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In vitro and in vivo evidence of neurotensin release from preganglionic axon terminals in the stellate ganglion of the cat
BRAIN RESEARCH ELSEVIER
Brain Research 640 (1994) 131-135
Research Report
In vitro and in vivo evidence of neurotensin release from preganglionic axon terminals in the stellate ganglion of the cat E. Maher, B. Bachoo, C. Polosa * Department of Physiology, McGill University, 3655 Drummond Street, Montreal H3G 1Y6, Que., Canada (Accepted 16 November 1993)
Abstract
We have previously shown that the neurotensin (NT) store in preganglionic axon terminals of the cat stellate ganglion (SG) is reversibly depleted by prolonged preganglionic stimulation. The present study addresses the questions of whether the preganglionic axon terminals release NT in response to depolarizing stimuli in vitro and whether in vivo NT is released by the tonic firing of the sympathetic preganglionic neurons. Slices of the SG of the anaesthetized cat, maintained in oxygenated Ringer solution, released NT. The efflux increased when the K concentration was increased from 5 to 25 or 45 mM or when veratridine was added to the medium. In Ca-free medium, efflux was suppressed. The effect of veratridine was blocked by tetrodotoxin (TI'X). In awake, freely moving cats, in which TFX was applied for 4 days to the preganglionic input of the right SG, the NT content of this ganglion doubled by comparison with the left SG. Since NT accumulates proximal to a ligature on the preganglionic input of the SG, the increased NT content is likely to result from suppression of action potential-dependent release while influx into the terminals persists. This result suggests that the steady state of the NT store in sympathetic preganglionic terminals is the result of a steady influx from the soma balanced by action potential-dependent loss, presumably release.
Key words: Preganglionic neuron; Sympathetic ganglion; Tonic activity; Neuropeptide turnover; Neuropeptide release
1. Introduction
The peptide neurotensin (NT) is present in preganglionic axons in the stellate ganglion (SG) of the cat [3,6]. N T undergoes complete processing in the sympathetic preganglionic neuron soma [15], is packaged in large dense-cored vesicles [19] and accumulates at the central but not at the distal side of a ligature on the preganglionic axons which project to the SG [15]. The accumulation suggests a steady net flux of N T from the preganglionic neuron soma to the terminals. If the accumulation rate at the ligature is an indication of the rate of N T delivery to the axon terminals, loss of N T by release a n d / o r intraterminal degradation must be postulated to maintain the steady state of the N T store in the terminals. The H P L C profile of extracts of control and stimulated SG showed a dominant immunoreactive peak, comprising 94% of the total immunoreactivity,
* Corresponding author. Fax: (1) (514) 398-7452. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 5 3 9 - F
which coeluted with synthetic N T (1-13) [15]. The lack of significant amounts of N T degradation products in ganglion extracts suggests that intraterminal degradation is not an important component of N T turnover. Taken together, these findings suggest that under physiological conditions the anterograde flux of N T in preganglionic axons may be balanced by release. Since tonic firing is a characteristic property of sympathetic preganglionic neurons [20], the release may be the result of the ongoing traffic of action potentials. The present report presents evidence of Ca-dependent release of N T from slices of the SG and describes the results of an in vivo test of the hypothesis that the tonic activity of preganglionic neurons releases NT. To this end, action potential conduction into the preganglionic terminals of the SG was blocked by chronic tetrodotoxin (T-FX) application to the intact preganglionic input of the ganglion in awake, freely moving cats. This procedure resulted in a marked increase in the N T store of the ganglion in the absence of evidence of sprouting. This finding suggests that N T is continu-
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ously r e l e a s e d by t h e t o n i c activity o f t h e p r e g a n g l i o n i c neurons.
2. Materials and methods Adult cats of either sex (3.1-5.8 kg) were anaesthetized with Na pentobarbital (35 mg/kg i.p. followed by 3 m g / k g / h i.v.). The general preparation of the animals was, as described in the preceding paper [16]. Heart rate was monitored with a tachograph triggered by the arterial pressure pulse and was continuously recorded, together with arterial pressure, on a Grass 7 polygraph. In the experiments testing the effect of chronically blocking the tonic firing of the sympathetic preganglionic neuron terminals in the SG on the NT store of this ganglion (n = 4), the right (RSG) and left SG (LSG) were approached retropleurally by bilaterally removing a short segment (1-2 cm) of the second and third rib close to the costovertebral junction. The tip of a silastic catheter (o.d. 1 mm) was positioned in the region where the preganglionic inputs to the SG (second thoracic white ramus (WRT2), WRT 3 and the sympathetic trunk) converge to enter the ganglion, The catheter, secured with several ligatures (10-0 suture) to the connective tissue sheath of the thoracic sympathetic trunk down to the level of WRT 4, was connected to a mini-osmotic pump (Alzet 2001; 1 p.l/h), containing a T r x solution (1/zg//zl) on the right side and the vehicle (citrate buffer, pH = 7.2) on the left side which was implanted s.c. in the mid-axillary region. After implanting the catheters and pumps, the chest was closed and the animal given postoperative care which included antibiotics. 4 days later, these cats were anaesthetized again, as described above, and the SG excised bilaterally to be assayed for NT content. Since previous data suggest that NT in the SG is exclusively in preganglionic axons [6,15], a change in ganglion content was assumed to be due to a change in preganglionic axon terminal content. Before excision, the osmotic pumps and catheters were removed and a bipolar silver hook electrode connected to a Grass $88 stimulator was placed on the WRT 3 as welt as on the sympathetic trunk between the caudal pole of the SG and WRT 4. These nerves were stimulated with trains of 2 Hz 30 s (pulse duration 0.5 ms, amplitude 10 V). On the right TTX-treated side, this stimulation caused no change in heart rate. After the preganglionic nerves, previously exposed to TTX, had been irrigated with warm sterile saline for 30 min, the stimulus train produced a brisk cardioacceleration (from 189_+ 8 to 227 + 12 bpm, n = 4). In the acute experiments of measurement of NT release, the RSG and LSG were excised after removal of the first three ribs bilaterally. Each ganglion was sliced into 10-12 slices of 500/~m thickness and placed in a 3-ml test tube containing Ringer solution at room temperature (22°C) of the following composition (in mM): NaCI 130, KC1 5, MgC12 1, CaCI 2 2, KH2PO 4 1, NaHCO 3 12 and glucose 11, which was bubbled with 95% 0 2 - 5 % CO 2 and contained the endopeptidase 24:11 inhibitor, thiorphan (10 p.M), to minimize NT degradation [7,9]. For measuring NT release, the slices from each ganglion were incubated in 1 ml of the Ringer solution (or of the modified Ringer solutions, see below) for 10 rain at 22°C. In 18 experiments, the K concentration of the solution was increased to 12 or 25 or 45 mM by iso-osmotic substitution of NaCI with KCI. In four experiments, veratridine (40 ~M) with or without TTX (100 /zM) was added. In five experiments, CaCI 2 was substituted with MgCI 2 (8 raM). At the end of the 10-min incubation period, the slices were placed on ice for later determination of NT content and the tube was spun at 2200 rpm for 5 min. The supernatant was evaporated to dryness in a Savant Speed Vac Concentrator and stored at -20°C until the radioimmmunoassay was performed, as described below. The release data are reported as fmol NT/ganglion/min. For determination of NT content of the slices or of the intact ganglion, the tissue was homogenized in ice-cold 0.5 M acetic acid and boiled for 20 min, followed by centrifugation for 20
min at 1000x g. The supernatant was evaporated to dryness and stored at -20°C until analysis. The radioimmunoassay was performed, as described in the preceding paper [16], on 0.1 ml of the ganglion extract (reconstituted in 1 ml assay buffer) or 0.1 ml of the reconstituted incubation medium of the slices or 0.1 ml of a NT standard solution (25-2500 pmol/l NTl_13; Sigma Chemical). In all experiments, one SG served as experimental tissue while the contralateral was used as control since, as reported previously, NT content in RSG and LSG is not significantly different [15]. Data shown are means+ S.E.M. Statistical analysis was performed using the computer program EPISTAT. A paired Student's t test was used to evaluate differences in NT content between tissue of the RSG and LSG sides. Group means were compared using one-way ANOVA. Significance was assigned to P levels of < 0.05.
3. Results 3.1. Release o f N T by S G in vitro I n sliced g a n g l i a , i n c u b a t e d in R i n g e r s o l u t i o n f o r u p to 30 m i n , t h e N T c o n t e n t was 1231 + 106 f m o l / g a n g l i o n ( n = 5). T h i s is s i m i l a r to t h e N T c o n t e n t o f whole, acutely excised ganglia reported previously (1292 + 71 f m o l / g a n g l i o n , P > 0.05) [15]. I n a b s e n c e o f t h i o r p h a n , N T was n o t d e t e c t e d in t h e i n c u b a t i o n m e d i u m . T h e e f f i c a c y o f t h i o r p h a n in p r e v e n t i n g N T b r e a k d o w n was m e a s u r e d by i n c u b a t i n g S G slices w i t h e x o g e n o u s N T (100 fmol): a f t e r s u b t r a c t i o n o f t h e spontaneous efflux (see below), recovery of the added N T i n c r e a s e d f r o m b e l o w t h e l i m i t o f d e t e c t i o n (10 f m o l ) in a b s e n c e o f t h i o r p h a n to 81 + 7 f m o l in p r e s e n c e o f t h i o r p h a n (10 / z M , n = 3, P < 0.05). I n sliced g a n g l i a i n c u b a t e d in n o r m a l R i n g e r s o l u t i o n , t h e r e w a s s p o n t a n e o u s e f f l u x o f N T o f 9.7 + 0.2 f m o l / g a n g l i o n / m i n ( n = 10). N T e f f l u x in 12 m M K ÷ was n o t signific a n t l y d i f f e r e n t f r o m t h a t o b t a i n e d in n o r m a l R i n g e r (10.3 + 0.4 f m o l / g a n g l i o n / m i n , n = 4, P > 0.05). W h e n t h e g a n g l i a w e r e i n c u b a t e d in R i n g e r s o l u t i o n c o n t a i n i n g 25 m M o r 45 m M K ÷, t h e e f f l u x i n c r e a s e d to 12.4 + 0.9 f m o l / g a n g l i o n / m i n ( n = 4, P < 0.05 vs. n o r m a l R i n g e r ) a n d 17.6 + 1.3 f m o l / g a n g l i o n / m i n (n = 7, P < 0.05 vs. n o r m a l R i n g e r ) , r e s p e c t i v e l y . T h e s e d a t a a r e s u m m a r i z e d in Fig. 1. S i m i l a r r e l e a s e r a t e s w e r e o b t a i n e d w i t h 45 m M K at 3 7 ° C (16.8 + 2.0 f m o l / g a n g l i o n / m i n , n = 3, P > 0.05 vs. 45 m M K ÷ at 22°C). W h e n t h e g a n g l i a w e r e i n c u b a t e d in R i n g e r s o l u t i o n c o n t a i n i n g v e r a t r i d i n e (40 / z M ) , N T e f f l u x i n c r e a s e d f r o m 8.6 + 0.8 to 22.6 + 1.6 f m o l / g a n g l i o n / m i n ( n = 4, P < 0.05 vs. n o r m a l R i n g e r ) . T h e i n c r e a s e in r e l e a s e p r o d u c e d by v e r a t r i d i n e w a s b l o c k e d by T T X ( 1 0 0 / x M ) (7.8 + 0.4 f m o l / g a n g l i o n / m i n , n = 4, P > 0.05 vs. n o r m a l R i n g e r ; Fig. 2). W h e n t h e g a n g l i a w e r e i n c u b a t e d in C a - f r e e R i n g e r , t h e s p o n t a n e o u s N T e f f l u x (0.8 + 0.03 f m o l / g a n g l i o n / m i n , P < 0.05 vs. n o r m a l R i n g e r , n = 5) as w e l l as t h e N T e f f l u x p r o d u c e d by 45 m M K ÷ (0.9 + 0.04 f m o l / g a n g l i o n / m i n , P < 0.05 vs. R i n g e r w i t h 45 m M K ÷, n = 5) w e r e m a r k e d l y r e d u c e d . T h e s e d a t a a r e s u m m a r i z e d in Fig. 3.
E. Maheret aL / Brain Research 640 (1994) 131-135 2o E "E
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Fig. 1. Spontaneous and K-evoked NT release from SG slices. Slices incubated for 10 rain at 220C in Ringer solution containing 2 mM Ca and increasing concentrations of K (in mM): 5 (n = 10), 12.5 (n = 4), 25 (n = 4), 45 (n = 7). Concentrations of > 5 mM were obtained by iso-osmotic substitution of NaCI with KCI. * P < 0.05 vs. 5 mM K.
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Fig. 4. Effect of chronic TI'X block of action potential conduction in preganglionic input on NT content of SG. Duration of block: 4 days. Control is sham-treated LSG (n = 4). TTX is SG ipsilateral to block (n = 4). * P < 0.05 vs. sham-treated control.
(n = 4, P < 0.05; Fig. 4). N T c o n t e n t in t h e s h a m t r e a t e d L S G was n o t significantly d i f f e r e n t ( P > 0.05) from the previously reported content of untreated SG [151.
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Fig. 2. Veratridine-evoked NT release from SG slices. Slices incubated for 10 min at 22°C in Ringer solution containing 2 mM Ca, 5 mM K (basal, n = 4) to which veratridine (40/xM, n = 4) or veratridine and TTX (100 ~M, n = 4) were added. * P < 0.05 vs. basal.
3.2. N T content in SG with chronic T T X block o f preganglionic input I n cats in which t h e intact p r e g a n g l i o n i c i n n e r v a t i o n o f t h e R S G was t r e a t e d with T T X for 4 days, t h e N T c o n t e n t o f t h e R S G was 2296 + 189 fmol while t h e N T c o n t e n t o f t h e s h a m - t r e a t e d L S G was 1196 + 149 fmol
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T h e d a t a p r e s e n t e d show t h a t K + - e v o k e d d e p o l a r ization o f slices o f t h e cat S G r e l e a s e s NT. This effect was s u p p r e s s e d in C a - f r e e m e d i u m . Since N T in t h e S G is in s y m p a t h e t i c p r e g a n g l i o n i c t e r m i n a l s [6,15], specifically l o c a l i z e d within l a r g e d e n s e - c o r e d vesicles [19], t h e s e findings i m p l y t h a t d e p o l a r i z a t i o n o f t h e p r e g a n g l i o n i c axon t e r m i n a l s causes exocytosis o f N T c o n t a i n i n g large d e n s e - c o r e d vesicles in a C a - d e p e n d ent manner. Morphological evidence of Ca-dependent, a c t i o n p o t e n t i a l - e v o k e d , exocytosis o f large d e n s e - c o r e d vesicles f r o m p r e g a n g l i o n i c axon t e r m i n a l s in cat superior cervical g a n g l i o n has r e c e n t l y b e e n r e p o r t e d [30]. The veratridine-evoked TTX-sensitive NT release from S G slices suggests t h a t m e c h a n i s m s a c t i v a t e d by action p o t e n t i a l s can c a u s e r e l e a s e o f N T - c o n t a i n i n g l a r g e d e n s e - c o r e d vesicles. R e l e a s e o f N T has b e e n previously shown from t h e p e r f u s e d a d r e n a l g l a n d o f t h e cat [10] a n d f r o m slices o f m o u s e h y p o t h a l a m u s [13]. T h e d a t a also show t h a t in awake, freely moving cats, t h e N T c o n t e n t o f t h e S G n e a r l y d o u b l e d a f t e r 4 days o f c o n t i n u o u s T T X infusion on t h e intact p r e g a n g l i o n i c input. T T X a p p l i e d locally to axons, at c o n c e n t r a t i o n s which b l o c k p r o p a g a t i o n o f a c t i o n p o t e n t i a l s , d o e s n o t affect axonal t r a n s p o r t m e c h a n i s m s o r p r o t e i n synthesis in t h e p e r i k a r y a [12,22]. T h e r e f o r e , t h e i n c r e a s e in N T c o n t e n t o f p r e g a n g l i o n i c t e r m i n a l s is unlikely d u e to i n c r e a s e d synthesis o r r a t e o f axonal t r a n s p o r t o f NT. N o e v i d e n c e has b e e n r e p o r t e d t h a t a c t i o n p o t e n tials m a y c a u s e i n t r a t e r m i n a l r e l e a s e o f large d e n s e c o r e d vesicle c o n t e n t followed by p r o t e o l y t i c d e g r a d a tion. A s m e n t i o n e d in t h e I n t r o d u c t i o n , a f t e r 20 m i n o f p r e g a n g l i o n i c s t i m u l a t i o n at 40 Hz, no significant a m o u n t o f N T d e g r a d a t i o n p r o d u c t s was i d e n t i f i e d by
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E. Maher et aL / Brain Research 640 (1994) 131-135
HPLC in SG extracts [15]. The possibility that the increased NT content of the ganglion with TTX-treated input was due to sprouting of the preganglionic axons, resulting in the addition of new varicosities with normal NT content, is made unlikely by the finding, in a study of the ultrastructure of the cat superior cervical ganglion, that the number and size of synaptic boutons were unchanged after 4 days of ] q ' X block of the preganglionic input [31]. Even if sprouting had occurred, an increase in NT content may not be a necessary consequence of it because, when sprouting was produced by partial denervation of the superior cervical ganglion, ChAT activity in the ganglion was unchanged, suggesting that the amount of enzyme present in each axon before sprouting was distributed over a greater number of varicosities after sprouting [11]. If the increased NT content of the SG is not the result of increased influx from the soma nor of suppression of action potential-dependent intraterminal degradation nor of sprouting, it may be the result of suppression by TTX of action potential-dependent ongoing release while supply by axonal transport remains constant. The in vitro data presented here show that NT is released by a Ca-dependent mechanism. Suppression by T T X of Ca-dependent exocytosis is, therefore presumably, the mechanism responsible for N T accumulation in the terminals. On this basis, it may be postulated, therefore, that one component of the turnover of NT in preganglionic axon terminals is release by the ongoing action potential traffic which is a characteristic property of preganglionic axons [20]. If it is assumed that the difference in NT content between the TTX-treated RSG and the sham-treated LSG (1087 5- 99 fmol) was the result of a constant rate of accumulation over the 4 days of TI'X treatment, one fourth of this amount (272 fmol or 23% of the ganglion content) would be the amount accumulated in 1 day. If it is assumed that, in absence of action potential traffic, there was no loss of NT by the terminals and that the accumulation in the presence of T'I'X equals the amount released per day in absence of TTX, this fraction of the ganglion content would turn over in 1 day. The mean rate of spontaneous firing of sympathetic preganglionic neurons in anesthetized or unanesthetized decerebrate cats is low, typically 1-2 Hz [20]. Electrical stimulation of the preganglionic input to the SG at 2 Hz for 100 min (12000 pulses) produced no depletion of NT content of the SG while stimulation with the same number of pulses at 40 Hz reduced content by 30% [15]. Thus, the frequency of the spontaneous firing of the sympathetic preganglionic neuron may seem inadequate to produce release and, by its suppression, accumulation of NT. This inconsistency may be reconciled in several ways. First, the firing frequency of sympathetic preganglionic neurons in awake, freely moving cats is not known: it may be
higher than in the anaesthetized or decerebrate, unanaesthetized, cat and be in a range which can cause release of NT from large dense-cored vesicles. Second, in the anaesthetized or decerebrate cat, a large fraction of the tonically active sympathetic preganglionic neurons fire in bursts, coincident with the inspiratory phase of the respiratory cycle, with instantaneous frequencies as high as 10-20 Hz [3]. Stimulation of the SG input at these frequencies depletes the NT content of the SG [15]. Third, the probability of release of N T may vary with impulse frequency: at frequencies of 1-2 Hz, the probability of release may be so low that loss (or accumulation when release is blocked by TTX) may only be measurable on the time scale of days. At the frog neuro-muscular junction, stimulation of the motor nerve with 2 Hz for 24 h produced a significant depletion of large dense-cored vesicle number in the motor axon terminals [14]. On the basis of the NT accumulation caused by TTX, it is estimated that 272 fmol NT are released by the SG over 24 h. By contrast, electrical stimulation at 40 Hz causes release of the same amount of NT by the SG in 6.5 min [15], suggesting a much higher probability of release at 40 Hz than at the frequency of the tonic activity of the sympathetic preganglionic neuron. Mechanisms which may encode frequency of action potentials at the nerve terminals into efficacy of stimulus-neuropeptide release coupling are activation of specific Ca channels [18] as well as activation of the second messengers protein kinase A [25], protein kinase C [23] and Ca-calmodulin kinase [17]. Fourth, in peptide-secreting endocrine cells, a very small proportion of secretory granules are found close to the cell membrane and may be readily released by single action potentials [5,8,24] while the majority of the secretory granules are linked to actin microfilaments and form a reserve pool. The mobilization of this reserve pool into the readily releasable pool is poorly understood. It may involve dissociation of the actin cytoskeleton by proteases activated by the high levels of cytosolic Ca achieved during high-frequency stimulation [26]. It is possible, therefore, that in the preganglionic axon terminal a small subset of NT-containing large dense core vesicles is docked near release sites and can readily undergo exocytosis in response to action potentials at low frequencies. Detection of the resulting NT release as a decrease in NT content of the ganglion extract may be difficult, however, because of the small amount released. Concerning the physiological consequences of NT release, activation of the NT receptor can lead to phosphoinositide synthesis [29] and inhibition of cAMP production [4]. Therefore, if the N T released by preganglionic axon terminals acts on pre- or postsynaptic receptors, in addition to its postsynaptic depolarizing action [2], it could modify functions of membranebound receptors [28] as well as the mechanism of
E. Maher et al. / Brain Research 640 (1994) 131-135
release of small molecule transmitters [21]. In addition, NT has been shown to have neurotrophic properties by increasing the survival of chick ciliary ganglion cells in culture [27]. Acknowledgements. This work was supported by the Quebec Heart Foundation and the Medical Research Council of Canada. E. Maher was the holder of a Medical Scientist Award of the Heart and Stroke Foundation of Canada. The preparation of the manuscript by C. Pamplin is gratefully acknowledged.
5. References [1] Bachoo, M., Ciriello, J. and Polosa, C., Effect of preganglionic stimulation on neuropeptide-like immunoreactivity in the stellate ganglion of the cat, Brain Res., 400 (1987) 377-382. [2] Bachoo, M. and Polosa, C., Cardioacceleration produced by close intra-arterial injection of neurotensin into the stellate ganglion of the cat, Can. J. Physiol. Pharmacol., 66 (1988) 408-412. [3] Bachoo, M. and Polosa, C., Properties of the inspiration-related activity of sympathetic preganglionic neurones of the cervical trunk in the cat, J. Physiol., 385 (1987) 545-564. [4] Bozou, J.C., de Nadai, F., Vincent, J.-P. and Kitabgi, P., Neurotensin, bradykinin and somatostatin inhibit cAMP production in neuroblastoma NIEll5 cells via both pertussis toxin sensitive and insensitive mechanisms, Biochem. Biophys. Res. Commun., 161 (1989) 1144-1150. [5] Burgoyne, R.D., Control of exocytosis in adrenal chromaffin cells, Biochern. Biophys. Acta, 1071 (1991) 174-202. [6] Caverson, M.M., Bachoo, M., Ciriello, J. and Polosa, C., Effect of preganglionic stimulation or chronic decentralization on neurotensin-like immunoreactivity in sympathetic ganglia of the cat, Brain Res., 482 (1989) 365-370. [7] Cheeler, F., Mazella, J., Kitabgi, P. and Vincent, J.P., Catabolism of neurotensin by neural (neuroblastoma clone NIEll5) and extraneural (MT29) cell lines, Peptides, 7 (1986) 1071-1077. [8] Chow, R.M., von Ruden, L. and Neher, E., Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells, Nature (London), 356 (1992) 60-63. [9] Coquerel, A., Dubuc, I., Menard, J.F., Kitabgi, P. and Costentin, J., Naloxone-insensitive potentiation of neurotensin hypothermic effect by the enkephalinase inhibitor thiorphan, Brain Res., 398 (1986) 386-389. [10] Corder, R., Mason, D.F.J., Perrett, D., Lowry, P.J., ClementJones, V., Linton, E.A., Besser, G.M. and Rees, L.H., Simultaneous release of neurotensin, somatostatin, enkephalins, and catecholamines from perfused cat adrenal glands, Neuropeptides, 3 (1982) 9-17. [11] Fonnum, F., Maehler, J. and Nja, A., Functional, structural and chemical correlations of sprouting of intact preganglionic sympathetic axons in the guinea-pig, J. Physiol. (London), 347 (1984) 741-749. [12] Kanda, K., Sato, M., Mashizume, K. and Yamada, J., The effect of blocking nerve conduction on retrograde HRP labeling of rat motoneuron, Neurosci. Lett., 99 (1989) 153-156. [13] Kitabgi, P., DeNadai, F., Cuber, J.C., Dubuc, I., Novel, D. and Costentin, J., Calcium-dependent release of neuromedin N and neurotensin from mouse hypothalamus, Neuropeptides, 15 (1990) 111-114. [14] Lynch, K., Stimulation-induced reduction of large dense core
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vesicle number in cholinergic motor nerve endings, Brain Res., 194 (1980) 249-254. [15] Maher, E., Bachoo, M., Cernacek, P. and Polosa, C., Dynamics of neurotensin stores in the stellate ganglion of the cat, Brain Res., 562 (1991) 258-264. [16] Maher, E., Bachoo, M. and Polosa, C., Role of neuron soma firing in the restoration of neurotensin store in sympathetic preganglionic neuron terminals after stimulus-evoked depletion, Brain Res., (1994). [17] Merritt, J.E., McNeil, S., Tomlinson, S. and Brown, B.L., The relationship between prolactin secretion and calmodulin activity, J. Endocrinol., 98 423-429. [18] Miller, R.J., Multiple Ca 2+ channels and neuronal function, Science, 235 (1987) 46-52. [19] Morales, M., Bachoo, M., Collier, B., Beaudet, A. and Polosa, C., Ultrastructural localization of neurotensin immunoreactivity in the stellate ganglion of the cat, J. Neurocytol., 22 (1993) 663-681. [20] Polosa, C., Mannard, A. and Laskey, W., Tonic activity in autonomic nervous system functions properties, origins. In C. McC Brooks et al. (Ed.), Integrative Functions of the Autonomic Nervous System, University of Tokyo Press, Tokyo, Japan, 1979, pp. 342-354. [21] Reyneke, L., Russell, V.A. and Taljaard, J.J., Regional effects of NT on the electrically stimulated release of [3H] dopamine and [14C] acetylcholine in the rat nucleus accumbens, Neurochem. Res., 17 (1992) 1143-1146. [22] Riccio, D.V. and Matthews, M.A., The effects of intraocular injection of tetrodotoxin on fast axonal transport of [3H] proline and [3H] fucose-labeled materials in the developing rat optic nerve, Neuroscience, 16 (1985) 1027-1039. [23] Strong, J.A., Fox, A.P., Tsien, R.W. and Kaczmarek, L.K., Stimulation of protein kinase C recruits covert-calcium channels in Aplysia bag cell neurons, Nature (London), 325 (1987) 714717. [24] Thomas, P., Wang, J.A. and Almeis, W., Millisecond studies of secretion in single rat pituitary cells stimulated by flash photolysis of caged Ca 2+, EMBO J., 12 (1993) 303-306. [25] Treiman, M., Allesoe, K. and von Spreckelsen, S., Facilitation by forskolin of electrically evoked vasopressin release in vitro requires bursting pattern of stimulation, Brain Res., 488 (1989) 260-264. [26] Trifaro, J.M., Vitale, M.-L. and DelCastillo, A.R., Cytoskeleton and molecular mechanisms in the neurotransmitter release by neurosecretory cells, Eur. J. Pharmacol., 225 (1992) 83-104. [27] Unsicker, K. and Stogbauer, F., Screening of adrenal medullary neuropeptides for putative neurotrophic effects, Int. J. Dev. Neurosci., 10 (1992) 171-179. [28] von Euler, G., van der Ploeg, I., Fredholm, B. and Fuxe, K., Neurotensin decreases the affinity of dopamine D-2 agonist binding by a G protein-independent mechanism, J. Neurochem., 56 (1991) 178-183. [29] Watson, M.A., Yamada, M., Yamada, M., Eusack, B., Ververka, K., Bolden-Watson, C. and Richelson, E., The rat neurotensin receptor expressed in Chinese hamster ovary cells mediates the release of inositol phosphates, J. Neurochem., 59 (1992) 19671970. [30] Weldon, P., Bachoo, M., Morales, M,A., Collier, B. and Polosa, C., Dynamics of large dense-cored vesicles in synaptic boutons of the cat superior cervical ganglion, Neuroscience, 55 (1993) 1045-1054. [31] Weldon, P., Bachoo, M. and Polosa, C., The role of tonic activity in maintaining steady-state levels of large dense-cored vesicles in sympathetic preganglionic nerve terminals, Physiol. Can., 23 (1992) 186 (Abstract).
Brain Research 640 (1994) 131-135
Research Report
In vitro and in vivo evidence of neurotensin release from preganglionic axon terminals in the stellate ganglion of the cat E. Maher, B. Bachoo, C. Polosa * Department of Physiology, McGill University, 3655 Drummond Street, Montreal H3G 1Y6, Que., Canada (Accepted 16 November 1993)
Abstract
We have previously shown that the neurotensin (NT) store in preganglionic axon terminals of the cat stellate ganglion (SG) is reversibly depleted by prolonged preganglionic stimulation. The present study addresses the questions of whether the preganglionic axon terminals release NT in response to depolarizing stimuli in vitro and whether in vivo NT is released by the tonic firing of the sympathetic preganglionic neurons. Slices of the SG of the anaesthetized cat, maintained in oxygenated Ringer solution, released NT. The efflux increased when the K concentration was increased from 5 to 25 or 45 mM or when veratridine was added to the medium. In Ca-free medium, efflux was suppressed. The effect of veratridine was blocked by tetrodotoxin (TI'X). In awake, freely moving cats, in which TFX was applied for 4 days to the preganglionic input of the right SG, the NT content of this ganglion doubled by comparison with the left SG. Since NT accumulates proximal to a ligature on the preganglionic input of the SG, the increased NT content is likely to result from suppression of action potential-dependent release while influx into the terminals persists. This result suggests that the steady state of the NT store in sympathetic preganglionic terminals is the result of a steady influx from the soma balanced by action potential-dependent loss, presumably release.
Key words: Preganglionic neuron; Sympathetic ganglion; Tonic activity; Neuropeptide turnover; Neuropeptide release
1. Introduction
The peptide neurotensin (NT) is present in preganglionic axons in the stellate ganglion (SG) of the cat [3,6]. N T undergoes complete processing in the sympathetic preganglionic neuron soma [15], is packaged in large dense-cored vesicles [19] and accumulates at the central but not at the distal side of a ligature on the preganglionic axons which project to the SG [15]. The accumulation suggests a steady net flux of N T from the preganglionic neuron soma to the terminals. If the accumulation rate at the ligature is an indication of the rate of N T delivery to the axon terminals, loss of N T by release a n d / o r intraterminal degradation must be postulated to maintain the steady state of the N T store in the terminals. The H P L C profile of extracts of control and stimulated SG showed a dominant immunoreactive peak, comprising 94% of the total immunoreactivity,
* Corresponding author. Fax: (1) (514) 398-7452. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 5 3 9 - F
which coeluted with synthetic N T (1-13) [15]. The lack of significant amounts of N T degradation products in ganglion extracts suggests that intraterminal degradation is not an important component of N T turnover. Taken together, these findings suggest that under physiological conditions the anterograde flux of N T in preganglionic axons may be balanced by release. Since tonic firing is a characteristic property of sympathetic preganglionic neurons [20], the release may be the result of the ongoing traffic of action potentials. The present report presents evidence of Ca-dependent release of N T from slices of the SG and describes the results of an in vivo test of the hypothesis that the tonic activity of preganglionic neurons releases NT. To this end, action potential conduction into the preganglionic terminals of the SG was blocked by chronic tetrodotoxin (T-FX) application to the intact preganglionic input of the ganglion in awake, freely moving cats. This procedure resulted in a marked increase in the N T store of the ganglion in the absence of evidence of sprouting. This finding suggests that N T is continu-
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E. Maher et al. / Brain Research 640 (1994) 131-135
ously r e l e a s e d by t h e t o n i c activity o f t h e p r e g a n g l i o n i c neurons.
2. Materials and methods Adult cats of either sex (3.1-5.8 kg) were anaesthetized with Na pentobarbital (35 mg/kg i.p. followed by 3 m g / k g / h i.v.). The general preparation of the animals was, as described in the preceding paper [16]. Heart rate was monitored with a tachograph triggered by the arterial pressure pulse and was continuously recorded, together with arterial pressure, on a Grass 7 polygraph. In the experiments testing the effect of chronically blocking the tonic firing of the sympathetic preganglionic neuron terminals in the SG on the NT store of this ganglion (n = 4), the right (RSG) and left SG (LSG) were approached retropleurally by bilaterally removing a short segment (1-2 cm) of the second and third rib close to the costovertebral junction. The tip of a silastic catheter (o.d. 1 mm) was positioned in the region where the preganglionic inputs to the SG (second thoracic white ramus (WRT2), WRT 3 and the sympathetic trunk) converge to enter the ganglion, The catheter, secured with several ligatures (10-0 suture) to the connective tissue sheath of the thoracic sympathetic trunk down to the level of WRT 4, was connected to a mini-osmotic pump (Alzet 2001; 1 p.l/h), containing a T r x solution (1/zg//zl) on the right side and the vehicle (citrate buffer, pH = 7.2) on the left side which was implanted s.c. in the mid-axillary region. After implanting the catheters and pumps, the chest was closed and the animal given postoperative care which included antibiotics. 4 days later, these cats were anaesthetized again, as described above, and the SG excised bilaterally to be assayed for NT content. Since previous data suggest that NT in the SG is exclusively in preganglionic axons [6,15], a change in ganglion content was assumed to be due to a change in preganglionic axon terminal content. Before excision, the osmotic pumps and catheters were removed and a bipolar silver hook electrode connected to a Grass $88 stimulator was placed on the WRT 3 as welt as on the sympathetic trunk between the caudal pole of the SG and WRT 4. These nerves were stimulated with trains of 2 Hz 30 s (pulse duration 0.5 ms, amplitude 10 V). On the right TTX-treated side, this stimulation caused no change in heart rate. After the preganglionic nerves, previously exposed to TTX, had been irrigated with warm sterile saline for 30 min, the stimulus train produced a brisk cardioacceleration (from 189_+ 8 to 227 + 12 bpm, n = 4). In the acute experiments of measurement of NT release, the RSG and LSG were excised after removal of the first three ribs bilaterally. Each ganglion was sliced into 10-12 slices of 500/~m thickness and placed in a 3-ml test tube containing Ringer solution at room temperature (22°C) of the following composition (in mM): NaCI 130, KC1 5, MgC12 1, CaCI 2 2, KH2PO 4 1, NaHCO 3 12 and glucose 11, which was bubbled with 95% 0 2 - 5 % CO 2 and contained the endopeptidase 24:11 inhibitor, thiorphan (10 p.M), to minimize NT degradation [7,9]. For measuring NT release, the slices from each ganglion were incubated in 1 ml of the Ringer solution (or of the modified Ringer solutions, see below) for 10 rain at 22°C. In 18 experiments, the K concentration of the solution was increased to 12 or 25 or 45 mM by iso-osmotic substitution of NaCI with KCI. In four experiments, veratridine (40 ~M) with or without TTX (100 /zM) was added. In five experiments, CaCI 2 was substituted with MgCI 2 (8 raM). At the end of the 10-min incubation period, the slices were placed on ice for later determination of NT content and the tube was spun at 2200 rpm for 5 min. The supernatant was evaporated to dryness in a Savant Speed Vac Concentrator and stored at -20°C until the radioimmmunoassay was performed, as described below. The release data are reported as fmol NT/ganglion/min. For determination of NT content of the slices or of the intact ganglion, the tissue was homogenized in ice-cold 0.5 M acetic acid and boiled for 20 min, followed by centrifugation for 20
min at 1000x g. The supernatant was evaporated to dryness and stored at -20°C until analysis. The radioimmunoassay was performed, as described in the preceding paper [16], on 0.1 ml of the ganglion extract (reconstituted in 1 ml assay buffer) or 0.1 ml of the reconstituted incubation medium of the slices or 0.1 ml of a NT standard solution (25-2500 pmol/l NTl_13; Sigma Chemical). In all experiments, one SG served as experimental tissue while the contralateral was used as control since, as reported previously, NT content in RSG and LSG is not significantly different [15]. Data shown are means+ S.E.M. Statistical analysis was performed using the computer program EPISTAT. A paired Student's t test was used to evaluate differences in NT content between tissue of the RSG and LSG sides. Group means were compared using one-way ANOVA. Significance was assigned to P levels of < 0.05.
3. Results 3.1. Release o f N T by S G in vitro I n sliced g a n g l i a , i n c u b a t e d in R i n g e r s o l u t i o n f o r u p to 30 m i n , t h e N T c o n t e n t was 1231 + 106 f m o l / g a n g l i o n ( n = 5). T h i s is s i m i l a r to t h e N T c o n t e n t o f whole, acutely excised ganglia reported previously (1292 + 71 f m o l / g a n g l i o n , P > 0.05) [15]. I n a b s e n c e o f t h i o r p h a n , N T was n o t d e t e c t e d in t h e i n c u b a t i o n m e d i u m . T h e e f f i c a c y o f t h i o r p h a n in p r e v e n t i n g N T b r e a k d o w n was m e a s u r e d by i n c u b a t i n g S G slices w i t h e x o g e n o u s N T (100 fmol): a f t e r s u b t r a c t i o n o f t h e spontaneous efflux (see below), recovery of the added N T i n c r e a s e d f r o m b e l o w t h e l i m i t o f d e t e c t i o n (10 f m o l ) in a b s e n c e o f t h i o r p h a n to 81 + 7 f m o l in p r e s e n c e o f t h i o r p h a n (10 / z M , n = 3, P < 0.05). I n sliced g a n g l i a i n c u b a t e d in n o r m a l R i n g e r s o l u t i o n , t h e r e w a s s p o n t a n e o u s e f f l u x o f N T o f 9.7 + 0.2 f m o l / g a n g l i o n / m i n ( n = 10). N T e f f l u x in 12 m M K ÷ was n o t signific a n t l y d i f f e r e n t f r o m t h a t o b t a i n e d in n o r m a l R i n g e r (10.3 + 0.4 f m o l / g a n g l i o n / m i n , n = 4, P > 0.05). W h e n t h e g a n g l i a w e r e i n c u b a t e d in R i n g e r s o l u t i o n c o n t a i n i n g 25 m M o r 45 m M K ÷, t h e e f f l u x i n c r e a s e d to 12.4 + 0.9 f m o l / g a n g l i o n / m i n ( n = 4, P < 0.05 vs. n o r m a l R i n g e r ) a n d 17.6 + 1.3 f m o l / g a n g l i o n / m i n (n = 7, P < 0.05 vs. n o r m a l R i n g e r ) , r e s p e c t i v e l y . T h e s e d a t a a r e s u m m a r i z e d in Fig. 1. S i m i l a r r e l e a s e r a t e s w e r e o b t a i n e d w i t h 45 m M K at 3 7 ° C (16.8 + 2.0 f m o l / g a n g l i o n / m i n , n = 3, P > 0.05 vs. 45 m M K ÷ at 22°C). W h e n t h e g a n g l i a w e r e i n c u b a t e d in R i n g e r s o l u t i o n c o n t a i n i n g v e r a t r i d i n e (40 / z M ) , N T e f f l u x i n c r e a s e d f r o m 8.6 + 0.8 to 22.6 + 1.6 f m o l / g a n g l i o n / m i n ( n = 4, P < 0.05 vs. n o r m a l R i n g e r ) . T h e i n c r e a s e in r e l e a s e p r o d u c e d by v e r a t r i d i n e w a s b l o c k e d by T T X ( 1 0 0 / x M ) (7.8 + 0.4 f m o l / g a n g l i o n / m i n , n = 4, P > 0.05 vs. n o r m a l R i n g e r ; Fig. 2). W h e n t h e g a n g l i a w e r e i n c u b a t e d in C a - f r e e R i n g e r , t h e s p o n t a n e o u s N T e f f l u x (0.8 + 0.03 f m o l / g a n g l i o n / m i n , P < 0.05 vs. n o r m a l R i n g e r , n = 5) as w e l l as t h e N T e f f l u x p r o d u c e d by 45 m M K ÷ (0.9 + 0.04 f m o l / g a n g l i o n / m i n , P < 0.05 vs. R i n g e r w i t h 45 m M K ÷, n = 5) w e r e m a r k e d l y r e d u c e d . T h e s e d a t a a r e s u m m a r i z e d in Fig. 3.
E. Maheret aL / Brain Research 640 (1994) 131-135 2o E "E
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Fig. 1. Spontaneous and K-evoked NT release from SG slices. Slices incubated for 10 rain at 220C in Ringer solution containing 2 mM Ca and increasing concentrations of K (in mM): 5 (n = 10), 12.5 (n = 4), 25 (n = 4), 45 (n = 7). Concentrations of > 5 mM were obtained by iso-osmotic substitution of NaCI with KCI. * P < 0.05 vs. 5 mM K.
25-
Control
TTX
Fig. 4. Effect of chronic TI'X block of action potential conduction in preganglionic input on NT content of SG. Duration of block: 4 days. Control is sham-treated LSG (n = 4). TTX is SG ipsilateral to block (n = 4). * P < 0.05 vs. sham-treated control.
(n = 4, P < 0.05; Fig. 4). N T c o n t e n t in t h e s h a m t r e a t e d L S G was n o t significantly d i f f e r e n t ( P > 0.05) from the previously reported content of untreated SG [151.
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Fig. 2. Veratridine-evoked NT release from SG slices. Slices incubated for 10 min at 22°C in Ringer solution containing 2 mM Ca, 5 mM K (basal, n = 4) to which veratridine (40/xM, n = 4) or veratridine and TTX (100 ~M, n = 4) were added. * P < 0.05 vs. basal.
3.2. N T content in SG with chronic T T X block o f preganglionic input I n cats in which t h e intact p r e g a n g l i o n i c i n n e r v a t i o n o f t h e R S G was t r e a t e d with T T X for 4 days, t h e N T c o n t e n t o f t h e R S G was 2296 + 189 fmol while t h e N T c o n t e n t o f t h e s h a m - t r e a t e d L S G was 1196 + 149 fmol
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T h e d a t a p r e s e n t e d show t h a t K + - e v o k e d d e p o l a r ization o f slices o f t h e cat S G r e l e a s e s NT. This effect was s u p p r e s s e d in C a - f r e e m e d i u m . Since N T in t h e S G is in s y m p a t h e t i c p r e g a n g l i o n i c t e r m i n a l s [6,15], specifically l o c a l i z e d within l a r g e d e n s e - c o r e d vesicles [19], t h e s e findings i m p l y t h a t d e p o l a r i z a t i o n o f t h e p r e g a n g l i o n i c axon t e r m i n a l s causes exocytosis o f N T c o n t a i n i n g large d e n s e - c o r e d vesicles in a C a - d e p e n d ent manner. Morphological evidence of Ca-dependent, a c t i o n p o t e n t i a l - e v o k e d , exocytosis o f large d e n s e - c o r e d vesicles f r o m p r e g a n g l i o n i c axon t e r m i n a l s in cat superior cervical g a n g l i o n has r e c e n t l y b e e n r e p o r t e d [30]. The veratridine-evoked TTX-sensitive NT release from S G slices suggests t h a t m e c h a n i s m s a c t i v a t e d by action p o t e n t i a l s can c a u s e r e l e a s e o f N T - c o n t a i n i n g l a r g e d e n s e - c o r e d vesicles. R e l e a s e o f N T has b e e n previously shown from t h e p e r f u s e d a d r e n a l g l a n d o f t h e cat [10] a n d f r o m slices o f m o u s e h y p o t h a l a m u s [13]. T h e d a t a also show t h a t in awake, freely moving cats, t h e N T c o n t e n t o f t h e S G n e a r l y d o u b l e d a f t e r 4 days o f c o n t i n u o u s T T X infusion on t h e intact p r e g a n g l i o n i c input. T T X a p p l i e d locally to axons, at c o n c e n t r a t i o n s which b l o c k p r o p a g a t i o n o f a c t i o n p o t e n t i a l s , d o e s n o t affect axonal t r a n s p o r t m e c h a n i s m s o r p r o t e i n synthesis in t h e p e r i k a r y a [12,22]. T h e r e f o r e , t h e i n c r e a s e in N T c o n t e n t o f p r e g a n g l i o n i c t e r m i n a l s is unlikely d u e to i n c r e a s e d synthesis o r r a t e o f axonal t r a n s p o r t o f NT. N o e v i d e n c e has b e e n r e p o r t e d t h a t a c t i o n p o t e n tials m a y c a u s e i n t r a t e r m i n a l r e l e a s e o f large d e n s e c o r e d vesicle c o n t e n t followed by p r o t e o l y t i c d e g r a d a tion. A s m e n t i o n e d in t h e I n t r o d u c t i o n , a f t e r 20 m i n o f p r e g a n g l i o n i c s t i m u l a t i o n at 40 Hz, no significant a m o u n t o f N T d e g r a d a t i o n p r o d u c t s was i d e n t i f i e d by
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E. Maher et aL / Brain Research 640 (1994) 131-135
HPLC in SG extracts [15]. The possibility that the increased NT content of the ganglion with TTX-treated input was due to sprouting of the preganglionic axons, resulting in the addition of new varicosities with normal NT content, is made unlikely by the finding, in a study of the ultrastructure of the cat superior cervical ganglion, that the number and size of synaptic boutons were unchanged after 4 days of ] q ' X block of the preganglionic input [31]. Even if sprouting had occurred, an increase in NT content may not be a necessary consequence of it because, when sprouting was produced by partial denervation of the superior cervical ganglion, ChAT activity in the ganglion was unchanged, suggesting that the amount of enzyme present in each axon before sprouting was distributed over a greater number of varicosities after sprouting [11]. If the increased NT content of the SG is not the result of increased influx from the soma nor of suppression of action potential-dependent intraterminal degradation nor of sprouting, it may be the result of suppression by TTX of action potential-dependent ongoing release while supply by axonal transport remains constant. The in vitro data presented here show that NT is released by a Ca-dependent mechanism. Suppression by T T X of Ca-dependent exocytosis is, therefore presumably, the mechanism responsible for N T accumulation in the terminals. On this basis, it may be postulated, therefore, that one component of the turnover of NT in preganglionic axon terminals is release by the ongoing action potential traffic which is a characteristic property of preganglionic axons [20]. If it is assumed that the difference in NT content between the TTX-treated RSG and the sham-treated LSG (1087 5- 99 fmol) was the result of a constant rate of accumulation over the 4 days of TI'X treatment, one fourth of this amount (272 fmol or 23% of the ganglion content) would be the amount accumulated in 1 day. If it is assumed that, in absence of action potential traffic, there was no loss of NT by the terminals and that the accumulation in the presence of T'I'X equals the amount released per day in absence of TTX, this fraction of the ganglion content would turn over in 1 day. The mean rate of spontaneous firing of sympathetic preganglionic neurons in anesthetized or unanesthetized decerebrate cats is low, typically 1-2 Hz [20]. Electrical stimulation of the preganglionic input to the SG at 2 Hz for 100 min (12000 pulses) produced no depletion of NT content of the SG while stimulation with the same number of pulses at 40 Hz reduced content by 30% [15]. Thus, the frequency of the spontaneous firing of the sympathetic preganglionic neuron may seem inadequate to produce release and, by its suppression, accumulation of NT. This inconsistency may be reconciled in several ways. First, the firing frequency of sympathetic preganglionic neurons in awake, freely moving cats is not known: it may be
higher than in the anaesthetized or decerebrate, unanaesthetized, cat and be in a range which can cause release of NT from large dense-cored vesicles. Second, in the anaesthetized or decerebrate cat, a large fraction of the tonically active sympathetic preganglionic neurons fire in bursts, coincident with the inspiratory phase of the respiratory cycle, with instantaneous frequencies as high as 10-20 Hz [3]. Stimulation of the SG input at these frequencies depletes the NT content of the SG [15]. Third, the probability of release of N T may vary with impulse frequency: at frequencies of 1-2 Hz, the probability of release may be so low that loss (or accumulation when release is blocked by TTX) may only be measurable on the time scale of days. At the frog neuro-muscular junction, stimulation of the motor nerve with 2 Hz for 24 h produced a significant depletion of large dense-cored vesicle number in the motor axon terminals [14]. On the basis of the NT accumulation caused by TTX, it is estimated that 272 fmol NT are released by the SG over 24 h. By contrast, electrical stimulation at 40 Hz causes release of the same amount of NT by the SG in 6.5 min [15], suggesting a much higher probability of release at 40 Hz than at the frequency of the tonic activity of the sympathetic preganglionic neuron. Mechanisms which may encode frequency of action potentials at the nerve terminals into efficacy of stimulus-neuropeptide release coupling are activation of specific Ca channels [18] as well as activation of the second messengers protein kinase A [25], protein kinase C [23] and Ca-calmodulin kinase [17]. Fourth, in peptide-secreting endocrine cells, a very small proportion of secretory granules are found close to the cell membrane and may be readily released by single action potentials [5,8,24] while the majority of the secretory granules are linked to actin microfilaments and form a reserve pool. The mobilization of this reserve pool into the readily releasable pool is poorly understood. It may involve dissociation of the actin cytoskeleton by proteases activated by the high levels of cytosolic Ca achieved during high-frequency stimulation [26]. It is possible, therefore, that in the preganglionic axon terminal a small subset of NT-containing large dense core vesicles is docked near release sites and can readily undergo exocytosis in response to action potentials at low frequencies. Detection of the resulting NT release as a decrease in NT content of the ganglion extract may be difficult, however, because of the small amount released. Concerning the physiological consequences of NT release, activation of the NT receptor can lead to phosphoinositide synthesis [29] and inhibition of cAMP production [4]. Therefore, if the N T released by preganglionic axon terminals acts on pre- or postsynaptic receptors, in addition to its postsynaptic depolarizing action [2], it could modify functions of membranebound receptors [28] as well as the mechanism of
E. Maher et al. / Brain Research 640 (1994) 131-135
release of small molecule transmitters [21]. In addition, NT has been shown to have neurotrophic properties by increasing the survival of chick ciliary ganglion cells in culture [27]. Acknowledgements. This work was supported by the Quebec Heart Foundation and the Medical Research Council of Canada. E. Maher was the holder of a Medical Scientist Award of the Heart and Stroke Foundation of Canada. The preparation of the manuscript by C. Pamplin is gratefully acknowledged.
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