Journal of NeurochemistryLippincott—Raven Publishers, Philadelphia© 1996 International Society for Neurochemistry

Cholera Toxin Induces Cyclic AMP—IndependentDown-Regulation of Gsa and Sensitization of

a2-Autoreceptors in Chick Sympathetic Neurons

Stefan Boehm, Sigismund Huck, Andrea Motejiek, *Helmut Drobny,

*Emst A. Singer, and *Mjchael Freissmuth

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

Abstract: The role of the stimulatory GTP-binding protein(G5) in the a2-autoinhibitory modulation of noradrenalinerelease was investigated in cultured chick sympatheticneurons. The ct2-adrenoceptor agonist UK14,304 causeda concentration-dependent reduction of electricallyevoked [

3H]noradrenalinerelease with half-maximal ef-fects at 14.0 ±5.5 nM. In neurons treated with 100 ng/ml cholera toxin for 24 h, the half-maximal concentrationwas lowered to 3.2 ± 1.4 nM without changes in themaximal effect of UK 14,304. The pretreatment with chol-era toxin also increased the inhibitory action of 10 nMUK 14,304 when compared with the inhibition of nor-adrenaline release in untreated cultures derived from thesame cell population. In cultures treated with either 10~.sMforskolin or 100 p~M8-bromo-cyclic AMP, neither thehalf-maximal concentration nor the maximal effect of UK14,304 was altered. Cholera toxin, forskolin, and 8-bromo-cyclic AMP all induced an increase in spontane-ous outflow and a reduction in electrically evoked over-flow, effects not observed after a pretreatment with di-deoxyforskolin. Exposure of neurons to cholera toxin, butnot to forskolin or 8-bromo-cyclic AMP, induced a trans-location of a-subunits of G

5 (G,~)from particulate to solu-ble fractions and led ultimately to a complete loss of G5,.from the neurons. In contrast, no effect was seen on thedistribution of either a-subunits of G1- or G0-type G pro-teins or of /3-subunits. These results indicate that choleratoxin causes a selective, cyclic AMP—independent down-regulation of G5~.This down-regulation of G5~is associ-ated with the sensitization of cs2-autoreceptors. KeyWords: a2-Adrenoceptor—Cholera toxin—Forskolin—Cyclic AMP—GTP-binding protein— Noradrenaline re-lease—Sympathetic neurons.J. Neurochem. 66, 1019—1026 (1996).

Noradrenaline (NA) release from central and pe-ripheral neurons is controlled by inhibitory a2-adreno-ceptors located at presynaptic nerve terminals (seeStarke, 1987). These receptors are linked to secondmessenger systems such as adenylyl cyclase and phos-pholipases as well as to ion channels, primarily viapertussis toxin—sensitive GTP-binding proteins (G

proteins) of the U, and/or G0 subtype (Summers andMcMartin, 1993; Ruffolo et a!., 1994). However, a2-adrenoceptors have also been shown to couple to thecholera toxin— sensitive stimulatory G protein (G0)after heterologous expression of the receptor (Chabreet al., 1994) and in reconstituted systems (see, e.g.,Freissmuth et al., 1989). cx2-Autoreceptors that regu-late stimulation-evoked NA release do so via pertussistoxin—sensitive (e.g., Lai et al., 1983; Allgaier et al.,1985; Boehm et al., 1992; Hill et al., 1993) and/or viapertussis toxin—insensitive G proteins (e.g., Musgraveet al., 1987; Murphy and Majewski, 1990; Murphy etal., 1992; Hill et al., 1993).

In the present study, we have used primary culturesof chick sympathetic neurons to investigate the func-tional role of G, in the a2-adrenoceptor—dependentcontrol of NA release. We have previously reportedthat in these neurons, a2-adrenergic agonists reducestimulation-evoked transmitter release (Boehm et al.,1991) and that this effect depends on G, and/or G0,because it is abolished by pretreatment with pertussistoxin (Boehm et a!., 1992). Here, we investigate theeffect of a long-term treatment with cholera toxin onthe a2-adrenergic inhibition of NA release. This bacte-rial toxin catalyzes an ADP ribosylation of a-subunitsof G~,leading first to a persistent activation (e.g.,Casey and Gilman, 1988; Freissmuth et a!., 1989) andthereafter to a loss of this U-protein subunit from

Received August 3, 1995; revised manuscript received September25, 1995; accepted September 25, 1995.

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

Abbreviations used: 8Br-cAMP, 8-bromo-cyclic AMP; DMSO,dimethyl sulfoxide; G protein, GTP-binding protein; G, and G0,pertussis toxin—sensitive G proteins; G~0,a-subunit of G,; G00, a-subunit of Go; G,, cholera toxin—sensitive stimulatory G protein;G,0, a-subunit of G,; G,0, and G,0L, the short and long splicevariant of the a-subunit of G,; Gfl, /3-subunit of G proteins; G~,y-subunit of G proteins; NA, noradrenaline; SDS, sodium dodecylsulfate; UK 14,304, 5-bromo-N- (4,5-dihydro-1H-imidazol-2-yl )-6-quinoxalinamine.

1019

1020 S. BOEHM ET AL.

plasma membranes (Chang and Bourne, 1989). Be-cause ADP ribosylation of U0,. by cholera toxin canenhance adenylyl cyclase activity, effects of a pretreat-ment with cholera toxin are compared with those of along-term exposure of the neurons to either forskolinor. 8-bromo-cyclic AMP (8Br-cAMP), reagents thatstimulate directly adenylyl cyclase and cyclic AMP—dependent protein kinase, respectively (e.g., Laurenzaet al., 1989).

MATERIALS AND METHODS

Cell culturesThe procedures used to dissociate andculture sympathetic

neurons have previously been described in detail (e.g.,Boehm et al., 1991, 1994). In brief, lumbosacral paraverte-bral sympathetic ganglia were derivedfrom 12-day-old chickembryos, trypsinized (0.1% for 30 mm at 36°C),triturated,and resuspended in Dulbecco’s modified Eagle’s medium(GIBCO no. 041-01885M) containing 2.2 g/L glucose, 10mg/L insulin, 25,000 IU/L penicillin, and 25 mg/L strepto-mycin (GIBCO), 10 j.sgIL nerve growth factor (GIBCO no.0436050), and 5% fetal calf serum (GIBCO no. 011-0620H). Cells were plated on tissue-culture dishes (diameter35 mm; Nunc) for the determination of 0 proteins, and onpolystyrol disks (diameter 5 mm) for superfusion experi-ments (Boehm et al., 1991). Culture dishes and disks werecoated with rat-tail collagen (Biomedical Technologies).

To obtain cultures for [3HINA release experiments, gan-

glia were dissectedfrom six to seven chick embryos, dissoci-ated simultaneously to give a homogenous population of~-~2x 106 cells, and plated into 24 microchambers, eachcontaining one collagen-coated disk. This procedure yields~-~4x l0~cells per disk, 50% of which are of nonneuralorigin (for details, see Boehm et al., 1991). In cultures forsuperfusion experiments, no precautions were taken to re-move these nonneural cells, which neither accumulate norrelease [3H]NA (Boehm et al., 1991). To prepare culturesfor U-protein assays, ganglia from up to 16 chicks weredissociatedsimultaneously, and subsequently nonneural cellswere reduced to <5% by differential plating, as described(Boehm et al., 1994). The resulting pure neuronal cell sus-pension was plated onto 35-mm culture dishes at a densityof 0.5—1 X 10’~neurons/dish.

Superfusion experimentsAfter 5 days in vitro, cultures were incubated for 60 mm

in 0.031.sM [

3H]NA (1,617 TBq/mmol) in culture mediumcontaining 1 mM ascorbic acid, at a temperature of 36°C.Thereafter, the cultures were transferred to small chamberswith volumes between 150 and 200 ftl and superfused at25°C,at a rate of 0.8 ml/min, with a buffer consisting of(mM)NaC1 (113), KC1 (4.8), CaC1

2(1.3), Mg504 (1.2),NaHCO3 (25), KH2PO4 (1.2), D-glucose (11), fumaric acid(0.5), sodium pyruvate (5.0), ascorbic acid (0.57), andEDTA-Na2 (0.03), at pH 7.4 after equilibration with 5%CO2 in 02. After a 1-h washout period, 4-mm fractions ofsuperfusate were collected, and electrical stimuli (36 mono-phasic rectangular pulses, 3 Hz, 0.5 ms, 50 V/cm, 60 mA)were applied after 72 mm (Si) and 100 mm (52) of superfu-sion. 5-Bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quin-oxalinamine (UK 14,304) was added 16 mm before S2 andkept at constant concentrations until the endof experiments,where the residual radioactivity of cultures was extracted by

immersion of disks in 2% (vol/vol) perchloric acid andsonication. Radioactivity in extracts and collected fractionswas determined by liquid scintillation counting.

The fractional rate of tritium outflow per 4 mm was esti-mated by calculating the amount of radioactivity in a 4-mmsuperfusate fraction as a percentage of the radioactivity inthe cells at the beginning of the respective collection period(L%; the radioactive contents of thecells at every time pointof superfusion experiments can be calculated as the sum ofthe radioactivity remaining in the cells at the end of experi-ments plus the amount of radioactivity that has been col-lected by superfusion). The amount of stimulation-evokedoverflow was calculated as the difference between the totaloutflow during thecollection period in whichthe stimulationhad been performed and the estimated basal outflow. Thisbasal outflow was calculated as the arithmetic mean of theoutflow during the collection period preceding stimulationand the outflow during the collection period that followedtheperiodof stimulation. This difference was then expressedas percentage of the total amount of tritium in cultures atthe beginning of stimulation (S~%,S2%).

Effects of pretreating neurons for 24 h with cholera toxin,forskolin, dideoxyforskolin, or 8Br-cAMPon stimulated andbasal outflow were evaluated by comparison ofthe overflowevoked by S~(S1 %) and of the outflow during the collectionperiod preceding S~(L, %), respectively. Effects of UK14,304 were evaluated by calculating the ratio between theoverflow evoked by S2 and the overflow evoked by S (S~/Si). For comparison of the effects of UK 14,304 in culturesthat had been subjected to different kinds of pretreatment,52/SI ratios obtained in the presence of UK 14,304 wereexpressed as a percentage of the corresponding mean S2/S1ratios of controls (S2/51, % of control).

Toobtain concentration—response relations for the inhibi-tion of evoked

3H overflow by UK 14,304 in either untreatedneurons or in neurons treated with cholera toxin, forskolin, or8Br-cAMP, cell cultures derived from different dissociationprocedures were not treated or pretreated (24 h) with either100 ng/ml cholera toxin, 10 pM forskolin, or 100 ,uM 8Br-cAMP. In one superfusion experiment, 12 culture disks pre-viously subjected to the same kind of pretreatment wereloaded with [3H]NAand superfused simultaneously. In eachof these experiments with 12 cultures, three cultures wereused as drug-free controls and the remaining nine culturedisks were exposed to three different concentrations of UK14,304; this was done again in groups of three, each of whichreceived one concentration of the a

2-adrenergic agonist.Thus, one data point in the concentration—response curvesfor UK 14,304 (after different kinds of pretreatment) wasobtained in cultures from at least two separate dissociationprocedures and in two independent superfusion experiments(if n 6) but more frequently in cultures from three dissoci-ations and in three superfusion experiments (n 9). Ifdatapoints comprise a number of culture disks not being amulti-ple of 3, cell cultures have been excluded, most frequentlybecause of a lack in stimulation-evoked overflow at SI and/or S2 (which happens, for example, if air bubbles are trappedin the superfusion chamber).

In a series of three separate experiments, 12 of 24 culturedisks obtained from one dissociation were pretreated for 24h with 100 ng/ml cholera toxin, 10 pM forskolin, or 100pM 8Br-cAMP. The remaining 12 culture disks of each dis-sociation were not treated. The effect of 10 nM UK 14,304was then compared in pretreated and nontreated neurons,

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G0,. AND a2-AUTORECEPTORS 1021

respectively, both derived from onehomogeneous cell popu-lation.

StatisticsData are given as arithmetic mean ±SEM values; n

= number of cell culture disks. Subsequent to a Kolmo-gorov—Smirnov test of normal distribution, the significanceof differences between the means of single data points (notincluded in concentration—response curves) was evaluatedby unpaired Student’s t test. On the other hand, concentra-tion—response curves were fitted to the experimentally ob-tained data points by using the ALLFIT program, whichcalculates estimates for the half-maximal concentration, forthe slope, and for the maximal effect, as well as correspond-ing standard errors. Furthermore, the program determinessignificances of differencesbetween concentration—responsecurves by simultaneous curve fitting with shared parametersand subsequent calculation of the F statistic on the resulting“extra sum of squares” (see DeLean et al., 1978).

Determination of G-protein levelsAfter 3—4 days in vitro, neuronal cell cultures derived

from one cell population were exposed to cholera toxin (100ng/ml), forskolin (10 pM), dideoxyforskolin (10 pM), 8Br-cAMP (100 pM), or to 0.1% dimethyl sulfoxide (DMSO)for various periods of time. Thereafter, cultures were washedwith superfusion buffer and quickly frozen by immersion inliquid nitrogen. After thawing, the cells were scraped fromthe culture dishes and subjected to a second freeze—thawcycle. The soluble and particulate fractions were separatedby centrifugation (50,000 g for 30 mm at 2°C).Theresultingpellet was dissolved immediately in Laemmli sample buffer~50 mM Tris-HC1, pH 6.8, 10% glycerol, 2% sodium dode-cyl sulfate (SDS), 0.1 mg/ml bromophenolblue] containing40 mM dithiothreitol. The soluble proteins were precipitatedby the addition of trichloroacetic acid (14% final concentra-tion) and subsequently dissolved in Laemmli sample buffer.Samples corresponding to 1.5—3 X l0~cells were appliedto SDS-polyacrylamide minigels (10% acrylamide, 0.26%N,N’-methylene bisacrylamide). No attempt was made toresolve the individual a-subunit subtypes of G~-or G-typeG proteins (G0,., ~ Proteins were subsequently trans-ferred to nitrocellulose and stained with Ponceau S to verifythat comparable amounts had been loaded. The nitrocelluloseblots were probed with the following antisera: antisera 11(anti-G~.com~,o~)and 7 (anti-G~.cemmon)raised against thepep-tides used to generate the original antisera A569 and K521,respectively (Mumby and Gilman, 1991), and antiserumCS 1, raised against the carboxy-terminal decapeptide of G00,,which was kindly provided by Graeme Milligan (Glasgow,U.K.). Due to the much higher levels of expression of G0~and G,~,antiserum 11 detects primarily these a-subunits inneuronal cell membranes. Immunodetection was performedwith a secondantibody conjugated to horseradish peroxidaseusing the Amersham ECL reagents (Amersham, Madison,WI, U.S.A.). Identity of immunoreactive bands was verifiedby comigration with purified G-protein subunit standardsprepared as in Freissmuth and Gilman (1989) or in Mumbyand Linder (1994).

Materials(—)-[ring-2,5,6-

3H]NA (1,617 TBq/mmol) was ob-tained from NEN, Dreieich, Germany; cholera toxin, for-skolin, 1,9-dideoxyforskolin, and 8Br-cAMP from Sigma,Munich, Germany; UK 14,304 from ResearchBiochemicals,

TABLE 1. Effect of pretreating chick sympatheticneurons with cholera toxin, forskolin, or 8Br-cAMP

for 24 h on basal 3H outflow and onelectrically evoked overflow

Basal outflow(L~%)

Stimulated overflow(S%) n

Control 0.54 ±0.02 1.49 ±0.04 133Cholera toxin 0.67 ±0.03” 0.83 ±0.04” 80Forskolin 0.64 ±0.02° 0.83 ±0.04” 74Dideoxyforskolin 0.48 ±0.04 (NS) 1.49 ±0.06 (NS) 298Br-cAMP 0.69 ±0.04” 1.08 ±0.04° 84

Cultures of chick sympathetic neurons were exposed for 24 h tocholera toxin (100 ng/ml), forskolin (10 MM), dideoxyforskolin (10

1iM), or 8Br-cAMP (100 1sM), labeled with [3H]NA, and subse-

quently superfused. After a I-h washout period, 4-mm fractions ofsuperfusate werecollected, and 3H overflow was triggered by electri-cal field stimulation after 72 mm of superfusion (S

1). Results areshown as percentage of the total cellular radioactivity (L1%, S1%;see Materials and Methods).

“p < 0.001 vs. corresponding controls; NS, not significantly dif-ferent from controls.

Natick, MA, U.S.A. Forskolin and dideoxyforskolin weredissolved at 10 mM, 8Br-cAMP at 100 mM, in DMSO;these solutions were diluted (1:1,000) to final concentrationswith culture medium; a 24-h treatment with DMSO (0.1%,vol/vol) affected neither results from release experimentsnor from G-protein determinations.

RESULTS

a~2-Adrenoceptor—mediatedmodulation of tritiumoverflow from chick sympathetic neurons labeledwith [

3H]NAPretreatment of chick sympathetic neurons with

cholera toxin (100 ng/ml), forskolin (10 tiM), or 8Br-cAMP (100 ~M) for 24 h reduced electrically evokedoverflow, which corroborates previous results (Boehmet al., 1994). In addition, these treatments enhancedbasal 3H outflow. Exposure of the neurons to dideoxy-forskolin, a forskolin analogue that does not activateadenylyl cyclase (Laurenza et al., 1989), had no effecton either spontaneous outflow or stimulation-evokedoverflow (Table 1).

The selective a2-adrenoceptor agonist UK 14,304

(Cambridge, 1981) caused a concentration-dependentreduction of electrically evoked overflow with half-maximal effects (obtained by curve fitting; see Fig. 1for corresponding data points and fitted curves) at 14.0±5.5 nM and a maximal inhibition to 54.6 ±2.9%of control. Neither a pretreatment of the neurons with10 1uM forskolin nor with 100 ,tiM 8Br-cAMP, both for24 h, altered the values of half-maximal concentration(forskolin, 11.5 ± 5.1 nM; 8Br-cAMP, 14.0 ±9.8nM) or of maximal effects (forskolin, 58.7 ±2.9% ofcontrol; 8Br-cAMP, 54.5 ±2.2% of control) of UK14,304. However, 8Br-cAMP decreased the slope ofthe concentration—response curve for the inhibition byUK 14,304. In neurons pretreated with cholera toxin,

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1022 S. BOEHM ET AL.

FIG. 1. Concentration—response curves for the inhibition ofelectrically induced 3H overflow by the a

2-adrenoceptor agonistUK 14,304 in cultures of chick sympathetic neurons pretreatedwith cholera toxin, torskolin, or 8Br-cAMP. After labeling with1

3H]NA, cultures were superfused, and subsequent to a 1-hwashout period, 4-mm samples of superfusate were collected.Electrical stimuli (36 pulses at 3 Hz) were applied after 72 (S

1)and 100 mm (S2) of superfusion. UK 14,304 was added to themedium 16 mm before S2 in cultures pretreated with either 100ng/ml cholera toxin (squares; top), with 10 p~Mforskolin (trian-gles; middle), or with 100 ~sM8Br-cAMP (diamonds; bottom),all for 24 h. For direct comparison, the effect of UK 14,304 inuntreated cultures is depicted by open circles. Results are shownas a percentage of mean S2/S1 ratios in the absence of UK14,304 (S2/S1 % of control), which amounted to 0.75 ±0.02(n = 26) in untreated cultures, to 0.74 ±0.03 (n = 17) in choleratoxin—treated cultures, to 0.71 ± 0.03 (n = 15) in forskolin-treated cultures, and to 0.90 ± 0.04 (n = 24) in 8Br-cAMP—treated cultures. n are indicated near the symbols.

cAMP, however, failed to alter the effect of the a2-adrenergic agonist (Fig. 2).

G-protein levels in chick sympathetic neuronsAfter a 24-h incubation of neurons with cholera

toxin (100 ng /ml), the particulate fraction was essen-tially devoid of G0,. (Fig. 3A, lane C). This effect wasspecific for G,,,; i.e., neither the bulk levels of U-pro-tein a-subunits (G0,,/G,.) detected with the ~antiserum (Fig. 3B) nor the G-protein /3-subunits (Ga;Fig. 3C) were affected by cholera toxin. In addition,the levels of Gb,. (Fig. 3A, lane F), of G00/G1,. (Fig.3B), and of G~(Fig. 3C) were not changed after a24-h incubation with 10 ,aM forskolin. In a similarmanner, neither 10 ,uM dideoxyforskolin nor 100 ,uM8Br-cAMP, both applied for 24 h, caused alterationsin the levels of G0,. (Fig. 3D).

the concentration—response curve was shifted to theleft with a significant reduction of the half-maximalconcentration (3.2 ±1.4 nM; p < 0.05 vs. untreatedas well as forskolin- or 8Br-cAMP—treated cultures),but without changes in the maximal effect (55.5±2.8% of control; Fig. 1).To rule out that differences between concentration—

response curves were caused by variations betweenseparate cell populations, the effects of 10 nM UK14,304 were investigated in neurons pretreated withcholera toxin, forskolin, or 8Br-cAMP and comparedwith results obtained in untreated neurons from thesame dissociation procedure; this series of experimentsclearly revealed that the exposure of neurons to 100ng/ml cholera toxin for 24 h enhanced the inhibitoryaction of 10 nM UK 14,304. The pretreatment of neu-rons with either 10 ,uM forskolin or 100 ,uM 8Br-

FIG. 2. Effect of a pretreatment of neurons with cholera toxin,forskolin, or 8Br-cAMP on the inhibition of electrically evoked3H overflow by 10 nM UK 14,304. One-half of each set of neu-ronal cell cultures derived from homogenous cell populationswas treated with 100 ng/ml cholera toxin (filled columns; top),with 10

1sMforskolin (hatched columns; middle), or with 100 1sM8Br-cAMP (lined columns; bottom), all for 24 h. Open columnsrepresent results obtained with the corresponding nontreatedcultures. After labeling with [

3H]NA, all cultures were superfused,and subsequent to a 1-h washout period, 4-mm samples of su-perfusate were collected. Electrical stimuli (36 pulses at 3 Hz)were applied after 72 (S

1) and 100 mm (S2) of superfusion. Thefour columns to the left of the axis break represent S2/S1 ratiosobtained in either the absence (ctl) or the presence of 10 nMUK 14,304 (UK; added to the medium 16 mm before S2). Forcomparison with the results of Fig. 1, the two columns to theright of the axis break show the inhibitory effect of 10 nM UK14,304 as a percentage of mean S2/S1 ratios obtained in theabsence of UK 14,304 (S2/S1, % of control). °p < 0.05; **p<0.01 vs. results obtained in corresponding untreated cultures;NS, no significant difference.

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Ge,. AND a2-AUTORECEPTORS 1023

GS,.L have previously been observed in primary cul-tures of human thyroid epithelial cells (Selzer et al.,1993). Irrespective of the levels of expression, bothG,,,.. and GS,,L were down-regulated by cholera toxin(Fig. 4A). Effects of cholera toxin were long lasting;24 h after removal of the toxin, membrane levels ofU0,. remained substantially reduced (Fig. 4D).

DISCUSSION

FIG. 3. Immunoblot analysis of the levels of G-protein subunitsin cultures of chick sympathetic neurons pretreated for 24 h withcholera toxin, forskolin, dideoxyforskolmn, or8Br-cAMP. Neuronswere pretreated for 24 h either with 10 ,iM forskolmn (F), 10pM dideoxyforskolin (ddF), or 100 pM 8Br-cAMP (

5Br) in 0.1 %DMSO, with 100 ng/ml cholera toxin (C), with vehicle (V), orwith 0.1% DMSO (0). Cells were lysed as outlined in Materialsand Methods, and aliquots of the particulate fraction corre-sponding to 3 x io~cells were applied to SOS-polyacrylammdegels. A: Immunoblot analysis was performed with antiserum CS1(anti-G

0~1:500 dilution). In lane a~//3y,purified recombinant~ (200 ng) and ~ (60 ng) and bovine brain G-protein /3’y-dimers (200 ng) were mixed. B: Immunoblots were probed withantiserum 11 (anti-G~00,,,,,~0~1:1,000 dilution). In lane a0/13y,

purified recombinant G0~.,(200 ng) and GS~.L(60 ng) and bovinebrain G-protein /3y-dimers (200 ng) were mixed; lane a~repre-sents purified recombinant ~ (10 ng) present at about thedetection limit. This low amount was chosen to illustrate thedifferent expression levels that can be readily estimated by acomparison of A (a0-blot) and B (a00,,,,,00-blot). Note that thisG~00,,,,,,0antiserum detects primarily a0 and a1, because theseare obviously expressed at much higher levels than a0. However,the antiserum 11 also detects a0 (as can be seen from the stan-dard lane labeled a01/3y) if the amount of the protein is suffi-ciently high. C: Immunoblots were performed with antiserum 7(anti-G~00,,,,,.,,01:1,000 dilution). In lane a0//3y, purified recom-binant G0~.,(200 ng) and G0~L(60 ng) and bovine brain G-protein /3y-dimers (200 ng) were mixed. D: Immunoblot analysiswas performed with antiserum CS1 (anti-G0,; 1:500 dilution). Inlane a0//3y, purified recombinant G~.(80 ng) and G0~.L(20 ng)and bovine brain G-protemn /3y-dimers (200 ng) were mixed.Similar results were obtained in two other cultures from twodifferent dissociations.

In spite of the almost complete elimination of U,,.from the particulate fraction, U,,, was not detected inthe soluble fraction (not shown). We therefore deter-mined the distribution of U,,. between soluble and par-ticulate fractions after shorter exposures to choleratoxin. Both 3 and 5 h after the addition of choleratoxin to the culture medium, substantial levels of G,,,were found in the soluble fraction (Fig. 4A, lanes 3SCand 5SC), but the distribution of G0,,/G1,. (Fig. 4B)and of G1~(Fig. 4C) was not affected. We noted thatthe relative abundanceof short and long splice variantsof U,,, (G,,., and G,,,L) varied substantially betweenindividual preparations of neurons (compare Fig. 3Aand Fig. 4A). Although the reason for this variabilityis not clear, variations in the expression of G,,,., and

Persistent activation of G,,. by cholera toxin (Changand Bourne, 1989), by constitutively activating muta-tions (Levis and Boume, 1992), and by prolongedexposure to neurotransmitter receptor agonists (Wede-gaertner and Bourne, 1994), induces a loss of G,,,from the plasma membrane. This is associated withdepalmitoylation of the protein (Wedegaertner and

FIG. 4. Immunoblot analysis of the levels of G-protemn subunitsin cultures of chick sympathetic neurons pretreated for variousperiods of time with cholera toxin. Neurons were treated for 3or 5 h with 100 ng/mI cholera toxin (3CS, 3CP, 5CS, 5CP) orvehicle (3V5, 3VP, 5VS, 5VP). After lysis and centrifugation, ali-quots of the particulate (3CP, 5CP, 3VP, 5VP) and soluble frac-tion (3CS, 5CS, 3VS, 5VS) corresponding to 3 x 10~cells wereapplied to SDS-polyacrylamide gels. Lane a,//3y shows a mix-ture of purified recombinant G0,., (200 ng) and G0~L(60 ng)with bovine brain G-protemn /3y-dimers (200 ng). A: Immunoblotswere probed with antiserum CS1 (anti-G,~1:500 dilution). B:Immunoblots were performed with antiserum 11 (anti G0,,,~1:1,000 dilution). Lane a011/3-y represents a mixture of purifiedrecombinant G0,, and G~3(100 ng each) and bovine brain G-protein /37-dimers (200 ng). C: Immunoblots were performedwith antiserum 7 (anti-G~00,,,,0001:1,000 dilution). D: Neuronswere pretreated for 24 h with 100 ng/ml cholera toxin (C), for24 h with 100 ng/ml cholera toxin followed by a 24-h incubationin medium (C24w) or with vehicle (V). Aliquots ofthe membranefraction corresponding to 3 x i0~cells were applied to SDS-polyacrylamide gels. Lane a,/fiy shows a combination of puri-fied recombinant G0~.,(100 ng) and G,~L(30 ng) and bovinebrain G-protein /3y-dimers (100 ng). Immunoblots were probedwith a combination of antiserum CS1 (anti-G~0)and ofantiserum7 (anti-G3.~,,,,,,,.,,)at a 1:500 and 1:1,000 dilution, respectively.Data are representative for two additional experiments per-formed on cultures from different dissociations.

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1024 S. BOEHM ET AL.

Bourne, 1994), which is thought to loosen the attach-ment of G,,, to the membrane (see review by Wede-gaertner et al., 1995). In addition, the degradation rateof G,,. is accelerated (Levis and Bourne, 1992; Wede-gaertner et al., 1995), although it is not clear whetherredistribution is a prerequisite for degradation. Our ob-servations obtained in primary cultures of chick sym-pathetic neurons reproduce the following characteristicfeatures: Cholera toxin induced both a redistributionof U,,. from the membrane to the cytosol at earlier timepoints and a reduction in the total level of the proteinafter long-lasting exposure. Currently, it is believedthat removal of G,,, from the membrane participates innegative feedback mechanisms that prevent excessivestimulation of cells by G,-coupled receptors and thatunderlie desensitization. Our present data point to anadditional, physiologically relevant role of down-regu-lated G,,.; i.e., the increased efficiency of another regu-latory pathway that is controlled by a distinct set of Uproteins. The cholera toxin treatment, which led toa loss of U,,. from neuronal membranes, induced anincreased potency of the a2-adrenoceptor agonist UK14,304, which inhibits stimulation-evoked NA releasevia G,- and/or G0-type G proteins (Boehrn et al.,1992). Hence, the down-regulation of G,,. was associ-ated with the sensitization of a 01/G,.-mediatedpathway.

A sensitization of regulatory pathways involving in-hibitory G proteins by a long-lasting stimulation of G,-linked signalingcascades has previously beenobservedin neuronal (Morton et al., 1992) and nonneural cells(Hadcock et al., 1990). In these reports, adenylyl cy-clase— and cyclic AMP—dependent mechanisms en-hanced the expression of G,. Hence, an increased levelof inhibitory U proteins, which couple to the a2-autore-ceptors of chick sympathetic neurons (Boehrn et al.,1992), might have caused the increased potency of thea2-adrenergic agonist in reducing transmitter release.However, changes in G,,,, U0,,, or in even /3-subunitswere not observed in the present study, neither after atreatment with cholera toxin nor with forskolin. Thus,the effect of cholera toxin was specific for U,,..

Cholera toxin, apart from inducing a degradation ofU,,, (Chang and Boume, 1989), activates U, proteinsand thereby enhances adenylyl cyclase activity (Caseyand Gilman, 1988; Laurenza et al., 1989). Long-termtreatment with cholera toxin causes a persistent en-hancement of adenylyl cyclase activity, as originallydescribed in a rat pituitary cell line (Chang andBoume, 1989). We have reported previously that a24-h exposure of chick sympathetic neurons to choleratoxin also induces an elevation of cyclic AMP levels(Boehm et al., 1994). Hence, effects of the choleratoxin treatment could have been mediated by an en-hanced activity in the adenylyl cyclase/cyclic AMP—dependent signaling cascade. In fact, long-lasting in-creases in cyclic AMP were previously found to sensi-tize a2-adrenoceptors, which inhibited adenylyl cy-clase in a human adenocarcinoma cell line (Sakaue

and Hoffmann, 1991). However, the following severallines of evidence challenge a role for elevated cyclicAMP in the sensitization of a2-autoreceptors describedhere: (1) The effect of cholera toxin on the UK14,304—induced reduction of transmitter release wasnot mimicked by 8Br-cAMP. (2) Even though the lev-els of cyclic AMP in chick sympathetic neurons areelevated after a 24-h exposure to cholera toxin (seeBoehm et al., 1994), elevations in cyclic AMP attenu-ate rather than enhance the a2-adrenergic inhibition ofNA release from sympathetic neurons (Schwartz andMalik, 1993; Boehm et al., 1994). (3) Previously,forskolin had been shown to decrease the messengerRNA levels of G,,. in cultured rat cortical neurons(Eriksson et al., 1992). In the present study, however,neither forskolin nor 8Br-cAMP affected the U,,. pro-tein. Thus, the effects of cholera toxin on both U,,, andthe a2-adrenergic inhibition of NA release appeared tobe independent of an increase in cyclic AMP.

The adenylyl cyclase/cyclic AMP—dependent sig-naling cascade has long been recognized as an im-portant modulator of neurotransmitter release. Short-term increases in cyclic AMP augment NA releasefrom central (e.g., Werner et al., 1982; Schoffelmeeret al., 1985) and peripheral (e.g., Schwartz and Malik,1993; Boehrn et al., 1994) neurons. In contrast, a long-lasting activation of adenylyl cyclase by 24-h exposureto cholera toxin or forskolin reduces stimulation-evoked NA release (this study and Boehm et al.,1994). One action shared by these two treatments isthe desensitization of adenylyl cyclase (Eriksson et al.,1992; Boehm et al., 1994). However, induction ofneurotransmitter release from chick sympathetic neu-rons by electrical field stimulation does not involve anactivation of adenylyl cyclase (Boehm et al., 1994).Changes in the levels of cyclic AMP due to 24-h expo-sure to cholera toxin or forskolin are also unlikely tobe responsible for the reduction of transmitter release,because cyclic AMP is increased (which should in-stead enhance electrically evoked release) after thetreatment with cholera toxin but not altered after theforskolin treatment (see Boehm et al., 1994). Thus,the inhibition of NA release after long-term exposureto forskolin or cholera toxin most probably is the resultof a phosphorylation process mediated by the cyclicAMP—dependent protein kinase subsequent to the ini-tial stimulation of adenylyl cyclase and the resultingincrease in cyclic AMP. The crucial role of adenylylcyclase is supported by the finding that a long-lastingtreatment with the forskolin analogue dideoxyfor-skolin, which fails to activate this enzyme (Laurenzaet al., 1989), did not mimick the effects of forskolinand cholera toxin. The involvement of the cyclicAMP—dependent protein kinase is indicated, becauseNA release was also reduced after a 24-h exposure to8Br-cAMP.

Ever since a2-adrenoceptors had first been demon-strated to inhibit adenylyl cyclase (Jakobs et al.,1976), a reduced activity in the adenylyl cyclase/cy-

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G,.,. AND a2-AUTORECEPTORS 1025

clic AMP—dependent signaling system has been sug-gested to form the grounds for the a2-adrenergic reduc-tion of NA release (e.g., Wemer et al., 1982; Schoffel-meer et al., 1986). However, evidence recentlyindicated that an inhibition of adenylyl cyclase is nota prerequisite for the a2-autoinhibitory regulation ofNA release (Ong et al., 1991; Schwartz and Malik,1993; Boehm et al., 1994). Although a2-adrenergicinhibition of NA release has been studied for two de-cades (Starke, 1987), the apparently high degree ofplasticity in this inhibitory feedback mechanism hasonly recently been appreciated. In chick sympatheticneurons, this plasticity includes an attenuation of a2-adrenergic effects by acutely elevated levels of cyclicAMP, even though the adenylyl cyclase/cyclic AMP—dependent protein kinase system is not part of the sig-naling cascade that links a2-adrenoceptor activation toan inhibition of transmitter release (Boehm et al.,1994). In addition, we have recently shown that thea2-autoreceptors undergo rapid, agonist-induced de-sensitization (Boehm et al., 1995). The present studydemonstrates receptor sensitization associated with theloss of membrane-bound U,,. and thereby reveals anovel kind of plasticity of a2-autoreceptors.

Taken together, our present and previous resultsshow that the signaling cascade G,,./adenylyl cyclase/cyclic AMP—dependent protein kinase contributes tothe modulation of sympathetic transmitter release atthe following three levels that differ both in their effectand in the time scale over which they occur: (1) Short-term elevation of cyclic AMP acts in a manner thatfacilitates neurotransmitter release and attenuates thea2-adrenergic inhibition; (2) persistent activation ofadenylyl cyclase restricts the release of transmitter;and (3) down-regulation of the membrane levels of0,,., a phenomenon frequently observed after long-lasting activation of G,-coupled neurotransmitter re-ceptors (see Wedegaertner et al., 1995), is associatedwith a sensitization of autoinhibitory feedback. Allthree mechanisms are obviously important in the fine-tuning of presynaptic modulation and will thus contrib-ute to synaptic plasticity.

Acknowledgment: We thank V. O’Connor for valuablecomments about the manuscript, G. Koth and K. Schwarzfor excellent technical assistance, and E. Tuisl for artwork.This study was supported by a grant from the Austrian Sci-ence Foundation (FWF) to M.F. (P10675).

REFERENCES

Allgaier C., Feuerstein T. J., Jackisch R., and Hertting G. (1985)Islet-activating protein (pertussis toxin) diminishes a2-adreno-ceptor-mediated effects on noradrenaline release. NaunynSchmiedehergs Arch. Pharmacol. 331, 235—239.

Boehm S., Huck S., Drobny H., and Singer E. A. (1991) Electricallyevoked noradrenaline release from cultured chick sympatheticneurons: modulation via presynapatic a2-adrenoceptors and lackof autoinhibition. Naunyn Schmiedebergs Arch. Pharmacol.

344, 130—132.Boehm S., Huck S., Drobny H., and Singer E. A. (1992) Pertussis

toxin abolishes the inhibition of Ca2~currents and of noradrena-

line release via a2-adrenoceptors in chick sympathetic neurons.

Naunyn Schmiedebergs Arch. Pharmacol. 345, 606—609.Boehm S., Huck S., Koth G., Drobny H., Agneter E., and Singer

E. A. (1994) a2-Adrenoceptor—mediated inhibition of electri-cally evoked [

3H]noradrenaline release from chick sympatheticneurons: role of cyclic AMP. J. Neurochem. 63, 146—154.

Boehm S., Huck S., Schwaj’z K., Agneter E., Drobny H., and SingerE. A. (1995) Rapid, agonist-induced desensitization of a

2-auto-receptors modulating transmitter release. Br. .1. Pharmacol. 114,1143—1148.

Cambridge D. (1981) UK 14,304, a potent and selective a5-agonistfor the characterization of a-adrenoceptor subtypes. Eur. J.Pharmacol. 72, 413—415.

Casey P. J. and Gilman A. G. (1988) G protein involvement inreceptor/effector coupling. J. Rio!. C/scm. 263, 2577—2580.

Chabre 0., Conklin B. R., Brandon S., Bourne H. R., and LimbirdL. E. (1994) Coupling of the a2-adrenergic receptor to multipleG proteins. J. Rio!. Chem. 269, 5730—5734.

Chang F. H. and Bourne H. R. (1989) Cholera toxin induces cAMP-independent degradation of G,,,. J. Biol. Chem. 264,5352—5357.

DeLean A., Munson P. J., and Rodbard D. (1978) Simultaneousanalysis of families of sigmoidal curves: application to bioassay,radioligand assay, and physiological dose-response curves. Am.J. Physjot. 235, E97—El02.

Eriksson P. S., NilssonM., Carlsson B., Isaksson 0. G. P., ROnnbäckL., and Hansson E. (1992) Chronic elevation of cAMP levelsinduces changes in the adenylate cyclase system, opiate receptorsensitivity and levels of G,,,-mRNA in cultured neurons. Neu-rosci. Lett. 135, 28—32.

Freissmuth M. and Gilman A. G. (l989)Mutations ofG,~designedto alter the reactivity of the protein with bacterial toxins: substi-tutions at Arg 87 result in loss ofGTPase activity. J. Riot. Chem.264, 21907—21914.

Freissmuth M., Casey P. J., and Gilman A. G. (1989) G proteinscontrol diverse pathways of transmembrane signaling. FASEBJ. 3,2125—2131.

Hadcock J. R., Ros M., Watkins D. C., and Malbon C. C. (1990)Cross-regulation between G-protein /mediated pathways. Stimu-lation of adenylcyclase increases expression of the inhibitoryG-protein G~2.J. Biol. Chem. 265, 14784—14790.

Hill C. E., Powis D. A., and Hendry I. A. (1993) Involvementof pertussis toxin-sensitive and -insensitive mechanisms in a-adrenoceptor modulation of noradrenaline release from rat sym-pathetic neurones in tissue culture. Br. J. Pharmacol. 110,281—288.

Jakobs K. H., Saur W., and Schultz G. (1976) Reduction of adenyl-ate cyclase activity of human platelets by the alpha-adrenergiccomponent of epinephrine. J. Cyclic Nucleotide Res. 2, 381—392.

Lai R.-T., Watanabe Y., and Yoshida H. (1983) Effect of islet-activating protein (lAP) on contractile responses of rat vasdeferens: evidence for participation of Ni (inhibitory GTP bind-ing regulating protein) in the a2-adrenoceptor-mediated re-sponse. Eur. J. Pharmacol. 90, 453—456.

Laurenza A., McHugh Sutkowski E., and Seamon K. B. (1989)Forskolin: a specific stimulator of adenyl cyclase or a diterpenewith multiple sites of action? Trends Pharmacol. Sd. 10, 442—447.

Levis M. J. and Bourne H. R. (1992) Activation of the a subunitof G, in intact cells alters its abundance, rate of degradation,and membrane avidity. J. Cell Biol. 119, 1297—1307.

Morton M. F., Street V. A., and Nathanson N. M. (1992) Selectiveregulation of G,~1expression and function in PCI2 cells bycAMP. J. Neurosci. 12, 1839—1846.

Mumby S. M. and Gilman A. G. (1991) Synthetic peptide antiserawith defined specificity for G protein a- and /J-subunits. Meth-ods Enzymol. 195, 215—233.

Mumby S. M. and Linder M. E. (1994) Myristoylation of G proteina-subunits. Methods Enzymol. 237, 254—267.

Murphy T. V. and Majewski H. (1990) Pertussis toxin differentiatesbetween a~and a2-adrenoceptor-mediated inhibition of nor-

J. Neurochem., Vol. 66, No. 3, 1996

1026 S. BOEHM ET AL.

adrenaline release from rat kidney cortex. Eur. J. Pharmacol.179, 435—439.

Murphy T. V., Foucart S., and Majewski H. (1992) Prejunctionala2-adrenoceptors in mouse atha function through G-proteinswhich are sensitive to N-ethylmaleimide, but not pertussis toxin.Br. J. Pharmacol. 106, 871—876.

Musgrave I., Marley P., and Majewski H. (1987) Pertussis toxindoes not attenuate a2-adrenoceptor-mediated inhibition of nor-adrenaline release in mouse atria. Naunyn Schmiedebergs Arch.

Pharmacol. 336, 280—286.Ong M.-L., Ball S. G., and Vaughan P. F. T. (1991) Regulation of

noradrenaline release from rat occipital cortex tissue chops bya2-adrenergic agonists. J. Neurochem. 56, 1387—1393.

RuffoloR. R. Jr., Stadel J. M., and Hieble J. P. (1994) a-Adrenocep-tors: recent developments. Med. Res. Rev. 14, 229—270.

Sakaue M. and Hoffmann B. (1991) cAMP regulates transcriptionof the

02A adrenergic receptor gene in HT-29 cells. J. Biol.C/scm. 266, 5743—5749.

Schoffelmeer A. N. M., Hogenboom F., and Mulder A. H. (1985)Evidence for a presynaptic adenylate cyclase system facilitating[3H]norepinephrine release from rat brain neocortex slices andsynaptosornes. J. Neurosci. 5, 2685—2689.

Schoffelmeer A. N. M., Wierenga E. A., and Mulder A. H. (1986)Role of adenylate cyclase in presynaptic a

2-adrenoceptor— andp-opioid receptor—mediated inhibition of [

3H]noradrenaline re-

lease from rat brain cortex slices. J. Neurochem. 46, 1711-1717.

Schwartz D. D. and Malik K. U. (1993) Cyclic AMP modulates butdoes not mediate the inhibition of [3H]norepinephrine releaseby activation of alpha-2 adrenergic receptors in cultured ratganglion cells. Neuroscience 52, 107—113.

Selzer E., Wilfing A., Schiferer A., Hermann M., Grubeck-Loebenstein B., and Freissmuth M. (1993) Stimulation of hu-man thyroid growth via the inhibitory guanine nucleotide bind-ing G protein G,: constitutive expression of the G-protein sub-unit C

1,,.1 in autonomous adenoma. Proc. Nati. Acad. Sci. USA90, 1609—1613.

Starke K. (1987) Presynaptic a-autoreceptors. Rev. Physiol. Bio-chem. Pharmacol. 107, 73—146.

Summers R. J. and McMartin L. R. (1993) Adrenoceptors and theirsecond messenger systems. J. Neurochem. 60, 10—23.

Wedegaertner P. B. and Boume H. R. (1994) Activation and depal-mitoylation of G,a. Cell 77, 1063—1070.

Wedegaertner P. B., Wilson P. T., and Bourne H. R. (1995) Lipidmodifications of trimeric C proteins. .1. Biol. Chem. 270, 503—506.

Werner J., Schoffelmeer A. N. M., and Mulder A. H. (1982) Effectsof cyclic AIvIP analogues and phosphodiesterase inhibitors onK + -induced [

3H]noradrenaline release from rat brain slices andon its presynaptic a-adrenergic modulation. J. Neurochem. 39,349—356.

J. Neurochem., Vol. 66, No. 3, 1996