Departments of 1 Oral Biology and 2 Pharmacology, University of Washington, Seattle, Washington 98195
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rap1 has
recently been identified on the secretory granule membrane and plasma
membrane of rat parotid acinar cells (N. J. D'Silva, D. DiJulio, C. B. Belton, K. L. Jacobson, and E. L. Watson. J. Histochem. Cytochem. 45: 965-973, 1997). In the
present study, we examined the cellular redistribution of Rap1
following treatment of acini with isoproterenol (ISO), the
-adrenergic agonist, and determined the relationship between
translocation and amylase release. In the presence of ISO, Rap1
translocated to the cytosol in a concentration- and time-dependent
manner; this effect was not mimicked by the muscarinic agonist,
carbachol. Translocation was maximal at 1 µM ISO and paralleled
amylase release immediately after ISO stimulation. Rap1 translocation
and amylase release were blocked by the
-adrenergic antagonist,
propranolol, whereas okadaic acid, a downstream secretory inhibitor,
significantly blocked amylase release but did not inhibit Rap1
redistribution. Results suggest that the translocation of Rap1 is
causally related to secretion and that the role of Rap1 in secretion is
at a site proximal to the exocytotic event.
small GTP-binding protein; translocation; salivary gland; exocrine secretion; amylase release
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN THE RAT PAROTID gland, protein secretion occurs via the regulated, constitutive, or paragranular pathways (39). In regulated secretion, secretory granules fuse with the plasma membrane and release secretory granule contents in response to cell stimulation. The actual fusion event (exocytosis) involves the fusion of the outer surface of the secretory granule with the inner surface of the plasma membrane and release of secretory granule contents. On the basis of results published by Gomperts (14), showing that guanosine 5'-O-(3-thiotriphosphate) can stimulate secretion when upstream signaling pathways are blocked, it has been suggested that downstream secretory events may be regulated by a GTP-binding protein, GE. However, it is not clear whether GE is a heterotrimeric or monomeric (small) GTP-binding protein (Smg) or represents more than one GTP-binding protein. The identification of Smgs on secretory granules of exocrine (9) and other secretory cells (7, 26) supports a role for these proteins in exocytosis.
Several Smgs have been implicated in exocytosis. For example, Rab3 proteins have been associated with regulated exocytosis in neuronal and neuroendocrine cells (7, 11, 12). Another Smg, Rap1, has also been linked to secretion in human neutrophils (26). Rap proteins are members of the Ras subfamily of the Ras superfamily of GTP-binding proteins (4). These Rap proteins have 50% homology to Ras oncoproteins and are found in nearly all tissues but differ from Ras in cellular and subcellular localization (4, 24, 26). Rap proteins are divided into two subgroups, Rap1 and Rap2, each of which has two subtypes: A and B (4). Evidence in yeast suggests that the secretory role of Rap1 is at the exocytotic step (27). In neutrophils, the identification of Rap1 primarily on the specific granules suggested that it plays a crucial role in phagocytosis, an exocytotic event that involves degranulation of the specific and azurophilic granules (26). This hypothesis was supported by studies that showed that, on stimulation of the Ca2+-dependent pathway, Rap1A translocated from the secretory granules to the plasma membrane with cytochrome b, a component of the nicotinamide adenine dinucleotide phosphate oxidase system (26, 31). The recent identification of Rap1 on secretory granules of rat parotid acinar cells (9) also suggests that it plays a role in secretion in a manner analogous to that postulated for Rap1A in neutrophils (26) and Rap1B in platelets (10, 28).
In general, Smgs regulate vesicular transport by cycling between membrane-bound and soluble forms, depending on the GTP- vs. GDP-bound state or modifications such as phosphorylation or isoprenylation (13, 15, 20). Translocation of a Smg in association with secretion has been used as evidence for its involvement in the latter (26), with the underlying assumption that proteins regulate secretion by shuttling between donor and acceptor compartments (39). For example, in nerve terminals, Rab3A and Rab3C dissociate from synaptic vesicles on Ca2+-dependent exocytosis (11, 12). This dissociation occurs during or after the exocytotic event and is dependent on the latter.
In the present study, we explored the secretory function of Rap1 by
examining the translocation of Rap1 in rat parotid acini in response to
isoproterenol (ISO) and its relationship to amylase release. Results
show that ISO causes the redistribution of Rap1 from the particulate to
the cytosolic fraction in a concentration- and time-dependent manner
and that this event is coincident with amylase release. Data also show
that both ISO-induced translocation and amylase release are blocked by
the -adrenergic receptor antagonist, propranolol, whereas okadaic
acid, a downstream inhibitor of secretion, inhibits amylase release
without an effect on translocation. Results suggest that Rap1 is
involved in secretion but at a site that is proximal to the exocytotic
event. Furthermore, data suggest that cAMP-mediated phosphorylation is
important for the translocation of Rap1.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of rat parotid acini. Rat parotid glands were isolated from male Sprague-Dawley rats (100-120 g), and parotid acini were prepared as described previously (9).
Preparation of rat parotid crude membrane fraction and cytosol. Rat parotid acini were homogenized with a glass Teflon pestle tube with a 0.012- to 0.014-in. clearance (Kontes, Vineland, NJ), and 250 g supernatant and pellet fractions were obtained as described by Robinovitch et al. (33). The cytosol was prepared from the 250 g supernatant, which was centrifuged at 12,000 g for 10 min and then followed by a centrifugation at 100,000 g for 1 h. The resultant supernatant was collected as the cytosolic fraction, and the pellet was discarded. For preparation of the crude membrane fraction, rat parotid acini were homogenized and centrifuged at 12,000 g for 10 min. The resultant pellet, which selected for organelles of high buoyant density including secretory granules, was designated the crude membrane fraction. Proteins were quantified using the Folin method of Hartree (16) with BSA as a standard.
Translocation studies. For these studies, cell aliquots were treated with an agonist in the presence or absence of an antagonist. Control samples were treated with distilled water and/or the appropriate vehicle of suspension for the antagonist. In all experiments, the cytosolic fraction was prepared from control and stimulated cells.
Amylase release. After treatment of parotid acini with the appropriate pharmacological agent, cell aliquots were retrieved for amylase determinations according to the method of Bernfeld (3). One-milliliter samples were centrifuged at 14,000 rpm for 30 s in a microcentrifuge. Amylase determinations were made for both the supernatant and pellet fractions. Amylase released into the supernatant was expressed as percent of total amylase, as described by Ito et al. (18). Data are expressed as means ± SE.
Immunochemiluminescent analysis. The cytosolic fractions were resolved by SDS-12% PAGE and transferred to 0.2-µm polyvinylidene difluoride (PVDF) (Novex, Encinatas, CA) or 0.1-µm Immobilon PVDF (Millipore) filters. For most of the studies, Novex PVDF filters were used. However, Immobilon filters, because of their smaller pore size, were utilized in the latter studies. The filters were blotted with rabbit anti-Rap1 affinity-purified polyclonal antibody, and antibody binding was detected with the enhanced chemiluminescence Western blotting detection system (Amersham, Arlington Heights, IL) using affinity-purified horseradish peroxidase-linked donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) as a secondary antibody.
Data analysis. Densitometric analysis of the chemiluminograms was done using Molecular Dynamics' ImageQuant software. Several chemiluminograms at different exposure durations were obtained for each experiment. The optical density obtained for the Rap1 signal on every chemiluminogram was plotted against time. Only data that were in the linear range of the film were accepted for further analysis. The average optical density for the linear exposures was calculated, and data from treated samples were expressed as percent of the corresponding control. Statistical analysis was done by a one-tailed Student's t-test. A one-tailed Student's t-test was used because a cytosolic increase in Rap1 was predicted, based on results from platelets (10). Data are presented as means ± SE unless otherwise indicated.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the rat parotid gland, significant amylase release has been shown to
occur on stimulation of the -adrenergic receptor acting
via the cAMP-protein kinase A (PKA) pathway (32). Hence, in initial
experiments, the translocation of Rap1 to the cytosol following
stimulation of parotid acini with ISO was determined. Rap1 was found to
translocate to the cytosol when rat parotid acinar cells were
stimulated with 1 µM ISO for 20 min (Fig.
1A), a
time frame in which a significant amount of amylase release occurs in
both glands and dispersed acini (Fig.
1A) (18, 25). The increase in
cytosolic Rap1 was 189 ± 44% (n = 4) of the corresponding control (Fig.
1A) and corresponds to a decrease
in Rap1 in the membrane fraction (Fig.
1B). In contrast to the results
obtained with ISO, there was no significant change in cytosolic Rap1 in cells treated for 20 min with the muscarinic receptor agonist, carbachol (50 µM) (Fig. 1A).
Increasing the Ca2+ concentration
in the incubation buffer from 0.2 to 1.28 mM had no effect on Rap1
translocation to the cytosol (data not shown).
|
In other studies, a concentration response to ISO at 20 min showed that maximal translocation to the cytosol was observed in rat parotid acinar cells stimulated with 1 µM ISO (Fig. 2). Occasionally (e.g., Fig. 2), in ISO-stimulated cells, cytosolic Rap1 resolved as a doublet and both signals increased in intensity. This doublet was thought to represent phosphorylated or unphosphorylated forms of Rap1 (20) or be due to the presence (24 kDa) or absence (22 kDa) of a reducing agent during Rap1 solubilization (6, 8, 30). The doublet was likely a function of incomplete reduction because the Rap1 antibody used in these studies did not predictably detect the low-molecular-mass Rap1 signal in cytosol samples solubilized in the absence of a reducing agent (data not shown). Translocation of Rap1 to the cytosol paralleled amylase release; maximal amylase secretion was also observed at a concentration of 1 µM ISO (Fig. 2) (1, 2).
|
On the basis of these findings, further studies were conducted to determine the time course of ISO-induced translocation of Rap1. The first time point taken was at 30 s because this was the earliest time at which samples could be collected. As shown in Fig. 3, translocation of Rap1 to the cytosol was time dependent and occurred as early as 30 s after stimulation with ISO. Translocation appeared to be maximal at 20 min and decreased slightly thereafter, consistent with the shuttling or recycling phenomenon of Smgs, described by Wagner and Williams (40). Amylase release, on the other hand, increased up to 40 min, the last time period monitored. It was also noted that there was a slight increase in the density of the Rap1 signal in control samples with time, which is likely due to basal secretion previously noted by Spearman et al. (34).
|
To further explore the involvement of Rap1 in amylase secretion, the
redistribution of Rap1 was investigated when amylase release was
blocked with propranolol, a -adrenergic antagonist that
competitively binds the
-adrenergic receptor, thereby inhibiting secretion by acting upstream in the secretory cascade. Rat parotid acini were preincubated with 10 µM propranolol or distilled water for
2 min followed by a 15-min incubation with 1 µM ISO. At 15 min,
sufficient amylase was released, and the cytosolic redistribution of
Rap1 was readily detectable (see Fig. 3). As shown in Fig. 4, ISO-induced translocation of Rap1 to the
cytosol was 167 ± 24% (n = 3) of
the control and was competitively inhibited by 10 µM propranolol.
ISO-induced amylase secretion, which was 159% greater than control,
was also completely inhibited in the presence of 10 µM propranolol.
|
Because results were consistent with a role for Rap1 in secretion, the next step was to investigate a possible site of action, i.e., downstream vs. upstream, in the secretory cascade. We rationalized that the best approach for determining whether Rap1 is directly involved in exocytosis would be to try to dissociate the translocation of Rap1 to the cytosol from the exocytotic event. Rat parotid acini were treated with 1 µM ISO in the presence or absence of okadaic acid, a phosphatase inhibitor that also inhibits secretion (35, 41). Because okadaic acid was found to inhibit cAMP-induced amylase release in rat parotid acini (35), Wagner et al. (41) suggested that it acts downstream in the secretory pathway, i.e., distal to cAMP-PKA. Okadaic acid at 10 and 1 µM significantly inhibited (~70 and 50%, respectively) amylase release from ISO-treated rat parotid acini (Fig. 5) (35) and was, therefore, used as a tool to inhibit secretion. It was predicted that, if Rap1 directly mediates exocytosis, then its translocation would be blocked when secretion is inhibited downstream in the secretion pathway, i.e., proximal to the exocytotic event. This was based on findings that the translocation of a Smg from the secretory vesicle during or immediately following the exocytotic event is supportive of its direct role in exocytosis (11, 12). For these studies, rat parotid acini were treated with 1 µM ISO for 15 min in the presence of 10 or 1 µM okadaic acid or ethanol (control). Studies were done at both concentrations of okadaic acid to test whether effects on translocation would also be concentration dependent. Densitometric quantification of the immunoblot data (Fig. 5) showed that Rap1 translocation was not inhibited by either 10 or 1 µM okadaic acid, thereby indicating that Rap1 translocates to the cytosol even when secretion is inhibited. The increase in cytosolic density of Rap1, expressed as percent of control, was 179 ± 11% with 1 µM ISO, 172 ± 3% with 10 µM okadaic acid + ISO, and 168 ± 10% with 1 µM okadaic acid + ISO.
|
Because 0.5-1 µM okadaic acid may fragment subcellular organelles after a 30-min incubation (42), cell viability and plasma membrane intactness were assessed by microscopic examination of trypan blue exclusion at the end of the ISO incubation period; >95% of the acini examined excluded the dye.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The parotid gland is an exocrine gland with two major signaling
pathways. The inositol
1,4,5-triphosphate-Ca2+ pathway
primarily mediates fluid and electrolyte release, whereas proteins
destined for secretion are released primarily via the -adrenergic-cAMP pathway (32). The localization and high levels of
Rap1 on the secretory granule membrane suggested that it has a role in
protein secretion in the parotid gland (9). The major findings of this
study are that Rap1 is redistributed to the cytosol in rat parotid
acini stimulated with the
-adrenergic agonist, ISO, and that Rap1
appears not to have a direct involvement in the exocytotic process.
However, because secretion represents a series of sequential steps,
Rap1 may play a direct role in secretion at a step proximal to the
exocytotic site. We used translocation as an indicator of the role of
Rap1 in secretion because Smgs have been shown to translocate in
association with the latter (12, 26, 28). Our results confirm and
extend the findings for a role for Rap1 in secretion in hematopoietic
cells to the parotid gland. A close parallel was established between
ISO-induced translocation of Rap1 and amylase release from both rat
parotid tissue slices and dispersed acini, reported here and previously (18, 25).
To further explore the relationship between ISO-induced translocation and amylase release (exocytosis), we sought to separate the two events. We investigated this by inhibiting secretion with propranolol and okadaic acid (35), proximal and distal secretory inhibitors, respectively. Studies with propranolol clearly showed that translocation is coincident with amylase release. However, studies with okadaic acid showed that translocation occurred even when amylase release was significantly blocked (70%). It was unlikely that the ~30% of secretion that was not inhibited was correlated with Rap1 translocation. As shown in Fig. 2, amylase release and translocation are closely correlated (r = 0.953; critical value is 0.874 at the 1% level of significance), i.e., translocation does not appear to be an all or none phenomenon. Thus results suggest that Rap1 has a role in secretion proximal to the exocytotic event but distal to cAMP-PKA (35, 41). Its function may precede or be parallel to the okadaic acid-sensitive step. Yamashina (43) characterized the effects of okadaic acid in the rat parotid gland and recently reported that 0.5-1 µM okadaic acid disrupted the lamellated structure of Golgi, replacing it with vesicles within 10 min of exposure. This disruption was rapidly reversed on withdrawal of okadaic acid. In the studies shown here, Golgi disruption would not have affected interpretation of the translocation data because particulate material was removed during isolation of the cytosol fraction.
The mechanism by which ISO induces Rap1 translocation was not established. However, because ISO acts via the cAMP pathway, a likely possibility is that cAMP-mediated phosphorylation is involved, as has been found in human platelets (28). A direct role for Ca2+ in mediating Rap1 translocation was ruled out by demonstrating that carbachol, at a concentration (50 µM) that significantly increases free intracellular Ca2+ levels and is optimum for amylase release from rat parotid acini (38), failed to affect the cytosolic translocation of Rap1. However, as suggested previously (6), Rap1 itself may play a role in Ca2+ regulation.
Given that the present data suggest a role for Rap1 upstream in the
secretory pathway, an important question relates to the potential
targets for Rap1. In platelets, studies suggest that Rap1 plays a role
in the regulation of phospholipase C (PLC) and metabolism of inositol
phospholipids (21, 22). In these cells, thrombin is a potent agonist
that acts via the PLC1-inositol 1,4,5-triphosphate-Ca2+ pathway to
mediate the release of
-granule contents (21). Torti and Lapetina
(37) suggested that during platelet activation Rap1, which binds Ras
GTPase-activating protein (RasGAP), recruits the RasGAP-PLC
1 complex
to the plasma membrane, thereby leading to PLC
1-induced hydrolysis
of membrane lipids and release of Ca2+, a signaling cascade that
culminates in secretion. Activation of the cAMP pathway, on the other
hand, inhibits secretion in thrombin-stimulated platelets (21) and
human erythroleukemia cells (22), which have characteristics similar to
platelets. Phosphorylation of Rap1B via cAMP appears to uncouple the
thrombin receptor from PLC by causing the translocation of Rap1 away
from the membrane to the cytosol (37), thereby preventing the formation of the Rap1B-RasGAP-PLC
1 complex at the plasma membrane. In
addition, Rap1 has also been shown to regulate
Ca2+ fluxes in platelets (6). In
the presence of GTP, phosphorylated Rap1 increases
Ca2+ uptake and intracellular
Ca2+ concentration. The role of
Rap1 in Ca2+ regulation is further
supported by data showing a correlation between the expression of Rap1B
and the 97-kDa sarco(endo)plasmic reticulum
Ca2+-ATPase in platelets,
hematopoietic cells, and some cancer lines (24). As suggested by
Magnier et al. (24), regulation of
Ca2+-ATPases may not be limited to
platelets but might be a more general process representative of other
cell types.
Rap1 has also been found to be associated with cytoskeletal assembly in human platelets (10). In the rat parotid gland, the presence of a cell web in proximity to the apical plasma membrane (17), where exocytosis occurs, suggests that cytoskeletal reorganization (29) and the accompanying amylase release in response to cell stimulation play a significant role in secretion, either by facilitating the movement of secretory granules or actually regulating the exocytotic event or both.
In summary, the studies shown here are significant in that a relationship between Rap1 and secretion, hitherto unexplored in exocrine glands, is established in the parotid gland. The unequivocal localization of Rap1 on the rat parotid secretory granule (9) and its time- and concentration-dependent translocation to the cytosol in ISO-stimulated cells in correlation with amylase release, as well as inhibition of this redistribution by propranolol and okadaic acid, suggest that this protein has a role in secretion that is distal to PKA stimulation and proximal to the exocytotic event. This secretory role may be via cytoskeletal interactions or via Ca2+ regulation and cross talk between the cAMP- and inositol 1,4,5-trisphosphate-Ca2+-dependent pathways.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Institute of Dental Research Grants DE-07023 and DE-10733.
![]() |
FOOTNOTES |
---|
Address for reprint requests: N. J. D'Silva, Dept. of Oral Biology, Box 357132, Univ. of Washington, Seattle, WA 98195.
Received 3 September 1997; accepted in final form 26 February 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arkle, S.,
R. Michalek,
and
D. Templeton.
The relationship of intracellular free calcium activity to amylase secretion in substance P- and isoprenaline-stimulated rat parotid acini.
Biochem. Pharmacol.
38:
1257-1261,
1989[Medline].
2.
Baum, B. J.,
J. M. Freiberg,
H. Ito,
G. S. Roth,
and
C. R. Filburn.
-Adrenergic regulation of protein phosphorylation and its relationship to exocrine secretion in dispersed rat parotid gland acinar cells.
J. Biol. Chem.
256:
9731-9736,
1981
3.
Bernfeld, P.
Amylases, and
.
Methods Enzymol.
1:
149-158,
1955.
4.
Bokoch, G. M.
Biology of the rap proteins, members of the ras superfamily of GTP-binding proteins.
Biochem. J.
289:
17-24,
1993[Medline].
5.
Cohen, P.,
C. Holmes,
and
Y. Tsukitani.
Okadaic acid: a new probe for the study of cellular regulation.
Trends Biochem. Sci.
15:
98-102,
1990[Medline].
6.
Corvazier, E.,
J. Enouf,
B. Papp,
J. de Gunzburg,
A. Tavitian,
and
S. Levy-Toledano.
Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes.
Biochem. J.
281:
325-331,
1992[Medline].
7.
Darchen, F.,
A. Zahraoui,
F. Hamme,
M. P. Monteils,
A. Tavitian,
and
D. Scherman.
Association of the GTP-binding protein rab3A with adrenal chromaffin granules.
Proc. Natl. Acad. Sci. USA
87:
5692-5696,
1990[Abstract].
8.
Darnanville, A.,
R. Bredoux,
K. J. Clemetson,
N. Kieffer,
N. Bourdeau,
S. Levy-Toledano,
J. P. Caen,
and
J. Enouf.
The phosphoprotein that regulates platelet Ca2+ transport is located on the plasma membrane, controls membrane-associated Ca2+-ATPase and is not glycoprotein Ib beta-subunit.
Biochem. J.
273:
429-434,
1991[Medline].
9.
D'Silva, N. J.,
D. DiJulio,
C. B. Belton,
K. L. Jacobson,
and
E. L. Watson.
Immunolocalization of rap1 in the rat parotid gland: detection on secretory granule membranes.
J. Histochem. Cytochem.
45:
965-973,
1997
10.
Fischer, T. H.,
M. N. Gatling,
J. C. Lacal,
and
G. C. White.
Rap1B, a cAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton.
J. Biol. Chem.
265:
19405-19408,
1990
11.
Fischer von Mollard, G.,
B. Stahl,
A. Khokhlatchev,
T. C. Sudhof,
and
R. Jahn.
Rab3C is a synaptic vesicle protein that dissociates from synaptic vesicles after stimulation of exocytosis.
J. Biol. Chem.
269:
10971-10974,
1994
12.
Fischer von Mollard, G.,
T. C. Sudhoff,
and
R. Jahn.
A small GTP-binding protein dissociates from synaptic vesicles during exocytosis.
Nature
349:
79-81,
1991[Medline].
13.
Goke, B.,
J. Williams,
J. Wishart,
and
R. deLisle.
Low molecular mass GTP-binding proteins in subcellular fractions of the pancreas: regulated phosphoryl G proteins.
Am. J. Physiol.
262 (Cell Physiol. 31):
C493-C500,
1992
14.
Gomperts, B. D.
GE: a GTP-binding protein mediating exocytosis.
Annu. Rev. Physiol.
52:
591-605,
1990[Medline].
15.
Hancock, J. F.,
A. I. Magee,
J. E. Childs,
and
C. J. Marshall.
All ras proteins are polyisoprenylated but only some are palmitoylated.
Cell
57:
1167-1177,
1989[Medline].
16.
Hartree, E. F.
Determination of protein: a modification of the Lowry method that gives a linear photometric response.
Anal. Biochem.
48:
422-427,
1972[Medline].
17.
Huleux, C.,
C. Creux,
M. Lemullois,
and
B. Rossignol.
Process of reestablishment of -adrenergic induced protein secretion after cytochalasin D inhibition in rat parotid gland. Effect of cholinergic agonist, phorbol ester and calcium.
Biol. Cell
73:
57-62,
1991[Medline].
18.
Ito, H.,
B. J. Baum,
and
G. S. Roth.
-Adrenergic regulation of rat parotid gland exocrine protein secretion during aging.
Mech. Ageing Dev.
15:
177-188,
1981[Medline].
19.
Itoh, T.,
K. Kaibuchi,
T. Sasaki,
and
Y. Takai.
The smg GDS-induced activation of smg p21 is initiated by cAMP-dependent protein kinase-catalyzed phosphorylation of smg p21.
Biochem. Biophys. Res. Commun.
177:
1319-1324,
1991[Medline].
20.
Lapetina, E. F.,
J. C. Lacal,
B. R. Reep,
and
L. Molina y Vedia.
A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets.
Proc. Natl. Acad. Sci. USA
86:
3131-3134,
1989[Abstract].
21.
Lapetina, E. G.
The signal transduction induced by thrombin in human platelets.
FEBS Lett.
286:
400-404,
1990.
22.
Lazarowski, E. R.,
D. A. Winegar,
R. D. Nolan,
E. Oberdisse,
and
E. G. Lapetina.
Effect of protein kinase A on inositide metabolism and rap1 G-protein in human erythroleukemia cells.
J. Biol. Chem.
265:
13118-13123,
1990
23.
Lerosey, I.,
V. Pizon,
A. Tavitian,
and
J. de Gunzburg.
The cAMP-dependent protein kinase phosphorylates the rap1 protein in vitro as well as in intact fibroblasts, but not the closely related rap2 protein.
Biochem. Biophys. Res. Commun.
175:
430-436,
1991[Medline].
24.
Magnier, C.,
R. Bredoux,
T. Kovacs,
R. Quarck,
B. Papp,
E. Corvazier,
J. de Gunzburg,
and
J. Enouf.
Correlated expression of the 97 kDa sarcoendoplasmic reticulum Ca(2+)-ATPase and rap1B in platelets and various cell lines.
Biochem. J.
297:
343-350,
1994[Medline].
25.
Mangos, J. A.,
N. R. McSherry,
T. Barber,
N. Arvanitakis,
and
V. Wagner.
Dispersed rat parotid acinar cells II: characterization of adrenergic receptors.
Am. J. Physiol.
229:
560-565,
1975[Medline].
26.
Maridonneau-Parini, I.,
and
J. de Gunzburg.
Association of rap1 and rap2 proteins with the specific granules of human neutrophils.
J. Biol. Chem.
267:
6396-6402,
1992
27.
McCabe, P. C.,
H. Haubruck,
P. Polakis,
F. McCormick,
and
M. A. Innis.
Functional interaction between p21(rap1A) and components of the budding pathway in Saccharomyces cerevisiae.
Mol. Cell. Biol.
12:
4084-4092,
1992[Abstract].
28.
Nagata, K.,
and
Y. Nozawa.
A low Mr GTP-binding protein, Rap1, in human platelets: localization, translocation and phosphorylation by cAMP-dependent protein kinase.
Br. J. Haematol.
90:
180-186,
1995[Medline].
29.
Perrin, D.,
K. Moller,
K. Hanke,
and
H. Soling.
cAMP and Ca2+-mediated secretion in parotid acinar cells is associated with reversible changes in the organization of the cytoskeleton.
J. Cell Biol.
116:
127-134,
1992[Abstract].
30.
Quarck, R.,
M. Bryckaert,
C. Magnier,
E. Corvazier,
R. Bredoux,
J. de Gunzburg,
M. Fontenay,
G. Tobelem,
and
J. Enouf.
Evidence for rap1 in vascular smooth muscle cells: regulation of their expression by platelet-derived growth factor BB.
FEBS Lett.
342:
159-164,
1994[Medline].
31.
Quinn, M.,
M. Mullen,
A. Jesaitis,
and
J. Linner.
Subcellular distribution of the rap1A protein in human neutrophils: colocalization and cotranslocation with cytochrome b559.
Blood
79:
1563-1573,
1992[Abstract].
32.
Quissell, D. O.,
E. Watson,
and
F. Dowd.
Signal transduction mechanisms involved in salivary gland regulated exocytosis.
Trends Biochem. Sci
19:
164-168,
1992.
33.
Robinovitch, M. R.,
P. J. Keller,
J. Iverson,
and
D. Kauffman.
Demonstration of a class of proteins loosely associated with secretory granule membranes.
Biochim. Biophys. Acta
382:
2640-2646,
1975.
34.
Spearman, T. N.,
J. P. Durham,
and
F. R. Butcher.
Cyclic AMP in the regulation of exocytosis in the rat parotid gland: evidence obtained with cholera toxin.
Biochim. Biophys. Acta
759:
117-124,
1984.
35.
Takuma, T.,
and
T. Ichida.
Okadaic acid inhibits amylase exocytosis from parotid acini stimulated by cyclic AMP.
FEBS Lett.
285:
124-126,
1991[Medline].
36.
Tau, S. C.,
and
H. H. Tai.
Intracellular translocation of rap1B G-protein induced prostaglandin E1 is blocked by phorbol ester in human platelets.
Prostaglandins Leukot. Essent. Fatty Acids
50:
299-302,
1994[Medline].
37.
Torti, M.,
and
E. G. Lapetina.
Role of rap1B and p21ras GTPase-activating protein in the regulation of phospholipase C1 in human platelets.
Proc. Natl. Acad. Sci. USA
89:
7796-7800,
1992[Abstract].
38.
Van Der Ven, P. F.,
T. Takuma,
and
B. J. Baum.
Chloropromazine inhibition of muscarinic-cholinergic responses in the rat parotid gland.
J. Dent. Res.
65:
382-386,
1986[Abstract].
39.
Von Zastrow, M.,
and
J. D. Castle.
Protein sorting among two distinct export pathways occurs from the content of maturing exocrine storage granules.
J. Biol. Chem.
105:
2675-2684,
1987.
40.
Wagner, A. C.,
and
J. A. Williams.
Pancreatic zymogen granule membrane proteins: molecular details begin to emerge.
Digestion
55:
191-199,
1994[Medline].
41.
Wagner, A.,
M. Wishart,
D. Yule,
and
J. Williams.
Effects of okadaic acid indicate a role for dephosphorylation in pancreatic stimulus-secretion coupling.
Am. J. Physiol.
263 (Cell Physiol. 32):
C1172-C1180,
1992
42.
Waschulewski, I. H.,
M. L. Kruse,
B. Agricola,
H. F. Kern,
and
W. E. Schmidt.
Okadaic acid disrupts Golgi structure and impairs enzyme synthesis and secretion in the rat pancreas.
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33):
G939-G947,
1996
43.
Yamashina, S.
Dynamic structure and function of Golgi apparatus in the salivary acinar cells.
J. Electron Microsc. (Tokyo)
44:
124-134,
1995[Medline].