| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, September
18, 1995; and in revised form, February 23, 1996) From the
Pituitary adenylate cyclase-activating polypeptide (PACAP) is
the most potent non-cholinergic neurotransmitter to stimulate
catecholamine secretion from rat chromaffin cells; however, the
mechanism of action is not clear. We used amperometric detection of
exocytosis and indo-1 monitoring of
[Ca There is convincing evidence that secretion of adrenal medullary
hormones in various species is regulated by peptidergic
neurotransmitters. Among a long list of peptides that exist in the
adrenal medulla, vasoactive intestinal polypeptide (VIP) ( At the same time that VIP was being
characterized as a medullary neurotransmitter, a new member of the
VIP/glucagon/secretin family of peptides (pituitary adenylate
cyclase-activating polypeptide, PACAP) was discovered and shown to be a
most potent activator of adenylyl cyclase(8) . PACAP occurs in
both a 27 (PACAP We undertook
the present work to identify the mechanism by which PACAP mobilizes
Ca
Figure 1:
Control of
external and internal Ca
Figure 2:
PACAP stimulation of exocytosis requires
external Ca
The role of
Ca
Figure 3:
PACAP-evoked increase in
[Ca
Figure 4:
ZnCl
When
[Ca
Figure 5:
Forskolin mimics PACAP-induced exocytosis.
Representative traces of exocytosis stimulated by forskolin (10
µM) applied for 10 s with Ca
When tested on indo-1 loaded chromaffin
cells, forskolin caused a rise in
[Ca
Figure 6:
ZnCl
To determine if the time required for cAMP
elevation could account for the latency of PACAP effects, we monitored
cAMP levels during PACAP exposure. PACAP required 5 s to significantly
increase cAMP levels compared with unstimulated controls (Fig. 7). The small and variable increase in cAMP observed at
2.5-s exposure to PACAP was not statistically significant. cAMP levels
appeared to reach a plateau at 5 s and remained at about the same level
during PACAP exposure for up to 30 s (Fig. 7).
Figure 7:
PACAP-stimulated elevation of cAMP.
Chromaffin cells were exposed to 100 nM PACAP for the time
indicated and cAMP content determined. Zero time represents untreated
controls. Symbols represent the mean (±S.E.) of three to four
observations at each time point. *, p < 0.05 compared with
control, unpaired Student's t test.
To further
implicate cAMP-dependent phosphorylation, PACAP effects were tested in
the presence of the protein kinase A inhibitor, adenosine 3`,5`-cyclic
monophosphorothioate R
Figure 8:
Inhibition of protein kinase A blocks
PACAP-evoked exocytosis. Chromaffin cells were stimulated by 10-s
application of PACAP (100 nM) or 500-ms application of
nicotine (10 µM) as indicated from an ejection pipette.
Exocytosis was detected amperometrically in control (solid
bars) and 300 µMR
Several lines of evidence have been presented to define the
secretory mechanism of PACAP, a non-cholinergic co-transmitter in the
adrenal medulla. The secretory mechanism of PACAP appears distinctly
different from acetylcholine acting through nicotinic and muscarinic
receptor stimulation. PACAP-evoked exocytosis and elevated
[Ca PACAP (or forskolin)
stimulated exocytosis and elevated
[Ca The mechanism of Ca The most intriguing
characteristic of PACAP stimulation was the approximate 7-s delay
between application and onset of action. Only a fraction of the latency
could be attributed to time required for the pipette solution to
displace bath solution around an individual cell. Delay intrinsic to
the protocol was less than 0.5 s as indicated by the latency when
depolarizing stimulus was administered. Receptor-mediated signal
transduction is unlikely to account for much of the latency, since
nicotinic receptor activation produced an almost immediate response.
The observed latencies appear to be closely related to the
intracellular mechanism involved in stimulating catecholamine
secretion. Agents that mobilize internal Ca In conclusion, the results
presented here demonstrate the importance of multiple transmitters
acting through cholinergic and non-cholinergic pathways in the adrenal
synapse. PACAP stimulation of catecholamine secretion is not redundant
or simply modulatory, but occurs independent of other secretagogues via
mechanisms distinct from cholinergic receptor coupled pathways. Thus
peptidergic transmission is likely to maintain catecholamine secretion
during periods of stress or situations where cholinergic receptors
desensitize or cholinergic transmission otherwise fails.
Volume 271,
Number 18,
Issue of May 3, 1996 pp. 10545-10550
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
]
to identify PACAP
actions in cultured chromaffin cells. PACAP (100 nM) required
external Ca to evoke secretion. However, unlike
nicotine and KCl which caused immediate and relatively brief secretion,
PACAP had a latency of 6.8 ± 0.96 s to the first secretory
response and secretion continued for up to 2 min. PACAP elevation of
[Ca] showed similar
latency and often remained above base line for several minutes
following a brief exposure. ZnCl
(100 µM)
selectively inhibited PACAP-stimulated secretion and
[Ca] with little
effect on nicotine-evoked responses. Nifedipine (10 µM)
had little effect on PACAP-evoked secretion but inhibited
nicotine-evoked secretion by more than 80%, while 
-conotoxin (100
nM) failed to affect either agonist. PACAP-stimulated cAMP
levels required 5 s to significantly increase, consistent with the
latency of exocytotic and Ca responses. Forskolin (10
µM) caused responses similar to PACAP. PACAP-evoked
exocytosis was blocked by the protein kinase A inhibitor adenosine
3`,5`-cyclic monophosphorothioate R
-diastereomer (R-cAMPS). These data show that PACAP stimulates
exocytosis by a mechanism distinctly different from cholinergic
transmitters that appears to involve cAMP-mediated Ca influx. Differences in receptor coupling mechanisms and
pharmacology of Ca entry stimulated by cholinergic
and peptidergic agonists support the idea that the peptidergic system
maintains catecholamine secretion under conditions where the
cholinergic system desensitizes or otherwise fails.
)satisfied several important criteria to be classified as a
non-cholinergic neurotransmitter in the rat adrenal gland(1) .
VIP-like immunoreactivity has been detected in nerve terminals of
adrenal glands of several
species(1, 2, 3, 4) . However, there
is also evidence against the existence of VIP immunoreactive fibers in
adrenal medulla (5) and splanchnic neurons(6) . While
VIP stimulates the cAMP as well as phosphatidylinositol
pathways(7) , it is difficult to draw firm conclusions about
the mechanism of peptide-evoked catecholamine secretion from studies of
intact adrenal gland.
) and 38 (PACAP
) amino acid
form and has about 70% homology with VIP(8, 9) . PACAP
immunoreactive fibers have been identified in the adrenal gland ()(but see (10) ), and PACAP receptors have been
demonstrated on chromaffin cells(10, 11) . Release of
PACAP-like material from perfused adrenal following splanchnic nerve
stimulation has been demonstrated(12) . Importantly, PACAP is
one of the most potent secretagogues in chromaffin cells in
culture(13, 14, 15) and in perfused adrenal
gland(16) . Finally, the adrenal gland has been reported to
contain the second highest concentration of PACAP among peripheral
organs of the rat(17) . Based on these findings, we have
suggested that both VIP and PACAP might function as non-cholinergic
neurotransmitters controlling catecholamine synthesis and secretion in
the rat adrenal medulla ( (12) and see also (18) and (19) ). The secretory properties of PACAP have been examined in
whole adrenal gland (16, 20) and mass cultures of
chromaffin cells(13, 14) ; however, the mechanism of
PACAP-evoked catecholamine secretion remains unclear. and stimulates secretion in primary cultures of
rat chromaffin cells. Amperometric detection of exocytosis and indo-1
fluorescence measurement of [Ca] were used to define PACAP effects on single chromaffin
cells. Our recently developed protocols were used to isolate and
identify internal versus external sources of Ca used in exocytosis. PACAP-evoked exocytosis was monitored in
cells during transient exposure to a Ca-free
environment and in cells in the presence of Ca but
depleted of internal Ca stores. Our findings show
that a peptidergic transmitter utilizes extracellular Ca to evoke catecholamine secretion, but by a mechanism distinctly
different from acetylcholine acting on nicotinic and muscarinic
receptors in this preparation. The data indicate that PACAP increases
Ca influx in association with cAMP production.
Primary Cultures of Rat Chromaffin
Cells
Chromaffin cells were cultured from 19- to 31-day-old
Sprague-Dawley rat pups as
described(21, 22, 23) . Briefly, medullary
fragments were incubated in 1.5 mg/ml type I collagenase and 0.5 mg/ml
DNase (Worthington) in phosphate-buffered saline, followed by trypsin
(1.25 mg/ml in PBS) and DNase (0.5 mg/ml), and cells dissociated by
trituration. Cells were washed by centrifugation (600 
g, 5 min) and plated on collagen-coated glass coverslips in
M-199 medium (Life Technologies, Inc.) plus serum and 0.1 µM dexamethasone and used after 2-7 days. Immunohistochemical
staining showed that cultures contained greater than 90% tyrosine
hydroxylase positive cells. (
)Coverslips with attached
chromaffin cells were transferred to HEPES-buffered bath solution for
monitoring of exocytosis or
[Ca]
. The solution contained
(in mM) 119 NaCl, 4.7 KCl, 1.2 MgSO
, 2.5
CaCl, 10 HEPES, 10 glucose, pH 7.4, with NaOH.
Ca-free solution had no added CaCl and
was supplemented with 1 mM EGTA.Electrochemical Detection of Exocytosis
Exocytosis
from single rat chromaffin cells was detected using established
micro-electrochemical
techniques(15, 24, 25, 26) .
Eight-µm carbon fibers were cannulated into polyethylene tubing,
pulled and cut (final tip diameter < 9 µm), and sealed into
glass capillaries. Capillaries were back-filled with 3 M KCl
and a Ag/AgCl wire used for connection to an EI 400 potentiostat
(holding potential: 650 mV) coupled to a personal computer for data
acquisition and analysis as described(15) . Carbon fiber
electrodes were positioned within 1 µm of a chromaffin cell to
record currents resulting from oxidation of exocytotically released
catecholamines(25, 26) .Control of Internal and External
Ca
Secretagogues in control or
Ca-free bath solutions were applied by pressure
ejection from pulled glass capillaries (
10 µm tip diameter)
positioned about 20 µm from the cell. Ca-free
solution had no added CaCl and was supplemented with 1
mM EGTA. Intracellular Ca stores were
depleted by 15-min pretreatment in Ca-free solution.
Control of the Ca environment was verified in two
ways. First, by showing that in a Ca-containing bath,
application of KCl (or nicotine) in Ca-free pipette
solution failed to produce exocytosis (Fig. 1a) or
elevate [Ca], (not shown) while
mobilization of internal Ca by caffeine under the
same conditions gave positive responses (Fig. 1a).
Second, in Ca-depleted cells, caffeine (applied with
Ca in the pipette) failed to stimulate the cells,
while KCl depolarization continued to give positive responses (Fig. 1b). In most experiments each cell was used as
its own control.
environment. a and b, amperometric recordings of exocytosis in chromaffin cells
in 2.5 mM Ca bath solution (a) and
in Ca-free (1 mM EGTA) solution after 15 min
pretreatment to deplete internal Ca stores (b). Horizontal bars show 10-s application of 35
mM KCl or 10 mM caffeine with or without
Ca from the ejection pipette as indicated. Note that
in a Ca-containing bath (a), removal of
Ca from the pipette eliminates the response to KCl
depolarization, but not to caffeine mobilization of internal stores,
while in Ca-depleted cells (b), exocytosis
is stimulated by KCl, but not by caffeine applied with Ca in the ejection pipette.
Measurement of
[Ca
[Ca]] was measured by ratiometric determination of in indo-1
fluorescence as described previously (27, 28, 29) using an ACAS 570, confocal
laser photometer (Meridian Instruments, Lansing, MI). Experimental
protocols for [Ca] determination paralleled those used during monitoring of
exocytosis.Measurement of Cellular cAMP Content
For
determination of cAMP content, chromaffin cells were plated at a
concentration of about 5 medullae (250,000 cells)/dish and used after 3
days. Culture medium was removed by washing with Krebs buffer solution,
and 100 nM PACAP in Krebs was added to the dish for various
time periods from 0 to 30 s. The reaction was stopped and cells
extracted in 1 ml of ice-cold trichloroacetic acid (6%). The extract
was centrifuged (3000 g, 15 min) and the pellet used
for protein determination as described(30) . The supernatant
was extracted with ethyl ether, evaporated to dryness, and the residue
used for cAMP determination using a DuPont NEN radioimmuno assay kit
according to the protocols supplied by the manufacturer.
PACAP Requires Extracellular Ca
Fig. 2a shows a representative
amperometric recording of exocytotic events stimulated in an individual
rat chromaffin cell by a 10-s application of 100 nM PACAP from
an ejection pipette aimed at the cell. Both the bath and pipette
solutions contained 2.5 mM Ca for
Exocytosis. Exocytosis
began 6.8 ± 0.96 s (25 cells) after the start of PACAP
application and continued for a minute or more after agonist
application. This pattern of stimulation is much different than
exocytosis produced by depolarization with KCl ( (16) and (31) and see Fig. 1a) or stimulation of
nicotinic receptors (below), both of which cause an almost immediate
and brief (20-30 s) burst of exocytotic events.
. Exocytotic events recorded from
chromaffin cells in 2.5 mM Ca-containing
bath solution were stimulated by a 10-s application of 100 nM PACAP plus Ca (a) or by 30-s
application of PACAP in Ca-free pipette solution (b). In c the records were obtained in
Ca-free (1 mM EGTA) bath solution after a
15-min pretreatment to deplete internal Ca stores. Horizontal bars indicate the period of PACAP application. The
traces in a and b are representative of 22
experiments and those in c from 7
observations.
entry from the external medium in PACAP-induced
exocytosis was examined by applying PACAP in a nominally
Ca-free pipette solution to chromaffin cells in a 2.5
mM Ca-containing bath solution. PACAP (100
nM for 30 s) produced no exocytosis during the application
period (Fig. 2b). However, immediately after the
cessation of peptide application, there was a massive exocytotic
response as the Ca-free pipette solution was
displaced by the Ca-containing bath solution.
Exocytosis continued for several minutes (not shown).Exocytosis by PACAP in Cells Depleted of Internal
Ca
To confirm the utilization of
external Ca Stores in PACAP-evoked exocytosis experiments
were performed with cells depleted of internal Ca by
maintaining cells in Ca-free (1 mM EGTA)
bath solution for 15 min prior to and during PACAP application. Ten
second application of PACAP plus Ca to
Ca-depleted cells caused a burst of exocytosis with
typical latency (Fig. 2c). The duration of PACAP-evoked
exocytosis in Ca-depleted cells was much less
compared with cells with normal Ca stores (compare Fig. 2, a and c).PACAP-evoked Elevation of
[Ca
The effects of
PACAP on [Ca]] were determined
in parallel with exocytosis experiments using identical protocols. Fig. 3a shows that a 10-s application of PACAP to an
indo-1-loaded chromaffin cell caused an increase in
[Ca] that exhibits a latency to
onset and prolonged duration consistent with PACAP stimulated
exocytosis. In some chromaffin cells, the initial rise in
[Ca] was followed by
fluctuating [Ca] after a brief
application of PACAP (see Fig. 4for example). In six cells
monitored for 1 min or longer after PACAP application, the average
latency to the beginning of [Ca] elevation was 7.9 ± 0.8 s. The mean peak was 398 ±
35 nM and in four of the six cells
[Ca] remained elevated for the
duration of recording (352 ± 29 nM at 60 s). The
remaining two cells exhibited Ca fluctuations
throughout the recording period.
].
[Ca] recorded from
indo-1-loaded chromaffin cells in 2.5 mM Ca-containing bath solution were stimulated by a
10-s application of 100 nM PACAP plus Ca (a) or by 30-s application of PACAP in
Ca-free pipette solution (b). In c the records were obtained in Ca-free (1 mM EGTA) bath solution after a 15-min pretreatment to deplete
internal Ca stores. Horizontal bars indicate
the period of PACAP application. Traces are representative of six to
eight observations under each condition.
selectively blocks
PACAP-stimulated exocytosis and elevated [Ca]. Exocytotic events stimulated
by 10-s application of 100 nM PACAP (a) or 500-ms
application of 10 µM nicotine (c) in the absence (open bars) and presence (filled bars) of 100
µM ZnCl were counted for 60 s beginning with
the onset of agonist application (zero time). Bars show number
of exocytotic events binned in consecutive 5-s intervals and represent
the mean ± S.E. of 12 experiments.
[Ca] was recorded in
indo-1-loaded chromaffin cells stimulated by 100 nM PACAP for
10 s (b) or 10 µM nicotine for 500 ms (d) in the absence and presence of 100 µM ZnCl as indicated. Traces are representative of 12 (b) and 7 (d)
observations.
] was monitored after
application of PACAP in a Ca-free pipette solution (Fig. 3b), there was no change in
[Ca] until after cessation of
peptide application, identical to the pattern of exocytosis produced
under the same conditions. Finally, the effects of PACAP on
[Ca] were determined in cells
depleted of internal Ca stores (Fig. 3c). The rapid decline of
[Ca] under these conditions is
consistent with a more rapid sequestration of Ca into
depleted internal stores and confirms the role of external
Ca entry in PACAP-evoked catecholamine secretion.Effects of Ca
We used a pharmacological approach to discriminate
between the delayed but long lasting responses stimulated by PACAP and
the immediate brief effects of depolarizing stimuli. Among several
Ca Channel Antagonists on
PACAP Action channel antagonists tested, only Zn exhibited differential effects on PACAP versus other
secretagogues. ZnCl (100 µM) almost completely
suppressed PACAP-evoked exocytosis (Fig. 4a) and
elevated [Ca] (Fig. 4b) with little or no effect on
nicotine-stimulated responses (Fig. 4, c and d) or KCl-evoked exocytosis (not shown). In the example in Fig. 4b, brief application of PACAP caused a
fluctuating rather than sustained elevation of
[Ca]. Nifedipine (10
µM) had no effect on PACAP-induced secretion of
catecholamines. PACAP stimulated 71 ± 7 and 72 ± 7
exocytotic events during 2-min recording in control and
nifedipine-treated cells (n = 9), respectively.
-Conotoxin produced an approximate 18% decline in secretion (58
± 6 events/2 min recording, n = 9), but the
difference was not significant. However, when tested against
nicotine-evoked secretion, nifedipine produced 83% inhibition (70
± 3 and 12 ± 2 events in control and 1 µM nifedipine, respectively, n = 5), and
-conotoxin had no effect (103 ± 22%) compared with
untreated controls (n = 5).Forskolin Effects on Exocytosis and
[Ca
Because PACAP is
established as a potent stimulator of adenylyl cyclase and cAMP
formation, we questioned whether forskolin, which also stimulates
adenylyl cyclase, would have secretory actions similar to PACAP.
Application of forskolin (10 µM for 10 s) produced a
long-lasting secretory response in 2.5 mM Ca] medium (Fig. 5a). The delay between beginning of
forskolin application and detection of the first exocytotic event was
7.5 ± 0.95 s (28 cells). When forskolin was administered in a
nominally Ca-free pipette solution, exocytotic events
were detected only after cessation of the Ca-free
solution (Fig. 5b). Application of forskolin plus
Ca to Ca-depleted chromaffin cells,
like PACAP, stimulated exocytosis only during the period when
Ca was available to the cells (Fig. 5c).
in the
pipette solution (a) and for 30 s without Ca (b) in cells maintained in 2.5 mM CaCl-containing medium and for 30 s plus
Ca in cells held in Ca-free EGTA
bath solution (c). Traces are representative of five to six
observations under each condition. Horizontal bars show the
period of forskolin application.
] that was similar to that
produced by PACAP. The average peak
[Ca] was 322 ± 29 nM with a latency of 6.7 ± 1 s (n = 5).
However, forskolin typically produced fluctuations in
[Ca] (see Fig. 6b for example) rather than the sustained elevation seen with PACAP.
In this regard, it was noted that forskolin-evoked exocytosis was
qualitatively similar to PACAP in terms of latency, use of external
Ca, and sensitivity to Zn (Fig. 6a). However, compared with PACAP,
forskolin-evoked exocytosis was less robust with fewer total exocytotic
events (compare Fig. 6a and Fig. 4a).
These data suggest that PACAP-evoked catecholamine secretion has a
prominent, but not exclusive, cAMP-mediated mechanism.
blocks
forskolin-stimulated exocytosis. Exocytotic events stimulated by 10 s
application of 10 µM forskolin (a) in the absence (open bars) and presence (filled bars) of 100
µM ZnCl were counted for 60 s, beginning with
the onset of agonist application (zero time). Bars show the
number of exocytotic events binned in consecutive 5-s intervals and
represent the mean ± S.E. of six experiments. b shows
[Ca] recorded in
indo-1-loaded chromaffin cells stimulated by 10 µM forskolin for 10 s in control 2.5 mM Ca bath solution.
Characteristic Delay in Onset of Exocytosis by
PACAP
In all tests performed, the latency between beginning of
agonist application and detection of the first exocytotic event
appeared to be most closely correlated to the mechanism of agonist
action (Table 1). Nicotine and excess KCl, which act by
depolarization and opening of voltage-dependent Ca channels, cause secretion to occur almost instantaneously. The
observed delay of a few hundred ms with these agonists reflects the
positioning of the ejection pipette 20 µm from the cells and the
resulting delay in changing the environment around the cell. Under
these same conditions, agonists which mobilize internal
Ca, muscarine, and caffeine, exhibited a latency
between 2 and 3 s, while PACAP (or forskolin) acting presumably through
a cAMP-dependent phosphorylation cascade, required approximately 7 s to
evoke secretion.
-diastereomer (R-cAMPS)(32) . R-cAMPS (300 µM for 30 min) caused a
significant reduction of PACAP-evoked exocytosis with little effect on
nicotine-evoked responses (Fig. 8).
-cAMPS
following 30-min pretreatment (shaded bars). The counting
period was 60 s for PACAP- and 30 s for nicotine-stimulated responses.
Note that nicotine-evoked responses were complete within 30 s, while
PACAP-evoked responses typically continued beyond the recording period. Bars represent the mean (±S.E.) of three experiments.
*, p < 0.05, unpaired Student's t test. n.s., not significant.
] occurred only after a
pronounced latency and required the presence of extracellular
Ca. The time course of PACAP-stimulated cAMP
elevation and the close correspondence between effects of PACAP and
forskolin support the conclusion that PACAP actions are mediated by
cAMP-dependent activation of protein kinases.] only when external
Ca was present, indicating that the peptide causes
Ca entry into chromaffin cells. The absence of
exocytosis and elevated [Ca] during 30-s application of PACAP without CaCl also
shows that Ca influx is required for PACAP-evoked
catecholamine secretion. The pronounced increase in
[Ca] and exocytosis upon
cessation of the stimulus was most likely due to stimulation of
intracellular second messengers during the application period, coupled
with high affinity binding of PACAP and slow dissociation from its
receptors, producing an exaggerated response as the pipette solution
was displaced by the Ca containing bath solution. entry following PACAP
application was different than that produced by acetylcholine.
Acetylcholine effects were sensitive to block of L-type Ca channels by nifedipine, but PACAP effects were not. This is
somewhat different from PACAP effects in mass cultures of porcine
chromaffin cells, which were reported to be inhibited by
nifedipine(14) . Neither agonist was opposed by block of N-type
channels by -conotoxin. The finding that 100 µM Zn discriminates between PACAP and other
secretagogues that stimulate Ca entry supports the
idea that PACAP promotes Ca entry by a distinct
mechanism. One possibility is that PACAP causes a
phosphorylation-dependent recruitment of channels not active during
nicotinic or KCl induced depolarization. While forskolin-evoked
exocytosis was qualitatively similar to that produced by PACAP,
forskolin consistently produced fewer exocytotic events. This may be
due to the reported ability of PACAP to stimulate the
inositol lipid cascade along with elevated cAMP levels(33) .
Multiple signaling pathways stimulated by PACAP but not forskolin would
account for the more robust secretion by PACAP. (muscarine and caffeine) acted within 2-3 s, while agents
which elevate cAMP (forskolin and PACAP) required about 7 s to produce
exocytosis. Receptor-mediated production of second messengers is not
likely to account for the latency for two reasons. First, both
muscarine and PACAP stimulate complex second messenger pathways, but
produced exocytosis with significantly different latencies. Second,
caffeine, which mobilizes internal Ca but does not
act through a plasma membrane receptor, acts with the same delay as
muscarine, while forskolin, which elevates cAMP independent of membrane
receptors, acts with the same delay as PACAP. These observations,
coupled with the instantaneous action of depolarizing agents, suggest
that the last step in the signaling pathway that directly causes
Ca influx or liberation of internal stores is the
factor controlling latency. The approximately 5-s latency for
PACAP-evoked elevation of cAMP levels in the present work is similar to
the latent period (about 5 s) before cAMP-dependent enhancement of
Ca current is observed in cardiac cells(34) .
The high density of chromaffin cells in the cultures (>90% tyrosine
hydroxylase positive) makes it unlikely that non-chromaffin
cells account for the observed changes in cAMP levels. Furthermore,
inhibition of protein kinase A by R-cAMPS
significantly reduced PACAP-evoked exocytosis without affecting
nicotine-evoked responses. These findings coupled with the observed 7-s
latency to PACAP-evoked exocytosis support the idea that cAMP
production and activation of protein kinase A are involved in
stimulating Ca entry and exocytosis. However, the
mechanism of this action remains unknown.
)))
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Cebolla, A. Fernandez-Perez, G. Perea, A. Araque, and M. Vallejo DREAM Mediates cAMP-Dependent, Ca2+-Induced Stimulation of GFAP Gene Expression and Regulates Cortical Astrogliogenesis J. Neurosci., June 25, 2008; 28(26): 6703 - 6713. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Valiante, M. Prisco, R. Sciarrillo, M. De Falco, A. Capaldo, F. Gay, P. Andreuccetti, V. Laforgia, and L. Varano Pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal polypeptide and their receptors: distribution and involvement in the secretion of Podarcis sicula adrenal gland J. Endocrinol., February 1, 2008; 196(2): 291 - 303. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Mustafa, M. Grimaldi, and L. E. Eiden The Hop Cassette of the PAC1 Receptor Confers Coupling to Ca2+ Elevation Required for Pituitary Adenylate Cyclase-activating Polypeptide-evoked Neurosecretion J. Biol. Chem., March 16, 2007; 282(11): 8079 - 8091. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Seino and T. Shibasaki PKA-Dependent and PKA-Independent Pathways for cAMP-Regulated Exocytosis Physiol Rev, October 1, 2005; 85(4): 1303 - 1342. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Carabelli, A. Giancippoli, P. Baldelli, E. Carbone, and A. R. Artalejo Distinct Potentiation of L-Type Currents and Secretion by cAMP in Rat Chromaffin Cells Biophys. J., August 1, 2003; 85(2): 1326 - 1337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Payet, L. Bilodeau, L. Breault, A. Fournier, L. Yon, H. Vaudry, and N. Gallo-Payet PAC1 Receptor Activation by PACAP-38 Mediates Ca2+ Release from a cAMP-dependent Pool in Human Fetal Adrenal Gland Chromaffin Cells J. Biol. Chem., January 10, 2003; 278(3): 1663 - 1670. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Cesetti, J. M. Hernandez-Guijo, P. Baldelli, V. Carabelli, and E. Carbone Opposite Action of beta 1- and beta 2-Adrenergic Receptors on CaV1 L-Channel Current in Rat Adrenal Chromaffin Cells J. Neurosci., January 1, 2003; 23(1): 73 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Morita, A. Sakakibara, S. Kitayama, K. Kumagai, K. Tanne, and T. Dohi Pituitary Adenylate Cyclase-Activating Polypeptide Induces a Sustained Increase in Intracellular Free Ca2+ Concentration and Catecholamine Release by Activating Ca2+ Influx via Receptor-Stimulated Ca2+ Entry, Independent of Store-Operated Ca2+ Channels, and Voltage-Dependent Ca2+ Channels in Bovine Adrenal Medullary Chromaffin Cells J. Pharmacol. Exp. Ther., September 1, 2002; 302(3): 972 - 982. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Hamelink, H.-W. Lee, Y. Chen, M. Grimaldi, and L. E. Eiden Coincident Elevation of cAMP and Calcium Influx by PACAP-27 Synergistically Regulates Vasoactive Intestinal Polypeptide Gene Transcription through a Novel PKA-Independent Signaling Pathway J. Neurosci., July 1, 2002; 22(13): 5310 - 5320. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Fukushima, H. Hikichi, K. Mizukami, T. Nagayama, M. Yoshida, M. Suzuki-Kusaba, H. Hisa, T. Kimura, and S. Satoh Role of endogenous PACAP in catecholamine secretion from the rat adrenal gland Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2001; 281(5): R1562 - R1567. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Machado, A. Morales, J. F. Gomez, and R. Borges cAMP Modulates Exocytotic Kinetics and Increases Quantal Size in Chromaffin Cells Mol. Pharmacol., September 1, 2001; 60(3): 514 - 520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Fukushima, T. Nagayama, H. Kawashima, H. Hikichi, M. Yoshida, M. Suzuki-Kusaba, H. Hisa, T. Kimura, and S. Satoh Role of calcium channels and adenylate cyclase in the PACAP-induced adrenal catecholamine secretion Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2001; 281(2): R495 - R501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Turquier, L. Yon, L. Grumolato, D. Alexandre, A. Fournier, H. Vaudry, and Y. Anouar Pituitary Adenylate Cyclase-Activating Polypeptide Stimulates Secretoneurin Release and Secretogranin II Gene Transcription in Bovine Adrenochromaffin Cells through Multiple Signaling Pathways and Increased Binding of Pre-Existing Activator Protein-1-Like Transcription Factors Mol. Pharmacol., July 1, 2001; 60(1): 42 - 52. [Abstract] [Full Text]
|
|
|
|
 |
|
 N. R. Mahapatra, M. Mahata, A. K. Datta, H.-H. Gerdes, W. B. Huttner, D. T. O'Connor, and S. K. Mahata Neuroendocrine Cell Type-Specific and Inducible Expression of the Chromogranin B Gene: Crucial Role of the Proximal Promoter Endocrinology, October 1, 2000; 141(10): 3668 - 3678. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Vaudry, B. J. Gonzalez, M. Basille, L. Yon, A. Fournier, and H. Vaudry Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: From Structure to Functions Pharmacol. Rev., June 1, 2000; 52(2): 269 - 324. [Abstract] [Full Text] [PDF]
|
|
ABSTRACT
