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Ionic mechanisms involved in the release of 3H-norepinephrine from the cat superior cervical ganglion
Life Sciences, Vol. 34, pp. 861-871 Printed in the U.S.A.
Pergamon Press
IONIC MECHANISMS INVOLVED IN THE RELEASE OF 3H-NOREPINEPHRINE FROM THE CAT SUPERIOR CERVICAL GANGLION E. Adler-Graschinsky, E.J. Filinger and A.E. Martfnez Instituto de Investigaciones Farmacol6gicas (CONICET) Junfn 956-5 ° Piso, 1113 Buenos Aires, Argentina (Received in final form December 14, 1983) Summary It has previously been reported that in the isolated cat superior cervical ganglion (SCG) labeled with tritiated norepinephrine (3H-NE), the stimulation of the preganglionic trunk at I0 Hz as well as the exposure to I00 pM exogenous acetylcholine (ACh), produced a Ca++-dependent release of 3H-NE. The present results show that a Ca++-dependent release of 3H-NE was produced also by exposure to either 50 pM veratridine or 60 mM KCI. Tetrodotoxin (0.5~M) abolished the release of 3H-NE induced by preganglionic stimulation, ACh and veratridine but did not modify the release evoked by KCI. The metabolic distribution of the radioactivity released by the different depolarizing stimuli showed that the 3H-NE was collected mainly unmetabolized. In the cat SCG neither the release of 3H-NE evoked by KCI nor the endogenous content of NE was modified by pretreatment with 6-OH-dopamine (6-OH-DA). On the other hand, this chemical sympathectomy depleted the endogenous content of NE in the cat nictitating membrane, whose nerve terminals arise from the SCG. The data presented suggest that the depolarization-coupled release of NE from the cat SCG involves structures that are different to nerve terminals and that contain Na + channels as well as Ca++ channels. It has been reported that in the isolated superior cervical ganglion(SCG) of the cat, the orthodromic stimulation of the cervical sympathetic preganglionic trunk evokes a release of norepinephrine (NE) which is dependent on the presence of extracellular Ca++ and is reduced by exposure to hexamethonium. Based on anatomical descriptions (I) and on the observation that the exogenous acetylcholine (ACh) had a releasing effect with similar characteristics to that evoked by the orthodromic stimulation, it was proposed elsewhere that the ACh released from preganglionic fibers evokes the output of NE through the activation of nicotinic receptors (2). The site from which NE originates in the cat ganglion has been proposed to be different from both the SIF cells and the nerve terminals and considered most probably as dendrites (2). The aim of the present study was to further investigate the ionic mechanisms that participate in the release of NE from sympathetic ganglia. Two depolarizing agents, veratridine and hypertonic KCI were employed to induce the overflow of radioactivity from the isolated cat SCG labeled in vitro with 3H-NE. The depolarization of nerve cell membranes caused by hypertonic KCI results from the reduction in the concentration gradient of potassium between the intr~ cellular and the extracellular spaces (3), whereas that elicited by veratridine is primarily due to the increase in the sodium permeability of the nerve membrane (4,5). Hence, the effects of tetrodotoxin (TTX) which selectively blocks 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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the mechanism whereby stimulation induces an increase in sodium permeability of the nerve membranes (6) were also assayed. In addition, in order to further analyze whether the sites from which NE originates in the ganglia are different from nerve terminals, the effect of KCI was tested on SCG isolated from adult cats pretreated with 6-OH-dopamine (6-OH-DA) to destroy nerve terminals (7). The latter results were compared with those provoked by depolarization with KCI of the nerve terminals of the nictitating membrane which arise from the postganglionic fibers of the SCG. Methods Animals: Adult cats of 1.5 to 3.0 kg of body weight and of either sex were anaesthetized with sodium pentobarbitone (35 mg/kg, i.p.) before use. Solutions: Modified Krebs'solution of the following composition (n~O was emplo~ ed: NaCI, 118,0; KCI, 4.7; CaCI2, 2.6; MgCI2, 1.2; NaH2PO4, 1.0; NaHCO3, 25.0; glucose, II.I; ethylenediamine tetraacetic acid (EDTA), 0.004 and ascorbic acid, 0.Ii. The solution was bubbled with a 95 % 02 : 5 % CO 2 mixture, so that its final pH was 7.4 and it was maintained at 37°C throughout the experiment. Both the addition of elevated concentrations of KCI and the omission of CaCI 2 from the Krebs'solution were performed without osmotic compensation. Superior cervical ~an~lion: Either the right or the left superior cervical ganglion (SCG) was placed into Krebs'solution and dissected together with 5 cm of the cervical sympathetic (preganglionic) nerve and about 0.5 cm of the carotid (postganglionic) trunk. The ganglion and the attached nerves were desheathed. The postganglionic fibers were cut close to the body of the ganglion and about 1 cm of the preganglionic trunk covered with silicone ~as employed to fix the preparation into an isolated organ bath of 3 ml capacity. After preincubation of the ganglion for 15 min in the Krebs'solution at 37°C, the NE stores were I~ beled b~ incubation of the tissue during 30 min with 5 ~Ci/ml (0.67 pM) of (~)-(7-3H)-NE (New England Nuclear, specific activity 7.5 Ci/mmole) as described elsewhere (8). At the end of the incubation period the tissue was submitted to eight consecutive l-min washes and then to twelve consecutive 5-min washes before the collection of the samples started. For the electrical stimulation the preganglionic sympathetic nerve was pulled through shielded bipolar platinum electrodes and square-wave pulses of 2 msec duration were delivered at I0 Hz and at supramaximal voltage (80 V) during 5 minutes. The stimulation by drugs was performed by exposure to either veratridine or ACh or elevated concentrations of potassium chloride. ACh was added together with 3.08 ~M eserine. Veratridine, as well as KCI, were employed simultaneously with both 1.0 mM hexamethonium and i0 ~M atropine. Two consecutive periods of stimulation (S 1 and $2) were performed 40 min apart; the first one was applied 80 min after the end of the incubation with 3M-NE. Aliquots of 0.5 ml of the bathing solution which had been in contact with the tissue for 2 to 5 min were taken for determination of total radioactivity in a liquid scintillation spectrometer. The overflow of radioactivity induced by stimulation was calculated by subtracting the spontaneous outflow assumed to have occurred in each sample during and after the period of stimulation and it was expressed as the fractional rate of loss (FR x 10-2), i.e. as the percentage of the tissue content released per minute. This last value was calculated by addition of the radioactivity lost during the successive washes to that mea~ ured in the tissue at the end of the experiment. The ratios $2/S 1 of the release of radioactivity induced by the two consecutive stimulations under control conditions were employed to compare the effect of drugs added during the second stimulation. At the end of the experiment the tissue was processed to determine endogenous NE according to Laverty and Taylor (9). In some experi-
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863
ments, 5.0 ml of the bathing solution pooled from two ganglia were employed for chromatographic separation of NE and its metabolites using alumina and Dowex 50x4 columns, according to the method described by Graefe, Stefano and Langer (I0). In separate experimental groups the cats were pretreated with 6-hydroxydopamine (6-OH-DA) 40 mg/kg, i.p., 40 hr before the isolation of the ganglia. The significance of differences between values was determined by Student's t test. A P value smaller than 0.05 was regarded as significant. Nictitatin~ membrane: The eyeball was excised and the nictitating membrane with all the adjoining tissue was removed from the orbit. The tissue was placed in Krebs'solution, the medial muscle was dissected and the cartilage on which the fibers of the medial muscle are inserted was attached to the bottom of a I0 ml organ bath. The upper end of the muscle was connected to a force-displacement transducer and the tension developed by the muscle was recorded with a Grass polygraph. The tissue was labeled with 3H-NE, submitted to depolarization with KCI and employed to determine endogenous NE as reported for the SCG. Nictitating membranes isolated from cats pretreated with 6-OH-DA were also employed. Further details are described under Results. Dru~s: The following drugs were used: acetylcholine hydrochloride, eserine sulfate, hexamethonium bromide and atropine sulfate (Sigma Chemical Co.); veratridine (Aldrich Chemical Co.); tetrodotoxin (Sankyo Co. Ltd.) and 6-hydroxydopamine (Regis). All drugs but veratridine were dissolved into distilled water and added to the organ bath in a volume up to I00 microliters. Veratridine was dissolved at a concentration of 20 mg/ml in dimethylsulfoxide (DMSO). When 50 ~LM veratridine was employed, the concentration of DMSO in the organ bath was 23 raM and it had no effect on either the rate of efflux or the endogenous content of NE in the isolated ganglia. Results Effects of Ca++-deprivation on the overflow of 3H-NE evoked by KCI and by veratridine in the isolated cat SCG As shown in Fig. I, the exposure to KCI produced a concentration-dependent release of 3H-NE from the isolated cat SCG and from the isolated cat nictitating membrane which is innervated by the nerve terminals of the postganglionic fibers that arise from the SCG. The concentration-dependent effect of KCI was observed within the range of 40 to 60 mM in the SCG and of 60 to I00 mM in the nictitating membrane. The magnitude of the releasing effect produced by KCI in both tissues was similar at 40, 80 and I00 mM KCI, but differed at 60 mM KCI. With this last concentration the fractional rate of loss of 3H-NE in the SCG (0.96 + 0.16, n = 6) was significantly higher than that obtained in the nictit~ ting m~mbrane (0.44 + 0.07, P < 0.001). Depolarization with veratridine has been employed to study mechanisms of transmitter release at central (II) and peripheral synapses (12). The present study shows that veratridine (Table I) evoked the overflow of 3H-NE in the isolated cat SCG as well. Veratridine was assayed in the presence of hexamethonium plus atropine to prevent from the NE-releasing effect caused by ACh (2). The overflow of ~H-NE induced by the concentration of veratridine employed (50 ~M) had a similar order of magnitude to that evoked by other depolarizing agents in the cat ganglion (between 0.5 and 1.0 % of the tissue content,Tablel) and it was not overcome by higher concentrations of veratridine, as determined in preliminary experiments. It has been reported that in the isolated cat SCG the omission of Ca ++ from the incubation medium prevented the release of 3H-NE induced by either exposure to ACh or electrical stimulation of the preganglionic fibers (2). Expe~ iments were performed in the present study to search whether the omission of
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60
80
100
K Ct (mMl FIG. 1 Release of 3H-NE induced by KCI from the isolated cat superior cervical ganglion (e) and the isolated cat nictitating membrane (O) Ordinate: increase in the release of radioactivity above basal levels expressed as the fractional rate of loss (FR x 10-2), i.e. as the percentage of the tissue content released per minute by the 2.5 min exposures to KCI. Abscissa (log scale): concentrations of KCI. Mean values are shown. Vertical lines indicate the S.E. of at least 6 experiments per group for the ganglion and 7 experiments per group for the nictitating membrane. Ca ++ modified the effects of KCI and of veratridine on the release of 3H-NE from the SCG of the cat. Both depolarizing agents have been shown to produce a Ca++-dependent release of NE from rat brain synaptosomes (13). As shown in Fig. 2, the release of 3H-NE induced by both KCI and veratridine in the presence of hexamethonium plus atropine, was almost entirely abolished when Ca++ was omitted from the incubation medium. The reductions observed in the Ca++-free medium were significant at P < 0.001. The values of the fractional release of 3H-NE (FR x 10 -2 ) obtained above basal levels when Ca++ was restored (0.88 + 0.ii, n = 8, for KCI and 0.79 ~ 0.20, n = 6, for veratridine) were similar ~n magnitude to those evoked by the same depolarizing agents under control conditions (see Table I). Effects of TTX on the overflow of 3H-NE and metabolic distribution of the 3H-NE released by the depolarizing stimuli Tetrodotoxin selectively blocks the mechanism whereby stimulation induces an increase in the sodium permeability of the membrane (6). As shown in Table I 0.5 ~M TTX abolished the increase in the release of 3H-NE induced by veratridine, preganglionic stimulation and acetylcholine but it had no effect on the overflow of 3H-NE evoked by 60 mM KCI.
5.0
5.0
2.0
Veratridine 50 ~M
Preganglionic nerve stimulation (I0 Hz, 2 msec, 80 Volts)
Acetylcholine I00 ~M
Eserine 3.08 ~M
None
Hexamethonium 1.0 mM plus atropine I0.0 ~M
Hexamethonium 1.0 mM plus atropine I0.0 ~M
Drugs present during stimulation
0.44+_0.11 (8)
1.15+--0.26 (9)
0.59+0.07 (i0)
1.14+__0.05(9) (c)
FR x 10 -2 (a)
1.38+-0.42 (8)
0.01+0.01 (6)
0.0_5~0.02
(0.025
(0.001
1.16+-0.12 (9)
(6)
N.S.
<0.005
0.92_+0.08 (7)
TTX
1.37+0.29 (10) 0.03+0.01 (6)
0.92+__0.12 (g)
Control
P (d)
(a) The increase in tritium release above basal levels elicited by the stimulation is expressed as a fraction of the tissue content released per minute. Hexamethonium and atropine were added 25 min before and during the stimulus. Eserine was added simultaneously with acetylcholine. (b) Two consecutive periods of stimulation (S 1 and S 2) were performed 40 min apart and the efflux of radioactivity was expressed as the ratio $2/S I. Values obtained under control conditions were compared with those obtained when the second stimulation was performed after 5 min incubation with 0.5 pM TTX. (c) Each value is the mean + S.E. of the number of experiments indicated between parentheses. (d) Values of P based on StUdent's t test. The TTX ratios were compared to the control ratios. N.S.: not significant.
2.5
Stimulation period (min)
Potassium chloride 60mM
Depolarizing stimulus
Ratios $2/51 (b)
Radioactivity released by the stimulation
Effects of TTX on the release of tritium elicited under different experimental conditions in the isolated cat superior cervical ganglion labeled with 3H-NE (Krebs'solution, 37°C)
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Release of 3H-NE From the Cat SCG
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FIG. 2
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Effects of Ca++-deprivation on the release of 3H-NE induced by 2.5 min exposures to KCI and by 5.0 min exposures to veratridine in the cat superior cervical ganglion. The ordinates indicate the release of radioactivity expressed as the fractional rate of loss (FR x 10-2), i.e. as the percentage of the tissue content released per minute. The open bars correspond to the spontaneous out flow of 3H-NE in consecutive samples. The -period of exposure to the depolarizing agent is shown by the black rectangle at the bottom of each panel and the increases in the release of radioactivity above basal levels are indicated by the cross-hatched bars. Hexamethonium (I mM) and atropine (I0 pM) were added 30 min before S I and were maintained in the organ bath up to the end of the experiment. Mean values are shown. Vertical lines indicate the S.E. of the mean of n experiments per group. In A (upper panel): consecutive 2.5 min samples were taken. S 1 was performed 85 min after the incubation in Ca++-free Krebs'solution and S 2 20 min after the incubation in the normal Krebs'solution (nffiS). In B (lower panel): consecutive 5 min samples were taken. S 1 was performed 40 min after the incubation in Ca++-free Krebs'solution and S 2 40 min after the addition of the normal Krebs'solution (n=5).
Fig. 3 shows that under depolarization with KCI, veratridine, preganglionic stimulation and ACh the unmetabolized 3H-NE was the main fraction released and the deaminated glycol 3H-DOPEG was the main metabolite collected. However, the percentage of unmetabolized 3H-NE released both by veratridine and by preganglionic stimulation was significantly higher than that observed under stimulation with either ACh or KCI. The lower panel of Fig. 3 shows that neither eserine nor hexamethonium plus atropine modified the metabolic distribution of the radioactivity released spontaneously. The absolute value for the spontaneous efflux of radioactivity in 5 min samples (23.00 + 2.44 nCi/100 mg tissue in the controls, n ffi 9) was not modified by TTX (23.79 + 2.09 nCi/100 mg tissue, n ffi 6), hexamethonium plus atropine (20.78 ~ 1.26 nCi/T00 mg tissue, n = 7) and eserine (18.88 ~ 3.26 nCi/ I00 mg tissue, n m 4). Effects of chemical sympathectomy As reported by Tranzer and Thoenen (7) chemical sympathectomy in adult cats destroys nerve terminals selectively without affecting cell bodies or dendrites. Fig. 4 B shows that the pretreatment with 6-OH-DA depleted the endoge nous content of NE in the nictitating membrane but not in the SCG. The 6-OH-DA treated ganglia also maintained their capacity to increase the overflow of 3H-NE in response to the depolarization with KCI (Fig. 4 A) as well as to store and to spontaneously release the 3H-transmitter. The radioactivity retained
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Release of 3H-NE From the Cat SCG
[]NE
~]DOPEO"DOMA []OMDA ONMN
A) Increase in 3H-release evoked by depotarizafion POTASSIUMCHLORIDE1~.)
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FIG. 3 Metabolism of 3H-norepinephrine released under different experimental conditions in the isolated cat superior cervical ganglion. The abbreviations correspond to unmetabolized 3H-norepinephrine (NE); 3H-3,4-dihydroxyphenylglycol (DOPEG); 3H-3,4-dihydroxymandelic acid (DOMA); 3H-O-methylated deaminated metabolites (OMDA) and 3Hnormetanephrine (NMN). Mean values of the number of experiments indicated in parentheses are shown. Vertical lines indicate the S.E. of the mean. In A (upper panel): increase above basal levels induced by different depolarizing stimuli. The conditions of stimulation are described in Table I. In B (lower panel): metabolic distr~ bution of the spontaneous output of 3H-NE in the presence of the different drugs added together with the depolarizing stimulus as described in Table I.
867
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Release of 3H-NE From the Cat SCG
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FIG. 4 Effects of the pretreatment with 6-OH-DA (40 mg/kg, i.p., 40 hr before the experiment) on the content of endogenous NE and on the K+-induced release of 3H-NE in the cat superior cervical ganglion (SCG) and nictitating membrane (NM). In A: increase in the radio activity above basal levels expressed as the fractional rate of loss (FR x 10-2), i.e. as the percentage of the tissue content released per minute by a 2.5 min exposure to 60 mM KCI. In B: en dogenous content of NE as micrograms per gram of wet tissue. Mean values of 6 experiments for the control group (open bars) and of 5 experiments for the 6-OH-DA treated-group (cross-hatched bars) are shown. Vertical lines indicate the S.E. of the mean. The asterisk denotes significance at P ~ 0.001 compared to the corresponding control. in the tissue at the end of the experiment was 18.58 ~ 2.22 ~Ci/g tissue, n=7, in the controls and 20.70 + 3.09 ~Ci/g tissue, n = 4, in the 6-OH-DA treated ganglia. The spontaneous outflow of tritium in 5 min samples collected 120 min after the incubation with the 3H-transmitter was 23.00 + 2.44 nCi/lO0 mg tissue in the controls, n = 9 and 22.90 + 4.10 nCi/lO0 mg tissue, n = 4, after 6-OH-DA. In addition, the metabolism of the 3H-NE released after sympathectomy did not differ from control values for either the spontaneous outflow or the K+-in duced release of the transmitter. Thus, unmetabolized 3H-NE was 15.9 + 1.4 % of the total spontaneous efflux in the controls (n = 4) and 14.1 + 2.3--% after 6-OH-DA (n = 4). Moreover, the increment in 3H-NE release above ~asal levels induced by 60 mM KCI was constituted by 38.5 + 3.6 % of unmetabolized 3H-NE in the controls (n = 4) and by 43.7 + 3 . 1 % after 6-OH-DA (n = 4).
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Discussion The present results show that in the isolated SCG of the cat the exposure to hypertonic KCI and the addition of veratridine produce a Ca++-dependent release of 3H-NE as reported previously for the release of 3H-NE induced by preganglionic stimulation and by ACh in the same tissue (2).
The e x t r a c e l l u l a r Ca++ i s one of t h e s p e c i f i c r e q u i r e m e n t s f o r t h e r e l e a s e o f NE from a d r e n e r g i c n e r v e s caused by d e p o l a r i z a t i o n o f t h e c e l l membranes (14). Under t h e p r e s e n t e x p e r i m e n t a l c o n d i t i o n s t h e a b s e n c e of e x t r a c e l l u l a r Ca++ p r e v e n t e d t h e r e l e a s e o f 3H-NE induced by e i t h e r v e r a t r i d i n e o r KC1-i n t h e c a t SCG i n c u b a t e d w i t h hexamethonium p l u s a t r o p i n e . This o b s e r v a t i o n a g r e e s w i t h t h e Cs++-dependence o b s e r v e d f o r t h e r e l e a s e o f 3H-NE induced by e i ther preganglionic stimulation or exogenous ACh in the cat SCG (2). The possibility that KCl had induced the release of 3H-NE through an osmotic effect was ruled out based on the results obtained by Haeusler and coworkers (15). They reported that when the osmotic pressure of the Krebs'solution was elevated by the addition of glucose, sucrose or LiC1 up to the level caused by 160 mM KC1, i.e. up to twice the normal pressure, only a negligible amount of 3H-NE was released by exposure to such solutions (15). The fact that the incubation with tetrodotoxin, which blocks the voltage dependent Na + channels (6), had abolished the overflow of 3H-NE induced in the cat ganglion by veratridine, preganglionic stimulation and ACh but not that evoked by KCI, indicates that the three first mechanisms but not the last one, involve the participation of the fast Na+-channels to evoke the overflow of the radioactive label as a result of the depolarization of the cell membrane. This observation agrees with the results obtained in synaptosomes from rat brain, which showed that the effects of veratridine but not those of KCI were blocked by TTX ( 1 3 ) . The complete blockade caused by TTX on the ACh-evoked release of 3H-NE di~ fers, however, from the results reported for the SIF-cells of the cat adrenal medulla (16,17). Although the authors employed the same TTX concentration (16) or a concentration ten times higher (17) than that used in the present work, they found that TTX does not block at all (17) or only partially blocks (16)the release of catecholamines induced by ACh. These observations give further support to the view that the structures of the cat ganglion from which NE originates are not identical to the SlF cells of the adrenal medulla (2). Hence, it is likely that in the structures from which NE originates in the cat SCG the s~ cretion of NE can be stimulated both through channels coupled to the nicotine ACh receptor (2) and through voltage-dependent Na + channels (present observations). The metabolic distribution of the 3H-NE released by the different depola_r izing stimuli resembled that reported for both the peripheral (18,19) and the central nervous system (20), where the unmetabolized 3H-NE and the metabolite 3H-DOPEG were the main fractions released in response to nerve stimulation. The fact that the proportion of unmetabolized 3H-NE was 40 % for either KCI or ACh and 70 % for either veratridine or preganglionic stimulation seems to be unrelated to the presence of hexamethonium plus atropine or of eserine, because these drugs did not affect by themselves the metabolic pattern of the 3H-NE spontaneously released. It is of interest to note that the cationic fluxes elicited by veratridine resembled more the fluxes occurring during physiological depolarization than those taking place during depolarization with high extracellular K+ concentrations (11,21,22). For this reason and also because media containing high K + are either hypertonic or partly Na+-deficient, the depolarization caused by veratridine has been considered more "physiological" than that obtained with high K+ (23). Although this conclusion agrees with the met-
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abolic similitude for the release of NE induced by both veratridine and preganglionic nerve stimulation, it does not fit with the proposal that ACh is the physiological substance involved in the release of NE caused by nerve stimulation. An alternative explanation for the differences in the proportion of unmetabolized 3H-NE may rely on the fact that for the metabolic studies no frequency-response curves or concentration-response curves were performed, i.e., that the results were obtained at either a unique frequency of stimulation or a unique concentration of the depolarizing agent. Dealing with it, differences in the percentage of unmetabolized 3H-NE have been reported by Dubocovich and Langer (24) for the perfused cat spleen submitted to different frequencies of nerve stimulation. The significantly higher amount of 3H-NE released by 60 m~ KCI in the SCG compared to the nictitating membrane could rely on the fact that in addition to a direct depolarization caused by KCI on the noradrenergic neuron of the SCG,an indirect effect of KCI mediated by ACh released from the preganglionic fibers had contributed to increase the overflow of NE. This last mechanism has been proposed for the overflow of NE induced by preganglionic stimulation of the cat SCG and it can be prevented by performing the experiments in the presence of i0.0 ~M atropine and 1.0 mM hexamethonium (2). Under these experimental conditions the overflow of 3H-NE induced by 60 mM KCI (Table I) had a similar magn~ tude to that observed in the absence of the cholinergic blockers (Fig. I). These observations suggest both that ACh has a negligible contribution to the observed overflow of ~H-NE evoked by KCI and that the structures from which NE is released in the ganglion are not identical to the nerve terminals in the nic titating membrane. The observation that chemical sympathectomy, which in adult cats destroys nerve terminals selectively without affecting cell bodies or dendrites (7) had not modified the overflow of 3H-NE induced by KCI in the cat SCG, gives further support to the view that this 3H-NE does not come from nerve terminals in the cat SCG (2). The possibility that the dose of 6-OH-DA employed had not reached the catecholaminergic structures of the SCG was discarded based on previous ev~ dence from Filinger and Rubio. They found that an identical schedule of 6-OH-DA pretreatment caused a significant fall in the endogenous content of dopamine in the SCG (25). The electrosecretory coupling in the adrenergic nerve endings is Ca++-de pendent and linked to the Na+ carrying mechanism (for review see 26). The data presented suggest that the depolarization-coupled release of NE from the cat SCG involves structures that also contain Na + channels as well as Caq-~ channels. If dendrites are the actual structures from which NE originates in the cat ganglia, the present results would indicate that dendrites are endowed with similar ionic mechanisms to those acting during the depolarization-coupled r~ lease of neurotransmitters in nerve terminals. A Ca++-dependent mode of neurotransmitter release from dendrites has also been proposed for the overflow of 3H-dopamine elicited by depolarization with hypertonic KCI in the rat substantia nigra (27). References I. T. CHIBA and T.H. WILLIAMS, Cell. Tissue Res. 162 331-341 (1975). 2. A.E. MARTINEZ and E. ADLER-GRASCHINSKY, J. Pharmacol. Exp. Ther. 212 527532 (1980). 3. A.M. SHANES, Pharmacol. Rev. 10 59-164 (1958). 4. W.A. CATTERALL and M. NIRENBERG, Proc. Natl. Acad. Sci. (USA) 70 3759-3763
(1973). 5. M. OHTA, T. NARAHASHI and R. KEELER, J. Pharmacol. Exp. Ther.184 143-154
(1973).
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6. T. NARAHASHI, J.N. MOORE and W.R. SCOTT, J. Gen. Physiol. 4 7 965-974(1964). 7. J.P. TRANZER and H. THOENEN, Experientia 24 155-156 (1968). 8. E. ADLER-GRASCHINSKY, S.Z. LANGER and M.C. RUBIO, J. Pharmacol. Exp. Ther. 180 286-301 (1972). 9. R. LAVERTY and K.M. TAYLOR, Anal. Biochem. 22 269-279 (1968). I0. K.-H. GRAEFE, F.J.E. STEFANO and S.Z. LANGER, Biochem. Pharmacol. 22 11471160 ( 1 9 7 3 ) . 11. M.P. BLAUSTEIN, J . P h y s i o l . 247 6 1 7 - 6 5 5 ( 1 9 7 5 ) . 12. N.B. THOA, F. WOOTEN, J . AXELROD a n d I . J . KOPIN, Mol. P h a r m a c o l . 11 1 0 - 1 8 (1975). 13. M.P. BLAUSTEIN, E.M. JOHNSON a n d P. NEEDLEV~N, P r o c . N a t l . Acad. S c i . (USA) 69 2 2 3 7 - 2 2 4 0 ( 1 9 7 2 ) . 14. W.W. DOUGLAS, B r . J . P h a r m a c o l . 34 4 5 1 - 4 7 4 ( 1 9 6 8 ) . 15. G. HAEUSLER, H. THOENEN, W. HAEFE-LY and A. HUERLIMANN, Naunyn-Schmiedeberg's Arch. Pharmacol. 261 389-411 (1968). 16. S.M. KIRPEKAR and J.C. PRAT, Proc. Natl. Acad. Sci. (USA) 76 2081-2083 (1979). 17. E. BLASCHKE and B. UVNgS, Acta Physiol. Scand. 113 267-269 (1981). 18. S.Z. LANGER and M.A. L~qERO, J. Pharmacol. Exp. Ther. 191 431-443 (1974). 19. M.A. LUCHELLI-FORTIS and S.Z. LANGER, Naunyn-Schmiedeberg's Arch.Pharmacol. 287 261-275 (1975). 20. M.B. FARAH, E. ADLER-GRASCHINSKY and S.Z. LANGER, Naunyn-Schmiedeberg's Arch. Pharmacol. 297 119-131 (1977). 21. G.A. GODDARD and J.D. ROBINSON, Brain Res. ii0 331-350 (1976). 22. P.P. LI and T.D. WHITE, J. Neurochem. 28 967-975 (1977). 23. G. LEVI, V. GALLO and M. RAITERI, Neurochem. Res. 5 281-295 (1980). 24. M.I. DUBOCOVlCH and S.Z. LANGER, J. Pharmacol. Exp. Ther. 198 83-I01(1976). 25. E.J. FILINGER and M.C. RUBIO, Acta physiol. Latinoam. 26 76 (1976). 26. W. HAEFELY, Catecholamines, Eds. H. Blaschko and E. Muscholl, pp. 661-725, Springer-Verlag, New York (1972). 27. L.B. GEFFEN, T.M. JESSEL, A.C. CUELLO and L.L. IVERSEN, Nature 260 259-260 (1976).
Pergamon Press
IONIC MECHANISMS INVOLVED IN THE RELEASE OF 3H-NOREPINEPHRINE FROM THE CAT SUPERIOR CERVICAL GANGLION E. Adler-Graschinsky, E.J. Filinger and A.E. Martfnez Instituto de Investigaciones Farmacol6gicas (CONICET) Junfn 956-5 ° Piso, 1113 Buenos Aires, Argentina (Received in final form December 14, 1983) Summary It has previously been reported that in the isolated cat superior cervical ganglion (SCG) labeled with tritiated norepinephrine (3H-NE), the stimulation of the preganglionic trunk at I0 Hz as well as the exposure to I00 pM exogenous acetylcholine (ACh), produced a Ca++-dependent release of 3H-NE. The present results show that a Ca++-dependent release of 3H-NE was produced also by exposure to either 50 pM veratridine or 60 mM KCI. Tetrodotoxin (0.5~M) abolished the release of 3H-NE induced by preganglionic stimulation, ACh and veratridine but did not modify the release evoked by KCI. The metabolic distribution of the radioactivity released by the different depolarizing stimuli showed that the 3H-NE was collected mainly unmetabolized. In the cat SCG neither the release of 3H-NE evoked by KCI nor the endogenous content of NE was modified by pretreatment with 6-OH-dopamine (6-OH-DA). On the other hand, this chemical sympathectomy depleted the endogenous content of NE in the cat nictitating membrane, whose nerve terminals arise from the SCG. The data presented suggest that the depolarization-coupled release of NE from the cat SCG involves structures that are different to nerve terminals and that contain Na + channels as well as Ca++ channels. It has been reported that in the isolated superior cervical ganglion(SCG) of the cat, the orthodromic stimulation of the cervical sympathetic preganglionic trunk evokes a release of norepinephrine (NE) which is dependent on the presence of extracellular Ca++ and is reduced by exposure to hexamethonium. Based on anatomical descriptions (I) and on the observation that the exogenous acetylcholine (ACh) had a releasing effect with similar characteristics to that evoked by the orthodromic stimulation, it was proposed elsewhere that the ACh released from preganglionic fibers evokes the output of NE through the activation of nicotinic receptors (2). The site from which NE originates in the cat ganglion has been proposed to be different from both the SIF cells and the nerve terminals and considered most probably as dendrites (2). The aim of the present study was to further investigate the ionic mechanisms that participate in the release of NE from sympathetic ganglia. Two depolarizing agents, veratridine and hypertonic KCI were employed to induce the overflow of radioactivity from the isolated cat SCG labeled in vitro with 3H-NE. The depolarization of nerve cell membranes caused by hypertonic KCI results from the reduction in the concentration gradient of potassium between the intr~ cellular and the extracellular spaces (3), whereas that elicited by veratridine is primarily due to the increase in the sodium permeability of the nerve membrane (4,5). Hence, the effects of tetrodotoxin (TTX) which selectively blocks 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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the mechanism whereby stimulation induces an increase in sodium permeability of the nerve membranes (6) were also assayed. In addition, in order to further analyze whether the sites from which NE originates in the ganglia are different from nerve terminals, the effect of KCI was tested on SCG isolated from adult cats pretreated with 6-OH-dopamine (6-OH-DA) to destroy nerve terminals (7). The latter results were compared with those provoked by depolarization with KCI of the nerve terminals of the nictitating membrane which arise from the postganglionic fibers of the SCG. Methods Animals: Adult cats of 1.5 to 3.0 kg of body weight and of either sex were anaesthetized with sodium pentobarbitone (35 mg/kg, i.p.) before use. Solutions: Modified Krebs'solution of the following composition (n~O was emplo~ ed: NaCI, 118,0; KCI, 4.7; CaCI2, 2.6; MgCI2, 1.2; NaH2PO4, 1.0; NaHCO3, 25.0; glucose, II.I; ethylenediamine tetraacetic acid (EDTA), 0.004 and ascorbic acid, 0.Ii. The solution was bubbled with a 95 % 02 : 5 % CO 2 mixture, so that its final pH was 7.4 and it was maintained at 37°C throughout the experiment. Both the addition of elevated concentrations of KCI and the omission of CaCI 2 from the Krebs'solution were performed without osmotic compensation. Superior cervical ~an~lion: Either the right or the left superior cervical ganglion (SCG) was placed into Krebs'solution and dissected together with 5 cm of the cervical sympathetic (preganglionic) nerve and about 0.5 cm of the carotid (postganglionic) trunk. The ganglion and the attached nerves were desheathed. The postganglionic fibers were cut close to the body of the ganglion and about 1 cm of the preganglionic trunk covered with silicone ~as employed to fix the preparation into an isolated organ bath of 3 ml capacity. After preincubation of the ganglion for 15 min in the Krebs'solution at 37°C, the NE stores were I~ beled b~ incubation of the tissue during 30 min with 5 ~Ci/ml (0.67 pM) of (~)-(7-3H)-NE (New England Nuclear, specific activity 7.5 Ci/mmole) as described elsewhere (8). At the end of the incubation period the tissue was submitted to eight consecutive l-min washes and then to twelve consecutive 5-min washes before the collection of the samples started. For the electrical stimulation the preganglionic sympathetic nerve was pulled through shielded bipolar platinum electrodes and square-wave pulses of 2 msec duration were delivered at I0 Hz and at supramaximal voltage (80 V) during 5 minutes. The stimulation by drugs was performed by exposure to either veratridine or ACh or elevated concentrations of potassium chloride. ACh was added together with 3.08 ~M eserine. Veratridine, as well as KCI, were employed simultaneously with both 1.0 mM hexamethonium and i0 ~M atropine. Two consecutive periods of stimulation (S 1 and $2) were performed 40 min apart; the first one was applied 80 min after the end of the incubation with 3M-NE. Aliquots of 0.5 ml of the bathing solution which had been in contact with the tissue for 2 to 5 min were taken for determination of total radioactivity in a liquid scintillation spectrometer. The overflow of radioactivity induced by stimulation was calculated by subtracting the spontaneous outflow assumed to have occurred in each sample during and after the period of stimulation and it was expressed as the fractional rate of loss (FR x 10-2), i.e. as the percentage of the tissue content released per minute. This last value was calculated by addition of the radioactivity lost during the successive washes to that mea~ ured in the tissue at the end of the experiment. The ratios $2/S 1 of the release of radioactivity induced by the two consecutive stimulations under control conditions were employed to compare the effect of drugs added during the second stimulation. At the end of the experiment the tissue was processed to determine endogenous NE according to Laverty and Taylor (9). In some experi-
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Release of 3H-NE From the Cat SCG
863
ments, 5.0 ml of the bathing solution pooled from two ganglia were employed for chromatographic separation of NE and its metabolites using alumina and Dowex 50x4 columns, according to the method described by Graefe, Stefano and Langer (I0). In separate experimental groups the cats were pretreated with 6-hydroxydopamine (6-OH-DA) 40 mg/kg, i.p., 40 hr before the isolation of the ganglia. The significance of differences between values was determined by Student's t test. A P value smaller than 0.05 was regarded as significant. Nictitatin~ membrane: The eyeball was excised and the nictitating membrane with all the adjoining tissue was removed from the orbit. The tissue was placed in Krebs'solution, the medial muscle was dissected and the cartilage on which the fibers of the medial muscle are inserted was attached to the bottom of a I0 ml organ bath. The upper end of the muscle was connected to a force-displacement transducer and the tension developed by the muscle was recorded with a Grass polygraph. The tissue was labeled with 3H-NE, submitted to depolarization with KCI and employed to determine endogenous NE as reported for the SCG. Nictitating membranes isolated from cats pretreated with 6-OH-DA were also employed. Further details are described under Results. Dru~s: The following drugs were used: acetylcholine hydrochloride, eserine sulfate, hexamethonium bromide and atropine sulfate (Sigma Chemical Co.); veratridine (Aldrich Chemical Co.); tetrodotoxin (Sankyo Co. Ltd.) and 6-hydroxydopamine (Regis). All drugs but veratridine were dissolved into distilled water and added to the organ bath in a volume up to I00 microliters. Veratridine was dissolved at a concentration of 20 mg/ml in dimethylsulfoxide (DMSO). When 50 ~LM veratridine was employed, the concentration of DMSO in the organ bath was 23 raM and it had no effect on either the rate of efflux or the endogenous content of NE in the isolated ganglia. Results Effects of Ca++-deprivation on the overflow of 3H-NE evoked by KCI and by veratridine in the isolated cat SCG As shown in Fig. I, the exposure to KCI produced a concentration-dependent release of 3H-NE from the isolated cat SCG and from the isolated cat nictitating membrane which is innervated by the nerve terminals of the postganglionic fibers that arise from the SCG. The concentration-dependent effect of KCI was observed within the range of 40 to 60 mM in the SCG and of 60 to I00 mM in the nictitating membrane. The magnitude of the releasing effect produced by KCI in both tissues was similar at 40, 80 and I00 mM KCI, but differed at 60 mM KCI. With this last concentration the fractional rate of loss of 3H-NE in the SCG (0.96 + 0.16, n = 6) was significantly higher than that obtained in the nictit~ ting m~mbrane (0.44 + 0.07, P < 0.001). Depolarization with veratridine has been employed to study mechanisms of transmitter release at central (II) and peripheral synapses (12). The present study shows that veratridine (Table I) evoked the overflow of 3H-NE in the isolated cat SCG as well. Veratridine was assayed in the presence of hexamethonium plus atropine to prevent from the NE-releasing effect caused by ACh (2). The overflow of ~H-NE induced by the concentration of veratridine employed (50 ~M) had a similar order of magnitude to that evoked by other depolarizing agents in the cat ganglion (between 0.5 and 1.0 % of the tissue content,Tablel) and it was not overcome by higher concentrations of veratridine, as determined in preliminary experiments. It has been reported that in the isolated cat SCG the omission of Ca ++ from the incubation medium prevented the release of 3H-NE induced by either exposure to ACh or electrical stimulation of the preganglionic fibers (2). Expe~ iments were performed in the present study to search whether the omission of
864
Release of 3H-NE From the Cat SCG
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¢'4
IO
1.50
m
0.75 lad
Z !
zIZ
O-
I
I
I
I
40
60
80
100
K Ct (mMl FIG. 1 Release of 3H-NE induced by KCI from the isolated cat superior cervical ganglion (e) and the isolated cat nictitating membrane (O) Ordinate: increase in the release of radioactivity above basal levels expressed as the fractional rate of loss (FR x 10-2), i.e. as the percentage of the tissue content released per minute by the 2.5 min exposures to KCI. Abscissa (log scale): concentrations of KCI. Mean values are shown. Vertical lines indicate the S.E. of at least 6 experiments per group for the ganglion and 7 experiments per group for the nictitating membrane. Ca ++ modified the effects of KCI and of veratridine on the release of 3H-NE from the SCG of the cat. Both depolarizing agents have been shown to produce a Ca++-dependent release of NE from rat brain synaptosomes (13). As shown in Fig. 2, the release of 3H-NE induced by both KCI and veratridine in the presence of hexamethonium plus atropine, was almost entirely abolished when Ca++ was omitted from the incubation medium. The reductions observed in the Ca++-free medium were significant at P < 0.001. The values of the fractional release of 3H-NE (FR x 10 -2 ) obtained above basal levels when Ca++ was restored (0.88 + 0.ii, n = 8, for KCI and 0.79 ~ 0.20, n = 6, for veratridine) were similar ~n magnitude to those evoked by the same depolarizing agents under control conditions (see Table I). Effects of TTX on the overflow of 3H-NE and metabolic distribution of the 3H-NE released by the depolarizing stimuli Tetrodotoxin selectively blocks the mechanism whereby stimulation induces an increase in the sodium permeability of the membrane (6). As shown in Table I 0.5 ~M TTX abolished the increase in the release of 3H-NE induced by veratridine, preganglionic stimulation and acetylcholine but it had no effect on the overflow of 3H-NE evoked by 60 mM KCI.
5.0
5.0
2.0
Veratridine 50 ~M
Preganglionic nerve stimulation (I0 Hz, 2 msec, 80 Volts)
Acetylcholine I00 ~M
Eserine 3.08 ~M
None
Hexamethonium 1.0 mM plus atropine I0.0 ~M
Hexamethonium 1.0 mM plus atropine I0.0 ~M
Drugs present during stimulation
0.44+_0.11 (8)
1.15+--0.26 (9)
0.59+0.07 (i0)
1.14+__0.05(9) (c)
FR x 10 -2 (a)
1.38+-0.42 (8)
0.01+0.01 (6)
0.0_5~0.02
(0.025
(0.001
1.16+-0.12 (9)
(6)
N.S.
<0.005
0.92_+0.08 (7)
TTX
1.37+0.29 (10) 0.03+0.01 (6)
0.92+__0.12 (g)
Control
P (d)
(a) The increase in tritium release above basal levels elicited by the stimulation is expressed as a fraction of the tissue content released per minute. Hexamethonium and atropine were added 25 min before and during the stimulus. Eserine was added simultaneously with acetylcholine. (b) Two consecutive periods of stimulation (S 1 and S 2) were performed 40 min apart and the efflux of radioactivity was expressed as the ratio $2/S I. Values obtained under control conditions were compared with those obtained when the second stimulation was performed after 5 min incubation with 0.5 pM TTX. (c) Each value is the mean + S.E. of the number of experiments indicated between parentheses. (d) Values of P based on StUdent's t test. The TTX ratios were compared to the control ratios. N.S.: not significant.
2.5
Stimulation period (min)
Potassium chloride 60mM
Depolarizing stimulus
Ratios $2/51 (b)
Radioactivity released by the stimulation
Effects of TTX on the release of tritium elicited under different experimental conditions in the isolated cat superior cervical ganglion labeled with 3H-NE (Krebs'solution, 37°C)
TABLE I
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Release of 3H-NE From the Cat SCG
Vol.
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FIG. 2
A) KCI 60mM 1.0
0.5
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Ca** free
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B) Verafridine 50~M
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,
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Effects of Ca++-deprivation on the release of 3H-NE induced by 2.5 min exposures to KCI and by 5.0 min exposures to veratridine in the cat superior cervical ganglion. The ordinates indicate the release of radioactivity expressed as the fractional rate of loss (FR x 10-2), i.e. as the percentage of the tissue content released per minute. The open bars correspond to the spontaneous out flow of 3H-NE in consecutive samples. The -period of exposure to the depolarizing agent is shown by the black rectangle at the bottom of each panel and the increases in the release of radioactivity above basal levels are indicated by the cross-hatched bars. Hexamethonium (I mM) and atropine (I0 pM) were added 30 min before S I and were maintained in the organ bath up to the end of the experiment. Mean values are shown. Vertical lines indicate the S.E. of the mean of n experiments per group. In A (upper panel): consecutive 2.5 min samples were taken. S 1 was performed 85 min after the incubation in Ca++-free Krebs'solution and S 2 20 min after the incubation in the normal Krebs'solution (nffiS). In B (lower panel): consecutive 5 min samples were taken. S 1 was performed 40 min after the incubation in Ca++-free Krebs'solution and S 2 40 min after the addition of the normal Krebs'solution (n=5).
Fig. 3 shows that under depolarization with KCI, veratridine, preganglionic stimulation and ACh the unmetabolized 3H-NE was the main fraction released and the deaminated glycol 3H-DOPEG was the main metabolite collected. However, the percentage of unmetabolized 3H-NE released both by veratridine and by preganglionic stimulation was significantly higher than that observed under stimulation with either ACh or KCI. The lower panel of Fig. 3 shows that neither eserine nor hexamethonium plus atropine modified the metabolic distribution of the radioactivity released spontaneously. The absolute value for the spontaneous efflux of radioactivity in 5 min samples (23.00 + 2.44 nCi/100 mg tissue in the controls, n ffi 9) was not modified by TTX (23.79 + 2.09 nCi/100 mg tissue, n ffi 6), hexamethonium plus atropine (20.78 ~ 1.26 nCi/T00 mg tissue, n = 7) and eserine (18.88 ~ 3.26 nCi/ I00 mg tissue, n m 4). Effects of chemical sympathectomy As reported by Tranzer and Thoenen (7) chemical sympathectomy in adult cats destroys nerve terminals selectively without affecting cell bodies or dendrites. Fig. 4 B shows that the pretreatment with 6-OH-DA depleted the endoge nous content of NE in the nictitating membrane but not in the SCG. The 6-OH-DA treated ganglia also maintained their capacity to increase the overflow of 3H-NE in response to the depolarization with KCI (Fig. 4 A) as well as to store and to spontaneously release the 3H-transmitter. The radioactivity retained
Vol. 34, No. 9, 1984
Release of 3H-NE From the Cat SCG
[]NE
~]DOPEO"DOMA []OMDA ONMN
A) Increase in 3H-release evoked by depotarizafion POTASSIUMCHLORIDE1~.)
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of 3H-products HEXAMETHONIUM
ESERINE (3)
0
FIG. 3 Metabolism of 3H-norepinephrine released under different experimental conditions in the isolated cat superior cervical ganglion. The abbreviations correspond to unmetabolized 3H-norepinephrine (NE); 3H-3,4-dihydroxyphenylglycol (DOPEG); 3H-3,4-dihydroxymandelic acid (DOMA); 3H-O-methylated deaminated metabolites (OMDA) and 3Hnormetanephrine (NMN). Mean values of the number of experiments indicated in parentheses are shown. Vertical lines indicate the S.E. of the mean. In A (upper panel): increase above basal levels induced by different depolarizing stimuli. The conditions of stimulation are described in Table I. In B (lower panel): metabolic distr~ bution of the spontaneous output of 3H-NE in the presence of the different drugs added together with the depolarizing stimulus as described in Table I.
867
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Release of 3H-NE From the Cat SCG
I.Z~-
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Vol. 34, No. 9, 1984
B
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FIG. 4 Effects of the pretreatment with 6-OH-DA (40 mg/kg, i.p., 40 hr before the experiment) on the content of endogenous NE and on the K+-induced release of 3H-NE in the cat superior cervical ganglion (SCG) and nictitating membrane (NM). In A: increase in the radio activity above basal levels expressed as the fractional rate of loss (FR x 10-2), i.e. as the percentage of the tissue content released per minute by a 2.5 min exposure to 60 mM KCI. In B: en dogenous content of NE as micrograms per gram of wet tissue. Mean values of 6 experiments for the control group (open bars) and of 5 experiments for the 6-OH-DA treated-group (cross-hatched bars) are shown. Vertical lines indicate the S.E. of the mean. The asterisk denotes significance at P ~ 0.001 compared to the corresponding control. in the tissue at the end of the experiment was 18.58 ~ 2.22 ~Ci/g tissue, n=7, in the controls and 20.70 + 3.09 ~Ci/g tissue, n = 4, in the 6-OH-DA treated ganglia. The spontaneous outflow of tritium in 5 min samples collected 120 min after the incubation with the 3H-transmitter was 23.00 + 2.44 nCi/lO0 mg tissue in the controls, n = 9 and 22.90 + 4.10 nCi/lO0 mg tissue, n = 4, after 6-OH-DA. In addition, the metabolism of the 3H-NE released after sympathectomy did not differ from control values for either the spontaneous outflow or the K+-in duced release of the transmitter. Thus, unmetabolized 3H-NE was 15.9 + 1.4 % of the total spontaneous efflux in the controls (n = 4) and 14.1 + 2.3--% after 6-OH-DA (n = 4). Moreover, the increment in 3H-NE release above ~asal levels induced by 60 mM KCI was constituted by 38.5 + 3.6 % of unmetabolized 3H-NE in the controls (n = 4) and by 43.7 + 3 . 1 % after 6-OH-DA (n = 4).
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Release of 3H-NE From the Cat SCG
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Discussion The present results show that in the isolated SCG of the cat the exposure to hypertonic KCI and the addition of veratridine produce a Ca++-dependent release of 3H-NE as reported previously for the release of 3H-NE induced by preganglionic stimulation and by ACh in the same tissue (2).
The e x t r a c e l l u l a r Ca++ i s one of t h e s p e c i f i c r e q u i r e m e n t s f o r t h e r e l e a s e o f NE from a d r e n e r g i c n e r v e s caused by d e p o l a r i z a t i o n o f t h e c e l l membranes (14). Under t h e p r e s e n t e x p e r i m e n t a l c o n d i t i o n s t h e a b s e n c e of e x t r a c e l l u l a r Ca++ p r e v e n t e d t h e r e l e a s e o f 3H-NE induced by e i t h e r v e r a t r i d i n e o r KC1-i n t h e c a t SCG i n c u b a t e d w i t h hexamethonium p l u s a t r o p i n e . This o b s e r v a t i o n a g r e e s w i t h t h e Cs++-dependence o b s e r v e d f o r t h e r e l e a s e o f 3H-NE induced by e i ther preganglionic stimulation or exogenous ACh in the cat SCG (2). The possibility that KCl had induced the release of 3H-NE through an osmotic effect was ruled out based on the results obtained by Haeusler and coworkers (15). They reported that when the osmotic pressure of the Krebs'solution was elevated by the addition of glucose, sucrose or LiC1 up to the level caused by 160 mM KC1, i.e. up to twice the normal pressure, only a negligible amount of 3H-NE was released by exposure to such solutions (15). The fact that the incubation with tetrodotoxin, which blocks the voltage dependent Na + channels (6), had abolished the overflow of 3H-NE induced in the cat ganglion by veratridine, preganglionic stimulation and ACh but not that evoked by KCI, indicates that the three first mechanisms but not the last one, involve the participation of the fast Na+-channels to evoke the overflow of the radioactive label as a result of the depolarization of the cell membrane. This observation agrees with the results obtained in synaptosomes from rat brain, which showed that the effects of veratridine but not those of KCI were blocked by TTX ( 1 3 ) . The complete blockade caused by TTX on the ACh-evoked release of 3H-NE di~ fers, however, from the results reported for the SIF-cells of the cat adrenal medulla (16,17). Although the authors employed the same TTX concentration (16) or a concentration ten times higher (17) than that used in the present work, they found that TTX does not block at all (17) or only partially blocks (16)the release of catecholamines induced by ACh. These observations give further support to the view that the structures of the cat ganglion from which NE originates are not identical to the SlF cells of the adrenal medulla (2). Hence, it is likely that in the structures from which NE originates in the cat SCG the s~ cretion of NE can be stimulated both through channels coupled to the nicotine ACh receptor (2) and through voltage-dependent Na + channels (present observations). The metabolic distribution of the 3H-NE released by the different depola_r izing stimuli resembled that reported for both the peripheral (18,19) and the central nervous system (20), where the unmetabolized 3H-NE and the metabolite 3H-DOPEG were the main fractions released in response to nerve stimulation. The fact that the proportion of unmetabolized 3H-NE was 40 % for either KCI or ACh and 70 % for either veratridine or preganglionic stimulation seems to be unrelated to the presence of hexamethonium plus atropine or of eserine, because these drugs did not affect by themselves the metabolic pattern of the 3H-NE spontaneously released. It is of interest to note that the cationic fluxes elicited by veratridine resembled more the fluxes occurring during physiological depolarization than those taking place during depolarization with high extracellular K+ concentrations (11,21,22). For this reason and also because media containing high K + are either hypertonic or partly Na+-deficient, the depolarization caused by veratridine has been considered more "physiological" than that obtained with high K+ (23). Although this conclusion agrees with the met-
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Release of 3H-NE From the Cat SCG
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abolic similitude for the release of NE induced by both veratridine and preganglionic nerve stimulation, it does not fit with the proposal that ACh is the physiological substance involved in the release of NE caused by nerve stimulation. An alternative explanation for the differences in the proportion of unmetabolized 3H-NE may rely on the fact that for the metabolic studies no frequency-response curves or concentration-response curves were performed, i.e., that the results were obtained at either a unique frequency of stimulation or a unique concentration of the depolarizing agent. Dealing with it, differences in the percentage of unmetabolized 3H-NE have been reported by Dubocovich and Langer (24) for the perfused cat spleen submitted to different frequencies of nerve stimulation. The significantly higher amount of 3H-NE released by 60 m~ KCI in the SCG compared to the nictitating membrane could rely on the fact that in addition to a direct depolarization caused by KCI on the noradrenergic neuron of the SCG,an indirect effect of KCI mediated by ACh released from the preganglionic fibers had contributed to increase the overflow of NE. This last mechanism has been proposed for the overflow of NE induced by preganglionic stimulation of the cat SCG and it can be prevented by performing the experiments in the presence of i0.0 ~M atropine and 1.0 mM hexamethonium (2). Under these experimental conditions the overflow of 3H-NE induced by 60 mM KCI (Table I) had a similar magn~ tude to that observed in the absence of the cholinergic blockers (Fig. I). These observations suggest both that ACh has a negligible contribution to the observed overflow of ~H-NE evoked by KCI and that the structures from which NE is released in the ganglion are not identical to the nerve terminals in the nic titating membrane. The observation that chemical sympathectomy, which in adult cats destroys nerve terminals selectively without affecting cell bodies or dendrites (7) had not modified the overflow of 3H-NE induced by KCI in the cat SCG, gives further support to the view that this 3H-NE does not come from nerve terminals in the cat SCG (2). The possibility that the dose of 6-OH-DA employed had not reached the catecholaminergic structures of the SCG was discarded based on previous ev~ dence from Filinger and Rubio. They found that an identical schedule of 6-OH-DA pretreatment caused a significant fall in the endogenous content of dopamine in the SCG (25). The electrosecretory coupling in the adrenergic nerve endings is Ca++-de pendent and linked to the Na+ carrying mechanism (for review see 26). The data presented suggest that the depolarization-coupled release of NE from the cat SCG involves structures that also contain Na + channels as well as Caq-~ channels. If dendrites are the actual structures from which NE originates in the cat ganglia, the present results would indicate that dendrites are endowed with similar ionic mechanisms to those acting during the depolarization-coupled r~ lease of neurotransmitters in nerve terminals. A Ca++-dependent mode of neurotransmitter release from dendrites has also been proposed for the overflow of 3H-dopamine elicited by depolarization with hypertonic KCI in the rat substantia nigra (27). References I. T. CHIBA and T.H. WILLIAMS, Cell. Tissue Res. 162 331-341 (1975). 2. A.E. MARTINEZ and E. ADLER-GRASCHINSKY, J. Pharmacol. Exp. Ther. 212 527532 (1980). 3. A.M. SHANES, Pharmacol. Rev. 10 59-164 (1958). 4. W.A. CATTERALL and M. NIRENBERG, Proc. Natl. Acad. Sci. (USA) 70 3759-3763
(1973). 5. M. OHTA, T. NARAHASHI and R. KEELER, J. Pharmacol. Exp. Ther.184 143-154
(1973).
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Release of 3H-NE From the Cat SCG
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6. T. NARAHASHI, J.N. MOORE and W.R. SCOTT, J. Gen. Physiol. 4 7 965-974(1964). 7. J.P. TRANZER and H. THOENEN, Experientia 24 155-156 (1968). 8. E. ADLER-GRASCHINSKY, S.Z. LANGER and M.C. RUBIO, J. Pharmacol. Exp. Ther. 180 286-301 (1972). 9. R. LAVERTY and K.M. TAYLOR, Anal. Biochem. 22 269-279 (1968). I0. K.-H. GRAEFE, F.J.E. STEFANO and S.Z. LANGER, Biochem. Pharmacol. 22 11471160 ( 1 9 7 3 ) . 11. M.P. BLAUSTEIN, J . P h y s i o l . 247 6 1 7 - 6 5 5 ( 1 9 7 5 ) . 12. N.B. THOA, F. WOOTEN, J . AXELROD a n d I . J . KOPIN, Mol. P h a r m a c o l . 11 1 0 - 1 8 (1975). 13. M.P. BLAUSTEIN, E.M. JOHNSON a n d P. NEEDLEV~N, P r o c . N a t l . Acad. S c i . (USA) 69 2 2 3 7 - 2 2 4 0 ( 1 9 7 2 ) . 14. W.W. DOUGLAS, B r . J . P h a r m a c o l . 34 4 5 1 - 4 7 4 ( 1 9 6 8 ) . 15. G. HAEUSLER, H. THOENEN, W. HAEFE-LY and A. HUERLIMANN, Naunyn-Schmiedeberg's Arch. Pharmacol. 261 389-411 (1968). 16. S.M. KIRPEKAR and J.C. PRAT, Proc. Natl. Acad. Sci. (USA) 76 2081-2083 (1979). 17. E. BLASCHKE and B. UVNgS, Acta Physiol. Scand. 113 267-269 (1981). 18. S.Z. LANGER and M.A. L~qERO, J. Pharmacol. Exp. Ther. 191 431-443 (1974). 19. M.A. LUCHELLI-FORTIS and S.Z. LANGER, Naunyn-Schmiedeberg's Arch.Pharmacol. 287 261-275 (1975). 20. M.B. FARAH, E. ADLER-GRASCHINSKY and S.Z. LANGER, Naunyn-Schmiedeberg's Arch. Pharmacol. 297 119-131 (1977). 21. G.A. GODDARD and J.D. ROBINSON, Brain Res. ii0 331-350 (1976). 22. P.P. LI and T.D. WHITE, J. Neurochem. 28 967-975 (1977). 23. G. LEVI, V. GALLO and M. RAITERI, Neurochem. Res. 5 281-295 (1980). 24. M.I. DUBOCOVlCH and S.Z. LANGER, J. Pharmacol. Exp. Ther. 198 83-I01(1976). 25. E.J. FILINGER and M.C. RUBIO, Acta physiol. Latinoam. 26 76 (1976). 26. W. HAEFELY, Catecholamines, Eds. H. Blaschko and E. Muscholl, pp. 661-725, Springer-Verlag, New York (1972). 27. L.B. GEFFEN, T.M. JESSEL, A.C. CUELLO and L.L. IVERSEN, Nature 260 259-260 (1976).