Stimulation of neuropeptide release from sensory and enteric neurons in the guinea-pig by α-latrotoxin

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Pergamon

0306-4522(95)00317-7

Neuroscience Vol. 69, No. 3, pp. 977 984, 1995 Elsevier Science Ltd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00

S T I M U L A T I O N OF N E U R O P E P T I D E R E L E A S E F R O M S E N S O R Y A N D E N T E R I C N E U R O N S IN THE G U I N E A - P I G BY e - L A T R O T O X I N S. A. W A T E R M A N * t a n d C. A. M A G G I : ~ tNeurosciences Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K. :~A. Menarini Farmaceutiche Riunite, Pharmacology Department, Via Sette Santi 3, Florence, 1-50131, Italy

Abstract--~-Latrotoxi from n, black widow spider venom, stimulates exocytosis of small synaptic vesicles at central and peripheral synapses. However, it is widely accepted that neuropeptide-containing large dense-core vesicles are insensitive to the toxin. In the present study, we investigated whether ~-latrotoxin releases neuropeptides from primary afferent and enteric neurons. The guinea-pig renal pelvis is innervated by primary sensory neurons containing tachykinins and calcitonin gene-related peptide, but has no functional cholinergic or noradrenergic motor innervation, ct-Latrotoxin increased the amplitude of spontaneous myogenic contractions of the renal pelvis, and this effect was prevented by prior capsaicin desensitization and by antagonists at neurokinin-I and neurokinin-2 receptors. In the presence of the latter antagonists, ct-latrotoxin decreased the amplitude of the contractions, and this is likely to be mediated by calcitonin gene-related peptide. Thus, ct-latrotoxin releases tachykinins and calcitonin gene-related peptide from capsaicin-sensitive sensory neurons in the renal pelvis. The circular muscle of the guinea-pig distal colon is innervated by excitatory and inhibitory motor neurons, which use a number of transmitters. In the presence of antagonists to block each of the known transmitters apart from the tachykinins, ct-latrotoxin increased the amplitude of spontaneous contractions; this effect was prevented by the prior addition of neurokinin-1 and neurokinin-2 receptor antagonists. Thus, ct-latrotoxin stimulates the release of tachykinins from excitatory motor neurons in the myenteric plexus of the distal colon. In conclusion, this study demonstrates that ~-latrotoxin is able to evoke the release of neuropeptides from both sensory and enteric neurons. This suggests that exocytosis of large dense-core vesicles shares more of the features of exocytosis of small synaptic vesicles than has previously been appreciated. Key words: autonomic, black widow spider venom, exocytosis, large dense-core vesicle.

ct-Latrotoxin, f r o m black w i d o w spider venom, evokes exocytosis of n e u r o t r a n s m i t t e r - c o n t a i n i n g small synaptic vesicles (SSVs) at central a n d peripheral synapses. 28 However, it is generally accepted t h a t n e u r o p e p t i d e - c o n t a i n i n g large dense-core vesicles ( L D C V s ) are insensitive to the toxin. 1'2'22'28 ~ - L a t r o toxin binds to a specific, high-affinity receptor o n the nerve terminal m e m b r a n e , neurexin I~. 37 N e u r e x i n Is also binds the synaptic vesicle m e m b r a n e protein, s y n a p t o t a g m i n . 8'25'26'29 The interaction between neurexin Ict a n d s y n a p t o t a g m i n has been p r o p o s e d as one o f a n u m b e r o f possible m e c h a n i s m s for docking synaptic vesicles at the presynaptic m e m b r a n e . 8,26,29

*To whom correspondence should be addressed. Abbreviations: CGRP, calcitonin gene-related peptide; LDCV, large dense-core vesicle; L-NOArg, NG-nitro-Larginine; NK, neurokinin; SR-48,968, (--)-N-methylN[4 - acetyamino - 4 - phenylpiperidino - 2 - (3,4 - dishlorophenyl)butyl]benzamide; SR- 140,333,(S)- l-[2-[3-(3,4dichlorophenyl)- l-(3-isopropox phenylacetyl)piperidin3 - yl ] ethyl] - 4 - phenyl - 1 - azoniabicyclo [ 2.2.2 ] octane chloride; SSV, small synaptic vesicle. 977

S y n a p t o t a g m i n is t h o u g h t to act as a calcium sensor a n d fusion clamp in regulated exocytosis; calcium entering the nerve terminal following depolarization a n d o p e n i n g of voltage-gated calcium c h a n n e l s binds to the C~ d o m a i n s of s y n a p t o t a g m i n , p r o d u c i n g a c o n f o r m a t i o n a l change a n d possibly dissociation of s y n a p t o t a g m i n from the d o c k i n g / f u s i o n complex. Dissociation of s y n a p t o t a g m i n would then allow fusion o f synaptic vesicles to proceedfl 4'3°'35 Synaptot a g m i n is able to form a ternary complex with N-type voltage-gated calcium channels, the nerve terminal p l a s m a m e m b r a n e protein, syntaxin, a n d neurexin Is. Such a complex would allow vesicles to dock at the sites of the highest calcium c o n c e n t r a t i o n s J 3,25,4~ A recent study suggests t h a t a l t h o u g h syntaxin binds to the N-type calcium channel, it does n o t interact with voltage-gated calcium c h a n n e l s of the P / Q or L subtypes. 34 It has been p r o p o s e d t h a t ~ - l a t r o t o x i n evokes t r a n s m i t t e r release by binding to neurexin I~ a n d subsequently inducing a c o n f o r m a t i o n a l change in the docking/fusion complex, bypassing the requirem e n t for calcium to b i n d to s y n a p t o t a g m i n . 8'26'29 If

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this is t h e case, t h e n o n e w o u l d p r e d i c t t h a t ~ - l a t r o t o x i n w o u l d e v o k e t h e release o f t r a n s m i t t e r s w h i c h (i) a r e s t o r e d in vesicles e x p r e s s i n g s y n a p t o t a g m i n in t h e i r m e m b r a n e s , a n d a r e t h e r e f o r e able to d o c k at t h e p r e s y n a p t i c m e m b r a n e via a n i n t e r a c t i o n with s y n t a x i n , N - t y p e c a l c i u m c h a n n e l s a n d n e u r e x i n I~, a n d (ii) a r e n o r m a l l y c o u p l e d to c a l c i u m influx t h r o u g h N - t y p e c a l c i u m c h a n n e l s . Since s y n a p t o t a g m i n is p r e s e n t o n L D C V s 14"27"33"36"41 a n d n e u r e x i n Ic~ is likely to be p r e s e n t in all n e r v e t e r m i n a l s , 28 ,z-latrot o x i n m a y be able to e v o k e t h e release o f p e p t i d e s c o n t a i n e d in L D C V s . F u r t h e r m o r e , t h e release o f peptides from a number of sensory and autonomic n e u r o n s r e q u i r e s c a l c i u m influx t h r o u g h N - t y p e c a l c i u m c h a n n e l s (e,g. s u b s t a n c e P release f r o m peri p h e r a l s e n s o r y e n d i n g s in g u i n e a - p i g u r e t e r a n d b r o n c h u s , 15'21 s u b s t a n c e P release f r o m e n t e r i c excita t o r y m o t o r n e u r o n s in t h e g u i n e a - p i g c o l o n , ~8 n e u r o p e p t i d e Y release f r o m s y m p a t h e t i c n e u r o n s in t h e h e a r t 5). In t h e p r e s e n t p h a r m a c o l o g i c a l s t u d y , we t h e r e f o r e i n v e s t i g a t e d w h e t h e r ~ - l a t r o t o x i n is a b l e to e v o k e t h e release o f n e u r o p e p t i d e s f r o m s e n s o r y a n d e n t e r i c neurons.

EXPERIMENTAl, PROCEDURES

Male albino guinea-pigs (250 350 g) were killed painlessly by stunning and bleeding via the carotid arteries. The kidneys and distal colon were excised and placed in Krebs solution at room temperature, gassed with 95% 0 2 / 5 % CO 2, pH 7.4, at 3 7 C . The composition of the Krebs solution was (raM): NaCI 119, KCI 4.7, KH2PO4 1.2, N a H C O 3 25, MgSO 4 1.5, o-glucose l l.0 and CaC12 2.5.

Renal pelvis experiments The renal pelvis was dissected from the renal parenchyma as described previously 16"~'2° and mounted in a 5 ml organ bath. Contractions in the circular axis were recorded by an isotonic transducer (load 2 mN). Electrical field stimulation was made by means of platinum wire electrodes placed at the top and bottom of the organ bath and connected to a Grass $88 stimulator. Square wave pulses (pulse width 0.5 ms, 60 V, 5 Hz) were delivered in single trains of 10 s duration. (S)-Ketoprofen (10/~M) was present in the organ bath throughout the experiments to block prostaglandin synthesis and increase the amplitude of stimulus-evoked contractions. 2°3z Sensory neurons in the renal pelvis release tachykinins (substance P and neurokinin A) and calcitonin gene-related peptide (CGRP). 2° The tachykinins produce contraction of the renal pelvis through an action on neurokinin-I (NKI) and N K 2 receptors, and this masks the relaxation produced by C G R P . ~9'2~J In half of the experiments, SR-140,333 and SR-48,968 (each 100nM; NK~ and NK2 receptor antagonist, respectively) 3~ were present throughout to reveal effects of C G R P . The experiments commenced after a 6 0 ~ 0 min equilibration period, when the amplitude and frequency of spontaneous contractions had reached a steady state. Three sets of experiments were performed. In the first set, the effect of ~-latrotoxin was compared to other stimuli. The preparations were electrically stimulated at the end of the equilibration period. After a 20 min rest, during which the contraction amplitude and frequency returned to control levels, l nM c~-latrotoxin was added. A further

50 min later, capsaicin was added. The preparations were electrically stimulated for a third time 20 min after capsaicin addition. In the second set of experiments, the effect of :~-latrotoxin on capsaicin-pretreated preparations was investigated. The preparations were electrically stimulated, and 20 min later capsaicin was added. After a further 20 min, the capsaicin was washed out and ~-latrotoxin added. Fifty minutes later, the preparations were electrically stimulated. The third set of experiments was identical to the second, except that u-latrotoxin was not added. The preparation was electrically stimulated 70 min after the addition of capsaicin.

Distal cohm experiments Segments of distal colon were opened longitudinally and the mucosa and submucosa carefully removed. Full circumference strips 3 m m wide were cut, mounted in 5 ml organ baths and connected to isometric transducers with thread at an initial tension of 10 mN. Spontaneous, circular muscle contractions developed within 10rain. Capsaicin (10/~M) was then added and left in contact with the tissue for 20 min to desensitize extrinsic sensory neurons. ~° After washing, the following drugs were added to block transmission from excitatory motor neurons (atropine, 1/~M), cholinergic interneurons (hexamethonium, 100 #M), noradrenergic nerve terminals (guanethidine, 3/~ M) and inhibitory motor neurons [apamin, 0.1 # M ; NO-nitro-L-arginine (L-NOArg), I00 #M]. In some experiments, SR-140,333 (100 nM) and SR-48,968 (100 nM) were also added to block N K t and N K 2 receptors, respectively. Indomethacin (10/~M) was present throughout the experiments to block prostaglandin synthesis.

Materials Drugs used were: :~-latrotoxin (Alomone Labs, Jerusalem), apamin, capsaicin, hexamethonium, indomethacin, L-NOArg (Sigma, St Louis, MO, U.S.A.), atropine hydrochloride (Serva, Heidelberg, Germany), and guanethidine (ICFI). (S)-Ketoprofen was synthesized by the Chemistry Department of Laboratorios Menarini, Spain. SR-140,333 or (S)-l-[2-[3-(3;4-dichlorophenyl)-l(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl]-4-phenyl- lazoniabicyclo[2.2.2]octane chloride and SR-48,968 or (--)N-methyl-N [4-acetylamino-4-phenylpiperidino-2-(3,4-dichlo rophenyl)butyl]benzamide were a kind gift of Dr X. Emonds-Alt, Sanofi Research, Montpellier, France. Stock solutions of SR-140,333, SR-48,968 and indomethacin were made in 100% dimethylsulphoxide. Dimethylsulphoxide at a final concentration of 0.01 O. 1% did not have any effect on the preparations. ~-Latrotoxin was used at concentration of 1 nM in all experiments. Previous studies have shown that at this concentration and in the presence of calcium, transmitter release from SSVs is sustained for long periods of time (see Ref. 38). Furthermore, our preliminary experiments indicated that this was the lowest concentration that consistently had an effect on the preparations.

Ana(vsis o[ results In experiments on the renal pelvis, the amplitude of contractions following drug treatment or electrical stimulation was measured and expressed relative to the amplitude of the contractions immediately preceding the treatment. For experiments on the distal colon, the contractile activity was integrated over periods of 5 min and expressed relative to the integrated contractile activity before addition of ~-latrotoxin. Repeated measures analysis of variance followed by N e w m a n Keuls tests were used to compare the treatment effects. A probability less than 0.05 was regarded as significant.

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~-Latrotoxin evokes neuropeptide release

RESULTS

Guinea -pig renal pelvis The guinea-pig renal pelvis is innervated by capsaicin-sensitive sensory neurons which release tachykinins and C G R P in response to electrical stimulation or acute capsaicin administration. Electrically evoked neuropeptide release is sensitive to tetrodotoxin and the N-type calcium channel blocker ~o-conotoxin GVIA. This preparation does not receive a functional cholinergic or noradrenergic motor innervation} ° We therefore used this preparation to investigate whether ~-latrotoxin evokes neuropeptide release from sensory neurons.

Effect of ~-latrotoxin on spontaneous activity The isolated guinea-pig renal pelvis preparations showed regular, spontaneous myogenic contractions, as described previously 16'19"2°(Fig. 1). Electrical stimulation for 10 s increased the amplitude of contractions to a maximum of 185 +_ 33% (n = 5) of the control, 1.63 +_ 0.24 rain after the stimulus onset. The contraction amplitude returned to control levels within 20 min. Subsequent addition of 1 nM ~-latrotoxin increased the amplitude of the contractions to a maximum of 136 +_ 14% of the control in a time-dependent manner (Figs 1, 2). The maximum effect of the toxin occurred after approximately 25 rain. Fifty minutes after the addition of the toxin, the amplitude of contractions was still increased (135+_ 15% of control). Addition of capsaicin at this time further increased the contraction amplitude to a maximum of 294 _+ 69% of control. The maximum effect of capsaicin occurred 3.25 +% . _0.75 min after its addition. Electrical field stimulation did not have any effect after capsaicin desensitization. Previous studies have demonstrated that the increase in contraction amplitude produced by electrical stimulation is mediated by tachykinins released from capsaicin-sensitive sensory neurons} ° To determine whether the effect of ~-latrotoxin was also due to transmitters released from these sensory neurons, the toxin was added to preparations which had been desensitized to capsaicin. Under these conditions, ~-latrotoxin did not cause an increase in contraction

amplitude (Fig. 2), suggesting that the increase is mediated by excitatory transmitter(s) from capsaicinsensitive neurons.

Effect of ot-latrotoxin on spontaneous activity in the presence of neurokinin-1 and neurokinin-2 receptor antagonists Electrical stimulation of sensory nerves in the guinea-pig renal pelvis preparation evokes the release of C G R P in addition to tachykinins} ° The inhibitory effect of released C G R P is revealed in the presence of antagonists to block NK~ and N K 2 receptors. The present study confirmed that electrical stimulation in the presence of SR-140,333 and SR-48,968 (NKj and N K z receptor antagonists, respectively) produced a decrease in contraction amplitude to 84_+ 3.5% of control. The contraction amplitude returned to control levels within 20 min. Subsequent addition of ~-latrotoxin decreased the contraction amplitude to a minimum of 79 +- 7.5% of the control. This effect was time-dependent (Figs 2, 3), with the maximum effect occurring approximately 30 min after the addition of the toxin. Fifty minutes after the toxin was added, the contraction amplitude was steady at 84_+ 8.3% of control levels. Addition of capsaicin at this time decreased the contraction amplitude further to 44+_9.4% of control. Electrical field stimulation no longer had any effect after capsaicin desensitization. To determine whether the inhibition of contraction produced by the toxin in the presence of NK~ and NK2 receptor antagonism was due to release of a transmitter from capsaicin-sensitive sensory neurons, capsaicin-pretreated tissues were used. Addition of ~-latrotoxin after capsaicin desensitization did not cause a decrease in contraction amplitude, indicating that the inhibitory transmitter was released from capsaicin-sensitive neurons. It has previously been demonstrated that the electrically evoked inhibition of contractions in the presence of tachykinin receptor antagonists is blocked by the CGRP receptor antagonist, C G R P 8 37"20 The effect of CGRP8 37 was not tested in the present study; however, based on this earlier study, it is likely that the inhibitory effect of

10 mN

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Fig. 1. Recording of contractions of the guinea-pig renal pelvis, showing the effect of ~-latrotoxin. The renal pelvis preparation was mounted in an organ bath and spontaneous myogenic contractions recorded with an isometric transducer. Electrical field stimulation (EFS; 5 Hz, 10 s) produced an increase in contraction amplitude which returned to baseline within 20 min. ~-Latrotoxin (1 nM) produced a slow increase in contraction amplitude which was maintained for 50 min. Capsaicin (10 #M) caused a large, rapid increase in contraction amplitude. An expanded time scale at the beginning of the trace shows individual contractions.

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capsaicin + alpha-latrotoxin+ SR-140,333 + SR-48,968

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alpha-latrotoxin+ SR-140,333 + SR-48,968 80.

capsaicin alpha-latrotoxin + capsaicin

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time (min) Fig. 2. Effect of ct-latrotoxin on the guinea-pig renal pelvis. The filled squares show the effect of ct-latrotoxin alone on renal pelvis preparations. In preparations previously desensitized to capsaicin (filled diamonds), ~-latrotoxin no longer evoked an increase in contraction amplitude. The slight decrease in amplitude of contractions under these conditions also occurred in time controls, in which ~-latrotoxin was not added after capsaicin desensitization (filled triangles). The amplitude of contractions in the presence of ~-latrotoxin alone is significantly greater than that in the latter two conditions for each time point (F~2a00)= 58.68; P <0.00l). Thus, ~t-latrotoxin evoked the release of excitatory transmitter(s) from capsaicin-sensitive primary afferent fibres in the renal pelvis. Whereas ~t-latrotoxin alone produced a large increase in contraction amplitude, in the presence of NK~ and NK 2 receptor antagonists, it produced a decrease in contraction amplitude (open squares). Thus, the toxin evoked the release of an inhibitory neurotransmitter, likely to be CGRP (see text). This decrease in contraction amplitude did not occur in capsaicin-pretreated preparations (open diamonds). This effect was time-dependent (Fls.1001= 1.938; P < 0.05), i.e. the differences were significant from 25 rain onwards. Each curve shows the average and S.E.M. of four to five experiments performed on preparations from different animals. For the sake of clarity, not all standard errors are shown. ~-latrotoxin revealed in the presence o f tachykinin receptor antagonists is mediated by C G R P .

Guinea-pig distal colon circular muscle The circular muscle o f the guinea-pig distal colon is innervated by excitatory and inhibitory m o t o r neurons whose cell bodies lie in the myenteric plexus.

These m o t o r neurons are the final neurons in reflex pathways running in the myenteric plexus and receive inputs from interneurons and sensory neurons. 4 In the present experiments, drugs were used to block transmission from all except the excitatory m o t o r neurons. Excitatory m o t o r neurons use both tachykinins and acetylcholine as transmitters. We

10 mN

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[ capsaicin 20 minutes

Fig. 3. Recording of contractions of the guinea-pig renal pelvis in the presence of NKt and NK 2 receptor blockade, showing the effect of ~-latrotoxin. SR-140,333 and SR-48,968 (each 100 nM) were used to block NK~ and NK 2 receptors. Under these conditions, electrical stimulation produced a decrease in the amplitude of spontaneous contractions, ct-Latrotoxin produced a sustained decrease in the contraction amplitude. Subsequent addition of capsaicin produced a marked decrease in contraction amplitude and frequency. EFS, electrical field stimulation.

~t-Latrotoxin evokes neuropeptide release

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2 mN

5 minutes Fig. 4. Recording of spontaneous contractions of the circular muscle of the guinea-pig distal colon circular muscle, demonstrating the effect of ~t-latrotoxin. Strips of circular, muscle and attached myenteric plexus were mounted in organ baths and circular muscle contraction measured with isometric transducers. A number of drugs was added to block transmission from all but the excitatory motor neurons (see Experimental Procedures). Atropine was also added to isolate the non-cholinergic component of transmission. In these conditions, c(-latrotoxin produced an increase in the amplitude of spontaneous conctractions after a delay of I0 min. added the muscarinic receptor antagonist, atropine, to block the cholinergic component of excitatory neuromuscular transmission and thus isolate the neuropeptide-mediated component, a-Latrotoxin was tested for its ability to evoke neuropeptide release from these autonomic neurons. The circular muscle of the distal colon contracts spontaneously and the amplitude of these contractions was increased significantly by ~t-latrotoxin (Figs 4, 5). The m a x i m u m effect of the toxin occurred after approximately 10min, and apparently disappeared after 20min. To determine whether the excitatory effect of a-latrotoxin was mediated by tachykinins, the experiments were repeated in the presence of antagonists to NK1 and N K 2 receptors.

U n d e r these conditions, a-latrotoxin no longer had an excitatory effect (Fig. 5). Instead, the toxin had an inhibitory effect which became evident 5-10 rain after toxin addition, and was maximal after 15 rain. The difference between the a-latrotoxin effect in the absence and presence of NK~ and NK2 receptor antagonists indicates firstly that the toxin evokes the release of tachykinins which act at N K l and/or N K 2 receptors and, secondly, that the apparently transient effect of the toxin is due to the release of an inhibitory transmitter which masks the effect of the tachykinins. The nature of this inhibitory transmitter iS unknown; however, it is not nitric oxide, since nitric oxide synthase inhibitors were present. Moreover, a-latrotoxin was not acting by opening an

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time (minutes) Fig. 5. Effect of ct-latrotoxin on the guinea-pig distal colon circular muscle, ct-Latrotoxin produced an increase in contraction amplitude which was maximal after 10 min and disappeared by 20 min. In the presence of antagonistsat NKj and NK 2 receptors, the toxin reduced contraction amplitude. This indicates that the increase in amplitude is due to the release of tachykinins. Each curve shows the average and S.E.M. of five experiments performed on preparations from different animals. There was a significant effect of the toxin (F(~.43)= 29.43; P < 0.01) which was time-dependent (F(4,43)= 3.539; P < 0.01).

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apamin-sensitive potassium channel, since apamin was present. The existence of a third inhibitory transmitter in the guinea-pig colon has been demonstrated previously. 17 ~-Latrotoxin is able to stimulate transmitter release in the absence of extracellular calcium.22"28The possibility that the toxin also stimulates neuropeptide release in a calcium-free medium could not be directly addressed in the present experiments involving measurement of smooth muscle contraction because the contractions are calcium-dependent. DISCUSSION

The present study has demonstrated that ~-latrotoxin evokes the release of neuropeptides from both sensory and enteric neurons. The toxin evoked the release of tachykinins and CGRP from capsaicin-sensitive sensory neurons in the guinea-pig renal pelvis and tachykinin release from motor neurons in the myenteric plexus of the guinea-pig distal colon. These results are contrary to the previously accepted notion 1'2'22'2s that neuropeptide-containing LDCVs are insensitive to ~-latrotoxin.

Neuropeptide release from primary affbrent neurons in the renal pelvis The present study has confirmed that electrical stimulation of the guinea-pig renal pelvis evokes the release of tachykinins from capsaicin-sensitive primary afferent neurons. Addition of antagonists at NKt and NK2 receptors unmasks an inhibitory effect of electrical stimulation28 (present study) which is mediated by CGRP co-released from the same population of neuronsfl8 Similarly, a-latrotoxin evoked the release of tachykinins and an inhibitory transmitter, probably CGRP, from these neurons. However, the maximal effect of ~-latrotoxin occurred after 25-30rain, compared with a maximal response to electrical stimulation (10 s train at 5 Hz) within 2 rain. In spite of the differing time courses, ~-latrotoxin appeared to evoke release of transmitter from the same pool as electrical field stimulation, since the magnitude of the response to electrical stimulation either during or after the addition of the toxin was reduced by more than half (results not shown). Acute administration of capsaicin to the renal pelvis preparation evokes massive and rapid release of tachykinins and CGRP from sensory neurons2° (present study). The magnitude of the effect of capsaicin was significantly greater than that of both ~-latrotoxin and electrical stimulation, suggesting that capsaicin is able to release transmitter from pools which are not releasable by ~-latrotoxin or by short-term electrical stimulation. Consistent with this, a number of studies have demonstrated that only a small proportion of the total neuropeptide content of nerve terminals is releasable by depolarization or calcium influx. For example, only 25% of cholecystokinin is releasable from rat hippocampal synaptosomes by

potassium depolarization or ionomycin39 and only 10% of cholecystokinin from rat cerebral cortical synaptosomes;9 only 17% of [Met]enkephalin can be released from crude rat striatal particulate fractions.12 Hfikanson et al. 6 described two releasable pools of tachykinins in sensory nerves of the rabbit iris. Release from the rapid-release pool could be triggered by electrical stimulation, capsaicin and bradykinin. Release from the other, slow-release pool occurred in response to capsaicin and prolonged electrical stimulation (2-3 h), but not to bradykinin. The mechanism(s) limiting the release of neuropeptides in response to certain stimuli is (are) unknown.

Neuropeptide release from autonomic neurons in the myenteric plexus o f the distal colon Enteric autonomic neurons comprise a number of different populations, each of which contains different combinations of transmitters and projects to different targets. 4 Excitatory motor neurons in the guinea-pig colon release acetylcholine and the tachykinins, substance P and neurokinin A, in response to reflex stimulation and electrical field stimulation. The present study has demonstrated that ~-latrotoxin also evokes the release of tachykinins from these neurons. It is conceivable that this was an indirect effect, secondary to the release of another transmitter from SSVs. However, in the present experiments, a number of drugs were present to block the release or effect of acetylcholine, ATP and noradrenaline. Furthermore, effects secondary to transmitter release from sensory neurons were eliminated by prior capsaicin treatment, and effects secondary to prostaglandins and nitric oxide were eliminated by the addition of indomethacin and L-NOArg, respectively. Although an indirect effect secondary to release of an unknown transmitter cannot be excluded, taken together with the results on the renal pelvis, it seems more likely that the toxin directly stimulated neuropeptide release from enteric neurons.

Exoo, tosis o f small synaptic vesicles and neuropeptidecontaining vesicles In recent years, numerous studies have characterized the proteins which are located in the membranes of SSVs and proposed roles for these proteins in exocytosis. The sequential formation of a number of protein complexes is believed to be involved.25'35The calcium-binding protein, synaptotagmin, which is present in some of these complexes, may act as a calcium sensor and as a fusion clamp (see Introduction). Synaptotagmin binds to a number of proteins, including N-type calcium channels, syntaxin and the ~-latrotoxin receptor (see Introduction). ~-Latrotoxin may act by bypassing the requirement for calcium to bind to synaptotagmin (see Introduction). The mechanism of exocytosis of SSVs and neuropeptide-containing LDCVs is usually considered to differ in a number of possibly interrelated ways, including (i) the subtype of voltage-gated calcium

c~-Latrotoxin evokes neuropeptide release channel coupled to exocytosis, (ii) the frequency of stimulation required to evoke exocytosis, (iii) the protein composition of vesicle membranes and (iv) the effect of ~-latrotoxin. These factors will be considered in turn. N-type calcium channels, blocked by co-conotoxin G V I A , are important in the release of non-peptide transmitter from a number of brain regions. On the other hand, it is c o m m o n l y cited that the release of neuropeptides is coupled to calcium influx through L-type calcium channels (e.g. Refs 2 and 22). Whilst this is sometimes the case, neuropeptide release is frequently sensitive to co-conotoxin G V I A , indicating the involvement of N-type calcium channels. In particular, peptide release from neurons in the preparations used in the present study require calcium influx through N-type channels. 18,2° Peptide release is considered to require higher frequencies of nerve stimulation than release of nonpeptide transmitters (e.g. Ref. 2). Never the less, peptide release from some sensory neurons occurs in response to stimulation frequencies as low as 1 Hz (e.g. Refs 15 and 20), and tachykinin release from enteric excitatory m o t o r neurons can be evoked by single pulses. TM M a n y of the proteins on SSVs are found in the membranes of neuropeptide-containing LDCVs. Thus, L D C V s contain synaptobrevin and synaptotagmin. 14'27'33'36'4° Consistent with this and with the involvement of these proteins in exocytosis from LDCVs, peptide release can be blocked by tetanus toxin and botulinum toxin B (which cleave synaptobrevin). 7AlA2,23 Furthermore, antibodies directed against synaptotagmin inhibit exocytosis of L D C V s in chromaffin cells. 3 A previous study found that ~-latrotoxin stimulated the release of acetylcholine but not C G R P from

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frog m o t o r nerve terminals. 22 This has been taken as evidence that exocytosis of SSVs and L D C V s is mediated by different biochemical mechanisms. However, whole black widow spider venom stimulates the release of [Met]enkephalin from crude rat striatal particulate fractions ~2 and, in the present study, we have demonstrated that ~-latrotoxin evokes neuropeptide release from the peripheral endings of sensory neurons and from autonomic neurons. The reason for the lack of effect of ~-latrotoxin on C G R P release in the frog neuromuscular junction is not clear. It would be interesting to test the effect of the toxin on this preparation using a functional measurement or by measuring C G R P release directly. It is possible, however, that there are differences in neuropeptide release from different neuronal types. CONCLUSION

The molecular and biochemical mechanisms involved in exocytosis have recently been a subject of intense debate. It is commonly believed that there are major differences in the exocytosis of SSVs versus LDCVs. Although there are differences between release characteristics in some preparations, there is a significant number of exceptions to this generalization, as we have reviewed above. The results of the present study demonstrate a further similarity in the release processes. The effect of ~-latrotoxin is clearly not restricted to SSVs, and ~-latrotoxin therefore cannot be used to evoke differential transmitter release. In conclusion, exocytoses of SSVs and neuropeptide-containing L D C V s share more features than has previously been recognized. Acknowledgements--This project has been assisted by funds

from The Queen's Trust of Australia. We are grateful to Dr P. Steele for critical discussion of the manuscipt.

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