cAMP Regulates Ca2+-dependent Exocytosis of Lysosomes and Lysosome-mediated Cell Invasion by Trypanosomes*

Ana RodríguezDagger , Iñigo Martinez, Albert Chung, Catherine H. Berlot§, and Norma W. Andrews

From the Departments of Cell Biology and § Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, 06520

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ca2+-regulated exocytosis, previously believed to be restricted to specialized cells, was recently recognized as a ubiquitous process. In mammalian fibroblasts and epithelial cells, exocytic vesicles mobilized by Ca2+ were identified as lysosomes. Here we show that elevation in intracellular cAMP potentiates Ca2+-dependent exocytosis of lysosomes in normal rat kidney fibroblasts. The process can be modulated by the heterotrimeric G proteins Gs and Gi, consistent with activation or inhibition of adenylyl cyclase. Normal rat kidney cell stimulation with isoproterenol, a beta -adrenergic agonist that activates adenylyl cyclase, enhances Ca2+-dependent lysosome exocytosis and cell invasion by Trypanosoma cruzi, a process that involves parasite-induced [Ca2+]i transients and fusion of host cell lysosomes with the plasma membrane. Similarly to what is observed for T. cruzi invasion, the actin cytoskeleton acts as a barrier for Ca2+-induced lysosomal exocytosis. In addition, infective stages of T. cruzi trigger elevation in host cell cAMP levels, whereas no effect is observed with noninfective forms of the parasite. These findings demonstrate that cAMP regulates lysosomal exocytosis triggered by Ca2+ and a parasite/host cell interaction known to involve Ca2+-dependent lysosomal fusion.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 beta -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-beta -D-glucosaminidase (beta -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-beta -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 beta -hexosaminidase, cell extracts prepared as described above were diluted 1:10, and 350 ml was used for beta -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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -hexosaminidase. As shown in Fig. 1a, 100 µM forskolin stimulates Ca2+-induced lysosomal exocytosis; an 82% increase in beta -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 beta -hexosaminidase release. b, dose-dependent effect of forskolin pretreatment on Ca2+-dependent beta -hexosaminidase release. c, effect of pretreatment with forskolin or its inactive analog dideoxyforskolin (ddForskolin) on Ca2+-dependent beta -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 beta -hexosaminidase activity was measured. The data represent the average of triplicate determinations ±S.D.

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 beta -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 beta -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 beta -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.

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 beta -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 beta -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 beta -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 beta -hexosaminidase release. b, effect of treatment with IBMX and 8-Br-cAMP on Ca2+-dependent beta -hexosaminidase release. c, effect of treatment with IBMX, forskolin (Fsk), 8-Br-cAMP, or IBMX plus 8-Br-cAMP on beta -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). beta -Hexosaminidase activity assays were performed on supernatants, and the data represent the average of triplicate determinations ±S.D.

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 beta -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 beta -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 beta -hexosaminidase release. NRK cells were incubated with cholera toxin and permeabilized with SLO before performing beta -hexosaminidase release assays. b, effect of treatment with mastoparan on Ca2+-dependent beta -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 beta -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.

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 beta -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 beta -hexosaminidase release (Fig. 5a). Treatment with these drugs had no significant effect on the base-line levels of beta -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 beta -hexosaminidase activity was measured. The data represent the average of triplicate determinations ±S.D.

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 beta -hexosaminidase release was reduced by 37% (Fig. 5b). In forskolin-treated cells, a more pronounced inhibition of Ca2+-induced beta -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 beta -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 beta -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 beta -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 beta -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 beta -hexosaminidase activity.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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 beta -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 beta -adrenergic receptors.

NRK cell exposure to isoproterenol triggered a small but dose-dependent lysosome exocytosis response, as reflected by the amount of beta -hexosaminidase released into the culture supernatant (Fig. 6b). This response was similar in magnitude to the beta -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 beta -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 beta -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 beta -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.

    ACKNOWLEDGEMENTS

We are grateful to A. Ma and H. Tan for excellent technical assistance and to B. Burleigh for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant RO1AI34867 (to N. W. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Institute Curie, Section de Recherche, 26, Rue d'Ulm, 75231 Paris Cedex 05, France.

To whom correspondence should be addressed: Dept. of Cell Biology, Yale University School of Medicine, 333 Cedar St. New Haven, CT 06520. Tel.: 203-737-2410; Fax: 203-737-2630; E-mail: norma.andrews{at}yale.edu.

    ABBREVIATIONS

The abbreviations used are: IBMX, 3-isobutyl-1-methylxanthine; [Ca2+]i, intracellular free Ca2+ concentration; Gs, stimulatory heterotrimeric G protein; Gi, inhibitory heterotrimeric G protein; NRK, normal rat kidney; SLO, streptolysin O; 8-Br-cAMP, 8-bromo-cyclic AMP.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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