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Sympathetic transmitter release in rat tail artery and mouse vas deferens: facilitation and depression during high frequency stimulation
Neuroscience Letters, 155 (1993) 37~,1 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940193/$ 06.00
37
NSL 09527
Sympathetic transmitter release in rat tail artery and mouse vas deferens: facilitation and depression during high frequency stimulation M. Msghina and L. Stj/irne Department of Physiology, Karolinska lnstitutet, Stockholm (Sweden) (Received 18 December 1992; Revised version received 25 February 1993; Accepted 26 February 1993)
Key words: Mouse vas deferens; Rat tail artery; Adenosine 5'-triphosphate; Noradrenaline; Cotransmission; Facilitation; Depression Electrophysiological and electrochemical methods were used to study the release of adenosine 5'-triphosphate (ATP) and noradrenaline (NA) from sympathetic nerves during stimulation with trains at 20 Hz (tetanus). In mouse vas deferens ATP release was mainly facilitated during the tetanus, but in rat tail artery progressively and reversibly depressed. In rat tail artery reduction of external calcium attenuated the depression and increased the facilitation during the tetanus, while increased external calcium accentuated the depression. Both ATP and NA release were depressed in parallel during the first 100 pulses of the tetanus. The depression of release was not due to action potential failure, or 0~:-adrenoceptor-mediated autoinhibition.
As shown by electrophysiological analysis, nerve stimulation with a train at high frequency ('tetanus') causes variable short term changes in transmitter release in different neurons. In some, release is mainly facilitated during a tetanus, in some mainly depressed and in others first facilitated then depressed [11]. In individual preparations, the pattern of release during a tetanus varies with the concentration of external calcium; at low calcium release is more strongly facilitated and at high calcium more strongly depressed [9]. The current knowledge about the release of sympathetic transmitters during a tetanus is based on analysis of excitatory junction potentials (EJPs), now known to be caused by adenosine 5'-triphosphate (ATP) [7]. In mouse vas deferens the EJPs were facilitated by the first few pulses at 10 Hz, but during longer trains increasingly depressed [1]. Addition of phenoxybenzamine enhanced the facilitation and alleviated the depression, which hence seemed to be due to activation of prejunctional ~t-adrenoceptors [1]. It is not known at present if ATP release from sympathetic nerves in other tissues is similarly affected during a tetanus, and if the release of noradrenaline (NA) during a high frequency train parallels that of ATP [4, 6, 10]. In the present paper we have addressed these questions by comparing the effects of nerve stimulation at 20 Hz (i) on ATP release in mouse Correspondence: M. Msghina, Department of Physiology, Karolinska Institutet, S-10401 Stockholm, Sweden.
vas deferens and rat tail artery, and (ii) on ATP and NA release in rat tail artery. Male C57 mice (20-30 g) or Sprague-Dawley rats (200-300 g) were stunned and bled to death and the vasa deferentia or 2-4 cm lengths of the proximal region of the central tail artery dissected out and pinned to the Sylgard layer covering the bottom of a 2-3 ml Perspex organ bath. The prostatic end of the vas deferens or the proximal part of the tail artery were drawn into a suction electrode for electrical stimulation via an NL 800 (Neurolog) constant-current isolation unit (rectangular pulses, 0.1-0.3 ms, 0.3 mA). The bath was perfused at 1-2 ml/min with modified Tyrode's solution of the following composition (mM): NaC1 136.9, KC1 2.7, CaCIz 1.3, MgCI2 0.5, glucose 5.6, prazosin 0.001 (added to block the neurogenic contraction) and Tris 20 (pH 7.4). The solution was gassed with 100% O2; the bath temperature held at 35-37°C. An internally perfused extracellular microelectrode (tip diameter of 80-150/lm) was used to record the nerve terminal spike (NTS) and the excitatory junction current (EJC), which reflect the compound nerve terminal action potential and the quantal release of ATE respectively [3, 8]. Differential pulse amperometry was used to study NA release by an electrochemically treated carbon fibre electrode (diameter and length of the active part: 8 pm and 50-120 pro, respectively) connected to a voltammetric instrument ('Biopulse', Solea Tacussel) [5]. The data were analysed statistically by one way analysis of variance (ANOVA), followed by Fischer test.
38
Results are expressed as mean + S.E.M.; P < 0.05 was considered significant. Fig. 1A shows the effects of nerve stimulation for 35 s at 20 Hz (tetanus), preceded and followed by pulse trains at 0.1 Hz, on the EJCs in mouse vas deferens and rat tail artery. During the tetanus, the EJCs in mouse vas deferens initially grew in amplitude to a peak value of 183.4 + 16.1% of the amplitude of control EJCs, then gradually declined towards a steady state level; at the 100th pulse the level was 81.3 + 8.5% of that of control EJCs (Fig. 1A,B; n = 7). In rat tail artery, the EJCs during the first few pulses of the tetanus grew but only to a peak level of 112.3 + 4.1% of that of control EJCs; this transient facilitation was followed by a progressive and profound depression; at the 100th pulse the level was 32.3 + 3.4% of that of control EJCs (Fig. 1A,B; n = 5). In most cases the EJCs recovered rapidly when stimulation was switched from 20 to 0.1 Hz. The size of the first EJC 10 s after the tetanus was 70 + 5.4% of that before the tetanus (n = 6). To find out if depression was due to activation of prejunctional 0~2-adrenoceptors, as earlier suggested [1], the tetanus was repeated in the same preparation with 1 /zM yohimbine (Fig. 1A,B) or 1 tiM
idazoxan (not shown) added to the bath. In mouse vas deferens, yohimbine enhanced the initial facilitation (the peak value was now 260.0_ 23.4% of that of control EJCs) and delayed the onset of the subsequent depression without much affecting the final EJC amplitude (which had a level of 86.1 __ 8.2% of that of control EJCs at the 100th pulse; n -- 5). In rat tail artery, these agents had no effect on the facilitation or depression of the EJCs during the tetanus, or the subsequent recovery (Fig. 1A,B). In the present study transmitter release during the tetanus was analysed in more detail in the rat tail artery. Reduction of the extracellular calcium, from 1.3 mM to 0.65 mM, greatly reduced the size of the EJCs at 0.1 Hz (not shown). The EJC amplitude during the tetanus was now initially markedly enhanced (to peak level of 173.2 __ 11.2% of that of control EJCs), then moderately depressed (to 84.8 __ 7.0% of control EJCs after 100 pulses, Fig. 1A; n = 4). A 4-fold increase in external calcium, to 2.6 mM enhanced the amplitude of the EJCs at 0.1 Hz by 374.5 _+ 27.3%. The EJCs during the tetanus were now profoundly reduced in size starting from the 2nd pulse (to 22.8 +_ 2.3% of control EJCs at the 100th
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Time(s) Fig. 1. A: representative experiments showing changes in the amplitude of excitatory junction currents (EJCs) during a tetanus (35 s at 20 Hz, preceded and followed by pulse trains at 0.1 Hz) in mouse vas deferens and rat tail artery, under different experimental conditions. The EJCs in each experiment are normalized to the first control EJC at 0.1 Hz. B: a s u m m a r y of 5 experiments showing the effects of yohimbine on the EJCs (averaged in groups of 4) during the first 100 pulses of the tetanus, in mouse vas deferens and rat tail artery. The EJCs are normalized to the average of the first 4 EJCs in each group. The effect of yohimbine in the mouse vas deferens was statistically significant for the indicated points (P < 0.05). Bars indicate standard error of mean (_+ S.E.M.). C: the current response to application by pressure ejection of I ,uM adenosine 5'-triphosphate (ATE upper panel) and the EJCs (lower panel) before (Ca), during (Cb) and after (Cc) the tetanus. Arrows indicate stimulation. MVD, mouse vas deferens; RTA, rat tail artery; YO, yohimbine.
39
the tetanus was due to reduced ATP release or to desensitization of postsynaptic P,-purinoceptors by which the EJCs are mediated, we examined the current response to local application of exogenous ATP (1 mM) by pressure ejection with a Picospritzer (20 psi, 3 ms; Fig. IC). The amplitude of the current .responses during and after the tetanus was 96.1 + 3.7% and 99.3 t 8.8%, respectively, of that before the tetanus (n = 4, P > 0.35) showing that
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Stimulus number Fig 2. A: original tracings from an experiment in rat tail arery showing the nerve terminal spike (NTS, l) and excitatory junction current (EJC) before (a), during the initial (b,c) and late phases (d) of the tetanus and IO s after the tetanus (e). B: relationship between the amplitude of the NTS (measured from the first positive to the first negative peak) and EJC during the first 40 pulses. The data are normalized to the NTS or EJC evoked by the first pulse at 0. I Hz before the tetanus.
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pulse; n = 4). The averaged EJC amplitude, 700 pulses at 20 Hz, at 0.65 mM calcium was 94.6 k 8.3% (n = 4) of that at 2.6 mM calcium, suggesting that the same total amount of ATP had been released under the two conditions, with different time-courses. At 0.65 mM calcium the size of the first EJC at 0.1 Hz, 10 s after the tetanus, was 74.5 f 11.8% and at 2.6 mM calcium 65.2 f 6.2% of that of control EJCs before the tetanus (n = 4). The timecourse of recovery of EJCs at 0.65 mM calcium was thus not significantly different from that at 2.6 mM calcium (P > 0.5). To decide whether the depression of the EJCs during
20
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Fig. 3. Original recordings of d[NA],, responses to nerve stimulation with trains of I-16 (A) and 2tLlOO pulses (B) at 20 Hz (the number of pulses in each train is indicated below respective recording). Our approach to assess the release of noradrenaline (NA) by stimuli 14; 5-8; 9-12 and I3 16. or l-20; 2140; 4160: 61-80 and 81 100 was the following: we assume that the d[NAkF responses to pulses IL4 or 1-20 directly reflect the release of NA caused by these pulse trains; the release caused by later 4- or 20-pulse sequences was derived by subtraction, as follows: “b” minus “a” gives the release by pulses 5-8, “c” minus “b” the release by pulses 9-12, etc. Similarly, “f’ minus “e” gives the release by pulses 2140. and “g” minus “f’ the release by pulses 41-60, etc.
40
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Stimulus number Fig. 4. A,B: changes in the amplitude of the excitatory junction current (EJC) and/I[NA]c v responses during nerve stimulation with 100 pulses at 20 Hz in rat tail artery in controls (1.3 mM external calcium and no drugs added). The approach used to calculate the NA release from the A[NA]cF responses is explained in the legend of Fig. 3; the EJCs represent averaged responses to the same stimuli. C,D: effects of 3/zM cocaine alone or together with 1 p M yohimbine on the EJC (C) and A[NA]cF (D) responses. COC, cocaine; COC + YO, cocaine together with yohimbine.
the tetanus did not desensitize the P2x-purinoceptors to exogenous ATE To find out if the depression of the EJCs was due to failure or changes in properties of the action potentials, we compared the nerve terminal spike (NTS) and the EJCs during the tetanus. As shown in Fig. 2, which is representative of 4 experiments, the NTS were not much affected in size or shape during the first 3 0 4 0 pulses, i.e. at a stage when the EJCs were markedly depressed. Later during the tetanus the NTS often became smaller and broader, but these changes were not correlated with the depression or recovery of the EJCs. Next, we compared in rat tail artery the release of ATP and NA during the first 100 pulses at 20 Hz, at 1.3 mM calcium. The amperometric response to nerve stimulation reflects changes in NA concentration at the carbon fibre electrode and is hence termed A[NA]cv. When occa-
sionally occurring in response to single pulse stimulation, the A[NA]cF response had a duration of roughly 5 s (Fig. 3A). The A[NA]cv response to a second pulse applied within 5 s would therefore merge with that caused by the first, precluding direct pulse by pulse analysis of NA release at frequencies higher than 0.2 Hz. To circumvent this difficulty, we used an indirect approach which is explained in Fig. 3 and legend. Since the per pulse release of NA could not be determined directly, we assessed the mean amount of NA released by consecutive sequences of 4 or 20 pulses during stimulation with a total of 16 (Fig. 3A) or 100 (Fig. 3B) pulses at 20 Hz. These calculated values for NA release per 4 or 20 pulse train were compared with the corresponding averaged EJCs, which directly reflect the mean ATP release to the same stimulus trains. The results, shown in Fig. 4A (4 pulse trains,
41 n = 16) and 4B (20 pulse trains, n = 23)iridicate that the release o f N A , similarly to that o f ATP, was slightly facilitated during the first few pulses o f the tetanus and increasingly depressed later. To ensure that the assessment o f N A release was not c o m p r o m i s e d by variable reuptake o f N A , we repeated these experiments in the presence o f the N A reuptake blocker cocaine ( 3 / I M ) . The results (Fig. 4C,D) show that cocaine by itself did not significantly affect the time-course or m a g n i t u d e o f the depression of the E J C (n = 11) or A[NA]cF response (n = 16). F u r t h e r addition o f 1 p M idazoxan was also without significant effect on the depression o f the E J C (n = 6) and A[NA]c F (n = 11) responses, or the recovery after the tetanus (tested only for the EJCs; n = 6, not shown). N o r did 1 / t M yohimbine, added together with cocaine, influence the time-course or m a g n i t u d e of depression o f the EJCs (Fig. 4C, n = 5), but in contrast to idazoxan, significantly delayed the onset o f the depression o f the A[NA]cF response (Fig. 4D, P < 0.05; n = 5). In conclusion, o u r results show that during a tetanus (i) A T P release f r o m sympathetic nerves in m o u s e vas deferens and rat tail artery is differently affected, (ii) A T P release in rat tail artery at ' n o r m a l ' calcium is progressively and p r o f o u n d l y depressed, (iii) the depression o f release is not due to ~2-adrenoceptor-mediated autoinhibition or action potential failure, (iv) external calcium concentration determines the rate o f A T P release during the tetanus but not the total a m o u n t released or the time course o f recovery after the tetanus, (vi) ATP and N A release mostly decline in a similar fashion during the first 100 pulses o f a tetanus at ' n o r m a l ' external calcium, but that for u n k n o w n reasons yohimbine (but not idazoxan) significantly delays the depression o f N A release but not o f ATP, and (vii) most o f the evidence still indicates that ATP and N A m a y be released in parallel, possibly f r o m the same vesicle, under these experimental conditions.
This w o r k w a s - s u p p o r t e d by Swedish Medical Research Council (project B91-14X-03027-22B) and K a r o linska Institutets Fonder. We t h a n k Dr. F. G o n o n for his valuable comments. 1 Bennett, M.R., An electrophysiological analysis of the uptake of noradrenaline at sympathetic nerve terminals, J. Physiol., 229 (1973) 533-546. 2 Betz, W.J., Depression of transmitter release at the neuromuscular junction of the frog, J. Physiol., 206 (1970) 629-644. 3 Brock, J.A. and Cunnane, T.C., Electrical activity at the sympathetic neuroeffector junction in the guinea-pig vas deferens, J. Physiol., 399 (1988) 607-632. 4 Ellis, J.L. and Burnstock, G., Angiotensin neuromodulation of noradrenergic and purinergic co-transmission in the guinea-pig vas deferens, Br. J. Pharmacol., 97 (1989) 1157-1164. 5 Mermet, C., Gonon, F. and Stj/irne, L., On-line electrochemical monitoring of the local noradrenaline release evoked by electrical stimulation of the sympathetic nerves in isolated rat tail artery, Acta Physiol. Scand., 140 (1990) 323-329. 6 Msghina, M., Mermet, C., Gonon, F. and Stj~rne, L., Electrophysiological and electrochemical analysis of the secretion of ATP and noradrenaline from the sympathetic nerves in rat tail artery: effects of ~2-adrenoceptor agonists and antagonists and noradrenaline reuptake blockers, Naunyn-Schmiedeberg's Arch. Pharmacol., 346 (1992) 173-186. 7 Sneddon, P. and Burnstock, G., ATP as a co-transmitter in rat tail artery, Eur. J. Pharmacol., 106 (1984) 149-152. 8 Stj/irne, L. and Stj/irne, E., Basic features of an extracellular recording method to study secretion of sympathetic co-transmitter, presumably ATP, Acta Physiol. Scand., 135 (1989) 217-226. 9 Swandulla, D., Hans, M., Zisper, K. and Augustine, G.J., Role of residual calcium in synaptic depression and posttetanic potentiation: fast and slow calcium signaling in nerve terminals, Neuron, 7 (1991) 915-926. 10 von Kugelgen, I. and Starke, K., Noradrenaline-ATP co-transmission in the sympathetic nervous system, Trends Pharmacol., 12 (1990) 319-324. 11 Zucker, R., Short term synaptic plasticity, Annu. Rev. Neurosci., 12 (1989) 13-31.
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NSL 09527
Sympathetic transmitter release in rat tail artery and mouse vas deferens: facilitation and depression during high frequency stimulation M. Msghina and L. Stj/irne Department of Physiology, Karolinska lnstitutet, Stockholm (Sweden) (Received 18 December 1992; Revised version received 25 February 1993; Accepted 26 February 1993)
Key words: Mouse vas deferens; Rat tail artery; Adenosine 5'-triphosphate; Noradrenaline; Cotransmission; Facilitation; Depression Electrophysiological and electrochemical methods were used to study the release of adenosine 5'-triphosphate (ATP) and noradrenaline (NA) from sympathetic nerves during stimulation with trains at 20 Hz (tetanus). In mouse vas deferens ATP release was mainly facilitated during the tetanus, but in rat tail artery progressively and reversibly depressed. In rat tail artery reduction of external calcium attenuated the depression and increased the facilitation during the tetanus, while increased external calcium accentuated the depression. Both ATP and NA release were depressed in parallel during the first 100 pulses of the tetanus. The depression of release was not due to action potential failure, or 0~:-adrenoceptor-mediated autoinhibition.
As shown by electrophysiological analysis, nerve stimulation with a train at high frequency ('tetanus') causes variable short term changes in transmitter release in different neurons. In some, release is mainly facilitated during a tetanus, in some mainly depressed and in others first facilitated then depressed [11]. In individual preparations, the pattern of release during a tetanus varies with the concentration of external calcium; at low calcium release is more strongly facilitated and at high calcium more strongly depressed [9]. The current knowledge about the release of sympathetic transmitters during a tetanus is based on analysis of excitatory junction potentials (EJPs), now known to be caused by adenosine 5'-triphosphate (ATP) [7]. In mouse vas deferens the EJPs were facilitated by the first few pulses at 10 Hz, but during longer trains increasingly depressed [1]. Addition of phenoxybenzamine enhanced the facilitation and alleviated the depression, which hence seemed to be due to activation of prejunctional ~t-adrenoceptors [1]. It is not known at present if ATP release from sympathetic nerves in other tissues is similarly affected during a tetanus, and if the release of noradrenaline (NA) during a high frequency train parallels that of ATP [4, 6, 10]. In the present paper we have addressed these questions by comparing the effects of nerve stimulation at 20 Hz (i) on ATP release in mouse Correspondence: M. Msghina, Department of Physiology, Karolinska Institutet, S-10401 Stockholm, Sweden.
vas deferens and rat tail artery, and (ii) on ATP and NA release in rat tail artery. Male C57 mice (20-30 g) or Sprague-Dawley rats (200-300 g) were stunned and bled to death and the vasa deferentia or 2-4 cm lengths of the proximal region of the central tail artery dissected out and pinned to the Sylgard layer covering the bottom of a 2-3 ml Perspex organ bath. The prostatic end of the vas deferens or the proximal part of the tail artery were drawn into a suction electrode for electrical stimulation via an NL 800 (Neurolog) constant-current isolation unit (rectangular pulses, 0.1-0.3 ms, 0.3 mA). The bath was perfused at 1-2 ml/min with modified Tyrode's solution of the following composition (mM): NaC1 136.9, KC1 2.7, CaCIz 1.3, MgCI2 0.5, glucose 5.6, prazosin 0.001 (added to block the neurogenic contraction) and Tris 20 (pH 7.4). The solution was gassed with 100% O2; the bath temperature held at 35-37°C. An internally perfused extracellular microelectrode (tip diameter of 80-150/lm) was used to record the nerve terminal spike (NTS) and the excitatory junction current (EJC), which reflect the compound nerve terminal action potential and the quantal release of ATE respectively [3, 8]. Differential pulse amperometry was used to study NA release by an electrochemically treated carbon fibre electrode (diameter and length of the active part: 8 pm and 50-120 pro, respectively) connected to a voltammetric instrument ('Biopulse', Solea Tacussel) [5]. The data were analysed statistically by one way analysis of variance (ANOVA), followed by Fischer test.
38
Results are expressed as mean + S.E.M.; P < 0.05 was considered significant. Fig. 1A shows the effects of nerve stimulation for 35 s at 20 Hz (tetanus), preceded and followed by pulse trains at 0.1 Hz, on the EJCs in mouse vas deferens and rat tail artery. During the tetanus, the EJCs in mouse vas deferens initially grew in amplitude to a peak value of 183.4 + 16.1% of the amplitude of control EJCs, then gradually declined towards a steady state level; at the 100th pulse the level was 81.3 + 8.5% of that of control EJCs (Fig. 1A,B; n = 7). In rat tail artery, the EJCs during the first few pulses of the tetanus grew but only to a peak level of 112.3 + 4.1% of that of control EJCs; this transient facilitation was followed by a progressive and profound depression; at the 100th pulse the level was 32.3 + 3.4% of that of control EJCs (Fig. 1A,B; n = 5). In most cases the EJCs recovered rapidly when stimulation was switched from 20 to 0.1 Hz. The size of the first EJC 10 s after the tetanus was 70 + 5.4% of that before the tetanus (n = 6). To find out if depression was due to activation of prejunctional 0~2-adrenoceptors, as earlier suggested [1], the tetanus was repeated in the same preparation with 1 /zM yohimbine (Fig. 1A,B) or 1 tiM
idazoxan (not shown) added to the bath. In mouse vas deferens, yohimbine enhanced the initial facilitation (the peak value was now 260.0_ 23.4% of that of control EJCs) and delayed the onset of the subsequent depression without much affecting the final EJC amplitude (which had a level of 86.1 __ 8.2% of that of control EJCs at the 100th pulse; n -- 5). In rat tail artery, these agents had no effect on the facilitation or depression of the EJCs during the tetanus, or the subsequent recovery (Fig. 1A,B). In the present study transmitter release during the tetanus was analysed in more detail in the rat tail artery. Reduction of the extracellular calcium, from 1.3 mM to 0.65 mM, greatly reduced the size of the EJCs at 0.1 Hz (not shown). The EJC amplitude during the tetanus was now initially markedly enhanced (to peak level of 173.2 __ 11.2% of that of control EJCs), then moderately depressed (to 84.8 __ 7.0% of control EJCs after 100 pulses, Fig. 1A; n = 4). A 4-fold increase in external calcium, to 2.6 mM enhanced the amplitude of the EJCs at 0.1 Hz by 374.5 _+ 27.3%. The EJCs during the tetanus were now profoundly reduced in size starting from the 2nd pulse (to 22.8 +_ 2.3% of control EJCs at the 100th
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Time(s) Fig. 1. A: representative experiments showing changes in the amplitude of excitatory junction currents (EJCs) during a tetanus (35 s at 20 Hz, preceded and followed by pulse trains at 0.1 Hz) in mouse vas deferens and rat tail artery, under different experimental conditions. The EJCs in each experiment are normalized to the first control EJC at 0.1 Hz. B: a s u m m a r y of 5 experiments showing the effects of yohimbine on the EJCs (averaged in groups of 4) during the first 100 pulses of the tetanus, in mouse vas deferens and rat tail artery. The EJCs are normalized to the average of the first 4 EJCs in each group. The effect of yohimbine in the mouse vas deferens was statistically significant for the indicated points (P < 0.05). Bars indicate standard error of mean (_+ S.E.M.). C: the current response to application by pressure ejection of I ,uM adenosine 5'-triphosphate (ATE upper panel) and the EJCs (lower panel) before (Ca), during (Cb) and after (Cc) the tetanus. Arrows indicate stimulation. MVD, mouse vas deferens; RTA, rat tail artery; YO, yohimbine.
39
the tetanus was due to reduced ATP release or to desensitization of postsynaptic P,-purinoceptors by which the EJCs are mediated, we examined the current response to local application of exogenous ATP (1 mM) by pressure ejection with a Picospritzer (20 psi, 3 ms; Fig. IC). The amplitude of the current .responses during and after the tetanus was 96.1 + 3.7% and 99.3 t 8.8%, respectively, of that before the tetanus (n = 4, P > 0.35) showing that
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pulse; n = 4). The averaged EJC amplitude, 700 pulses at 20 Hz, at 0.65 mM calcium was 94.6 k 8.3% (n = 4) of that at 2.6 mM calcium, suggesting that the same total amount of ATP had been released under the two conditions, with different time-courses. At 0.65 mM calcium the size of the first EJC at 0.1 Hz, 10 s after the tetanus, was 74.5 f 11.8% and at 2.6 mM calcium 65.2 f 6.2% of that of control EJCs before the tetanus (n = 4). The timecourse of recovery of EJCs at 0.65 mM calcium was thus not significantly different from that at 2.6 mM calcium (P > 0.5). To decide whether the depression of the EJCs during
20
40
60
80
100 pulses
Fig. 3. Original recordings of d[NA],, responses to nerve stimulation with trains of I-16 (A) and 2tLlOO pulses (B) at 20 Hz (the number of pulses in each train is indicated below respective recording). Our approach to assess the release of noradrenaline (NA) by stimuli 14; 5-8; 9-12 and I3 16. or l-20; 2140; 4160: 61-80 and 81 100 was the following: we assume that the d[NAkF responses to pulses IL4 or 1-20 directly reflect the release of NA caused by these pulse trains; the release caused by later 4- or 20-pulse sequences was derived by subtraction, as follows: “b” minus “a” gives the release by pulses 5-8, “c” minus “b” the release by pulses 9-12, etc. Similarly, “f’ minus “e” gives the release by pulses 2140. and “g” minus “f’ the release by pulses 41-60, etc.
40
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Stimulus number Fig. 4. A,B: changes in the amplitude of the excitatory junction current (EJC) and/I[NA]c v responses during nerve stimulation with 100 pulses at 20 Hz in rat tail artery in controls (1.3 mM external calcium and no drugs added). The approach used to calculate the NA release from the A[NA]cF responses is explained in the legend of Fig. 3; the EJCs represent averaged responses to the same stimuli. C,D: effects of 3/zM cocaine alone or together with 1 p M yohimbine on the EJC (C) and A[NA]cF (D) responses. COC, cocaine; COC + YO, cocaine together with yohimbine.
the tetanus did not desensitize the P2x-purinoceptors to exogenous ATE To find out if the depression of the EJCs was due to failure or changes in properties of the action potentials, we compared the nerve terminal spike (NTS) and the EJCs during the tetanus. As shown in Fig. 2, which is representative of 4 experiments, the NTS were not much affected in size or shape during the first 3 0 4 0 pulses, i.e. at a stage when the EJCs were markedly depressed. Later during the tetanus the NTS often became smaller and broader, but these changes were not correlated with the depression or recovery of the EJCs. Next, we compared in rat tail artery the release of ATP and NA during the first 100 pulses at 20 Hz, at 1.3 mM calcium. The amperometric response to nerve stimulation reflects changes in NA concentration at the carbon fibre electrode and is hence termed A[NA]cv. When occa-
sionally occurring in response to single pulse stimulation, the A[NA]cF response had a duration of roughly 5 s (Fig. 3A). The A[NA]cv response to a second pulse applied within 5 s would therefore merge with that caused by the first, precluding direct pulse by pulse analysis of NA release at frequencies higher than 0.2 Hz. To circumvent this difficulty, we used an indirect approach which is explained in Fig. 3 and legend. Since the per pulse release of NA could not be determined directly, we assessed the mean amount of NA released by consecutive sequences of 4 or 20 pulses during stimulation with a total of 16 (Fig. 3A) or 100 (Fig. 3B) pulses at 20 Hz. These calculated values for NA release per 4 or 20 pulse train were compared with the corresponding averaged EJCs, which directly reflect the mean ATP release to the same stimulus trains. The results, shown in Fig. 4A (4 pulse trains,
41 n = 16) and 4B (20 pulse trains, n = 23)iridicate that the release o f N A , similarly to that o f ATP, was slightly facilitated during the first few pulses o f the tetanus and increasingly depressed later. To ensure that the assessment o f N A release was not c o m p r o m i s e d by variable reuptake o f N A , we repeated these experiments in the presence o f the N A reuptake blocker cocaine ( 3 / I M ) . The results (Fig. 4C,D) show that cocaine by itself did not significantly affect the time-course or m a g n i t u d e o f the depression of the E J C (n = 11) or A[NA]cF response (n = 16). F u r t h e r addition o f 1 p M idazoxan was also without significant effect on the depression o f the E J C (n = 6) and A[NA]c F (n = 11) responses, or the recovery after the tetanus (tested only for the EJCs; n = 6, not shown). N o r did 1 / t M yohimbine, added together with cocaine, influence the time-course or m a g n i t u d e of depression o f the EJCs (Fig. 4C, n = 5), but in contrast to idazoxan, significantly delayed the onset o f the depression o f the A[NA]cF response (Fig. 4D, P < 0.05; n = 5). In conclusion, o u r results show that during a tetanus (i) A T P release f r o m sympathetic nerves in m o u s e vas deferens and rat tail artery is differently affected, (ii) A T P release in rat tail artery at ' n o r m a l ' calcium is progressively and p r o f o u n d l y depressed, (iii) the depression o f release is not due to ~2-adrenoceptor-mediated autoinhibition or action potential failure, (iv) external calcium concentration determines the rate o f A T P release during the tetanus but not the total a m o u n t released or the time course o f recovery after the tetanus, (vi) ATP and N A release mostly decline in a similar fashion during the first 100 pulses o f a tetanus at ' n o r m a l ' external calcium, but that for u n k n o w n reasons yohimbine (but not idazoxan) significantly delays the depression o f N A release but not o f ATP, and (vii) most o f the evidence still indicates that ATP and N A m a y be released in parallel, possibly f r o m the same vesicle, under these experimental conditions.
This w o r k w a s - s u p p o r t e d by Swedish Medical Research Council (project B91-14X-03027-22B) and K a r o linska Institutets Fonder. We t h a n k Dr. F. G o n o n for his valuable comments. 1 Bennett, M.R., An electrophysiological analysis of the uptake of noradrenaline at sympathetic nerve terminals, J. Physiol., 229 (1973) 533-546. 2 Betz, W.J., Depression of transmitter release at the neuromuscular junction of the frog, J. Physiol., 206 (1970) 629-644. 3 Brock, J.A. and Cunnane, T.C., Electrical activity at the sympathetic neuroeffector junction in the guinea-pig vas deferens, J. Physiol., 399 (1988) 607-632. 4 Ellis, J.L. and Burnstock, G., Angiotensin neuromodulation of noradrenergic and purinergic co-transmission in the guinea-pig vas deferens, Br. J. Pharmacol., 97 (1989) 1157-1164. 5 Mermet, C., Gonon, F. and Stj/irne, L., On-line electrochemical monitoring of the local noradrenaline release evoked by electrical stimulation of the sympathetic nerves in isolated rat tail artery, Acta Physiol. Scand., 140 (1990) 323-329. 6 Msghina, M., Mermet, C., Gonon, F. and Stj~rne, L., Electrophysiological and electrochemical analysis of the secretion of ATP and noradrenaline from the sympathetic nerves in rat tail artery: effects of ~2-adrenoceptor agonists and antagonists and noradrenaline reuptake blockers, Naunyn-Schmiedeberg's Arch. Pharmacol., 346 (1992) 173-186. 7 Sneddon, P. and Burnstock, G., ATP as a co-transmitter in rat tail artery, Eur. J. Pharmacol., 106 (1984) 149-152. 8 Stj/irne, L. and Stj/irne, E., Basic features of an extracellular recording method to study secretion of sympathetic co-transmitter, presumably ATP, Acta Physiol. Scand., 135 (1989) 217-226. 9 Swandulla, D., Hans, M., Zisper, K. and Augustine, G.J., Role of residual calcium in synaptic depression and posttetanic potentiation: fast and slow calcium signaling in nerve terminals, Neuron, 7 (1991) 915-926. 10 von Kugelgen, I. and Starke, K., Noradrenaline-ATP co-transmission in the sympathetic nervous system, Trends Pharmacol., 12 (1990) 319-324. 11 Zucker, R., Short term synaptic plasticity, Annu. Rev. Neurosci., 12 (1989) 13-31.