Journal of NeurochemistrjLippincott—Raven Publishers, Philadelphia© 1998 International Society for Neurochemistry

Presynaptic Inhibition by Concanavalin A: Are a-LatrotoxinReceptors Involved in Action Potential-Dependent

Transmitter Release?

Stefan Boehm and Sigismund Huck

Department of Neuropharmacology, University of Vienna, Vienna, Austria

Abstract: Effects of concanavalin A on transmitter re-leasewere investigated in primary cultures of chick sym-pathetic neurons. The lectin reduced electrically evoked[3H]noradrenalinerelease by up to 30% with half-maxi-mal inhibition at 0.16 bLM. Concanavalin A also reducedthe release triggered by extracellular Ca2~in neurons de-polarized by 25 mM K~or rendered Ca2~-permeable bythe ionophore A231 87. The inhibitory action of concanav-aIm A on electrically evoked release was additive to thatof the a

2-adrenergic agonist UK 14,304. Inactivation ofG~and GIG0 type G proteins by either cholera orpertus-sis toxin did not alter the inhibitory effect of the lectin.Concanavalin A failed to affect the resting membrane po-tential, action potential waveforms, orvoltage-dependentK~and Ca

2~currents. In contrast, the lectin efficientlyblocked both the Ca2~-dependent and -independent a-latrotoxin-induced transmitter release, but only when ap-plied before the toxin. The reduction of electricallyevoked, as well as a-latrotoxin-evoked, release by conca-navalin A was attenuated in the presence of glucose andabolished by methyl a-D-mannopyranoside. The dimericderivative, succinyl-concanavalin A, was significantly lessactive than tetrameric concanavalin A. In bovine adrenalchromaffin cells, which displayed only weak secretoryresponses to a-latrotoxin, concanavalin A failed to alterK~-evokedcatecholamine secretion. These results showthat concanavalin A causes presynaptic inhibition in sympathetic neurons and indicate that cross-linking of a-la-trotoxin receptors may reduce action potential-depen-dent transmitter release. Key Words: ConcanavalinA—a-Latrotoxin—Noradrenaline release—Chick sym-pathetic neurons—Bovine chromaffin cells—Presynap-tic inhibition.J. Neurochem. 71, 2421 —2430 (1998).

latrotoxin (Grasso et al., 1978, 1980; Rubin et a!.,1978; Meldolesi, 1982), a neurotoxin derived from theblack widow spider venom (Meldolesi et al., 1986).This inhibitory effect of the lectin involves binding toa-latrotoxin receptors, which hampers the subsequentassociation of the toxin (Meldolesi, 1982). a-Latro-toxin stimulates neurotransmitter release via at leasttwo different receptors, neurexin Ta, which binds a-latrotoxin in a Ca2+ -dependent manner (Geppert et al.,1998), and a Ca 2+ -independent receptor of a-latro-toxin (CIRL), also named latrophilin (Krasnoperov etal., 1997; Lelianova et al., 1997). Both neurexin Ia(Ushkaryov et al., 1992) and CIRL/latrophilin (Kras-noperov et al., 1997; Lelianova et al., 1997) are glyco-proteins and can thus bind lectins.

Although ConA prevents a-latrotoxin-induced exo-cytosis, unequivocal effects of the lectin on depolariza-tion-dependent neurotransmitter release have not beendetected. The release of GABA from rat brain synapto-somes (Grasso et al., 1978), of acetylcholine at theneuromuscular junction (Rubin et al., 1978), and ofcatecholamines from bovine adrenal chromaffin cells(Trifaro and Bourne, 1981), each due to K~depolar-ization, is not altered by ConA. Furthermore, ConAwas found to either potentiate (Thio et al., 1993) orreduce (Mayer and Vyklicky, 1989) excitatory synap-tic transmission in the hippocampus. Although the po-tentiating effect is likely to be related to the reductionof desensitization of ionotropic glutamate receptors byConA (Thio et al., 1993), the mechanism underlyingthe reductionof synaptic currents in the presence of thelectin remained obscure (Mayer and Vyklicky, 1989).Primary cultures of chick sympathetic neurons provide

The lectin from Canavalia ensiformis, concanavalinA (ConA), has been used frequently to study variousaspects of neuronal function, in particular, neuronalplasticity (for review, see Lin and Levitan, 1991).Despite the multitude of different effects ConA exertsat postsynaptic sites, just one prominent action of thislectin has been described at presynaptic specializa-tions: ConA prevents transmitter release evoked by a-

Received April 27, 1998; revised manuscript received July 20,1998; accepted July 20, 1998.

Address correspondence and reprint requests to Dr. S. Boehm atDepartment of Neuropharmacology, University of Vienna, Waeh-ringerstrasse I 3a, A- 1090 Vienna, Austria.

Abbreviations used: CIRL, Ca2~-independent receptor of a-latro-toxin; ConA, concanavalin A; G protein, GTP-binding protein; NA,noradrenaline; UK 14,304, 5-bromo-N- (4,5-dihydro-1H-imidazol-2-yl) -6-quinoxalinamine.

2421

2422 S. BOEHM AND S. HUCK

an experimental model to investigate mechanisms oftransmitter release, in particular presynaptic modula-tion (for review, see Boehm and Huck, 1997a). Theseneurons release noradrenaline (NA) exclusively atgrowth cones and/or axon terminals, but not at neu-ronal somata (Przywara et al., 1993). NA release fromthese presynaptic specializations can be induced byaction potentials triggered via electrical field stimula-tion, by K~depolarization, by Ca2~ionophores, andby a-latrotoxin (Boehm and Huck, 1996). Electricallyevoked NA release occurs in the absence of autoinhibi-tory feedback, but can be inhibited by activation ofpresynaptic a

2-autoreceptors (Boehm et a!., 1991).This inhibition viaa2-adrenoceptors displays rapid, ag-onist-induced desensitization (Boehm et al., 1995). Toinvestigate the mechanisms underlying this desensiti-zation, we applied ConA, as this lectin has been usedto characterize the desensitization of GTP-binding pro-tein (G protein) -coupled receptors, such as /3-adrener-gic receptors (Lohse et al., 1990). Surprisingly, wefound that the lectin itself caused a significant reduc-tion of electrically evoked [

3HINA overflow. Wetherefore addressed the question as to how ConA mightaffect transmitter release. The results indicate thatConA reduces action potential-dependent transmitterrelease most likely by cross-linking a-latrotoxin recep-tors.

MATERIALS AND METHODS

Neuronal cell culturesThe procedures for dissociating and culturing chick sym-

pathetic neurons have been described in more detail else-where (e.g., Boehm et al., 1991, 1994). Cells were platedon poly-D-lysine-coated (Sigma, no. 1149) tissue culturedishes (Nunc, no. 153066) for electrophysiological re-cordings and on collagen-coated (Biomedical Technologies,no. BT-274) polystyrol discs (diameter 5 mm) for superfu-sion experiments (—~8X i0~cells per disc).

Superfusion experimentsAfter 5 days in vitro, cultures were incubated in 0.03 ,aM

[3H]NA (1,617 GBq/mmol) for 60 mm at 36°C.Thereafter,the culture discs were placed in superfusion chambers asdescribed (Boehm et al., 1991). A buffer consisting of (inmM) NaC1 (120), KC1 (3.0), MgC1

2 (2.0), sucrose (20),HEPES (10), fumaric acid (0.5), sodium pyruvate (5.0),and ascorbic acid (0.57), adjusted to pH 7.4 with NaOH,was continuously passed through these chambers at a rateof 1 ml/min by using a peristaltic pump. The temperatureof the buffer was kept constantly at 25°C.CaC12 (2.0 mM)was either present throughout superfusion or applied as asecretory stimulus as specified in Table 1. The buffer con-tained 20 mM sucrose, which was replaced by either a-D-glucose or methyl a-D-mannopyranoside to investigate thesugar specificity of the actions of ConA. Thetritium releasedfrom cultures of chick sympathetic neurons labelled with[3H]NA consists mainly of unmetabolized neurotransmitter

and parallels the release of endogenous NA (Wakade andWakade, 1988).

Subsequent to a 1-h superfusion period, 4-mm fractionsof superfusate were collected. The manipulations indicated

in Table 1 were performed to induce stimulation-evokedtritium overflow after 72 mm (S

1), 92 mm (S2), and occa-sionally 112 mm (S3) of superfusion. Unless indicatedother-wise, ConA was added to the buffer 4 mm before S2 andkept at a constant concentration until the end of experiments,where the residual radioactivity of cultures was extracted byimmersion of discs in 2% (vol/vol) perchloric acid andsonication. Radioactivity in extracts and collected fractionswas determined by liquid scintillation counting (PackardTriCarb 2100 TR).

The rate of tritium outflow per 4 mm (unstimulated, basaloutflow)was estimated by calculating the amount of radioac-tivity in a 4-mm superfusate fraction as a percentage of theradioactivity in the cells at the beginning of the respectivecollection period (L%; the amount of radioactivity in thecultures at any time during superfusion experiments can becalculated as the sum of the radioactivity remaining in thecells at the end of experiments and that retrieved duringthe collection of superfusate fractions). Stimulation-evokedoverflow of radioactivity corresponded to the difference be-tween total outflow during the collection period of stimula-tion and the estimated basal outflow, which declined linearlythroughout experiments (see, e.g., Fig. 1A); therefore, basaloutflow during periods of stimulation was assumed to beequal to the arithmetic mean of the samples preceding andthose following stimulation. The stimulation-evoked over-flow (in analogy to thebasal outflow) was calculated as thepercentage of the total amount of tritium in cultures at thebeginning of the periods of stimulation, S~,S2. and S3 (S1%,S2%, and S3%).

Most commonly, overflow of tritium was triggered at leasttwice, and ConA was added before S2 (see above). In thesecases, effects were evaluated by calculating theratio betweenthe overflow evoked by S2 and the overflow evoked by S1(S2/S1), and by comparing this parameter to the S2/S1 ratioobtained in the absence of any drug [S1% and S2% valuesmay show considerable variability between different cul-tures, whereas S2/S ratios are remarkably constant (seeBoehm and Huck, 1996)1. The effect of ConA on basaloutflow of tritium was evaluated accordingly, by comparingL% values during the 4-mm periods preceding S2 (L2%) andS (L~%),respectively (L2/L1). In most of the experimentswith a-latrotoxin as secretory stimulus, ConA was appliedbefore the toxin (see Fig. 5A). In these cases, the effect ofConA was quantified by comparing L1% and S~%valuesobtained in the presence of the lectin with the same valuesunder control conditions. Ca

2~-induced tritium overflowafter pretreatment of neurons with the calcium ionophoreA23187 (calcimycin) is poorly reproducible (Boehm andHuck, 1996). Therefore, with this stimulation paradigm,overflow was elicited once only, and S

1% values in the pres-ence of ConA were again compared with those in the absenceof the lectin. To facilitate the comparison of ConA-inducedeffects under theconditions of different kinds of stimulation,values in the presence of the lectin were expressed as apercentage of the corresponding mean control values.

ElectrophysiologyVoltage-activated K~or Ca

2~currents, as well as actionpotential waveforms, were recorded by the whole-cell vari-ant of the patch-clamp technique (Hamill et al., 1981), atroom temperature (20—24°C), from cell bodies of sympa-thetic neurons, as described previously (Boehm et al., 1992;Boehm and Huck, 1996). Patch pipettes were made fromglass capillaries (Science Products, Frankfurt/Main, Ger-

.1. Neurochem., Vol. 71, No. 6, 1998

PRESYNAPTIC INHIBITION BY CONCANAVALIN A 2423

TABLE 1. Basal and stimulated tritium overflow from chick sympatheticneurons labelled with [

3H]NA

Basal Stimulated

Condition L1% Stimulus S1% n

2 rni’vI Ca2~ 0.87 ±0.02 30 pulses, 50 mA, 3 Hz 2.02 ±0.05 151

0 Ca2~/25mAl K~ 0.90 ±0.05 2 mM Ca2~ 0.85 ±0.05 240 Ca2~after LTX0 Ca2~after A23187

2.781.91

±0.27±0.17

2 mM Ca2~2 mM Ca2~

4.96 ±0.421.90 ±0.18

186

0 Ca~ 0.81 ±0.06 0.3 M sucrose 1.28 ±0.10 22

Cultures of chick sympathetic neurons were labelled with [3H]NA and subsequently superfused as described inMaterials and Methods. When appropriate, cultures were exposed to 1 nM a-latrotoxin (LTX) or to 3

1iM A23 187from minutes 48 to 52 of superfusion. Thereafter, the buffer contained either no or 2 mM CaCl2 and 25 mM K~(Na~was reduced accordingly) where indicated. After 60 mm of superfusion, the collection of 4-mm superfusatefractions was started. After 72 mm of superfusion, tritium overflow was triggered by electrical field stimulation,by the addition of Ca

2~,or by the inclusion of sucrose in a Ca2~-freebuffer. Results are shown as percentage ofthe total cellular radioactivity (L

1%, S~%;see Materials and Methods). The basal outflow in the presence of 2 mMCa

2~corresponded to 0.19 ±0.01 nCi (n = 151), and the overflow due to electrical field stimulation correspondedto 0.42 ±0.01 nCi (n = 151).

many) using a Flaming—Brown type micropipette puller(Sutter Instruments). The mntemal (pmpette) solution con-tained (mn mM) KC1 (140), CaCl

2 (1.6), EGTA (10),HEPES (10), Mg-ATP (2), and Li-GTP (2), adjusted topH 7.3 wmth KOH, for the measurement of action potentialsand K~ currents, and (in mM) N-methyl-D-glucamine(115), tetraethylammonium chloride (20), CaC12 (1.6),EGTA (10), glucose (10), HEPES (20), Mg-ATP(2), andLm-GTP (2), adjusted to pH 7.3 with HC1, for the determina-tion of Ca

2~currents. The external bathmng solution wasidentical to the buffer used in superfusion expermments (seeabove) and contained 1 ,aM tetrodotoxin to block voltage-activated Na ± currents when Ca2~or K + currents were re-corded. Action potentials were elicited in current clampmode by the injection of suprathreshold depolarizing cur-rents for I ms. Ca2~currents were elicited in voltage clampmode by depolarizations from a holding potential of —80mV to 0 mY, whereas K~currents were evoked by 1-s rampdepolarizations from —70 to +100 mV. All measurementswere performed at a frequency of 2—3 mm -l by using theCLAMPEX software (pCLAMP, Axon Instruments) and anL/M EPC-7 amplifier (List Medical). Drugs were appliedto the cells under investigation via a five-barrelled needledevice previously described in more detail (Boehm andHuck, 1991).

Catecholamine secretion from bovine adrenalchromaffin cells

Chromaffin cells were isolated from bovine adrenal glandsas described (Boehm and Huck, 1993). Cells were resus-pended in Dulbecco’s modified Eagle’s medium (GIBCO,no. 041-01885M), supplemented as above with the excep-tion of nerve growth factor, which was omitted, and platedon 24-multiwell plates (Nunc, no. 143982) coated with rattail collagen (Biomedical Technologies, no. BT-274). Onday 2 mn vitro, the culture medium was changed, and cytosinearabinoside (final concentration, 10~M) was added.

After 7 days in vmtro, the catecholammne secretion fromchromaffin cells was determined by measuring the releaseoftritium after the cells were loaded with [3H]NA as described(Boehmand Huck, 1993). This procedure accurately reflectsthe release of endogenous catecholamines (Kilpatrick et al.,

1980). After loading (2 h at 36°C)wmth 0.01 ~M [3H]NAand subsequent to three washes, the cultures were incubatedfor 2 h in culture medium at 36°C.All subsequent steps wereperformed at room temperature (20—24°C)with culturessubmerged in the buffer used for superfusion experiments(see above). After a 30-mm adaptation period, the experi-ments were carried out as follows: the basal release duringa 5-mm period was determined in the absence or presenceof 2 mM Ca2 and 3 ,aM ConA, respectmvely. The stmmulatedrelease was then induced by incubating the cultures for 5mm in buffer containing either 1 nM a-latrotoxin or 25 mMKC1 (NaC1 wasreduced accordingly) and 3 fLM ConA, whenappropriate. Finally, the radioactive contents of the cellswere extracted by an incubation in 2% (vol/vol) perchloricacid for 5 mm. Radioactivmty was determined by liquid scm-tillatmon counting. Basal and stimulated tritium release werecalculated as a percentage of the total radioactivity of thecultures at the beginning of the corresponding incubationperiod.

StatisticsAll data are given as arithmetic means ±SEM; n is the

number of cell culture discs in release experiments and thenumber of individual cells in whole-cell recordings. All datapoints were obtained in cultures from at least two differentdissociation procedures (in superfusion experiments, sets ofthree culture discs were treated identically; therefore, n val-ues are multiples of 3, unless a culture disc was lost duringan experiment). Subsequent to a Kolmogorov—Smirnov testof normal distribution, the significance of differences be-tween the means of single data points was evaluated bythe unpaired Student’s t test. Concentration—response curveswere fitted to the experimentally obtained data points byusing the ALLFIT program (DeLean et al., 1978).

DrugsThe following drugs were obtained as indicated: (—)-

[ring-2,5,6- 3H I noradrenaline (1,617 GBq/mmol) fromNEN (Vienna, Austria); a-latrotoxin from Alomone Labs(Jerusalem, Israel); ConA, succinyl-ConA, cholera toxin,pertussis toxin, A23 187 (calcimycin), and tetrodotoxin fromSigma (Vienna, Austria); and5-bromo-N-(4,5-dihydro-lH-

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2424 S. BOEHM AND S. HUCK

FIG. 1. Inhibition of electrically evoked [3HINAre-lease from cultured chick sympathetic neurons byConA. Cultures were labelled with [3H]NAand sub-sequently superfused. After 60 mm of superfusion,the collection of 4-mm superfusate fractions wasstarted. A: Time course of tritiumoutflow in an exper-iment with two periods of electrical field stimulation,S

1 and S2. In cultures represented by squares, ConAwas present as indicated by the shaded bar. Thedata depict tritium outflow as percentage of totalradioactivity in the cells. n = 6. B: Tritium overflowwas triggered by three consecutive periods of electrical field stimulation, S1, S2, and S3, which were started after 72, 92, and 112 mmof superfusion, respectively. ConA (1 ~tM)was added to the cultures represented by open squares 4 mm before, and was removed 8mm after, the beginning of S2. At this latter point of time, sucrose was replaced by methyl a-D-mannopyranoside in the culturesrepresented by filled symbols. Cultures indicated by circles were not exposed to ConA and served as controls. Results are shown asS~/S~,S~/S~,and 53/S1 ratios, respectively; n = 12 at S2 and n = 6 at S3. ***p < 0.001, versus control; **p < 0.01, versus control.C: Concentration—response curve for the inhibition of electrically evoked overflow by ConA (squares) and succinyl-ConA (triangles)as evaluated by changes in S2/S~ratios; experiments were performed as shown in A. n = 6—9.

imidazol-2-yl) -6-quinoxalinamine (UK 14,304) from Re-search Biochemicals Inc. (Natick, MA, U.S.A.). Bulk chem-icals were from Merck (Vienna, Austria).

RESULTS

Inhibition of electrically evoked [3H] NA overflow

from chick sympathetic neurons by ConASubsequent to a 60-mm washout period, tritium out-

flow under continuous superfusion of chick sympa-thetic neurons previously labelled with Ii3H]NA de-clined linearly. Application of electrical fields to trig-ger action potentials caused reproducible increases intritium outflow (Table 1), and the resulting S

2/51 val-ues amounted to 0.80 ±0.01 (n = 17; Fig. 1A). Inclu-sion of ConA (1 jsM) in the superfusion buffer 4 mmbefore the second stimulation (S2) reduced this S2/ S1value to 0.59 ±0.02 (n = 9; Fig. 1A). The inhibitionby ConA (1 ~tM) was irreversible when the buffercontained sucrose as the sugar component. However,the inhibition could be reversed by replacing sucroseby methyl a-D-mannopyranoside (Fig. 1B). The inhib-itory action of ConA on electrically evoked overflowwas concentration-dependent with half-maximal ef-fects at 156.9 ±34.5 nM ConA and a maximum of30.4 ±2.1% inhibition (Fig. 1C). In contrast, unstimu-lated (basal) tritium outflow was not altered by ConA(Fig. 1A). L2/L1 values in the absence of ConAamounted to 0.95 ±0.02 (n = 17) and were not af-fected by the application of ConA (e.g., 3 ,aM ConA,L2/L1, 0.94 ±0.02; 10 1uM ConA, L2/L1, 0.99 ±0.05;p > 0.05 versus control in both cases). When dimericsuccinyl-ConA was applied instead of tetrameric ConA(see Gunther et al., 1973), neither basal (data notshown) nor electrically evoked overflow was altered(Fig. 1C).

The inhibitory action of ConA on electricallyevoked tritium overflow from chick sympatheticneurons does not involve G~,G1, or G0 typeG proteins

Inhibition of transmitter release from sympatheticneurons via presynaptic receptors most commonly in-

volves U proteins of the G~/G0family (see Boehmand Huck, 1997a, for an overview). Therefore, theneuronal cell cultures were treated with pertussis toxin(100 ng/ml for 24 h) to prevent the activation of G1/G0type G proteins. This treatment abolished the inhibitioncaused by the a2-adrenoceptor agonist UK 14,304 (10

as shown before (Boehm et a!., 1992). However,pertussis toxin did not alter the inhibitory action ofConA (Fig. 2A), nor did it affect the amount of electri-cally evoked tritium overflow (S1%; control: 1.93±0.09, n = 12; pertussis toxin: 1.95 ±0.07; n = 12;p > 0.05).

To find out whether proteins of the G5 family mightbe involved in the effect of ConA, cultures were treatedwith cholera toxin (100 ng/ml for 24 h), which elimi-nates G5a from the neurons (Boehm et al., 1996). Thistreatment did not alter the inhibition by ConA, nor did

FIG. 2. Comparison of the inhibitory effects of ConA and thea2-adrenergic agonist UK 14,304 on electrically evoked [

3H]NArelease from chick sympathetic neurons. Cultures were labelledwith [3H]NAand subsequently superfused. After 60 mm of su-perfusion, the collection of 4-mm superfusate fractions wasstarted. ConA (1 or 3

1~M)and/or UK 14,304 (10 riM) wereapplied, and electrical stimulation was performed, as shown inFig. 1A. The changes in tritium overflow were evaluated by S2/S~ratios. A: Neurons were treated with either cholera toxin(ChTX) or pertussis toxin (PTX), both at 100 ng/ml, for 24 hbefore superfusion experiments. *p < 0.05, versus correspond-ing controls; n = 5—9. B: ConA (1 pM) and UK 14,304 (10 pM)were applied either separately or together to untreated cultures;**p < 0.01, versus control; ##p < 0.01, versus both ConA andUK 14,304, applied separately. n = 6.

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PRESYNAPTIC INHIBITION BY CONCANAVALIN A 2425

it affect the reduction of overflow by UK 14,304 (Fig.2A). However, in line with previous results (Boehmet al., 1996), the cholera toxin treatment reduced theamount of electrically evoked tritium overflow (S1%;control: 1.64 ±0.08, n = 12; cholera toxin: 0.53±0.04; n = l2;p <0.001).

The inhibitory actions of ConA and the ~

adrenoceptor agonist UK 14,304 on electricallyevoked tritium overflow are additive

The finding that pertussis toxin abolished the inhibi-tion by UK 14,304, but not the effect of ConA, indi-cated that these two agents reduced tritium overflowvia distinct mechanisms. To corroborate this assump-tion, ConA (1 1.tM) and UK 14,304 (10 ,uM) wereapplied either alone or in combination. ConA reducedelectrically induced tritium overflow by 26.0 ±3.4%(n = 6), and UK 14,304 reduced the overflow by 39.9±2.5% (n = 6). Together, ConA and UK 14,304caused an inhibition by 68.1 ± 4.7% (n = 6; Fig.2B). Hence, the actions of ConA and UK 14,304 areadditive.

ConA does not alter the resting membranepotential, action potential waveforms, or voltage-dependent Ca2+ or K± currents of chicksympathetic neurons

Electrically evoked NA release from sympatheticneurons relies on the generation and propagation ofaction potentials, as evidenced by its tetrodotoxin sen-sitivity (e.g., Boehm eta!., 1991). Hence, ConA mightinhibit transmitter release by altering action potentials.Nevertheless, action potential waveforms were notchanged in the presence of 10 ~uMConA (Fig. 3A).An alternative mechanism to reduce transmitter releaseis a drug-induced hyperpolarization (e.g., Bug et al.,1986), but the resting membrane potential remainedunaltered in the presence of the lectin (Fig. 3A). Like-wise, K~currents induced by ramp depolarizationsfrom —70 to +100 mV were not affected by the lectin(Fig. 3C).

Presynaptic inhibition of sympathetic transmitter release due to the activation of G protein-coupled recep-tors relies on the inhibition of neuronal Ca 2+ channels(Boehm and Huck, 1996). Accordingly, Ca

2~currentstriggered by depolarizations from —80 to 0 mV wereclearly reduced by the a

2-adrenergic agonist UK14,304, but remained unaltered in the presence of 10pM ConA (Fig. 3B). Hence, the electrophysiologicalexperiments failed to detect an ionic basis for the inhib-itory action of ConA.

ConA reduces exclusively Ca2tdependent tritium

overflow from chick sympathetic neurons, but thiseffect is independent of the route oftransmembrane Ca2~entry

To find out whether the inhibition of tritium over-flow by ConA depends on the mechanisms underlyingstimulation-evoked overflow, different stimulation par-adigms were used: (a) neurons were predepolarized

FIG. 3. Lack of effect of ConA on voltage-gated ion channelsin chick sympathetic neurons. All recordings were performed inthe whole-cell configuration ofthe patch-clamp technique (Ham-ill et al., 1981). A: Action potentials. In neurons in current clampmode, 1-ms depolarizing currents were injected to elicit actionpotentials. Traces obtained in the presence of 1 pM tetrodotoxinwere subtracted from traces recorded in the absence of thistoxin to show exclusively active membrane responses. Valuesof the resting membrane potential determined before the actionpotentials are indicated. B: Ca2~currents. Neurons were volt-age-clamped at —80 mV and depolarized to 0 mV for 30 ms. C:K~currents. Neurons were voltage-clamped at —70 mV, and 1-s ramp depolarizations to +100 mV were applied. All the re-cordings shown were obtained before (control), at the end of a2-mm exposure to 10 pM ConA (or 10 pM UK 14,304), and afterwashout of the lectin. The traces shown here are representativeof results obtained in five cells for each type of recording.

by 25 mM K~in the absence of Ca2~,and overflowwas then triggered by the addition of Ca2~(b) neu-rons were rendered Ca2~-permeableby exposure tothe Ca2~ionophore A23187 in the absence of Ca2~,and overflow was again triggered by the addition ofCa2~and (c) neurons were superfused with Ca2~-free buffer, and 300 mM sucrose was included to evokeCa2~-independent tritium overflow due to hypertonic-ity of the buffer (see Table 1). ConA (1

1uM) reducedCa

2~-triggered overflow, whether neurons had beenpredepolarized by K + or treated with the Ca2~iono-phore A23 187, whereas overflow due to sucrose-medi-ated hypertonicity of the buffer was not affected byConA (Fig. 4).

ConA reduces a-latrotoxin-evoked tritiumoverflow from chick sympathetic neurons whenapplied before the toxin

We found recently that a-latrotoxin efficientlyevokes NA release from chick sympathetic neurons invitro (Boehm and Huck, 1996). ConA has long beenknown to prevent the binding of a-latrotoxin to itsreceptors and to reduce the secretagogue action of thistoxin (e.g., Meldolesi, 1982). In the present experi-ments, exposure of neurons to 1 nM a-latrotoxin for4 mm caused a significant increase in tritium outflowin the absence of extracellular Ca2~,and this effectwas potentiated by the addition of 2 mM Ca2~to the

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2426 S. BOEHM AND S. HUCK

FIG. 4. Effects of ConA on [3HINArelease from cultured chicksympathetic neurons evoked by the application of 2 mM Ca2~in neurons predepolarized by 25 mM K~or rendered Ca2~-permeable by exposure to 3 pM A23187, and by 300 mM su-crose in the absence of Ca2~.Cultures were labelled with [3H]NAand subsequently superfused with a Ca2~-free buffer. When ap-propriate, A231 87 was present from minute 48 to 52 of superfu-sion. After 60 mm of superfusion, the collection of 4-mm superfu-sate fractions was started. Stimulations (see Table 1) with su-crose and with Ca2~in K~-depolarizedcells were performedafter 72 (S

1) and 92 mm (S2) of superfusion, and 3 pM ConAwas applied 4 mm before S2. In contrast, the stimulation withCa

2~in A231 87-treated cells was performed once only (after72 mm of superfusion; Si), because Ca2~-triggered overflow ispoorly reproducible in this case (see Boehm and Huck, 1996),and 3 pM ConA was applied 4 mm before S~.Accordingly, ef-fects of ConA were evaluated by comparing S

2/S1 ratios and bycomparing S~%values, respectively. To facilitate the comparisonof ConA effects, all results are given as percentage of corre-sponding controls. *p < 0.05, versus corresponding controls. n= 12 for K~-dependentoverflow, n = 6 for A231 87-dependentoverflow, and n = 11 for sucrose-dependent overflow.

buffer (Fig. 5A; see also Boehm and Huck, 1996).Inclusion of 3 ~uMConA in the superfusion mediumbefore the application of a-latrotoxin reduced thetoxin-induced overflow in the absence as well as pres-ence of Ca

2~,and the overflow amounted to 36.8±3.9% of control (n = 6; p < 0.001 versus control)and to 2.2 ±0.3% of control (n = 6; p <0.001 versuscontrol), respectively (Fig. 5A). The inhibitory actionof 3

1uM dimeric succinyl-ConA in the absence (68.1±5.3% of control, n = 5; p < 0.01 versus control)or presence (73.5 ±4.1% of control, n = 5; p < 0.01versus control) of extracellular Ca

2~was significantlysmaller than that of tetrameric ConA (Fig. 5A). WhenConA (3

1iM) was applied subsequently to a-latro-toxin, the lectin failed to affect the toxin-induced in-crease in tritium outflow, whether extracellular Ca

2~was absent or present, and the values obtained in thepresence of the lectin amounted to 88.1 ±4.6% (n= 6; p > 0.1 versus control) and to 84.1 ±7.4% ofcontrol (n = 6; p > 0.1 versus control), respectively.

For a direct comparison of the effects of ConA onelectrically evoked (Fig. 1A) and a-latrotoxin-induced(Fig. 5B) tritium overflow, neurons were first exposedto the toxin and then overflow was triggered by twosubsequent exposures (each for 60 5; see Table 1) to2 mM Ca2~.ConA (1 poll) was applied 4 mm beforethe second stimulation period (S

2), but failed to altertritium overflow (Fig. SB). S2/S1 ratios of Ca

2~-trig-

gered overflow in a-latrotoxin-treated neurons in thepresence of 1 ~M ConA amounted to 90.8 ±6.7% ofcontrol (n = 6; p > 0.1). For comparison, S

2/S1 ratiosof electrically evoked tritium overflow in the presenceof 1 ~sMConA amounted to 69.6 ±2.1% of control(n = 9; p < 0.001; see Fig. 1A).

The inhibitory actions of ConA on electricallyevoked and a-latrotoxin-induced tritium overflowfrom sympathetic neurons display similarsugar specificities

To obtain evidence as to whether ConA might reduceelectrically evoked and cr-latrotoxin-induced overflow viaa common binding site, we compared the sugar specifici-ties of these two inhibitory actions of the lectin. Concen-tration—response curves for the ConA-induced inhibitionof electrically evoked (Fig. 6A) and a-latrotoxin-evoked(Fig. 6B) overflow revealed that the lectin was somewhatmore potent and more efficacious in reducing a-latro-toxin-induced tritium overflow. However, the effect ofreplacing sucrose, which was normally included in thesuperfusion buffer (20 mM), by either glucose or methyla-D-mannopyranoside altered the two concentration—re-sponse curves in the same way: replacement of the disac-charide sucrose by glucose caused a rightward shift ofboth concentration—response curves, whereas methyl a-D-mannopyranoside abolished all inhibitory actions ofConA (Fig. 6).

ConA does not alter tritium outflow from bovineadrenal chromaffin cells labelled with [~HI NA

To obtain independent evidence that ConA may in-fluence depolarization-evoked NA release through an

FIG. 5. Effects of ConA on a-latrotoxin-evoked [3H]NArelease

from cultured chick sympathetic neurons. Cultures were labelledwith 13H]NA and subsequently superfused with a Ca2~-freebuffer. After 60 mm ofsuperfusion, the collection of4-mm super-fusate fractions was started. From minute 48 to 52 (B) or fromminute 72 to 76 (A) of superfusion, neurons were exposed to 1nM ct-latrotoxin. Ca2~was included in the buffer for 60 s, asindicated by the black bars. The data depict tritium outflow aspercentage of total radioactivity in the cells. A: Time course oftritium outflow: 3 pM ConA or 3 pM succinyl-ConA was included4 mm before a-latrotoxin, as indicated by the shaded bar. °Frac-tions in which p < 0.05 for ConA versus succinyl-ConA and forConA versus control; ~fractionsin which p < 0.05 for ConAversus control only. n = 6. B: Time course of tritium outflow: 1pM ConA was included in the buffer 4 mm before S

2 in thecultures represented by squares, as indicated by the shadedbar. n = 6.

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PRESYNAPTIC INHIBITION BY CONCANA VALIN A 2427

FIG. 6. Comparison of the inhibition of electrically evoked (A)and a-latrotoxin-evoked (B) [3H]NArelease from chick sympa-thetic neurons by ConA: effects of saccharides. Experimentswere performed as shown in Figs. 1A and 5A, respectively. Cul-tures were labelled with [3H]NAand subsequently superfusedwith a buffer containing 20 mM sucrose, 20 mMglucose, or 20mM methyl a-D-mannopyranoside (a-MMP). After 60 mm ofsuperfusion, the collection of 4-mm superfusate fractions wasstarted. A: The buffer contained 2 mM Ca2~,and two periodsof electrical field stimulation, S

1 and S2, were applied after 72and 92 mm of superfusion, respectively. The indicated concen-trations of ConA were applied 4 mm before S2. Results showS2/S1 ratios expressed as percentage of corresponding controls.*p < 0.05, between the ConA-induced inhibition in sucrose andglucose. n = 6—9. B: From minute 72 to 76 of superfusion, theneurons were exposed to 1 nM cr-latrotoxin in Ca

2~-free buffer.The indicated concentrations of ConA were applied 4 mm beforea-latrotoxin. The outflow of tritium subsequentto the applicationof the toxin was evaluated and expressed as percentage of cor-responding controls. *p < 0.05, between the ConA-induced inhi-bition in sucrose and glucose. n = 6.

action on a-latrotoxin receptors, we investigated itseffect on K ±-evoked tritium outflow from bovine adre-nal chromaffin cells labelled with [3HINA. In thesecells, secretory responses to a-latrotoxin are muchsmaller than those of depolarizing stimuli, such as highK~,and require the presence of extracellular Ca2~(Michelena et a!., 1997). Accordingly, a-latrotoxinfailed to trigger tritium outflow from chromaffin cellsin the absence of extracellular Ca2~and exerted onlya weak secretagogue action in the presence of Ca2~(Fig. 7). The secretory effect of a-latrotoxin in 2 mMCa2~was reduced by 98.9 ±0.9% (n = 6;p <0.001)in the presence of 1

1~MConA. K~at 25 mM stimu-lated tritium outflow in the presence of Ca

2~moreefficiently than a-latrotoxin, but 3 ,uM ConA failedto affect K + -induced tritium outflow from chromaffincells (Fig. 7).

DISCUSSION

Despite the plethora of effects the plant lectin ConAhas been reported to exert on various neuronal func-tions, this is the first report to provide direct evidencethat ConA may cause presynaptic inhibition of trans-mitter release. The lectin reduced electrically evoked[3HI NA release from chick sympathetic neurons ina concentration-dependent manner with half-maximaleffects at 0.16

1.tM. This effect arose at a presynapticsite of action, as evidenced by the following data: (a)Sites of transmitter release in primary cultures of chicksympathetic neurons are located exclusively at axons

and axon terminals, and not at the somatodendriticregion (Przywara et al., 1993). (b) Neither the restingmembrane potential nor action potentials that link elec-trical stimuli to the Ca

2~-dependent release of NA [asindicated by tetrodotoxin sensitivity (e.g., Boehm etal., 1991)1 were altered by ConA. (c) Tetrodotoxin-insensitive [and thus action potential-independent (seeBoehm and Huck, 1996)1 NA release due to uniformdepolarization of all parts of the neurons with 25 mMK + was also reduced by ConA. Taken together, ConAacted at or near the active zones to reduce transmitterrelease.

ConA may exert its biological effects either by bind-ing to carbohydrate residues or via saccharide-indepen-dent hydrophobic interactions (Edelman and Wang,1978). In the present experiments, the inhibitory actionof ConA depended on the sugars present in the buffer:(a) the inhibition by ConA was irreversible in sucrose-containing buffer, but could be reversed by methyl a-D-mannopyranoside; (b) replacing sucrose by glucoseattenuated the inhibitory action of ConA; and (c) re-placing sucrose by methyl a-D-mannopyranoside abol-ished the ConA-induced reduction of NA release.Hence, the presynaptic inhibition by ConA was medi-ated by carbohydrate binding and not by alternativehydrophobic interactions. At physiological pH, ConAforms tetramers, and via these tetramers glycoproteinstethered to the lectin become cross-linked. Succinyl-ConA binds the same glycoconjugates as ConA, butfails to induce cross-linkage, because this ConA deriv-ative forms dimers instead of tetramers (Gunther etal., 1973). In the present study, succinyl-ConA failedto alter electrically evoked transmitter release. Thus,

FIG. 7. Effects of ConA and a-latrotoxin on catecholamine se-cretion from bovine adrenal chromaffin cells. Chromaffin cellswere labelled with [3HINAand after a washout period incubatedfor two subsequent periods of 5 mm in buffer containing 2 mMCa2, 1 nMcs-latrotoxin (csLTX), 25 mMK~,and/or 3pM ConA.The left column within each pair represents the first, and theright column represents the second, 5-mm incubation period.Results depict the amount of radioactivity released into the bufferduring one incubation period, expressed as percentage of radio-activity in the cells at the beginning of this period. n = 12. Theamounts of radioactivity released by 25 mM K~in the presenceand absence of ConA are not significantly different from eachother (p > 0.05).

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2428 S. BOEHM AND S. HUCK

the presynaptic inhibition due to ConA required cross-linking of the lectin binding sites.

Previously, activation of U protein-coupled recep-tors was found to cause presynaptic inhibition of trans-mitter release from chick sympathetic neurons in pri-mary cell culture (Boehm et a!., 1991; Boehm andHuck, 1996). Most commonly, G protein-coupled re-ceptors are glycosylated and may thus represent bind-ing sites for ConA. Moreover, multimerization of suchreceptors may result in their activation (Hebert et al.,1996). Hence, activation of presynaptic receptorsmight have mediated the inhibitory action of ConA.However, results obtained with cholera or pertussistoxin argue against such a mechanism: in cultures ofchick sympathetic neurons, pretreatment with thesetoxins prevents the function of receptors linked to U.or G1/G0 type U proteins, respectively (Boehm et a!.,1992, 1996). Accordingly, after pertussis toxin treat-ment, the inhibitory action of an a2-adrenergic agonistwas lost, but the inhibition by ConA remained un-changed. Furthermore, the inhibitory actions of an a2-adrenergic agonist and ConA were additive, which alsopoints to distinct signal transduction mechanisms.Cholera toxin, albeit reducing the amount of electri-cally evoked release (see Boehm et a!., 1996), alsofailed to alter the ConA-mediated inhibition. Havingexcluded G. and G1/G0 proteins from the signallingmechanisms of ConA, members of the Gq/U11 familyremain as possible alternatives. Gq-coupled receptorshave been reported to stimulate NA release from sym-pathetic neurons (Boehm and Huck, 1997b). Hence,an activation of such receptors by ConA should in-crease rather than reduce transmitter release. Alterna-tively, ConA might block Gq-coupled receptors andthereby antagonize the stimulatory action of a pre-viously unrecognized endogenous transmitter thatcould be released in response to electrical field stimula-tion. However, under our experimental conditions, en-dogenously released transmitters are rapidly removeddue to continuous superfusion, and autoregulatorymechanisms are thus unlikely to occur (Boehm et a!.,1991).

In sympathetic neurons, G protein-coupled receptorsregulate excitability and transmitter release most com-monly via a modulation of ion channels, most fre-quently of voltage-activated Ca

2~or K~channels(Hille, 1994). ConA has also beenreported to increasethe activity of K~channels (Lin et al., 1989) and toeither enhance (Mironov and Lux, 1991) or reduce(Golard, 1995) Ca2~currents. However, in our experi-ments neither Ca2~nor K + currents were altered inthe presence of ConA. In addition, action potentialwaveforms, as well as the resting membrane potential,were not affected by the lectin. Taken together, thenegative results obtained in electrophysiological exper-iments indicate that ConA causes presynaptic inhibi-tion without altering the activity of voltage-gated ionchannels.

One well documented action of ConA at presynaptic

sites is its binding to a-latrotoxin receptors and theresulting abolition of a-latrotoxin-induced transmitterrelease (Rubin et al., 1978; Grasso et al., 1978, 1980;Meldolesi, 1982). Accordingly, ConA virtually abol-ished the secretagogue action of a-latrotoxin in chicksympathetic neurons. This result raises the question asto whether the inhibition of electrically evoked releasewas also mediated by binding of ConA to a-latrotoxinreceptors, and our data indicate that this was the case.This conclusion is based on the following observations:(a) ConA reduced transmitter release caused by a-latrotoxin only when applied before the toxin. Once theneurons had been exposed to the toxin, ConA affectedneitherNA release in the absence of extracellular Ca 2+

nor the overflow triggered by the subsequent applica-tion of Ca2~.For comparison, NA release triggeredby the application of Ca2~to neurons rendered Ca2~-permeable by the ionophore A23 187 was reduced sig-nificantly by the lectin. (b) The dimeric analogue, suc-cinyl-ConA, failed to reduce electrically evoked re-lease and was much less efficient than tetrameric ConAin inhibiting a-latrotoxin-induced release. (c) The af-finity of ConA for glycopeptides changes by up tothree orders of magnitude when the configuration ofmannose-containing sugar residues is altered (Baen-ziger and Fiete, 1979; Ohyama et al., 1985). The factthat ConA inhibited both electrically evoked anda-latrotoxin-evoked transmitter release at similar con-centrations indicates that binding to similar, if not iden-tical, carbohydrate residues was involved. This inter-pretation is also supported by the finding that theConA-mediated inhibition of both types of evoked re-lease was attenuated by glucose and abolished bymethyl a-D-mannopyranoside. (d) Alternative mecha-nisms that might underlie the presynaptic inhibitionby ConA, such as the activation of G protein-coupledreceptors or the modulation of ion channels (seeabove), had been excluded. (e) In bovine adrenal chro-maffin cells, a-latrotoxin caused catecholamine secre-tion only in the presence of extracellular Ca2~,andthis secretagogue effect was much smaller than that of25 mM K ~ Furthermore, ConA did not reduce K + -

induced catecholamine secretion from chromaffin cells(see also Trifaro and Bourne, 1981). In contrast, K~-evoked NA release from sympathetic neurons wasclearly reduced by the lectin, and in these cells, a-latrotoxin was the most efficient secretagogue, even inthe absence of Ca2~.Hence, the ability of ConA toinhibit transmitter release correlates with the secreta-gogue action of a-latrotoxin.

The above observations provide evidence that ConAreduces action potential-dependent transmitter releaseby binding to a-latrotoxin receptors. However, the linkbetween binding of ConA to these receptors and thereduction in transmitter release is less clear. Both a-latrotoxin receptors, neurexin Ia (Ushkaryov et al.,1992) and CIRL/latrophilin (Krasnoperov eta!., 1997;Lelianova et al., 1997), are glycosylated and can thusbind ConA. However, the current data are not apt to

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PRESYNAPTIC INHIBITION BY CONCANAVALIN A 2429

reveal whether neurexins, CIRL/latrophi!in, or both a-latrotoxin receptors were involved in the ConA-in-duced presynaptic inhibition. On one hand, the inhibi-tory action of ConA on depolarization-evoked releasewas not found in chromaffin cells (even though thelectin antagonized the secretagogue action of a-latro-toxin in this preparation); this would favor a role ofneurexin Ia, because adrenal chromaffin cells, unlikeneurons, do not express neurexins (Ushkaryov et a!.,1992), whereas they do express CIRL/latrophilin(Krasnoperov et a!., 1997). On the other hand, ConAinhibited a-latrotoxin-evoked release not only in thepresence of Ca2~,but also in its absence; this wouldfavor CIRL/latrophilin as the site of action for thelectin. Regardless of which of the two a-latrotoxinreceptors was involved in presynaptic inhibition, theintrinsic ability of ConA to form multimers was a pre-requisite for its effect on action potential-dependenttransmitter release (as evidenced by the lack of effectof succinyl-ConA). In this context, it is interesting tonote that the interaction of a-latrotoxin with its recep-tors also leads to their multimerization, which is be-lieved to play a role in the action of the toxin ontransmitter release (Petrenko et al., 1993). Further-more, a-latrotoxin not only causes massive vesicle ex-ocytosis, but also reduces stimulation-evoked transmit-ter release from central (Capogna et a!., 1996) andperipheral (Ceccarelli and Hurlbut, 1980) neurons.Hence, cross-linkage of a-latrotoxin receptors,whether through toxin or ConA binding, appears suf-ficient to disorganize the process of Ca2~-dependenttransmitter release in order to cause presynaptic inhibi-tion.

Acknowledgment: The excellent technical assistance ofG. Koth, A. Motejlek, and K. Schwarz is gratefully acknowl-edged. The authors thank M. Freissmuth and V. O’Connorfor helpful comments about the manuscript. This study wassupported by grants from the Austrian Science Foundation(FWF; P12997-MED) and from the “Jubilhumsfonds derOsterreichischen Nationalbank” (no. 6821), both to SB..

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