from Sigma; mastoparan and MDL-12,330A were
from Calbiochem. Latranculin A was from Molecular Probes (Eugene,
Oregon). Reduced streptolysin-O (SLO) was from Murex Diagnostics
(Dartford, United Kingdom).
Cell Culture--
NRK cells were grown at 37 °C with 5%
CO2 in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum. Confluent monolayers containing 6 × 104 cells/cm2 were used for all experiments.
Drug Treatments--
Cells were treated with 100 µM forskolin or dideoxyforskolin for 15 min at 37 °C
or as indicated, with 4 µM isoproterenol for 15 min at
37 °C or as indicated, with 100 µM MDL-12,330A for 30 min at 37 °C or as indicated, with 500 µM 8-Br-cAMP
and 500 µM IBMX for 15 min at 37 °C or as indicated,
with 0.4 µg/ml cholera toxin overnight at 37 °C, with 20 or 40 µg/ml mastoparan for 15 min at 37 °C, with 2 µM
cytochalasin D for 10 min at 37 °C, with 1 µM
latranculin A for 10 min at 37 °C, and with 10 µM
phalloidin for 10 min at 37 °C.
SLO Permeabilization and Exocytosis Assay--
Confluent
monolayers of NRK cells in 60-mm culture dishes were washed twice with
1 ml of ice-cold buffer A (20 mM Hepes, 110 mM
NaCl, 5.4 mM KCl, 0.9 mM
Na2HPO4, 10 mM MgCl2, 2 mM CaCl2, 11 mM glucose, pH 7.4)
and incubated for 10 min at 4 °C with SLO at 0.5 units/ml in buffer
A. Cells were washed once with 1 ml of ice-cold buffer B (20 mM Hepes, 100 mM potassium glutamate, 40 mM KCl, and 5 mM EGTA, pH 7.2) containing 2 mM MgATP and 5 mM free Mg2+ (added
as MgCl2). 0.5 ml of buffer B with or without 1 µM Ca2+ was added to the cells at 37 °C
for 10 min. The concentrations of free Mg2+ and
Ca2+ were maintained using a Ca2+ or
Mg2+/EGTA buffering system, calculated using the software
developed by Foehr and Warchol (Ulm, Germany). Incubation buffer was
collected for each sample and centrifuged for 5 min at 11,000 ×g before performing
-hexosaminidase activity assays.
Total extracts were obtained by incubation of culture dishes with 0.5 ml of buffer B containing 1% Nonidet P-40, followed by a 5-min
centrifugation of the extract at 11,000 ×g.
N-Acetyl-
-D-glucosaminidase (
-Hexosaminidase)
Activity Assay--
For each sample, 350 ml of the incubation buffer
was incubated for 15 min at 37 °C with 50 ml of 6 mM
4-methyl-umbellyferyl-N-acetyl-
-D-glucosaminide in sodium citrate-phosphate buffer, pH 4.5. The reaction was stopped by
the addition of 100 ml of 2 M
Na2CO3, 1.1 M glycine, and the fluorescence was measured in a F-2000 spectrofluorimeter (Hitachi Instruments, Inc., Danbury, CT) at excitation 365 nm/emission 450 nm.
To determine the total cellular content of
-hexosaminidase, cell
extracts prepared as described above were diluted 1:10, and 350 ml was
used for
-hexosaminidase activity assays.
Determination of Intracellular cAMP Levels--
Confluent
monolayers of NRK cells in 24-well dishes, prelabeled for 24 h
with [3H]adenine (22 Ci/mmol, 5 µCi/ml), were incubated
with the indicated drugs in Dulbecco's modified Eagle's medium 10%
fetal calf serum at 37 °C, washed once with Hepes-buffered
Dulbecco's modified Eagle's medium, and incubated for 15 min at
37 °C in the same medium containing 1 mM IBMX with or
without 50 µM forskolin. Reactions were terminated by the
addition of 1 ml/well of 5% trichloroacetic acid containing 1 mM ATP and 1 mM cAMP. Acid-soluble nucleotides were separated on ion-exchange columns as described previously (23). In
the mastoparan treatment experiments, [3H]adenine-labeled
cells were permeabilized with SLO before exposure to 20 µg/ml
mastoparan for 15 min at 37 °C. In the experiments with live
T. cruzi, [3H]adenine-labeled NRK cells plated
in 6-well dishes were exposed to 5 × 107
trypomastigotes or epimastigotes in 1 ml of Ringer's buffer (24) for
30 min at 37 °C.
T. cruzi Cell Invasion Assay--
NRK cells were plated at a
density of 2.5 × 104 cells/cm2 in
Dulbecco's modified Eagle's medium, 10% fetal bovine serum on 12-mm round coverslips placed in 6-cm plastic tissue culture dishes and grown
for 48 h at 37 °C in a humidified atmosphere containing 5%
CO2. Coverslips with attached cells were washed briefly
with Ringer's immediately before incubation for 30 min at 37 °C
with 5 × 107 T. cruzi trypomastigotes/ml
of Ringer's. Infected cells were washed and fixed in 2% (w/v)
paraformaldehyde, phosphate-buffered saline, and the number of
intracellular parasites was determined by immunofluorescence as
described previously (11).
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RESULTS |
Ca2+-evoked Exocytosis of Lysosomes Is Modulated by
Intracellular cAMP Levels--
To investigate whether
Ca2+-dependent exocytosis of lysosomes in
fibroblasts could also be potentiated by elevation in cytosolic cAMP
levels, NRK cells were pretreated with forskolin, an activator of
adenylyl cyclase, before permeabilization with SLO and detection of
released
-hexosaminidase. As shown in Fig.
1a, 100 µM
forskolin stimulates Ca2+-induced lysosomal exocytosis; an
82% increase in
-hexosaminidase release was observed after a 4-min
exposure to 1 µM free Ca2+, with a 70%
increase still detected after 10 min. The stimulatory effect of
forskolin is dose-dependent (Fig. 1b) and
specific, because the inactive analog dideoxyforskolin has no effect
(Fig. 1c).

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Fig. 1.
Stimulation of adenylyl cyclase enhances
Ca2+-induced exocytosis of lysosomes. a,
effect of pretreatment with forskolin on the kinetics of
Ca2+-dependent -hexosaminidase release.
b, dose-dependent effect of forskolin
pretreatment on Ca2+-dependent
-hexosaminidase release. c, effect of pretreatment with
forskolin or its inactive analog dideoxyforskolin
(ddForskolin) on Ca2+-dependent
-hexosaminidase release. NRK cells were permeabilized with SLO, and
buffer B containing 1 µM Ca2+ was added for
variable periods of time (a) or for 10 min (b and
c) at 37 °C. Supernatants were collected, and
-hexosaminidase activity was measured. The data represent the
average of triplicate determinations ±S.D.
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We next investigated whether pretreatment of NRK cells with
MDL-12,330A, an irreversible inhibitor of adenylyl cyclase (25), affected Ca2+-dependent lysosomal exocytosis.
Fig. 2a shows that inhibition of adenylyl cyclase activity results in a dose-dependent
reduction in
-hexosaminidase release from NRK cells after exposure
to 1 µM Ca2+, reaching about 70% reduction
at concentrations between 75 and 100 µM. To detect
inhibition of adenylyl cyclase, intracellular cAMP levels were measured
after forskolin stimulation. Treatment with 100 µM
MDL-12,330A reduced intracellular cAMP levels in forskolin-stimulated NRK cells by 43% (Fig. 2b).

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Fig. 2.
Inhibition of adenylyl cyclase decreases
Ca2+-induced exocytosis of lysosomes. a,
dose-dependent effect of pretreatment with MDL-12,330A on
Ca2+-dependent -hexosaminidase release.
Cells were permeabilized with SLO, and buffer B, containing 1 µM Ca2+, was added for 10 min at 37 °C.
Supernatants were collected, and -hexosaminidase activity was
measured. b, intracellular cAMP levels measured in NRK cells
exposed or not to forskolin, after treatment with MDL-12,330A. The data
represent the average of triplicate determinations ±S.D.
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As an alternative method for increasing cytosolic levels of cAMP, NRK
cells were exposed to the phosphodiesterase inhibitor IBMX (30), a
treatment that also resulted in a dose-dependent increase
in Ca2+-induced
-hexosaminidase release (Fig.
3a). To obtain direct evidence
implicating cAMP in the potentiation of Ca2+-induced
lysosomal exocytosis, NRK cells were incubated with 8-Br-cAMP, a
membrane-permeant analog of cAMP. Cells were preincubated with IBMX to
avoid degradation of 8-Br-cAMP during the assay (18). Under these
conditions, 8-Br-cAMP enhanced Ca2+-induced
-hexosaminidase release in a dose-dependent fashion, reaching a 40% increase over the level induced by IBMX alone at 500 µM (Fig. 3b). The addition of forskolin, IBMX,
or 8-Br-cAMP to NRK cells in the absence of Ca2+ did not
increase
-hexosaminidase release above base-line levels (Fig.
3c), indicating that elevated cytosolic cAMP has a
regulatory role but is not sufficient to trigger exocytosis of
lysosomes in NRK cells.

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Fig. 3.
Elevation in cytosolic cAMP levels enhances
Ca2+-dependent exocytosis of lysosomes.
a, dose-dependent effect of treatment with the
phosphodiesterase inhibitor IBMX on
Ca2+-dependent -hexosaminidase release.
b, effect of treatment with IBMX and 8-Br-cAMP on
Ca2+-dependent -hexosaminidase release.
c, effect of treatment with IBMX, forskolin
(Fsk), 8-Br-cAMP, or IBMX plus 8-Br-cAMP on
-hexosaminidase release in the absence of free Ca2+.
After pretreatment with the indicated drugs for 15 min at 37 °C, NRK
cells were permeabilized with SLO and exposed to buffer B containing 1 µM Ca2+ for 10 min at 37 °C, and the
supernatant was collected (a and b) or not
permeabilized, and the supernatant was directly collected
(c). -Hexosaminidase activity assays were performed on
supernatants, and the data represent the average of triplicate
determinations ±S.D.
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Ca2+-dependent Exocytosis of Lysosomes Can
Be Modulated by Trimeric G Proteins That Regulate Adenylyl Cyclase
Activity--
Constitutive and regulated exocytic pathways are
regulated by heterotrimeric G proteins in several cell types (18,
26-27). Gs and Gi can also regulate adenylyl
cyclase, resulting in stimulation or inhibition of cAMP synthesis,
respectively. In view of the results above, implicating intracellular
cAMP levels on the regulation of lysosome exocytosis, we investigated
whether the process could be regulated by heterotrimeric G proteins
through stimulation of adenylyl cyclase. NRK cells were pretreated with
cholera toxin, which ADP-ribosylates and activates the a subunit of
Gs (28). After cholera toxin treatment,
Ca2+-induced
-hexosaminidase release was elevated
approximately 25% (Fig. 4a),
whereas the intracellular levels of cytosolic cAMP showed a
significantly larger increase (Fig. 4c). The reason for this
discrepancy is unclear, but it may be a consequence of the activation
of additional cholera toxin targets, such as XLas (29). To determine
whether Gi, conversely, could regulate lysosomal exocytosis
by inhibiting adenylyl cyclase, cells were pretreated with mastoparan,
a tetradecapeptide that activates Gi but not Gs
(30). NRK cells were permeabilized with SLO and incubated with
mastoparan for 15 min before the addition of
Ca2+-containing buffer. Ca2+-induced
-hexosaminidase release was reduced by approximately 35% in the
mastoparan-treated cells (Fig. 4b), consistent with the
similarly reduced (40%) levels of cytosolic cAMP detected in response
to forskolin after mastoparan treatment (Fig. 4d). In
agreement with the results shown above (Fig. 3c), incubation of cells with either cholera toxin or mastoparan in the absence of
Ca2+ had no significant effect on lysosomal exocytosis
(Fig. 4, a-b).

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Fig. 4.
Heterotrimeric G protein activators regulate
Ca2+-induced exocytosis of lysosomes. a,
effect of pretreatment with cholera toxin (CTx) on
Ca2+-dependent -hexosaminidase release. NRK
cells were incubated with cholera toxin and permeabilized with SLO
before performing -hexosaminidase release assays. b,
effect of treatment with mastoparan on
Ca2+-dependent -hexosaminidase release. NRK
cells were permeabilized with SLO and incubated with mastoparan. Buffer
B containing 1 µM Ca2+ was added, and after
10 min at 37 °C, supernatants were collected, and -hexosaminidase
activity was measured. c-d, intracellular cAMP levels were
measured in NRK cells after treatment with cholera toxin
(c), permeabilization with SLO and treatment with 20 µg/ml
mastoparan (d), and exposure or not to forskolin. The lower
values in d when compared with c reflect an
inhibitory effect of the SLO permeabilization procedure on adenylyl
cyclase activity. The data represent the average of triplicate
determinations ±S.D.
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Microfilament Structure Modulates
Ca2+-dependent Exocytosis of
Lysosomes--
There is a significant amount of evidence suggesting
that the cortical actin network must be rearranged to allow secretory vesicles to contact and fuse with the plasma membrane (22, 31). To
investigate if the actin cytoskeleton also played a role in the
regulation of lysosomal exocytosis, NRK cells were treated with agents
that either disrupt (cytochalasin D, latranculin A) or stabilize
(phalloidin) actin microfilaments, and Ca2+-induced
-hexosaminidase release was assessed.
NRK cells were incubated or not with forskolin, followed by the
addition of cytochalasin D or latranculin A before permeabilization with SLO and exposure to Ca2+-containing buffer.
Cytochalasin D or latranculin A caused an enhancement of 60-70% for
control cells and of 30-35% for forskolin-treated cells in the levels
of Ca2+-induced
-hexosaminidase release (Fig.
5a). Treatment with these drugs had no significant effect on the base-line levels of
-hexosaminidase release observed in the absence of Ca2+
stimulation (Fig. 5a), indicating that microfilament
disruption, similarly to cAMP elevation, is not sufficient to promote
exocytosis of lysosomes.

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Fig. 5.
Microfilament disruption or stabilization
affects Ca2+-induced exocytosis of lysosomes.
a, NRK cells were treated or not with forskolin
(Fsk) for 20 min, followed or not by the addition of
cytochalasin D (Cyt-D) or latranculin A (Lat-A)
for 10 min before and during the SLO permeabilization step.
b, NRK cells were pretreated or not with forskolin,
permeabilized with SLO, incubated in buffer B containing or not
phalloidin for 10 min, followed by addition of buffer B containing 0 or
1 µM Ca2+ in the presence or absence of
phalloidin. Supernatants were collected after 10 min at 37 °C, and
-hexosaminidase activity was measured. The data represent the
average of triplicate determinations ±S.D.
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To investigate whether stability of microfilaments affected lysosomal
exocytosis and its potentiation by cAMP, NRK cells stimulated or not
with forskolin were exposed to the F-actin-stabilizing agent
phalloidin. Because phalloidin is membrane-impermeable, it was added to
the cells at the time of SLO permeabilization and kept during the
exocytosis assay period. After exposure to phalloidin, the level of
Ca2+-induced
-hexosaminidase release was reduced by 37%
(Fig. 5b). In forskolin-treated cells, a more pronounced
inhibition of Ca2+-induced
-hexosaminidase release was
observed (51%). These results indicate that stabilization of the actin
cytoskeleton, in addition to reducing the levels of
Ca2+-dependent lysosome exocytosis under normal
conditions, prevents the potentiation induced by cAMP.
The
-Adrenergic Agonist Isoproterenol Stimulates cAMP Production
and Lysosome Exocytosis in NRK Cells--
Forskolin and cholera toxin
are potent activators of adenylyl cyclase, which induce the
accumulation of very high levels of cAMP in NRK cells (Figs. 2 and 4).
We were thus interested in investigating the effect on lysosome
exocytosis of cAMP elevation triggered by cell stimulation under more
physiological conditions. For this we treated NRK cells with
isoproterenol, an agonist that binds to
-adrenergic receptors in
many cell types, activating adenylyl cyclase (32). As shown in Fig.
6a, cAMP levels in NRK cells
after exposure to isoproterenol were elevated, although not to the same
degree observed after forskolin treatment. Interestingly, a
dose-dependent stimulatory effect of isoproterenol on
lysosome exocytosis was detected when the culture supernatant of intact NRK cells was assayed for released
-hexosaminidase (Fig.
6b). The enhancement in exocytosis triggered by 2-4
µM isoproterenol was about 50%, a value comparable with
what is induced by forskolin or 8-Br-cAMP on SLO-permeabilized cells in
the presence of Ca2+ (Figs. 1 and 3). The overall
exocytosis levels in the experiment shown in Fig. 6a were,
however, lower than what is observed in NRK cells after
permeabilization and exposure to Ca2+-containing buffers.
Taken together, these results suggest that stimulation of the
-adrenergic receptor in intact cells provides sufficient stimuli to
trigger exocytosis of a small fraction of the lysosome population.

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Fig. 6.
Isoproterenol stimulates cAMP production and
exocytosis of lysosomes in intact cells. a,
intracellular cAMP levels were measured in NRK cells after treatment
with IBMX alone, IBMX and isoproterenol, or IBMX and forskolin.
b, NRK cells were exposed for 15 min at 37 °C to the
indicated concentrations of isoproterenol, and the supernatant was
collected and assayed for -hexosaminidase activity.
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Modulation in cAMP Levels Regulates Lysosome-mediated Cell Invasion
by T. cruzi--
Previous work showed that the intracellular protozoan
T. cruzi invades mammalian cells by an unusual mechanism,
which involves recruitment and fusion of host cell lysosomes at the
parasite entry site (11, 12). T. cruzi entry is
significantly enhanced by previous disassembly of host cell actin
filaments as well as by host cell treatment with membrane-permeant
analogs of cAMP (11). In addition, cell invasion by T. cruzi
requires parasite-induced [Ca2+]i transients (13,
14, 33). Taken together with the observations reported here, these
findings demonstrate that there are important parallels between the
T. cruzi cell invasion mechanism and the process of
Ca2+-dependent lysosome exocytosis. We were
thus interested in verifying if modulation of cAMP levels in host cells
under physiological conditions could also affect the T. cruzi invasion process. As shown in Fig.
7a, pretreatment of NRK cells
with the adenylyl cyclase inhibitor MDL-12,330A reduced invasion
levels, whereas exposure to isoproterenol had a stimulatory effect.
Furthermore, a consistent elevation in cAMP levels was detected in NRK
cells after exposure to the infective T. cruzi
trypomastigote stages but not after exposure to the noninfective
epimastigotes (Fig. 7b). These findings indicate that cAMP
generated in host cells in response to trypomastigotes may play a role
in the process of lysosome recruitment and fusion with the plasma
membrane that occurs during parasite invasion (11, 12).

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Fig. 7.
Modulation of adenylyl cyclase activity
regulates cell invasion by T. cruzi.
a, cell invasion by T. cruzi trypomastigotes was
quantitated after pretreatment or not of the host cells with 100 µM MDL-12,330A or 10 µM isoproterenol for
30 min at 37 °C. b, intracellular cAMP levels were
measured in NRK cells after exposure to IBMX alone, IBMX and T. cruzi epimastigotes, or IBMX and T. cruzi
trypomastigotes for 30 min.
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DISCUSSION |
Potentiation of both constitutive and regulated secretion by
elevation in cytosolic cAMP levels has been reported previously in
several cell types (15-19, 34). The cAMP-dependent
mechanisms responsible for enhanced exocytosis are unknown but may
include direct phosphorylation of secretory vesicle membrane components by the cAMP-dependent protein kinase A (35),
phosphorylation-dependent disassembly of the cortical actin
cytoskeleton (36-38), or modulation of
microtubule-dependent vesicular transport (39-41). Our
results show that Ca2+-dependent exocytosis of
lysosomes in NRK fibroblasts can be modulated by agents that regulate
cAMP levels and also provide evidence in support of a regulatory role
of the actin cytoskeleton in this process. Because a link between
[Ca2+]i, actin cytoskeleton rearrangements, and
lysosome fusion was demonstrated previously for the cell invasion
mechanism of the protozoan parasite T. cruzi (11-14), we
investigated if the signaling events known to be involved in this
process included host cell cAMP responses. Previous work had shown that
membrane-permeant analogs of cAMP enhance T. cruzi entry
into NRK fibroblasts (11). Here we show that enhancement in T. cruzi invasion is also observed upon host cell stimulation with
isoproterenol, an agonist that binds to
-adrenergic G
protein-coupled receptors, activating adenylyl cyclase. The smaller
levels of enhancement in T. cruzi entry observed after
isoproterenol treatment (Fig. 6a), when compared with
previous findings using membrane-permeant analogs of cAMP (11),
probably reflect the more physiologically significant and possibly
localized cAMP elevation generated by binding of this agonist to
-adrenergic receptors.
NRK cell exposure to isoproterenol triggered a small but
dose-dependent lysosome exocytosis response, as reflected
by the amount of
-hexosaminidase released into the culture
supernatant (Fig. 6b). This response was similar in
magnitude to the
-hexosaminidase exocytic response we previously
observed after cell stimulation with bombesin, which is another agonist
that activates intracellular signaling pathways by binding to G
protein-coupled receptors (6). Because in these experiments with
agonists the cells were not permeabilized and exposed to
Ca2+-containing buffers, the low lysosome exocytosis levels
observed were not surprising, reflecting a more physiologically
significant situation that may involve a concerted action of locally
generated Ca2+ and cAMP. Indeed, in neurons and myocytes,
activation of the
-adrenergic receptor-adenylyl cyclase-cAMP cascade
results in potentiation of Ca2+ channel activity (42-43).
Although similar studies have not been performed in NRK cells, our cAMP
measurements clearly indicate that stimulation of the
-adrenergic
receptor in these cells also results in adenylyl cyclase activation.
Elevated cAMP was observed in NRK cells after exposure to
trypomastigotes, the infective stages of T. cruzi, but not
after exposure to the noninfective epimastigote forms. This is a
significant finding, because trypomastigotes are the T. cruzi life cycle stages that are capable of invading host cells
through a Ca2+-dependent lysosome recruitment
process, which involves parasite-mediated signaling (11, 33).
Epimastigotes, on the other hand, are the insect vector stages that are
not capable of cell invasion or of triggering Ca2+
signaling in mammalian cells (13). It is thus conceivable that the
signaling events triggered by T. cruzi trypomastigotes in mammalian cells involve adenylyl cyclase activation and that this is an
important component of the parasite invasion mechanism. This conclusion
is reinforced by our finding that inhibition of NRK cell adenylyl
cyclase activity by MDL-12,330A has a strong inhibitory effect on
T. cruzi invasion (Fig. 7a). Taken together with
the finding that isoproterenol can trigger exocytosis of lysosomes, our
results provide evidence indicating that physiological cell stimulation
in conjunction with cAMP production facilitates mobilization of
lysosomes for fusion with the plasma membrane.
Receptor-mediated activation of the heterotrimeric GTPases
Gs and Gi or the expression of their active
subunits stimulate or inhibit adenylyl cyclase activity, respectively
(44). Ligation of the
-adrenergic receptor by isoproterenol, which
we showed to stimulate both lysosome exocytosis and T. cruzi
invasion, activates adenylyl cyclase through Gs (32).
Several lines of evidence point to the involvement of Gs
and Gi in the regulation of the exocytic pathway. In
epithelial cells, apical transport requires a Gs class of
heterotrimeric G protein (26) acting through cAMP elevation and
activation of protein kinase A (18, 19). Regulated exocytosis in
chromaffin cells appears to be controlled by at least two trimeric G
proteins acting in series, with a secretory granule-associated
Go protein controlling an ATP-dependent priming reaction and a plasma membrane-bound Gi3 involved in a late
Ca2+-dependent fusion step (27).
Gi, and to a lesser extent Go, were implicated
in the regulated exocytosis of insulin (45). As predicted, we observed
an enhancement in Ca2+-dependent exocytosis of
lysosomes in NRK cells after treatment with cholera toxin, which
activates Gs. Conversely, mastoparan, which activates
Gi or Go, has an inhibitory effect. As
discussed above, our results with the Gs activator
isoproterenol directly demonstrate that stimulation of a
receptor-mediated signaling pathway coupled to adenylyl cyclase can
modulate both lysosome exocytosis and host cell invasion by the
protozoan parasite T. cruzi.
It is well documented that cAMP induces
microtubule-dependent anterograde vesicle transport in a
variety of cell types (46, 47). Lysosomes in particular redistribute
from the perinuclear area to the cell periphery in response to
forskolin or to membrane-permeant analogs of cAMP (11, 24). The
cAMP-induced redistribution of lysosomes was attributed to cytosolic
acidification, also known to promote microtubule-dependent
anterograde movement of lysosomes (24, 48). Elevation in cAMP has been
linked to intracellular acidification in some cell types, such as rat
inner medullary-collecting duct cells, where increased cAMP levels
activate HCO3
exit pathways (49). It is thus
conceivable that acidification-dependent redistribution of
lysosomes to the cell periphery plays a central role in the
potentiation of both Ca2+-dependent exocytosis
and cell invasion by T. cruzi, observed after elevation in
cytosolic levels of cAMP.
The cortical actin cytoskeleton has long been proposed to act as a
physical barrier to exocytosis (20-22). From studies on adrenal
chromaffin cells, it was concluded that reorganization of the cortical
actin network is necessary to allow granules to reach exocytic sites in
stimulated cells (50, 51). Actin filament disassembly is sufficient for
triggering exocytosis in pancreatic acinar cells (40), although in
other cell types drugs that depolymerize actin do not elicit exocytosis
but can potentiate agonist-evoked responses (52, 53). Our observations
on exocytosis of lysosomes in NRK cells are consistent with these
findings; cytochalasin D or latranculin A do not trigger exocytosis in
the absence of Ca2+ but significantly potentiate the
Ca2+-evoked response. Furthermore, the enhancing effect of
microfilament-disrupting drugs is additive to the effect of forskolin.
When actin filaments are stabilized by exposure to phalloidin, the
opposite is observed. Ca2+-dependent exocytosis
of lysosomes is inhibited in both control and forskolin-treated cells.
The simplest interpretation of these results is that disassembly of the
cortical actin barrier facilitates access of lysosomes to the plasma
membrane for subsequent fusion. The observation that
Ca2+-induced lysosome exocytosis is impaired when
depolymerization of microfilaments is blocked by phalloidin is
consistent with this view, and because phalloidin reduces lysosome
exocytosis in forskolin-treated cells to a greater extent than in
control cells, it is conceivable that actin filaments act as an
obstacle to cAMP-induced anterograde movement of lysosomes on microtubules.
The results discussed above are fully consistent with previous findings
demonstrating that disruption of the actin cytoskeleton of mammalian
cells significantly facilitates invasion by T. cruzi (11).
Host cell [Ca2+]i transients induced by T. cruzi trypomastigotes trigger transient rearrangements in the
cortical actin cytoskeleton (14), and this event is required for
invasion (33). Because cell entry by T. cruzi involves
recruitment and fusion of host cell lysosomes, it is significant that
the cortical actin cytoskeleton also acts as a barrier for
Ca2+-dependent lysosome exocytosis. Taken
together with the effects of modulation in cAMP levels described in
this work, our observations make it increasingly clear that there are
important parallels between the mammalian signaling pathways that
trigger lysosome exocytosis and the mechanism of cell invasion utilized
byT. cruzi.