Introduction

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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 G_q/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

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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, R_p 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.

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 K_d 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

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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).

View larger version (27K): [in this window] [in a new window]   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.

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.

View larger version (13K): [in this window] [in a new window]   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.

View larger version (37K): [in this window] [in a new window]   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.

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.

View larger version (27K): [in this window] [in a new window]   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.

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, (R_p)-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, (R_p)-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.

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.

View larger version (30K): [in this window] [in a new window]   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+.

	Discussion

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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.