From the Department of Physiology and
Biophysics, ¶ Service of Endocrinology, Department of Medicine,
Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec J1H
5N4, Canada, ** Institut National de la Recherche
Scientifique Santé, University of Quebec, Pointe-Claire,
Quebec H9R 1G6, Canada, and the
European
Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and
Molecular Neuroendocrinology, INSERM U413, UA CNRS, University of
Rouen, Mont-Saint-Aignan 76821, France
Received for publication, June 28, 2002, and in revised form, November 11, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous studies have shown
that human fetal adrenal gland from 17- to 20-week-old fetuses
expressed pituitary adenylate cyclase-activating polypeptide (PACAP)
receptors, which were localized on chromaffin cells. The aim of the
present study was to identify PACAP receptor isoforms and to determine
whether PACAP can affect intracellular calcium concentration
([Ca2+]i) and catecholamine secretion.
Using primary cultures and specific stimulation of chromaffin cells, we
demonstrate that PACAP-38 induced an increase in
[Ca2+]i that was blocked by PACAP (6-38), was
independent of external Ca2+, and originated from
thapsigargin-insensitive internal stores. The PACAP-triggered
Ca2+ increase was not affected by inhibition of PLC Pituitary adenylate cyclase-activating polypeptide is a 38-residue
The effects of PACAP are mediated through interaction with two types of
high affinity receptors: type I receptors are selectively activated by
PACAP, whereas type II receptors bind PACAP and VIP with similar
affinity (3). Three isoforms of PACAP receptors have now been cloned
and designated as PACAP-specific receptor I (PAC1-R) (4, 5)
and VIP/PACAP mutual receptors 1 and 2 (VPAC1-R and
VPAC2-R) (6, 7). Both PAC1-R (type 1 receptors) and VPAC1-R/VPAC2-R (type 2 receptors) belong
to the seven-transmembrane domain, G-protein-coupled receptor family,
and are all positively coupled to adenylyl cyclase (2). Eight
isoforms of PAC1-R, resulting from alternative splicing,
have been characterized to date. These variants display differential
signal transduction properties with regard to adenylyl cyclase and
phospholipase C (PLC) stimulation (1, 2). In addition to these
classical signaling pathways, PACAP has been found to stimulate a
Ca2+-calmodulin nitric oxide synthase (8) and
mitogen-activated protein kinase activity (9). These various
transduction mechanisms are involved in the neurotrophic activities
exerted by PACAP (i.e. inhibition of apoptosis and
stimulation of neurite outgrowth) during development (9-11).
PACAP and its receptors are actively expressed in the adrenal medulla
(12-14). In particular, we have previously demonstrated the occurrence
of PACAP-38 (15) and PACAP binding sites (16) in chromaffin cells from
16- to 20-week-old fetal human adrenal glands. Activation of these
receptors by PACAP-38 causes stimulation of cAMP production and induces
a modest increase in inositol 1,4,5-triphosphate (IP3) formation (16), suggesting a role for the
neuropeptide in the developing adrenal gland. During the process of
adrenal gland development, pheochromoblasts originating from the neural crest migrate throughout the fetal cortex, acquiring progressive differentiation through contact with the steroidogenic cells (17, 18)
(for review see Ref. 19). However, the neuroendocrine regulation of
catecholamine release by chromaffin cells in the human fetus has not
been investigated.
Although PACAP is known to be a potent activator of catecholamine
secretion from rat and porcine adrenochromaffin cells (20, 21), the
differential coupling of PACAP receptor variants to the various
Ca2+ sources and to the adenylyl cyclase and PLC signaling
pathways is still poorly understood. In particular, the effect of PACAP on fetal chromaffin cells has never been investigated. The aim of the
present study was therefore to identify the PACAP receptor isoforms
expressed in the human fetal adrenal gland and to analyze the signaling
pathways responsible for PACAP-evoked [Ca2+]i
increase and catecholamine secretion.
Chemicals--
The chemicals used in the present study were
obtained from the following sources: RNAqueousTM-4PCR
purchased from Ambion (Austin, TX); dithiothreitol,
p(dT)12-18, rRNasin Ribonuclease Inhibitor, Moloney
murine leukemia virus reverse transcriptase (RT) and DNA ladder from
Promega (Madison, WI); deoxy-NTPs and Taq DNA polymerase
from Amersham Pharmacia Biotech (Piscataway, NJ); Xestospongin C,
U73122, forskolin, thapsigargin, and H-89 from Calbiochem-Novabiochem
Corp. (San Diego, CA); methacholine, sodium orthovanadate, nicotine,
and DNase from Sigma-Aldrich Canada Ltd (Oakville, Ontario, Canada); collagenase, MEM Eagle's medium and OPTI-MEM from Invitrogen
(Burlington, Ontario, Canada); (Rp)-cAMPS from
Biomol (Plymouth Meeting, PA); Fluo-4 from Molecular Probes (Eugene,
OR); PACAP-38 was synthesized by the solid phase methodology as
previously described (22); and PACAP (6-38) was from American Peptide
Co. (Sunnyvale, CA). All other chemicals were of A grade purity.
Retrieval and Preparation of Glands--
Fetal adrenal glands
were obtained from fetuses aged 14-20 weeks (post fertilization) at
the time of therapeutic abortion. Fetal ages were estimated by foot
length and time after menstruation, according to Steeter et
al. (23). The project was approved by the human subject review
committee of our institution. After retrieval, glands were cleansed of
fat and processed immediately for cellular or RNA preparation.
RT-PCR--
RNA isolation and complementary DNA (cDNA)
synthesis: total RNA was isolated from whole human fetal adrenal glands
(14, 17, and 20 weeks of gestation), and fetal human brain (18 weeks)
using RNAqueousTM-4PCR, according to the manufacturer's
recommendations. RNA content and quality were determined
photometrically. 5 µg of total RNA was denatured (70 °C, 10 min)
and reverse-transcribed in the presence of 200 µM
p(dT)12-18 at 42 °C for 50 min in 20 µl of 1× RT
buffer (25 mM Tris-HCl, pH 8.3, 37.5 mM KCl,
1.5 mM MgCl2) containing: 15 mM
dithiothreitol, 200 µM deoxy-NTPs, 25 units of rRNasin
ribonuclease inhibitor, and 200 units of Moloney murine leukemia virus
RT. Inactivation of the enzyme (70 °C, 10 min) was followed by
glyceraldehyde-3-phosphate dehydrogenase PCR to assess the quality of
the cDNA template (24).
PCR Amplifications--
cDNA samples (2 µl) were used for
subsequent PCR amplifications in 50 µl of 1× PCR buffer (10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM
MgCl2) containing 200 µM deoxy-NTPs, 10 pmol
of each of the sense and antisense primers, and 2.5 units of
Taq DNA polymerase. Primers used for the
amplification of PAC1-R splice variant cDNAs were sense, 5'-CTTGTGCAGAAACTTCAGTCTCCAGACATG, and antisense, 5'-TCGGTGCTTGAAGTCCACAGCGAAGTAACGGTTCACCTT (25), corresponding to base
sequences 1235-1264 and 1499-1537 of human PAC-R (4). These primers
flank the insertion site of 84-bp cassettes (5) and were designed to
give a 303-bp amplicon for the normal (null) PACAP receptor, a 387-bp
amplicon for the SV-1 or SV-2 splice variants (insertion of one 84-bp
cassette), and a 471-bp amplicon for the insertion of two cassettes.
PCR was carried out in a PerkinElmer Life Sciences (geneAmp PCR System
2400) thermocycler at 94 °C for 2 min followed by 35 cycles at
94 °C for 30 s, 61 °C for 30 s, 72 °C for 1 min, and
a final extension at 72 °C for 5 min. Primers used for the
amplification of VPAC1-R cDNA were the sense, 5'-ATGTGCAGATGATCGAGGTG, and antisense, 5'-TGTAGCCGGTCTTCACAGAA (26),
corresponding, respectively, to base sequences 127-146 and 431-450 of
human VPAC1-R (6). PCR was carried out at 94 °C for 2 min followed by 30 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 5 min, giving a 324-bp amplicon. Primers used for the amplification of VPAC2-R cDNA were the sense, 5'-TCAAACAGAAAAACACAAAGC,
and antisense, 5'-ACCTGTTCCTGTCCTTCATCC (7), corresponding,
respectively, to base sequences 294-314 and 653-673 of human
VPAC2-R (7). PCR was carried out at 95 °C for 2 min,
followed by 35 cycles at 95 °C for 1 min, 58 °C for 1 min,
72 °C for 1 min, and a final extension at 72 °C for 5 min, giving
a 380-bp amplicon.
In every PCR experiment, amplification in the absence of cDNA and
in the presence of 2 µg of RNA was performed as a control (not
shown). Negative controls using cDNA templates from NCI-H295R and
Chinese hamster ovary cells were performed (not shown). PCR products
(10 µl) were analyzed on 2% (w/v) agarose gel and visualized by
ethidium bromide staining. Length of PCR products was estimated using a
100-bp DNA ladder. Identification of the PCR products was confirmed by
enzymatic digestion and electrophoresis on 3% (w/v) agarose with a
25-bp DNA ladder (not shown). Experiments were performed with RNA
isolated from three different adrenal glands from 14-, 17-, and
20-week-old human fetuses. Three experiments were performed for each age.
Cell Culture--
Glands were processed as described previously
(16). Whole tissues from one or two glands were used for each cell
preparation, without separation of fetal zone, neocortex, or chromaffin
cells. Briefly, small portions of glands (1-2 mm3) were
dissociated with collagenase (2 mg/ml) and DNase (25 µg/ml) in
Eagle's minimal essential medium containing 2% antibiotics. After
three 20-min incubations, cells were dissociated, filtered, and
centrifuged for 10 min at 100 × g. The cell pellet was
suspended in OPTI-MEM medium containing 2% fetal calf serum,
antibiotics, and antimycotics. Cells were plated at a density of
~2 × 105 on plastic coverslips (25 mm). Cells were
grown for 3 days in a humidified atmosphere of 95% air/5%
CO2, at 37 °C.
Calcium Measurement--
For dye loading, cells were incubated
for 30 min at 37 °C in the physiological medium OPTI-MEM containing
4 µM of the fluorescent calcium indicator Fluo-4/AM.
Hydrolysis was performed for 30 min at 37 °C in a medium
containing: 140 mM NaCl; 5.4 mM KCl; 2 mM CaCl2; 1 mM MgCl2;
10 mM HEPES, pH 7.4; and 1 g/liter glucose. The coverslips
were then mounted on the stage of an inverted microscope (Nikon
Diaphot, Mississauga, Ontario, Canada). The light source was generated
by a 100-watt mercury lamp. Band-pass filters (450-490 nm) and
(520-560 nm) were used for excitation and emission, respectively. The
emitted light was recorded by a photon-counting unit. Calcium calibration was performed as described previously (27). However, because calcium dye properties may be different in cytoplasmic and
nucleoplasmic compartments (28), [Ca2+]i should
be considered only as semiquantitative data (29).
Catecholamine Secretion--
An amperometric technique was used
to record secretion from chromaffin cells as described previously (30).
Briefly, a 5-µm carbon fiber electrode (Ala Scientific Instrument
Inc., Westbury, NY) connected to a Patch Clamp PC-501A amplifier
(Warner Instrument Corp., Hamden, CT) modified for voltametry was
positioned in close proximity of a cell. The potential of the electrode
was fixed to 800 mV, and current traces were filtered at 4 kHz and
recorded on a DAS-75 digital recorder (Dagan Corp., Minneapolis, MN).
Data Analysis--
Curves were fitted with SigmaPlot (version
7.0, Chicago, IL). The data are presented as means ± S.E.
from the number of experiments indicated in the legends or in the text.
Statistical analyses of the data were performed using the one-way
analysis of variance test. Homogeneity of variance was assessed by
Bartlett's test, and p values were obtained from Dunnett's tables.
Molecular Identification of PACAP Receptors--
Using
autoradiography, we have previously shown that PACAP receptors in the
human fetal adrenal gland were localized only on chromaffin cells (16).
An RT-PCR approach was used to identify the PACAP receptor subtypes
expressed in adrenal glands from 14- to 20-week-old fetuses. As
indicated under "Materials and Methods," PAC1-R splice
variants were discriminated using primers flanking the insertion site
of the 84-bp cassettes. As shown in Fig.
1A, two bands were detected: a
303-bp band corresponding to the short form of the receptor and a
387-bp band corresponding to an isoform containing a single insertion
cassette. Amplicons of 324 and 380 bp corresponding, respectively, to
VPAC1-R and VPAC2-R sequences were also
detected in all fetal adrenal glands. However, based on our previous
results, these receptors have a more diffuse distribution throughout
the adrenal gland, whereas PAC1-Rs are only detected in
chromaffin cells (16). Expression of the PAC1-R,
VPAC1-R, and VPAC2-R RNAs was also observed in
18-week-old fetal brains. Amplification of glyceraldehyde-3-phosphate
dehydrogenase (Fig. 1D) confirmed that cDNA was present
in each reaction.
Effect of PACAP on Cytosolic Ca2+
Concentration--
Chromaffin cells, which gather into small clusters
with numerous processes, could be easily distinguished from the large
individual fetal steroidogenic cells (Fig.
2, white arrows). Measurements of [Ca2+]i were performed on peripheral cells of
the clusters, selected by a pinhole placed in the optical path (Fig. 2,
asterisk). Mean [Ca2+]i in chromaffin
cells incubated in medium containing 2 mM Ca2+
was 77 nM ± 7 (n = 6) in resting
conditions. Application of 1 × 10 Nature of the Intracellular Ca2+ Pool--
We have
previously shown that PACAP-38 induces a 3.4-fold increase in cAMP
production and a modest increase in IP3 formation in fetal
human chromaffin cells (16). In the present study, we first
investigated whether or not the PACAP-sensitive Ca2+ pool
was responsive to thapsigargin (TG), a known inhibitor of the
sarco(endo)plasmic reticulum Ca2+-ATPase pumps but without
effect on plasma membrane Ca2+-ATPase activity (33). In one
series of experiments, the cells were bathed in Ca2+-free
medium. Application of PACAP-38 (1 × 10
In some other cell types, it has been shown that PACAP receptors are
coupled to PLC through a Gq/11 protein to produce
diacylglycerol and IP3 (34). In the specific case of human
fetal chromaffin cells, we previously found that IP3
production is relatively low (16). Hence, experiments were performed to
assess the putative role of IP3-sensitive calcium pools in
the PACAP-induced increase in [Ca2+]i. Cells were
treated with U-73122 compound, a specific PLC
Involvement of a cAMP-sensitive calcium pool in the PACAP response was
tested by directly activating cAMP production with forskolin (FSK).
Fig. 7A demonstrates that FSK
(1 × 10
Caffeine is known to activate ryanodine channels and to
induce [Ca2+]i increase in numerous cell types
(37). Application of caffeine (20 mM) to chromaffin cells
induced an increase in [Ca2+]i (Fig.
8A). More importantly, the
Ca2+ response to PACAP-38 (1 × 10
A recent study reported that neuroendocrine cells contained dense core
secretory vesicles that could constitute a dynamic Ca2+
store, whereby the P-type Ca2+ pump was responsible for
Ca2+ uptake in these secretory vesicles (38). To assess
such putative participation, we used orthovanadate at 100 µM, a concentration known to inhibit the
ATP-dependent P-type Ca2+ pump. In
Ca2+-free medium, cells were first challenged with PACAP-38
(1 × 10 Functional Properties of Chromaffin Cells--
Activation of
cholinergic receptors in chromaffin cells is the main stimulus for
mobilizing Ca2+ to induce catecholamine secretion (39).
Hence, experiments were undertaken to determine if chromaffin cells
from 17- to 20-week-old human fetuses express functional cholinergic
receptors. Release of Ca2+ from IP3-sensitive
Ca2+ pools was tested using methacholine, a muscarinic
receptor agonist. Fig. 9A
shows that application of methacholine (10 µM) to
chromaffin cells induced a large, transient increase of
[Ca2+]i followed by a plateau indicating an
influx of Ca2+as previously shown for guinea pig adrenal
chromaffin cells (40). Moreover, voltage-dependent
Ca2+ channels are functional in 17- to 20-week-old human
fetal adrenal chromaffin cells. Indeed, membrane depolarization caused
by activation of nicotinic receptors with nicotine (10 µM) induced a [Ca2+]i increase
(Fig. 9B) similar to that obtained after depolarization
using KCl (30 mM) (Fig. 9C).
Because PACAP-38 increased [Ca2+]i in human
chromaffin cells, the question arises as to whether these cells are
capable of catecholamine secretion. A carbon fiber (5 µm) for
amperometric detection (41) was used to monitor catecholamine secretion
from cell clusters. Stimulation with PACAP-38 (5 × 10 Our study demonstrates that the fetal human adrenal gland
expresses both type I and type II PACAP receptors. Activation of chromaffin PACAP receptors with PACAP-38 induced a transient increase in [Ca2+]i originating exclusively from
intracellular calcium pools and did not involve Ca2+ influx
from the external medium. Moreover, the PACAP-sensitive Ca2+ pool was not mobilized by IP3 or TG. More
importantly, PACAP-38 activated a ryanodine/caffeine-sensitive pool,
which involved cAMP and a phosphorylation step by PKA. In addition, we
were able to demonstrate that activation of the PAC1
receptor induced secretion of catecholamine by the chromaffin cells.
The results presented herein indicate that, among the various isoforms
of the PACAP receptors, the short fragment and one isoform A of an
hip-hop insertion cassette of the PAC1 receptors are
present in the human fetal adrenal gland at the second trimester of
gestation. By using PACAP (6-38), a PAC1 receptor-specific antagonist (32), we demonstrate that the Ca2+ increase
induced by PACAP-38 is mediated by the activation of PAC1-R
coupled to adenylyl cyclase (2).
One important finding of this study is the observation that the
PACAP-induced Ca2+ rise was not affected by the absence of
Ca2+ in the external medium. This clearly indicated that
PACAP-38 does not trigger a Ca2+ influx through channels or
exchangers, in agreement with data obtained in rat hippocampal neurons
(31). However, a number of reports indicate that PACAP activates a
Ca2+ influx through various pathways, including
Ca2+ channels (29, 42-44). In chromaffin cells from adult
human adrenal glands, voltage-dependent Ca2+
channels have been described (45), but no electrophysiological studies
have been performed in fetal cells. KCl depolarization, or application
of nicotine, were able to induce an increase in [Ca2+]i in human fetal chromaffin cells
suggesting the presence of functional voltage-dependent
Ca2+ channels. However, our results clearly show that these
Ca2+ channels were not involved in PACAP-induced
Ca2+ increase.
The second most important feature of the present data was that
PACAP-responsive Ca2+ pools were insensitive to TG. It has
been reported that TG triggers the release of Ca2+ from
major nonmitochondrial Ca2+ stores (46), including the
IP3-sensitive Ca2+ pool (47). Our data
conclusively demonstrate that preincubation of the cells with TG or
chronic application of high concentrations of TG (8 µM)
have no effect on the Ca2+ response triggered by PACAP-38.
Previous experiments have shown that, in human fetal chromaffin cells,
PACAP-38 triggered low production of IP3 (16). However, the
present results, using either the PLC inhibitor U73132 (35) or
Xestospongin C, a blocker of IP3 receptors (36),
demonstrated that Ca2+ release from
IP3-sensitive Ca2+ stores did not contribute to
the increase in [Ca2+]i triggered by
PACAP-38.
Several lines of evidence pointed toward involvement of the cAMP/PKA
pathway in the [Ca2+]i increase triggered by
PACAP-38 in human fetal chromaffin cells. FSK, which directly activates
adenylyl cyclase to produce cAMP, triggered an increase in
Ca2+ that was similar in amplitude and kinetics to that
induced by PACAP-38. Moreover, like PACAP-38, the response triggered by
FSK was independent of the presence of Ca2+ in the external
medium. Additionally, blocking PKA using H-89 or
(Rp)-cAMPS inhibited the effects of both
PACAP-38 and FSK on [Ca2+]i. Opposite results
were found in hippocampal neurons where PACAP-induced Ca2+
response was not triggered by FSK or (Bu)2cAMP and
insensitive to H-89 (31). Cyclic AMP-sensitive Ca2+ stores
have been described in numerous cell types, but their relationship with
the IP3- and the ryanodine/caffeine Ca2+ stores
is still a matter of debate. In several cell types, it has been shown
that the ryanodine receptor is subject to phosphorylation by several
kinases, including PKA (48, 49). Our results clearly indicated that
PACAP- and FSK-induced Ca2+ increases were sensitive to
PKA-dependent phosphorylation. This, together with the
effect of ryanodine/caffeine, could indicate that PACAP-38 mobilizes
Ca2+ from caffeine-sensitive stores in human fetal
chromaffin cells.
Application of caffeine to human fetal chromaffin cells triggered a
moderate increase in [Ca2+]i confirming the
presence of caffeine-sensitive Ca2+ stores. In bovine adult
chromaffin cells, PACAP released Ca2+ from a
ryanodine/caffeine store (50), which was independent of
IP3, as observed for our cell model. However, it was also
reported that Ca2+ rise was insensitive to
(Rp)-cAMPS contrary to human fetal chromaffin cells where H-89, or (Rp)-cAMPS, completely
inhibited both PACAP- and FSK-induced responses. The role and
weightiness of the caffeine-dependent Ca2+
pools in chromaffin cells have been outlined by several authors (51)
who report that caffeine-dependent Ca2+ pools
release more Ca2+ than
IP3-dependent Ca2+ pools in
permeabilized chromaffin cells (52, 53). The signaling pathway has not
yet been fully characterized, although several possibilities have been
proposed (54). More recently, secretory vesicles in neuroendocrine
cells have been demonstrated to constitute a dynamic Ca2+
pool (38). Several features of the vesicle-Ca2+ stores are
similar to those defined by PACAP-dependent stores. Indeed,
these pools are insensitive to TG, are not mobilized by IP3, and are sensitive to caffeine and ryanodine. As
proposed by Mitchell et al. (38), cytosolic Ca2+
is pumped into the deep vesicles by an ATP-dependent P-type
Ca2+ pump. Ca2+ is released by primed vesicles
located near the plasma membrane when [Ca2+]i
concentrations increase due to Ca2+ channel opening (55).
Our data indicate that treatment of chromaffin cells with
Na3VO4 did not empty the PACAP-sensitive
Ca2+ pools, as illustrated by identical levels of
Ca2+ increase obtained before and after
Na3VO4 application. However, the fact that the
amplitude of the subsequent responses to PACAP-38 decreased as a
function of the number of PACAP-38 applications indicated that
re-pumping of Ca2+ may have been impaired by
Na3VO4.
By using amperometry, we also demonstrated that human fetal chromaffin
cells release catecholamine in response to PACAP-38. [Ca2+]i increase is a requirement, as outlined by
the absence of secretion in cells treated with PACAP (6-38) or
(Rp)-cAMPS. Catecholamine secretion could be
linked to the paracrine control of secretion of
dehydroepiandrosterone sulfate and cortisol by fetal steroidogenic
cells. Indeed, in cell cultures, PACAP-38 stimulates DHEA and cortisol
secretion, an effect abolished by preincubation with the
In summary, stimulation of chromaffin cells with PACAP-38 induced a
cAMP-dependent increase in intracellular Ca2+
whose properties and regulation exhibit particular features. This model
has thus enabled the identification of a novel pathway in the
regulation of cAMP-dependent Ca2+ release,
which could be specific to fetal tissues. In addition to its role in
catecholamine secretion, PACAP-38 could act as a survival factor for
chromaffin cells, because, in contrast to fetal cells, chromaffin cells
do not undergo apoptosis, either in vivo or in
vitro (56, 57). Recent studies conducted in knock-out mice for
PACAP, revealed that PACAP was not essential for normal development of
the adrenal gland, nor for basal catecholamine secretion; however, its
presence was essential for adaptive responses. Indeed, mice lacking
PACAP were unable to survive in response to a metabolic stress (58).
Taken together, these results clearly indicate that PACAP is one of the
important factors necessary in maintaining adequate interaction between
catecholamine and steroid-producing cells, an interaction that occurs
during normal development.
(preincubation with U-73122) or by pretreatment of cells with
Xestospongin C, indicating that the inositol
1,4,5-triphosphate-sensitive stores were not mobilized. However,
forskolin (FSK), which raises cytosolic cAMP, induced an increase in
Ca2+ similar to that recorded with PACAP-38. Blockage of
PKA by H-89 or (Rp)-cAMPS suppressed
both PACAP-38 and FSK calcium responses. The effect of PACAP-38 was
also abolished by emptying the caffeine/ryanodine-sensitive Ca2+ stores. Furthermore, treatment of cells with
orthovanadate (100 µM) impaired Ca2+
reloading of PACAP-sensitive stores indicating that PACAP-38 can
mobilize Ca2+ from secretory vesicles. Moreover, PACAP
induced catecholamine secretion by chromaffin cells. It is concluded
that PACAP-38, through the PAC1 receptor, acts as a
neurotransmitter in human fetal chromaffin cells inducing catecholamine
secretion, through nonclassical, recently described,
ryanodine/caffeine-sensitive pools, involving a cAMP- and
PKA-dependent phosphorylation mechanism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amidated neuropeptide
(PACAP-38)1 originally
isolated from the ovine hypothalamus for its ability to stimulate cAMP
formation in rat anterior pituitary cells. Processing of PACAP-38 can
generate a 27-amino acid amidated peptide (PACAP-27) that exhibits 68%
sequence identity with vasoactive intestinal polypeptide (VIP), thus
identifying PACAP as a member of the VIP/secretin/glucagon superfamily
of regulatory peptides (1, 2).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (60K):
[in a new window]
Fig. 1.
Expression of PACAP receptor isoforms in the
human fetal adrenal gland. cDNA from adrenal glands 14-, 17-, and 20-week-old fetuses and from 18-week-old human fetal brain
(HFB) were amplified using oligonucleotide primers encoding:
A, nucleotide sequences 1235-1264 and 1499-1537 of
the PAC1 receptor, designed to give three possible
amplicons of 303, 387, and 471 bp; B, nucleotide sequences
127-146 and 431-450 of the human VPAC1 receptor; and
C, nucleotide sequences 294-314 and 653-673 of the human
VPAC2 receptor. D, amplification of
glyceraldehyde-3-phosphate dehydrogenase (192 bp) was used to ensure
RNA quality and amounts.
9 M
PACAP-38 induced a transient elevation of [Ca2+]i
(Fig. 3A). This [Ca2+]i increase was
characterized by a rapid upstroke followed by a slower decrease to
basal levels. The time course of the decrease could be fitted by a
monoexponential function. At a concentration of 1 × 10
9 M PACAP-38, the time constant
was
57.3 ± 7.4 s (n = 9). In Ca2+-free medium (0 Ca2+ plus 1 mM
EGTA), application of 1 × 10
9 M
PACAP-38 provoked an increase in [Ca2+]i similar
to that recorded in 2 mM Ca2+-containing medium
(Fig. 3B) with a time constant
(49.7 ± 3.5 s; n = 11) that was not
significantly different (p = 0.34), suggesting that
PACAP-38 causes mobilization of intracellular Ca2+ stores.
Indeed, application of Ni2+, a known Ca2+
channel blocker, during the decreasing phase of the Ca2+
response, had no effect on the kinetics of the Ca2+ spike
induced by application of PACAP-38 (1 × 10
9
M) in 2 mM Ca2+-containing medium,
whereas a second application of PACAP-38 in the presence of
Ni2+ provoked a Ca2+ response similar to that
obtained in the absence of the blocker (Fig. 3C).
Application of increasing concentrations of PACAP-38 resulted in a
dose-dependent rise in the amplitude of the
[Ca2+]i response with an ED50 value
of 5 nM (Fig. 3D), a value similar to that
obtained (2.6 nM) by measuring the
[Ca2+]i increase on hippocampal neurons (31).
Administration of repeated pulses of PACAP-38 (1 × 10
9 M) at various time intervals induced a
reproducible [Ca2+]i increase, even when applied
at very short intervals (Fig.
4A). The amplitudes of the
responses to any of the four pulses applied were not significantly
different (Fig. 4B). To further confirm that the
PACAP-induced [Ca2+]i increase was due to the
activation of the PAC1-R type receptor, we used PACAP
(6-38), a PAC1 receptor-specific antagonist shown to
inhibit the activation of adenylyl cyclase with a Ki of 7 nM (32). A first application of PACAP-38 (1 × 10
7 M) triggered a Ca2+ response
as previously described. The antagonist PACAP (6-38) was then applied
(10 µM) for a 10-min period and followed by a second
application of PACAP-38, at the same concentration, which induced a
lower Ca2+ increase (Fig. 4C). For
concentrations of PACAP (6-38) of 0.3, 1, and 10 µM, the
Ca2+ response to PACAP-38 (1 × 10
7
M) was reduced to 48.5 ± 12% (n = 4), 25 ± 2% (n = 2), and 6.9 ± 3.4%
(n = 4) of the control, respectively (Fig.
4D).
View larger version (131K):
[in a new window]
Fig. 2.
Phase-contrast morphology of a human fetal
adrenal gland cell culture. A and B,
representative illustrations from two different cell cultures. After
24 h in culture, chromaffin cells (CC) were gathered in
small clusters that exhibited several long processes. Cells from the
fetal zone (white arrows) were easily identified by their
size and their polygonal morphology. Black arrows indicate
contact between a chromaffin cell extension and a fetal cell. The
asterisk indicates a cell selected by a pinhole
placed in the optical path. Scale bars represent 10 µm.
View larger version (16K):
[in a new window]
Fig. 3.
Effects of PACAP-38 on
[Ca2+]i in human fetal chromaffin cells.
A, PACAP-38 (P) 1 × 10 9
M was applied in a medium containing 2 mM of
Ca2+ (representative of 9 cells from 9 different cell
cultures). Scale: vertical, 25 nM;
horizontal, 50 s. B, PACAP-38 (P)
1 × 10
9 M was applied in a
Ca2+-free medium containing 1 mM EGTA
(representative of 11 cells from 9 different cell cultures). Scale:
vertical, 20 nM; horizontal, 50 s. C, application of Ni2+ (500 µM)
during the falling phase of the Ca2+ response induced by
PACAP-38 (P) 1 × 10
9 M had
no effect on the time course of the response nor on the subsequent
response induced by a new application of PACAP-38 (representative of 5 cells from 5 different cell cultures). Scale: vertical, 11 nM; horizontal, 100 s. D,
relationship between the concentration of PACAP-38 and the
Ca2+ increase (n = 3, 5, 15, 3, 3, and 3 for 0.01, 0.1, 1, 40, 200, and 1000 nM of PACAP-38,
respectively). The experimental points were fitted by the logistic
function with an ED50 of 5 nM.
View larger version (37K):
[in a new window]
Fig. 4.
Frequency dependence of the PACAP-induced
Ca2+ increase in the human fetal adrenal gland.
A, four identical concentrations of PACAP-38 (P)
1 × 10 9 M were applied on the same
cell. Note that the four Ca2+ increases are identical with
no apparent desensitization. Scale: vertical, 20 nM; horizontal, 100 s. B,
compilation of data obtained in three different cells from three
different glands where PACAP-38 was applied four successive times
indicated by 1, 2, 3, and 4 corresponding to the first, second, third, and fourth PACAP-38
applications. Data are normalized to the first response. C,
effect of PACAP (6-38) on the PACAP-induced Ca2+ increase
in the human fetal adrenal gland. A first application of PACAP-38
(P) (1 × 10
7 M), which
elicited a Ca2+ increase, was followed by application of 10 µM of the PAC1-R antagonist PACAP (6-38)
(I). After 10 min, PACAP-38 (P) (1 × 10
7 M) was further applied. Scale:
vertical, 13 nM; horizontal, 20 s. D, inhibition of the PACAP-38 (1 × 10
7 M) Ca2+ responses by PACAP
(6-38) applied during 10 min; 1, normalized
Ca2+ increase in control condition; 2, blockage
of the Ca2+ response by 0.3 µM PACAP (6-38)
obtained in four different cells from three different glands;
3, blockage of the Ca2+ response by 1 µM of PACAP (6-38) obtained in two different cells from
two different glands; 4, blockage of the Ca2+
response by 10 µM PACAP (6-38) obtained in four
different cells from three different glands. *, significantly different
at the p = 0.05 level.
9
M) produced an increase in Ca2+ as described
previously (Fig. 5A). The
subsequent application of TG (4 µM) triggered an
additional increase in [Ca2+]i resulting from
blockage of the sarco(endo)plasmic reticulum Ca2+-ATPase
pumps. When PACAP-38 was applied further, the amplitude and kinetics of
the Ca2+ increase were similar to those obtained prior to
TG application, indicating that TG and PACAP-38 did not mobilize the
same Ca2+ pool(s). In a second series of experiments, cells
were preincubated for 30 min in a calcium-free medium containing 8 µM TG. Under these conditions, the Ca2+
response to TG (4 µM) was greatly reduced, whereas the
response to PACAP-38 (2.5 × 10
9 M) was
not affected (Fig. 5B).
View larger version (15K):
[in a new window]
Fig. 5.
Lack of sensitivity of the PACAP-induced
Ca2+ increase to thapsigargin in the human fetal adrenal
gland. A, a first application of PACAP-38
(P) 1 × 10 9 M, which
elicited a Ca2+ increase, was followed by application of
4 × 10
6 M of thapsigargin
(TG) to deplete the TG-sensitive Ca2+ pools. A
subsequent application of PACAP-38 at the same concentration triggered
a Ca2+ response, which was not affected by TG
(representative of three cells, three different cells cultures);
experiments conducted in a Ca2+-free medium. Scale:
vertical, 14 nM; horizontal, 100 s. B, the cells were preincubated in a medium containing
8 × 10
6 M of TG during 30 min.
Application of PACAP-38 (P) 2.5 × 10
9
M elicited a Ca2+ increase similar to the
response obtained in control conditions, whereas TG (4 × 10
6 M) elicited a slight increase indicating
that the TG-sensitive Ca2+ stores are depleted
(representative of four cells, three different cells cultures). Scale:
vertical, 38 nM; horizontal, 100 s.
inhibitor (35), at a
concentration of 1 µM for 18 h. PACAP-38 (1 × 10
9 M) was then added in the presence or
absence of Ca2+ in the external medium as described above.
In five different cells, the amplitude as well as the kinetics of the
Ca2+ response to PACAP-38 were similar to those obtained in
control cells (Fig. 6A),
indicating that the Ca2+ increase was not dependent on
IP3-sensitive pools. Further confirmation of these results
was provided from experiments using Xestospongin C (XeC), a potent
specific blocker of the inositol 1,4,5-triphosphate (IP3)
receptors (36). In this experimental design, PACAP-38 (1 × 10
9 M) was first applied to induce a
[Ca2+]i increase. XeC (20 µM) was
then applied for 10 min prior to a second application of PACAP-38. Fig.
6B shows that the amplitude of the Ca2+ increase
was not affected by XeC, thus indicating that IP3-sensitive Ca2+ pools are not involved. Similar results were obtained
from five different cells.
View larger version (17K):
[in a new window]
Fig. 6.
PACAP-38 does not mobilize
IP3-sensitive Ca2+ stores in the human fetal
adrenal glands. A, cells were preincubated for 18 h in
the presence of the PLC inhibitor U73122 (1 × 10 6
M). Experiments were conducted in a Ca2+-free
medium. This treatment did not modify the PACAP-38 (P)
1 × 10
9 M-induced Ca2+
increase (representative of five cells, three different cell cultures).
Scale: vertical, 20 nM; horizontal,
50 s. B, Xestospongin C (XeC) 20 × 10
6 M was applied after a first response
elicited by PACAP-38 (P) 1 × 10
9
M. The second application of PACAP-38 (P)
triggered a similar Ca2+ increase (representative of five
cells, five different cells cultures). Scale: vertical, 14 nM; horizontal, 100 s.
5 M) and PACAP-38 (1 × 10
9 M) triggered identical calcium increases.
Similar results were obtained in the absence of Ca2+ in the
bathing medium (data not shown, n = 3). The involvement of a PKA-dependent phosphorylation step in the increase in
Ca2+ triggered by PACAP was assessed by using H-89, an
inhibitor of PKA. When cells were pretreated with H-89 (10 µM) for 15 min, subsequent stimulation with PACAP-38
failed to trigger a Ca2+ increase (Fig. 7B,
n = 7). The FSK-triggered Ca2+ increase was
also abolished (Fig. 7B, n = 4). When
(Rp)-cAMPS (1 mM), a more specific
membrane-permeant inhibitor of PKA was used, the response to PACAP-38
(2 × 10
7 M), but not to TG, was
abolished (Fig. 7C, n = 4).
View larger version (16K):
[in a new window]
Fig. 7.
PACAP-38 uses the cAMP/PKA pathway to trigger
Ca2+ increase in the human fetal adrenal gland.
A, forskolin (FSK) 1 × 10 5
M triggered a Ca2+ increase similar to that
evoked by PACAP-38 (P) 1 × 10
9
M (representative of three cells, three different cell
cultures). Scale: vertical, 38 nM;
horizontal, 100 s. B, cells were treated
with H-89 (10 × 10
6 M) for 15 min.
Application of PACAP-38 (P) 2 × 10
7
M as well as FSK (1 × 10
6
M) were not able to trigger Ca2+ increase
(representative of seven cells, five different cell cultures). Scale:
vertical, 20 nM; horizontal, 50 s. C, a first application of PACAP-38 (P) 2 × 10
7 M elicited a Ca2+
response. The second application, after
(Rp)-cAMPS (1 × 10
3
M) did not induce any response. In all panels, thapsigargin
(TG) is always able to elicit a large increase in
intracellular Ca2+ (representative of four cells, four
different cell cultures). Scale: vertical, 100 nM; horizontal, 200 s.
9
M) was significantly decreased following caffeine
application to chromaffin cells (
51 ± 6.7%, n = 5). Similar results were obtained with ryanodine (20 µM, n = 4, data not shown).
View larger version (21K):
[in a new window]
Fig. 8.
PACAP-38 mobilizes Ca2+ from a
ryanodine/caffeine-sensitive stores in the human fetal adrenal
gland. A, addition of caffeine (20 mM)
triggered a Ca2+ increase, revealing the presence of a
ryanodine/caffeine store. Emptying this store blunted the
Ca2+ response to PACAP-38 (P) 1 × 10 9 M (representative of five cells, three
different cell cultures). Scale: vertical, 11 nM; horizontal, 100 s. B, cells
were treated for 10-15 min with Na3VO4 (100 µM) and challenged several times by PACAP-38
(P) 1 × 10
9 M. The amplitude
of the Ca2+ response decreased as a function of the number
of trials (representative of six cells from four different cell
cultures). Scale: vertical, 10 nM;
horizontal, 50 s. C, plot of the amplitude
of the Ca2+ responses for subsequent trials. 1,
response in control conditions; 2, first response after
treatment with Na3VO4; 3, second
response after Na3VO4; 4, third
response after Na3VO4; data are normalized to
the amplitude of the first response after
Na3VO4 (bar number 2). Compilation
of data from B. *, significantly different at the
p = 0.05 level.
9 M) followed by application of
Na3VO4 (100 µM) for 10 min.
Thereafter, the first addition of PACAP-38 induced a Ca2+
response not significantly different in amplitude (9.2 ± 9%
decrease, n = 6) from that obtained prior to adding
Na3VO4. However, subsequent applications of
PACAP-38 gave rise to a lower amplitude of Ca2+ increase
(
63% and
86% for the second and third PACAP-38 applications, respectively) (Fig. 8B). Data obtained from three different
cell cultures are summarized in Fig. 8C.
View larger version (9K):
[in a new window]
Fig. 9.
Cholinergic agonists and KCl increase
[Ca2+]i in fetal human chromaffin cells in the
human fetal adrenal gland. A, methacholine (10 × 10 6 M), a muscarinic agonist, induced a
Ca2+ increase when applied in the bath (representative of
three cells, three different cell cultures). Scale:
vertical, 50 nM; horizontal, 200 s. B, Ca2+ increase upon addition of nicotine
(10 × 10
6 M) (representative of three
cells, three different cell cultures). Scale: vertical, 38 nM; horizontal, 200 s. C,
depolarization by 30 mM KCl induced a Ca2+
increase (representative of three cells, three different cell
cultures). Scale: vertical, 14 nM;
horizontal, 50 s.
7 M) in a 2 mM Ca2+
medium, triggered the quantal release of catecholamine by chromaffin cells as demonstrated by the occurrence of spikes under the carbon electrode (Fig. 10A,
n = 5). If the cells are preincubated for 20 min with
the PAC1-R antagonist PACAP (6-38) (10 µM),
application of PACAP-38 (5 × 10
7 M)
failed to stimulate catecholamine release (Fig. 10B,
n = 4). Similar results were obtained if the cells are
preincubated with (Rp)-cAMPS (1 mM,
10 min) prior application of PACAP-38 (data not shown,
n = 2).
View larger version (15K):
[in a new window]
Fig. 10.
Amperometric recordings of catecholamine
secretion by fetal human chromaffin cells. Current spikes were
recorded by a carbon electrode positioned near the cell. A,
secretion induced by application of PACAP-38 (P) 5 × 10 7 M. Scale: vertical, 0.8 pA;
horizontal, 625 s. (representative of five cells, three
different cells cultures). B, cells were preincubated for 20 min in the presence of PACAP (6-38) 1 × 10
5
M and challenged with PACAP-38 (P) (5 × 10
7 M). Scale: vertical, 0.8 pA;
horizontal, 800 s (representative of four cells, two
different cells cultures).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-adrenoreceptor antagonist, propranolol (15).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Estelle Chamoux and Lucie Chouinard for their expert experimental assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Canadian Institute of Health Research (Grants MOP-37891 and MT-6813 to N. G. P. and M. D. P.) and by a France-Quebec Exchange Program with Hubert Vaudry.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.
§ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, 3001 12th Ave. North, Sherbrooke, Quebec J1H 5N4, Canada. Tel.: 819-564-5305; Fax: 819-564-5399; E-mail: Marcel.Payet@USherbrooke.ca.
A recipient of a studentship from the Medical Research Council
of Canada.
§§ Holder of The Canadian Research Chair in Endocrinology of the Adrenal Gland.
Published, JBC Papers in Press, November 11, 2002, DOI 10.1074/jbc.M206470200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PACAP-38, pituitary adenylate cyclase-activating polypeptide of 38 residues; VIP, vasoactive intestinal polypeptide; PAC1-R, PACAP-specific receptor I; VPAC, VIP/PACAP mutual receptor; R, receptor; PLC, phospholipase C; RT, reverse transcriptase; cDNA, complementary DNA; IP3, inositol 1,4,5-triphosphate; TG, thapsigargin; XeC, Xestospongin C; FSK, forskolin; PKA, protein kinase A; MEM, minimal essential medium; (Rp)-cAMPS, Rp-adenosine 3',5'-cyclic monophosphorothioate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Arimura, A. (1998) Jpn. J. Physiol. 48, 301-331[Medline] [Order article via Infotrieve] |
2. |
Vaudry, D.,
Gonzalez, B.,
Basille, M.,
Yon, L.,
Fournier, A.,
and Vaudry, H.
(2000)
Pharmacol. Rev.
52,
269-324 |
3. |
Harmar, A.,
Arimura, A.,
Gozes, I.,
Journot, L.,
Laburthe, M.,
Pisegna, J.,
Rawlings, S.,
Robberecht, P.,
Said, S.,
Sreedharan, S.,
Wank, S.,
and Waschek, J.
(1998)
Pharmacol. Rev.
50,
265-270 |
4. | Ogi, K., Miyamoto, Y., Masuda, Y., Habata, Y., Hosoya, M., Ohtaki, T., Masuo, Y., Onda, H., and Fujino, M. (1993) Biochem. Biophys. Res. Commun. 196, 1511-1521[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Pisegna, J.,
and Wank, S.
(1996)
J. Biol. Chem.
271,
17267-17274 |
6. | Couvineau, A., Rouyer-Fessard, C., Darmoul, D., Maoret, J., Carrero, I., Ogier-Denis, E., and Laburthe, M. (1994) Biochem. Biophys. Res. Commun. 200, 769-776[CrossRef][Medline] [Order article via Infotrieve] |
7. | Svoboda, M., Tastenoy, M., Van Rampelbergh, J., Goossens, J., De, Neef, P., Waelbroeck, M., and Robberecht, P. (1994) Biochem. Biophys. Res. Commun. 205, 1617-1624[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Murthy, K.,
and Makhlouf, G.
(1994)
J. Biol. Chem.
269,
15977-15980 |
9. |
Villalba, M.,
Bockaert, J.,
and Journot, L.
(1997)
J. Neurosci.
17,
83-90 |
10. | Gonzalez, B., Basille, M., Vaudry, D., Fournier, A., and Vaudry, H. (1997) Neuroscience 78, 419-430[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Vaudry, D.,
Gonzalez, B.,
Basille, M.,
Fournier, A.,
and Vaudry, H.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
9415-9420 |
12. | Tabarin, A., Chen, D., Hakanson, R., and Sundler, F. (1994) Neuroendocrinology 59, 113-119[Medline] [Order article via Infotrieve] |
13. | Frodin, M., Hannibal, J., Wulff, B., Gammeltoft, S., and Fahrenkrug, J. (1995) Neuroscience 65, 599-608[CrossRef][Medline] [Order article via Infotrieve] |
14. | Moller, K., and Sundler, F. (1996) Regul. Pept. 63, 129-139[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Breault, L.,
Yon, L.,
Montero, M.,
Chouinard, L.,
Contesse, V.,
Delarue, C.,
Fournier, A.,
Lehoux, J. G.,
Vaudry, H.,
and Gallo-Payet, N.
(2000)
Ann. N. Y. Acad. Sci.
921,
429-433 |
16. |
Yon, L.,
Breault, L.,
Contesse, V.,
Bellancourt, G.,
Delarue, C.,
Fournier, A.,
Lehoux, J.,
Vaudry, H.,
and Gallo-Payet, N.
(1998)
J. Clin. Endocrinol. Metab.
83,
1299-1305 |
17. | Michelsohn, A., and Anderson, D. (1992) Neuron 8, 589-604[Medline] [Order article via Infotrieve] |
18. | Ehrhart-Bornstein, M., Breidert, M., Guadanucci, P., Wozniak, W., Bocian-Sobkowska, J., Malendowicz, L. K., and Bornstein, S. R. (1997) Horm. Metab. Res. 29, 30-32[Medline] [Order article via Infotrieve] |
19. |
Mesiano, S.,
and Jaffe, R.
(1997)
Endocr. Rev.
18,
378-403 |
20. | Chowdhury, P., Guo, X., Wakade, T., Przywara, D., and Wakade, A. (1994) Neuroscience 59, 1-5[CrossRef][Medline] [Order article via Infotrieve] |
21. | Guo, X., and Wakade, A. (1994) J. Physiol. (Lond.) 475, 539-545[Abstract] |
22. | Chartrel, N., Conlon, J. M., Danger, J. M., Fournier, A., Tonon, M. C., and Vaudry, H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3862-3866[Abstract] |
23. | Streeter, G. (1920) Contr. Embryol. 11, 143-179 |
24. | Basille, M., Vaudry, D., Coulouarn, Y., Jegou, S., Lihrmann, I., Fournier, A., Vaudry, H., and Gonzalez, B. (2000) J. Comp. Neurol. 425, 495-509[CrossRef][Medline] [Order article via Infotrieve] |
25. | Olianas, M., Ingianni, A., Sogos, V., and Onali, P. (1997) J. Neurochem. 69, 1213-1218[Medline] [Order article via Infotrieve] |
26. | Solano, R., Carmena, M., Carrero, I., Cavallaro, S., Roman, F., Hueso, C., Travali, S., Lopez-Fraile, N., Guijarro, L., and Prieto, J. (1996) Endocrinology 137, 2815-2822[Abstract] |
27. | Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450[Abstract] |
28. | Perez-Terzic, C., Stehno-Bittel, L., and Clapham, D. E. (1997) Cell Calcium 21, 275-282[Medline] [Order article via Infotrieve] |
29. |
Kopp, M. D.,
Schomerus, C.,
Dehghani, F.,
Korf, H. W.,
and Meissl, H.
(1999)
J. Neurosci.
19,
206-219 |
30. | Wightman, R. M., Jankowski, J. A., Kennedy, R. T., Kawagoe, K. T., Schroeder, T. J., Leszczyszyn, D. J., Near, J. A., Diliberto, E. J., Jr., and Viveros, O. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10754-10758[Abstract] |
31. | Tatsuno, I., Yada, T., Vigh, S., Hidaka, H., and Arimura, A. (1992) Endocrinology 131, 73-81[Abstract] |
32. | Robberecht, P., Gourlet, P., De, Neef, P., Woussen-Colle, M. C., Vandermeers-Piret, M. C., Vandermeers, A., and Christophe, J. (1992) Mol. Pharmacol. 42, 347-355[Abstract] |
33. |
Lytton, J.,
Westlin, M.,
and Hanley, M. R.
(1991)
J. Biol. Chem.
266,
17067-17071 |
34. | Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P., and Journot, L. (1993) Nature 365, 170-175[CrossRef][Medline] [Order article via Infotrieve] |
35. | Salari, H., Bramley, A., Langlands, J., Howard, S., Chan-Yeung, M., Chan, H., and Schellenberg, R. (1993) Am. J. Respir. Cell Mol. Biol. 9, 405-410[Medline] [Order article via Infotrieve] |
36. |
Ma, H. T.,
Patterson, R. L.,
van Rossum, D. B.,
Birnbaumer, L.,
Mikoshiba, K.,
and Gill, D. L.
(2000)
Science
287,
1647-1651 |
37. | Kramer, R., Mokkapatti, R., and Levitan, E. (1994) Pflugers Arch. 426, 12-20[Medline] [Order article via Infotrieve] |
38. |
Mitchell, K. J.,
Pinton, P.,
Varadi, A.,
Tacchetti, C.,
Ainscow, E. K.,
Pozzan, T.,
Rizzuto, R.,
and Rutter, G. A.
(2001)
J. Cell Biol.
155,
41-52 |
39. | Burgoyne, R. (1991) Biochim. Biophys. Acta 1071, 174-202[Medline] [Order article via Infotrieve] |
40. | Ohta, T., Asano, T., Ito, S., Kitamura, N., and Nakazato, Y. (1996) Cell Calcium 20, 303-314[Medline] [Order article via Infotrieve] |
41. | Zhou, Z., and Misler, S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6938-6942[Abstract] |
42. |
Osipenko, O.,
Barrie, A.,
Allen, J.,
and Gurney, A.
(2000)
J. Biol. Chem.
275,
16626-16631 |
43. | Hahm, S., Hsu, C., and Eiden, L. (1998) J. Mol. Neurosci. 11, 43-56[Medline] [Order article via Infotrieve] |
44. |
Przywara, D.,
Guo, X.,
Angelilli, M.,
Wakade, T.,
and Wakade, A.
(1996)
J. Biol. Chem.
271,
10545-10550 |
45. | Gandia, L., Mayorgas, I., Michelena, P., Cuchillo, I., de Pascual, R., Abad, F., Novalbos, J., Larranaga, E., and Garcia, A. (1998) Pflugers Arch. 436, 696-704[CrossRef][Medline] [Order article via Infotrieve] |
46. | Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205[CrossRef][Medline] [Order article via Infotrieve] |
47. | Gouy, H., Cefai, D., Christensen, S. B., Debre, P., and Bismuth, G. (1990) Eur. J. Immunol. 20, 2269-2275[Medline] [Order article via Infotrieve] |
48. | Hohenegger, M., and Suko, J. (1993) Biochem. J. 296, 303-308[Medline] [Order article via Infotrieve] |
49. | Suko, J., Maurer-Fogy, I., Plank, B., Bertel, O., Wyskovsky, W., Hohenegger, M., and Hellmann, G. (1993) Biochim. Biophys. Acta 1175, 193-206[Medline] [Order article via Infotrieve] |
50. | Tanaka, K., Shibuya, I., Uezono, Y., Ueta, Y., Toyohira, Y., Yanagihara, N., Izumi, F., Kanno, T., and Yamashita, H. (1998) J. Neurochem. 70, 1652-1661[Medline] [Order article via Infotrieve] |
51. | Liu, P. S., Lin, Y. J., and Kao, L. S. (1991) J. Neurochem. 56, 172-177[Medline] [Order article via Infotrieve] |
52. | Robinson, I. M., and Burgoyne, R. D. (1991) J. Neurochem. 56, 1587-1593[Medline] [Order article via Infotrieve] |
53. | Stauderman, K. A., McKinney, R. A., and Murawsky, M. M. (1991) Biochem. J. 278, 643-650[Medline] [Order article via Infotrieve] |
54. | Guse, A. H., da Silva, C. P., Weber, K., Armah, C. N., Ashamu, G. A., Schulze, C., Potter, B. V., Mayr, G. W., and Hilz, H. (1997) Eur. J. Biochem. 245, 411-417[Abstract] |
55. | Neher, E. (1998) Neuron 20, 389-399[Medline] [Order article via Infotrieve] |
56. |
Chamoux, E.,
Breault, L.,
LeHoux, J.-G.,
and Gallo-Payet, N.
(1999)
J. Clin. Endocrinol. Metab.
84,
4722-4730 |
57. |
Chamoux, E.,
Narcy, A.,
Lehoux, A.,
and Gallo-Payet, N.
(2002)
J. Clin. Endocrinol. Metab.
87,
1819-1828 |
58. |
Hamelink, C.,
Tjurmina, O.,
Damadzic, R.,
Young, W.,
Weihe, E.,
Lee, H.,
and Eiden, L.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
461-466 |