Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulation of Sympathetic Neuron Neuropeptide Y and Catecholamine Expression

Journal of NeurochemistryLippincott-Raven Publishers, Philadelphia© 1995 International Society for Neurochemistry

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP )Regulation of Sympathetic Neuron Neuropeptide Y and

Catecholamine Expression

Department ofAnatomy and Neurobiology, University of Vermont College of Medicine, Burlington, Vermont, U.S.A.

Abstract : Two forms of pituitary adenylate cyclase-acti-vating polypeptide (PACAP), the 38- and 27-amino-acidforms (PACAP38 and PACAP27, respectively), whichshare amino acid sequence homology with vasoactiveintestinal peptide (VIP), were evaluated for their abilitiesto regulate sympathetic neuron catecholamine and neu-ropeptide Y (NPY) expression . PACAP38 and PACAP27potently and efficaciously stimulated NPYand catechola-mine secretion in primary cultured superior cervical gan-glion (SCG) neurons; 100- to 1,000-fold higher concen-trations of VIP were required to modulate secretion, sug-gesting that SCG neurons express the PACAP-selectivetype I receptor . PACAP38 elicited a sustained seven- toninefold increase in the rate of NPY secretion and three-fold stimulation in the rate of catecholamine release.PACAP38 and PACAP27 produced parallel neuronal NPYand catecholamine release, but cellular levels of NPY andcatecholamines were differentially regulated. Sympa-thetic neuron NPY content was decreased, whereas cel-lular total catecholamine levels were elevated by thePACAP peptides ; total NPY and catecholamine levels(secreted plus cellular content) were increased. In con-cert with the increased total peptide and transmitter pro-duction, pro-NPY and tyrosine hydroxylase mRNA levelswere elevated . Furthermore, PACAP38 was more effica-cious than PACAP27 in regulating pro-NPY and tyrosinehydroxylase mRNA. SCG neuronal expression of mRNAencoding the type I PACAP receptor further supportedthe studies demonstrating that sympathetic neuronal lev-els of NPY and catecholamine content and secretion andmRNA are differentially regulated by thePACAP peptides .Key Words: Pituitary adenylate cyclase-activating poly-peptide-Vasoactive intestinal peptide-Superior cervi-cal ganglion-Cell culture-Neuropeptide-Tyrosine hy-droxylase.J. Neurochem. 65,978-987 (1995) .

Pituitary adenylate cyclase-activating polypeptides(PACAPs) belong to the vasoactive intestinal peptide(VIP)/secretin/glucagon family of bioactive peptidesand mediate various physiological functions in neuro-endocrine tissues . Isolated originally from ovine hypo-

Victor May and Karen M. Braâs

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thalamic extracts based on ability to stimulate anteriorpituitary adenylyl cyclase activity, PACAPs regulatehormone production and secretion in the pituitarygland, thyroid gland, and pancreas (Miyata et al ., 1989 ;Goth et al ., 1992 ; Chen et al ., 1993 ; Tsuji et al ., 1994 ;Yada et al ., 1994) . These peptides also regulate en-dothelium-independent vasodilation, gastrointestinalfunctions, and neuroendocrine cell calcium flux andneurite outgrowth (Warren et al ., 1991 ; Abssod et al.,1992 ; Canny et al ., 1992 ; Deutsch and Sun, 1992 ;Mungan et al ., 1992 ; Tatsuno et al ., 1992 ; Braas et al .,1994a ; Shioda et al ., 1994) .

There are two structural forms of PACAP that ex-hibit distinct bioactivities : PACAP38 is a 38-amino-acid form of PACAP [pro-PACAP (131-168) ] , andPACAP27 is a 27-amino-acid form, corresponding tothe 27-amino-terminal residues of PACAP38 [pro-PA-CAP (131-157) ] . Both peptides arise from a commonprecursor following alternative posttranslational pro-cessing (Miyata et al ., 1990 ; Ogi et al., 1990) . PA-CAP27 exhibits 68% homology with VIP, and the 10amino-terminal residues of PACAP are 60% homolo-gous to growth hormone-releasing hormone (Miyataet al ., 1989) . Although the physiological activities ofPACAP and VIP are similar in some tissues, PACAPis -1,000-fold more potent than VIP in many neuroen-docrine systems (Miyata et al ., 1989 ; Deutsch and Sun,1992) .

Received October 28, 1994 : final revised manuscript receivedMarch 14, 1995 ; accepted March 20, 1995 .

Address correspondence and reprint requests to Dr . V. May atDepartment of Anatomy and Neurobiology, University of VermontCollege of Medicine, Given Health Science Complex, Burlington,VT 05405, U.S .A .

Abbreviations used: CSFM, complete serum-free medium ; DA,dopamine ; DMEM, Dulbecco's modified Eagle's medium ; DOPAC,3,4,dihydroxyphenylacetic acid; E, epinephrine; HVA, homovanillicacid; NE, norepinephrine (noradrenaline); NPY, neuropeptide Y;PACAP, pituitary adenylate cyclase-activating polypeptide; PA-CAP38 and PACAP27, 38- and 27-amino-acid forms of pituitaryadenylate cyclase-activating polypeptide, respectively ; PRP, pitu-itary adenylate cyclase-activating polypeptide-related peptide; SCG,superior cervical ganglion ; VIP, vasoactive intestinal peptide.

PACAP REGULATES NEURONAL NPY AND CATECHOLAMINE

The diverse activities of PACAP have been postu-lated to be mediated by multiple target tissue receptors(Shivers et al ., 1991 ; Ishihara et al ., 1992 ; Morrow etal ., 1993 ; Spengler et al ., 1993 ; Hamar and Lutz,1994) . The type I PACAP receptor exhibits high affin-ity for both PACAP peptides but binds VIP with 1,000-fold lower affinity . The type II PACAP receptor bindsPACAPs and VIP with apparent equal affinity and maybe the classical VIP receptor (Ishihara et al ., 1992 ;Spengler et al ., 1993) . The cDNA encoding the typeI PACAP receptor has been cloned (Hashimoto et al .,1993 ; Hosoya et al ., 1993 ; Pisegna and Wank, 1993 ;Spengler et al ., 1993 ; Svoboda et al ., 1993) ; alterna-tively spliced variants of the PACAP type I receptordisplay differential patterns of adenylyl cyclase andphospholipase C stimulation, suggesting that the tis-sue-specific expression of different PACAP receptorisoforms may provide a means of modulating cellularsignal transduction (Hosoya et al ., 1993 ; Spengler etal ., 1993 ; Svoboda et al ., 1993) .

In the PNS, the differentiation and secretory profileof mammalian cells of the sympathoadrenal lineagehave been shown to be modulated by VIP . For manycell types, however, the concentrations of VIP neces-sary for these responses are uncharacteristically highfor specific peptide actions (Ip et al ., 1982, 1985;Tischler et al ., 1985 ; Audigier et al ., 1986 ; Houchi etal ., 1987 ; Malhotra et al ., 1988, 1989 ; Pincus et al .,1989, 1990) . Micromolar levels of VIP, for example,are necessary to increase neuroblast mitosis, survival,neurite outgrowth, and cyclic AMP production in supe-rior cervical ganglion (SCG) cultures from embryonicrats (Pincus et al ., 1989, 1990) . High concentrationsof VIP are necessary to stimulate L-tyrosine, tetrahy-drobiopterin:oxygen oxidoreductase (EC 1.14.16.2 ; ty-rosine hydroxylase) activity, cyclic AMP production,and phospholipid breakdown in adult SCG cultures (Ipet al ., 1982, 1985 ; Audigier et al., 1986) ; similarlyhigh levels of VIP are required to augment secondmessenger production, calcium uptake, and catechola-mine secretion and production in primary and neoplas-tic adrenal medullary chromaffin cell cultures (Tischleret al ., 1985 ; Houchi et al ., 1987 ; Malhotra et al ., 1988,1989) . In some cases, VIP may be simulating the activ-ities of other structurally related physiological ligandsat sympathoadrenal cell receptors, and the PACAPsmay be among the regulators of sympathetic and ad-renal function . PACAPs, for instance, enhance sym-pathetic neuroblast survival (DiCicco-Bloom andDeutsch, 1992) and are more potent than VIP in stimu-lating catecholamine secretion from bovine adrenalchromaffin cells (Watanabe et al ., 1991 ; Isobe et al .,1993) . Only PACAP38 and PACAP27 activate ade-nylyl cyclase activity and cyclic AMP production atnanomolar concentrations in PC12 rat pheochromocy-toma cells (Deutsch and Sun, 1992) .SCG neuronal neurotransmitter and neuropeptide

content and release are differentially regulated by acti-

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vation of intracellular signaling pathways (May et al .,1995) . Activators of the protein kinase A pathwayproduce sustained neuropeptide Y (NPY) and cate-cholamine release . By contrast, maximal stimulationdecreases cellular NPY content, whereas total cellularcatecholamine content is increased . Regulation of theprotein kinase C pathway stimulates NPY secretionwithout altering cellular NPY levels, whereas both cat-echolamine release and content are increased . The en-dogenous activators of these intracellular signalingpathways in sympathetic neurons involved in regula-tion of neurotransmitter and neuropeptide expressionhave not been fully established .To evaluate directly the roles of PACAPs on sympa-

thetic neuronal function and to compare the relativeinfluences of PACAP and VIP, we have evaluated theeffects of pro-PACAP-derived peptides on primarycultured rat sympathetic neurons . In particular, we ex-amined PACAP regulation of NPY and catechola-mines, the primary neuropeptide and neurotransmitter,respectively, produced by the principal neurons of theSCG . We have demonstrated that PACAP38 and PA-CAP27, acting through PACAP type I receptors, arepotent and efficacious regulators of SCG neuronalNPY and catecholamine content and secretion andmRNA levels, suggesting that these peptides are physi-ological regulators of sympathetic neuronal function .

MATERIALS AND METHODS

Cell culturePrimary SCG neuron cultures were prepared as previously

described (May et al ., 1995) . In brief, SCG tissues fromneonatal rat from three or four litters, generally 35-50 ani-mals (70-100 ganglia), were dissociated to produce apooled population of cells . The tissues were dissociated inDulbecco's modified Eagle's medium (DMEM)-air (Mayand Eipper, 1986) containing 4 mg/ml of collagenase (CLS2, 171 U/mg; Worthington Biochemical Corp ., Freehold, NJ,U.S.A .), 1 mg/ml of hyaluronidase (1,897 U/mg ; Worthing-ton), 10 /.tg/ml of DNase (type 1, 2,000 Kunitz units/mg ;Sigma Chemical Co., St . Louis, MO, U.S .A .), and 10 mg/ml of bovine serum albumin for 20 min at 37 °C and subse-quently incubated with 3 mg/ml of trypsin (225 U/mg; Wor-thington) for 3 min . The cells were plated at an initial densityof 1 .5 X 104 neurons/cm 2 and cultured in DMEM-Ham's F-12 medium (1 :1 vol/vol) containing 5% rat serum (GIBCO-BRL Life Technologies, Grand Island, NY, U.S.A .), 10%Nu-Serum (Collaborative Research, Bedford, MA, U.S .A .),and 32 ng/ml of 2.5S nerve growth factor (CollaborativeResearch) . At 36 h following plating, the cultures weretreated with 10 jLM cytosine /3-D-arabinofuranoside for 24 hand subsequently maintained in a modified complete serum-free medium [CSFM; 250 u1 of CSFM/1.5 X 104 cells(May and Eipper, 1986)] consisting of DMEM-Ham's F-12 medium (1 :1 vol/vol) containing 10 /Lg/ml of insulin,20 leg/ml of transferrin, 1 mg/ml of bovine serum albumin,10 nM T3 , 32 ng/ml of 2.5S nerve growth factor, and traceelements (May and Eipper, 1986 ; Higgins et al ., 1991 ; Mayet al ., 1995) ; the culture medium was replaced completelyevery 48 h .

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For all studies, replicate wells from a single dissociationwere used for control and treated neurons. Each experimentwas repeated two to five times, and each trial used five to10 individual cultures per data point. PACAP38, PACAP27,VIP, and PACAP-related peptide (PRP) were obtained fromPeninsula Laboratories (Belmont, CA, U.S.A .) and BachemCalifornia (Torrance, CA, U.S.A .) and were prepared as10 ° M stocks in 0.01 M HCl and stored at -85°C.

Peptide level analysisThe levels of neuronal and secreted peptide were evalu-

ated using double-antibody radioimmunoassays in 50 mMsodium phosphate (pH 7 .6) containing 0.025% Triton X-100 at a final assay volume of 200 pl with a 1 :100,000dilution of rabbit anti-NPY JH3 (Braas et al ., 1994b ; Mayet al., 1995) . The samples were incubated for 24 h at 4°C;iz-'I-NPY (Amersham Corp ., Arlington Heights, IL, U.S .A .)was added, and competitive binding proceeded for 24 h at4°C. Bound '2'I-NPY was separated from free by double-antibody immunoprecipitation (May and Eipper, 1986 ;Braas et al ., 1994a,b ; May et al ., 1995) . The cumulativeamount of secreted peptide was calculated as the sum of theNPY immunoreactive material secreted per well for each 24-or 48-h period within the specific treatment period .

Catecholamine level analysisCell culture medium and cells were prepared as previously

described (May et al ., 1995) . Samples were fractionatedusing a SMART microanalytical system (Pharmacia LKBBiotechnology, Piscataway, NJ, U .S.A .) with a Pharmacia5-pm (particle size) reversed-phase Sephasil Cjs SC 2.1/10 column using 75 mM monochloroacetic acid (pH 3.1)containing 0.67 mM Na2EDTA, 0.93 mM octanesulfonicacid, and 0.5% tetrahydrofuran as the mobile phase (May etal ., 1995) . Catecholamine and metabolite levels were quanti-fied by peak area using the peak integration data evaluationprogram of SMART Manager operating software (version1 .41) following amperometric detection at an oxidation po-tential of 0.70 V (50 nA full scale) . Sample amine levelswere determined by comparison with standard curves con-structed from the evaluation of five to eight known concen-trations of each standard . The accumulation of the dopamine(DA) metabolites 3,4-dihydroxyphenylacetic acid (DO-PAC) and homovanillic acid (HVA) in conditioned mediumwas used as an index of catecholamine release (May etal ., 1995) . The cumulative medium metabolite levels werecalculated as the sum of material (DOPAC + HVA) perculture for each 24- or 48-h period within the specific treat-ment period . The level of total cellular catecholamines andmetabolites was calculated as the sum of the norepinephrine(NE; noradrenaline), DA, epinephrine (E), DOPAC, andHVA levels (NE + DA + E + DOPAC + HVA) .

ImmunocytochemistrySympathetic neurons were cultured in CSFM containing

vehicle or 180 nM PACAP38 for 96 h, fixed with 4% para-formaldehyde, and stained for NPY immunoreactivity usingthe avidin-biotin-peroxidase technique as described pre-viously (Braas et al ., 1994b ; May et al ., 1995) . Morphomet-ric analysis was performed as previously described (Braaset al ., 1994b; May et al ., 1995) .

Northern blot analysisNorthern blot analysis was performed as previously de-

scribed (Braas et al., 1994a,b) . In brief, total RNA from

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individual control and treated SCG cultures (16-mm-di-ameter culture wells) was prepared using RNA STAT-60total RNA/mRNA isolation reagent (Tel-Test "B,"Friendswood, TX, U.S.A .)

Total RNA from individual wells was electrophoresed on1 .5% agarose gels containing 2 .2 M formaldehyde, 20 mM3-(N-morpholino)propanesulfonic acid, 5 mM sodium ace-tate, and 1 mM EDTA, pH 7 .0 . The RNA was transferredto Nytran (Schleicher and Schnell, Keene, NH, U.S.A .) bycapillary action in 20X SSC (3 M NaCl and 0.3 M sodiumcitrate, pH 7.0) ; the blots were baked, prehybridized, hybrid-ized, and washed as previously described (Braas et al ., 1989,1994a,b) . NPY cDNA probes (Dickerson et al ., 1987 ; Mayet al ., 1995) were labeled with [a-32p]deoxy-CTP to a spe-cific activity of 350 pCi/pg by random prime synthesis(Amersham) and used (10 6 cpm/ml) in the northern analysis .To assess corresponding changes in catecholamine biosyn-thetic enzyme mRNA expression, blots were rehybridized totyrosine hydroxylase cDNA probes (300 pCi/pg) (Lewiset al ., 1983) . The blots were exposed to a phosphor screenin an imaging plate (Bio-Rad Laboratories, Hercules, CA,U.S.A .) for 4 h at room temperature . The imaging plateswere scanned using a Bio-Rad Phosphorlmager ; quantitativeanalysis was performed using Bio-Rad PhosphorAnalystsoftware . To correct for the actual amount of RNA appliedto each lane, the blots were stripped and hybridized to cDNAprobes (570 pCi/pg) derived from 18S and 28S frog rRNA(3.8 and 4.2 kB Ncol fragments derived from pXlrlOla) .The changes in NPY and tyrosine hydroxylase mRNA ex-pression were normalized to 18S rRNA levels in eachsample .

Reverse transcription-PCRTotal RNAfrom individual SCG neuron cultures was used

to synthesize first-strand cDNA using SuperScript 11 reversetranscriptase and oligo dT primers with the SuperScript Pre-amplification System (GIBCO-BRL Life Technologies) ina final volume of 20 pl . Amplification was performed in a50-Ml reaction volume consisting of 12 .5 mM Tris-HC1 (pH8.3) containing 62.5 mM KCI, 2.5 MM MgC12 , 200 pMdeoxynucleotide triphosphates, 20 pmol of primers, I MI ofcDNA, and 1 .25 U of AmpliTaq DNA polymerase (PerkinElmer, Norwalk, CT, U.S .A .) using the AmpliWax PCRgem-facilitated hot start (Perkin Elmer) . Reverse transcrip-tion-PCR was conducted using primers specific for thePACAP type I receptor [upper, CTTGTACAGAAGCTG-CAGTCCCCAGACATG; lower, CCGGTGCTTGAAGTC-CATAGTGAAGTAACGGTTCACCTT (Spengler et al .,1993)] . PCR was conducted using a Perkin Elmer DNAThermal Cycler model 480 with the following cycle parame-ters (30 cycles) : initial denaturation, 94°C, 5 min; denatur-ation, 94°C, 30 s; annealing, 60 °C, 30 s; extension, 72°C, 45s; final extension, 72°C, 5 min. Twenty percent of the ampli-fied products were resolved on 1.6% agarose gels and visu-alized by UV illumination following ethidium bromidestaining .

Data analysisANOVA was used to determine differences among treat-

ments, and Newman-Keuls test was used in post hoc analy-sis ; analysis was performed using the SigmaStat for Win-dows version 1 .0 statistics software (Jandel Scientific, SanRafael, CA, U.S.A .) . The significance ofchanges in neuronalpeptide or catecholamine levels or secretion was evaluated


FIG . 1 . PACAP38 stimulates sympathetic neuron secretion .SCG neurons were plated at an initial density of 1 .5 x 10' cells/cm' and incubated on day 9 of culture in 250 pl of mediumcontaining vehicle (control ; 0) or 180 nM PACAP38 (" ) . At theindicated times, the conditioned medium was collected for assayof secreted NPY immunoreactivity, and fresh medium containingvehicle or peptide was replaced . The cumulative amount of se-creted NPY was determined by summation of neuropeptide lev-els in the conditioned medium per well for each period withinthe indicated time . Data are mean - SEM values (in pmol) ofNPY immunoreactivity secreted per 10' cells from five cultures(the error bars are within the symbols) .

using a p value of <0.05 . All data are expressed as mean± SEM values . For data points without apparent error bars,the error bars are within the symbols .

RESULTS

PACAPs regulate sympathetic ganglion neuronalNPY secretion and contentWe previously demonstrated that depolarization and

activation of the protein kinase A or protein kinase Cintracellular signaling pathways differentially regulateSCG neuronal NPY and catecholamine secretion andcellular content (May et al ., 1995) . Although severalligand-receptor systems, including VIP, may activatethe sympathetic adenylyl cyclase signaling cascade,many of the regulators remain to be established . Therecent identification and characterization of PACAPssuggested that PACAP27 and PACAP38 may beamong the endogenous ligands interacting at sympa-thoadrenal cells .To determine whether PACAP peptides exert regu-

latory effects on SCG neurons, NPY secretion wasevaluated in control and PACAP38-treated cultures .PACAP38 elicited a sustained sevenfold stimulationof NPY secretion (Fig . l ) . The rate of NPY secretionincreased from a basal value of 2.4 fmol/104 cells1hto 17 fmol/10 4 cells/h with PACAP38 treatment .PACAP38 and PACAP27 elicited similar maximally

stimulated SCG NPY secretion at nanomolar concen-trations (Fig . 2) . Approximately 100-1,000-foldhigher concentrations of VIP were required to stimu-late NPY secretion, suggesting that SCG neuronsexpress type 1 PACAP receptors . The effects of thePACAP were not cell density dependent. Similar stim-ulated NPY secretion (picomoles of NPY immunore-

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activity per neuron) was observed in cultures with ini-tial SCG cell plating densities of 1 .8 x 10'-1 .5 x 10'cells/cmz (data not shown) .PACAP38 elicited differential effects on SCG cul-

ture NPY content and release . PACAP38 produced aconcentration-dependent stimulation of NPY secretion(Fig . 3A) . Neuronal NPY content was unchanged overa wide range of PACAP concentrations ; at concentra-tions of PACAP38 >100 nM NPY content was de-creased (Fig . 3B), suggesting that secretory rates ex-ceeded neuropeptide biosynthetic rates, leading to de-creased intracellular NPY stores . Total sympatheticneuron NPY production in PACAP38-treated cultures(cumulative NPY secreted + neuronal NPY content)was greater than control cultures and suggested thatPACAP38 elevated NPY biosynthesis . Similar in-creased NPY secretion and decreased NPY contentwere observed in SCG neurons in which protein kinaseA was stimulated by cyclic AMP analogues or adenylylcyclase activators (May et al ., 1995) . PACAP27 alsoelicited similar concentration-dependent increasedSCG cellular NPY secretion and decreased NPY con-tent (data not shown) . In contrast, PRP [pro-PACAP(82-110)], a potentially processed peptidefrom the PACAP precursor molecule, had no effectson either NPY release or culture NPY content over thesame concentration range as PACAP38 and PACAP27 .To assess whether the increased NPY production

represented changes in the population of SCG neuronsexpressing NPY immunoreactivity, cells were culturedin the presence and absence of 180 nM PACAP38 for96 h and immunocytochemically stained for NPY . Incontrol cultures, 57% of the neurons expressed NPYimmunoreactivity (data not shown), which was com-parable to the population of NPY SCG neurons in bothintact adult SCG and cultured SCG neurons (Jarvi etal ., 1988 ; Marek and Mains, 1989 ; May et al ., 1995) .

FIG . 2 . PACAP38 and PACAP27 elicit potent and efficaciousstimulation of SCG neuronal NPY secretion . Primary culturedSCG neurons were incubated in 250 pi of CSFM/1 .5 x 10' cellscontaining 10 -"-10 - ' M PACAP38 ( " ), PACAP27 (0), or VIP(A) for two 48-h periods beginning on day 9 of culture . Theconditioned culture medium from the final 48-h period was ana-lyzed for secreted NPY . Data are mean ± SEM (bars) values (inpmol) of NPY immunoreactivity per 10' cells (n = 5-6 culturesfor each peptide concentration) .

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The same number of neurons was stained for NPY incultures treated with PACAP38 . Furthermore, becausePACAP38 did not alter the number of total or immuno-reactive neurons, the increase in NPY production mostlikely reflected a stimulation of NPY expression in afixed population of NPY neurons .

Because the PACAPs stimulate neuritogenesis, pro-mote neurite development in rat PC 12 pheochromocy-toma cells, and increase neuroblast survival (Deutschand Sun, 1992; DiCicco-Bloom and Deutsch, 1992),the ability of PACAP38 and PACAP27 to supplantnerve growth factor in SCG cultures was evaluated .Sympathetic neurons were cultured in CSFM in theabsence of nerve growth factor containing 0.1 nM-1 MM PACAP38 or PACAP27 . Neither peptide wascapable of replacing nerve growth factor in promotingneuronal survival in the neonatal SCG cultures (datanot shown) . The peptides also had no apparent effectson neuronal cell density or neurite outgrowth in cellscultured in CSFM in the presence of 32 ng/ml of nervegrowth factor .

PACAP stimulates sympathetic neuroncatecholamine release and contentWe have previously shown that the DA metabolites

DOPAC and HVA accumulated linearly with time inconditioned culture medium and could be used as anindex of SCG neuron catecholamine release (May etal ., 1995) . As with NPY secretion, PACAP38 eliciteda concentration-dependent increase in catecholaminerelease (Fig . 3C) . Total medium catecholamine metab-olite levels (DOPAC + HVA) were maximally stim-ulated approximately threefold . In contrast to thedecreased cellular NPY levels, PACAP produced aconcentration-dependent increase in cellular catechol-amine levels (Fig . 3D) . Total cellular catecholamineand catecholamine metabolite levels (NE + DA + E

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FIG . 3 . PACAP38 differentially regulatesSCG neuronal NPY and catecholamine con-tent and secretion . Sympathetic neuronsplated at an initial density of 1 .5 x 10 4 cells/cm 2 were incubated on day 9 of culture in250 pl of medium containing vehicle (0) or10 10-10 -6 M PACAP38 for 96 h . The condi-tioned medium (A and C) from the final 48 hand cell extracts (B and D) were assayed forboth NPY immunoreactivity (A and B) andtotal catecholamines/metabolites (C and D) .Data are mean i- SEM (bars) values (in pmol)of secreted NPY immunoreactivity (A) or (innmol) of total DOPAC plus HVA (C) per 10"cells or the cellular content (in pmol) of NPY(B) or total catecholamine (D) per 10" cells(n = 7-10 cultures for each treatment) .

+ DOPAC + HVA) were increased threefold withconcentrations of PACAP38 of >10 nM. In addition,the PACAP peptides appeared to be more potent ateliciting catecholamine than NPY release .

PACAP38 stimulates sustained SCG neuron NPYand catecholamine secretionTo determine whether PACAP38 stimulation of

sympathetic neuronal NPY and catecholamine secre-tion was sustained, cultures were incubated in the pres-ence or absence of 100 nM PACAP38 beginning onday 3 of culture . Medium containing fresh PACAP38or vehicle was replaced each 48 h, and the levels ofreleased NPY or catecholamines were measured inthe conditioned medium . The stimulatory effects ofPACAP38 on SCG NPY and catecholamine releasewere observed within the first 48-h period and weremaintained throughout chronic stimulation (Fig . 4) .PACAP38 elicited a sustained ninefold increase inNPY secretion ; neuronal NPY release was increasedfrom a basal rate of 2 .7 fmol/104 cells/h to a stimu-lated rate of 25 fmol/104 cells/h with PACAP treat-ment (Fig . 4A) . The stimulated release of catechola-mines was also sustained, and the rate of DOPAC andHVA accumulation increased from a basal value of 18pmol/10 4 cells/h to 59 pmol/10 4 cells/h with chronicPACAP38 treatment (Fig . 4B) . The efficacy ofPACAP38 to stimulate NPY and catecholamine releasewas lower in the less mature (3- and 5-day) neuronalcultures .

Sympathetic neuron NPY and catecholaminecontent and secretion are differentially regulatedby PACAP peptides and VIPTo compare directly the regulation of NPY and cate-

cholamine release from SCG neurons, cultures weretreated with vehicle (control) or 100 nM PACAP38,


FIG . 4. PACAP38 elicits sustained stimulated NPY and cate-cholamine secretion . SCG neurons were incubated in CSFMcontaining vehicle (9) or 100 nM PACAP38 (" ) beginning onday 3 of culture . Each 48 h conditioned medium was collectedfor assay of secreted NPY (A) and catecholamines (B) . Thecumulative NPY or catecholamine metabolite levels representthe summation of the peptide or transmitter levels in the condi-tioned medium per well for each 48-h period within the indicatedtime . Data are mean ± SEM values (in pmol) of secreted NPYimmunoreactivity (A) or (in nmol) of total metabolites (B) per10° cells from five or six cultures (the error bars are within thesymbols) .

PACAP27, or VIP . The conditioned medium from in-dividual cultures was assayed for both NPY and cate-cholamines . Treatment with either PACAP38 orPACAP27 elicited parallel neuronal NPY and cate-cholamine release (Fig . 5A and C) . Activation of theprotein kinase A second messenger pathway producedsimilar increases in NPY immunoreactivity and DAmetabolite levels in SCG neuron conditioned medium(May et al ., 1995) . In contrast, VIP increased NPYsecretion to 260% of control values, but catecholaminemetabolite levels were unchanged (Fig . 5A and C) .The three peptides had very different effects on cel-

lular NPY and catecholamine levels . Sympathetic neu-ronal NPY content was decreased to -45% of controllevels by PACAP38 and PACAP27 ; VIP decreasedcellular NPY content to -80% of control levels (Fig .5B) . In contrast, treatment with either PACAP38 orPACAP27 resulted in increased neuronal catechola-mine content (Fig. 5D) . PACAP38 and PACAP27 in-creased total cellular catecholamine and metabolite(NE + DA + E + DOPAC + HVA) levels to 240and 220% of control values, respectively, whereas VIPincreased neuronal total catecholamine/metabolites to130% of control values . Furthermore, PACAP38,PACAP27, and VIP differentially regulated the levelsof individual catecholamines and metabolites (Fig . 6) .

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FIG . 5 . PACAPs differentially regulate sympathetic neuron NPYand catecholamine content and secretion . Dissociated SCG neu-rons were cultured in CSFM containing vehicle (control ; CTL) or100 nM PACAP38 (P38), PACAP27 (P27), or VIP for 96 h begin-ning on day 9 of culture . Conditioned medium (A and C) andcell extracts (B and D) were assayed for NPY (A and B) andtotal catecholamine/metabolites (C and D) . Data are meanSEM (bars) values (in pmol) of secreted NPY immunoreactivity

(A) or (in nmol) of total DOPAC plus HVA (C) per 10 ° cells orthe cellular content (in pmol) of NPY (B) or total catecholamine(D) per 10 ° cells (n = 6 cultures for each treatment) . ANOVAdemonstrated a significant effect of treatment on NPY immuno-reactivity and total catecholamine/metabolite levels (p

0.0001) : `significantly different by ANOVA and Newman-Keuls test (p < 0.05) compared with CTL and VIP ; "significantlydifferent from CTL, P27, and P38 (p --_ 0 .05) .

The changes in cellular NE levels produced by thethree peptides were modest, but the levels of DA wereincreased to >250% of control values by PACAP38and PACAP27 . This may have reflected increased DA

FIG . 6. PACAPs differentially regulate sympathetic neuron cate-cholamine and metabolite levels . The cell extracts of the sympa-thetic neuronal cultures treated with vehicle (control) or 100 nMPACAP38, PACAP27, or VIP, as described in Fig . 5, were as-sayed for catecholamine/metabolite levels . Data are mean" SEM (bars) values (in pmol) of catecholamine or metaboliteper 10 ° cells (n = 5 for each treatment) . One-way ANOVA dem-onstrated a significant effect of treatment on catecholamine/metabolite levels (p < 0.0001) : `significantly different from con-trol by ANOVA and Newman-Keuls test (p < 0.05) .

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FIG. 7. PACAP38 and PACAP27 increase sympathetic neuronpro-NPY and tyrosine hydroxylase (TH) mRNA expression . SCGneurons were plated at an initial density of 1 .5 x 10° cells/cm' into 16-mm-diameter wells and were incubated with vehicle(control ; CTL) or 100 nM PACAP38 (P38), PACAP27 (P27), orVIP for 96 h. Total RNA extracts were evaluated for pro-NPY (A)and TH (B) mRNA expression by northern blot analysis ; autora-diograms were generated by storage phosphor imaging. Dataare relative mean - SEM (bars) mRNA levels normalized to rRNA(n = 3 for each treatment) . ANOVA demonstrated a significanteffect of treatment on mRNA expression (p < 0.0001) : `signifi-cantly different from CTL, P27, and VIP by ANOVA and New-man-Keuls test (p < 0.05) ; ""significantly different from CTL,P38, and VIP (p < 0.05) .

biosynthesis in neurons cultured in the absence ofascorbate, a cofactor for the catecholamine biosyn-thetic enzyme 3,4-dihydroxyphenethylamine, ascor-bate:oxygen oxidoreductase (EC1 .14.17.1 ; DA /ß-hy-droxylase) . E and DOPAC were increased >500% byPACAPs. In comparison, VIP stimulated catechol-amine and metabolite levels to <160% of controlvalues .

PACAP38 and PACAP27 increase pro-NPY andtyrosine hydroxylase mRNA expressionTo determine whether changes in SCG NPY and

catecholamine secretion and content elicited byPACAPs were reflected in the biosynthetic capabilityof the neurons, expression of pro-NPY rnRNA and thecatecholamine biosynthetic enzyme tyrosine hydroxy-lase mRNA was evaluated. PACAP38 and PACAP27increased levels of both neuronal pro-NPY and tyro-sine hydroxylase mRNA (Fig . 7) . Pro-NPY mRNAcontent was increased to 140% of control values byPACAP38 but only to 120% of control values byPACAP27 (Fig . 7A) . Differential increases in tyrosinehydroxylase mRNA expression by the two peptideswere also observed (Fig . 7B) . PACAP38 increasedtyrosine hydroxylase mRNA expression to 180% ofcontrol levels, whereas PACAP27 increased expres-

1. Neurorhem ., Vol. 65, No. 3, 1995


sion to 150% of control levels . The mechanisms under-lying the differential increases in mRNA expressionelicited by PACAP38 and PACAP27 may be reflectedin the differential stimulation ol'intracellular signalingpathways by the two peptide~ . VIP, at the same concen-tration as PACAPs, did not alter either pro-NPY ortyrosine hydroxylase niRNA expression .

SCG neurons express mRNA encoding PACAPtype I receptorsThe potent effects ol'PACAP38 and PACAP27 com-

pared with VIP on SCG NPY and catecholamine re-lease, content, and rnRNA suggested that these sympa-thetic neurons express PACAP type 1 receptors. Splicevariants of the PACAP type I receptor are divergentfrom each other by the absence or presence of eitherone or two 84-bp cassettes [ HIP and HOP cassettes(Spengler et al ., 1993 ) ] . To substantiate further thephysiological importance of` PACAP actions on SCG,the expression of niRNA encoding PACAP type 1 re-ceptor isoforms was examined using reverse transcrip-tion-PCR . Three products of different sizes were ob-tained following amplification of cDNA transcribedfrom total RNA ol'cultured sympathetic neurons usingprimers to differentiate the receptor splice variants(Fig . 8) . The predominant 387-bp product representedexpression of the PACAP type I receptor mRNA con-taining one cassette . Minor 303- and 487-bp productscorresponded to the expression of PACAP receptormRNA without either the HIP or HOP cassette andwith the isoforrn with both cassettes, respectively .

DISCUSSION

Many studies have shown that VIP exerts importantphysiological effects on sytnpathoadrenal cell function .In primary bovine adrenal medullary cells and PC12pheochromocytotna cells, VIP stimulated neuritogen-esis and increased tyrosine hydroxylase phosphoryla-tion, thereby augmenting cellular tyrosine hydroxylase

FIG. 8. Cultured SCG neurons express mRNA encoding type IPACAP receptors (PACAP-R). Total RNA from individual sympa-thetic neuronal cultures (16-mm-diameter wells) was reverse-transcribed, and the cDNA was amplified using primers flankingthe insertion site of the HIP and HOP cassettes of the type IPACAP-R. The amplified products and a 100-bp DNA ladderwere resolved on 1 .6% agarose gels, stained with ethidium bro-mide, and visualized by UV illumination . The predicted sizes ofproducts containing neither (303 bp), one (387 bp), and both(471 bp) cassettes are indicated.


activity and catecholamine production (Tischler et al .,1985 ; Houchi et al ., 1987 ; Malhotra et al ., 1988, 1989 ;Zigmond et al ., 1989) . VIP mediated many of theseeffects by stimulating cyclic AMP production and acti-vating cyclic AMP-dependent processes, although VIPalso appeared to induce chromaffin catecholamine syn-thesis and secretion by activation of the phosphatidyl-inositol signaling cascade (Tischler et al ., 1985 ; Hou-chi et al ., 1987 ; Malhotra et al ., 1988, 1989) . Similarresults were also obtained in studies examining theregulatory roles of VIP on sympathetic neuronal func-tion . VIP enhanced embryonic neuroblast mitosis andsurvival, increased sympathetic neuron cyclic AMPcontent, increased SCG phospholipid breakdown, andactivated SCG tyrosine hydroxylase activity (Ip et al .,1982, 1985 ; Audigier et al ., 1986 ; Pincus et al ., 1989,1990 ; Zigmond et al ., 1989) . These results implicatedthe VIP/secretin/glucagon family of peptides as non-cholinergic preganglionic neurotransmitters involvedin the transsynaptic regulation of SCG function .The studies presented here directly compared

PACAP38, PACAP27, and VIP regulation of SCGneuropeptide and neurotransmitter expression using aprimary neuronal culture system . Consistent with thepotent effects of PACAP38 and PACAP27 in stimulat-ing PC12 adenylyl cyclase activity and cyclic AMPproduction (Deutsch and Sun, 1992), both peptidesdemonstrated high potency and efficacy in stimulatingSCG neuronal NPY and catecholamine secretion com-pared with VIP . Half-maximal NPY neurosecretory ef-fects were elicited at < 10 nM PACAP, and maximalresponses were observed at 100 nM peptide ; PACAPexhibited slightly higher potency in eliciting SCG cate-cholamine secretion . These results suggested that SCGneurons exhibited high-affinity type I receptors forPACAP rather than type II PACAP receptors, whichdemonstrate equal affinity for PACAP and VIP(Gottschall et al ., 1990 ; Spengler et al ., 1993 ; Hamarand Lutz, 1994) . Both PACAP38 and PACAP27 failedto produce significant changes in culture NPY contentover a broad range of peptide concentrations, sug-gesting that neuronal NPY biosynthesis may be tightlycoupled to neurosecretory rates . Only high concentra-tions of PACAPs modulated cellular neuropeptide lev-els, diminishing SCG NPY content and suggesting thatthe neuropeptide secretory rates may have exceededcellular production rates . These results are consistentwith our previous studies examining the regulatoryroles of cyclic AMP-mediated activation of SCG NPYproduction (May et al ., 1995) . These results differedfrom our studies with AtT-20/D16v cells, in whichPACAPs and VIP augmented both proopiomelanocor-tin hormone secretion and cellular content (Brags etal ., 1994a) .

In contrast to cellular NPY levels, PACAPs stimu-lated SCG catecholamine content . Cellular catechola-mine and catecholamine metabolite levels were aug-mented two- to fivefold, indicating an increase in cate-

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cholamine biosynthesis, turnover, and metabolism inresponse to PACAP. These increases were comparableto the protein kinase A-mediated elevation of SCGneuronal catecholamine expression (May et al ., 1995) .The differential PACAP regulation of cellular NPYand catecholamine levels is not unexpected becauseneuronal biosynthesis of neuropeptides and neurotrans-mitters is fundamentally different ; neuropeptide pro-duction requires de novo synthesis and transport tonerve terminals, whereas neurotransmitter levels relyon not only biosynthesis in nerve terminals but alsoneurotransmitter uptake and metabolism . Each step ofthe two pathways is subject to regulation and mayprovide the regulatory mechanisms that produce theobserved differences in SCG NPY and catecholaminecontent . Furthermore, coexisting neurotransmitters andneuropeptides are compartmentalized into two separatevesicle populations : Neurotransmitter-containing syn-aptic vesicles make up --95% of the population,whereas neuropeptide-containing secretory granulesrepresent only 5% of the vesicle population (Thureson-Klein, 1983) . In the splenie nerve, higher-frequencystimulation is required for release of NPY than cate-cholamines (Lundberg et al ., 1986), which may relateto the differences in PACAP potency in eliciting NPYand catecholamine release . The differences in the magnitudes of the PACAP-induced NPY and catechola-mine release may correspond to the higher basal trans-mitter versus peptide secretory rates (May et al .,1995), which may also reflect the separate compart-mentalization, vesicle population sizes, and biosyn-thetic pathways.The total increase in SCG NPY and catecholamine

production (content plus cumulative secreted) byPACAP38 and PACAP27 paralleled the induction ofpro-NPY and tyrosine hydroxylase mRNA expression .The magnitude of the changes in pro-NPY and tyrosinehydroxylase niRNA expression, however, was smallerthan those of the changes in either content or secretion .It is interesting that PACAP38 was more efficaciousthan PACAP27 in stimulating mRNA levels . This mayreflect differences between the coupling of the twopeptides to intracellular signaling pathways : PACAP38and PACAP27 both stimulated adenylyl cyclase,whereas only PACAP38 potently stimulated phospho-lipase C in PC] 2 cells and LLC PK1 cells transfectedwith cDNA encoding isoforms of the type I PACAPreceptor (Deutsch and Sun, 1992 ; Spengler et al .,1993) . VIP stimulated SCG neuronal cyclic AMP pro-duction and phospholipid turnover but was not verypotent (Ip et al ., 1985 ; Audigier et al ., 1986) .

The PACAP receptors belong to the seven-trans-membrane domain family of G protein-coupled recep-tors ; the type I PACAP receptor exhibits high affinityfor both PACAP38 and PACAP27 but not VIP,whereas the type 11 receptor exhibits equally high af-finity for PACAP38, PACAP27, and VIP (Spengler etal ., 1993 ; Harnar and Lutz, 1994) . The type II receptor

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is thought to represent the prototypic VIP receptor .The type I PACAP receptor has multiple isoforms,depending on the alternative splicing of two 84-bpcassettes in the PACAP receptor gene (Hosoya et al .,1993 ; Spengler et al ., 1993 ; Svoboda et al ., 1993) .The cassettes, HIP and HOP, encode potential aminoacid additions in the third cytoplasmic loop of thePACAP receptor, which represents one of the primaryinteractive sites with G proteins . Using primers to thenucleotide sequence of the rat PACAP receptor andreverse transcription-PCR, we have confirmed the ex-pression of the type I PACAP receptor in SCG neurons .The predominant mRNA expressed by cultured SCGneurons contained one cassette, whereas minor levelsof other splice variant receptor mRNAs were also pres-ent. The specific isoforms of the PACAP type I recep-tor expressed by SCG neurons and the coupling tointracellular signaling pathways are currently under in-vestigation . Cultured sympathetic neuron expressionof VIP receptor subtypes and the population of SCGneurons responding to VIP have not been fully investi-gated, although VIP immunoreactivity has been identi-fied in some preganglionic neurons innervating theSCG (Baldwin et al ., 1991) .We have identified PACAP38 and PACAP27 as po-

tential endogenous regulators of SCG neuronal peptideand transmitter expression . Both bioactive peptides dif-ferentially regulated sympathetic neuronal NPY andcatecholamine content and secretion . PACAP38 ap-peared more efficacious than PACAP27 in regulatingpro-NPY and tyrosine hydroxylase mRNA. VIP, onthe other hand, was 100-1,000-fold less potent thaneither PACAP38 or PACAP27 in regulating neuronalpeptide and transmitter expression . Moreover, culturedsympathetic neurons expressed mRNAs encoding iso-forms of the type I PACAP receptor . Out - results coin-cide with studies of primary and neoplastic chromaffincells and suggest that the two peptidergic forms ofPACAP represent physiological regulators of sympa-thoadrenal cell development and function .

Acknowledgment : The authors are deeply indebted to Dr .Richard Mains for the JH3 NPY antiserum and NPY plas-mid . We are very grateful to Drs . Dona Chikaraishi andBarbara Sollner-Webb for the tyrosine hydroxylase andrRNA plasmids, respectively . We also thank Cynthia Bran-denburg for technical assistance and critical reading of themanuscript and Susan Harakall for technical help with themRNA analysis . The numerous helpful discussions with Drs .Robert Hamill and Carson Cornbrooks during these studiesare greatly appreciated . This work was supported by grantsHD-27468 and NS-01636 (to V .M .) from the National Insti-tutes of Health and grant DIR-9116229 (to V .M. andK.M.B .) from the National Science Foundation .

Abssod A ., Chen D ., Wang Z . Y ., and Hakanson R . ( 1992) Vasculareffects of pituitary adenylate cyclise activating polypeptide : a

J. Neurochenx ., Vo/. 65, No. 3, 1995

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Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulation of Sympathetic Neuron Neuropeptide Y and Catecholamine Expression