effects of pacap 127 on the canine endocrine pancreas in vivo:...

Effects of PACAP1–27 on the canine endocrinepancreas in vivo: interaction with cholinergicmechanism

Nobuharu Yamaguchi, Tamar Rita Minassian, and Sanae Yamaguchi

Abstract: The aim of the present study was to characterize the effects of pituitary adenylate cyclase activating poly-peptide (PACAP) on the endocrine pancreas in anesthetized dogs. PACAP1–27 and a PACAP receptor (PAC1) blocker,PACAP6–27, were locally administered to the pancreas. PACAP1–27 (0.005–5 µg) increased basal insulin and glucagonsecretion in a dose-dependent manner. PACAP6–27 (200 µg) blocked the glucagon response to PACAP1–27 (0.5 µg) byabout 80%, while the insulin response remained unchanged. With a higher dose of PACAP6–27 (500 µg), both responsesto PACAP1–27 were inhibited by more than 80%. In the presence of atropine with an equivalent dose (128.2 µg) ofPACAP6–27 (500 µg) on a molar basis, the insulin response to PACAP1–27 was diminished by about 20%, while theglucagon response was enhanced by about 80%. The PACAP1–27-induced increase in pancreatic venous blood flow wasblocked by PACAP6–27 but not by atropine. The study suggests that the endocrine secretagogue effect of PACAP1–27 isprimarily mediated by the PAC1 receptor, and that PACAP1–27 may interact with muscarinic receptor function inPACAP-induced insulin and glucagon secretion in the canine pancreas in vivo.

Key words: atropine, PACAP, PAC1, muscarinic, interaction.

Résumé : La présente étude a eu pour but de caractériser les effets du polypeptide pituitaire activateur de l’adénylatecyclase (PACAP) sur le pancréas endocrine de chiens anesthésiés. Le PACAP1–27 et un bloqueur du récepteur duPACAP (PAC1), PACAP6–27, ont été administrés localement dans le pancréas. Le PACAP1–27 (0,005–5 µg) a augmentéla sécrétion basale d’insuline et de glucagon de manière dose dépendante. Le PACAP6–27 (200 µg) a bloqué la réponsedu glucagon au PACAP1–27 (0,5 µg) d’environ 80 %, mais il n’a pas modifié la réponse de l’insuline. La plus fortedose de PACAP6–27 (500 µg) a inhibé les deux réponses au PACAP1–27 de plus de 80 %. En présence d’atropine etd’une dose équivalente (128,2 µg) de PACAP6–27 (500 µg) sur leur base molaire, la réponse de l’insuline au PACAP1–27

a diminué d’environ 20 %, alors que celle du glucagon a augmenté de près de 80 %. L’augmentation induite par lePACAP1–27 du débit sanguin veineux pancréatique a été bloquée par le PACAP6–27 mais pas par l’atropine. L’étudedonne à penser que l’effet sécrétagogue endocrine du PACAP1–27 est principalement véhiculé par le récepteur PAC1, etque le PACAP1–27 pourrait interagir avec la fonction du récepteur muscarinique dans les sécrétions d’insuline et de glu-cagon induites par le PACAP dans le pancréas canin in vivo.

Mots clés : atropine, PACAP, PAC1, muscarinique, interaction.

[Traduit par Rédaction] Yamaguchi et al. 729

Introduction

Pituitary adenylate cyclase activating polypeptide(PACAP) is a ubiquitous neuropeptide having two isomers,(i) the 38 amino acid residues (PACAP1–38) and (ii) the N-terminal-amidated 27 residues (PACAP1–27), originally iso-lated from the ovine hypothalamus (Miyata et al. 1990).Both PACAP1–38 and PACAP1–27 exhibit highly selective af-finity for PACAP receptors in membranes from various tis-sues, including the endocrine pancreas (Arimura and Shioda1995; Harmar et al. 1998). Pancreatic B cells express twoPACAP receptor subtypes, the PACAP-specific (PAC1) re-ceptor and the PACAP – vasoactive intestinal peptide (VIP)

shared receptor (VPAC2) (Harmar et al. 1998). However, thePAC1 receptor, particularly its PAC1TM4 variant, is the pri-mary PACAP-selective receptor in the endocrine pancreas(Chatterjee et al. 1996; Muroi et al. 1998; Yada et al. 1997).A recent study, however, could not confirm the expressionof the PAC1TM4 variant either in rat or mouse islets butrather indicated that the PAC1-short and -hop splice variantsare the most abundant in islets (Jamen et al. 2002). EitherPACAP1–27 or PACAP1–38 stimulates insulin and glucagonsecretion from the isolated, perfused rat pancreas (Yokota etal. 1993). While the insulin response to PACAP1–38 was sig-nificantly reduced by a PAC1 antagonist (PACAP6–38), theglucagon response remained unchanged in the isolated,

Can. J. Physiol. Pharmacol. 81: 720–729 (2003) doi: 10.1139/Y03-067 © 2003 NRC Canada

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Received 25 November 2002. Published on the NRC Research Press Web site at http://cjpp.nrc.ca on 12 June 2003.

N. Yamaguchi,1 T.R. Minassian, and S. Yamaguchi. Groupe de recherche sur le système nerveux autonome (GRSNA), Faculté depharmacie, Université de Montréal, Montréal, QC H3C 3J7, Canada.

1Corresponding author (e-mail: [email protected]).

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perfused porcine pancreas (Tornoe et al. 1997). This maysuggest involvement of a different receptor in the glucagonresponse. On the other hand, intravenous PACAP1–38 en-hanced both the basal and carbachol-induced glucagon se-cretion in mice (Fridolf et al. 1992), suggesting aninteraction between the muscarinic cholinergic effect and thesecretagogue effects of the peptide. These previous observa-tions prompted us to further characterize the secretagogueeffects of PACAP1–27 on the endocrine pancreas in vivo,with special reference to the functional implications of thePAC1 receptor and the potential interaction with muscariniccholinergic receptor function in the endocrine pancreatic re-sponse to PACAP1–27 in anesthetized dogs.

Materials and methods

The local animal research committee at the Université deMontréal has approved the experimental protocol. The ani-mals used in this study have been cared for and used in ac-cordance with the principles and guidelines of the CanadianCouncil on Animal Care (1993, 1984).

Preparation of animalsAdult mongrel dogs, fasted overnight but allowed free ac-

cess to water, were anesthetized with pentobarbital sodium(30 mg/kg i.v., followed by 4 mg/kg as needed). Artificialrespiration (room air) was maintained through anendotracheal tube connected to a Harvard pump (model 607,Harvard Apparatus, Inc., Holliston, Mass.). The rectal tem-perature of each dog was monitored and kept constant at37.5 ± 0.5°C by means of a thermoregulator (model 74; Yel-low Springs Instruments, Yellow Springs, Ohio) connectedto a heating pad. Both femoral arteries were cannulated; theright femoral artery was used to measure aortic pressure, andthe left femoral artery was used to obtain aortic blood sam-ples.

Preparation of pancreas for local intra-arterial drugadministration and extracorporeal venous circuit

Following a median laparotomy, the superior pancreatico-duodenal (SPD) artery was dissected free from the surround-ing tissues. Fine polyethylene tubing (PE-50) was inserted tothe SPD artery through an adjacent small branch so that theSPD arterial blood flow remained unobstructed. Local drugadministrations to the pancreas were made through this cath-eter. The volume of this catheter was fixed to be 0.5 mL inthe first group and 0.25 mL in the other groups, and thecatheter was connected to an infusion pump (model 55-2226, Harvard Apparatus, Inc.).

Soft, flexible silicon tubing (3 mm i.d.) was inserted in aretrograde manner into the SPD vein, so that a major part ofthe venous blood draining the pancreas could be obtained(Havel et al. 1996; Yamaguchi and Fukushima 1998). Ve-nous blood from the pancreas was directly drained as itpassed through an electromagnetic flow probe into a smallreservoir through this catheter, from which pancreatic ve-nous blood was sampled. The venous blood volume in thereservoir was kept as small as possible with an automaticblood-level controller and continuously returned to the dogby a perfusion pump (Masterflex® model 7016-52; Cole-Parmer Instrument Co., Vernon Hills, Ill.) through a silicon

tubing (3 mm i.d.) inserted into the portal vein (Yamaguchiand Fukushima 1998). After all surgical procedures werecompleted; sodium heparin (200 U/kg, i.v.) was adminis-tered, followed by 100 U/kg every hour thereafter. The dogwas then allowed a stabilization period of about 60 min. Theblood volume withdrawn for sample collections was re-placed with an intravenous injection of the same volume ofsaline (0.9%) in addition to its continuous infusion at a slowrate during the whole period of the surgery and the experi-ment.

Measured parametersPancreatic venous blood flow, mean aortic pressure, and

heart rate were recorded by means of a polygraph system(model RM-6000, Nihon Kohden, Tokyo, Japan). The SPDvenous blood flow was measured with an electromagneticflow probe (3 mm i.d., model FF-030T, Nihon Kohden) con-nected to the SPD venous catheter (Yamaguchi andFukushima 1998). SPD venous and aortic blood (3 mL ofeach) was simultaneously sampled, and an aliquot of 1.5 mLwas immediately transferred to chilled tubes. Plasma con-centrations of immunoreactive insulin and glucagon weredetermined by means of commercially available radio-immunoassay kits (ICN Pharmaceuticals, Inc., Costa Mesa,Calif.). Blood was immediately centrifuged at 4°C for 5 minat 14 000 rpm (15 800 × g) with a refrigerated centrifuge(model 5402, Eppendorf AG, Hamburg, Germany) and theplasma was stored at –80°C until assayed. Hematocrit wasmeasured in all pancreatic venous blood samples. At the endof each experiment, the pancreas was removed and weighed.For evaluating the basal and PACAP1–27-evoked secretion ofinsulin, the net output of insulin from the pancreas was cal-culated as follows. Net output of insulin (µU·min–1·g–1 pan-creas) = [([INS]SPDV – [INS]AO)([BF]SPDV)(1 – [Hct]SPDV)]/(wet weight of pancreas), where [INS]SPDV and [INS]AO arethe plasma immunoreactive insulin concentrations in thesuperior pancreaticoduodenal venous and aortic blood, respec-tively. [BF]SPDV and [Hct]SPDV are superior pancreatico-duodenal venous blood flow and its hematocrit, respectively.The net output of pancreatic glucagon was obtained in thesame way as described for the insulin output.

Experimental protocolsThe present study consisted of five groups of anesthetized

dogs. The first group (27.7 ± 1.4 kg, n = 7) served to obtaindose-related insulin and glucagon responses to PACAP1–27,and, thereby, to determine the dose used for the subsequentexperiments. This group received PACAP1–27 (Sigma Chemi-cal, St. Louis, Mo.) with four different concentrations of0.01, 0.1, 1.0, and 10 µg·mL–1 (0.00318, 0.0318, 0.318, and3.18 µM, respectively). Each solution was locally infused ata rate of 0.5 mL·min–1 for precisely 1 min. The total dosesdelivered to the pancreas during each infusion were there-fore 0.005, 0.05, 0.5, and 5 µg. The dead volume of the SPDarterial catheter (0.5 mL) was taken into account in relationto the infusion rate. After taking the initial control samplefrom the SPD vein and the aorta, the vehicle (saline 0.9%,pH 7.38) was infused for 1 min and samples were obtained1, 3, and 5 min after the onset of infusion. This procedurewas repeated every 15 min for the four different doses ofPACAP1–27. The sample obtained at 15 min after the onset of

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each infusion served as the control for the subsequent inter-vention.

The second group (24.2 ± 1.2 kg, n = 6) served to ensurethe reproducibility of the insulin and glucagon responses toPACAP1–27 administered twice with an interval of 60 min.Following the initial control sample, the vehicle was locallyinfused to the pancreas for a period of 4 min at a rate of0.25 mL·min–1. At the beginning of the fourth minute, im-mediately after taking the second control sample, PACAP1–27(0.5 µg with 2 µg·mL–1, or 0.636 µM solution) was added tothe vehicle infusion line at the same infusion rate. Thus, thevehicle and PACAP1–27 were simultaneously conveyed to thepancreas during the fourth minute. Then, both infusionswere discontinued at the end of 4 min. A sample was ob-tained during this simultaneous infusion period (designatedas sample 1 min), followed by samples 2, 3, 5, 10, 15, and30 min after the onset of the PACAP1–27 infusion. After a re-covery period of 30 min (i.e., 60 min after the beginning ofthe first block), the second block started, following exactlythe same procedures described for the first block.

In the third (23.9 ± 1.3 kg, n = 6) and fourth groups(22.8 ± 3.0 kg, n = 6), the effect of a selective PAC1 receptorantagonist (PACAP6–27) (Sigma Chemical) (Robberecht et al.1991, 1992) on the insulin and glucagon responses toPACAP1–27 was tested following exactly the same protocolused in the second group. Instead of the vehicle, however,the third and fourth groups received total dose of 200 and500 µg of PACAP6–27, respectively, during the second blockof the protocol. The doses of PACAP6–27 were selected onthe basis of our previous study on the adrenal medulla(Lamouche and Yamaguchi 2001).

The fifth group (22.2 ± 1.8 kg, n = 6) served to verify ifthere is any interaction between the secretagogue effects ofPACAP1–27 and muscarinic cholinergic mechanisms in theendocrine pancreas, following the same protocol describedfor the two preceding groups. Instead of PACAP6–27 admin-istered during the second block, the fifth group received128.2 µg of atropine (Sigma Chemical), which is the equiva-lent molar dose (189 nmol) of 500 µg of PACAP6–27.

Statistical analysesThe statistical evaluations were carried out using a statisti-

cal software package (SigmaStat for Windows, v. 2.03,SPSS, Chicago, Ill.). Differences over a given experimentalperiod within the same subjects were assessed by the analy-sis of variance for repeated measures followed by multiplecomparisons with the Dunnett test or the Student–Newman–Keuls test. The net maximum increments of insulin andglucagon outputs in response to PACAP1–27 in the absenceand the presence of PACAP6–27 or atropine in the same sub-ject were analyzed using the paired t test. When applicable,a preliminary logarithmic transformation was used to satisfythe condition of a normal distribution of variance(Wallenstein et al. 1980). All results are expressed as means ±SE, and P < 0.050 was considered statistically significant.

Results

Basal values for the measured parametersInitial resting values for plasma concentrations of insulin

and glucagon in SPD venous and aortic blood, as well as the

basal data for SPD venous blood flow and hematocrit, meanaortic pressure, heart rate and postmortem wet weight of thepancreas, are summarized in Table 1. These initial valueswere not statistically different between groups. Mean aorticpressure, heart rate, and hematocrit remained relatively sta-ble during a given period of experiment, and the observedvariations in those parameters were not statistically signifi-cant.

Dose-dependent increase in the basal secretion of insulinand glucagon in response to various doses of PACAP1–27

The local administrations of PACAP1–27 to the pancreasresulted in significant increases in the basal plasma concen-trations of insulin and glucagon in SPD venous blood. SPDvenous blood flow also increased significantly. Conse-quently, both insulin and glucagon outputs significantly in-creased following a dose-dependent manner (Fig. 1).However, the amplitude of the maximum response ofglucagon output was significantly smaller, being roughly onefourth that of the insulin response (Fig. 1). Within the doserange tested, the minimum effective dose of PACAP1–27,judged as a dose that caused the first statistically significantresponse, was 0.005 µg for insulin, while it was 0.5 µg forglucagon output (Fig. 1). The latter dose (0.5 µg) ofPACAP1–27 was, therefore, selected for the subsequent ex-periments. The maximum response of insulin output andSPD venous blood flow was observed 1 min after the onsetof PACAP1–27 infusion, and the increased insulin output re-turned to the corresponding preinfusion control level by5 min (Figs. 2A and 2C). However, glucagon output in-creased rather slowly, reaching its maximum level 3 min af-ter the administration of PACAP1–27 (Fig. 2B). Plasmaconcentration of insulin and glucagon in aortic blood in-creased slightly at higher doses of PACAP1–27, but thesevariations were subtracted when calculating the net output ofthese hormones.

Reproducibility of insulin and glucagon responses toPACAP1–27

In the control group receiving the vehicle, both insulinand glucagon outputs were reproducible when the same dose(0.5 µg) of PACAP1–27 was administered twice with an inter-val of 60 min (Figs. 3A and 3B). This holds true for the in-creasing responses of the SPD venous blood flow (Fig. 4A),as well as plasma concentrations of insulin and glucagon inthe SPD venous blood. There was no statistically differencebetween the first and the second responses when comparedwith values expressed as net (∆) maximum increments.

Effect of PACAP6–27 on the secretion of insulin andglucagon induced by PACAP1–27

In the group receiving 200 µg of PACAP6–27, the basal out-put of insulin remained unchanged in the presence ofPACAP6–27 alone, but that of glucagon increased from 0.172 ±0.058 to 0.566 ± 0.177 ng·min–1 (P < 0.05, n = 6). The netmaximum increase in insulin output induced by 0.5 µg ofPACAP1–27 did not change significantly in the presence ofPACAP6–27 (Fig. 5A). However, the net increase in the maxi-mum glucagon output was significantly diminished by about80% (Fig. 5B). Similarly, the maximum response of insulinconcentration in the SPD venous blood remained statistically

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unchanged, whereas that of glucagon concentration wasabolished (Table 2). The response of SPD venous bloodflow remained unaffected in this group (Fig. 4B).

In the group treated with 500 µg of PACAP6–27, the basaloutputs of insulin and glucagon did not change significantlyby PACAP6–27 alone. The net increase in the maximum insu-lin output in response to PACAP1–27 was inhibited by morethan 80% (Fig. 6A), and that of glucagon output was almostcompletely blocked (Fig. 6B). The net responses of plasmaconcentrations of insulin and glucagon in the SPD venousblood were also diminished to an extent similar to that ob-served in their output responses (Table 2). The SPD venousblood flow response was blocked by 500 µg of PACAP6–27(Fig. 4C).

Effect of atropine on the secretion of insulin andglucagon induced by PACAP1–27

The secretagogue effect of 0.5 µg of PACAP1–27 wastested in a separate group receiving 128.2 µg of atropine, a

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Parameter Group 1 (n=7) Group 2 (n=6) Group 3 (n=6) Group 4 (n=6) Group 5 (n=6)

[INS]SPDV (µU/mL) 165.5±35.7 283.7±90.4 278.9±114.0 176.3±43.6 212.3±36.6

[INS]AO (µU/mL) 8.9±1.9 23.6±8.4 16.5±2.8 17.7±1.5 17.7±1.6

[GGN]SPDV (ng/mL) 0.528±0.095 0.490±0.043 0.488±0.069 0.753±0.200 0.557±0.086

[GGN]AO (ng/mL) 0.325±0.063 0.320±0.042 0.316±0.045 0.498±0.114 0.347±0.056

[BF]SPDV (mL/min) 35.3±2.6 54.0±5.1 54.1±2.3 54.4±5.0 41.3±3.8

[Htc]SPDV (%) 44.5±1.5 44.5±1.0 43.3±2.0 43.2±1.8 43.4±1.6

MAP (mmHg) 123.9±6.4 128.2±2.5 137.7±4.9 125.1±2.9 126.6±8.4

HR (beats/min) 139.0±3.3 163.3±4.7 164.3±11.1 162.8±6.0 153.0±7.9

PCR (g) 57.9±2.4 51.9±4.5 47.5±3.0 43.3±8.0 43.9±2.3

Note: Values are means ± SE. n, number of dogs tested; SPDV, superior pancreaticoduodenal venous; AO, aortic; INS, insulin; GGN, glucagon;BF, blood flow; Htc, hematocrit; MAP, mean aortic pressure; HR, heart rate; PCR, wet weight of postmortem pancreas.

Table 1. Initial values for plasma concentrations of insulin and glucagon in pancreatic venous and aortic blood, pancreatic venousblood flow and hematocrit, mean aortic pressure, heart rate, and postmortem wet weight of pancreas in anesthetized dogs.

Fig. 1. Effect of various doses of PACAP1–27 locally adminis-tered to the pancreas through the superior pancreatico-duodenalartery on the basal secretion of insulin (filled columns) andglucagon (open columns) in anesthetized dogs. The results areexpressed as ratios above the corresponding control values ob-served immediately before the administration of the vehicle (VH,saline) and each dose of PACAP1–27. *, P < 0.05 vs. VH; **,P < 0.001 vs. VH; †, P < 0.05 vs. the corresponding insulin re-sponse (ANOVA, n = 7).

Fig. 2. Effect of PACAP1–27 (0.5 µg) on the basal output of insu-lin (A, [INS]OP) and glucagon (B, [GGN]OP), as well as on thebasal superior pancreatico-duodenal venous blood flow (C,[BF]SPDV), after the local administration of PACAP1–27. The vehi-cle (VH1, saline) and PACAP1–27 were administered at the corre-sponding arrows. C1 and C2 represent the first and secondcontrol value obtained immediately before VH1 and PACAP1–27

administration, respectively. Each column represents the mean± SE (n = 24) obtained from 4 groups, each of which consistedof 6 dogs. The data from the first block of the protocol in eachgroup (groups 2, 3, 4, and 5) were pooled, because the experi-mental procedures were the same in these four groups. *, P <0.05 vs. C2; **, P < 0.001 vs. C2 (ANOVA).



dose equivalent to 500 µg of PACAP6–27 on their molar ba-sis. The basal output of either insulin or glucagon was notaffected by atropine alone. In the presence of atropine, thePACAP1–27-induced increase in insulin output was inhibitedby about 20% (Fig. 7A). By contrast, the glucagon responsewas enhanced by about 80% (Fig. 7B). The similar modulat-ing effects of atropine were observed in plasma concentra-tions of insulin and glucagon in SPD venous blood(Table 2). However, atropine did not affect to any signifi-cant extent the increasing response of SPD venous bloodflow induced by PACAP1–27 (Fig. 4D).

Discussion

The present study was carried out to characterize thesecretagogue effects of exogenous PACAP1–27 on the canineendocrine pancreas in vivo, with special reference to thefunctional implications of PAC1 receptor and the potentialinteraction with muscarinic receptor-mediated function. Theresults suggest the functional existence of the PAC1 receptorthat principally mediates the secretion of insulin andglucagon. The study showed that the blockade of muscarinicreceptors with atropine resulted in the significant reductionand enhancement of PACAP1–27-induced insulin andglucagon secretion, respectively.

Within the dose range tested, the basal secretion of bothinsulin and glucagon was significantly increased by

PACAP1–27 in a dose-dependent manner without major sys-temic effect. However, the insulin response was about 100-fold more sensitive than that of glucagon as judged by theminimum effective dose for stimulating each hormone secre-tion. In addition, the onset of the secretagogue effect wasimmediate for insulin, reaching its maximum response in1 min, while it was delayed for glucagon, resulting in itsmaximum response 3 min after the administration of thepeptide. Furthermore, the maximum insulin response toPACAP1–27 was roughly four times greater than the

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Fig. 3. Effect of repeated administrations of PACAP1–27 (0.5 µg)on the basal output of insulin (A, [INS]OP) and glucagon (B,[GGN]OP). PACAP1–27 was administered with an interval of60 min, with the control group receiving vehicle (VH1 and VH2),as indicated by the corresponding arrows. The horizontal linesattached to the arrows indicate the infusion period of 4 min forVH1 and VH2 and 1 min for the first and second infusion ofPACAP1–27. Open circles indicate the control values obtained im-mediately before the administration of PACAP1–27. *, P < 0.05vs. the corresponding control values indicated by open circles(ANOVA, n = 6).

Fig. 4. Effect of repeated administrations of PACAP1–27 (0.5 µg)on basal pancreatic blood flow as measured in the superiorpancreaticoduodenal vein ([BF]SPDV). PACAP1–27 and vehiclewere administered with an interval of 60 min in various groups.The control group (panel A) received the vehicle (VH1 andVH2), each at the corresponding arrows, and PACAP1–27 twice,each at the corresponding arrows. The group in panel B received200 µg of PACAP6–27 instead of VH2. The group in panel C re-ceived 500 µg of PACAP6–27 instead of VH2. The group in panelD received 128.2 µg of atropine instead of VH2. The horizontallines attached to the arrows indicate the infusion period of 4 minfor VH1 and VH2 or drug and 1 min for the first and second in-fusion of PACAP1–27. Open circles indicate the control valuesobtained immediately before the administration of PACAP1–27. *,P < 0.05 vs. the corresponding control values indicated by opencircles (ANOVA, n = 6).



glucagon response, when comparing the ratios above theirbasal secretion.

Such differences in the pattern of insulin and glucagon se-cretions in response to blood-borne PACAP1–27 may be ac-counted for by the morphological nature of the islet celldistribution and the intraislet microcirculation. It has beenshown that, in the canine pancreas, the islet B cells weremainly localized in the central area and being the most nu-merous and that A cells were often localized at the periphery(Hawkins et al. 1987). Similar observations have been re-ported by Wieczorek et al. (1998). With respect to theintraislet microcirculation in the canine pancreas, the direc-tion of intraislet blood flow was found to be from the B cellcore outward to the A-cell-containing mantle (Stagner andSamols 1986), and the B cell is perfused before the A cellduring normal arterial perfusion in the rat and the dog mod-

els (Stagner et al. 1988). Thus, insulin secreted by B cells inthe islet core appears to be able to reach the A cells of themantle via the intraislet portal vasculature (Weir andBonner-Weir 1990). In addition, there is an intraislet inhibi-tory mechanism by insulin regulating the secretion ofglucagon (Samols and Harrison 1976; Maruyama et al.1984). Taken together, these observations suggest that thedelayed and the less potent glucagon response to PACAP1–27observed in the present study may have resulted most proba-bly from the potent preceding increase in insulin secretion.In this context, it is of interest to note that the peak glucagonresponse was observed when the insulin response returnedalmost to its basal value, a phenomenon that can be ex-plained by the intraislet vascular communications (Stagneret al. 1988; Weir and Bonner-Weir 1990) and the intraislet

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Fig. 5. Net maximum increment in insulin (A, ∆ Max [INS]OP)and glucagon output (B, ∆ Max [GGN]OP) in response toPACAP1–27 (0.5 µg) in the absence (VH1) and presence of200 µg of PACAP6–27. PACAP1–27 was administered twice withan interval of 60 min in the same dog. *, P = 0.028 vs. the cor-responding control value (VH1) (paired t test, n = 6).

Drug Hormone Before After P

PACAP6–27, 200 µg [INS]SPDV (µU/mL) 1071.2±249.5 907.8±232.2 0.281[GGN]SPDV (ng/mL) 0.619±0.262 –0.027±0.267 0.034

PACAP6–27, 500 µg [INS]SPDV (µU/mL) 1237.9±266.9 302.7±105.3 0.023[GGN]SPDV (ng/mL) 0.685±0.053 –0.137±0.140 0.006

Atropine, 128.2 µg [INS]SPDV (µU/mL) 1326.6±273.9 1059.2±237.2 0.028[GGN]SPDV (ng/mL) 1.224±0.296 2.114±0.645 0.006

Table 2. Maximum net changes in plasma insulin and glucagon concentrations in pancreatic venous blood inducedby 0.5 µg of PACAP1–27 before and after treatment with PACAP6–27 or atropine.

Fig. 6. Net maximum increment in insulin (A, ∆ Max [INS]OP)and glucagon output (B, ∆ Max [GGN]OP) in response toPACAP1–27 (0.5 µg) in the absence (VH1) and presence of500 µg of PACAP6–27. PACAP1–27 was administered twice withan interval of 60 min in the same dog. *, P = 0.028 vs. corre-sponding control value (VH1); **, P = 0.031 vs. correspondingcontrol value (VH1) (paired t tests, n = 6).



negative insulin-glucagon regulatory mechanism (Samolsand Harrison 1976; Maruyama et al. 1984). Another possi-bility may be a different distribution of PAC1 receptors inthe islets. Indeed, the expression level of the PACAP-specific receptor has been shown to follow the order of insu-lin B cells ≥ glucagon A cells > acinar cells, although suchan order of the labeling intensity may differ depending onthe different methodology including the specific antibodyused (Emami et al. 1998). Moreover, the cellular specificityof the signaling protein localization, such as the distributionof different adenylyl cyclase isomers being dependent on is-let cells and species, may also account for the differencesbetween insulin and glucagon secretion induced byPACAP1–27 (Emami et al. 1998).

It has been observed that desensitization of the PAC1 re-ceptor or tachyphylaxis of PACAP-induced response devel-oped relatively rapidly in certain tissues, such aspheochromocytoma cells (Taupenot et al. 1999), sheep pitu-itary cells (Sawangjaroen et al. 1996), and human retinoblas-toma cells (Dautzenberg and Hauger 2001) under in vitroconditions. In the present study, however, both insulin andglucagon responses to 0.5 µg of PACAP1–27 were reproduc-ible upon the second administration with an interval of60 min. This holds true for the pancreatic vasodilator re-sponse to PACAP1–27, as measured in the SPD venous bloodflow. Thus, the exposure to local blood-borne PACAP1–27 ofthe insulin B and glucagon A cells, as well as the pancreaticvasculature, for a relatively short period of time (1 min) in

vivo did not cause tachyphylaxis in the endocrine pancreasunder the present experimental conditions.

In the isolated, perfused porcine pancreas, the PACAP1–38-induced insulin secretion was highly significantly reducedby PACAP6–38, another PAC1 antagonist, while the simulta-neous glucagon response remained unaltered (Tornoe et al.1997). In the present study, however, we observed that thehigher dose of PACAP6–27 significantly attenuated the net in-creases in both insulin and glucagon secretions induced byPACAP1–27. Inconsistency between these findings may be duemost probably to species differences or different experimentalconditions. Indeed, it has been suggested that several PACAPreceptor subtypes with varying affinity for PACAP6–38 mayexist in the porcine pancreas in vitro (Tornoe et al. 1997).

The vasodilatory effect of PACAP has been well docu-mented in various organs, including the pancreas (Vaudry etal. 2000). PACAP1–27 increases pancreatic blood flow fol-lowing a dose-dependent manner in either anesthetized(Yamaguchi 2001) or conscious dogs (Ito et al. 1998). In thepresent study, the PACAP1–27-induced increase in the SPDvenous blood flow was not affected by the low dose ofPACAP6–27. Therefore, the inhibition of glucagon secretionobserved in the presence of the low dose of PACAP6–27 re-sulted from its direct action on the glucagon A cells and notfrom other secondary effects, such as decreased blood flow.The finding that the insulin response was not significantlyaffected in the presence of the low dose of PACAP6–27 maybe due most probably to the very potent insulin response toPACAP1–27, rather than a possible low affinity of PACAP6–27for the insulin B cells. Indeed, in the presence of the highdose of PACAP6–27, the net insulin response also becamesignificantly diminished, suggesting a competitive antago-nism at the level of insulin B cells. Nevertheless, the pancre-atic blood flow response to PACAP1–27 was also markedlyblocked with the high dose of PACAP6–27, a possible sec-ondary factor that may have significantly diminished the in-sulin output. This possibility, however, may not be the case,because the PACAP1–27-induced increase in insulin concen-tration in the SPD venous blood was also significantly inhib-ited with the high dose of PACAP6–27 to an extent similar tothat expressed in its output.

PACAP6–27, a selective PAC1 receptor antagonist, has beenshown to specifically antagonize the increase in adenylatecyclase activity induced by PACAP1–27 in the rat pancreatic(Robberecht et al. 1991) and human neuroblastoma cellmembranes (Robberecht et al. 1992). Moreover, it has beenwell documented that the effect of PACAP is mediatedthrough PAC1 receptors and results mostly from the activa-tion of the adenylyl cyclase system in the isolated rat pan-creatic islets (Borboni et al. 1999). Furthermore, certainbinding studies indicate that the PAC1 receptor predominatesover VPAC2 receptor in the insulin B cells (Borboni et al.1999), and that the PAC1 receptor is the primary PACAP-selective receptor in the endocrine pancreas (Chatterjee et al.1996; Muroi et al. 1998; Yada et al. 1997). In the presentstudy, the PACAP1–27-induced increases in the insulin andglucagon secretions were blocked by more than 80% in thepresence of PACAP6–27 with the doses tested. Taken to-gether, it appears likely that, in the canine endocrine pan-creas in vivo, the inhibition by PACAP6–27 of the insulin andglucagon responses to PACAP1–27 may have resulted primar-

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Fig. 7. Net maximum increment in insulin (A, ∆ Max [INS]OP)and glucagon output (B, ∆ Max [GGN]OP) in response toPACAP1–27 (0.5 µg) in the absence (VH1) and presence of128.2 µg of atropine. PACAP1–27 was administered twice with aninterval of 60 min in the same dog. *, P = 0.028 vs. correspond-ing control value (VH1); **, P = 0.002 vs. corresponding controlvalue (VH1) (paired t tests, n = 6).



ily from the specific antagonism at the PAC1 receptor. Thisinterpretation is consistent with recent studies in PAC1receptor-deficient mice showing that glucose-stimulated in-sulin secretion was significantly reduced in vitro and in vivo(Jamen et al. 2000), and that the glucagon response to insulin-induced hypoglycemia was markedly impaired (Persson andAhren 2002), both of which indirectly indicate that theVPAC2 receptor is not functionally implicated in the endo-crine pancreatic responses to pathophysiological glucose al-terations. Nevertheless, the possible PACAP-induced VIPrelease, which in turn stimulates insulin and glucagon secre-tion (Tornoe et al. 1997), cannot completely be ruled out inthe present study, if PACAP6–27 may have certain affinityfor the VPAC2 receptor, as in the case of PACAP6–38(Harmar et al. 1998).

To investigate if there are any functional interactions be-tween muscarinic receptors and the PACAP-mediated re-sponses, the local effects of PACAP1–27 were further testedin the presence of atropine with an equivalent dose ofPACAP6–27 on their molar basis. The results indicated thatatropine slightly inhibited PACAP1–27-induced insulin secre-tion. Since PACAP6–27 almost completely blocked thePACAP1–27-induced pancreatic vasodilator effect, and be-cause atropine did not at all affect the pancreatic vascular re-sponse to PACAP1–27 as observed in this and other studies(Ito et al. 1998), it is clear that PACAP1–27 by itself did notexert any direct cholinergic effect in the canine pancreas invivo. Therefore, the atropine-sensitive inhibition of the insu-lin response to PACAP1–27 was not related to the local pan-creatic blood flow. Since PACAP increases exocrinepancreatic secretion through the activation of vagalcholinergic neurons (Onaga et al. 1997), and because PAC1receptor immunoreactivity was abundant in the pancreaticganglia (Kirchgessner and Liu 2001), it is plausible that thecholinergic neural component may have contributed at leastin part to the insulin response to PACAP1–27 via muscarinicreceptor stimulation by acetylcholine released from thesplanchnic nerve terminals. This interpretation is compatiblewith other studies showing the existence of direct or indirectfacilitating interactions between exogenous PACAP and pe-ripheral cholinergic neurotransmission in the heart (Hirose etal. 1997; Runcie et al. 1995; Seebeck et al. 1996; Yonezawaet al. 1996) and the adrenal medulla (Lamouche et al. 1999;Lamouche and Yamaguchi 2003).

In contrast to the insulin secretion, the PACAP1–27-induced glucagon response was significantly enhanced in thepresence of atropine. It has been indicated that insulin main-tains an ongoing restraint upon glucagon secretion, and thata decrease in insulin secretion causes hypersecretion ofglucagon (Maruyama et al. 1984). It therefore appears likelythat, in the present study, the increase in PACAP1–27-inducedinsulin secretion may have restrained the glucagon response.It is then conceivable that the actual decrease of the insulinresponse by atropine may have reduced inhibition of theglucagon response, resulting in the enhancement of glucagonsecretion. As discussed above, however, the possibility thatthe neural acetylcholine could be released by PACAP1–27cannot completely be ruled out. Because cholinergic stimu-lation usually increases glucagon secretion via muscarinicreceptor activation (Holst et al. 1981; Havel and Taborsky1989; Yamaguchi 1992), the present finding of the enhanced

glucagon response in the presence of atropine rathersuggests the existence of an atropine-sensitive inhibitorymechanism operating in the cascade of the PAC1-mediatedglucagon secretion.

In conclusion, the local administration of PACAP1–27 to thepancreas resulted in increases in insulin and glucagon secre-tion in a dose-dependent manner in anesthetized dogs. Thepeak insulin response was roughly 4 times greater than that ofthe glucagon response. Both the insulin and glucagon re-sponses were markedly inhibited by PACAP6–27. PACAP1–27-induced insulin secretion was reduced by atropine, while theglucagon response was enhanced significantly. The studysuggests that the PAC1 receptor primarily mediates thePACAP1–27-induced secretion of both insulin and glucagonin the canine endocrine pancreas in vivo. It is also suggestedthat the muscarinic component in the endocrine pancreasmay interact with the PACAP-mediated mechanisms in regu-lating insulin and glucagon secretion.

Acknowledgement

This work was supported by grants from the Canadian In-stitutes of Health Research (MT-10605).

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