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* Laboratory of Molecular Immunology and
Light Microscopy Core Facility, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892; and
Department of Pharmacology and Center for Developmental Genetics, State University, Stony Brook, NY 11794
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies demonstrate a close relationship between secretion and PLD activation in the rat mast cell line RBL-2H3. For example, treatment of these cells with cholera toxin enhances PLD activation and secretion to the same extent without affecting the activation of other PLs (1). Also, inhibitors of PKs that regulate PLD activity suppress both PLD activation and secretion similarly (7). Finally, primary alcohols that divert production of phosphatidic acid by PLD to phosphatidylalcohol (referred to as a transphosphatidylation reaction) suppress secretion as well (1, 2). The transphosphatidylation reaction is used to assay PLD activity in vivo (see Materials and Methods) because, unlike phosphatidic acid, the phosphatidylalcohol is produced specifically by PLD, is relatively inert as a signaling lipid, and accumulates within the cell (8).
Two mammalian isoforms have been cloned, namely PLD1 and PLD2
(9, 10, 11). PLD1 can be activated by several mechanisms.
These include activation by small GTPases (12, 13, 14, 15, 16), Rho
kinase (17),
Ca2+/calmodulin-dependent (CaM) kinase II
(18), and PKC in a catalytically dependent and independent
manner (12, 19, 20). For example, recombinant PLD1 is
stimulated in vitro in the presence of phosphatidylinositol
4,5-bisphosphate by ADP-ribosylation factor (ARF), Rho proteins (i.e.,
RhoA, Rac1, and Cdc42), and PKC
by direct interaction with this PLD
(12, 13, 14, 15, 16). PLD2 also requires phosphatidylinositol
4,5-bisphosphate for expression of activity, but unlike PLD1 this
activity is not affected by the small GTPases or PKC
(10), although modest stimulation by ARF has been reported
by some workers (21, 22).
PLD is activated in isolated mast cells (23) and cultured mast cell lines such as the RBL-2H3 cell (1, 24, 25) by receptor ligands (23, 26), calcium ionophores (24), thapsigargin (1, 27), and PMA (24) to cause sustained production of phosphatidic acid and diglycerides (23, 24, 25). PLD is responsible for much of the increase in diglycerides in stimulated mast cells (23). The secretory response to Ag is lost on permeabilizing mast cells but can be fully restored by provision of ARF1 or phosphatidylinositol transfer protein, both of which increase levels of phosphatidylinositol 4,5-bisphosphate and restore the PLD and secretory responses to Ag (2). RBL-2H3 cells express message for PLD1b (one of the two variant forms of PLD1) and, to a much greater extent, PLD2 (A. Vaid, P. M. Holbrook, and M. A. Beaven, unpublished data). Subcellular fractionation studies indicate that both granule and plasma membrane fractions contain PLD activity (A. Chahdi, P. F. Fraundorfer, and M. A. Beaven, unpublished data). Although the type of PLD associated with these fractions is unclear, expressed enhanced green fluorescent protein (EGFP)-tagged PLD1 localizes primarily with the secretory granule (28) and endogenous PLD2 protein is located primarily in plasma membrane fractions (A. Chahdi, P. F. Fraundorfer, and M. A. Beaven, unpublished data). Our studies with expressed EGFP-tagged PLD1 and PLD2 also indicate that both isoforms are activated by the PLD stimulants noted above and that activation is markedly enhanced by prior treatment of RBL-2H3 cells with cholera toxin (7).
In this paper, we have taken advantage of the presence of both PLD1 and PLD2 in RBL-2H3 cells to determine the role of each isoform in the secretory process. Specifically, we have investigated the disposition of EGFP-tagged PLDs during cell stimulation and show that, while PLD1 is essential for the translocation of granules to the plasma membrane, PLD2 is essential for fusion of the granules to the plasma membrane and secretion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thapsigargin, PMA, and Ro31-7549 were purchased from LC
Laboratories (Woburn, MA); KN-92 and KN-93 were purchased from
Calbiochem (La Jolla, CA); H-89 was from Alexis Biochemicals (San
Diego, CA); GTP and GTP
S were from Boehringer Mannheim
(Indianapolis, IN); radiolabeled compounds were from DuPont-NEN
(Boston, MA); cholera toxin was from List Biologicals (Campbell, CA);
anti-rat mast cell protease II Ab was from Moredun Animal Health
(Midlothian, U.K.); cell culture reagents were from Life
Technologies (Rockville, MD); Tris-glycine polyacrylamide gels
were from NOVEX (San Diego, CA). DNP-BSA and DNP-specific monoclonal
IgE were purchased from Sigma-Aldrich (St. Louis, MO). The Ab against
the Fc
RII subunit was kindly supplied by Dr. J. Rivera (National
Institute of Arthritis and Musculoskeletal and Skin Diseases, National
Institutes of Health, Bethesda, MD).
Cell culture and experimental conditions
RBL-2H3 cells were grown as monolayers in MEM with Earle’s
salts, supplemented with glutamine, antibiotics, and 15% FBS. For each
experiment cells were harvested by trypsinization, transferred to
24-well (2 x 105 cells/0.4 ml/well) cluster
plates (29), and incubated overnight in complete growth
medium with 25 ng/ml DNP-specific IgE to achieve 100% occupancy of
FcRI. Where indicated, 1 µg/ml cholera toxin was added to cell
cultures 4 h before the experiment. Cultures were washed and the
required buffered solution was added (0.2 ml/well). Experiments on
intact cells were performed in a PIPES-buffered medium (25 mM PIPES (pH
7.2), 159 mM NaCl, 5 mM KCl, 0.4 mM MgCl2, 1 mM
CaCl2, 5.6 mM glucose, and 0.1% fatty acid-free
fraction V from bovine serum) or in calcium-free PIPES-buffered
medium (as above except 0.1 mM EGTA replaced 1 mM
CaCl2). Unless stated otherwise, cultures were
incubated for 30 min with or without inhibitors before addition of
stimulants for 15 min. Also, the concentrations of inhibitors and
stimulants used were those found to produce maximal effects, although a
suboptimal dose (3 ng/ml) of Ag was used in some experiments as
noted.
Assay of PLD by the transphosphatidylation assay and inhibition of phosphatidic acid formation by use of butanol
RBL-2H3 cells were incubated overnight with DNP-specific IgE in 24-well plates and, when required, exposed to cholera toxin as described above. [3H]Myristic acid (2 µCi/ml) was added for the final 90 min of incubation to label cellular phospholipids. Cells were then incubated in the PIPES-buffered medium in the presence of 1% ethanol for 10 min before stimulation. Under these conditions, [3H]phosphatidylethanol is formed by a PLD-specific transphosphatidylation reaction and accumulates within the cell (30). Radiolabeled phosphatidic acid and phosphatidylethanol were isolated by solvent extraction and quantified by TLC as described previously (26). The total amount of [3H]phosphatidylethanol and [3H]phosphatidic acid was expressed as a percentage of total [3H]phospholipid in Triton X-100 (Sigma-Aldrich) extracts of unstimulated cultures.
In experiments where butanol was used to suppress phosphatidic acid formation by PLD, 50 mM 1-butanol was added to cultures in place of ethanol. tert-Butanol, which is not a substrate for transphosphatidylation, was added to additional cultures as a control. [3H]Phosphatidylbutanol and [3H]phosphatidic acid were assayed as described above.
Construction of plasmids and techniques for transient transfection
Full-length cDNA were excised from hemagglutinin-tagged plasmids
by SmaI and HpaI for PLD1b and PLD1bK898R and
SmaI and XbaI for PLD2 and PLDK758R and subcloned
into a pEGFP-C expression vector (Clontech Laboratories, Palo Alto,
CA). The sequences (Fig. 1
A)
were confirmed by DNA sequence analysis. Cells were transiently
transfected with each DNA preparation (25 µg/2 x
107 cells) by electroporation (Gene Pulser, 960
µF, 250 V; Bio-Rad, Hercules, CA). Successful transfection was
confirmed by fluorescence microscopy and Western blotting. Cells were
used within 24 h of transfection. All expressed PLDs were EGFP
fusion proteins of PLD1b, PLD2, PLD1bK898R, and PLD2K758R, even when
referred to in the text without the EGFP designation.
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RBL-2H3 cells were transfected with EGFP vector or the EGFP-tagged PLDs by electroporation as described above and then suspended in complete growth medium before transfer to Lab-Tek chambered coverslips (Nalge Nunc International, Naperville, IL). The chambered coverslips were then incubated overnight (16 h) at 37°C. Stimulants and other reagents were added directly to the chambers for the times indicated in the text. The cultures were then washed three times with PBS immediately before microscopic examination. For double labeling of cells, all incubations were performed at room temperature and samples were washed three times with PBS as indicated. Cultures were fixed with 4% formaldehyde in PBS for 10 min, washed, permeabilized with 0.5% Triton X-100 for 15 min, and then washed before incubation for 1 h with a blocking reagent, 1% BSA in PBS. The samples were then incubated for 2 h with a solution of the primary Ab in 1% BSA in PBS and, after washing, with the secondary Ab conjugated with rhodamine for a further 45 min. After washing, samples were mounted using the Prolonged Antifade kit (Molecular Probes, Eugene, OR).
All confocal images were obtained using a Zeiss LSM410 confocal laser scanning microscope and a Fluar x100 objective (1.3 numerical aperture; Zeiss, Oberkochen, Germany). EGFP fluorescence images were acquired by use of the 488-nm excitation line of an argon laser, a LP 505-nm emission filter, and a pinhole set to produce a 1.25-µm slice. For transmitted light images, the 543-nm line of a HeNe laser was used. The images were collected at a size of 103 x 103 pixels and a data depth of 8 bits. Custom-written programs in the IDL programming language (Research Systems, Boulder, CO) were used for morphometric analysis. These programs evaluated the average distance of all fluorescent pixels from the nearest outer edge of the cell perimeter. This program may underestimate actual distance of remaining granules when these are obscured by reuptake of EGFP-tagged PLD into Golgi (see Results).
Presentation of results
Individual experiments were performed with three to four cultures for each data point. The data are presented as the mean ± SEM from three or more separate experiments as indicated. Statistical significance was determined by use of the SigmaPlot 2000 (Jandel Scientific, Corte Madera, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EGFP-tagged PLDs (Fig. 1
A) were expressed in RBL-2H3
cells by electroporation. Consistent with the extent of expression of
endogenous mRNA for PLD1 and PLD2, EGFP-PLD2 was expressed more
efficiently (
7-fold by densitometric measurement) than EGFP-PLD1
(Fig. 1B). Examination of the cells by phase contrast and
confocal microscopy revealed that PLD1 was localized with intracellular
structures and that PLD2 was localized in the vicinity of the plasma
membrane (Fig. 1C). As shown in Fig. 1D,
EGFP-PLD1 colocalized with the granule marker, rat mast cell protease
II (31), although some granules were not associated with
the EGFP label. PLD2, in contrast, colocalized with the subunit of
FcRI in the plasma membrane. These data confirm previous findings
that EGFP-PLD1 is associated with secretory granules in RBL-2H3 cells
(28) and show for the first time that EGFP-PLD2 is
expressed exclusively on the plasma membrane in these cells. In
addition, stimulation of cells with Ag or thapsigargin resulted in
translocation of most if not all PLD1-labeled structures to the cell
periphery and the relocation of PLD1 to the plasma membrane, probably
as a consequence of fusion of the labeled structures with the plasma
membrane (Fig. 2). The doses of
stimulants used in this experiment produced maximal secretion of
another granule marker, hexosaminidase (40% release, data not
shown).
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) and secretion (data not
shown). As seen in Fig. 2, these structures remained intact and there
was little, if any, transfer of EGFP-PLD1 to the plasma membrane.
The EGFP-tagged catalytically inactive mutant of PLD1 (PLD1K898R) also
associated with granule-like structures (Fig. 2). However, in cells
expressing this mutant there was no apparent translocation of these
granules after cell stimulation. Therefore, the catalytic activity of
PLD1 is necessary for translocation of granules, and expression of
PLD1K898R blocks this translocation.
In contrast to EGFP-PLD1, wild-type EGFP-PLD2 and its catalytically inactive mutant, PLD2K758R, were expressed on the plasma membrane. No marked changes in this distribution occurred whether cells were stimulated with Ag, thapsigargin, or PMA.
Recycling of PLD1 to secretory granules after cell stimulation
The translocation and fusion of EGFP-PLD1-labeled granules with
the plasma membrane was evident 5 min after addition of Ag (Fig. 3A). Few intact labeled
granules remained after 15 min. In accord with these results, release
of hexosaminidase was apparent by 5 min and largely complete by 15 min
(data not shown). At 15 min almost all of the EGFP label was
located in the plasma membrane. Thereafter, EGFP begun to appear in
extranuclear (possibly perinuclear) structures (i.e., 30- and 60-min
time points in Fig. 3). By 240 min the label had mostly disappeared
from the plasma membrane and was now again associated with granule-like
structures. These results indicated that PLD1 did not remain in the
plasma membrane but was recycled to residual or newly matured granules.
As noted previously, there was no apparent change in the disposition of
PLD2 during the course of stimulation (Fig. 3A). The
movement of granule-associated PLD1 to the cell membrane and the
reassociation of PLD1 with granules was verified by morphometric
analysis of data from several experiments (Fig. 3B).
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Another indication that movement of granules to the cell periphery
was dependent on PLD1 activity came from studies with 1-butanol which,
as noted earlier, diverts production of phosphatidic acid by PLD to
phosphatidylbutanol. tert-butanol (2-methyl-2-propanol),
which is not a substrate for this transphosphatidylation reaction, was
used as a control reagent (for examples, see Refs. 33 and
34). The presence of 50 mM 1-butanol suppressed the
translocation of EGFP-labeled granules (Fig. 4A, with morphometric data
shown in Fig. 4B) and secretion of hexosaminidase (Fig. 4C), possibly through a decreased production of
[3H]phosphatidic acid (60% reduction, data
not shown). However, tert-butanol had minimal or no
discernable effect on these responses. The inhibitory effects of
1-butanol on translocation were consistently observed in all
experiments but were contrary to an earlier report (35)
for reasons that are unclear.
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We next examined stimulants of PLD, such as PMA and cholera toxin,
that do not elicit an increase in
[Ca2+]i (32, 36) or secretion (7) in RBL-2H3 cells.
Interestingly, both PMA (see Fig. 2) and more so cholera toxin (Fig. 5A, with morphometric data
shown in Fig. 5B) induced movement of granules to the cell
periphery. Treatment with cholera toxin induced movement of granules to
the cell periphery without apparent fusion of granules with the plasma
membrane (Fig. 5A, second panel), and 1-butanol retards this
movement (Fig. 5B, third panel). However, additional studies
revealed that treatment with cholera toxin substantially enhanced the
extent of fusion of granules with the plasma membrane (Fig. 5A, final panel), activation of PLD (Fig. 5C),
and secretion (Fig. 5D) when the cells were subsequently
stimulated with a low dose of Ag (3 ng/ml). Cholera toxin by itself did
not induce secretion (Fig. 5D). Cholera toxin treatment
enhanced not only the extent of secretion (i.e., as in Fig. 5D) but also the rate of the secretory response to low-dose
Ag (Fig. 5E). This accelerated response was attenuated in
the presence of 1-butanol but not in the presence of
tert-butanol (Fig. 5F). Studies with a minimal
stimulatory dose (75 nM) of thapsigargin produced virtually identical
results to those shown in Fig. 5 (data not shown). These results
suggested that, after treatment cholera toxin, granules were poised for
rapid fusion with the plasma membrane once
[Ca2+]i increased upon
addition of Ag or thapsigargin.
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A). However, the PKC
inhibitor, Ro31-7549, had unexpected effects. Although Ro31-7549
blocked fusion of PLD1-labeled granules with the plasma membrane (Fig. 6A), effects on translocation were unclear because this
inhibitor perturbed intracellular structure in both stimulated and, to
a lesser extent, unstimulated cells (data not shown). We found no
previous reports of disruption of intracellular structure by PKC
inhibitors. All three kinase inhibitors suppressed secretion (Fig. 6B).
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A)
and maximal (Fig. 7B) inhibition of secretion. Secretion was
still blocked by all three kinase inhibitors in cholera toxin-treated
cells. Nevertheless, cholera toxin-treated cells were more resistant
than control cells to the inhibitory effects of KN93, especially with
the high dose of this inhibitor (Fig. 7B). This increased
resistance was verified by additional experiments with various doses of
KN93 (Fig. 7, C and D). Similar results were
obtained in studies with thapsigargin (data not shown). These studies
indicated that all three PKs participated at some stage of the
secretory process and, possibly, that CaM kinase II plays a less
prominent role than PKC and PKA in the final fusion events.
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The preceding findings strongly indicated that PLD1 was essential
for translocation of granules to the cell periphery before their
eventual fusion with the plasma membrane. We next investigated whether
expressed PLD2 participates in the secretory process, because its
location in the plasma membrane might facilitate membrane fusion. The
secretory response to thapsigargin was enhanced by expression of either
EGFP-tagged PLD1 or PLD2 and suppressed by expression of the
catalytically inactive mutants of PLD1 and PLD2 (Fig. 8A). Similar but more modest
effects on secretion were observed in Ag-stimulated cells (Fig. 8B). Analysis by flow cytometry indicated that 20–30% of
cells expressed EGFP-tagged PLDs. Experiments with cells selected for
expression of the EGFP-tagged PLDs (5) were unsuccessful
because of poor responses to Ag after the protracted selection
procedure.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this paper we show that both isoforms of expressed PLD can participate in the secretory process and may do so at different steps, namely PLD1 in the movement of secretory granules to the periphery and PLD2 (and possibly PLD1) in the fusion of granules with the plasma membrane. The evidence for these conclusions is the following: 1) PLD1 is expressed predominantly on secretory granules and PLD2 on plasma membrane; 2) both PLD isoforms probably participate in the secretory process, as expression of the catalytically inactive form of either PLD1 or PLD2 suppresses thapsigargin-induced and, to a lesser extent, Ag-induced secretion; and 3) a role for PLD1 in the movement of granules to the cell periphery is suggested by the suppression of this phase of secretion in cells expressing the catalytically inactive form of PLD1. The location of PLD2 implies that its role is restricted to the plasma membrane, where it presumably facilitates docking or fusion of the granule with the plasma membrane. However, the data do not exclude a role for PLD1 as well as PLD2 in these fusion events.
It would appear that Ag-induced secretion is less dependent on PLD than
thapsigargin-induced secretion (Fig. 8). The reason for this is
unknown, but, unlike Ag, thapsigargin weakly stimulates PLC
(1), and activation of PKC by thapsigargin (Z. Peng and M.
A. Beaven, unpublished observations) may depend primarily on
PLD-mediated generation of PKC activators. However, PLD could regulate
secretory events other than PKC, as will be discussed later.
If PLD1 regulates translocation of granules and PLD2, alone or in combination with PLD1, regulates fusion of translocated granules with the plasma membrane, the activation of these two isoforms must then be coordinated to maintain effective coupling of the two events. Both PLD1 and PLD2 when expressed in RBL-2H3 cells respond to the same external stimulants (7). They are both activated in a synergistic manner by any combination of PMA, thapsigargin, and cholera toxin and by Ag (7). These and other studies with inhibitors in RBL-2H3 cells suggest that both expressed isoforms of PLD as well as endogenous PLD may be regulated by PKC, CaM kinase II, and PKA, although it is unknown whether these kinases regulate PLD directly or indirectly. The fact that expressed PLD1 and PLD2 respond in tandem to the same stimulants means that either both isoforms are directly regulated by the same stimulatory signals or activation of PLD1 leads to activation of PLD2 as granules begin to fuse with the plasma membrane. PLD2 requires phosphatidylinositol 4,5-bisphosphate for its activity (10), but unlike PLD1 no other regulatory factor has been described to date for PLD2 (37). Phosphatidylinositol 4,5-bisphosphate production is increased following PLD activation (2, 38, 39), probably as a consequence of stimulation of type I phosphatidylinositol 4 phosphate 5-kinase (40) by the PLD product, phosphatidic acid. Thus, one possible scenario to account for the coactivation of both isoforms of PLD is that, once granules and their associated PLD1 begin to fuse with the plasma membrane, PLD1-stimulated phosphatidylinositol 4,5-bisphosphate production leads to activation of PLD2 in the plasma membrane. This scenario extends the notion of a positive feedback loop between phosphatidylinositol 4 phosphate 5-kinase and PLD1 (39) to include PLD2.
Further studies are necessary to elucidate the exact roles of the PKs and calcium at different steps of the secretory process in mast cells. The studies with inhibitors suggest that in Ag-stimulated cells PKA, PKC, and CaM kinase II all participate at one or more stages of the secretory process and that translocation of PLD1-labeled granules is regulated by PKA and CaM kinase II. The role of PKC in the latter process is uncertain. Nevertheless, the ability of cholera toxin, and possibly PMA, to induce movement of granules to the cell periphery suggests that this process can be mediated via a single kinase and can occur regardless of changes in [Ca2+]i. Therefore, there may be some redundancy in the regulation of PLD and secretion by the various PKs.
Many studies of exocytosis have focused on the interaction of phosphatidylinositol 4,5-bisphosphate and PKs with proteins such as synaptotagmin and the calcium-dependent activator protein for secretion that are thought to promote docking and fusion of granules with the plasma membrane. PKC, CaM kinase II, and cAMP-dependent kinases have been implicated in the regulation of recruitment of secretory granules in various types of secretory cells (reviewed in Ref. 41). Phosphatidylinositol 4,5-bisphosphate is required for successful exocytosis of granules in neuroendocrine and chromaffin cells, where it may recruit and regulate the interaction of the calcium sensor, synaptotagmin (42), and the calcium-dependent activator protein for secretion with the fusion apparatus (43). The connections of PLD to these events have not been determined, but regulation of exocytosis and PLD by the same PKs and the stimulation of phosphatidylinositol 4,5-bisphosphate synthesis via PLD point to possible connections. Other possible connections include recruitment and activation of essential components to membranes by virtue of the charged nature of phosphatidic acid (44) and the promotion of membrane fusion by phosphatidic acid itself. PLA2-mediated fusion of neutrophil membranes is augmented by phosphatidic acid and, of relevance to this study, stimulation of intact neutrophils leads to activation of PLD in granules as well as plasma membrane (6).
Although we believe from our data that the two isoforms of PLD act cooperatively in coordinating translocation and fusion events in RBL-2H3 cells, it is likely that the exact role of PLD in secretion may vary from one cell type to another. A recent report, also based on studies with expressed PLDs, indicates an essential role for PLD1 but not PLD2 in fusion events in chromaffin cells (5). In these cells, PLD2 was not detectable and PLD1 was located primarily in the plasma membrane.
An interesting detail in the present studies is the recycling of PLD1
from the plasma membrane to granules. The phenomenon points to possible
reuse of this enzyme by the cell. It incidentally reassured us that
EGFP-PLD1 was not initially expressed at an inappropriate site within
the cell (45). Another interesting detail is that only a
fraction of rat mast cell protease II-positive granules acquired PLD1
(Fig. 1C), yet the majority of PLD1-labeled granules
appeared to be secreted in all cells. A consistent feature of RBL-H3
cells is that secretion of other granule constituents such as
histamine, 5-hydroxytryptamine, and hexosaminidase rarely exceeds 50%
of that originally present in the cells (for examples, see Refs.
4, 29 , and 32). This is probably due to
incomplete secretion of granules from individual cells, as almost all
cells (90%) secrete granules in response to Ag (Fig. 5 in Ref.
46). Therefore, it is possible that only granules
associated with PLD1 are competent to secrete, but this remains to be
determined.
In summary, PLD1 in RBL-2H3 cells appears to play an essential role in translocation of secretory vesicles/granules to the plasma membrane through recruitment of newly synthesized and recycled PLD1, although the molecular details of how this is accomplished requires further study. In addition, PLD1 and PLD2 appear to serve distinct but complementary roles in secretion when both are expressed within the cell.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael A. Beaven, National Institutes of Health, Room 8N109, Building 10, Bethesda, MD 20892-1760. E-mail address: beavenm{at}nhlbi.nih.gov
3 Abbreviations used in this paper: PLD, phospholipase D; ARF, ADP-ribosylation factor; CaM, calmodulin/Ca2+ dependent; [Ca2+]i, concentration of cytosolic free calcium; EGFP, enhanced green fluorescent protein; PK, protein kinase.
Received for publication June 22, 2001. Accepted for publication March 22, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Biol. Chem. 272:3860.
-granule transport and exocytosis. Semin. Cell Dev. Biol. 11:53.[Medline]
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R. Galandrini, F. Micucci, I. Tassi, M. G. Cifone, B. Cinque, M. Piccoli, L. Frati, and A. Santoni Arf6: a new player in Fc{gamma}RIIIA lymphocyte-mediated cytotoxicity Blood, July 15, 2005; 106(2): 577 - 583. [Abstract] [Full Text] [PDF]
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P. Huang, Y. M. Altshuller, J. C. Hou, J. E. Pessin, and M. A. Frohman Insulin-stimulated Plasma Membrane Fusion of Glut4 Glucose Transporter-containing Vesicles Is Regulated by Phospholipase D1 Mol. Biol. Cell, June 1, 2005; 16(6): 2614 - 2623. [Abstract] |
, Ag plus cholera
toxin; •, Ag; 
, cholera toxin). The effects of 1- and
tert-butanol on secretion in cholera toxin-treated cells
were determined in three additional experiments (F). The
data show mean ± SEM except where values for SEM fall within the
data points. Significant differences in values are indicated by
asterisks (*, p < 0.05; **,
p < 0.01).
