Department of Dental Pharmacology, Division of Integrated Medical
Science (K.M., S.K., K.K., T.D.), and Department of Orthodontics and
Craniofacial Developmental Biology, Division of
Cervico-Gnathostomatology (A.S., K.T.), Graduate School of Biomedical
Sciences, Hiroshima University, Hiroshima, Japan
Characteristics of pituitary adenylate cyclase-activating
polypeptide (PACAP)-induced increase of Ca2+ entry and
catecholamine (CA) release were studied in bovine adrenal medullary
chromaffin cells. PACAP induced intracellular free Ca2+
concentration ([Ca2+]i), showing an initial
transient [Ca2+]i rise followed by a
sustained rise and CA release, which were not blocked by the blocking
agents for nicotinic acetylcholine receptor (nAChR) channel, the
voltage-dependent Ca2+ channel (VOC), or the
Na+ channel. The sarcoendoplasmic Ca2+-ATPase
inhibitors thapsigargin and cyclopiazonic acid did not affect the
PACAP-induced sustained rise of [Ca2+]i, but
did inhibit the initial [Ca2+]i rise. In
cells pretreated with cyclopiazonic acid or membrane-permeable, low-affinity Ca2+ chelator
N',N',N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, PACAP further stimulated the entry of Ca2+ or
Mn2+, whereas these treatments masked
[Ca2+]i dynamics induced by bradykinin.
PACAP-induced sustained [Ca2+]i rise and
Mn2+ entry were enhanced by acidic extracellular solution
and reduced by alkalinization, whereas thapsigargin-induced
Mn2+ entry was regulated by the opposite. PACAP-induced
[Ca2+]i rise and Mn2+ entry were
not affected by blockers of cAMP-dependent protein kinase,
phospholipase C, or protein kinase C. All store-operated Ca2+ channel (SOC) blocking agents tested inhibited
thapsigargin-induced Mn2+ entry.
1{
-[3-(4-Methoxyphenyl)-propoxy]-4-methoxyphenylethyl}-1H-imidazole hydrochloride (SK&F 96365),
(R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide, and econazole inhibited PACAP-induced Ca2+ or
Mn2+ entry, whereas GdCl3, 7,8-benzoflavone,
nor-dihydroguaiaretic acid, 5-nitro-2-(3-phenylpropylamino)benzoic
acid, fulfenamic acid, and niflumic acid did not. SK&F 96365 and
econazole but not GdCl3 inhibited PACAP-induced CA release.
These results suggest that PACAP activates a novel Ca2+
entry pathway associated with sustained CA release independent of the
nAChR channel, VOC and SOC, activated by acid pH, with different
sensitivity to blockers of SOC. This pathway may provide a useful model
for the study of receptor-operated Ca2+ entry.
 |
Introduction |
Pituitary
adenylate cyclase-activating polypeptide (PACAP) is a member of the
family of vasoactive polypeptides. Two distinct biologically active
forms, PACAP-38 and PACAP-27, have been described. PACAP, widely
distributed in the central nervous system and peripheral organs, is
present in large amounts in adrenal glands (Arimura et al., 1991
). It
is a very potent secretagogue for catecholamine (CA) from the adrenal
medulla (Watanabe et al., 1992
; Isobe et al., 1993
; Chowdhury et al.,
1994
; Guo and Wakade, 1994
; Perrin et al., 1995
; Babinski et al., 1996
;
Neri et al., 1996
; Przywara et al., 1996
; Tanaka et al., 1996
; Geng et
al., 1997
). PACAP immunoreactive fibers innervate adrenal chromaffin
cells (Tornøe et al., 2000
), and splanchnic nerve stimulation
increases PACAP-(1-38) release from perfused porcine adrenal gland
(Tornøe et al., 2000
). The PACAP type 1 receptor antagonist
PACAP-(6-38) inhibits electrical stimulation-evoked CA release from
perfused rat adrenal gland (Fukushima et al., 2001a
). Thus, PACAP is
thought to function as a noncholinergic neurotransmitter in adrenal
glands (Przywara et al., 1996
; Lamouche et al., 1999
; Fukushima et al.,
2001b
).
An increase in intracellular free Ca2+
concentration ([Ca2+]i)
is generally essential for CA release by various secretagogues in
adrenal chromaffin cells and PACAP also increases
[Ca2+]i in the cells; an
initial sharp rise followed by a sustained rise. When extracellular
Ca2+ was omitted, both the sustained
[Ca2+]i rise and CA
release in response to PACAP were eliminated, although PACAP still
induced an initial transient
[Ca2+]i rise (Isobe et
al., 1993
; Przywara et al., 1996
). Thus, the initial sharp rise in
[Ca2+]i mainly reflects a
rapid Ca2+ release from intracellular
Ca2+ stores, whereas the sustained
[Ca2+]i rise is due to
Ca2+ entry through the plasma membrane, which is
essential for CA release in response to PACAP. The various pathways
through which the regulated entry of Ca2+ occurs
are found in adrenal chromaffin cells; nicotinic acetylcholine (ACh)
receptor (nAChR) channels, voltage-operated Ca2+
channels (VOCs), store-operated channels (SOCs), and other unidentified channels. SOC is regulated by the discharge/repletion of intracellular Ca2+ stores and may be functionally related to
receptor-operated Ca2+ channels, which couple to
the G protein/phospholipase C-inositol trisphosphate
(IP3) pathway.
However, unrelated mechanisms underlying the increase in
Ca2+ entry in response to PACAP have been
reported. For example, several studies have shown that PACAP activates
VOC in porcine (Isobe et al., 1993
), bovine (Tanaka et al., 1996
;
O'Farrell and Marley, 1997
), and canine (Geng et al., 1997
) chromaffin
cells. On the other hand, PACAP stimulated cAMP-mediated
Ca2+ influx in rat (Przywara et al., 1996
) and
bovine (Perrin et al., 1995
) adrenal chromaffin cells and activated
Ca2+ influx through a pathway coupled with
phospholipase C (Taupenot et al., 1999
) with both adenylate cyclase and
phospholipase C in PC12 cells (Osipenko et al., 2000
). The involvement
of distinct mechanisms for the time-dependent responses to PACAP have
also been reported; the initial response is mediated by L-type VOC and
the sustained response may involve the activation of the
Gq/11/phospholipase C-
/phosphoinositide-signaling pathway in PC12 cells (Taupenot et
al., 1999
). The L-type VOC is responsible for CA release induced by
PACAP, and activation of adenylate cyclase is involved in the PACAP-induced release of epinephrine, but not norepinephrine, in
perfused rat adrenal glands (Fukushima et al., 2001b
).
Aside from the confirmation by all studies that the secretory response
to PACAP is long-lasting and entirely dependent on an influx of
extracellular Ca2+, the detailed nature of
PACAP-induced Ca2+ entry remains to be
elucidated. It will be beneficial to analyze PACAP-induced
Ca2+ entry to get new insight into the mechanisms
of [Ca2+]i signaling. The
aim of the present study is to verify the characteristics of
PACAP-induced [Ca2+]i
dynamics, focusing on whether PACAP activates nAChR channel, VOC, SOC,
or other pathways to regulate
[Ca2+]i signaling and CA
release. The present results demonstrated that PACAP is coupled with a
unique Ca2+ entry pathway distinguishable from
nAChR channel, VOC, and SOC that is not associated with adenylate
cyclase, phospholipase C, or protein kinase C (PKC).
 |
Materials and Methods |
Reagents.
The following reagents were obtained as indicated:
-agatoxin (
-AgTx), IVA, bradykinin,
-conotoxin (
-CgTx)
GVIA,
-CgTx MVIIC, PACAP38 (human), and PACAP-(6-38) were from
Peptide Institute, Inc. (Osaka, Japan); 7,8-benzoflavone, caffeine,
GdCl3, fulfenamic acid, staurosporine,
thapsigargin, and U-73122 were from Wako Pure Chemicals (Osaka, Japan);
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfon-amide (H-89) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide
hydrochloride (HA1004) were from Seikagaku Corporation (Tokyo, Japan);
cyclopiazonic acid, diltiazem hydrochloride, econazole, nicardipine
hydrochloride, nifedipine, and SK&F 96365 were from Sigma-Aldrich (St.
Louis, MO); fura-2, fura-2 acetoxymethylester, and
N',N',N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) were from Dojindo Laboratories (Kumamoto, Japan); nor-dihydroguaiaretic acid (NDGA) and
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) were from BIOMOL
Research Laboratories (Plymouth Meeting, PA); niflumic acid was from
Cayman Chemicals (Ann Arbor, MI); adenosine-3',5'-cyclic
monophosphotioate, Rp diastereomer
sodium salt was from BioLog Life Science Institute (Bremen, Germany); and tetrodotoxin was from Sankyo Co., Ltd. (Tokyo, Japan).
(R,S)-(3,4-Dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphen-yl)ethyl]-acetamide (LOE 908) was kindly provided by Boehringer Ingelheim Pharma KG (Biberach, Germany). All other chemicals were reagent grade.
Cyclopiazonic acid, H-89, HA1004, LOE 908, diltiazem, nicardipine,
nifedipine, NPPB, staurosporine, thapsigargin, and U-73122 were
dissolved in dimethyl sulfoxide to make stock solutions. Econazole,
fulfenamic acid, NDGA, and niflumic acid were dissolved in ethanol to
make stock solutions. Dimethyl sulfoxide (0.1%) or ethanol (0.1%) at
the highest final concentrations used had no effects on
[Ca2+]i,
Mn2+ entry, and CA release, respectively (data
not shown).
Cell Preparation.
Chromaffin cells of bovine adrenal glands
were isolated enzymatically as described previously (Morita et al.,
1987
). Cells were maintained in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum, penicillin G (100 U/ml), streptomycin
(100 µg/ml), ascorbate (0.1 mM), and HEPES (5 mM) at 37°C in a
humidified incubator under 5% CO2, 95% air
atmosphere in suspension culture for 24 to 72 h for measurement of
[Ca2+]i or in 35-mm
tissue culture dish (106 cells/dish) for 3 to 6 days for CA release assay. Cells were washed and suspended in either
medium before use: normal medium containing 150 mM NaCl, 5 mM KCl, 1 mM
MgSO4, 1.3 mM CaCl2, 5 mM
glucose, 10 mM HEPES-Tris buffer, and 0.5% BSA, pH 7.4;
Ca2+-deficient medium containing 150 mM NaCl, 5 mM KCl, 1 mM MgSO4, 5 mM glucose, 0.1 mM EGTA, 10 mM HEPES-Tris buffer, and 0.5% BSA, pH 7.4; or
Ca2+-sucrose medium containing 1.3 mM
CaCl2, 5 mM glucose, 340 mM sucrose, 10 mM
HEPES-Tris buffer, and 0.5% BSA, pH 7.4.
Estimation of [Ca2+]i.
[Ca2+]i was estimated
with the use of the calcium-sensitive dye fura-2 as described
previously (Morita et al., 1997
). Briefly, the cells were incubated at
32°C with 1 µM fura-2 acetoxymethylester for 30 min for loading the
dye. Cells were then centrifuged at 10g for 10 min and
resuspended to yield 3 × 106 cells. Cells
were washed with normal medium, Ca2+-deficient
medium, or Ca2+-sucrose medium with rapid
centrifugation then resuspended in the medium immediately before use.
Fluorescence was measured with a dual wavelength fluorescence
spectrophotometer with excitation at 340 and 380 nm and emission at 510 nm. [Ca2+]i was
calculated from the fluorescence ratio at 340 and 380 nm using the
equation of Grynkiewicz et al. (1985)
and the value of 224 nM for
Kd of fura-2. To monitor
Ca2+ entry, some of the experiments were carried
out according to the
Ca2+-free/Ca2+
reintroduction protocol (Clementi et al., 1992
). To this end, fura-2-loaded chromaffin cells were washed with
Ca2+-deficient medium with rapid centrifugation
before use. After stimulants were added, the first
[Ca2+]i peak was recorded
and then Ca2+ was reintroduced into the medium
and the ensuring second
[Ca2+]i peak was recorded.
Measurement of Mn2+ Entry.
Divalent cation entry
was monitored by the fura-2 Mn2+-quenching
technique. Fura-2 has a higher affinity for Mn2+
than Ca2+ (Grynkiewicz et al., 1985
). Cells
loaded with fura-2 as described above were suspended in
Ca2+-deficient medium. Two minutes after the
addition of stimulants, Mn2+ (0.25 mM, final
concentration) was added. Fluorescence was exited at 360 nm. Emission
was recorded at 510 nm. Maximal Mn2+-quenching
values were estimated in each preparation at the end of the recording
by permeabilization of the cells with 10 µM digitonin.
CA Assay.
For the measurement of release of CA, incubations
were carried out as described previously (Morita et al., 1987
). The
incubation medium was separated from the cells, mixed with perchloric
acid (5% of final concentration), and centrifuged at 4500g
for 15 min. The total CA content in the resultant supernatant was
measured fluorometrically by the method of von Euler and Lishajko
(1961)
with adrenaline as a standard. Statistical analysis was
performed by Student's t test.
 |
Results |
PACAP Induced [Ca2+]i Rise and CA
Release.
PACAP (PACAP-38) induced a biphasic
[Ca2+]i rise; an initial
sharp rise followed by a sustained rise in chromaffin cells. The PACAP
receptor antagonist PACAP-(6-38), blocked the
[Ca2+]i rise by
pretreatment and sharply reduced the
[Ca2+]i rise when added
at the sustained phase, suggesting that continuous stimulation of PACAP
receptors is required to maintain the
[Ca2+]i rise (Fig.
1A). Concentration-response curves for
the [Ca2+]i rise and CA
release show that PACAP as low as 10 and 100 pM initiated the
[Ca2+]i rise and CA
release, respectively, and produced almost maximal effects at 10 nM
(EC50 values for the
[Ca2+]i rise and CA
release were 0.20 and 0.38 nM, respectively) (Fig. 1B). Adrenal
chromaffin cells develop several types of VOC; P/Q-type, N-type, and
L-type. To determine whether these channels are involved in the
PACAP-induced [Ca2+]i
rise, chromaffin cells were pretreated with
-AgTx IVA, a blocker of
P/Q-type VOC,
-CgTx GVIA, a blocker of N-type VOC, and diltiazem, nicardipine, and nifedipine, which are L-type channel blockers. Pretreatment of cells with diltiazem did not affect the shape of the
[Ca2+]i rise induced by
PACAP (Fig. 1C), whereas diltiazem blocked almost completely the
ACh-induced peak and sustained
[Ca2+]i rise. No
individual blocker of VOC affected both the initial peak and sustained
rise of [Ca2+]i induced
by 10 nM PACAP (Table 1). Some studies
have reported the involvement of VOC in PACAP-induced
[Ca2+]i rise using a
higher concentration of PACAP (100 nM), which is the supramaximal
concentration for CA release and
[Ca2+]i rise at the
present study. In the present study, nicardipine and nifedipine, but
not
-AgTx IVA and
-CgTx GVIA, slightly inhibited the
[Ca2+]i rise induced by
100 nM of PACAP. Peak and sustained
[Ca2+]i rise induced by
ACh and excess KCl were effectively blocked by nifedipine and
nicardipine, and slightly reduced by
-AgTx IVA (Table 1).

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Fig. 1.
PACAP-induced increase in
[Ca2+]i and CA release from bovine adrenal
chromaffin cells. A, fura-2-loaded adrenal chromaffin cells were
incubated at 32°C. Typical patterns of the rise in
[Ca2+]i induced by various concentrations of
PACAP are shown. The rise in [Ca2+]i was
determined fluorometrically using fura-2 dye as described under
Materials and Methods. PACAP receptor antagonist (1 µM) was added 5 min before or 15 min after the addition of 10 nM
PACAP. B, concentration-response curves for PACAP-induced
[Ca2+]i rise and CA release. Sustained
[Ca2+]i was the value at 10 min after the
addition of PACAP. Total CA released in the medium was assayed
fluorometrically. Values are the mean ± S.E.M. of the net
increase in [Ca2+]i and CA release for three
to six experiments using triplicate assays. C and D, effects of
diltiazem on [Ca2+]i rise induced by PACAP
(C) or ACh (D) in bovine adrenal chromaffin cells. Fura-2-loaded
chromaffin cells were incubated at 32°C either in the presence of
diltiazem (30 µM) or vehicle for 5 min then PACAP (3 nM) or ACh (5 µM) was added. Typical patterns for the rise in
[Ca2+]i are shown. These and the following
experiments are representative of experiments performed with very
similar results in at least three different batches of cells.
|
|
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TABLE 1
Effects of various treatments on PACAP-induced
[Ca2+]i rise in adrenal chromaffin cells
The experimental design was similar to that described in Fig. 1. Drugs
were applied 3 min before stimulation. Peak rise of
[Ca2+]i was obtained during 10 to 15 and 2 to 5 s after the addition of PACAP and 22.5 mM KCl, respectively. Sustained
rise of [Ca2+]i by PACAP and excess KCl were obtained
at 10 and 3 min after the addition of the stimulants, respectively.
Values are the mean ± S.E.M. of the net increase in peak and
sustained phase of [Ca2+]i for six to twelve
experiments.
|
|
In agreement with these results on
[Ca2+]i rise, VOC
blockers did not affect CA release induced by 10 nM PACAP (Table
2). Nifedipine and nicardipine slightly
reduced CA release induced 100 nM PACAP (Table 2). Nifedipine,
nicardipine, and diltiazem effectively blocked the secretory response
to ACh and excess KCl (Table 2). The PACAP-induced
[Ca2+]i rise and CA
release were resistant to tetrodotoxin (TTX), an Na+ channel blocker (Tables 1 and 2). The
PACAP-induced sustained [Ca2+]i rise and CA
release were eliminated in the Ca2+-deficient
medium and entirely restored in the Ca2+-sucrose
medium, with rather larger increases in
[Ca2+]i than with the
normal medium (Tables 1 and 2). These results suggest that VOC and
Na+ are not involved in the
[Ca2+]i rise and CA
release induced by PACAP, except in very high concentrations.
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TABLE 2
Effects of various treatments on PACAP-induced CA release from adrenal
chromaffin cells
The experimental design was similar to that described in Table 1. Drugs
were applied 3 min before stimulation. Values are the mean ± S.E.M. of the net increase in CA release for six to twelve experiments
of triplicate assays. Significantly different from the corresponding
control at * p < 0.05 and
** p < 0.01.
|
|
Diltiazem has been shown to effectively block the nAChR. It is more
potent than the L-type Ca2+ channel (Lopéz
et al., 1993
), in a noncompetitive manner at concentrations far lower
than those needed to inhibit binding of
-bungarotoxin in chromaffin
cells (Houlihan et al., 2000
). These results suggest that diltiazem
blocks the nAChR channel with less affinity to ACh binding sites.
Therefore, the lack of diltiazem effect on PACAP-induced
[Ca2+]i rise and CA
release at the concentration that blocked ACh-induced [Ca2+]i rise and CA
release almost completely (when both nAChR and VOC were blocked) also
suggest that PACAP does not seem to act to nAChR to stimulate the
[Ca2+]i rise and CA release.
PACAP-Induced Ca2+ Entry.
SOC has been shown to be
an another important pathway permitting Ca2+
entry through plasma membrane. This pathway is activated by mobilizing Ca2+ from intracellular
Ca2+ stores in various cells (Barritt, 1999
;
Hofmann et al., 2000
). Bradykinin has been shown to induce a
[Ca2+]i rise mainly by
mobilizing Ca2+ from intracellular
Ca2+ stores through the phospholipase
C/IP3-signaling pathway and the subsequent
Ca2+ entry through SOC in bovine chromaffin cells
(O'Sullivan and Burgoyne, 1989
; Cheek and Barry, 1993
; Castro et al.,
1995
). Bradykinin was used as a reference agonist for the
IP3-mediated Ca2+
release/SOC activation signaling pathway. It has been shown that the
mobilization of Ca2+ from
Ca2+ stores by pharmacological manipulation
(e.g., inhibition of Ca2+-ATPase in the stores by
thapsigargin or cyclopiazonic acid) activates Ca2+ entry through SOC (Takemura et al., 1989
;
Berridge, 1995
; for review, see Fasolato et al., 1994
). Whether SOC is
involved in PACAP-induced Ca2+ entry was examined
in cells pretreated with thapsigargin and cyclopiazonic acid. In these
cells, SOC was expected to be preactivated. Thus, the effects of PACAP
and bradykinin should be masked if these agonists activate
Ca2+ entry by the same mechanism. Figure
2, A and B, shows the results of the
changes in PACAP- and bradykinin-induced
[Ca2+]i rise in cells
pretreated with thapsigargin or cyclopiazonic acid in normal medium.
Whereas the initial sharp rise of
[Ca2+]i induced by PACAP
disappeared in thapsigargin- and cyclopiazonic acid-pretreated cells,
the sustained phase remained similar to the control (Fig. 2A). On the
other hand, both the initial and sustained rises of
[Ca2+]i induced by
bradykinin were almost eliminated by these treatments (Fig. 2B). These
results suggest that the bradykinin-induced sustained [Ca2+]i rise is due to
the activation of SOC, but PACAP induces the sustained
[Ca2+]i rise by
stimulating Ca2+ entry through a mechanism
different from SOC.

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Fig. 2.
Effects of thapsigargin (TG) or cyclopiazonic acid
(CPA) on PACAP- and bradykinin (BK)-induced
[Ca2+]i rise in adrenal chromaffin cells. A
and B, after 45-min pretreatment with TG (2 µM), CPA (45 µM), or
vehicle in the dark at 32°C in normal medium, cells were washed
rapidly with fresh medium before fluorescence measurement. Then cells
were stimulated with PACAP (10 nM) (A) or BK (3 nM) (B) at the times
indicated. Typical patterns of the rise in
[Ca2+]i are shown. These and the following
experiments are representative of experiments performed with very
similar results in at least three different batches of cells.
|
|
To confirm this, Ca2+ entry induced by PACAP was
evaluated by comparing various treatments to bradykinin. To evaluate
Ca2+ entry, changes in
[Ca2+]i rise were
measured by the introduction of Ca2+ into the
medium after the treatment of cells with Ca2+
mobilization in Ca2+-deprivated medium. To see
whether these changes in
[Ca2+]i induced by the
addition of Ca2+ are due to
Ca2+ entry from extracellular space into the
cells, the Mn2+-quenching technique was used.
This cation has been used as a surrogate for Ca2+
entry, given its quenching effect on fura-2 (Grynkiewicz et al., 1985
;
Clementi et al., 1992
). After the transient rise in
[Ca2+]i induced by PACAP
in a Ca2+-deprived medium, the addition of
Ca2+ into the medium caused larger increases in
[Ca2+]i than in
vehicle-treated cells, and the increase gradually declined (Fig.
3A). Bradykinin also increased
[Ca2+]i with the
introduction of [Ca2+]i
into the medium, and the increase was smaller than that of PACAP,
although the bradykinin-induced transient increment of [Ca2+]i was similar to
that of PACAP. The addition of Mn2+ to PACAP- and
bradykinin-treated cells resulted in a sustained quenching of fura-2
fluorescence compared with that in vehicle-treated cells, and the
effect of PACAP was greater than that of bradykinin, as observed in the
rise of [Ca2+]i (Fig.
3B). Figure 3, C and D, shows Ca2+ entry induced
by PACAP and bradykinin in cyclopiazonic acid-pretreated cells in
Ca2+-deprived medium. Cyclopiazonic acid caused a
sharp transient increase in
[Ca2+]i in the absence of
extracellular Ca2+ (Fig. 3C). The addition of
PACAP and bradykinin showed no further increase in
[Ca2+]i after treatment
with cyclopiazonic acid. In this condition, the introduction of
Ca2+ into the medium failed to cause an
[Ca2+]i rise in
bradykinin-treated cells (cyclopiazonic acid + bradykinin) over control
(cyclopiazonic acid) but further increased
[Ca2+]i in PACAP-treated
cells (cyclopiazonic acid + PACAP) (Fig. 3C). Similarly, Fig. 3D shows
that bradykinin produced no further effect on
Mn2+ entry in cells pretreated with cyclopiazonic
acid, but PACAP did increase Mn2+ entry in these
cells. There is a difference in the sensitivity of sarcoendoplasmic
reticulum Ca2+-ATPase to these
Ca2+-ATPase inhibitors (Morita et al., 1997
), and
it is difficult to control the concentration of
Ca2+ in stores at a constant level by
Ca2+-ATPase inhibitors. To control
Ca2+ concentrations in Ca2+
stores at low levels, TPEN, a membrane-permeant multivalent cation chelator with moderate affinity for Ca2+
(Kd for Ca2+ of
~130 µM) (Arslan et al., 1985
; Hofer et al., 1998
) was used. There
are advantages to using TPEN over Ca2+-ATPase
inhibitor because TPEN is assumed to have no significant influence on
[Ca2+]i in the cytoplasm
or any other cell compartment where the steady-state concentration is
in the nanomolar or low micromolar range. In compartments where the
concentration of Ca2+ is comparable to
Kd, TPEN should bind
Ca2+ and rapidly reduce its concentration,
thereby reducing [Ca2+]i
in sarcoendoplasmic reticulum (Arslan et al., 1985
; Hofer et al.,
1998
). In TPEN-pretreated cells, neither PACAP nor bradykinin produced
an increase in [Ca2+]i
(Fig. 3E). Thus, the concentration of Ca2+ in the
pools was considered to be low enough to rule out the involvement of
release of Ca2+ from pools. The introduction of
Ca2+ into the medium did not cause an
[Ca2+]i rise over the
control in bradykinin-treated cells (Fig. 3E). However, a larger
increase in [Ca2+]i rise
was observed in PACAP-treated cells than in vehicle- or bradykinin-treated cells (Fig. 3E).

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Fig. 3.
PACAP- and bradykinin (BK)-induced
Ca2+ entry and Mn2+ entry in adrenal chromaffin
cells and effects of various treatments. A and B, PACAP- and bradykinin
(BK)-induced Ca2+ entry (A) and Mn2+ entry (B).
Fura-2-loaded chromaffin cells were incubated at 32°C either in the
presence of PACAP (10 nM), BK (3 nM), or vehicle in
Ca2+-deprived medium for the indicated time. Then
CaCl2 (1.3 mM) (A) or MnCl2 (0.25 mM) (B) was
added to the suspension to initiate Ca2+ entry and
Mn2+ entry. [Ca2+]i was measured
as described in Fig. 1. Mn2+ entry was monitored by the
fura-2/Mn2+-quenching technique, as described under
Materials and Methods. Fluorescence was excited at 360 nm. Emission was recorded at 510 nm. Maximal Mn2+-quenching
values were estimated in each preparation at the end of the recording
by permeabilization of the cells with 10 µM digitonin. C and D,
effects of cyclopiazonic acid (CPA) on PACAP- and BK-induced
Ca2+ entry and Mn2+ entry. Fura-2-loaded
chromaffin cells were incubated at 32°C in the presence of vehicle or
CPA (45 µM) in Ca2+-deprived medium for 3 min then cells
were stimulated with vehicle, PACAP (10 nM), or BK (3 nM). Two minutes
later, CaCl2 (1.3 mM) or MnCl2 (0.25 mM) was
added to the medium to initiate Ca2+ entry (C) and
Mn2+ entry (D). Maximal Mn2+-quenching values
were estimated in each preparation at the end of the recording by
permeabilization of the cells with 10 µM digitonin. E, effects of
TPEN on PACAP- and BK-induced Ca2+ entry in adrenal
chromaffin cells. Fura-2-loaded chromaffin cells were incubated in
Ca2+-deprived TPEN (2 mM)-containing medium either in the
presence of PACAP (10 nM), BK (3 nM), or vehicle. Then
CaCl2 (final concentration of 2.6 mM) was added.
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|
pH Sensitivity.
It has been shown that acidification of
extracellular solutions inhibits SOC, and raising the pH increases SOC
activity in various cell types (Malayev and Nelson, 1995
; Iwasawa et
al., 1997
; Kerschbaum and Cahalan, 1998
; Khoo et al., 2001
). The
effects of extracellular acidification on PACAP-induced
Ca2+ entry were examined in bovine chromaffin
cells. It was found that the sustained rise of
[Ca2+]i induced by PACAP
was enhanced at pH 6.6 and reduced at 8.4 compared with pH 7.4, whereas
the initial peak rise of
[Ca2+]i was only slightly
affected (Fig. 4A).
Mn2+ entry induced by PACAP was also enhanced in
acidic solutions and reduced in alkaline solutions over the range from
pH 6.2 to 8.4, whereas thapsigargin-induced Mn2+
entry was reduced in acidic solutions and enhanced in alkaline solutions over these ranges (Fig. 4). These results also suggest that
PACAP-induced Ca2+ entry is distinguishable from
SOC in chromaffin cells.

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Fig. 4.
Effects of changes in extracellular pH on
[Ca2+]i rise and Mn2+ entry
induced by PACAP and thapsigargin. A, fura-2-loaded chromaffin cells
were incubated at 32°C in normal medium for 5 min at indicated pH
then PACAP (10 nM) was added to the suspension. The rise in
[Ca2+]i was determined as described under
Materials and Methods. The experiments are
representative of experiments performed with very similar results in at
least three different batches of cells. B, cells were incubated in
Ca2+-deprived medium for 5 min at the indicated pH,
followed by incubation for 3 min in the presence of PACAP (10 nM),
thapsigargin (TG, 1 µM), or vehicle. MnCl2 (0.25 mM) was
then added to the suspension and incubated for 1 min to initiate
Mn2+ entry. The PACAP- and TG-induced Mn2+
entry in the indicated external pH is shown as the ratio to those in pH
7.4. The external pH was shifted by changing the ratio HEPES/Tris.
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Effects of Inhibitors of Signal Transduction.
Whether
cAMP-dependent protein kinase (PKA), PKC, and phospholipase C were
involved in the regulation of PACAP-induced Ca2+
entry was studied using the respective inhibitors (Table
3). We have previously shown that
adenosine-3',5'-cyclic monophoshotioate, (Rp)-cAMPS, a PKA inhibitor,
significantly reduced at 0.5 mM ACh-induced CA release and completely
blocked the potentiation by 8-bromo-cAMP and forskolin of ACh-induced
CA release in bovine chormaffin cells (Morita et al., 1995
).
Adenosine-3',5'-cyclic monophoshotioate, (Rp)-cAMPS had no effect on
PACAP-induced Mn2+ entry. Other inhibitors of
PKA, H-89 and HA1004, were also without effect. When cells were
pretreated with 3 µM U-73122, an inhibitor of phospholipase C, the
bradykinin-induced
[Ca2+]i rise was blocked
(the peak rise of [Ca2+]i
induced by 1 nM bradykinin was 136.3 ± 11.7 nM in control and 9.1 ± 1.1* nM in 3 µM U-73122-treated cells, respectively, and the sustained rise was abolished; *p < 0.001).
However, U-73122 at this concentration had no effect on PACAP-induced
Mn2+ entry. An inhibitor of PKC, staurosporine,
or the combination of H-89 and U-73122 was also without effect on
Mn2+ entry. These inhibitors did not affect
PACAP-induced [Ca2+]i
rise.
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TABLE 3
Effects of inhibitor of PKA, PKC and phospholipase C on Mn2+
entry and [Ca2+]i rise induced by PACAP in adrenal
chromaffin cells
Basal Mn2+ entry (% fura-2 quench) at 1 and 5 min after the
addition of Mn2+ were 25.44 ± 1.05 and 55.11 ± 0.96, respectively. Values represent percentage of control calculated
by D/C × 100 (C = Mn2+ entry in the presence of 10 nM PACAP at 1 and 5 min in the absence of drugs minus basal
Mn2+ entry were 25.54 ± 1.64 and 23.61 ± 1.53, respectively. D = Mn2+ entry in the presence of PACAP
minus basal Mn2+ entry in the presence of drugs. Basal
Mn2+ entry was not altered by drugs tested. PACAP-induced
[Ca2+]i rise was similar to that described in Table
1.
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Effects of Ca2+ Entry Blockers.
The pretreatment
of cells in normal incubation medium with 10 µM SK&F 96365, one of
the most popular inhibitors of Ca2+ entry through
SOC, did not affect the PACAP-induced initial peak rise of
[Ca2+]i but diminished
the sustained rise (Fig. 5A). The
addition of SK&F 96365 during the sustained rise of
[Ca2+]i sharply
attenuated the rise (Fig. 5A). In the
Ca2+-deprived medium, pretreatment with SK&F
96365 did not affect the transient increase in
[Ca2+]i induced by PACAP
but blocked Ca2+ entry in PACAP-treated cells
induced by reintroducing Ca2+ into the medium
(Fig. 5B). SK&F 96365 did not affect the small rise of
[Ca2+]i but blocked
Ca2+ entry induced by thapsigargin (Fig. 5C). On
the other hand, GdCl3, a blocker of nonselective
cation currents, including SOC in various cells (Schumann et al.,
1994
), affected neither the initial nor the sustained
[Ca2+]i rise induced by
PACAP (Fig. 5D). GdCl3 inhibited the
Ca2+ entry induced by thapsigargin but failed to
inhibit PACAP-induced Ca2+ entry (Fig. 5E).
Effects of other compounds having Ca2+entry
blocking action LOE 908 (Ca2+ antagonists),
econazole, and 7,8-benzoflavone (cytochrome P-450 inhibitors), NDGA
(lipoxygenase inhibitors), and NPPB, fulfenamic acid, and niflumic acid
(cyclooxygenase inhibitors) on PACAP-induced Ca2+entry were examined (Table
4). SK&F 96365, LOE 908, and econazole significantly inhibited PACAP-induced Mn2+ entry,
whereas 7,8-benzoflavone, NDGA, NPPB, fulfenamic acid, and niflumic
acid did not block PACAP-induced Mn2+ entry. All
of these compounds effectively blocked thapsigargin-induced Mn2+ entry at these concentrations.

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Fig. 5.
Effects of SK&F 96365 and GdCl3 on
[Ca2+]i rise and Ca2+ entry
induced by PACAP and thapsigargin (TG). A, fura-2-loaded chromaffin
cells were incubated at 32°C in normal medium for 5 min then PACAP (3 nM) was added to the suspension. SK&F 96365 (10 µM) was added 3 min
before or 5 min after the addition of PACAP. B and C, fura-2-loaded
chromaffin cells were incubated at 32°C either in the presence of
SK&F 96365 (10 µM) or vehicle in Ca2+-deprived medium for
3 min then PACAP (10 nM) (B) or TG (1 µM) (C) was added.
CaCl2 (1.3 mM) was added 3 min after the addition of PACAP
or TG to the suspension to initiate Ca2+ entry. D,
fura-2-loaded chromaffin cells were incubated at 32°C in normal
medium for 5 min then PACAP (3 nM) was added to the suspension.
GdCl3 (10 µM) was added 3 min before the addition of
PACAP. E, cells were incubated in Ca2+-deprived medium in
the presence of PACAP (10 nM), TG (1 µM), or vehicle for 3 min then
CaCl2 (1.3 mM) was added to the suspension to initiate
Ca2+ entry. GdCl3 (10 µM) was added 1.5 min
after the addition of Ca2+.
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TABLE 4
Effects of various store-operated Ca2+ entry blocker on
Mn2+ entry induced by PACAP and thapsigargin (TG) in adrenal
chromaffin cells
Values represent percentage of control calculated as described in Table
3, except that Mn2+ entry induced by PACAP and TG at 1 and 5 min were 25.54 ± 1.64 and 23.61 ± 1.53 for PACAP and
14.89 ± 0.78 and 16.58 ± 1.52 for TG, respectively, and the
basal Mn2+ entry in the presence of drugs tested (basal values
at 1 and 5 min were 25.44 ± 1.05 and 55.11 ± 0.96; SK&F
96365, 14.70 ± 1.85 and 38.08 ± 4.48; LOE 908, 15.98 ± 0.73 and 46.55 ± 3.16; econazole, 16.51 ± 1.22 and
45.55 ± 3.16; 7,8-benzoflavone, 25.45 ± 4.24 and 57.10 ± 4.47; NDGA, 19.95 ± 1.45 and 46.48 ± 0.62; NPPB,
20.87 ± 2.83 and 46.66 ± 3.50; fulfenamic acid, 25.08 ± 2.86 and 54.27 ± 2.70; niflumic acid, 13.37 ± 2.63 and
37.50 ± 4.23, respectively).
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SK&F 96365 and econazole, which inhibited the sustained
[Ca2+]i rise but not the
initial [Ca2+]i rise
induced by PACAP, inhibited PACAP-induced CA release with the similar
range of concentrations to block Ca2+ entry
(Table 5). SK&F 96365 and econazole did
not block CA release induced by ionomycin, a Ca2+
ionophore, suggesting that the inhibition of PACAP-induced CA release
by these agents was not due to the inhibition of the steps leading to
CA release subsequent to Ca2+ entry
(ionomycin-induced CA release in the absence and the presence of 10 µM SK&F 96365 and 5 µM econazole were 0.56 ± 0.04, 0.56 ± 0.02, and 0.59 ± 0.03 µg/106 cells,
respectively). PACAP-induced CA release was resistant to
GdCl3.
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TABLE 5
Effects of SK&F 96365, econazole, and GdCl3 on PACAP-induced
[Ca2+]i rise and CA release from bovine adrenal
chromaffin cells
The experimental design was similar to that described in Tables 1 and
2. Drugs were applied 3 to 10 min before the addition of 10 nM PACAP.
Values of the mean ± S.E.M. of the net increase in
[Ca2+]i and CA release for three to eight experiments
using triplicate assays. Significantly different from the corresponding
control at * p < 0.05 and
** p < 0.01.
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Discussion |
The ACh-stimulated increase in
[Ca2+]i in bovine adrenal
chromaffin cells is mainly triggered by an influx of
Ca2+ through the nAChR channel, VOC, and the
subsequent activation of Ca2+-induced
Ca2+ release, all of which contribute to CA
release. These events in response to ACh are of short duration, whereas
PACAP induces large and sustained increases in
[Ca2+]i and CA release.
The present study sought to elucidate which pathways (nAChR channel,
VOC, SOC, or an unidentified channel) contribute to this peculiar
Ca2+ and secretory response to PACAP.
Reports vary concerning the effect of VOC blockers on PACAP-induced
rise in [Ca2+]i and CA
release. For example, Przywara et al. (1996)
showed that in rat
cultured adrenal chromaffin cells, neither L- nor N-type VOC
participates in the PACAP-induced CA release. On the other hand,
Fukushima et al. (2001b)
showed that nifedipine, L-type VOC antagonist,
reduced PACAP-induced CA release in isolated perfused rat adrenal
gland. Tanaka et al. (1996)
reported that the activation of L-type
Ca2+ channels by PACAP is due to a membrane
depolarization that depends on PKC-mediated Na+
influx in bovine adrenal chromaffin cells. O'Farrell and Marley (1997)
analyzed the contributions of various types of VOC to PACAP-induced CA
release using the specific blockers of VOC subtypes, and suggested the
involvement of L-, N-, and Q-type of VOC. Thus, the contribution of VOC
to the PACAP-induced Ca2+ influx was considered a
possible mechanism for the Ca2+ influx induced by
PACAP in adrenal chromaffin cells. However, all the blockers of VOC
(L-type, P/Q-type, and N-type) failed to affect the PACAP-induced
[Ca2+]i rise and CA
release in the present study, whereas ACh- and excess KCl-evoked
[Ca2+]i rise and CA
release were effectively blocked by the VOC blockers, showing that the
treatment was enough to block VOC under these experimental conditions.
In addition, the PACAP-induced
[Ca2+]i rise did not
require extracellular Na+ because these responses
to PACAP were not attenuated in the presence of TTX or in
Ca2+-sucrose medium. However, the
[Ca2+]i rise induced by a
supramaximal concentration of PACAP, 100 nM, was partially sensitive to
L-type VOC blockers nicardipine and nifedipine. Thus, the effects of
Ca2+ antagonists differ in part depending on
PACAP concentration. Although the reason for the discrepancy in the
sensitivity to VOC blockers is unclear, Ca2+
antagonists, at least at concentrations that fully inhibited VOC, had
no effect on the responses to submaximal concentration of PACAP in the
present study. Therefore, any involvement of VOC in the
[Ca2+]i rise and CA
release induced by PACAP seems unlikely. Ca2+
antagonists are well known to block L-type VOC. However, these drugs
may interact with sites other than L-type VOC. Several studies have
shown that L-type Ca2+ antagonists inhibit the
nAChR channel of chromaffin cells (Gandía et al., 1991
;
Lopéz et al., 1993
; Houlihan et al., 2000
). The fact that
PACAP-induced [Ca2+]i
rise and CA release were not modified by diltiazem may also help to
rule out the possibility that PACAP interacts with nAChR channel to
stimulate Ca2+ entry. In addition, nonexcitable
cells, which do not possess VOC, are reported to possess a
dihydropyridine-sensitive Ca2+ entry pathway (Ma
et al., 1995
). Therefore, it may be that the results from the
Ca2+ antagonists alone are insufficient to prove
any contribution of VOC to
[Ca2+]i rise and CA release.
It has been postulated that SOCs are activated by the mobilization of
Ca2+ from its stores by physiological stimulation
(IP3-mediated release) or pharmacological
manipulation (inhibitors of sarcoendoplasmic reticulum
Ca2+-ATPase). The present results would seem to
exclude the possibility that PACAP-induced Ca2+
entry is mediated by SOCs, based on the following observations. Cells
pretreated with Ca2+-ATPase inhibitors
(thapsigargin and cyclopiazonic acid) showed no greater
Ca2+ entry stimulated by bradykinin, which is
thought to mediate Ca2+ entry by SOC. SOC was
apparently sufficiently activated by these manipulations to mask SOC
activation by bradykinin. Still, PACAP produced considerable
Ca2+ entry into these cells, suggesting that
PACAP activates a different Ca2+ entry pathway
from the SOC activated by thapsigargin or cyclopiazonic acid. To
examine another means of reducing Ca2+
concentration in the pools, we used TPEN, a membrane-permeable, low-affinity Ca2+ chelator, by which the
concentration of Ca2+ in pools can be kept low
and constant without release of Ca2+ into cytosol
and without increasing
[Ca2+]i (Arslan et al.,
1985
; Hofer et al., 1998
). When cells were treated with TPEN, both
bradykinin and PACAP failed to produce the initial transient increase
in [Ca2+]i in the
Ca2+-deprived medium, suggesting that the
concentration of Ca2+ in the stores was kept low.
In these cells, bradykinin caused no further increase in
Ca2+ entry, whereas PACAP did increase
Ca2+ entry, confirming the results obtained using
Ca2+-ATPase inhibitors.
The unique character of PACAP-activated Ca2+
entry was further demonstrated by changing extracellular pH.
Acidification of the solution enhanced and alkalinization inhibited
PACAP-induced Ca2+ entry, without affecting
Ca2+ mobilization from intracellular stores.
Several studies have shown that acidification inhibits SOC (Malayev and
Nelson, 1995
; Iwasawa et al., 1997
; Kerschbaum and Cahalan, 1998
; Khoo
et al., 2001
). Thapsigargin-induced Mn2+ entry
was reduced in acidic solutions and enhanced in alkaline solutions over
the range from 6.2 to 8.4 in bovine chromaffin cells in the present
study. Reducing pH has also been reported to reduce
Ca2+ current through VOC (Kaibara and Kameyama,
1988
; Klöckner and Isenberg, 1994
). Thus, pH-induced changes in
these channels have been examined in relation to the causes of
pathophysiological conditions. Extracellular acidosis decreases the
contractile force of isolated preparations of vascular smooth muscle
and induces vasodilation in vivo (Rinaldi et al., 1987
; Loutzenhiser et
al., 1990
). It might be of interest to consider the activation of the PACAP-activated Ca2+ channel by acid pH in
relation to the pathophysiological role of PACAP-induced CA release
versus ACh-induced release in acidosis or alkalosis, which remains to
be elucidated.
PACAP type 1 receptor is expressed predominantly in rat adrenal
medullary chromaffin cells (Shivers et al., 1991
; Spengler et al.,
1993
; Babinski et al., 1996
; Nogi et al., 1997
). It couples with G
protein and potently activates intracellular second messenger systems,
including cAMP/PKA, phospholipase C/IP3, and PKC.
Osipenko et al. (2000)
recently reported that PACAP requires the
activation of adenylate cyclase coupled with phospholipase C to
activate Ca2+ influx through a pathway inhibited
by Ras in PC12 cells. We have previously shown that cAMP plays a
critical role in ACh-induced CA release by inhibiting
Na+,K+-ATPase in bovine
chromaffin cells, resulting in an increase in intracellular
Na+ concentration accompanied by the activation
of VOC and Na+-Ca2+
exchange, thus increasing
[Ca2+]i (Morita et al.,
1987
, 1991a
,b
, 1995
). However, this mechanism does not seem to be
involved in the PACAP-induced
[Ca2+]i rise because the
PACAP-induced [Ca2+]i
rise and Mn2+ entry were not blocked by PKA
inhibitors, and PACAP-induced Ca2+ rise was not
blocked by TTX or a Ca2+-sucrose medium,
suggesting no Na+ requirement. PKC is activated
by PACAP and participates in the [Ca2+]i rise in
neuroepithelial cells (Zhou et al., 2001
). However, regulation by PKC
of PACAP-induced [Ca2+]i
signaling in bovine adrenal chromaffin cells seems unlikely because the
phospholipase C inhibitor U-73122 and staurosporine, an inhibitor of
PKC, did not affect the PACAP-induced
[Ca2+]i rise or
Mn2+ entry in chromaffin cells in the present study.
GdCl3 is a blocker of nonselective cation
currents, including SOC (Schumann et al., 1994
). We have recently
identified trps (trp3 and trp6) from
rat brain homologous to Drosophila trp, which is thought to
encode the SOC (Mizuno et al., 1999
). In COS cells transfected with
these trps, thapsigargin increased whereas
GdCl3 blocked the Ca2+
entry (Mizuno et al., 1999
). In the present study,
GdCl3 also blocked thapsigargin-induced
Ca2+ entry in chromaffin cells but did not block
PACAP-induced Ca2+ entry. These results suggest
that PACAP-activated Ca2+ channels have a
different sensitivity to SOC blockers. This finding is in agreement
with the following pharmacological properties. PACAP-activated
Ca2+ entry was resistant to such SOC inhibitors
as 7,8-benzoflavone, NDGA, NPPB, fulfenamic acid, and niflumic acid,
although at higher concentrations than IC50 in
some cells (Clementi and Meldolesi, 1996
), which all effectively
blocked thapsigargin-induced Ca2+ entry. The
finding that SK&F 96365, LOE 908, and econazole, not entirely selective
inhibitors of SOC (Leung and Kwan, 1999
), blocked both PACAP-activated
Ca2+ entry and thapsigargin-activated SOC
suggests that these blockers do not distinguish between PACAP-activated
Ca2+ entry and SOC. No specific inhibitor has yet
been found for PACAP-activated Ca2+ entry that
seems promising for investigation of a receptor-operated Ca2+ channel distinct from SOC.
In agreement with the effects of SK&F 96365, econazole, and
GdCl3 on PACAP-induced Ca2+
entry, PACAP-induced CA release was inhibited by SK&F 96365 and econazole but resistant to GdCl3. That
Ca2+ entry is essential for PACAP-induced CA
release is further confirmed by the fact that SK&F 96365 and econazole
blocked sustained [Ca2+]i
rise and Ca2+ entry without blocking the initial
[Ca2+]i rise.
Among the pathways by which extracellular Ca2+
can enter the cells, receptor-activated Ca2+
entry is the least understood. The present results suggest that PACAP
activates Ca2+ entry through plasma membrane in a
unique way characterized by independence from the depletion of store
Ca2+, differing sensitivity to blockers for SOC
and VOC, activation by acidic pH, and independence of adenylate
cyclase/cAMP and phospholipase C pathways. This pathway may prove to be
a useful model for studying receptor-activated
Ca2+ channels and for developing selective
blockers of receptor-activated Ca2+ channels
distinct from VOC and SOC.
We are grateful to Boehringer Ingelheim Pharma KG (Biberach,
Germany) for providing the LOE 908.
Accepted for publication April 24, 2002.
Received for publication January 22, 2002.
This work was supported in part by Grants-in-Aid for Scientific
Research from the Ministry of Education, Science and Culture, Japan.
PACAP, pituitary adenylate cyclase-activating
polypeptide;
CA, catecholamine;
[Ca2+]i, intracellular free Ca2+ concentration;
ACh, acetylcholine;
nAChR, nicotinic acetylcholine receptor;
VOC, voltage-dependent
Ca2+ channel;
SOC, Ca2+ store
depletion-operated Ca2+ channel;
IP3, inositol
trisphosphate;
PKC, protein kinase C;
U-73122, 1-[6-[[(17
)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione;
-CgTx,
-conotoxin;
H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfon-amide;
HA1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide
hydrochloride;
SK&F 96365, 1{
-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenylethyl}-1H-imidazole
hydrochloride;
TPEN, N',N',N',N'-tetrakis(2-pyridylmethyl)ethylenediamine;
NDGA, nor-dihydroguaiaretic acid;
NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid;
LOE 908, (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)
ethyl]-acetamide;
BSA, bovine serum albumin;
-AgTx IVA,
-agatoxin IVA;
TTX, tetrodotoxin;
PKA, cAMP-dependent protein
kinase;
TG, thapsigargin.