| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Anatomisches Institut der Technischen Universität München (B.H.-Z., M.G.), 80802 Munich; and II Medizinische Klinik der Technischen Universität München (A.G., W.S., C.P.), 81675 Munich, Germany
Address all correspondence and requests for reprints to: Prof. Dr. Manfred Gratzl, Anatomisches Institut der Technischen Universität München, Biedersteiner Strasse 29, 80802 Munich, Germany. E-mail: gratzl{at}lrz.tu-muenchen.de
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Electron microscopic evaluation of gastric ECL cells revealed characteristic cytoplasmic vesicles containing a small electron-dense core surrounded by a translucent halo (for references, see Ref.1). Recent reports suggest that histamine is taken up into vesicles by a specific monoamine transporter driven by a proton gradient (6, 7). Therefore, ECL cells display similarity to chromaffin cells of the adrenal gland or to neurons. In all of these cell types, amines or neurotransmitters are accumulated in cytoplasmic vesicles by transporters dependent on the establishment of a proton gradient across the vesicular membrane by V-type adenosine triphosphatases (8).
We have previously established a model to study histamine secretion in vitro using highly enriched ECL cells in primary culture (4, 5, 9). Histamine secretion was stimulated by gastrin in the presence of extracellular calcium. Calcium entry across the plasmalemma was of critical importance for histamine secretion after receptor and postreceptor activation (5). However, the intracellular mechanisms resulting in fusion of histamine vesicles with the plasma membrane after calcium entry have not been defined.
On the other hand, the calcium-dependent intracellular secretory apparatus of neurons and endocrine cells has been analyzed in detail by numerous recent investigations. According to current models, docking and fusion of synaptic vesicles or endocrine secretory vesicles are mediated by three membrane-bound SNARE [soluble NSF (N-ethylmaleimide sensitive fusion protein) attachment protein receptor] proteins, termed syntaxin, synaptobrevin, and SNAP-25. As cleavage of SNARE proteins by clostridial neurotoxins results in blockade of exocytosis by neurons or endocrine cells, the SNARE proteins are regarded as key players in exocytosis (10, 11, 12, 13, 14, 15, 16, 17, 18, 19). In addition to the SNARE proteins, vesicular membrane proteins such as synaptotagmin and synaptophysin, have been suggested to participate in the exocytosis of synaptic vesicles in neurons (20, 21).
We attempted to examine the presence of SNARE proteins and vesicular synaptotagmin and synaptophysin in ECL cells. It was our aim to obtain straightforward evidence for the neuroendocrine nature of these cells. In fact, we have identified five of these proteins in ECL cells by immunoblotting and immunocytochemistry characterizing ECL cells as a neuroendocrine cell type. Moreover, we have functionally related the expression of two of these proteins to histamine secretion by specific cleavage and blockade of exocytosis by clostridial neurotoxins. Our data establish that these proteins are functionally involved in exocytosis, and that ECL cells display a great similarity to neurons and endocrine cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Monoclonal antibodies directed against synaptobrevin II (Cl. 69.1) and synaptotagmin I (Cl. 41.1) and a polyclonal antibody (rabbit) directed against synaptophysin were provided by Reinhard Jahn (New Haven, CT), and a polyclonal antibody against histidine-decarboxylase (rabbit) was supplied by Tsutomu Chiba (Kobe, Japan). Monoclonal SNAP-25 antibody (SMI81) and monoclonal antisyntaxin antibody (Cl. HPC-1) were purchased from Sternberger Monoclonals (Baltimore, MD) and Sigma, respectively. Peroxidase-labeled, biotinylated, Cy3-labeled, and fluorescein isothiocyanate-labeled antimouse (or antirabbit) IgG antibodies were obtained from Dianova (Hamburg, Germany). Tetanus toxin light (TeTxL) chain was a gift from Ulrich Weller (Mainz, Germany), and botulinum neurotoxin A (BoNT/A) was supplied by Bibhuti R. DasGupta (Madison, WI) and Clifford C. Shone (Porton Down, Salisbury, UK).
Isolation and cultivation of ECL cells
Mucosal cells from the stomachs of fed female Sprague-Dawley
rats (total of 53 preparations; n = 5 rats/experiment; 180–200 g
BW; Charles River, Sulzfeld, Germany) were isolated by pronase E
digestion (1.3 mg/ml) using the everted sac technique (described in
detail in Refs. 4 and 5). The resulting mucosal cell preparation was
fractionated by counterflow elutriation using the JE 6B rotor (Beckman
Instruments, Glenrothes, UK) run in a J2–21M/E Beckman centrifuge.
The original methods (4) and modifications (5) were described
previously. Briefly, 108 mucosal cells were loaded into a
standard chamber at a flow rate of 16 ml/min and a rotor velocity of
2300 rpm. Small cells (diameter, <10 µm) were collected at 2000 rpm
and a flow rate of 22–23 ml/min. For further purification, 7 ml of
this cell fraction were overlaid above two different layers of Nycodenz
[supplemented with 1.2 mM MgCl2, 10
mM HEPES (pH 7.4) and 10 mg/ml BSA] diluted 1:1 or 1:2
with 140 mM NaCl, 1.2 mM MgSO4, 1
mM CaCl2, 11 mM
D-glucose, 0.5 mM dithiothreitol, 10 mg/ml BSA,
and 10 mM HEPES, pH 7.4. After centrifugation for 8 min at
210 x g, the cells were collected from the interface
between the top and second layers of the density gradient. The fraction
contained more than 95% ECL cells determined by acridine orange uptake
and immunostaining for histidine decarboxylase (HDC). Cell viability
(trypan blue exclusion) exceeded 95%. The average cell yield from 5
rats in 60 experiments was 3.5 x 105 ECL cells.
Immunohistochemistry of rat gastric mucosa
Rat stomach was fixed by immersion in 4% (wt/vol)
paraformaldehyde in PBS, pH 7.4, for 2 days at 4 C, subsequently washed
for 12 h in PBS, pH 7.4, supplemented with 6.8% (wt/vol) sucrose
and embedded in paraffin. Sections were deparaffinized, rehydrated,
incubated for 30 min with 10% methanol and 0.3%
H2O2 in PBS, and heated in 10 mM
citrate (pH 6) three times for 5 min each time in a microwave oven.
Then the sections were washed with PBS, incubated with 10% (vol/vol)
goat serum in PBS for 30 min, and incubated overnight at 4 C with
anti-HDC (1:1000) in 2% (vol/vol) goat serum in PBS. After washing
with PBS, the slides were incubated with biotinylated antirabbit
secondary antibodies [1:500 in 2% (vol/vol) goat serum PBS] for
2 h at room temperature. After washing with PBS, avidin-labeled
peroxidase (Vectastain, Vector Laboratories, Burlingame, CA) and
detection by diaminobenzidene were employed.
For colocalization studies immunostaining was performed on thin sections. Tissue was fixed as described above, dehydrated for 1 h at 4 C in 100% acetone, and embedded in polyacrylate (Technovit 8100, Heraues-Kulzer, Wehrheim, Germany) according to the manufacturer’s recommendations. Serial sections (1 µm) were placed on Poly-Prep slides (Sigma), dried for 2 h, and incubated for 10 min at 37 C with 0.1% (wt/vol) trypsin in 0.1% (wt/vol) CaCl2. Endogenous peroxidase was blocked as described above, followed by incubation with 10% (vol/vol) goat serum in PBS for 30 min. Serial sections were incubated overnight at 4 C with anti-SNAP-25 (1:200), antisynaptobrevin II Cl. 69.1 (1:200), and anti-HDC (1:2000) in 2% goat serum in PBS. After washing with PBS, the slides were incubated with biotinylated antimouse or antirabbit secondary antibodies (1:500 in 2% goat serum PBS) for 2 h. Immune complexes were visualized with avidin labeled peroxidase and diaminobenzidene (see above).
Immunocytochemistry of isolated and cultured ECL cells
Purified cells (4 x 104) were placed on
sterile glass coverslips, each coated with Cell-Tak (diluted 1:2 with
0.5 M NaHCO3) and cultured at 37 C for 48
h in DMEM-Ham’s F-12 supplemented with 0.2% (wt/vol) BSA, 5%
(vol/vol) FBS (Life Technologies, Paisley, Scotland), 5 mg/liter
insulin, 5 mg/liter transferrin, 5 g/liter sodium selenite, 10
nM hydrocortisone, 1 pM gastrin, and 50
mg/liter gentamicin sulfate. After culture, the cells were washed with
PBS, fixed for 20 min in 3.7% formalin, washed twice in PBS, and
blocked in 10% (vol/vol) goat serum in PBS for 30 min before
incubation first with anti-SNAP-25, antisynaptobrevin, or anti-HDC
antibody (all diluted 1:1000 in 2% goat serum in PBS) overnight at 4
C. After washing twice in PBS, cells were incubated with Cy3 (1:500)-
or FITC (1:100)-conjugated antimouse and antirabbit secondary
antibodies. Slides were examined with an Axioplan fluorescence
microscope (Zeiss, Oberkochen, Germany) with suitable filters
(excitation/emission, 546/590 or 450–490/520). Controls were treated
identically without addition of the first antibody and did not show any
staining (n = 3).
Histamine release from ECL cells after short term culture,
permeabilization, and incubation with neurotoxins
Purified ECL cells were placed on culture wells (4 x
104 cells/well) coated with Matrigel at a 1:5 dilution and
cultured at 37 C for 48 h in DMEM-Ham’s F-12 supplemented with
5% FBS (see above). The culture medium was removed and replaced by
DMEM-Ham’s F-12 medium without FBS but supplemented with 0.2% BSA for
3 h at 37 C to remove the influence of growth factors. The medium
was removed, and ECL cells were permeabilized with 8 µM
digitonin (in 0.1% dimethylsulfoxide) in potassium glutamate medium
(150 mM potassium glutamate, 5 mM EDTA, 0.5
mM EGTA, 5.7 mM magnesium acetate, 2
mM Mg2+-ATP, and 10 mM
1,4-piperazinediethanesulfonic acid, pH 7.2) for 2 min at 30 C. This
treatment permeabilized 85–95% of the cells, as determined by trypan
blue uptake. The medium was then renewed without or with TeTxL
containing 0.1% BSA or with or without BoNT/A containing 20
mM DTT, followed by incubation for 15 min at 30 C.
Histamine secretion by ECL cells was determined by incubation with
potassium glutamate buffer without (control) or with 10–100
µM free Ca2+ at 30 C for 10 min. This
interval was found to be optimal for the histamine release experiments.
Free Ca2+ concentrations in potassium glutamate buffer were
calculated and monitored with a Ca2+-selective
minielectrode (22). Histamine was measured in the supernatant and in
cell lysates. Cells were lysed with 3% acetic acid, boiled for 2 min,
and stored at -20 C. Histamine was measured in all samples by RIA
using a commercial kit (Dianova).
SDS-PAGE and immunoblotting
Samples (8 µg protein/lane) of purified ECL cells were
separated on 12.5% gels by SDS-PAGE (23) and blotted onto
nitrocellulose (24). Binding of monoclonal antisynaptobrevin II
antibody (Cl. 69.1; diluted1:5,000) (25), monoclonal
antisynaptotagmin I antibody (Cl. 41.1; diluted 1:2,000) (26),
monoclonal anti-SNAP-25 antibody (1:2,000, SMI81, Sternberger
Monoclonals, Baltimore, MD), monoclonal antisyntaxin-1 antibody
(1:2,000; Cl. HPC-1; Sigma Immunochemicals, Deisenhofen, Germany),
and polyclonal rabbit anti-synaptophysin antibody (1:70,000) was
detected by peroxidase-labeled antimouse (or antirabbit) IgG antibodies
(1:3,000; Dianova) and the enhanced chemiluminescence method (Amersham
Buchler, Braunschweig, Germany).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
shows that small ECL cells in the basal
part of the gastric glands are labeled, demonstrating the specificity
of the antibody. Controls performed in the absence of the first
antibody did not show any staining.
|
). In
consecutive thin sections, antibodies directed against SNAP-25 and
synaptobrevin were applied. As shown in Fig. 2, immunolabeled ECL cells
(B and D) contained HDC as well as the characteristic neuronal and
endocrine marker proteins SNAP-25 and synaptobrevin/VAMP (A and C).
Other adjacent mucosal cells did not show any staining. No staining was
observed when sections were incubated with the secondary antimouse or
antirabbit antibodies without addition of the primary antibody (E and
F).
|
, AB double
immunostaining of purified ECL cells for SNAP-25 and HDC is shown.
Figure 3, C and D, shows colocalization for synaptobrevin and HDC. In
control cells that were treated identically except that the first
antibody was omitted, no staining was observed. In all cell
preparations, more than 95% of the cells were labeled with HDC and
SNAP-25 or synaptobrevin/VAMP, corresponding to the degree of
enrichment. Similar values were obtained with antibodies directed
against synaptotagmin I and synaptophysin (data not shown). Despite the
low amounts of ECL cells present in the gastric mucosa (see Fig. 1
),
the present method of purification yielded a small, but highly
enriched, preparation of ECL cells, which has already successfully been
used in previous studies to investigate histamine release (4, 5).
Subsequently, we attempted to analyze SNAP-25 and synaptobrevin/VAMP
and related proteins by immunoblotting. In addition, we used the cell
preparations (
3.5 x 105 highly purified ECL cells
were isolated from five rats) for functional studies to investigate
whether two of the detected proteins are involved in the exocytosis of
histamine.
|
, A and B,
shows the results of two single experiments. Figure 4C gives an
overview of all the proteins observed during our experiments. The
proteins were detected by specific antibody binding and by the
established size determined by comparison with marker proteins
indicated on the left. All immunoblots showed specific
single bands. Synaptobrevin corresponded to 18 kDa, SNAP-25 to 25 kDa,
syntaxin to 35 kDa, synaptophysin to 38 kDa, and synaptotagmin to 65
kDa, values obtained previously in SDS gels.
|
shows histamine
secretion by the permeabilized ECL cells after 15-min exposure to
increasing concentrations of free Ca2+. The concentrations
tested were 0, 10, 30, and 100 µM free Ca2+.
The free Ca2+ concentrations were calculated and controlled
with a Ca2+-selective minielectrode (22). Basal release (no
Ca2+ added) in the different experiments was stimulated
approximately 2.5-fold by 30 µM free Ca2+. As
shown in Fig. 5, Ca2+ concentrations above 30
µM did not further enhance histamine release (n = 5
independent experiments). Therefore, this calcium concentration was
used for stimulation of the cells in the experiments described
below.
|
). Inactivation of TeTxL by boiling
abolished the attenuating effect of TeTxL on histamine release (n
= 5 experiments).
|
, bands at
the bottom). In contrast, the amounts of synaptobrevin were
greatly decreased in cells incubated with TeTxL (Fig. 6B, top). Thus, it may be concluded that inhibition of
exocytosis by TeTxL is associated with specific cleavage of
synaptobrevin within gastric ECL cells.
Effect of BoNT/A
We also examined the effect of BoNT/A on permeabilized ECL cells.
Again, purified and cultured ECL cells were permeabilized by 2-min
exposure to 8 µM digitonin. Subsequently, cells were
incubated with or without 100 nM BoNT/A for 15 min.
Histamine release of permeabilized ECL cells was elicited by 30
µM free Ca2+ during a 10-min incubation
period. This effect was almost completely inhibited by 100
nM BoNT/A (Fig. 7A; n = 3
experiments).
|
, see bands at the top and
densitometric evaluation), whereas syntaxin and synaptobrevin remained
intact (Fig. 7B, bands at the bottom), indicating the
specificity of our experiments. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Calcium entry across the plasma membrane is of critical importance for histamine secretion. In the present study, addition of molar concentrations of free calcium to permeablized ECL cells was found to elicit histamine release. These data prove that calcium by itself triggers exocytosis in ECL cells, similar to observations in adrenal chromaffin and other endocrine cells and in neurons. Previous studies have shown that isolated ECL cells typically exhibit a membrane potential of about -50 mV and have voltage-gated K+ and Ca2+ channels (9, 27) similar to neurons and chromaffin cells of the adrenal gland. An average cytoplasmic calcium concentration of about 250 nM was determined in stimulated single ECL cells after gastrin stimulation (5). In contrast to these experiments, a concentration of 10–30 µM calcium was necessary to stimulate exocytosis of histamine vesicles in permeabilized cells. This observation may indicate that calcium influx into intact ECL cells elicited by gastrin actually yields calcium concentrations about 100 times higher in the subplasmalemmal space than in the overall cell body. Similar conclusions have been drawn from observations in nerve terminals and adrenal chromaffin cells (28, 29).
Although it is generally accepted that docking and fusion of secretory vesicles to the plasma membrane depend on the presence of intracellular calcium, proteins participating in exocytosis have been identified in neurons and a few endocrine cell types (10, 11, 12, 13, 14, 15, 16, 17, 18, 19). SNAP-25, synaptobrevin, and syntaxin are regarded as key proteins of the exocytosis apparatus and were initially thought to be unique to neurons and endocrine cells. These SNARE proteins have also been detected in various other cell types, including muscle cells and adipocytes. The distribution of characteristic subtypes of the SNARE proteins, however, exhibits a tissue-specific pattern (for references, see Refs. 30–34).
Proteins of the exocytosis apparatus of ECL cells are not only of special interest to delineate the relation of this cell type to the endocrine system, but are also of pharmacological interest in attempts to block histamine secretion. Here we show that ECL cells of the gastric mucosa, which are characterized by the presence of the marker enzyme HDC, also contain synaptobrevin and SNAP-25. Moreover, both SNARE proteins were colocalized in isolated and highly purified ECL cells by simultaneous immunostaining for HDC. Finally, immunoblotting revealed synaptobrevin and SNAP-25 in the isolated ECL cells and, in addition, syntaxin, which strongly suggests the presence of these proteins within ECL cells. The antibodies used for immunocytochemistry and immunoblotting were specific for SNAP-25, syntaxin-1, and synaptobrevin II (VAMP-2), subtypes of these proteins present preferentially in neurons and endocrine cells. Therefore, our results obtained by double immunostaining and immunoblotting clearly show that the SNARE proteins SNAP-25, synaptobrevin II (VAMP-2), and syntaxin-1 are present in ECL cells. In a recent study, SNAP-25, syntaxin-1, and VAMP-2 have been detected in populations of gastric mucosal cells (35). Specifically, VAMP-2 was colocalized with isolated tubovesicular membranes of gastric parietal cells. In the present study, cells neighboring ECL cells in the mucosal sections did not show VAMP-2 staining. As we did not characterize these cells with appropriate antibodies, it is possible that these cells represent chief cells rather than parietal cells, which are both present in the basal part of gastric glands. An alternative explanation for the lack of immunostaining for VAMP-2 in adjacent cells may be due to the low abundance of VAMP-2 in parietal cells compared with that in ECL cells or in masking of VAMP-2 by formation of complexes with other components of the exocytosis apparatus preventing binding with the antibody.
According to a current model, the SNARE proteins synaptobrevin,
syntaxin, and SNAP-25 first form a heterotrimeric complex. Binding of
soluble 
-SNAP and N-ethylmaleimide-sensitive fusion
protein to the docking complex and ATP hydrolysis by
N-ethylmaleimide-sensitive fusion protein cause disassembly
of the SNARE complex, which is followed by exocytosis (36, 37). It
appears likely that syntaxin-1, SNAP-25, and VAMP-2 also form a complex
in ECL cells. However, due to the low abundance of ECL cells in the
gastric mucosa and the small number of highly purified ECL cells
obtained during isolation, we were unable to study the
formation of a protein complex in ECL cells by
immunoprecipitation.
Syntaxins are SNARE proteins that constitute a large family of proteins differentially expressed in several mammalian cells (38). Syntaxin-1 is expressed in neurons and adrenal chromaffin cells, where it is located in both the plasma membrane and the membrane of secretory vesicles (18, 39, 40). A role for syntaxin-1 in synaptic vesicle docking and fusion is suggested by its association with calcium channels and the SNARE proteins (41, 42). The simultaneous presence of syntaxin-1, SNAP-25, and VAMP-2 in ECL cells may indicate that the mechanism of exocytosis in ECL cells closely resembles that of the adrenal chromaffin cells and neurons.
Furthermore, we identified two vesicle-associated proteins in ECL cells that have been previously implicated in exocytosis by neurons and endocrine cells. Synaptotagmin I is a 65-kDa integral membrane protein present in synaptic vesicles and endocrine secretory vesicles and has been identified as a vesicular Ca2+ receptor (26, 43). The antibody used in this study selectively detected synaptotagmin I but no other isoforms of this protein, which have a wide distribution in nonneuronal cell types (44, 45). Therefore, synaptotagmin I can be regarded as a marker for neuronal and endocrine cells. Synaptophysin, a 38-kDa protein found typically in synaptic vesicles, was also detected in ECL cells. The protein exhibits structural homology to gap junction proteins and participates in pore formation during exocytosis (46). The association of synaptophysin with the complex of SNARE proteins occurs via its specific association with the SNARE protein synaptobrevin/VAMP (25, 47, 48). Again, synaptophysin represents the neuronal form of the pantophysin family with other members present in a wide variety of cells (49). The presence of synaptotagmin I and synaptophysin in ECL cells directly underlines the neuroendocrine nature of this cell type.
Permeabilized ECL cells were attacked by clostridial neurotoxins. In general, intact endocrine cells, unlike neurons, are not susceptible to clostridial neurotoxins such as tetanus and BoNT/A. However, after permeabilization, exocytosis of endocrine cells can be blocked by the neurotoxins. This effect is caused by proteolytic cleavage of synaptobrevin and SNAP-25 within endocrine cells by the neurotoxins (13, 14, 18, 19, 50). Thus, the same SNARE proteins are specifically attacked by clostridial neurotoxins in endocrine cells and neurons (for references, see Refs. 20 and 21). Using clostridial neurotoxins, we found that in rat gastric ECL cells, two of the neuroendocrine marker proteins, synaptobrevin II/VAMP-2 and SNAP-25, are functionally involved in histamine release. Tetanus toxin inhibited histamine secretion by only 50%, whereas BoNT/A completely blocked histamine secretion. These results are in contrast to those in chromaffin cells of the adrenal gland, where catecholamine secretion is only partially blocked by BoNT/A, although the extent of protein cleavage with 100 nM BoNT/A was identical in both preparations (18). The exact reason for this difference is unknown. However, it may be speculated that functional and inactive pools of synaptobrevins and SNAP-25 exist in ECL cells. These forms could display different susceptibilities to the neurotoxins, as previously reported to occur during complex formation (51, 52).
Our present results show that histamine is released from permeabilized
ECL cells upon stimulation with calcium, similar to previous studies
using chromaffin cells (for references, see Refs. 13, 18, and 50). As
the process of histamine release from permeabilized ECL cells was
mediated by proteins previously reported to participate in exocytosis,
it can be concluded that 1) histamine in ECL cells is released from
membrane-bound structures and not from the cytoplasm; and 2) that
histamine-containing vesicles undergo exocytosis upon stimulation with
micromolar concentrations of Ca2+. Thus, although
ultrastructural evidence for the localization of histamine within
cytoplasmic vesicles of ECL cells remains to be presented (1), it is
very likely that it is released from vesicles, as is known to be the
case for neurotransmitters and hormones. Interestingly, basal histamine
secretion was higher in permeabilized ECL cells than in intact cells
(4) and, in addition, varied significantly among several sets of
experiments. One possible explanation for this phenomenon may be the
unusually high numbers of docked and peripheral secretory vesicles that
we observed by electron microscopy in ECL cells. Therefore, addition of
digitonin to the ECL cells not only permeabilizes the plasma membrane,
but diffuses in various extents deeper into the cell, and the detergent
may hit docked vesicles (diameter, 300 nm). The differences in basal
release may reflect differences in the range of digitonin diffusion in
the cortex of the ECL cells that cannot be exactly controlled at
present.
Recent observations suggest the presence of the vesicular monoamine transporter type 2 VMAT2 in ECL cells, which may serve as a transporter for histamine into the storage organelles (6, 7). Furthermore, histamine secretion is inhibited by bafilomycin (4, 5), suggesting that a proton gradient across the vesicular membrane generated by V-type adenosine triphosphatases (8) may exist in ECL cells. Both transports may act together during the uptake of histamine by cytoplasmatic vesicles, which is a common feature of amine and neurotransmitter uptake by vesicles of endocrine cells and neurons.
In summary, our data provide convincing evidence that the exocytotic apparatus for histamine secretion in ECL cells shares many properties with neurons and endocrine cells. Now, with these additional criteria met, little doubt remains about the neuroendocrine nature of ECL cells. Moreover, functional analysis of the proteins involved in exocytosis demonstrates that SNAP-25 and synaptobrevin II are cleaved by neurotoxins and mediate histamine release, which is of considerable pharmacological interest.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received May 27, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-SNAP, 14–3-3 proteins and calmodulin on
priming and triggering of regulated exocytosis. J Cell Biol 130:1063–1070This article has been cited by other articles:
 |
|
 |
|
  R. Zanner, M. Gratzl, and C. Prinz Expression of the endocytic proteins dynamin and amphiphysin in rat gastric enterochromaffin-like cells J. Cell Sci., May 1, 2004; 117(11): 2369 - 2376. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. D. Burgoyne and A. Morgan Secretory Granule Exocytosis Physiol Rev, April 1, 2003; 83(2): 581 - 632. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. ZANNER, M. GRATZL, and C. PRINZ Circle of Life of Secretory Vesicles in Gastric Enterochromaffin-Like Cells Ann. N.Y. Acad. Sci., October 1, 2002; 971(1): 389 - 396. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Gouraud, A. Laera, G. Calamita, M. Carmosino, G. Procino, O. Rossetto, R. Mannucci, W. Rosenthal, M. Svelto, and G. Valenti Functional involvement of VAMP/synaptobrevin-2 in cAMP-stimulated aquaporin 2 translocation in renal collecting duct cells J. Cell Sci., September 15, 2002; 115(18): 3667 - 3674. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Gerhard, N. Neumayer, E. Presecan-Siedel, R. Zanner, E. Lengyel, T. Cramer, M. Hocker, and C. Prinz Gastrin Induces Expression and Promoter Activity of the Vesicular Monoamine Transporter Subtype 2 Endocrinology, August 1, 2001; 142(8): 3663 - 3672. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Feng, R. Flaumenhaft, C. Bandeira–Melo, P. F. Weller, and A. M. Dvorak Ultrastructural Localization of Vesicle-associated Membrane Protein(s) to Specialized Membrane Structures in Human Pericytes, Vascular Smooth Muscle Cells, Endothelial Cells, Neutrophils, and Eosinophils J. Histochem. Cytochem., March 1, 2001; 49(3): 293 - 304. [Abstract] [Full Text]
|
|
|
|
 |
|
 L. Graff, F. Castrop, M. Bauer, H. Höfler, and M. Gratzl Expression of Vesicular Monoamine Transporters, Synaptosomal-associated Protein 25 and Syntaxin1: A Signature of Human Small Cell Lung Carcinoma Cancer Res., March 1, 2001; 61(5): 2138 - 2144. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. Grosse, A. Bulling, C. Brucker, U. Berg, A. Amsterdam, A. Mayerhofer, and M. Gratzl Synaptosome-Associated Protein of 25 Kilodaltons in Oocytes and Steroid-Producing Cells of Rat and Human Ovary: Molecular Analysis and Regulation by Gonadotropins Biol Reprod, August 1, 2000; 63(2): 643 - 650. [Abstract] [Full Text]
|
|
|
|
 |
|
 N. Zeng, C. Athmann, T. Kang, J. H. Walsh, and G. Sachs Role of neuropeptide-sensitive L-type Ca2+ channels in histamine release in gastric enterochromaffin-like cells Am J Physiol Gastrointest Liver Physiol, December 1, 1999; 277(6): G1268 - G1280. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Prinz, R. Zanner, M. Gerhard, S. Mahr, N. Neumayer, B. Hohne-Zell, and M. Gratzl The mechanism of histamine secretion from gastric enterochromaffin-like cells Am J Physiol Cell Physiol, November 1, 1999; 277(5): C845 - C855. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Banerjee, T. Shih, E. A. Alexander, and J. H. Schwartz SNARE Proteins Regulate H+-ATPase Redistribution to the Apical Membrane in Rat Renal Inner Medullary Collecting Duct Cells J. Biol. Chem., September 10, 1999; 274(37): 26518 - 26522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Flaumenhaft, K. Croce, E. Chen, B. Furie, and B. C. Furie Proteins of the Exocytotic Core Complex Mediate Platelet alpha -Granule Secretion. ROLES OF VESICLE-ASSOCIATED MEMBRANE PROTEIN, SNAP-23, AND SYNTAXIN 4 J. Biol. Chem., January 22, 1999; 274(4): 2492 - 2501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Mahr, N. Neumayer, H. J. Kolb, W. Schepp, M. Classen, and C. Prinz Growth Factor Effects on Apoptosis of Rat Gastric Enterochromaffin-Like Cells Endocrinology, October 1, 1998; 139(10): 4380 - 4390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Zanner, G. Hapfelmeier, M. Gratzl, an |
