Pituitary Adenylate Cyclase-Activating Polypeptide and Vasoactive Intestinal Peptide-Stimulated Cyclic AMP

Synthesis in Rat Cerebral Cortical SlicesInteraction with Noradrenaline, Adrenaline, and Forskolin

Jerzy Z. Nowak* and Katarzyna Kuba

Department of Biogenic Amines, Polish Academy of Sciences, P-225, 90-950 Lodz, Poland

Received July 25, 2001; Accepted December 4, 2001

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP; 0.001–1 µM) and vasoactive intestinal peptide(VIP; 0.01–1 µM) produced a concentration-dependent stimulation of cyclic AMP (cAMP) formation in rat cere-bral cortical slices prelabeled with [3H]adenine. The effects of PACAP38 and PACAP27 were similar, and moreefficacious (at 0.1 and 1 µM) than those of VIP. Adrenaline and noradrenaline (each at 100 µM) also stimulatedcAMP formation, with the latter compound being more effective. Combination of PACAP38, PACAP27 (each at0.1 µM) and VIP (1 µM) with adrenaline or noradrenaline resulted in most cases in additive effects, with somesupraadditive (PACAP27 plus adrenaline) or subadditive (PACAP38 or VIP plus noradrenaline) fluctuations. Incontrast, combination of each of the three peptides with 3 µM forskolin resulted in synergistic effects. Theseresults indicate that in rat cerebral cortex there is no synergism between PACAP or VIP with noradrenaline oradrenaline; however, based on the forskolin data, it seems likely that synergistic effects may take place withVIP or PACAP and other cAMP-stimulating neuroregulators.

Index Entries: PACAP; VIP; noradrenaline; adrenaline; cAMP, rat cerebral cortex.

Journal of Molecular NeuroscienceCopyright © 2002 Humana Press Inc.All rights of any nature whatsoever reserved.ISSN0895-8696/02/18:47–52/$11.50

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Introduction

Pituitary adenylate cyclase-activating polypep-tide (PACAP) and vasoactive intestinal polypeptide(VIP) are biologically active peptides that display ahigh degree of structural and functional similarities.PACAP exists in two biologically equipotent shortand long forms, consisting, respectively, of 27 and38 amino acid residues, i.e., PACAP 1-27 (PACAP27)and PACAP 1-38 (PACAP38). VIP is a 28 amino acidpeptide, whose primary structure shows 68% homol-ogy with the short PACAPform. PACAP(both forms)and VIP equipotently interact with VPAC1 andVPAC2 receptors (VIP ≈ PACAP38 ≈ PACAP27).PACAP also interacts with its specific PAC1 recep-tors, which under physiological conditions are

poorly recognized by VIP (PACAP38 ≈ PACAP27 >>VIP) (Harmar et al., 1998; Vaudry et al., 2000).

PACAP, VIP, and their receptors are widely dis-tributed in brain and peripheral tissues, indicatinga pleiotropic nature of the peptides’ activity (Ros-tene, 1984; Gozes and Brenneman, 1989; Arimura,1998; Gozes et al., 1999; Vaudry et al., 2000). In thecentral nervous system (CNS), PACAP and VIPare thought to play a neurotransmitter and/or neu-romodulator role; one of the most frequentlyreported biochemical effects of these peptides is their ability to stimulate adenylyl cyclase activityand cAMP accumulation (e.g., Borghi et al., 1979;Etgen and Browning, 1983; Rostene, 1984; Gozes and Brenneman, 1989; Vaudry et al., 2000; Tatsunoet al., 2001).

*Author to whom all correspondence and reprint requests should be addressed. E-mail: jerzyn@amina1.zabpan.lodz.pl

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Earlier studies have shown that in its biologicalactions, VIP plays in concert with some other neu-roregulators (e.g., Fredholm and Lundberg, 1982;Emami et al., 1983; Magistretti and Schorderet, 1985;Karbon et al., 1986; Durroux et al., 1987; Schaad etal., 1989; Fatatis et al., 1994; Nowak et al., 1998; Pel-legri et al., 1998). Of particular interest is its syner-gistic interaction with noradrenaline, a phenomenonfirst observed in slices of cerebral cortex of mouse(Magistretti and Schorderet, 1984, 1985) and rat(Ferron et al., 1985), using, respectively, biochemi-cal and electrophysiological approaches. This func-tionally important interaction has subsequently been described to occur in rat pineal gland wherethe two regulators synergistically act to enhance the activity of both the cAMP- and melatonin-generating systems (Yuwiler, 1987; Chick et al., 1988).Recently, Shioda et al. (2000) reported on PACAP-noradrenaline synergistic interplay with respect tocalcium signaling in rat hypothalamus. However, itis unknown whether the described interaction ofPACAPand noradrenaline does affect the cAMPgen-erating system, and, if so, whether it can be extendedto other brain regions of rat, e.g., cerebral cortex. Thiswork was aimed at elucidating this issue.

Materials and MethodsAnimals

Experiments were carried out on male albinoWistar rats weighing 180–220 g. The animals weremaintained under a 12 h light/12 h dark lightingschedule (lights on between 06.00 and 18.00) withstandard food and water available ad libitum. Theexperiments were carried out in strict accordancewith the Polish governmental regulations concern-ing experiments on animals.

Assay of cAMP FormationOn the day of experiment, the animals were killed

by decapitation between 9.00–9.30, brains wereremoved, and cerebral cortex (without white matter)isolated and processed for the measurement of cAMPgeneration. In brief, the tissue pieces (consisting ofa part of parietal cerebral cortex) were rapidly cross-sliced (0.25 mm) with the aid of a McIlwain tissuechopper and suspended in cold, O2/CO2 (95�5)-gassed, glucose containing modified Krebs-Henseleit medium (KHM; mmol/L): 118, NaCl; 5, KCl; 1.3, CaCl2; 1.2, MgSO4; 25, NaHCO3; 11.7, D-glucose; pH 7.4).

The formation of [3H]cyclic AMP in [3H]adenine-prelabeled tissues was assayed according to Shimizu

et al. (1969). The formed [3H]cyclic AMP was iso-lated by sequential Dowex-alumina column chro-matography according to Salomon et al. (1974). Theresults were individually corrected for a percentagerecovery with the aid of [14C]cyclic AMP added to each column system prior to the nucleotide extrac-tion. The accumulation of cyclic AMP during a 10-min stimulation period was assessed as a per-centage of the conversion of [3H]adenine to [3H]cyclicAMP. Details of the whole procedure were describedby us earlier (Nowak and Sek, 1994).

ChemicalsThe following drugs were used: (±)-adrenaline

hydrochloride, forskolin, L-noradrenaline (arterenol)bitartrate, forskolin, PACAP38 (Sigma, St. Louis, MO), PACAP27 (RBI, Natick, MA). Radioactive compounds were: (2,8-3H)adenine (specific activity26.9 Ci/mmol) and [14C]cyclic AMP (specific activity52.3 mCi/mmol), both from DuPont-NEN (Bad Hom-burg, Germany).

Data AnalysisAll data are expressed as mean ± SEM values. For

statistical evaluation of results, analysis of variance(ANOVA) was used followed by the Newman-Keulstest.

ResultsAs shown in Fig. 1, PACAP27, PACAP38 (0.001–

1 µM) and VIP(0.01–1 µM) produced a concentration-dependent stimulation of cAMP formation in rat cerebral cortex. Both forms of PACAP were equipo-tent, whereas VIP appeared to be less effective thanPACAP, especially when used at higher concentra-tions. The data expressed as “net increases,” i.e., dif-ferences between peptide-stimulated values andrespective basal values, were (in percent conversion):at 0.1 µM– 3.55 ± 0.47 (n = 9) for VIP, 5.43 ± 0.88 (8) forPACAP27, and 5.68 ± 0.92 (8) for PACAP38; at 1 µM–5.11 ± 0.92 (17) for VIP, 8.00 ± 0.55 (18) for PACAP27,and 8.20 ± 1.23 (10) for PACAP38.

The study of an interaction between the three pep-tides and noradrenaline or adrenaline on cAMP for-mation was carried out using selected concentrationsof the compounds, i.e., 0.1 µM for both forms ofPACAP, 1 µM for VIP, and 100 µM for catecholamines.At the concentration used, the two catecholaminesclearly stimulated cAMP production, however, theeffects evoked by adrenaline were always smaller:0.68 vs 2.89, 0.44 vs 1.24, and 1.52 vs 4.08% conver-sion (data represent mean values showing net

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increases of cAMP taken from three different paral-lel experiments) (Fig. 2). Simultaneous applicationto the incubation medium of PACAP27, PACAP38, orVIP with noradrenaline or adrenaline resultedmostly in an additive effect; yet, a combination ofPACAP27 plus adrenaline showed a tendency to asupraadditive interaction, while combinations ofPACAP38 or VIP with noradrenaline resulted in asubadditive interaction (Fig. 2).

A combination of both PACAP forms (each usedat 0.1 µM) or VIP (1 µM) with 3 µM forskolin resultedin a synergistic interaction (Fig. 3). The net increasevalues produced by each combination were signifi-cantly larger (p < 0.05) than calculated sums of netincreases produced by respective compounds: 9.73vs 7.41 for PACAP27, 8.34 vs 6.98 for PACAP38, and9.23 vs 7.75 for VIP.

DiscussionAlthough PACAP and VIP acting via a receptor-

dependent way are capable of stimulating phos-pholipase C activity, with subsequent generation oftwo signaling molecules, such as inositol trisphos-phate (IP3) and diacylglycerol (DAG), the adenylylcyclase/cAMP pathway seems to be a major effec-tor system of both PAC1- and VPAC-type receptors

(Harmar et al., 1998; Vaudry et al., 2000). Actingthrough PAC1 receptors, which are specific forPACAP, this peptide strongly stimulates productionof cAMP in various cells/tissues of mammals (e.g.,Schomerus et al., 1994; Basille et al., 1995; Tatsunoet al., 2001), avians (Nowak et al., 1999, 2000), andlower vertebrates (Yon et al., 1992; Wong et al., 1998).Under physiological or experimental conditions,

Fig. 1. The effect of PACAP27, PACAP38, and VIP on cAMPproduction in rat cerebral cortical slices. The points repre-sent the means ± SEM of 7–18 experiments. The data showsnet increases in cAMP production (i.e., differences betweenpeptide-stimulated values and basal values) expressed aspercent conversion. The control (basal) value expressed inpercent conversion was: 3.45 ± 0.26(14). *, p < 0.05 forVIP vs PACP38; **, p < 0.05 for VIP vs PACAP38 and PACAP27.

Fig. 2. Interactions of PACAP27, PACAP38, and VIP withnoradrenaline or adrenaline on cAMP production in rat cerebral cortical slices. The peptides were applied at 0.1 µM and catecholamines at 100 µM concentration. Thebars represent means ± SEM; the number of experiments ineach series was: 10–16 for A (PACAP27), 10–17 for B(PACAP38), and 13–17 for C (VIP). The respective control(basal) vales expressed in percent conversion were: 2.25 ±0.39(10), 2.03 ± 0.18(10), and 3.68 ± 0.38(13) for (A), (B),and (C).

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PAC1 receptors are either not recognized, or onlyweakly (as compared to PACAP) influenced by VIP.VIP and PACAP can, however, similarly stimulatecAMP production when these two peptides interactwith VPAC-type receptors occurring in an array ofbiological systems (Arimura, 1998; Vaudry et al.,2000).

The present parallel study with PACAP27,PACAP38, and VIP has shown that all three peptidessignificantly stimulated cAMPformation in cerebralcortical slices of rat. Both PACAPforms were equipo-tent; whereas VIP, when used at 0.1 and 1 µM con-centrations, was clearly less effective in evoking thecAMP response than any PACAP form, with a dif-ference reaching one order of magnitude. Theobserved rank order of potency of the tested pep-tides, i.e., PACAP38 ≈ PACAP27 > VIP, suggests therole of adenylyl cyclase-linked PAC1 receptors inmediating the action of PACAP on cAMP genera-tion. Yet, at 0.01 µM concentration, all three peptidesinduced similar responses, as indicated by compa-rable net increases in the cAMP effect evoked by VIP,PACAP27, and PACAP38 (respective mean valueswere 2.89, 2.51, and 3.62% conversion; Fig. 1). Thismay suggest that cerebral cortex of rat contains bothPAC1- and VPAC-type receptors, whose activationcontributes to the observed cAMP responses.

Earlier reports demonstrated that in some bio-logical systems, e.g., mouse cerebral cortex (Mag-istretti and Schorderet, 1985) or rat pineal gland or cerebral cortex (Duman and Enna, 1987; Yuwiller,1987; Chick et al., 1988), the ability of VIP to stimu-late cAMP formation can be markedly increased by simultaneous activation of α1-adrenergic recep-tors. A convincing evidence indicates that theobserved VIP-noradrenaline synergism involves α1-adrenoceptor-driven mechanism linked to phospholipase C/IP3/DAG pathway, with a subse-quent activation by DAG of protein kinase C(PKC)-dependent phosphorylation (Karbon et al., 1986; Yuwiler, 1987; Chik et al., 1988; Schaad et al.,1989).

Although Duman and Enna (1987) demon-strated a potentiation by 6-fluoronoradrenaline (an α-adrenoceptor agonist) of the VIP-driven cAMPresponse in rat cerebral cortical slices, we were unableto reproduce these data testing a similar region ofrat brain, and using physiological adrenergic agonists, i.e., noradrenaline and adrenaline. In fact,in our hands a combination of VIP with noradrena-line (but not with adrenaline) gave the cAMP valueeven lower than the value reflecting a sum of par-ticular net effects of the two neuroregulators (5.33vs 7.85% conversion). An obvious disagreementbetween the data coming from two laboratories is difficult to explain at present; both research groupsused similar methodology, yet, the animals were not the same, i.e., Sprague-Dawley (Duman andEnna, 1987) and Wistar rats (the present study). Thus,it seems possible that some species- or evenintraspecies-dependent differences in the cerebralcortical network of both VIP-ergic and adrenergicnerve fibers (enabling a functional interplay betweendifferent neuroregulators) may be of crucial signif-icance in providing anatomical substrate for anyinteraction to occur.

Further results of this study, showing mainlyadditive effects on cAMP formation evoked by com-binations of PACAP27 or PACAP38 with noradrena-line or adrenaline, indicate that there is nosynergistic interaction with respect to the rat cere-bral cortical cAMP generating system betweenPACAP and the two catecholamines. These resultsare not in line with recently described data by Shiodaet al. (2000) which demonstrated in adult maleSprague-Dawley rats a colocalization of PACAPandnoradrenaline in catecholaminergic nerve terminalsof medullary neurons projecting to the hypothala-mus, and their action (when released together) insynergy to evoke calcium signaling and secretion

Fig. 3. Interactions of PACAP27, PACAP38, and VIP withforskolin. PACAP (both forms) was used at 0.1 µM, VIP at 1 µM, for forskolin at 3 µM. The bars represent means ±SEM of 8–12 experiments. Control (basal) value expressedin percent conversion was: 1.91 ± 0.27(7). Note that com-binations of the peptides with forskolin gave values (exp-resesed as net increases) significantly larger (*; p < 0.05)than those representing the calculated sums of net increasesproduced by particular compounds.

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of arginine vasopressin. The mechanism of this syn-ergistic interaction is unclear, and the Japaneseauthors proposed that, in addition to the role ofcAMP/PKA pathway, an unidentified factor “X”linked to L-type Ca2+ channels possibly takes partin the observed phenomenon.

In contrast to our findings showing the lack of asynergistic interaction between PACAP or VIP withnoradrenaline or adrenaline, the other findings ofthis study did show a synergism between these neuropeptides and forskolin. Forskolin is a directactivator of adenylyl cyclase, and thus, its actiondoes not involve any membrane receptor-relatedmechanism; however, forskolin often potentiates Gs-protein-linked receptor-driven signals to producemore cAMP inside target cells (Seamon and Daly,1986). In other words, the ability of forskolin to stim-ulate adenylyl cyclase activity in a given cell/tissuetells us that such cells or tissue contain responsivecAMPgenerating system(s), which may be activatedby endogenous agonists of various Gs-coupledmembrane receptors. Thus, our results on a syner-gistic PACAP/VIP-forskolin interaction stronglysuggest that in the rat cerebral cortex, an unrecog-nized neurotransmitter(s) or neuroregulator(s) otherthan (or in addition to) noradrenaline or adrenalinemay play in concert with PACAP (PAC1-receptor-related signal) and VIP(VPAC-receptor-related signal)to activate the adenylyl cyclase/cAMPsignaling path-way in either a neuronal or glial compartment, orboth. The presence in the cerebral cortex of (inter)neu-rons containing VIP and PACAP (e.g., Morrison et al.,1984; Koves et al., 1991), as well as PAC1 and VPACtype receptors (e.g., Vaudry et al., 2000), strengthensthe view that biochemical effects of PACAP or VIP,together with their interactions with other neuroreg-ulators, observed under experimental conditions byus and others, may be a part of a complex mechanism,essential for fine tuning at least some physiologicalprocesses in living systems.

AcknowledgmentsThe results described in this paper were presented

on Joint Meeting of the 11th Annual Meeting of theEuropean Neuropeptide Club (ENC) and AmericanSummer Neuropeptide Conference (May 6–11, 2001,Jerusalem-Tel Aviv, Israel).

The authors thank Prof. Jolanta B. Zawilska forher helpful discussion on the data and critical read-ing of the manuscript. This research was supportedby the grant No 6P04C037-17 from the State Com-mittee for Scientific Research (KBN) in Poland.

ReferencesArimura A. (1998) Perspectives on pituitary adenylate

cyclase activating polypeptide (PACAP) in the neu-roendocrine, endocrine, and nervous systems. Japn. J.Physiol. 48, 301–331.

Borghi C., Nicosia S., Giachetti A., and Said S. I. (1979)Vasoactive intestinal polypeptide (VIP) stimulatesadenylate cyclase in selected areas of rat brain. Life Sci.24, 65–70.

Basille M., Gonzalez B. J., Desrues L., Demas M., FournierA., and Vaudry H. (1995) Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates adenylylcyclase and phospholipase C activity in rat cerebellarneuroblasts. J. Neurochem. 65, 1318–1324.

Chick C. L., Ho A. K., and Klein D. C. (1988) α1-Adrenergicpotentiation of vasoactive intestinal peptide stimula-tion of rat pinealocyte adenosine 3′,5′-monophosphateand guanosine 3′,5′-monophosphate: evidence for a roleof calcium and protein kinase C. Endocrinology 122,702–708.

Duman R. S. and Enna S. J. (1987) Modulation of receptor-mediated cyclic AMP production in brain. Neurophar-macology 26, 981–986.

Durroux T., Barberies C., and Jard S. (1987) Vasoactiveintestinal polypeptide and carbachol act synergisticallyto induce hydrolysis of inositol containing phospho-lipids in the rat superior cervical ganglion. Neurosci.Lett. 75, 211–215.

Emami S., Gespach C., Forgue-Lafitte M. E., Broer Y., andRosselin G. (1983) Histamine and VIPinteractions withreceptor-cyclic AMP systems in the human gastriccancer cell line HGT-1. Life Sci. 33, 415–423.

Etgen A. M. and Browning E. T. (1983) Activators of cyclicadenosine 3′,5′-monophosphate accumulation in rathippocampal slices: action of vasoactive intestinal pep-tide (VIP). J. Neurosci. 3, 2487–2493.

Fatatis A., Holtzclaw L. A., Avidor R., and Brenneman D. E. (1994) Vasoactive intestinal peptide increasesintracellular calcium in astroglia: synergism with α-adrenergic receptors. Proc. Natl. Acad. Sci. USA 91,2036–2040.

Ferron A., Siggins G. R., and Bloom F. E. (1985) Vasoac-tive intestinal polypeptide acts synergistically with nor-epinephrine to depress spontaneous discharge rate incerebral cortical neurons. Proc. Natl. Acad. Sci. USA 82,8810–8812.

Fredholm B. B. and Lundberg J. M. (1982) VIP-inducedcyclic AMP formation in the cat submandibular gland.Potentiation by carbacholine. Acta Physiol. Scand. 114,157–159.

Gozes I. and Brenneman D. E. (1989) VIP: molecular biol-ogy and neurobiological function. Mol. Neurobiol. 3,201–236.

Gozes I., Fridkin M., Hill J. M., and Brenneman D. E. (1999)Pharmacological VIP: prospects and problems. Curr.Med. Chem. 6, 1019–1034.

Harmar A. J., Arimura A., Gozes I., Journot L., LaburtheM., Pisegna J. R., et al. (1998) International Union ofPharmacology. XVIII. Nomenclature of receptors for

52 Nowak and Kuba

Journal of Molecular Neuroscience Volume 18, 2002

vasoactive intestinal peptide and pituitary adenylatecyclase-activating polypeptide. Pharmacol. Rev. 50,265–270.

Ho A. K., Chick C. L., and Klein D. C. (1987) Trans-membrane receptor cross-talk: concurrent VIP and α1-adrenergic activation rapidly elevates pinealocytecGMP > 1000-fold. Biochem. Biophys. Res. Commun. 146,1478–1484.

Karbon E. W., Shenolikar S., and Enna S. J. (1986) Phor-bol esters enhance neurotransmitter-stimulated cyclicAMP production in rat brain slices. J. Neurochem. 47,1566–1575.

Koves K., Arimura A., Gorcs T. G., and Somogyvari-VighA. (1991) Comparative distribution of immunoreactivepituitary adenylate cyclase activating polypeptide andvasoactive intestinal polypeptide in rat forebrain. Neu-roendocrinology 54, 159–169.

Magistretti P. J. and Schorderet M. (1984) VIP and nora-drenaline act synergistically to increase cyclic AMP incerebral cortex. Nature 308, 280–282.

Magistretti P. J. and Schorderet M. (1985) Norepinephrineand histamine potentiate the increases in cyclic adeno-sine 3′,5′-monophosphate elicited by vasoactive intesti-nal polypeptide in mouse cerebral cortical slices:mediation by α1-adrenergic and H1-histaminergicreceptors. J. Neurosci. 5, 362–368.

Morrison J. H., Magistretti P. J., Benoit R., and Bloom F. E.(1984) The distribution and morphological character-istics of the intracortical VIP-positive cells: an immuno-histochemical analysis. Brain Res. 292, 269–282.

Nowak J. Z. and Sek B. (1994) Stimulatory effect of hista-mine on cyclic AMP formation in chick pineal gland.J. Neurochem. 63, 1338–1345.

Nowak J. Z., Kuba K., and Zawilska J. B. (1999) PACAP-induced formation of cyclic AMP in the chicken brain:regional variations and the effect of melatonin. BrainRes. 830, 195–199.

Nowak J. Z., Kuba K., and Zawilska J. B. (2000) Effects ofpituitary adenylate cyclase-activating polypeptide(PACAP) on cyclic AMP formation in the duck andgoose brain. Acta Neurobiol. Exp. 60, 209–214.

Nowak J.Z., Woldan-Tambor A., Kuba K. and ZawilskaJ.B. (1998) A synergistic interaction between histamineand vasoactive intestinal peptide (VIP) on cyclic AMPproduction in the chick pineal gland. J. Physiol. Phar-macol. 49, 377–386.

Pellegri G., Magistretti P. J., and Martin J. L. (1998) VIPand PACAPpotentiate the action of glutamate on BDNFexpression in mouse cortical neurons. Eur. J. Neurosci.10, 272–280.

Rostene W. H. (1984) Neurobiological and neuroendocrinefunctions of the vasoactive intestinal peptide (VIP).Prog. Neurobiol. 22, 103–129.

Salomon Y., Londos C., and Rodbell M. (1974) A highlysensitive adenylate cyclase assay. Anal. Biochem. 58,541–548.

Schaad N. C., Schorderet M., and Magistretti P. J. (1989)Accumulation of cyclic AMP elicited by vasoactiveintestinal peptide is potentiated by noradrenaline, his-tamine, adenosine, baclofen, phorbol esters, andouabain in mouse cerebral cortical slices: studies on therole of arachidonic acid and protein kinase C. J. Neu-rochem. 53, 1941–1951.

Schomerus E., Poch A., Bunting R., Mason W. T., and McAr-dle C. A. (1994) Effects of pituitary adenylate cyclase-activating polypeptide in the pituitary: activation oftwo signal transduction pathways in the gonadotrope-derived αT3-1 cell line. Endocrinology 134, 315–323.

Seamon K. B. and Daly J. W. (1986) Forskolin: its biolog-ical and chemical properties. Adv. Cyclic Nucleotide Pro-tein Phosphorylation Res. 20, 1–150.

Shimizu H., Daly J. W., and Creveling C. R. (1969) Aradioisotopic method for measuring the formation ofadenosine 3′5′-cyclic monophosphate in incubatedslices of brain. J. Neurochem. 16, 1609–1619.

Shioda S., Yada T., Muroya S., Uramura S., Nakajo S.,Ohtaki H., et al. (2000) Functional significance of colo-calization of PACAP and catecholamine in nerve ter-minals. Ann. NY Acad. Sci. 921, 211–217.

Tatsuno I., Uchida D., Tanaka T., Saeki N., Hirai A., SaitoY., et al. (2001) Maxadilan specifically interacts withPAC1 receptor, which is a dominant form ofPACAP/VIP family receptors in cultured rat corticalneurons. Brain Res. 889, 138–148.

Vaudry D., Gonzales B. J., Basille M., Yon L., Fournier A.,and Vaudry H. (2000) Pituitary adenylate cyclase-activating polypeptide and its receptors: from struc-ture to functions. Pharmacol Rev. 52, 269–324.

Wong A. O. L., Leung M. Y., Shea W. L. C., Tse L. Y., ChangJ. P., and Chow B. K. C. (1998) Hypophysiotropic actionof pituitary adenylate cyclase-activating polypeptide(PACAP) in the goldfish: immunohistochemicaldemonstration of PACAPin the pituitary, PACAPstim-ulation of growth hormone release from pituitary cells,and molecular cloning of pituitary type I PACAPrecep-tor. Endocrinology 139, 3465–3479.

Yon L., Feuilloley M., Chartrel N., Arimura A., Conlon J.M., Fournier A., and Vaudry H. (1992) Immunohisto-chemical distribution and biological activity of pitu-itary adenylate cyclase-activating polypeptide(PACAP) in the central nervous system of the frog Ranaridibunda. J. Comp. Neurol. 324, 485–499.

Yuwiler A. (1987) Synergistic action of postsynaptic α-adrenergic receptor stimulation on vasoactive intestinal polypeptide-induced increases in pineal N-acetyltransferase activity. J. Neurochem. 49, 806–811.