(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]
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.
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) ()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.
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) 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.
We undertook
the present work to identify the mechanism by which PACAP mobilizes
Ca 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.
Figure 1:
Control of
external and internal Ca 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.
Figure 2:
PACAP stimulation of exocytosis requires
external Ca. 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.
The role of
Ca 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).
Figure 3:
PACAP-evoked increase in
[Ca]
.
[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.
Figure 4:
ZnCl 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.
When
[Ca]
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.
Figure 5:
Forskolin mimics PACAP-induced exocytosis.
Representative traces of exocytosis stimulated by forskolin (10
µM) applied for 10 s with Ca 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.
When tested on indo-1 loaded chromaffin
cells, forskolin caused a rise in
[Ca]
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.
Figure 6:
ZnCl 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.
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-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).
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-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.
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]
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.
PACAP (or forskolin)
stimulated exocytosis and elevated
[Ca]
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.
The mechanism of Ca 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.
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 (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.
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.