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J. Physiol. (I957) I35, 412-
CHANGES IN CONFIGURATION OF SPONTANEOUSLY DISCHARGED SPIKE POTENTIALS FROM SMOOTH MUSCLE OF THE GUINEA-PIG'S TAENIA COLI. THE EFFECT OF ELECTROTONIC CURRENTS AND (^) OF ADRENALINE, ACETYLCHOLINE^ AND HISTAMINE
BY EDITH BtFLBRING
From the Department of Pharmacology, University of Oxford
(Received 27 September (^) 1956)
Autorhythmicity can^ be^ produced in^ many excitable^ tissues^ by introducing
conditions in which the membrane potential becomes unstable. The most
extensive studies have^ been carried out on nerve and on striated muscle, both
of which normally have a very stable membrane but which in a calcium-
deficient medium become rhythmically active. In this condition the behaviour
of striated muscle^ resembles in some respects that of a continuously dis-
charging sensory organ. The^ smooth muscle of the longitudinal layer of the
intestine shows the same behaviour normally. Its membrane potential is very
unstable, there is a spontaneous rhythm of spike (^) potentials, and it (^) responds to
a number of stimuli by varying the frequency of its spontaneous discharge of
impulses. The tension which the muscle produces in (^) isometric conditions is directly proportional to^ the spike frequency, and the rate of discharge depends on the state of polarization of the membrane. A good correlation exists between the three variables: tension, membrane potential and spike frequency, but little is known about the (^) size and the configuration of the spike potentials. A study has therefore been made to see (^) whether the duration of the individual spike bears^ any relation to the tension produced, and whether the (^) spike configuration (^) undergoes changes which can be related to stimulation or inhibition of activity. A short report of the results obtained was given to the XXth International Physiological (^) Congress in Brussels (Biilbring, 1956).
METHODS All experiments were done on isolated smooth muscle strips taken from the taenia coli of (^) the guinea-pig, in most experiments not exceeding 3 mm length in situ. The volume of the bath was 2 ml., and bathing solutions flowed continuously at a rate of 2 ml./min. Thus any substances
SPIKE CONFIGURATION IN SMOOTH MUSCLE
added were washed away. This method and that of (^) taking intracellular records was the same (^) as those described earlier by Builbring & Hooton (1954), and (^) Builbring (1954). The method ofrecording the (^) tension and of applying electrotonic currents was described (^) by Builbring (1955).
RESULTS Spike potentials during spontaneous (^) activity All preparations were set (^) up under 3-5 (^) g initial tension and showed (^) spontaneous
activity. The action potentials which were recorded varied in size from less
than 1 mV to 35 mV. They never caused a reversal of the membrane potential,
and the percentage depolarization from the starting level, produced by the
spike, was not proportional to the absolute membrane potential. However,
the spikes (^) arising from a low membrane potential were (^) usually smaller (^) than those arising from a high potential.
a
. (^) sec
5 mV
Fig. 1. Taenia coli. Intracellular records: (^) spontaneous discharge of spike potentials (a) and (b) from the same (^) preparation; (c) from another muscle.
The duration of the spike potentials and the slope of the rising and (^) falling phase were (^) equally variable. While the rise time to (^) an average peak of 10 mV was between 7 and 35 msec the return to the base line lasted from (^7) to 300 msec. The (^) rate of repolarization could be (^) either greater or less than the rate of rise.
If it was fast it led to a phase of after-hyperpolarization lasting several hundred
msec. As a (^) rule the spikes were preceded by a slow (^) depolarization which varied in slope and in degree from (^) being scarcely perceptible to a well-defined (^) prepoten- tial (^) (Fig. 1 a, b). Slow waves of depolarization (^) of the membrane were of three kinds. The fastest changes occurred at a rate of about 1/sec and they (^) usually led to the (^) discharge of a spike potential, i.e. they were (^) the prepotentials. The second type of slow potential changes was rarely seen; in this the waves were not regular, they (^) occurred at intervals of 2-10 sec and had apparently no relation (^) to the spike discharge (Fig. 1 c). (^) The third type of slow fluctuations of the membrane (^) potential was associated with the pendular activity, the full cycle (^) lasting 1-3 min (see Fig. 3). These (^) waves were definitely correlated with the rate and configuration of (^) the spikes.
SPIKE CONFIGURATION IN SMOOTH MUSCLE 415
During the relaxation shown in the lower record, which is the direct continua-
tion of the upper part, the spikes were more widely spaced and not every one
was followed by an increase in tension which thus no longer summated.
(^2 4 6 8) 10sec Fig. 3. Record of tension and electrical activity (intracellular electrode). Upper record rising phase, lower record falling phase of (^) pendular cycle: the two records are continuous; for 4escription see text. (Broken line in (^) upper record when tension record was reset to bottom of screen.)
0~~ ~~~~~ 0~~. -
Fig. 4. Effect of electrotonic currents (100 ,uA) on spike frequency and configuration (^) in relation to the tension. Changes of polarity every 20 to 60 sec: upper; (1) normal, (^) (2) 20 sec -, (3) 30 sec +; middle; 60 sec + and reversal of polarity; (^) lower; (1) 30 sec +, (2) 5 sec off.
The effect of changes in polarization of the membrane
When the muscle was exposed to electrotonic currents the size and con- figuration of the spike potentials underwent similar changes as during spon- taneous fluctuation of (^) the membrane potential. In the experiment from which records (^) were taken for Fig. 4 weak electrotonic currents (^) were applied. An attempt
was made to imitate the spontaneous fluctuations by reversing the polarity
every 20-60 (^) sec. The (^) change in the appearance (^) of the spikes was a decrease (^) in
416 EDITH BULBRING
size and a slowing of the rate of repolarization when the membrane was
depolarized; this was associated with a rise in tension. On the other hand,
when the membrane was polarized, the spike height increased and the rate of
repolarization became faster; each spike was followed by a phase of hyper-
polarization; this was associated with a (^) fall in tension. The (^) tendency to
discharge multiple spikes was increased during cathodal stimulation. The
L....j50 msec
J10 mV
a b Fig. 5. Spike potentials (^) recorded at 20 sec intervals (read from below upwards). (^) In (a) - (^25) 'A was applied for (^60) sec; in (b), 15 min later, +50 (^) pA was applied (^) for 60 sec (two records without spikes (^) omitted).
spikes changed to plateaus of depolarization lasting up to 500 msec on top of
which there were double or triple spikes. The delay in repolarization resulting from application (^) of cathodal currents as compared (^) with the faster rate of repolarization as a result of (^) anodal currents is shown on a faster time base in Fig. 5.
Strong anodal currents stopped the spike discharge. Preceding their
reappearance slow waves of potential changes were sometimes observed (see
Fig. 6). These were (^) presumably prepotentials due to electrotonic (^) spread from neighbouring already active fibres, as will be (^) discussed below.
The effect of adrenaline
Adrenaline slowed or stopped the spike discharge. The height of the spike
potentials was (^) reduced until they finally disappeared, but (^) this effect was some-
418 EDITH^ BULBRING
(b) 2,ag^ Adr.
a bc^ d e
k (^) m L.' 100 msec Fig. 7. The effect of adrenaline, initial concentration 1 x^ 10-6, (a) on membrane potential, spike frequency and spike size, (b) on spike configuration at the corresponding points on the graph (no discharge omitted).
SPIKE CONFIGURATION IN SMOOTH MUSCLE
After the administration of histamine the (^) gradually (^) progressing delay in repolarization is shown in five successive (^) spikes, changing the^ configuration to one not normally seen. As the histamine effect (^) passed off and (^) spike discharge slowed (third spike in Fig. 10b) the duration became (^) distinctly shorter. (^) This was followed by a period when double (^) spikes were (^) discharged. Only 17 min after histamine was given the (^) preparation reverted to (^) single spikes, which were briefer (^) than initially. 5sug Adr.
(^2) ~~~~~~~~~~1sec g (^10) mV
(^30) sec-
2min 3 mi 5min Fig. 8.^ Records^ of^ electrical (^) activity and tension. The effect of adrenaline (^) (initial concentration 2-25 x (^) 10-6) (^) abolishing spike potentials and (^) shortening their duration when (^) they (^) reappear; this is (^) preceded by slow (^) waves. Same (^) experiment as (^) Fig. 11.
The (^) striking similarity between the effect of (^) acetylcholine and that of cathodic (^) polarization is (^) seen (^) by comparing Fig. 4 with (^) Fig. 1I. The (^) depolariza- tion and increase in (^) spike frequency often (^) led to (^) irregular discharges, the duration of (^) depolarization was (^) prolonged to form a (^) plateau which sometimes lasted as (^) long as 1 sec and on which (^) multiple spikes occurred. While (^) adrenaline changed the condition^ of^ a^ preparation from^ producing paired spikes to (^) firing single ones, acetylcholine had^ the^ opposite effect and (^) changed single spikes to pairs. (^) Repolarization was (^) delayed and the next (^) spike arose before the mem- brane (^) potential had returned (^) to its (^) previous level1. In (^) Fig. 12 the (^) administra- tion of (^) acetylcholine led to (^) depolarization and increased (^) frequency of (^) spike discharge. While the^ tension^ increased, the rate of (^) repolarization of the (^) spike potentials was^ slowed. Plateaus were seen (^) carrying two or more (^) spikes (tension
off screen). At the peak of the tension (adjusted, to be visible on screen) the
spike frequency already diminished; repetitive (^) firing ceased and after-
hyperpolarization appeared as the muscle relaxed. There followed a short
(^27) PHYSIO. CXXXV
419
SPIKE CONFIGURATION IN SMOOTH MUSCLE
10 mV 0i1pg.ACh
. (^6)
25 sec 135 sec (^70) sec 90 sec (^4)
Fig. 11. Records of electrical (^) activity and tension. The effect ofacetylcholine, initial concentration 5 x (^) 10-8, prolonging the (^) spike duration to a (^) plateau carrying several spikes. Same (^) experiment as Fig. 8.
u- 1 I^2 I 3 I^4 5 sec^ 2/,gACh I-n
min
3 min
LL11LU~UU&
[1g
2 min
{IOmV
_ (^4) mi Fig. 12. The effect of acetylcholine on (^) electrical activity and tension. 1 min after the injection of ACh tension record off the screen; it (^) was readjusted so that the record shown at 2 min is 1 (^) g higher than the corresponding levels on the other records.
I V-
I-
1-
422 EDITH BULBRING
period during the 4th minute when the size of the spikes was increased. This
period of increased spike height was usually of short duration; it happened
during (^) the 3rd-5th minutes and, probably because of its (^) transience, was (^) not
seen every time. In Fig. 13 the increase in spike height took place already
during the period of repetitive firing. Spike potentials varied between 3 and
6 mV before acetylcholine was (^) given. While the frequency increased (^) they be-
came very small and of long duration, producing irregular plateaus. During
the 3rd minute they grew in size while the frequency was still fast, and finally,
during the 5th minute, as the impulse discharge slowed, the spike height was
16 mV. It then declined rapidly.
IlOOmsec. 1m
ACh^ AChI1min^3 min^4 min^ Smin^ 6min Fig. 13. The change in spike (^) configuration caused by acetylcholine, initial (^) concentration 8 x 1O-7: read from below upwards. Note reduced spike size, increased duration and repeti- tive firing, followed (^) by a (^) large increase in spike height.
DISCUSSION The spike potentials which intestinal smooth muscle (^) discharges spontaneously resemble those of other tissues with (^) autorhythmicity in that they are preceded by a phase of (^) slow depolarization of the membrane. (^) If the prepotential is the characteristic of a pacemaker (^) every smooth muscle cell appears to have (^) this quality. Bozler (1948) has pointed out (^) that most visceral muscles behave (^) like single muscular (^) units, and this view has recently been once more substantiated for the dog's stomach (Ichikawa & (^) Bozler, 1955). According to Prosser, Sperelakis & Bergman (^) (1955) intestinal smooth muscle does not (^) constitute a syncytium and intercellular bridges shown in electron-micrographs have double (^) cell membranes and (^) no fibrillar continuity. Nevertheless, muscular conduction proceeds in plexus-free circular intestinal muscle of the cat at 5 (^) cm/sec, and in
EDITH BLLBRING
continuous with the slow depolarization leading up to the next spike. The greater
the after-hyperpolarization the more the next (^) spike will be (^) delayed and thus
the rate is slowed. Each spike may still be followed by a production of tension,
but this disappears before the next one arrives, consequently the tension does
not build up. The degree of relaxation is a function of the intervals between
spikes. In heart muscle the changes in configuration of the action potential
produced by stimulation of autonomic nerves (Hutter & Trautwein, 1955) or
by transmitter substances (Webb & Hollander, 1956) are in the opposite
direction to those in smooth muscle. Acetylcholine and vagal stimulation
which depress the (^) contractions of the heart muscle decrease the duration (^) of
the action potential and accelerate repolarization, while adrenaline, in general,
has the (^) opposite effect and slows repolarization as it increases (^) muscular contraction. It is (^) tempting to speculate that (^) recovery processes concerned with active
ion transport are already in progress during the falling phase of the action
potential. Studies of the rate of uptake of 42K (Born & Biilbring, 1956) have
shown that this is reduced in those conditions when the rate (^) of repolarization is slowed, and (^) that potassium influx is increased in those conditions in which
the rate of repolarization is fast and the spike is followed by prolonged
hyperpolarization. As the mechanical change appears also to be related to the
rate of (^) repolarization of the spike potentials, it may be that the (^) metabolic processes involved in active ion (^) transport are also playing a part in the (^) linkage between the membrane and the mechanical manifestations.
SUMMARY
- The configuration of the spike potentials discharged by the isolated taenia
coli of the guinea-pig has been studied using intracellular electrodes.
- During spontaneous activity the (^) spikes were preceded by a slow mem-
brane depolarization (prepotentials) varying in slope and in degree. They were
often followed by a (^) prolonged period of after-hyperpolarization.
- (^) Spike size and configuration (^) depended on the state of polarization of the membrane.
- The spontaneous (^) fluctuations of the membrane potential, the application of opposite (^) electronic circuits, and the administration of (^) antagonistic pharma- cological substances produced (^) qualitatively similar effects.
- A delay in (^) the repolarization of the spike potential occurred during
spontaneous phases of depolarization as well as during cathodic current
stimulation and after the (^) administration of acetylcholine or histamine. The longer duration (^) of the spike potentials was associated with a (^) faster rate of
discharge and with the development of tension.
- A fast (^) rate of repolarization and the appearance (^) of an after-hyper- polarization following each spike occurred during spontaneous phases of rising
SPIKE CONFIGURATION IN SMOOTH MUSCLE 425
membrane potential, as a result of anodic polarization and after the admini-
stration of adrenaline. The shorter duration of the spike potentials was
associated with a (^) slow rate of discharge and with muscular relaxation. I wish to thank Mr 0. B. (^) Saxby and Mr (^) D. Groves for their most careful technical assistance.
REFERENCES ADRIAN, E. D. & GELFAN, S. (1933). (^) Rhythmic activity in skeletal muscle fibres. J. Phy8iol. 78, 271-287. ARVANITAKI, A. & (^) CHIATAzoNITIs, N. (1955). Potentiels d'activit6 du soma neuronique (^) g6ant (Aplysia). Arch. Sci. phy8iol. 9, 115-144. BORN, G. V. R. & BULBRING, E. (1956). The movement of potassium between smooth muscle (^) and the surrounding fluid. J. Phy8iol. 131, 690-703. BOZLER, E. (1948). Conduction, automaticity and tonus of visceral (^) muscles. Experientia, 4, 213-218. BRuNE, H. F. & KOTowsKi, H. (1956). Die Erregungsleitung in der glatten Muskulatur des Meerschweinchen-Dickdarms. (^) Pflag. Arch. gms. Physiol. 262. 484-493. BULBRING, E. (1954). Membrane potentials of smooth muscle fibres of the taenia coli of the guinea-pig. J.^ Phy8iol. 125, 302-315. BULBRING, E. (^) (1955). Correlation between membrane potential, spike discharge and tension in smooth muscle. (^) J. Phy8iol. 128, 200-221. B#LBRING, E.^ (1956). Electrophysiology of smooth muscle with (^) autorhythmicity. XX (^) int. phyaiol. (^) Congr., Brumsel8 (Abstr. Rev.), pp. 230-238. BfLBRING, E., HOLMAN, M. & LVJLLMANN, H. (1956). Effects of (^) calcium deficiency on striated muscle of (^) the frog. J. Physiol. 133, 101-117. B#LBRING, E. &^ HOOTON, I. N. (1954). Membrane (^) potentials ofsmooth muscle fibres in the rabbit's sphincter pupillae. J. Phy8iol. 125, 292-301. HOYLE, G. & LowY, J. (1956). The (^) paradox of Mytilus muscle. A new interpretation. J. exp. Biol. 33, 295-310. HUTTER, 0. F. & (^) TRAUTWEIN, W. (1956). Vagal and sympathetic effects on the (^) pacemaker fibres in the sinus venosus of the heart. (^) J. gen. Physiol. 39, 715-733. ICIKAWA, S. & BOZLER, E. (1955). Monophasic and diphasic action (^) potentials of the stomach. Amer. J. (^) Physiol. 182, 92-96. JUNG, H. (1955). tber die Aktionspotentiale am schwangeren (^) und nicht schwangeren Uterus. Pflfig. Arch. ges. Physiol. 262, 13-22. PROSSER, C. L. (1956). Conduction in non-striated (^) muscles. XX int. physiol. Congr., Brussels. (No abstract.) PROSSER, C. L., SMITH, C. E. & MELTON, C. E. (^) (1955). Conduction of action potentials in the ureter of the rat. Amer. J. Physiol. 181, (^) 651-660. PROSSER, C. L., SPERELAKIS, N. & BERGMAN, R. A. (1955). Conduction in intestinal circular muscle. Amer. J. Phy8iol. 183, 652. WEBB, J. L. & HOLLANDER, P. B. (1956). The action (^) of acetylcholine and epinephrine on the cellular membrane potentials and (^) contractility of rat atria. Circulation (^) Re8. 4, 332-336.
