Journal of Neurot hemi.su'yRaven Press, Ltd., New York

') 1994 International Society for Neurochemistry

a2-Adrenoceptor-Mediated Inhibition of Electrically Evoked[3H]Noradrenaline Release from Chick Sympathetic

Neurons : Role of Cyclic AMP

Stefan Boehm, Sigismund Huck, Gabriele Koth, *Helmut Drobny,*Ernst Agneter, and *Ernst A. Singer

Departments of Neuropharmacology and *Pharmacology, University q/ Vienna, Vienna, Austria

Abstract: This study explores the role of cyclic AMP inelectrically evoked [H]noradrenaline release and in thea2-adrenergic modulation of this release in chick sympa-thetic neurons. Along with an increase in stimulation-evoked tritium overflow, applications of forskolin en-hanced the formation of intracellular cyclic AMP. Botheffects of forskolin were potentiated by the phosphodies-terase inhibitor 3-isobutyl-1-methylxanthine . The for-skolin-induced increase in overflow was abolished by theRp-diastereomer of cyclic AMP-thioate, an antagonist atcyclic AMP-dependent protein kinases, and 1,9-dideoxy-forskolin, an inactive analogue at adenylyl cyclase, hadno effect on the evoked overflow . A 24-h pretreatmentwith either cholera toxin or forskolin reduced the subse-quent forskolin-induced accumulation of cyclic AMP andinhibited the stimulation-evoked release. Basal cyclicAMP production, however, remained unaltered after for-skolin treatment and was enhanced after 24 h of choleratoxin exposure . The a2-adrenergic agonist bromoxidinedid not affect the formation of cyclic AMP stimulated byforskolin but reduced electrically evoked release. How-ever, effects of bromoxidine on 3H overflow were attenu-ated by forskolin as well as by 8-bromo-cyclic AMP. Ef-fects of bromoxidine on [H]noradrenaline release wereparalleled by an inhibition of voltage-activated Ca2+ cur-rents, primarily through a delayed time course of currentactivation . This effect was abolished when either forskolinor 8-bromo-cyclic AMP was included in the pipette solu-tion . Both substances, however, failed to affect Ca2+ cur-rents in the absence of bromoxidine. These results sug-gest that the signaling cascade of thea2-adrenergic inhi-bition of noradrenaline release involves voltage-activatedCat ' channels but not cyclic AMP. Elevated levels of cy-clic AMP, however, antagonize this a2-adrenergic reduc-tion, apparently through a disinhibition of Ca21 channels .Key Words: Cyclic AMP-a2-Adrenoceptors-Nor-adrenaline release-Ca2+ currents-Chick sympatheticneurons.J. Neurochern . 63, 146-154 (1994) .

Protein phosphorylation plays a crucial role in thephysiological regulation of neurotransmitter release

146

(for review, see Walaas and Greengard, 1991) . Thesephosphorylations are often catalyzed by cyclic AMP-dependent protein kinases . Treatments that lead to anincrease of intracellular cyclic AMP have consistentlybeen reported to enhance the release of noradrenaline(NA) from central and peripheral neuronal sources(Werner et al ., 1982 ; Schoffelmeer et al ., 1985 ; Onget al ., 1991 ; Schwartz and Malik, 1993) . Activation ofpresynaptic a,-adrenergic receptors, on the other hand,reduces stimulation-evoked NA release from noradren-ergic neurons (for review, see Starke, 1987) . Since thedemonstration that a-adrenoceptors in human plateletsinhibit the activity of adenylyl cyclase (Jakobs et al .,1976), it has been thought that az-adrenergic effectsin neurons could also be mediated through regulationof this enzyme . However, studies in this area yieldedconflicting results . a2-Adrenergic agents were shownto either reduce (Schwartz and Malik, 1993), enhance(Wakade et al ., 1988), or have no effect (Ong et al .,1991) on the formation of cyclic AMP. On the basisof indirect evidence without cyclic AMP measurement,it was concluded that a2-adrenoceptor activation con-strains NA release by an inhibition of presynaptic ade-nylyl cyclase (Werner et al ., 1982 ; Schoffelmeer et al .,1986) . However, evidence to the contrary has also beenpresented supporting the idea that the a_-adrenergicmodulation of NA release is not mediated throughmodulation of cyclic AMP (Ong et al ., 1991 ; Schwartzand Malik, 1993) .

Besides being linked to adenylyl cyclase, a-adren-ergic receptors use other cellular effector systems, such

Received September 17, 1993 ; revised manuscript received No-vember 10, 1993 ; accepted November 15, 1993 .

Address correspondence and reprint requests to Dr . S. Boehm atDepartment of Neuropharmacology, University of Vienna, Waeh-ringerstrasse 13a, A-1090 Vienna, Austria.

Abbrevations used : 8Br-CAMP, 8-bromo-cyclic AMP; Rp-cAMPSand Sp-cAMPS, Rp- and Sp-diastereomers of adenosine-3',5'-cyclicmonophosphothioate ; G protein, GTP-binding protein ; GTP-y-S,guanosine-5'-O-(3-thiotriphosphate) ; IBMX, 3-isobutyl-I-methyl-xanthine ; NA, noradrenaline .

as voltage-dependent Cat+ and K+ channels (for re-view, see Brown and Birnbaumer, 1990). Thus, thea-adrenergic modulation of NA release may well bemediated by mechanisms other than the alteration ofintracellular cyclic AMP, as has been pointed out pre-viously by several authors (e.g ., Kato et al ., 1985 ;Bucher et al ., 1990).

In chick sympathetic neurons, we found a,-adrener-gic agents inhibited transmembrane Ca" currents(Boehm and Huck, 1991) and the release of [3HINAelicited by electrical field stimulation (Boehm et al .,1991). Both effects were pertussis toxin sensitive(Boehm et al ., 1992), indicating that the signaling cas-cade includes G;- or G�-type GTP-binding proteins (Gproteins) (for review, see Birnbaumer, 1990). How-ever, adding to the complexity, one G protein mayregulate more than one effector system and thus, inaddition to its negative coupling to adenylyl cyclase,G;, as G,� was found to be linked to Cat ` and K+channels (Birnbaumer, 1990).The function of voltage-gated Ca2+ channels criti-

cally depends on phosphorylation/dephosphorylationreactions that are catalyzed by cyclic AMP-dependentprotein kinases, among other enzymes (see Walaas andGreengard, 1991). Hence, the a2-adrenergic inhibitionof NA release may involve one or more of the follow-ing intracellular signaling pathways : first, a decrease incyclic AMP, which in turn might reduce Ca" currents ;second, a direct blockade of the Ca-+ channel by acti-vated G protein (by reducing the Ca-+ influx, bothmechanisms would decrease the levels of cytoplasmicCat+ essential for transmitter release) ; third, dimin-ished activity of phosphoproteins involved in presyn-aptic function due to lowered levels of cyclic AMP.

The present study investigates the role of cyclicAMP in the release of NA and its modulation via a2-adrenoceptors in chick sympathetic neurons by a three-fold approach, as follows: determination of cyclicAMP formation in cultures preloaded with [3Hj-adenosine (according to Nees et al ., 1985), measure-ment of electrically evoked [3HINA release from su-perfused cultures (according to Boehm et al ., 1991),and recording of voltage-activated Ca2+ currents usingthe whole-cell variant of the patch-clamp technique(according to Hamill et al ., 1981). Our experimentsindicate that although the cyclic AMP system effec-tively antagonizes the a2-adrenoceptor-mediated re-duction of NA release and Ca2+ currents, a2-adrenergicagents do not impair NA release via inhibition of ade-nylyl cyclase. We also conclude that the relief of aZ-adrenoceptor-mediated inhibition by cyclic AMP doesnot adequately explain the enhancement of NA release,which can be observed after treatments that raise thelevels of intracellular cyclic AMP.

MATERIALS AND METHODS

Cell culturesThe procedures of dissociating and culturing sympathetic

neurons have been described in detail previously (Boehm et

CYCLIC AMP AND NORADRENALINE RELEASE 14 7

al ., 1991). In brief, paravertebral sympathetic ganglia weredissected from 12-day-old chick embryos, trypsinized (0, I %for 30 min at 36°C), subsequently triturated, and finally re-suspended in Dulbecco's modified Eagle's medium (G1BC0no . 041-01885M) containing 2,2 g/L glucose, 10 mg/L insu-lin, 25,000 IU/L penicillin, and 25 mg/L streptomycin(GIBCO no . 043-05140D), 100 mg/L gentamicin (Seromedno . A2712), 5% fetal calf serum (GIBCO no . 011-0620H),and 10 Ng/L nerve growth factor (GIBCO no . 0436050) .Cells were plated on poly-D-lysin-coated (Sigma no . 1149)tissue culture dishes (Nunc no . 153066) for electrophysiolo-gical recordings, on collagen-coated (Biomedical Techno-logies no . BT-274) polystyrol discs (diameter 5 mm) forsuperfusion experiments (8 X 10' cells per disc), or on colla-gen-coated 24-multiwell culture plates (Nunc no . 14 .3982)to determine the accumulation of cyclic AMP (2X 10' cells per well) . In cultures meant for electrophysiologi-cal recordings or superfusion experiments, no precautionswere taken to remove nonneuronal cells (-50% at plating,see Boehm et al ., 1991) . We have shown previously thatupon electrical stimulation, fibroblasts do not release ['H]NA(Boehm et al ., 1991). However, nonneural cells were reducedto <5% by differential plating as described elsewhere (Wa-kade et al ., 1988) when measurements of the formation ofcyclic AMP were performed.

Measurement of cyclic AMP accumulationFormation of cyclic AMP was determined essentially as

described by Nees et al . (1985) . In brief, cultures 3 days invitro were incubated for 3 h with ['H]adenosine (60.7 Ci/mmol ; 10 pCi/500 pI of culture volume). The cells werethen washed three times with superfusion medium (see be-low) and incubated for 30 min with 10 -' M 3-isobutyl-l-methylxanthine (IBMX) in 1 ml of superfusion medium .Thereafter, solutions were changed either to a challenge ofvarious concentrations of forskolin for another 30 min in thecontinued presence of 10 ' M IBMX, or alternatively cul-tures were stimulated by electrical pulses (36 or 360 mono-phasic rectangular pulses, 0.5 ms, 40 V/cm, 60 mA, 3 Hz;see also Wakade et al ., 1988), or depolarizing K' concentra-tions (30 or 100 mM KCI, matched by an equimolar reduc-tion of NaCl) added for 6 min. Both stimuli, the electricalpulses and the elevated K', consistently induce the release ofI1 HINA, in multiwell culture dishes as well as in superfusionexperiments (Boehm and Huck, 1993). a-Adrenoceptor ago-nists, when used, were present during the entire preincuba-tion and stimulation periods . All steps were performed at36°C . Supernatants were then removed and cells were lysedin the presence of 400 pI of ice-cold 2% (vol/vol) perchloricacid, to which 20 pl of a standard mixture of carrier (adeno-sine 2 mM, cyclic AMP 1 .25 mM, ATP 1 mM) was finallyadded. The extract was transferred to Eppendorf vials, neu-tralized with 20 P,l of 4 M KZC03 , and centrifuged at 12,000g for 10 min . Supernatants were kept frozen at -80°C untilanalysis .The contents of radioactive cyclic AMP were separated

by HPLC on a C, H reverse-phase column equipped with UVdetection (254 run) . The column was washed for 5 min witha 0.1 M K2HPO, buffer (pH 5.8) and eluted at a flow rateof I ml/min on a linear gradient against 57% (vol/vol) metha-nol for 40 min. Fractions (1 min) were collected and theirradioactive contents were determined by liquid scintillationcounting . The amount of tritium eluting together with theadded standard of cyclic AMP was expressed as a percentageof the total radioactivity retrieved from the column .

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Superfusion experimentsCultures were used for superfusion experiments after 5

days in vitro. Labeling of the cells with [ZH]NA (43.7 Ci/mmol) and superfusion was performed as described pre-viously (Boehm et al ., 1992). After a l-h washout period,4-min fractions were collected, and electrical stimuli (36monophasic rectangular pulses, 3 Hz, 0.5 ms, 50 V/cm, 60mA) were applied at 12 min (S,) and 40 min (S2) (StimulatorT, Hugo Sachs Elektroniks, Hugstetten, F.R.G .) . Unlessstated otherwise, test drugs were added 16 min before SZand kept at constant concentrations until the end of the exper-iment. The superfusion medium contained (mmol/L) NaCl113, KCl 4.8, CaC12 1 .3, MgS04 1 .2, NaHC03 25, KH2PO41 .2, D-glucose 11, fumaric acid 0.5, sodium pyruvate 5.0,ascorbic acid 0.57, EDTA-Na2 0.03, at pH 7.4, after equili-bration with 5% C02 in 02 at 25°C (see Boehm et al ., 1991).At the end of the experiment the residual radioactivity ofcultures was extracted by immersion of discs in 1 .2 ml of2% (vol/vol) perchloric acid and sonication . Radioactivityin extracts and collected fractions was determined by liquidscintillation counting (Packard Tri-Carb 460 CD; countingefficiency 24%) .The fractional rate of tritium outflow per 4 min was esti-

mated by calculating the amount of radioactivity in a 4-minsuperfusate fraction as a percentage of the radioactivity inthe cells at the beginning of the respective collection period(for example, as L,%) . Stimulation-evoked overflow of ra-dioactivity was calculated as the difference between totaloutflow during and after stimulation and the estimated basaloutflow, which was assumed to decline linearly from thesample before stimulation to that 4-8 min after commence-ment of stimulation ; the stimulation-evoked overflow wasset in relation to the total amount of tritium in cultures atthe beginning of stimulation (S,%). Drug effects were evalu-ated by calculating the ratio between the overflow evokedby S= and the overflow evoked by S, (S2/S,).S2/S, ratioswere expressed as a percentage of the corresponding meanS2/S, control ratios (S2/S, , percentage of control) . Effects ofdrugs on the basal outflow of tritium were estimated eitherby calculating the ratio between the fractional outflow duringthe 4-min collection period preceding S2 (L2%) and the 4-min collection period preceding S, (L,% ; L2/L,), or by com-paring L,% values of cultures subjected to different kindsof treatment.

ElectrophysiologyWhole-cell Ca" currents were recorded at room tempera-

ture (20-24°C) from cell bodies of sympathetic neurons after24 h in vitro, as described previously (Boehm and Huck,1991). The internal (pipette) solution contained (mM) N-methyl-D-glucamine 115, tetraethylammonium chloride 20,CaC12 1 .6, Mg-ATP 2, Li-GTP 2, EGTA 10, glucose 10,HEPES 20, adjusted to pH 7.3 with HCI. The external bath-ing solution consisted of (m* choline chloride 120, CaCl z5, glucose 20, HEPES 10, adjusted to pH 7.3 with KOH.To enable complete equilibration of the cytoplasm with theinternal solution of the pipette (Pusch and Neher, 1988),current recordings were started >5 min after the establish-ment of the whole-cell configuration. Ca 21 currents wereelicited by depolarizations from a holding potential of -80to 0 mV at a frequency of 2-3 min- ' . To account for thelime-dependent rundown of Ca21 currents, drug effects wereevaluated by measuring current amplitudes in the presence oftest drugs (B) and by comparing them with control currentsrecorded before (A) and after (washout, C) the application

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S. BOEHM ET AL.

of the drugs (Boehm and Huck, 1991), according to thefollowing equation : 200 x B/(A + C) =percentage ofcontrolcurrent.

StatisticsAll data are given as arithmetic mean ± SEM; n = number

of culture wells in cyclic AMPformation experiments, num-ber of cell culture discs in release experiments, and numberof individual cells in whole-cell recordings . Concentration-response curves were fitted to the experimentally obtaineddata points by using the ALLFIT program (DeLean et al .,1978), which calculates estimates for the IC;,) , the slope, andthe maximal effect as well as appropriate standard errors .Moreover, the program determines significances of differ-ences between single concentration-response curves by si-multaneous fitting with shared parameters and subsequentcalculation of the F statistic on the resulting "extra sum ofsquares" (DeLean et al ., 1978). Significance of differencesbetween single data points not wrapped by curves was evalu-ated by unpaired Student's t test .

MaterialsSubstances were obtained from the following sources:

(-)-[ring-2,5,6-ZH]NA (43 .7 Ci/mmol) and [2,8,5 1_3H]-adenosine (60.7 Ci/mmol) from NEN, Dreieich, F.R.G . For-skolin, 1,9-dideoxyforskolin, 8-bromo-cyclic AMP (8Br-cAMP), cholera toxin, IBMX, (-)-NA hydrochloride fromSigma, Munich, F.R.G . Adenosine, ATP, cyclic AMP, theRp- and Sp-diastereomers of adenosine-3',5'-cyclic mono-phosphothioate (Rp-cAMPS, Sp-cAMPS) from BoehringerMannheim, F.R.G . Bromoxidine (UK 14,304) was kindlydonated by Pfizer, New York (NY, U.S .A .) .

Water-insoluble drugs (IBMX, forskolin, 1,9-dideoxyfor-skolin) were initially dissolved in dimethyl sulfoxide anddiluted to final concentrations with the respective buffers .Appropriate controls ensured that dimethyl sulfoxide [maxi-mal final concentration, 0.1% (vol/vol)] affected neither re-sults from release experiments nor from Ca21 current mea-surements.

RESULTS

Accumulation of cyclic AMPCultures loaded with ['H]adenosine as described in

Materials and Methods accumulated 0.103 ± 0.008%(n = 26) cyclic AMP during a 1-h incubation periodwith 10-3 M of the cyclic nucleotide phosphodiesteraseinhibitor IBMX (Thompson, 1991). These basal valuesof cyclic AMP formation were not significantly af-fected by either electrical field stimulation (36 pulsesat 3 Hz : 0.105 ± 0.046%, n = 5; 360 pulses at 3 Hz :0.079 ± 0.011 %, n = 5) or depolarizing K+ concentra-tions applied for 6 min (30 mMK+: 0.096 ± 0.017%, n= 3 ; 100 mMK+ : 0.068 ± 0 .012%, n = 3) . Moreover, a60-min exposure to 10' M of either bromoxidine(0.103 -!- 0.018%, n = 4) or NA (0.116 ± 0.008%,n = 4), selective and unselective agonists at the a2-adrenergic receptor subtype, did not alter the basalaccumulation of cyclic AMP. On the other hand, a 30-min challenge by forskolin (10-6 to 10-4 M) increasedcyclic AMPformation > 100-fold at maximal forskolinconcentrations (Fig . 1 A) . Likewise, a 30-min treatmentwith cholera toxin (100 ng/ml) significantly increased

FIG . 1 . Formation of cyclic AMP . Ac-cumulation of cyclic AMP due to 30-min stimulations with forskolin pre-sented as a percentage of the totalradioactivity (TA) of the cultures . Neu-rons were preloaded with [3 H]-adenosine for 3 h and supplementaldrugs were added 30 min before andduring the stimulation with forskolin .A : Cyclic AMP accumulation inducedby the indicated concentrations offorskolin in the presence of 10 s MIBMX . n indicated in parentheses . B :Formation of cyclic AMP induced by10 s M forskolin in the presence andabsence of IBMX, in the presence ofthe 1x2 -adrenergic agonists bromoxi-dine (Uk) or NA, and subsequent toa 24-h pretreatment of neurons witheither cholera toxin (ChTX) or for-skolin . With the exception of choleratoxin (ng/ml) applied drug concentra-tions are indicated as log M; n indi-cated in the bars . .p - 0.05, vs . theeffect of forskolin in the presence ofIBMX.

cellular production of cyclic AMP (0.368 ± 0.108%,n = 6, p < (1 .001 vs . control) . Omission of the phos-phodiesterase inhibitor IBMX during the stimulationwith forskolin decreased cyclic AMP accumulation by>50% (Fig. 1B) .The forskolin-induced increase of cyclic AMP was

significantly reduced by a 24-h pretreatment of cultureswith either 100 ng/ml cholera toxin or 10-s Mforskolin(Fig . 1 B) . Basal accumulation of cyclic AMP remainedunaltered after 24 h of treatment with 10-5 M forskolin(0.091 - 0.003, n = 3), whereas 24-h incubations with100 ng/ml cholera toxin significantly elevated basalcyclic AMP formation (0.236 ± 0.034, n = 5, p< 0.001 vs . control) . This finding is in line with aprevious report indicating a long-lasting activation ofadenylyl cyclase after chronic cholera toxin treatment(Chang and Bourne, 1989) . In the presence ofbromoxi-dine or NA (l0 'or 10-4 M, the cyclic AMP produc-tion induced by 10-5 M forskolin was not significantlyaffected (Fig . 113) .[`H]NA release

Inclusion of 10- ' to 10 -4 Mforskolin in the superfu-sion medium caused concentration-dependent in-creases of stimulated ['HINA release (Fig . 2A) . 1,9-Dideoxyforskolin, a forskolin analogue without effecton adenylyl cyclase (for review, see Laurenza et al .,1989), failed to enhance the stimulation-induced tri-tium overflow (Fig . 2A) . The effects of forskolin weresignificantly augmented by IBMX (Fig . 2B) but abol-ished by Rp-cAMPS, an antagonist of intracellular cy-clic AMP (Botelho et al ., 1988 ; Fig . 2B) . IBMX alonedid not significantly affect the stimulated tritium over-flow (e.g ., 10 3 M: 95.4 ± 1 .5% of control ; n = 4) .

Unlike forskolin, the cyclic AMP analogue 8Br-

CYCLIC AMP AND NORADRENALINE RELEASE 149

cAMP did not alter electrically evoked 3H overflow(judged by S2/S, values, Fig. 2A) when added 16 minbefore S2 . Likewise, inclusion of 10-4 M 8Br-cAMPin the superfusion medium 20 min before starting thesample collection (i .e ., 32 min before S,) did not affectS, % values (Table 1) . No significant effect on the elec-trically induced overflow was seen in the presence ofthe Sp- or Rp-diastereomers of cyclic AMP-thioate (Sp-cAMPS, 30 ACM: 95 .3 - 7 .5% of control, n = 6 ; Rp-cAMPS, 30 pM: 97.2 ± 2 .4% of control, n = 7), whichare agonists and antagonists at cyclic AMP-dependentprotein kinases, respectively (Botelho et al ., 1988) .

Basal tritium efflux was enhanced in a concentra-tion-dependent manner after addition of forskolin (16min before S2, Table 2) and in the presence of highconcentrations of 8Br-cAMP (10 -4 M, added 32 minbefore S, ; Table 1), IBMX, or Rp-cAMPS (added 16min before S2 ; Table 2) .

Pretreatment of cultures with either 100 ng/ml chol-era toxin or 10-5 M forskolin for 24 h markedly re-duced basal and electrically evoked 'H release (Table1) . In line with our previous observation (Boehm etal ., 1991), the a2-adrenoceptor agonist bromoxidineinhibited the electrically induced 'H overflow in a con-centration-dependent manner with an IC50 of 0.02

0.007 pM and a maximal effect of 53.9 ± 2.8% ofcontrol (Fig . 2C) . When 10-4 M8Br-cAMP was addedto the medium 32 min before S, , it significantly re-duced the apparent maximal effect of bromoxidine(64.8 + 16.8% of control) and shifted its IC 5() valueto 0.26 ± 0.7 pM (p ~ 0.001 vs . control ; Fig . 2C) .Likewise, effects of bromoxidine were attenuated andthe concentration-response curve was modified whenapplied in combination with 10-5 M forskolin 16 min

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FIG . 2. [3H]NA release. Effect of cx 2 -adrenoceptor activation and of sub-stances acting on the cyclic AMPsystem on the electrically induced[3H]NA release from superfused cul-tures of chick sympathetic neurons.After preloading with [3H]NA and sub-sequent to a 1-h washout period, 4-min samples of the superfusate werecollected, and electrical stimuli wereapplied at 12 (S,) and 40 min (S 2 ) afterthe start of fraction collection . Drugswere added 16 min before S2 (unlessstated otherwise) and effects wereevaluated by S2 /S, ratios . A: Effect offorskolin (filled circles), 1,9-dideoxy-forskolin (open circles), and 8Br-cAMP (filled squares) on electricallyinduced release. The dashed line indicates the control S2/S, value of 0.74 ± 0.01 (n = 150) ; n shown in parentheses. ..`p - 0.001, vs . control. B: Effect of IBMX and Rp-cAMPS on the forskolin-induced enhancement of stimulated [3H]NA release. Drug concentrations are shown as log M. Results aredepicted as a percentage of the corresponding control S2/S, value (0 .737 -- 0.014 ; n = 27), which is indicated by the dashed line ; nindicated in the bars . `.`p = 0.001, vs . control; x1) - 0.05, vs . 10 s M forskolin . C: Concentration-response curves for the inhibitionof electrically evoked [3H]NA release by the a2-adrenergic agonist bromoxidine (Uk) obtained under various experimental conditions .Bromoxidine was added to the superfusion media 16 min before S2, either alone (control ; open circles) or together with 10 sM forskolin(filled circles) . Filled squares: The superfusion media contained 813r-cAMP from 32 min before S, and bromoxidine from 16 min beforeS2 onward . Results are shown as a percentage of corresponding S2/S, control values ; n = 5-10 .

before S Z (ICs(,, 0.007 ± 0.004 ItM ; maximal effect,68 .9 -!- 2.5% of control ; p :!~ 0.05 vs . control ; Fig . 2C) .However, the effects of saturating (10-6 M) bromoxi-dine concentrations (S,/S, , 54.52 ± 2 .11 % of control ;n = 9) were not altered when neurons were pretreatedfor 24 h with either 100 ng/ml cholera toxin (S=/S,,56.73 -!- 3 .86% of control ; n = 5) or 10 5 M forskolin(S4S,, 55.25 _- 3 .24% of control ; n = 7) .

Ca2+ currentsAmplitudes of whole-cell Ca" currents elicited by

depolarizations from -80 to 0 mV were not signifi-cantly altered when either 10-5 M forskolin (105.7±- 2 .0% of control ; n = 6) or 10-4 M 8Br-cAMP (99.9-!- 2.05% of control ; n = 6) was externally applied

TABLE 1 . Values of basal (L,%) and electrically evoked(S,%) ['H]NA release from superfused chick

sympathetic neurons

Values are under control conditions, in the presence of 8Br-cAMP(10 ' M) added to the superfusion medium 32 min before S,, andafter a 24-h pretreatment with either cholera toxin (100 ng/ml ; ChTx)or forskolin (10 ' M; For) . The fractional basal 3H efflux undercontrol conditions corresponds to 0.294 ± 0.009 nCi (n = 150) per4-min fraction ; the stimulated overflow equals 0.805 - 0.031 nCi(n = 150) . NS, not significantly different from control .

"h - 0.001, vs. control .

1 . Nenrorhc"m ., Vo/ . 03, No . 1, 1994

through a perfusion system for 3 min (Fig . 3A) . Like-wise, the inclusion of either 10 -5 M forskolin (1 .37- 0.13 nA ; n = 9) or 10-4 M 8Br-cAMP (0.89 - 0.11nA; n = 7) in the internal (pipette) solution did notsignificantly alter Ca2+ current amplitudes (control,0.99 ± 0.17 nA ; n = 10) .As reported previously (Boehm and Huck, 1991),

TABLE 2 . 8ffécts of,forskolin, the phosphodiesteraseinhibitor IBMX, and the Rp- as well as the

Sp-diastereomers of cyclic AMP-thioate on basal1 3H]NA release.from superfused cultures of

chick .sympathetic neurons

NS, not significantly different from control ."p - 0.05,''1) - 0.01,'1) - 0.001, vs . control, respectively .

DrugConcentration

(PM) L, /L, n

Forskolin 0.0 0.884 ± 0.024 90.1 0.965 ± 0.026" 61 .0 1 .008 ± 0.026" 6

10 .0 1.021 - 0.023' 9100.0 1 .050 ± 0.039" 6

IBMX 0.0 0.917 ± 0.016 91 .0 0.871 - 0.022" 6

10 .0 0.948 ± 0.038" 6100.0 0.907 -- 0.021" 6

1,000.0 1 .086 ± 0.034' 4

Rp-cAMMS 0.0 0.891 - 0.022 7I .0 0.936 ± 0.044"s 3

30 .0 0.972 ± 0.020" 7

Sp-CAMPS 0.0 0.933 ± 0.029 630 .0 0.996 ± 0.018"S 6

nBasal release

(L,%)Stimulated release

(S I%)

Control 150 0.65 -!- 0.02 1 .66 ± 0.038ßr-cAMP 48 0.84 ± 0.06" 1 .72 + 0.04NS

ChTx (24 h) 56 0.54 -!- 0.02" 0.92 ± 0.04"For (24 h) 50 0.63 ± 0.02"S 0.80 ± 0.03"

the a_-adrenergic agonist bromoxidine reduced Ca"currents in a concentration-dependent manner (Fig .3D), though less effectively (ICs() , 0.64 - 0.15 pM)than the release of NA (IC 5( ,, 0.02 -- 0.007 NM). Dueto what appears to be delayed time course of activation]see Fig . 3B(a), upper trace], the effects of bromoxi-dine on current amplitudes were significantly greaterwhen measured 10 ms after the beginning of the depo-larization (initial component) compared with the end(sustained component) of a 135-ms test pulse (Fig .3C) . Whereas amplitudes at the end of the test pulseare apparently not influenced by current activation(which has reached equilibrium by then), changes mea-sured at 10 ms primarily reflect the modulation of acti-vation kinetics . These phenomena have previouslybeen described by complex kinetic models for NA(Pollo et al ., 1992) and other neurotransmitters (e.g .,Boland and Bean, 1993) .The effects of bromoxidine were strikingly modi-

fied when either 10-5 M forskolin or 10-4 M 8Br-cAMP was added to the internal (pipette) solution .Under these conditions bromoxidine reduced the ini-tial component (measured 10 ms after the beginningof the depolarizing test pulse) just as much as thesustained component (measured after 135 ms) (Fig .3C) . In fact, when compared with the control group,

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FIG . 3. Ca ? ' currents . Effect of a2-adreno-ceptor activation and of substances actingat the cyclic AMP system on whole-cellCa t ' currents of chick sympathetic neu-rons elicited by depolarizations from -80to 0 mV . A : Effect of bath application of10 5 M forskolin (filled circles) and 10-4 M813r-cAMP (filled squares) on Cat ' currentpeak amplitudes . Currents were evoked ata frequency of 2 min ' in the presence orabsence of drugs (open circles) . The insetshows original current traces ; calibration,0 .4 nA/0 .04 s . B : Effect of bromoxidine(Uk) on Cat- currents (/c_ upper traces)under control conditions (a), or with either10 ' M forskolin (b) or 10 4 M 8Br-cAMP(c) added to the internal (pipette) solution .The current traces were obtained before,after (control), and during the applicationof bromoxidine (Uk 10 e M. The lowertraces depict the currents recorded in thepresence of bromoxidine [l cjUk)] as afraction of the averaged control currents[/c,,(ctl)] obtained 20 s before and 20 s afterthe application of bromoxidine . C : Reduc-tion of Cat current amplitudes (Ica) by10 s M bromoxidine measured 10 ms(open columns) or 130 ms (hatched col-umns) after the beginning of the depolariz-ing test pulses under control conditions(ctl) or with either 10-5 M forskolin (for) or10 -4 M 813r-cAMP (cAMP) added to theinternal solution . . .p - 0 .01, vs . the measurement after 10 ms ; n = 7-9 . D : Concentration-response curves for the inhibition of Ca2-

currents by bromoxidine (Uk) under the following experimental conditions : control (open circles), 10 5 M forskolin (filled circles), 10 4

M 813r-cAMP (filled squares), or 5 x 10-5 M Rp-cAMPS (filled diamonds) added to the internal solution . Current amplitudes (/ca ) weremeasured 10 ms after the beginning of the depolarization . The results are shown as a percentage of control current amplitudes ; n -7-9 .

the effect of bromoxidine upon the sustained compo-nent was not significantly modified by either for-skolin or 8Br-cAMP (p > 0.05), whereas these twosubstances markedly affected the reduction of theinitial component (Fig . 3C, D) . Hence, the ratio1(-,,(Uk)/I~,, (control) during the time course of depo-larization, which in the absence of forskolin or 8Br-cAMP can be fitted to a single exponential function(see Kasai and Aosaki, 1989), now appears almostas a straight horizontal line [Fig . 3B(b and c)] . Interms of the definition given above it appears thatforskolin or 8Br-cAMP alleviates the effects of bro-moxidine on current activation but has no effect onthe sustained component of the current .

Forskolin and 8Br-cAMP, when included in the pi-pette solution, conspicuously leveled off the concentra-tion-response curve of bromoxidine effects on the ini-tial Ca" current component (measured at 10 ms afterthe start of the depolarization) (Fig . 3D) . Hence, ICs �values and maximal effects of bromoxidine (ICS� , 0 .64- 0.15 pM; maximum, 57.4 -- 2.4% of control) shiftedin the presence of 10-4 M 8Br-cAMP (ICs(,, 1 .9 ± 1 .2NM; maximum, 70.8 -!- 3.7% of control ; p - 0 .05 vs .control) or 10-5 M forskolin (ICs( ,, 0.45 -} 0.23 p,M;maximum, 66.1 - 3.0% of control ; P - 0.05 vs . con-trol) . Rp-cAMPS (5 x 10-5 NI), on the other hand,

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did not significantly affect the concentration-responsecurve of bromoxidine (IC5o , 0.65 -- 0.27 pM; maxi-mum, 57.5 ± 3.3% of control ; Fig . 3D) .When forskolin (10-5 M) was applied externally to-

gether with bromoxidine (10-5 M), Caz+ current activa-tion was initially delayed but recovered to a normalrate of activation within 5 min (not shown) . Appar-ently, bromoxidine acted immediately, whereas sometime was needed for a sufficient buildup of cellularcyclic AMP by forskolin to antagonize this effect .

DISCUSSION

Most, if not all, of the actions of cyclic AMP inmammalian tissues are believed to be mediated by cy-clic AMP-dependent protein kinases (Ogreid et al .,1983) . In this study we explored the role of the cyclicAMP-dependent protein kinase system in two phe-nomena that are potentially related, i .e., the electricallyinduced release of [ jH]NA from chick sympatheticneurons and the inhibition of this release by a2-adren-ergic (auto)receptor stimulation .

Our data on the electrically induced [ 3H]NA releasefrom chick sympathetic neurons agree with previousreports that forskolin facilitates the release of NA fromcentral (Werner et al ., 1982 ; Schoffelmeer et al ., 1985 ;Ong et al ., 1991) and peripheral (Schwartz and Malik,1993) sources . In addition to stimulating adenylyl cy-clase, forskolin has been shown to act through a mech-anism that does not involve cyclic AMP production(for review, see Laurenza et al ., 1989) . However, sev-eral arguments support our conclusion that the effectsof forskolin are indeed mediated through the produc-tion of cyclic AMP. Thus, as well as enhancing releaseof NA, the exposure of cultures to forskolin also in-creased cyclic AMP production . The forskolin deriva-tive 1,9-dideoxyforskolin, which does not stimulate ad-enylyl cyclase, had no effect on the electrically evokedrelease of NA. The enhancement of release by for-skolin was abolished by Rp-cAMPS, an antagonist ofintracellular cyclic AMP (Botelho et al ., 1988) . Finally,the phosphodiesterase inhibitor IBMX augmented theeffects of forskolin both on the production of cyclicAMP and on the release of NA . Because IBMX andrelated compounds also act as adenosine receptor an-tagonists (Stiles, 1986), we conducted experimentsdemonstrating that adenosine receptor activation didnot alter the release of NA in our culture system (un-published observation) . We may therefore excludesuch a mechanism from the effect of IBMX on NArelease .

Considering the observations listed above, in partic-ular, the blockade of forskolin effects by Rp-cAMPS,it is puzzling that the cyclic AMP agonists Sp-cAMPSand 8Br-cAMP did not enhance electrically inducedNA release . However, this discrepancy may reflect thecomplexity of the cyclic AMP-dependent protein ki-nase system, which has two major types of the enzyme,each equipped with two interactive binding sites for

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cyclic AMP at their regulatory subunits (Ogreid et al .,1983 ; Robinson-Steiner and Corbin, 1983), and eachwith different properties and tissue distributions (seeWalaas and Greengard, 1991) . In slices of neocorticalrat brain, 8Br-CAMP has been reported to augment NArelease induced by high potassium (Werner et al .,1982) or electrical stimulation (Schoffelmeer et A .,1985, 1986) .

In contrast to the potentiation of forskolin effectsmentioned above, IBMX alone did not enhance theelectrically induced release of NA . It appears that inour culture system, synthesis rather than degradationregulates the range of endogenous cyclic AMP at levelsthat affect neuroscretory mechanisms . In support ofthis interpretation we found Rp-cAMPS had no effecton the NA release induced by electrical field stimula-tion even though it abolished the enhancement of therelease due to forskolin .

Twenty-four-hour exposure of cultures to either for-skolin or cholera toxin significantly diminished electri-cally evoked ['H]NA release . This effect could be at-tributed to a down-regulation of adenylyl cyclase activ-ity, particularly because forskolin-stimulated cyclicAMP production was also reduced . However, basalcyclic AMP production remained unaltered after 24 hof forskolin treatment and was actually increased bytwofold after treatment with cholera toxin . Hence, theimpaired NA release more likely reflects initial effectsthat are due to the increase of cyclic AMP rather thanthe actual activity of adenylyl cyclase at the time ofelectrical stimulation . Enhanced levels of cyclic AMPdue to stimulation of adenylyl cyclase by forskolinhave been shown to have a significant impact on generegulation, such as the expression of G;- and G,-type Gproteins (Hadcock et a1 ., 1990) or of a-adrenoceptors(Sakaue and Hoffman, 1991) .Whereas preincubation with forskolin increased bro-

moxidine effects in the human colonic adenocarcinomacell line HT-29 by an enhanced expression of a,-adre-noceptors (Sakaue and Hoffman, 1991), we observedunaltered effects of bromoxidine in cultures pretreatedwith forskolin or cholera toxin . Likewise, bromoxidineconcentrations that were fully active in inhibition ofNA release did not affect the production of cyclicAMP, leading us to conclude that bromoxidine effectswere not mediated through a regulation of intracellularcyclic AMP levels . Recently, Schwartz and Malik(1993) reached the same conclusion, even though intheir experiments on cultured rat superior cervical gan-glion cells bromoxidine did cause inhibition of cyclicAMP production . Nonetheless, we found the a,-adre-noceptor-mediated inhibition of'H overflow to be sus-ceptible to treatments known to enhance intracellularlevels of cyclic AMP, because both forskolin and 8Br-cAMP significantly attenuated the action of the a_-adrenergic agonist bromoxidine . These data are inagreement with several reports showing that either cy-clic AMP analogues or forskolin counteracted the inhi-bition of NA release induced by a-adrenergic agonists

(Werner et al ., 1982 ; Schoffelmeer et al ., 1985, 1986) .We explored this phenomenon by looking at Ca2+ cur-rents recorded with patch-clamp techniques . As notedpreviously (Boehm and Huck, 1991), bromoxidine in-hibited Ca2+ currents primarily through a delayed timecourse of activation . This delay of Ca21- current activa-tion was abolished by forskolin as well as by 8Br-cAMP, thus providing an explanation for the attenua-tion of a-adrenergic effects on NA release by for-skolin or 8Br-CAMP mentioned above . Internal appli-cations of the cyclic AMP antagonist Rp-cAMPS didnot affect the bromoxidine-induced inhibition of Ca21current, indicating on one hand that tonically producedcyclic AMP did not reach levels sufficient to interferewith the a_-adrenergic response and, on the other hand,that the signaling cascade of a 2-adrenoceptor activa-tion did not depend on cyclic AMP.

The inclusion of the nonhydrolyzable GTP analoguegttanosine-5'-O-(3-thiotriphosphate) (GTP-y-S) in thepipette solution mimicked the delayed activation kinet-ics of Ca-+ currents due to bromoxidine (Huck andBoehm, 1992) . This effect was overcome by externallyapplied forskolin or 8Br-cAMP (unpublished observa-tion) . This observation is in line with a recent reporton rat sensory neurons, where the effect of intracellularGTP-y-S was offset by forskolin and 8Br-cAMP (Dol-phin, 1992) . However, in contrast to our data, no antag-onism of cyclic AMP against a,-adrenoreceptor activa-tion could be observed in rat sympathetic neurons(Schofield, 1991) and in a human neuroblastoma cellline (Polio et al ., 1992) .

At present, we know little about the molecular mech-anism by which elevated cyclic AMP levels might at-tenuate the effect of a 2-adrenoreceptor activation . Ourprevious observations that bromoxidine effects werepertussis toxin sensitive (Boehm et al ., 1992) implyinvolvement of G;- or G,,-type G proteins (see Birn-baumer, 1990), but the specific type of G protein andthe exact mode of signal transduction remain unclear .Recent data on the noradrenergic inhibition of neuronalCat ' currents favor a membrane-delimited signal trans-duction pathway independent of diffusible second mes-sengers (Beech et al ., 1992), probably involving G � -type G proteins (Kleuss et al ., 1991 ; Taussig et al .,1992) . G ; has recently been shown to be less sensitiveto activation by GTP-y-S after cyclic AMP-dependentphosphorylation (Imaizumi et al ., 1991), and a similarmechanism could be operative on Go-type G proteinsas well . It is also conceivable that Ca24- channels be-come phosphorylated, thereby changing their bindingaffinity for the G protein .

It is tempting to speculate that forskolin enhancesthe release of NA through the removal of a tonic aZ -adrenoceptor-mediated inhibition . However, unlikecortical slices that when treated with yohimbine mani-fest a multifold increase of stimulus-induced NA re-lease, in our culture system the a 2-adrenoceptor antag-onist yohimbine did not significantly enhance NA re-lease, most likely because the transmitter is too rapidly

CYCLIC AMP AND NORADRENALINE RELEASE

washed away to become active at the presynaptic site(Boehm et al ., 1991) . Likewise, the pretreatment ofcultures with pertussis toxin, which by inactivation ofG� - and G i-type G proteins (Birnbaumer, 1990) wouldremove any negative feedback, enhanced the electri-cally induced NA release by just 19% (Boehm et al .,1992), whereas the release of NA increased by > 100%in the presence of forskolin .

Due to its crucial role in excitation-secretion cou-pling (Augustine et al ., 1987), a modulation of Ca2+

influx would subsequently affect the release of trans-mitter . However, in the absence of bromoxidine, wefound neither forskolin nor 8Br-cAMP increased orattenuated Ca2+ currents, corroborating a recent publi-cation (Przywara et al ., 1991) . These data indicate thatthe enhancement of electrically evoked NA release byforskolin is mediated by an action downstream of Ca2+

influx, possibly at the level of synaptic vesicle proteinphosphorylation (see review by Greengard et al .,1993) . Agreeing with previous data on chick (Przywaraet al ., 1991) and rat sympathetic neurons (Schwartzand Malik, 1992), neither electrical field stimulationnor K + depolarization affected the accumulation ofcyclic AMP in our culture system, indicating that thesecretory process in chick sympathetic neurons maybe modulated by, but does not depend on, the cyclicAMP-dependent protein kinase system .

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Acknowledgment: We thank W. Schtitz for valuablecomments on the manuscript. The perfect technical assis-tance of A . Motejlek and K . Schwarz is gratefully acknowl-edged .

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