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MS 10989 Received 14 April 2000; accepted after revision 7 July 2000.
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ABSTRACT |
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INTRODUCTION |
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Pituitary adenylate cyclase-activating polypeptide (PACAP) was first isolated from ovine hypothalamus and was found to be present as two forms of 27 and 38 amino acids in the brain and other organs (Arimura, 1998). Among the peripheral tissues, adrenal medullae contain the second highest level of PACAP (Arimura et al. 1991), and it has been shown to be a potent secretagogue for catecholamines in cultured rat (Watanabe et al. 1992) and porcine (Isobe et al. 1993) adrenal medullary cells and rat (Watanabe et al. 1995) and dog (Lamouche et al. 1999) adrenal glands in vivo. Immunohistochemical studies combined with immunological sympathectomy revealed that the peptide was localized in preganglionic sympathetic nerve fibres surrounding chromaffin cells in rats (Holgert et al. 1996). This finding is consistent with findings in in situ hybridization studies which showed that PACAP mRNA was present in intermediolateral cell column neurones of the rat spinal cord (Beaudet et al. 1998), but not in adrenal medullary cells (Nielsen et al. 1998). These anatomical findings, taken together with its actions as a potent secretagogue, suggest that PACAP functions as a neurotransmitter or a neuromodulator of preganglionic sympathetic fibres. In fact, application of PACAP27 increased the intracellular Ca2+ concentration ([Ca2+]i) through mobilization of intracellular Ca2+ ions and facilitated Ca2+ influx in bovine adrenal medullary cells; the latter was suggested to be mediated by protein kinase C (PKC) (Tanaka et al. 1996). These effects were generally elicited by 0·1 µM PACAP and there was no difference in potency between PACAP27 and PACAP38 in producing the [Ca2+]i increase. Similar concentrations of PACAP were used to evoke an increase in [Ca2+]i in rat chromaffin cells (Przywara et al. 1996). Catecholamine secretion, however, was evoked by 100-fold lower concentrations of PACAP in rat (Watanabe et al. 1992) and porcine (Isobe et al. 1993) chromaffin cells. The potency required to induce secretion did not differ between PACAP27 and PACAP38, whereas the latter was 100-fold more potent in generating inositol phosphates than the former in PC12 cells (Deutsch & Sun, 1992) and chromaffin cells (Watanabe et al. 1992; Tanaka et al. 1998). While PACAP was considered to produce an increase in [Ca2+]i through phosphoinositide turnover in various types of cells including adrenal chromaffin cells (Rawlings et al. 1994; Tanaka et al. 1996; Kopp et al. 1999), the signal transduction mechanism for actions of PACAP released from sympathetic nerve terminals in adrenal medullary cells might not be mediated by messengers resulting from phosphoinositide turnover. We examined the role of PACAP in catecholamine secretion in dissociated guinea-pig chromaffin cells using amperometry and the perforated patch method, and then investigated the distribution of PACA1 receptors (PAC1Rs) in chromaffin cells using immunocytochemistry and confocal microscopy. The present results suggest that PACAP functions as a neuromodulator in adrenal medullae.
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METHODS |
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Animals
Female guinea-pigs weighing 250-300 g were housed in a light- and temperature-controlled room, and standard chow and tap water were always freely available. All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committees of Fukuoka and Showa Universities.
Amperometry
Experiments were performed on chromaffin cells enzymatically isolated from adrenal medullae. Guinea-pigs were killed by a blow to the neck. The adrenal glands were excised and immediately put into ice-cooled nominally Ca2+-free (i.e. Ca2+-deficient) balanced salt solution for which 1·8 mM CaCl2 was simply removed from the standard saline (see below). Adrenal medullae were cut into three to six pieces and incubated for 30 min with 0·25 % collagenase dissolved in Ca2+-deficient solution. After incubation, tissues were washed three times in Ca2+-deficient solution and left in this solution at 23-25°C until commencement of the experiments. Before the start of the experiments, one or two pieces of tissue were placed in the bath apparatus on an inverted microscope and chromaffin cells were dissociated mechanically using fine needles. Dissociated chromaffin cells were left for a few minutes to facilitate attachment to the bottom of the bath before being constantly perfused with standard saline at a rate of 1 ml min-1. Chemicals were bath applied and, unless otherwise noted, PACAP was added for 2 min. Catecholamine release from dissociated cells was measured using amperometry (Chow et al. 1992). A carbon-fibre electrode (ProCFE; Axon Instruments) was carefully placed on a chromaffin cell and +600 mV was applied to the electrode under voltage clamp conditions. The current due to oxidation of catecholamines at the tip of the electrode was recorded using an Axopatch 200A amplifier (Axon Instruments), and data were stored on a DAT data recorder (RD-120TE, TEAC, Japan) and fed to a brush recorder after low-pass filtering at 5 Hz. For quantitative analysis, the signals were low-pass filtered at 100 or 500 Hz and digitized at a sampling interval of 5 or 1 ms, respectively, using AxoData software (version 1.2, Axon Instruments). The total charge of evoked currents was measured using AxoGraph software (version 3.0, Axon Instruments), and nicotine- or muscarine-induced secretion was obtained by subtraction of the secretion recorded before stimulation (usually for 50-60 s) from that observed during stimulation (for the initial 50-60 s of stimulation). The standard saline contained (mM): 137 NaCl, 5·4 KCl, 1·8 CaCl2, 0·5 MgCl2, 0·53 NaH2PO4, 5 D-glucose, 5 Hepes and 4 NaOH (pH 7·4). To study the effects of Ca2+ mobilized from intracellular store sites, high concentrations of muscarine were applied in a Ca2+-deficient solution, in which 1·8 mM CaCl2 in the standard saline was replaced with 3·6 mM MgCl2.
Whole-cell recordings
Whole-cell currents were recorded using the perforated patch method (Horn & Marty, 1988). Currents recorded using an Axopatch 200A amplifier were fed into a brush recorder after low-pass filtering at 5 Hz, and onto a data recorder. The pipette solution contained (mM): 120 potassium isethionate, 20 KCl, 10 NaCl, 10 Hepes and 2·6 KOH (pH 7·2). On the day of the experiments, nystatin was added to the pipette solution at a final concentration of 100 ng ml-1. The membrane potential was corrected for a liquid junction potential of -12 mV between the nystatin solution and the standard saline.
The patch clamp and amperometric experiments were carried out at 23-25°C. Data are expressed as means ± S.E.M., and statistical significance was determined using Student's t test unless otherwise noted.
Immunochemistry
The animals were anaesthetized with pentobarbital (50 mg kg-1), blood was flushed out from the blood vessels with 50-100 ml of 0·9 % NaCl (37°C) via the ascending aorta, then the vessels were perfused for 30 min with 200-300 ml of a fixative consisting of 2 % paraformaldehyde and 2·5 % acrolein (Sigma) in 0·1 M phosphate buffer (pH 7·2) for 20-30 min. The adrenal glands, removed immediately after perfusion-fixation, were postfixed in 2 % paraformaldehyde for 12 h at 4°C. Sections of adrenal gland 7 µm thick were cut on a cryostat (Micron, Heidelberg, Germany) and picked up on gelatin-coated slides. The sections were pretreated with 0·5 % sodium borohydride solution for 30 min and endogenous peroxidase activity was blocked with 0·01 M phosphate-buffered saline (PBS, pH 7·2) that contained 0·5 % hydrogen peroxide. The sections were then placed in PBS containing 0·2 % Triton X-100 for 30 min, preincubated with 10 % normal horse serum in PBS for 1 h and then incubated with rabbit primary antiserum (see below) at a dilution of 1:1000 to 1:2000 for 2 days at 4°C. After incubation, tissue sections were immunostained by the avidin-biotin complex method (Vector Laboratories, Burlingame, CA, USA) and subsequently incubated for 5-6 min with 20 mg 3,3'-diaminobenzidine-4 HCl (Pierce, Rockford, IL, USA) dissolved in 0·1 ml of 5 % hydrogen peroxide diluted in 0·05 M Tris-HCl buffer (pH 7·6). The sections were counterstained with veronal acetate-buffered 1 % Methyl Green (pH 4·0), dehydrated in a graded series of ethanol and coverslipped with Malinol (Muto Chemicals, Tokyo, Japan).
Dissociated adrenal chromaffin cells were fixed in 2 % paraformaldehyde in PBS for 2 h at 4°C, preincubated in 5 % goat or rabbit serum in PBS for 30 min at 23-25°C, and then incubated with mouse nicotinic ACh receptor (nAChR) antibody (Ab) (MAB 398: Chemicon, Temecula, CA, USA) at a dilution of 1:10 for 1 day at 4°C, rabbit PAC1R Ab at a dilution of 1:2500 for 2 days at 4°C, or guinea-pig 
COP Ab for the identification of the Golgi complex (Duden et al. 1991; Misumi et al. 1997) at a dilution of 1:100 for 30 min at 23-25°C. After the incubation, the cells were washed three times in PBS and then treated with the respective secondary Ab conjugated with fluorescein isothiocyanate (FITC) or rhodamine at a dilution of 1:25 or 1:50 (goat anti-rabbit IgG Ab, goat anti-guinea-pig IgG Ab and rabbit anti-mouse IgG Ab; Cappel, Aurora, OH, USA). The fluorescence was observed using laser confocal microscopy (Zeiss LSM 410; Inoue et al. 2000). The objective lens was an oil-immersion lens with a magnification of × 63 and a numerical aperture of 1·25. For FITC, the 488 nm laser was illuminated and 510-525 nm emission was observed, whereas for rhodamine, the 543 nm laser was used and emission above 570 nm was observed. Whole-cell images were acquired with illumination of the 488 nm laser and emission of all wavelengths. To study the specificity of the immunoreaction, the preparation was treated with secondary Ab alone, and almost no immunoreactivity was observed under the same conditions as used for receptor Abs and COP Ab.
The primary antiserum against PACAP27 (no. 88121-5) was raised in a rabbit. Details on the production and characterization of the antiserum have been described elsewhere (Köves et al. 1990). An enzyme-linked immunosorbent assay indicated that the antiserum recognized both PACAP27 and PACAP38, but did not cross-react with other peptides, including porcine vasoactive intestinal polypeptide, secretin, glucagon, thyrotropin-releasing hormone, ovine corticotrophin-releasing factor and growth hormone-releasing hormone. Primary antisera (nos 93093-2 and 93094-2) were raised in rabbits against a synthetic peptide corresponding to the sequence starting at Lys411 of PAC1R, which is presumed to be its carboxy-terminal intracellular domain (Spengler et al. 1993). Details regarding acquisition of the antisera and the specificity have been described elsewhere (Li et al. 1997). Immunoblot analysis yielded a single protein band of 57 kDa corresponding to PAC1R in extracts of most of the tissues including the adrenal gland, thus indicating that the antisera reacted specifically only with PAC1R.
Electron microscopy
Adrenal glands of the guinea-pig were fixed with 2 % glutaraldehyde in 0·1 M phosphate buffer (pH 7·4) for 2 h at room temperature. Specimens were post-fixed in 4 % OsO4, dehydrated and embedded in Epon. The sections were doubly stained with uranyl acetate and lead citrate and examined under a Hitachi H-7000 electron microscope.
Chemicals
(±)-Muscarine chloride, methoxyverapamil (D-600), forskolin, phorbol 12,13-dibutyrate, 4
-phorbol 12,13-didecanoate and nystatin were obtained from Sigma; N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) were from Seikagaku (Tokyo, Japan); 3-isobutyl-1-methylxanthine (IBMX) was from Biomol (Plymouth Meeting, PA, USA); collagenase was from Yakult (Tokyo, Japan); nicotine was from Nacalai (Kyoto, Japan); PACAP27 and PACAP38 were from Peptide Institute (Osaka, Japan).
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RESULTS |
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PACAP-induced concentration-dependent enhancement of catecholamine secretion
In the majority of adrenal chromaffin cells, spontaneous catecholamine secretion rarely occurred and application of 10 µM nicotine for about 1 min at intervals of 2-3 min induced similar amounts of catecholamine secretion in a repeatable manner (Fig. 1A). Administration of 1 nM PACAP38 (Fig. 1A) or PACAP27 (e.g. Fig. 2) for about 2 min did not induce secretion but did enhance the subsequent secretion in response to nicotine. In 5 of 38 cells (13 %) exposed to PACAP38 or PACAP27, the increase in secretion evoked by nicotine continued after washout of nicotine (Fig. 1A), whereas in the remaining cells (87 %), the increase in secretion terminated on washout, irrespective of the extent of enhancement by PACAPs (e.g. Fig. 2). Figure 1B shows cumulative curves of the nicotine-induced secretion before, just after and 18 min after PACAP38 application. It is evident that exposure to PACAP38 enhanced the nicotine-induced secretion reversibly, and the extent of enhancement did not depend on the time of the nicotinic stimulation. Since PACAP38 and PACAP27 have essentially the same actions in guinea-pig adrenal chromaffin cells, detailed properties of the actions were studied using PACAP27 (PACAP).
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A, amperometric recordings of catecholamine secretion in response to 10 µM nicotine. Nicotine (N) and 1 nM PACAP38 were added to the perfusate for the periods indicated (bars for N and double line for PACAP38). Chart records were interrupted for the indicated times. B, cumulative curves of secretion; a-c shown in A correspond to a-c in B. Zero on the abscissa represents the time when nicotine was applied. | ||
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A, amperometric recordings of catecholamine secretion in response to 10 µM nicotine. Nicotine was bath applied during the periods indicated (bars) before (con) and after treatment with 10 nM (upper traces) and 0·3 nM (lower traces) PACAP27 (PACAP; P, double lines). PACAP was applied for about 2 min in this and the following figures. Some of the recordings during PACAP pretreatment are not shown. Upper and lower records are from the same cell. B, amount of nicotine-induced secretion, expressed as a fraction of that just before PACAP pretreatment (see Methods), plotted against time after PACAP pretreatment; a-e shown in A correspond to a-e in B. Squares represent nicotine-induced secretion following 0·1 nM PACAP pretreatment. C, relative amount of nicotine-induced secretion plotted against the concentration of PACAP applied for pretreatment. Means ± S.E.M. of 4-15 cells for each concentration of PACAP ( | ||
Application of PACAP at nanomolar concentrations for 2 min induced no change in basal catecholamine secretion in 87 % of the cells tested (28 of 31 cells with 1 nM, 12 of 15 cells with 3 nM, and 5 of 6 cells with 10 nM), but did produce a marked enhancement of the subsequent nicotine-induced secretion (Fig. 2). This action of PACAP was concentration dependent (Fig. 2C), and was elicited in 2 of 6 cells tested with 0·3 nM PACAP (> 15 % increase in secretion being regarded as an enhancement), 14 of 15 cells with 1 nM PACAP, and in all cells exposed to higher concentrations. As is evident in Fig. 2B, the PACAP-induced enhancement was a long-lasting effect and this was also concentration dependent. On average, 1 and 10 nM PACAP-induced enhancements persisted for 9·3 ± 2·0 (n = 6) and 16·8 ± 3·3 min (n = 5), respectively, and in 3 of 26 cells exposed to 1-10 nM PACAP, a maximum enhancement occurred not just after exposure, but about 10 min later (Fig. 2A and B). The concentration dependence of the PACAP38-induced enhancement (Fig. 2C, 
) coincided with that for PACAP27 (Fig. 2C, 
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The possible sites of action of PACAP are nAChRs, voltage-dependent Ca2+ channels or the secretory machinery. To explore these possibilities, the effects of PACAP pretreatment on secretion evoked by muscarinic receptor stimulation in standard saline and Ca2+-deficient solution were investigated. Low concentrations of muscarine produce catecholamine secretion through activation of non-selective cation channels (Inoue & Kuriyama, 1991) and the subsequent opening of voltage-dependent Ca2+ channels (Inoue et al. 1998). This muscarine-induced secretion was also enhanced by nanomolar concentrations of PACAP. Figure 3A shows that pretreatment with 1 nM PACAP increased the 3 µM muscarine-induced secretion by 126 % and this secretion reverted to the control level about 11 min after washout of PACAP. In seven cells, 1 nM PACAP enhanced muscarine-induced secretion by 115 ± 56 %, a value which did not differ from that (98 ± 21 %, n = 15) for nicotine-induced secretion. A similar increase in muscarine-induced secretion (120 ± 64 %, n = 3) was obtained with application of 1 nM PACAP38. These results suggest that voltage-dependent Ca2+ channels are a possible target, because these channels play a primary role in both secretions (Inoue et al. 1998) and were enhanced in bovine chromaffin cells by protein kinase A (PKA) activation (Artalejo et al. 1990). Therefore, to elucidate whether the Ca2+ channel activity is essential for PACAP-induced enhancement of secretion, the effects on secretion induced by Ca2+ mobilized from intracellular store sites in response to high concentrations of muscarine (Inoue et al. 1995) were investigated.
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A-C, amperometric recordings of secretion in response to 3 µM muscarine (M), 10 µM muscarine in Ca2+-deficient solution, and 10 µM nicotine (N). Chemicals were bath applied during the periods indicated (bars for M and N, double line for PACAP (P)) and Ca2+-deficient solution was substituted for standard saline during the periods indicated by the dotted line. Some of the recordings during exposure to 1 nM PACAP are not shown. Numbers represent approximate times (min) after PACAP pretreatment. B and C were from the same cell (different from that in A). D, relative amount of secretion in response to 10 µM nicotine, 3 µM muscarine and 10 µM muscarine in Ca2+-deficient solution (M Ca(-)). The amount of secretion is expressed as a fraction of that before 1 nM PACAP pretreatment. Means and S.E.M. of 15 cells for N, 7 cells for M and 6 cells for M Ca(-). * P = 0·023, ** P = 0·101 (Mann-Whitney test). | ||
Application of 10 µM muscarine in Ca2+-deficient solution transiently induced catecholamine secretion and subsequent restoration of standard saline containing Ca2+ also resulted in transient secretion in some cells (Fig. 3B). This second secretion was probably due to Ca2+ influx through voltage-dependent Ca2+ channels, but not to so-called 'capacitative Ca2+ entry' (Putney, 1986). This is suggested for two reasons. First the secretion was blocked by the Ca2+ channel blocker D-600 (10 µM) or by 0·1 mM Cd2+ ions. Second, when muscarine-containing Ca2+-deficient solution was replaced first with Ca2+-deficient solution, then with standard saline, the transient secretion did not occur. Thus, the secretion may have been due to a transient depolarization and the subsequent opening of voltage-dependent Ca2+ channels during washout of 10 µM muscarine. Pretreatment with 1 nM PACAP enhanced Ca2+ mobilization-induced secretion by 237 ± 69 % (n = 6), a value which was not smaller than that for the nicotine or 3 µM muscarine-induced secretion. Furthermore, enhancement of the Ca2+ mobilization-induced secretion diminished over a time course similar to that seen with nicotine-induced secretion (Fig. 3B and C) and the putative muscarine-induced secretion (Fig. 3B). Therefore, we suggest that nanomolar concentrations of PACAP mainly act at a site downstream of an increase in [Ca2+]i.
Differential facilitation of AChR-mediated secretion by PKC and PKA activators
The splice variants hop1 and hop2 of the PAC1R present in rat (Nogi et al. 1997) and bovine adrenal medullary cells (Tanaka et al. 1998) were found to be coupled with adenylate cyclase and phospholipase C in various cells, including chromaffin cells (Spengler et al. 1993). Furthermore, stimulation of PKA or PKC is known to enhance secretion of neurotransmitters (Chavez-Noriega & Stevens, 1994; Trudeau et al. 1998) and hormones (Gillis et al. 1996; Renström et al. 1997). Thus we used protein kinase activators to study the possible involvement of PKA or PKC in PACAP-induced enhancement of catecholamine secretion. Figure 4 shows the effects of 0·1 µM phorbol 12,13-dibutyrate, a PKC activator, and 10 µM forskolin, an adenylate cyclase activator, on nicotine-induced secretion. Application of 0·1-1 µM phorbol ester for 2 min itself increased basal secretion in 5 of 8 cells tested, of which two cells showed a transient secretion (Fig. 4A), and conspicuously enhanced the subsequent nicotine-induced secretion in all five cells tested (254 ± 77 % increase). The maximum enhancement was observed just after a 2 min pretreatment. On the other hand, 2 min exposure to 10 µM forskolin induced secretion in 1 of 5 cells, but enhanced the subsequent nicotine-induced secretion in all three cells tested (96 ± 25 % increase). In contrast to the enhancement by the phorbol ester, the maximum facilitation occurred 5-10 min after forskolin washout in two cells (Fig. 4, see d and e).
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A, amperometric recordings of secretion in response to 10 µM nicotine. Nicotine (N, bar), 0·1 µM phorbol 12,13-dibutyrate (PB, double line) or 10 µM forskolin (F, double line) was bath applied during the indicated periods. Some of the recordings during 2 min exposure to forskolin are not shown. B, amount of secretion relative to that evoked by the first application of nicotine, plotted against recording time; a-f shown in A correspond to a-f in B. Bars indicate exposure to PB or F. | ||
What was conspicuously different between the effects of PKC and PKA activation was the suppression of muscarine-induced secretion by the phorbol ester (Fig. 5). In the cell in which 10 nM PACAP enhanced secretion in response to 3 µM muscarine, a 2 min exposure to 1 µM phorbol ester abolished the subsequent muscarine-induced secretion, and this secretion was restored to 61 ± 8 % (n = 3) of control around 20 min after washout of the phorbol ester. Suppression of the muscarine-induced secretion and enhancement of the nicotine-induced secretion may be mediated by PKC, since 1 or 0·1 µM 4-phorbol 12,13-didecanoate, an inactive phorbol ester (Castagna et al. 1982), had no such actions. On the other hand, a 2 min exposure to forskolin resulted in 118 ± 38 % (n = 4) enhancement of the muscarine-induced secretion, a value which did not differ from that of the nicotine-induced secretion. Therefore, PKA, but not PKC, is probably responsible for the PACAP-induced enhancement of secretion, if phosphorylation is involved.
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A-C, amperometric recordings of secretion in response to 3 µM muscarine. Muscarine (M) and 10 nM PACAP, 1 µM phorbol 12,13-dibutyrate (Phorbol) or 10 µM forskolin were bath applied during the periods indicated (M, bar; PACAP, phorbol or forskolin, double line). A and B were obtained from the same cell, but C was from a different cell. Approximate times after the pretreatment are indicated. D, relative amount of nicotine (N)- or muscarine (M)-induced secretion after pretreatment with 10 µM forskolin or 1 or 0·1 µM phorbol 12,13-dibutyrate. The amount of nicotine- or muscarine-evoked secretion after the pretreatment is expressed relative to that before pretreatment. Means and S.E.M. of 3 cells for N and 4 cells for M with forskolin, and 5 cells for N and 4 cells for M with phorbol. | ||
Involvement of PKA in PACAP-induced enhancement of secretion
To elucidate the participation of PKA in the PACAP-induced enhancement of catecholamine secretion, the effects of the phosphodiesterase inhibitor IBMX on this enhancement were investigated. In Fig. 6A, a 2 min exposure to 1 nM PACAP or 0·5 mM IBMX resulted in enhancement of the subsequent nicotine-induced secretion to a similar extent, but the simultaneous application of PACAP and IBMX did not produce further enhancement of the secretion. Figure 6B summarizes the relative amounts of nicotine-induced secretion after pretreatment with PACAP, IBMX and a combination of PACAP and IBMX in four cells. The absence of a further increase in secretion with the application of PACAP and IBMX together suggests that the effect of PACAP is mediated by a cyclic nucleotide-dependent pathway.
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A, amperometric recording of secretion in response to 10 µM nicotine. Nicotine (N) and 0·1 mM IBMX, 1 nM PACAP (P) or IBMX + PACAP were bath applied during the periods indicated (N, bar; IBMX, PACAP or PACAP and IBMX, double line). Since addition of IBMX induced an upward shift of the basal current level, IBMX was washed out in standard saline prior to the application of nicotine. B, increase in nicotine-induced secretion after pretreatment with IBMX, PACAP, and PACAP and IBMX. IBMX solutions were sonicated, using an ultrasonic homogenizer. Means and S.E.M. of 4 cells. | ||
The effect of protein kinase inhibitors on the PACAP-induced enhancement of secretion was then examined (Fig. 7). The simultaneous application of PACAP and 100 µM HA1004, a general kinase inhibitor (Hidaka et al. 1984), abolished the enhancement of nicotine-induced secretion by PACAP (n = 3) and this suppressing action did not occur with 10 µM HA1004 (n = 2; not shown). If the target of HA1004 is PKA, then 2 µM H89, a specific inhibitor of PKA (Chijiwa et al. 1990), would be expected to abolish the PACAP-induced enhancement (the inhibition constant of H89 for PKA is 50-fold lower than that of HA1004). This inference was first examined for nicotine-induced secretion. The amount of nicotine-induced secretion just after pretreatment with 1 nM PACAP and 2 µM H89 was, unexpectedly, 50 ± 4 % (n = 4) of that prior to exposure (not shown). This diminution might have been due to block of the nAChR channel by H89 since the chemical is lipophilic, and the putative channel-blocking action of H89 seemed to disappear 2-5 min after washout. Thus, the effect of pretreatment with PACAP or PACAP and H89 was examined on the nicotine-induced secretion about 5 min after pretreatment. The secretion after the application of PACAP and H89 was 108 ± 15 % of controls, whereas that after pretreatment with PACAP alone in the same four cells was 233 ± 20 % of controls. These results suggest that PACAP-induced enhancement of secretion is sensitive to 2 µM H89. Since 2 µM H89 did not seem to interfere with muscarinic receptor-activated channels, the effect of H89 on enhancement by PACAP was further studied on 3 µM muscarine-induced secretion. As expected, the enhancement of the 3 µM muscarine-induced secretion just after pretreatment with PACAP was almost suppressed by the simultaneous application of H89 (Fig. 7Ab). Figure 7B summarizes the relative amount of nicotine- and muscarine-induced secretion just after pretreatment with PACAP and with PACAP and the kinase inhibitors: a similar enhancement of the nicotine- and muscarine-induced secretion by PACAP was abolished by the simultaneous application of PACAP and HA1004 (n = 3) or H89 (n = 4), respectively. These results with IBMX and the protein kinase inhibitors suggest the involvement of PKA in PACAP-induced enhancement of catecholamine secretion.
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A, amperometric recordings of 10 µM nicotine (N; a)- and 3 µM muscarine (M; b)-induced secretions. Upper and lower traces, pretreatment with 1 nM PACAP (P) and with PACAP and 100 µM HA1004 (HA; a) or 2 µM H89 (b), respectively. a and b were from different cells. Chemicals were bath applied during the periods indicated (N and M, bar; PACAP or PACAP and HA or H89, double line). Some of the recordings during pretreatment are not shown. B, relative amount of nicotine- and muscarine-induced secretion after pretreatment with 1 nM PACAP, 1 nM PACAP and 100 µM HA1004, and 1 nM PACAP and 2 µM H89. Means and S.E.M. of 3 cells for HA and 4 cells for H89. | ||
Effects of PACAP on whole-cell currents
Activation of PKA was reported to suppress the Na+ pump in bovine chromaffin cells with the consequent enhancement of catecholamine secretion (Morita et al. 1995). Thus, we investigated the effects of PACAP on the whole-cell current using the perforated patch method. If the Na+ pump is suppressed in the presence of PACAP, then an inward current would probably develop (Inoue et al. 1999) and the time course of decay of the Ca2+-dependent outward current (IO) induced by Ca2+ mobilization would be retarded due to an increase in the Na+ concentration just beneath the plasma membrane and the consequent impairment of Ca2+ extrusion through the Na+-Ca2+ exchanger. Figure 8A and B shows that a prior exposure to 1 nM PACAP for about 2 min did not alter the peak amplitude of IO induced by 10 µM muscarine in Ca2+-deficient solution or its decay time course, whereas the subsequent inward current attributed to non-selective cation channel activation was significantly augmented. In addition, the application of PACAP did not notably alter the holding current level: in 8 of 11 cells it did not induce a current and in the remaining three cells it induced inward currents of 1-3 pA, which diminished gradually over a time course of 5-10 min after washout. Next, we asked whether the augmentation was selective for muscarinic non-selective cation channels. Figure 8C demonstrates that pretreatment with PACAP enhanced the 3 µM muscarine-induced inward current, but not the 10 µM nicotine-induced current in the same cell in the presence of external Ca2+. This enhancement of muscarinic currents was not related to the development of the inward current, and the time course of deactivation of muscarinic currents after stimulation was not affected by PACAP pretreatment, regardless of whether or not PACAP induced an inward current. Figure 8D summarizes the amplitudes of muscarine-induced currents in standard saline (n = 6) and Ca2+-deficient solution (n = 5) and those of nicotinic currents in standard saline (n = 4). It is evident that exposure to PACAP selectively enhanced muscarinic inward currents by about 60 %.
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A and C, traces of whole-cell current recorded at -60 mV with the perforated patch method. Chemicals (10 µM muscarine in Ca2+-deficient solution, M Ca(-); 3 µM muscarine, M; 10 µM nicotine, N; 1 nM PACAP) were bath applied during the indicated periods. B, half-decay times (T½ ) and amplitudes (IO) of 10 µM muscarine-induced outward currents in Ca2+-deficient solution after exposure to PACAP, expressed as fractions of those before exposure. Means and S.E.M. of 5 cells. D, amplitudes of muscarine- and nicotine-induced inward currents (IM and IN, respectively) after exposure to PACAP, expressed as fractions of those prior to exposure. Means and S.E.M. of 11 cells for IM and 4 cells for IN. The relative amplitudes of currents induced by 3 µM muscarine (1·39 ± 0·21, n = 6) and 10 µM muscarine in Ca2+-deficient solution (1·84 ± 0·11, n = 5) were pooled. | ||
Immunochemistry
The studies outlined above suggest that PACAP functions as a neuromodulator rather than a neurotransmitter, if the peptide is released from nerve terminals. Thus, we asked which structure(s) in guinea-pig adrenal medullae contains PACAP and how PAC1Rs are distributed in chromaffin cells. PACAP-like immunoreactivity was found in nerve fibres, but not in ganglion cells or chromaffin cells (Fig. 9A). Most PACAP-like immunoreactive nerve fibres were closely associated with putative chromaffin cells and occasionally appeared to terminate on the cells. In addition, PACAP-like immunoreactive dense nerve fibres were present around some of the capillaries in the medulla, as was noted in the lateral septum of sheep brain (Köves et al. 1990). Only a few PACAP-like immunoreactive nerve fibres were observed in the adrenal cortex (not shown). These properties of PACAP-like immunoreactivity differed from those in the case of staining with PAC1R antisera. PAC1R-like immunoreactive materials were diffuse in putative chromaffin cells and the adrenal cortex lacked any detectable PAC1R-like immunoreactivity (not shown). To elucidate the detailed distribution of PAC1Rs, PAC1R-like immunoreactivity in dissociated adrenal chromaffin cells was observed using laser confocal microscopy.
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A, section of adrenal medulla immunostained for PACAP. PACAP-like immunoreactive nerve fibres (arrows) with varicosities (arrowhead) were distributed in close proximity to chromaffin cells. PACAP-positive nerve fibres were also visible around capillaries. B, confocal microscopic images of cells immunostained for PAC1R. Images represent superimposition of whole-cell images and PAC1R-like immunoreactivity (yellow) (see Methods). Z-axis analysis was performed with a full width at half-maximal intensity of ca 0·7 µm. Arrows indicate sites for XZ (a) and YZ (b) images. Asterisks in this and the following figures indicate the location of the nucleus. C, histogram of the distribution of PAC1R-like immunoreactivity. N and Non indicate the presence of PAC1R-like immunoreactivity in the plasma membrane of the nuclear and non-nuclear portions of the cell, respectively, and M indicates its presence in both compartments. | ||
When the three-dimensional structure of chromaffin cells was analysed using confocal microscopy, the majority of cells were oval shaped and the nucleus was displaced to one side in one or two of XY, XZ and YZ images. Thus, the cell can be divided into two portions, nuclear and non-nuclear, in such images by the straight line which is drawn tangentially to the rim of the nucleus and perpendicularly to the long axis of the cell, and the distribution of PAC1R-like immunoreactivity was evaluated in such images. PAC1R-like immunoreactive materials were mainly localized at the periphery of the nuclear portion shown in Fig. 9Ba, whereas in Fig. 9Bb they were present at the periphery of the non-nuclear portion and at the cell centre adjoining the nucleus. Figure 9C summarizes the membrane distribution of immunoreactive materials in 17 chromaffin cells: the PAC1R-like immunoreactivity was mainly confined to the plasma membranes of the nuclear portion (N) in four cells and the non-nuclear portion (Non) in six cells and present in both portions (M) in seven cells, suggesting that PAC1Rs are distributed diffusely in the plasma membrane. In contrast to the case of cardiac parasympathetic ganglion cells (Braas et al. 1998), however, PAC1R-like immunoreactivity did not circumscribe all the cell periphery without interruption. The occupation rate of the material was ca 31 % of the perimeter measured in XY images, irrespective of the type of distribution.
To determine whether the stained structure adjacent to the nucleus was the Golgi complex, dissociated chromaffin cells were successively incubated with PAC1R Ab and COP Ab, and immunoreactive materials were visualized by making use of FITC-conjugated anti-rabbit IgG Ab and rhodamine-conjugated anti-guinea-pig IgG Ab, respectively. Figure 10 shows that the portion next to the nucleus in the right-hand cell was stained with both PAC1R Ab and COP Ab, whereas part of the plasma membrane in the nuclear portion of the left-hand cell was stained only with PAC1R Ab. Therefore, PAC1R is apparently present not only in the plasma membrane, but also in the Golgi complex.
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Immunostainings for PAC1R (upper image) and | ||
The diffuse distribution of PAC1R-like immunoreactivity in the plasma membrane led to the notion that the distribution of nAChRs in the chromaffin cell should be investigated since PACAP may be present in cholinergic preganglionic nerve fibres in guinea-pig adrenal medulla. Figure 11A demonstrates that nAChR-like immunoreactive materials were present in a spot-like fashion in the plasma membrane, but were diffuse in the cytoplasm. The number of such spots in each cell ranged from one to four, consistent with the number of synaptic buttons in rat adrenal medullae (Kajiwara et al. 1997), and these 'hot spots' of immunoreactivity were mainly localized in the plasma membrane near the nucleus. To study the localization in detail, the distance between the spots and the nearest rim of the nucleus was expressed as a fraction of the longest diameter of the cell, determined in XY, XZ or YZ images of the cell, and the results are summarized in Fig. 11B. It is evident that the majority of the spots were localized to the plasma membrane near the nucleus. Thus, we investigated sites of synapses in the chromaffin cell using electron microscopy in order to confirm the findings obtained with confocal microscopy, since the majority of synapses are presumably formed by preganglionic sympathetic nerve terminals (Coupland, 1972). Figure 11C demonstrates that the neuronal component with small vesicles formed an asymmetrical synapse with the plasma membrane near the nucleus, whereas the non-nuclear portion was occupied by secretory vesicles. Similar electron micrographs were consistently obtained (n = 4).
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A, confocal microscopic images of nAChR-like immunoreactivity. Upper and lower images correspond to YZ and XY images. Z-axis analysis was similar to that in Fig. 9. Arrowheads represent spots of nAChR-like immunoreactivity. Arrow indicates the site of the YZ image. B, histogram of nAChR-like immunoreactive spots with various distances between spots and the nearest rim of the nucleus. Distances are expressed as fractions of the longest diameter of the cell (see text). C, electron micrograph of chromaffin cells. Arrow indicates an asymmetrical synapse; the inset shows this area at a higher magnification. | ||
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DISCUSSION |
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Mechanism for PACAP-induced enhancement of secretion
The main findings in the present study are that nanomolar concentrations of PACAP peptides enhanced nicotine- and muscarine-induced secretions and this enhancement was not accompanied by either an increase in basal secretion or a change in the resting membrane current in most cells. In 27 % of cells, an inward current of 1-3 pA gradually developed during exposure to PACAP, and the current diminished gradually over 5-10 min after washout. We have not investigated the ionic mechanism for the slow PACAP-induced current as part of the present study. However, our results suggest that the generation of this inward current may be involved in an increase in basal secretion observed in 13 % of cells, since enhancement of basal secretion often continued for 5-10 min after washout of PACAP and the secretion depended on external Ca2+ ions. Thus, the increase in basal secretion is probably due to depolarization and the consequent activation of voltage-dependent Ca2+ channels, as was found in porcine adrenal chromaffin cells (Isobe et al. 1993). In the majority of cells, exposure to PACAP enhanced subsequent nicotine- and muscarine-induced secretions with no change in basal secretion; therefore, production of the inward current is not likely to play a primary role in PACAP-induced enhancement of secretion.
Three lines of evidence indicate that PACAP-induced enhancement of secretion is mediated by a cAMP-PKA-dependent pathway. First, PACAP facilitated nicotine- and muscarine-induced secretions to a similar extent. This PACAP action was mimicked by application of forskolin, but not by a phorbol ester. Pretreatment with the phorbol ester enhanced the nicotine-induced secretion, but abolished the muscarine-induced secretion in a reversible manner. This abolition is consistent with the finding that stimulation of PKC suppressed the muscarinic activation of a non-selective cation channel in chromaffin cells (Inoue & Imanaga, 1995). Second, the extent of enhancement of the nicotine-induced secretion by the simultaneous application of PACAP and IBMX did not exceed that by PACAP alone, although exposure to IBMX alone did enhance the subsequent nicotine-induced secretion by 65 %. This apparent occlusion of the enhancement by IBMX may be accounted for by saturation of cAMP production. Third, enhancement of nicotine- and muscarine-induced secretions by PACAP was abolished by application of 100 µM HA1004 or 2 µM H89. This 50-fold difference in potency between HA1004 and H89 is consistent with the difference between the inhibition constants of both inhibitors for PKA, whereas the inhibition constant of the former for PKC was comparable to that of the latter. These results indicate that PKA-mediated phosphorylation is involved in the PACAP-induced enhancement of secretion. This notion is compatible with biochemical findings that the EC50 for production of cAMP by PACAP was 0·3 nM whereas that for the generation of inositol phosphates was 300 nM in the rat adrenal medulla (Watanabe et al. 1992).
Pretreatment with PACAP selectively augmented the activation of non-selective cation channels in response to muscarine. This finding was unexpected since PACAP enhanced external Ca2+-dependent secretions induced by nicotine and muscarine to a similar extent. This apparent discrepancy might be explained by the notion that enhancement of muscarinic currents is not large enough to cause a significant difference in the extent of depolarization. The extent of PACAP-induced enhancement of the internal Ca2+-dependent secretion in response to high concentrations of muscarine was not less than that of the external Ca2+-dependent secretion in response to nicotine or muscarine. These results clearly indicate that the major site for PACAP action is located downstream of an increase in [Ca2+]i, although stimulation of PKA was reported to increase voltage-dependent Ca2+ channel activity in bovine chromaffin cells (Artalejo et al. 1990). Application of PACAP did not consistently produce an inward current, and thus the inhibition of the Na+ pump activity and consequent impairment of the Na+-Ca2+ exchanger may not be involved in PACAP-induced enhancement of catecholamine secretion. This notion was further supported by the finding that PACAP did not retard the falling phase of IO in response to Ca2+ mobilization. We conclude that impairment of Ca2+ handling mechanisms is not responsible for PACAP-induced enhancement of secretion. Hence, the remaining possibilities are an increase in the size of the readily releasable pool of vesicles (Gillis et al. 1996; Renström et al. 1997) or an increase in the Ca2+ sensitivity of the secretory machinery (Lonart et al. 1998; Trudeau et al. 1998).
Physiological implications
To elucidate the potential role of PACAP in guinea-pig adrenal medullae, it is important to identify associated structures that contain the peptide. PACAP-like immunoreactivity was found in nerve fibres, which run between putative chromaffin cells and occasionally appeared to terminate on cells, whereas chromaffin cells and ganglion cells entirely lacked PACAP-like immunoreactivity. These findings are consistent with those in the rat adrenal medulla in that PACAP-like immunoreactivity was exclusively present in preganglionic sympathetic fibres. The notion that PACAP in adrenal medullae is present in preganglionic sympathetic fibres is further supported by data obtained in in situ hybridization experiments in which PACAP mRNA was found in intermediolateral cell column neurones in the spinal cord (Beaudet et al. 1998), but not in chromaffin cells of the rat (Nielsen et al. 1998). Since adrenaline cells comprise the majority of adrenal medullary cells in the guinea-pig (Eränkö, 1955), it seems reasonable to assume that PACAP fibres innervate adrenaline cells in the guinea-pig. In rat adrenal medullae, a dense network of PACAP-positive fibres surround noradrenaline cells and lower numbers of often more weakly fluorescent PACAP-positive fibres were associated with adrenaline cells (Holgert et al. 1996). Compared with PACAP-positive fibres in rat adrenal medullae, those in guinea-pig appeared to be sparse. This sparsity might be related to the fact that guinea-pig adrenal medullae have almost no noradrenaline cells. If PACAP fibres are in fact sparse in guinea-pig adrenal medullae, then chromaffin cells lacking terminals of PACAP nerve fibres may be stimulated by PACAP which diffuses from nearby nerve terminals, and the concentration of PACAP will consequently be diluted. This notion is consistent with the diffuse distribution of PAC1R-like immunoreactivity.
PAC1R-like immunoreactive materials were found in the plasma membrane of the non-nuclear as well as that of the nuclear portion, where the synapses are mainly located. On the other hand, the majority of nAChR-like immunoreactive materials were present as 'hot spots' in the plasma membrane near the nucleus. These spots may correspond to the subsynaptic membrane since synapses were localized to the plasma membrane in the nuclear portion and nAChRs are known to be concentrated at the neuromuscular junction of the innervated skeletal muscle (Apel & Merlie, 1995) and at postsynaptic membranes in brain neurones (Arroyo-Jiménez et al. 1999). Although we did not clarify the neurochemical properties of the synapses, those we observed are likely to have been made by preganglionic sympathetic nerve terminals. First, brain synapses in which the presence of nAChRs was confirmed using immunoelectron microscopy were of an asymmetrical type. Second, the majority of synapses in adrenal chromaffin cells are thought to be formed by preganglionic sympathetic nerve terminals (Coupland, 1972).
The different distribution of nAChRs and PAC1Rs may reflect differences in the physiological roles that nAChRs and PAC1Rs play in adrenal chromaffin cells. The finding that PACAP itself did not induce secretion, but rather enhanced nAChR-mediated secretion, would fit with the different distribution of the receptors. The nAChRs need to be concentrated at the subsynaptic membrane in order to mediate a rapid synaptic transmission, whereas PACAP is not involved in such a transmission and therefore PAC1Rs need not be concentrated at synapses.
In summary, PACAP enhanced nicotine- and muscarine-induced catecholamine secretion with no increase in basal secretion and no change in membrane current in most of the cells tested, and PAC1R-like immunoreactivity was distributed diffusely in the plasma membrane of chromaffin cells. These results suggest that PACAP functions as a neuromodulator in guinea-pig adrenal medullae to facilitate ACh-mediated secretion. To our knowledge, this is the first study to report that receptors for a neuromodulator and a neurotransmitter, which are presumably released from the same nerve terminals, are distributed differently in the plasma membrane.
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This study was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Brain Science Foundation (Japan). Thanks are due to Dr Y. Misumi (Fukuoka University) for the kind gift of anti-COP Ab.
Corresponding author
M. Inoue: Department of Physiology, School of Medicine, Fukuoka University, Fukuoka 814-0180, Japan.
Email: minoue{at}fukuoka-u.ac.jp
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