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Two-component responses to sympathetic nerve stimulation in the rat tail artery
Camp. Biochem.
Physid.
Vol. 81C, No. 2, pp. 311-317, 1985
0306-4492/85 $3.00 + 0.00 0 1985 Pergamon Press Ltd
Printed in Great Britain
TWO-COMPONENT RESPONSES TO SYMPATHETIC NERVE STIMULATION IN THE RAT TAIL ARTERY T. NEILD* and N. KOTECHA Neuropharmacology
Group,
Department of Physiology, Monash Australia. Telephone: 03-541-08 (Received 13 December
University,
Clayton,
Victoria
3168,
1I
1984)
Abstract-l.
Membrane potential and tension were recorded simultaneously from the smooth muscle of the rat tail artery. 2. A single stimulus to the perivascular nerves caused a tension transient. 3. The tension transient had two components, one due to a muscle action potential and one due to a-adrenoceptor activation. 4. During trains of stimuli most of the tension was due to a-receptor activation, even when every stimulus ca&ed a smooth muscle action potential.
was coupled to a force transducer (compliance 1.8pm/mN, resonant frequency 120 Hz). The distance between the wires was set to 800 pm. A pair of platinum wires was positioned parallel to the artery and used to electrically stimulate the perivascular nerves. The impedance of the stimulating electrodes in physiological saline was 100 n (0.1 msec pulse), which is much lower than the output impedance of most commercial isolated stimulators. To overcome this problem an isolated buffer stage was constructed and placed between an isolated stimulator (Devices 2533) and the stimulating electrodes to provide the required stimulus current. The circuit diagram is shown in Fig. 2. It consisted of a unity gain amplifier with a high power Darlington transistor inside the feedback loop. In practice the output impedance was limited only by the positive power supply, which must be isolated. The positive supply consisted of five Polapulse (Polaroid Corporation, Cambridge, MA, USA) batteries which are designed to give high currents for short periods. The negative side of the supply was not critical, and could be any 9 V battery. The buffer would handle oulses of 0.5-26V and maintain the voltage into loads as small as 10 R. When connected to the stimulating electrodes a voltage of 18 V through the buffer was equivalent to the maximum output (nominal 150 V) of a Grass S88 stimulator with SIUS isolator. The artery was immersed in continuously flowing physiological saline at 32”C, which was the temperature recorded from a thermocouple placed next to the tail artery in an anaesthetized rat with a core temperature of 38°C. The composition of the saline (mmol/l) was Na 145, K 5, Ca 2.5, Mg 2, Cl 134, HCO, 25, H,PO, 1, glucose 11, and it was equilibrated with 95% 0,/S% CO,. Intracellular recordings were made using conventional glass microelectrodes filled with 2 M KCI with resistances of 60-120 MQ. In all figures force is expressed as force per mm length of artery to compensate for preparations of different length. No correction has been applied for variation in wall thickness of arteries taken from rats of different weights.
INTRODUCTION
In most arteries and arterioles, stimulation of the perivascular nerves produces an excitatory junction potential (e.j.p.) in the arterial smooth muscle, and this may lead to an action potential and vasoconstriction. These events do not seem to involve a-adrenoceptors, perhaps because the nerve-released noradrenaline acts on a specialized junctional receptor (Hirst and Neild, 1981; Neild and Zelcer, 1982) or perhaps because the neurotransmitter is not noradrenaline (Sneddon and Westfall, 1984). In many arteries, however, it is clear that some noradrenaline also reaches a-adrenoceptors, particularly during prolonged trains of nerve stimulation (Medgett and Langer, 1984; Cheung, 1982; Nilsson, 1984; Owen et al., 1983). In the rat tail artery Cheung (1982b) showed that some noradrenaline could reach cr-adrenoceptors following a single stimulus to the nerves, and that this caused a small depolarization of the smooth muscle. The aim of the experiments described here was to measure the membrane potential and tension generated by the smooth muscle simultaneously, and to determine when the tension was due to action potentials and when it was due to a-receptor activation. MATERIALS AND METHODS
Male Wistar rats weighing 180-360 g were killed by stunning and exsanguination and the central artery dissected from the region 30-60 mm from the base of the tail. A piece of artery 2-2.5 mm long was mounted on an apparatus (Fig. 1) similar to that described by Mulvany and Halpern (1977) which permitted measurement of force developed by the arterial muscle. Two molybdenum wires 35 pm in diameter were threaded through the artery and each held tightly stretched on a supporting pillar. The distance between wires could be adjusted by the micrometer screw, and one wire
*National Health and Medical Australia Research Fellow. C.BP 81,2C E
Research
Council
RESULTS
The responses to single stimuli of 0.1 msec duration are shown in Fig. 3. Low voltage stimuli gave an e.j.p. and no perceptible tension. Slightly increasing the stimulus voltage gave a membrane potential response
of 311
312
T. NEILD and N. KOTECHA
STIMUATING ’
ELECTRODE
Fig. 1. Diagram of the apparatus used to record tension from the artery. The artery was held on two tightly stretched wires (supporting wires) threaded through the lumen. The supporting pillar for one wire incorporated a force transducer. The perivascular nerves were stimulated using a pair of platinum wires aligned parallel to the artery. The tension transducer and the opposite supporting pillar were angled at 30” from vertical to facilitate access for the microelectrode (not shown) for membrane potential recording.
which differed from the e.j.p. in that it had a more rapid initial decay. This response was probably due to the partial activation of voltage-dependent membrane conductances, and was associated with some tension development in the muscle. A further increase in stimulating voltage produced an action potential and a large tension transient. Increases in stimulus voltage beyond this level always increased the size of the tension transient, but the effect on the membrane potential responses varied. In Fig. 3 the action potential amplitude was constant but the highest stimulus voltage produced a slow depolarization of several seconds duration. In other arteries (Fig. 4) the amplitude of the action potential increased with increasing stimulus voltage. The peak of tension following a single stimulus occurred 1.55 set after the stimulus (mean of 63 records, SEM 0.029 set). There was no correlation between the amplitude of the tension transient and the time to peak (correlation coefficient-O.33). The responses evoked by stimuli of 0.1 msec duration were due to transmitter release from nerves and not direct muscle stimulation. Figure 4 shows the relationship between stimulus voltage and peak tension or membrane potential change for one artery. Addition of a high concentration (3 x 10m6M) of tetrodotoxin to block nerve conduction caused some reduction in the responses to low voltage stimuli, but had little effect on the response to the higher voltages. This was not surprising in view of the type of stimulating electrodes used, which probably caused some depolarization of the nerve terminals (Keef and Neild, 1982). Guanethidine (lo-’ M) greatly reduced
the responses to electrical depended on the number stimulated in the presence always possible to abolish maximum stimulus, and (Fig. 4).
stimulation. The reduction of times the nerves were of guanethidine, but it was the responses to all but the that response was reduced
Eflects of cc-adrenoceptor antagonists The specific q-adrenoceptor antagonist prazosin reduced the amplitude of the tension transient caused by single stimuli without reducing the amplitude of the action potential (Figs 5, 6). Figure 5 shows the effect of 10-6M prazosin on the tension transients and membrane potential changes.
+3ov
Fig. 2. Circuit diagram of the high current output buffer for isolated stimulators. The operational amplifier was a high voltage integrated circuit type (LM 344) and the slew rate was limited by the 100 pF external frequency compensation capacitor. The NPN Darlington transistor in the output stage was type BD 649. The positive power supply consisted of five Polapulse batteries, and the negative supply was a single PP3 battery.
Stimulation
of rat tail artery
313
mv -LO -6O-
--?----
Fig. 3. Membrane potential changes and tension transients recorded from the rat tail artery in response to single stimuli to the perivascular nerves. Upper records: membrane potential. Middle: tension on the same time scale as the membrane potential records. Lower: tension on a slower time scale. A. Response to a low voltage stimulus gave an e.j.p. and no tension. B. Response to a large stimulus that evoked an e.j.p. that activated some voltage dependent conductances but did not lead to an action potential. This caused some tension development. A still larger stimulus (C) produced an action potential and more tension. The largest stimulus (D) gave an action potential of the same amplitude, but did give an increased tension.
In the control records the tension transients consisted of two components. At low stimulus voltages the initial component was small and the peak of the response was determined by the second slower component. As the stimulus voltage was increased the initial component grew more rapidly than the second component and at the highest stimulus voltage it dominated the response. Prazosin ( 10e6 M) abolished the second component. This reduced the peak amplitude of all the responses, although the reduction was greatest at the lower voltages. There was no effect of prazosin on the e.j.p. or action potential (Fig. 6). There was, however, a depolarization of much slower time course that was abolished by prazosin (Fig. 5). The amplitude of this slow depolarization depended on the stimulus voltage, and it was rarely greater than 5 mV. Its time to peak varied from artery to artery and was in the range 12-30 sec. The peak of the slow depolarization was always later than the peak of tension. The records shown in Fig. 5 were chosen because the two components of the tension transient were particularly clear. This was not always the case, as illustrated by the records from a different preparation shown in Fig. 7. In this preparation the tension transients appeared to have only one component, with perhaps an inflection on the rising phase. These tension transients were considerably reduced by phentolamine (10m6 M) showing that they were partly due to a-adrenoceptor activation. Phentolamine also abolished the slow change in membrane potential without affecting the e.j.p. or action potential. Presumably, the two components of tension could not be distinguished because the a receptor dependent component reached its peak at almost the same time as the action potential dependent component. It is interesting that the slow depolarization also peaks in a relatively short time in this preparation. In two preparations phentolamine increased the amplitude of the e.j.p. (see Holman and Surprenant,
1980) and this led to action potential generation at low stimulus voltages that had not caused action potentials before phentolamine was added. In these arteries the tension transient caused by low voltage stimuli were also larger in the presence of phentolamine, but the responses to higher voltages were always reduced. Trains of stimulation The contribution of a-receptor activation to the tension was greatest when trains of stimuli were given. Figure 8 shows the effect of trains of 20 pulses at 1 Hz. A low stimulus voltage that caused e.j.p.s without action potentials gave a small slowlydeveloping increase in tension that was completely abolished by prazosin (10m6 M). A higher stimulus voltage gave an initial small peak of tension that was similar to the response obtained with a single stimulus, followed by a steadily increasing tension as the stimulus train continued. Most of this later tension was abolished by a-adrenoceptor antagonists. The membrane potential changes during trains of stimulation consisted of e.j.p.s and action potentials superimposed on an underlying depolarization. This depolarization was greatly reduced by a-receptor antagonists, as previously reported by Cheung (1982b). During the high voltage stimulation the amplitude of the action potential usually declined, and the decline in tension after the initial peak seemed to be related to this. In preparations in which the action potential amplitude was constant for the first three or four stimuli the tension rose monotonically without an initial peak. DISCUSSION
A single stimulus to the perivascular nerves gave rise to a wide variety of membrane potential changes. Small stimuli gave e.j.p.s which did not activate any of the voltage dependent membrane conductances
T. NEILD and N. KOTECHA
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Fig. 4. Right: amplitude of the tension transient caused by single stimuli of increasing voltage. Tension has been standardized to give tension per mm length of artery. Left: amplitude of the membrane potential change (e.j.p., intermediate response or action potential) in the same artery in response to the same stimuli. There are no points for membrane potential change at some of the higher stimulus voltage because the large tension transients dislodged the microelectrode. Filled circles: control. Open circles: in the presence of 3 x 10-6M tetrodotoxin. Stars: after 640 stimuli at maximum voltage in 10d5 M guanethidine. Triangles: after a further 230 stimuli in guanethidine.
CONTROL
1
-50 mV -60
4
6
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1; Stimulus
PRAZOSIN
li3
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Voltage
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] 60OtJN/mm
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Fig. 5. Tension transients and membrane potential change in the rat tail artery caused by single stimuli of increasing voltage given at 1 min intervals. The membrane potential records are shown at high gain to show the slow depolarization, and the action potentials are truncated. The membrane potential changes in response to the 6, 14 and 22 V stimuli are shown in Fig. 6. Prazosin (1O-6 M) abolished the second component of the tension transient and greatly reduced the slow depolarization.
Stimulation
315
of rat tail artery
CONTROL -20 -
PRAZO!S.IN
lO+U
-20 _
4
I
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6V
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Fig. 6. Action potentials in response to single stimuli at 6, 14 and 22 V. Prazosin (10v6M) had no effect on these responses. Data from the same experiments as Fig. 5.
associated with the action potential and did not lead to tension generation. Cheung (1984) has suggested that the membrane potential must change to a level more positive than -49mV before depolarization alone can activate tension generation, and the e.j.p.s we observed generally did not reach that level. A slight increase of stimulus voltage above the strength that gave an e.j.p. produced membrane potential changes that were larger and appeared to be due to partial activation of the action potential mechanism. These responses are characteristic of the rat tail artery (Surprenant, 1980; Cheung, 1982a, 1984), although
they do occur in other arteries to some extent (Keef and Neild, 1982). There was some tension generation associated with these responses, and there was presumably some influx of calcium into the cells even when the action potential was not fully developed. Higher stimulus voltages produced responses that could properly be called action potentials because of their rapid rate of rise and their regenerative nature, and they were associated with the largest tension transients. It was noted, however, that the tension produced could be graded by altering the stimulus voltage even when the action potential amplitude had
CONTROL
-50
mV -60 1min PHENTOLAMINE
lIZi6 M
6
10
-50 mV -60 6
14 Stimulus
18
22
Voltage
Fig. 7. Tension transients and membrane potential changes caused by single stimuli of increasing voltage given at 1 min intervals. The membrane potential records are shown at high gain to show the slow depolarization, and the action potentials are truncated. The tension transients from this artery could not be resolved into two components. Phentolamine (lOmoM) reduced the amplitude of the slow depolarization and the tension transients.
T.
316
PRAZOSIN
NEILD
and N. KOTECHA
lob M 3OOpN/mm
-
I
Fig. 8. Tension and membrane potential changes caused by a train of 20 stimuli at 1 Hz. A. A low stimulus voltage caused e.j.p.s with an underlying depolarization. The dotted line indicates resting membrane potential. Force developed slowly during the stimulus train. The force and the steady depolarization were abolished by prazosin (10e6 M). B. A higher stimulus voltage gave action potentials and a greater steady depolarization. The large action potential at the beginning of the train caused a brief peak of force, but most of the force developed later in the train was abolished by prazosin and was therefore due to x-receptor activation.
reached a maximum (Fig. 3) showing that there was no simple relationship between tension and the membrane potential change associated with it. The component of the tension transients that depended on the action potential reached its peak 1.55 set after the stimulus, and this time did not depend on the amplitude or time course of the action potential. A few comparable records in a preliminary communication by Cheung (1984) have similar characteristics. There was a delay of approximately 400 msec between the action potential and the onset of force generation. This is slightly longer than the delay of 170 msec found by Fay (1977) using direct electrical stimulation of isolated toad stomach muscle cells. This delay is unlikely to be due simply to the rate at which the free calcium entering the cell becomes available to the contractile system, as experiments with freeze-glycerinated vascular smooth muscle have shown that this is not the rate-limiting step (Peterson, 1982). Furthermore, recent direct measurements of the time course of intracellular free calcium changes following electrical stimulation of ferret portal vein showed that the free calcium peaks
in 6.5 set for a tension transient that peaks in 48 set (Morgan and Morgan, 1984). It seems, therefore, that the action potential in the rat tail artery is a brief event compared to the tension transient it produces, and variations in its time course do not affect the time course of the tension. The experiments with a-adrenoceptor antagonists showed that the relative contribution of a-receptor activation to the tension developed from a single stimulus could not be estimated from the peak amplitude of the tension because of the different timing of the components of the tension transient. Whereas the action potential dependent component always had the same time course the a-receptor component varied considerably in its time course. In the records of Fig. 7 the time course of the two components seems similar, whereas in Fig. 5 the a-receptor dependent component is clearly slower. The difference in the time courses was not constant for any one artery, and sometimes changed during the experiment. The a-receptor dependent tension was always in its declining phase when the cc-receptor dependent slow depolarization reached its peak, clearly showing
Stimulation
of rat tail artery
that this depolarization was not the cause of the tension. During trains of stimulation it was clear that the majority of tension developed was due to a-receptor activation as reported by other workers using this artery (e.g. Medgett and Langer, 1984). There was an a-receptor dependent depolarization of several millivolts as previously reported by Cheung (1982b), but further experiments would be needed to determine whether this depolarization contributed to the tension generated. It was clear when cc-receptors were blocked that continuous action potentials in the muscle did not produce a large tension (Fig. 8), suggesting that action potentials alone would not be a very effective mechanism for maintaining arterial tone. Acknowledgemenfs-We are most grateful to Dr R. M. Wadsworth for his helpful criticism of the manuscript. This work was supported by the National Health and Medical Research Council. REFERENCES
Cheung D. W. (1982a) Spontaneous and evoked excitatory junction potentials in rat tail arteries. J. Physiol. 328, 449-459. Cheung D. W. (1982b) Two components in the cellular response of rat tail arteries to nerve stimulation. J. Physiol. 328, 46 1468. Cheung D. W. (1984) Neural regulation of electrical and mechanical activities in the rat tail artery. PfIiisers Arch. 400, 335-337. Fay F. S. (1977) Mechanics of single isolated smooth muscle cells. In Excitation-contraction Coupling in Smooth Muscle (Edited by Casteels R., Godfraind T. and Ruegg). Elsevier/North Holland, Amsterdam. Hirst G. D. S. and Neild T. 0. (1981) Localization of specialized noradrenaline receptors at neuromuscular
317
junctions on arterioles of the guinea-pig. J. Physiol. 313, 343-350. Holman M. E. and Surprenant A. (1980) An electrophysiological analysis of the effects of noradrenaline and alpha-receptor antagonists on neuromuscular transmission in mammalian muscular arteries. Er. J. Pharmacol. 71, 651-661. Keef K. and Neild T. 0. (1982) Modification of the response to nerve stimulation in small arteries of the guinea-pig ear caused by distension of the artery. J. Physiol. 331, 355-365. Medgett I. C. and Langer S. Z. (1984) Heterogeneity of smooth muscle alpha adrenoceptors in rat tail artery in vitro. J. Pharmac. exp. Ther. 229, 823-830. Morgan J. P. and Morgan K. G. (1984) Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein. J. Physiol. 351, 155-167. Mulvany M. J. and Halpern W. (1977) Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circularion Res. 41, 19-26. Neild T. 0. and Zelcer E. (1982) Noradrenergic neuromuscular transmission with special reference to arterial smooth muscle. Prog. Neurobiol. 19, 141-158. Nilson M. (1984) Different nerve responses in consecutive sections of the arterial system. Acta physiol. stand. 121, 353-361. Owen M. P., Walmsley J. G., Mason M. F., Bevan R. D. and Bevan J. A. (1983) Adrenergic control in three artery segments of diminishing diameter in rabbit ear. Am. J. Physiol. 245, H320-326. Peterson J. W. (1982) Rate-limiting steps in the tension development of freeze-glycerinated vascular smooth muscle. J. gen. Physiol. 79, 437-452. Sneddon P. and Westfall D. P. (19841 Pharmacological . evidence that adenosine triphosphate and noradrenaline are co-transmitters in the guinea-pig vas deferens. J. Physiol. 347, 561-580. Surprenant A. (1980) A comparative study of neuromuscular transmission in several mammalian arteries. Pjiigers Arch. 386, 85-9 1. I
Physid.
Vol. 81C, No. 2, pp. 311-317, 1985
0306-4492/85 $3.00 + 0.00 0 1985 Pergamon Press Ltd
Printed in Great Britain
TWO-COMPONENT RESPONSES TO SYMPATHETIC NERVE STIMULATION IN THE RAT TAIL ARTERY T. NEILD* and N. KOTECHA Neuropharmacology
Group,
Department of Physiology, Monash Australia. Telephone: 03-541-08 (Received 13 December
University,
Clayton,
Victoria
3168,
1I
1984)
Abstract-l.
Membrane potential and tension were recorded simultaneously from the smooth muscle of the rat tail artery. 2. A single stimulus to the perivascular nerves caused a tension transient. 3. The tension transient had two components, one due to a muscle action potential and one due to a-adrenoceptor activation. 4. During trains of stimuli most of the tension was due to a-receptor activation, even when every stimulus ca&ed a smooth muscle action potential.
was coupled to a force transducer (compliance 1.8pm/mN, resonant frequency 120 Hz). The distance between the wires was set to 800 pm. A pair of platinum wires was positioned parallel to the artery and used to electrically stimulate the perivascular nerves. The impedance of the stimulating electrodes in physiological saline was 100 n (0.1 msec pulse), which is much lower than the output impedance of most commercial isolated stimulators. To overcome this problem an isolated buffer stage was constructed and placed between an isolated stimulator (Devices 2533) and the stimulating electrodes to provide the required stimulus current. The circuit diagram is shown in Fig. 2. It consisted of a unity gain amplifier with a high power Darlington transistor inside the feedback loop. In practice the output impedance was limited only by the positive power supply, which must be isolated. The positive supply consisted of five Polapulse (Polaroid Corporation, Cambridge, MA, USA) batteries which are designed to give high currents for short periods. The negative side of the supply was not critical, and could be any 9 V battery. The buffer would handle oulses of 0.5-26V and maintain the voltage into loads as small as 10 R. When connected to the stimulating electrodes a voltage of 18 V through the buffer was equivalent to the maximum output (nominal 150 V) of a Grass S88 stimulator with SIUS isolator. The artery was immersed in continuously flowing physiological saline at 32”C, which was the temperature recorded from a thermocouple placed next to the tail artery in an anaesthetized rat with a core temperature of 38°C. The composition of the saline (mmol/l) was Na 145, K 5, Ca 2.5, Mg 2, Cl 134, HCO, 25, H,PO, 1, glucose 11, and it was equilibrated with 95% 0,/S% CO,. Intracellular recordings were made using conventional glass microelectrodes filled with 2 M KCI with resistances of 60-120 MQ. In all figures force is expressed as force per mm length of artery to compensate for preparations of different length. No correction has been applied for variation in wall thickness of arteries taken from rats of different weights.
INTRODUCTION
In most arteries and arterioles, stimulation of the perivascular nerves produces an excitatory junction potential (e.j.p.) in the arterial smooth muscle, and this may lead to an action potential and vasoconstriction. These events do not seem to involve a-adrenoceptors, perhaps because the nerve-released noradrenaline acts on a specialized junctional receptor (Hirst and Neild, 1981; Neild and Zelcer, 1982) or perhaps because the neurotransmitter is not noradrenaline (Sneddon and Westfall, 1984). In many arteries, however, it is clear that some noradrenaline also reaches a-adrenoceptors, particularly during prolonged trains of nerve stimulation (Medgett and Langer, 1984; Cheung, 1982; Nilsson, 1984; Owen et al., 1983). In the rat tail artery Cheung (1982b) showed that some noradrenaline could reach cr-adrenoceptors following a single stimulus to the nerves, and that this caused a small depolarization of the smooth muscle. The aim of the experiments described here was to measure the membrane potential and tension generated by the smooth muscle simultaneously, and to determine when the tension was due to action potentials and when it was due to a-receptor activation. MATERIALS AND METHODS
Male Wistar rats weighing 180-360 g were killed by stunning and exsanguination and the central artery dissected from the region 30-60 mm from the base of the tail. A piece of artery 2-2.5 mm long was mounted on an apparatus (Fig. 1) similar to that described by Mulvany and Halpern (1977) which permitted measurement of force developed by the arterial muscle. Two molybdenum wires 35 pm in diameter were threaded through the artery and each held tightly stretched on a supporting pillar. The distance between wires could be adjusted by the micrometer screw, and one wire
*National Health and Medical Australia Research Fellow. C.BP 81,2C E
Research
Council
RESULTS
The responses to single stimuli of 0.1 msec duration are shown in Fig. 3. Low voltage stimuli gave an e.j.p. and no perceptible tension. Slightly increasing the stimulus voltage gave a membrane potential response
of 311
312
T. NEILD and N. KOTECHA
STIMUATING ’
ELECTRODE
Fig. 1. Diagram of the apparatus used to record tension from the artery. The artery was held on two tightly stretched wires (supporting wires) threaded through the lumen. The supporting pillar for one wire incorporated a force transducer. The perivascular nerves were stimulated using a pair of platinum wires aligned parallel to the artery. The tension transducer and the opposite supporting pillar were angled at 30” from vertical to facilitate access for the microelectrode (not shown) for membrane potential recording.
which differed from the e.j.p. in that it had a more rapid initial decay. This response was probably due to the partial activation of voltage-dependent membrane conductances, and was associated with some tension development in the muscle. A further increase in stimulating voltage produced an action potential and a large tension transient. Increases in stimulus voltage beyond this level always increased the size of the tension transient, but the effect on the membrane potential responses varied. In Fig. 3 the action potential amplitude was constant but the highest stimulus voltage produced a slow depolarization of several seconds duration. In other arteries (Fig. 4) the amplitude of the action potential increased with increasing stimulus voltage. The peak of tension following a single stimulus occurred 1.55 set after the stimulus (mean of 63 records, SEM 0.029 set). There was no correlation between the amplitude of the tension transient and the time to peak (correlation coefficient-O.33). The responses evoked by stimuli of 0.1 msec duration were due to transmitter release from nerves and not direct muscle stimulation. Figure 4 shows the relationship between stimulus voltage and peak tension or membrane potential change for one artery. Addition of a high concentration (3 x 10m6M) of tetrodotoxin to block nerve conduction caused some reduction in the responses to low voltage stimuli, but had little effect on the response to the higher voltages. This was not surprising in view of the type of stimulating electrodes used, which probably caused some depolarization of the nerve terminals (Keef and Neild, 1982). Guanethidine (lo-’ M) greatly reduced
the responses to electrical depended on the number stimulated in the presence always possible to abolish maximum stimulus, and (Fig. 4).
stimulation. The reduction of times the nerves were of guanethidine, but it was the responses to all but the that response was reduced
Eflects of cc-adrenoceptor antagonists The specific q-adrenoceptor antagonist prazosin reduced the amplitude of the tension transient caused by single stimuli without reducing the amplitude of the action potential (Figs 5, 6). Figure 5 shows the effect of 10-6M prazosin on the tension transients and membrane potential changes.
+3ov
Fig. 2. Circuit diagram of the high current output buffer for isolated stimulators. The operational amplifier was a high voltage integrated circuit type (LM 344) and the slew rate was limited by the 100 pF external frequency compensation capacitor. The NPN Darlington transistor in the output stage was type BD 649. The positive power supply consisted of five Polapulse batteries, and the negative supply was a single PP3 battery.
Stimulation
of rat tail artery
313
mv -LO -6O-
--?----
Fig. 3. Membrane potential changes and tension transients recorded from the rat tail artery in response to single stimuli to the perivascular nerves. Upper records: membrane potential. Middle: tension on the same time scale as the membrane potential records. Lower: tension on a slower time scale. A. Response to a low voltage stimulus gave an e.j.p. and no tension. B. Response to a large stimulus that evoked an e.j.p. that activated some voltage dependent conductances but did not lead to an action potential. This caused some tension development. A still larger stimulus (C) produced an action potential and more tension. The largest stimulus (D) gave an action potential of the same amplitude, but did give an increased tension.
In the control records the tension transients consisted of two components. At low stimulus voltages the initial component was small and the peak of the response was determined by the second slower component. As the stimulus voltage was increased the initial component grew more rapidly than the second component and at the highest stimulus voltage it dominated the response. Prazosin ( 10e6 M) abolished the second component. This reduced the peak amplitude of all the responses, although the reduction was greatest at the lower voltages. There was no effect of prazosin on the e.j.p. or action potential (Fig. 6). There was, however, a depolarization of much slower time course that was abolished by prazosin (Fig. 5). The amplitude of this slow depolarization depended on the stimulus voltage, and it was rarely greater than 5 mV. Its time to peak varied from artery to artery and was in the range 12-30 sec. The peak of the slow depolarization was always later than the peak of tension. The records shown in Fig. 5 were chosen because the two components of the tension transient were particularly clear. This was not always the case, as illustrated by the records from a different preparation shown in Fig. 7. In this preparation the tension transients appeared to have only one component, with perhaps an inflection on the rising phase. These tension transients were considerably reduced by phentolamine (10m6 M) showing that they were partly due to a-adrenoceptor activation. Phentolamine also abolished the slow change in membrane potential without affecting the e.j.p. or action potential. Presumably, the two components of tension could not be distinguished because the a receptor dependent component reached its peak at almost the same time as the action potential dependent component. It is interesting that the slow depolarization also peaks in a relatively short time in this preparation. In two preparations phentolamine increased the amplitude of the e.j.p. (see Holman and Surprenant,
1980) and this led to action potential generation at low stimulus voltages that had not caused action potentials before phentolamine was added. In these arteries the tension transient caused by low voltage stimuli were also larger in the presence of phentolamine, but the responses to higher voltages were always reduced. Trains of stimulation The contribution of a-receptor activation to the tension was greatest when trains of stimuli were given. Figure 8 shows the effect of trains of 20 pulses at 1 Hz. A low stimulus voltage that caused e.j.p.s without action potentials gave a small slowlydeveloping increase in tension that was completely abolished by prazosin (10m6 M). A higher stimulus voltage gave an initial small peak of tension that was similar to the response obtained with a single stimulus, followed by a steadily increasing tension as the stimulus train continued. Most of this later tension was abolished by a-adrenoceptor antagonists. The membrane potential changes during trains of stimulation consisted of e.j.p.s and action potentials superimposed on an underlying depolarization. This depolarization was greatly reduced by a-receptor antagonists, as previously reported by Cheung (1982b). During the high voltage stimulation the amplitude of the action potential usually declined, and the decline in tension after the initial peak seemed to be related to this. In preparations in which the action potential amplitude was constant for the first three or four stimuli the tension rose monotonically without an initial peak. DISCUSSION
A single stimulus to the perivascular nerves gave rise to a wide variety of membrane potential changes. Small stimuli gave e.j.p.s which did not activate any of the voltage dependent membrane conductances
T. NEILD and N. KOTECHA
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Fig. 4. Right: amplitude of the tension transient caused by single stimuli of increasing voltage. Tension has been standardized to give tension per mm length of artery. Left: amplitude of the membrane potential change (e.j.p., intermediate response or action potential) in the same artery in response to the same stimuli. There are no points for membrane potential change at some of the higher stimulus voltage because the large tension transients dislodged the microelectrode. Filled circles: control. Open circles: in the presence of 3 x 10-6M tetrodotoxin. Stars: after 640 stimuli at maximum voltage in 10d5 M guanethidine. Triangles: after a further 230 stimuli in guanethidine.
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Fig. 5. Tension transients and membrane potential change in the rat tail artery caused by single stimuli of increasing voltage given at 1 min intervals. The membrane potential records are shown at high gain to show the slow depolarization, and the action potentials are truncated. The membrane potential changes in response to the 6, 14 and 22 V stimuli are shown in Fig. 6. Prazosin (1O-6 M) abolished the second component of the tension transient and greatly reduced the slow depolarization.
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Fig. 6. Action potentials in response to single stimuli at 6, 14 and 22 V. Prazosin (10v6M) had no effect on these responses. Data from the same experiments as Fig. 5.
associated with the action potential and did not lead to tension generation. Cheung (1984) has suggested that the membrane potential must change to a level more positive than -49mV before depolarization alone can activate tension generation, and the e.j.p.s we observed generally did not reach that level. A slight increase of stimulus voltage above the strength that gave an e.j.p. produced membrane potential changes that were larger and appeared to be due to partial activation of the action potential mechanism. These responses are characteristic of the rat tail artery (Surprenant, 1980; Cheung, 1982a, 1984), although
they do occur in other arteries to some extent (Keef and Neild, 1982). There was some tension generation associated with these responses, and there was presumably some influx of calcium into the cells even when the action potential was not fully developed. Higher stimulus voltages produced responses that could properly be called action potentials because of their rapid rate of rise and their regenerative nature, and they were associated with the largest tension transients. It was noted, however, that the tension produced could be graded by altering the stimulus voltage even when the action potential amplitude had
CONTROL
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14 Stimulus
18
22
Voltage
Fig. 7. Tension transients and membrane potential changes caused by single stimuli of increasing voltage given at 1 min intervals. The membrane potential records are shown at high gain to show the slow depolarization, and the action potentials are truncated. The tension transients from this artery could not be resolved into two components. Phentolamine (lOmoM) reduced the amplitude of the slow depolarization and the tension transients.
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Fig. 8. Tension and membrane potential changes caused by a train of 20 stimuli at 1 Hz. A. A low stimulus voltage caused e.j.p.s with an underlying depolarization. The dotted line indicates resting membrane potential. Force developed slowly during the stimulus train. The force and the steady depolarization were abolished by prazosin (10e6 M). B. A higher stimulus voltage gave action potentials and a greater steady depolarization. The large action potential at the beginning of the train caused a brief peak of force, but most of the force developed later in the train was abolished by prazosin and was therefore due to x-receptor activation.
reached a maximum (Fig. 3) showing that there was no simple relationship between tension and the membrane potential change associated with it. The component of the tension transients that depended on the action potential reached its peak 1.55 set after the stimulus, and this time did not depend on the amplitude or time course of the action potential. A few comparable records in a preliminary communication by Cheung (1984) have similar characteristics. There was a delay of approximately 400 msec between the action potential and the onset of force generation. This is slightly longer than the delay of 170 msec found by Fay (1977) using direct electrical stimulation of isolated toad stomach muscle cells. This delay is unlikely to be due simply to the rate at which the free calcium entering the cell becomes available to the contractile system, as experiments with freeze-glycerinated vascular smooth muscle have shown that this is not the rate-limiting step (Peterson, 1982). Furthermore, recent direct measurements of the time course of intracellular free calcium changes following electrical stimulation of ferret portal vein showed that the free calcium peaks
in 6.5 set for a tension transient that peaks in 48 set (Morgan and Morgan, 1984). It seems, therefore, that the action potential in the rat tail artery is a brief event compared to the tension transient it produces, and variations in its time course do not affect the time course of the tension. The experiments with a-adrenoceptor antagonists showed that the relative contribution of a-receptor activation to the tension developed from a single stimulus could not be estimated from the peak amplitude of the tension because of the different timing of the components of the tension transient. Whereas the action potential dependent component always had the same time course the a-receptor component varied considerably in its time course. In the records of Fig. 7 the time course of the two components seems similar, whereas in Fig. 5 the a-receptor dependent component is clearly slower. The difference in the time courses was not constant for any one artery, and sometimes changed during the experiment. The a-receptor dependent tension was always in its declining phase when the cc-receptor dependent slow depolarization reached its peak, clearly showing
Stimulation
of rat tail artery
that this depolarization was not the cause of the tension. During trains of stimulation it was clear that the majority of tension developed was due to a-receptor activation as reported by other workers using this artery (e.g. Medgett and Langer, 1984). There was an a-receptor dependent depolarization of several millivolts as previously reported by Cheung (1982b), but further experiments would be needed to determine whether this depolarization contributed to the tension generated. It was clear when cc-receptors were blocked that continuous action potentials in the muscle did not produce a large tension (Fig. 8), suggesting that action potentials alone would not be a very effective mechanism for maintaining arterial tone. Acknowledgemenfs-We are most grateful to Dr R. M. Wadsworth for his helpful criticism of the manuscript. This work was supported by the National Health and Medical Research Council. REFERENCES
Cheung D. W. (1982a) Spontaneous and evoked excitatory junction potentials in rat tail arteries. J. Physiol. 328, 449-459. Cheung D. W. (1982b) Two components in the cellular response of rat tail arteries to nerve stimulation. J. Physiol. 328, 46 1468. Cheung D. W. (1984) Neural regulation of electrical and mechanical activities in the rat tail artery. PfIiisers Arch. 400, 335-337. Fay F. S. (1977) Mechanics of single isolated smooth muscle cells. In Excitation-contraction Coupling in Smooth Muscle (Edited by Casteels R., Godfraind T. and Ruegg). Elsevier/North Holland, Amsterdam. Hirst G. D. S. and Neild T. 0. (1981) Localization of specialized noradrenaline receptors at neuromuscular
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junctions on arterioles of the guinea-pig. J. Physiol. 313, 343-350. Holman M. E. and Surprenant A. (1980) An electrophysiological analysis of the effects of noradrenaline and alpha-receptor antagonists on neuromuscular transmission in mammalian muscular arteries. Er. J. Pharmacol. 71, 651-661. Keef K. and Neild T. 0. (1982) Modification of the response to nerve stimulation in small arteries of the guinea-pig ear caused by distension of the artery. J. Physiol. 331, 355-365. Medgett I. C. and Langer S. Z. (1984) Heterogeneity of smooth muscle alpha adrenoceptors in rat tail artery in vitro. J. Pharmac. exp. Ther. 229, 823-830. Morgan J. P. and Morgan K. G. (1984) Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein. J. Physiol. 351, 155-167. Mulvany M. J. and Halpern W. (1977) Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circularion Res. 41, 19-26. Neild T. 0. and Zelcer E. (1982) Noradrenergic neuromuscular transmission with special reference to arterial smooth muscle. Prog. Neurobiol. 19, 141-158. Nilson M. (1984) Different nerve responses in consecutive sections of the arterial system. Acta physiol. stand. 121, 353-361. Owen M. P., Walmsley J. G., Mason M. F., Bevan R. D. and Bevan J. A. (1983) Adrenergic control in three artery segments of diminishing diameter in rabbit ear. Am. J. Physiol. 245, H320-326. Peterson J. W. (1982) Rate-limiting steps in the tension development of freeze-glycerinated vascular smooth muscle. J. gen. Physiol. 79, 437-452. Sneddon P. and Westfall D. P. (19841 Pharmacological . evidence that adenosine triphosphate and noradrenaline are co-transmitters in the guinea-pig vas deferens. J. Physiol. 347, 561-580. Surprenant A. (1980) A comparative study of neuromuscular transmission in several mammalian arteries. Pjiigers Arch. 386, 85-9 1. I