49
J. Physiol. (1975), 245, pp. 49-62 With 8 text-figureB Printed in Great Britain
MEMBRANE PROPERTIES OF THE SMOOTH MUSCLE CELLS OF THE RAT ANOCOCCYGEUS MUSCLE
BY KATE E. CREED* From the Department of Pharmacology, University of Glasgow, Glasgow G12 8QQ
(Received 10 January 1974) SUMMARY
1. The membrane properties of the rat anococcygeus muscle, during rest and activity, were investigated with micro-electrodes and partition stimulation. 2. Intercellular current spread occurred within the muscle and the mean length constant was 2-7 mm. The membrane showed rectification to depolarizing pulses. 3. The mean resting potential was - 62-1 mV and the input resistance was 23-0 MQ. Stimulation of intramural nerves produced depolarization to -21 mV and a 10 % reduction in input resistance. Displacement of the membrane potential indicated that the transmembrane potential at the peak of the response was independent of the membrane potential. 4. Noradrenaline also produced depolarization and this was accompanied by a decrease in membrane resistance as indicated by a reduction in amplitude of the electrotonic potential. 5. It was concluded that the muscle possesses cable properties and that the action of the transmitter, noradrenaline, is to increase membrane permeability so that the membrane potential moves towards an equilibrium potential. INTRODUCTION
Experiments with micro-electrodes have indicated that the anococcygeus muscle of the rat has no spontaneous electrical activity (Creed, Gillespie & Muir, 1975). Direct electrical stimulation of the muscle is not normally possible but contraction can be evoked by noradrenaline and other drugs (Gillespie, 1972). Field stimulation with pulses of 1 msec duration also produces contraction which is blocked by phentolamine, suggesting that noradrenaline released by nerve activity is responsible for excitation of the muscle and that myogenic activity and conduction *
M.R.C. Research Fellow.
KATE E. CREED within the muscle does not occur. This corresponds to Bozler's description of a multiunit muscle (1948). Maximal field stimulation of the motor nerves or noradrenaline produces depolarization of the membrane to the same value of about -20 mV. In other smooth muscles the ax-action of noradrenaline is to increase membrane ionic permeability (guinea-pig taenia coli, Bulbring & Tomita, 1969; rabbit common carotid artery, Mekata & Niu, 1972; oestrogen-dominated guinea-pig uterus, Szurszewski & BUlbring, 1973). The present experiments were in part intended to determine whether the a-action of noradrenaline in the anococcygeus involves a similar increase in ionic permeability. In addition the passive properties of the muscle at rest and during activity were examined. In spite of indirect evidence of a multi unit organization, it was found that intercellular current spread can occur. It was confirmed that excitation is associated with a decrease in membrane resistance and a noradrenaline equilibrium potential of about -21 mV was indicated. Some of these results have been presented to the Physiological Society (Creed & Gillespie, 1974). 50
METHODS
Male Wistar rats weighing 230-270 g were stunned and bled. The anococcygeus muscle was removed and set up as previously described (Creed et at. 1975). The muscle was stimulated by passage of current between Ag-AgCl plates, 10 mm apart, through which the caudal end of the muscle passed (partition stimulating method Abe & Tomita, 1968). This method was also used to displace the membrane potential in order to study the effect of this on the response to field stimulation. Intracellular stimulation was carried out through the recording microelectrode with the WPI current injection pre-amplifier (Model M4A). The distance of the micro-electrode from the nearest stimulating plate was measured through a binocular microscope. In order to reduce movement the muscle was progressively stretched until contraction to repetitive stimulation at 30 Hz, seen in the binocular microscope, was
just abolished. RESULTS
Passive electrical properties Passage of current across the cell membrane was used to study the passive properties of single cells. Intracellular current pulses of 10 msec duration were applied through the same electrode that was used for measurement of the transmembrane potential. Micro-electrodes were chosen so that the pulse recorded in response to passage of current was approximately square when the tip of the electrode was extracellular. It was then assumed that, when the tip was intracellular, that part of the voltage with a slow time course of onset was due to the voltage drop across the membrane and the balance adjusted accordingly. The amplitude of the recorded pulse was linearly proportional to the intensity of the applied
MEMBRANE PROPERTIES OF RAT ANOCOCCYGEUS 51 current although slight rectification to depolarizing currents was seen in a few cells. No active response could be evoked in response to depolarizing currents. From the relationship between the intensity of the applied current and the amplitude of the recorded pulse, the input resistance at
21 mm
50 mV
3-7 mm
61 mm
-I
I
I sec
40 1=243 mm
20 1-
E
-o
10
E.
E
4
2
I
I
1
2
L
I
3 4 Distance (mm)
I
5
6
7
Fig. 1. Electrotonic potentials recorded in the rat anococcygeus muscle at four distances from the stimulating plate are shown in the top four panels. In each record the middle trace is the membrane potential. The bottom trace indicates the passage of current between the two stimulating plates and in the record at 6-1 mm the gain was reduced by half. The steadystate amplitude of the electrotonic potential has been plotted against distance (below) and indicates that the decay was exponential, with a length constant (A) of 2-43 mm in this preparation.
KATE E. CBEED 52 rest was 18-28 MQ (mean + S.D. = 23-0 + 3-6 mQ, n = 12). The rise time of the potential was approximately exponential and gave a time constant of about 2-5 msec. In order to study the spread of current within the tissue, square pulses of 1 see duration were applied to the tissue through extracellular plate electrodes and recorded at various distances from the stimulating partition. Although the smooth muscle cells are orientated in a longitudinal direction and bundles of two to eight cells can be seen in the electron 40
008 Current (V/cm)
-20 E ._ 0 c-
0~ -40
Fig. 2. The relationship between the amplitude of the electrotonic potential recorded from the rat anococcygeus at four distances from the stimulating plate and the intensity of the applied current, expressed as a voltage gradient. For hyperpolarizing currents the relationship was linear but some rectification occurred to depolarizing currents.
microscope, these bundles are not obvious in the dissecting microscope. Therefore to ensure, as far as possible, that recordings were from functionally related cells, the electrode was inserted into cells lying approximately the same distance in from the side of the muscle for each series of measurements. Electrotonic potentials could be recorded up to 7 mm from the stimulating plate and were found to decay exponentially with distance (Fig. 1). There was some variation from one preparation to another in the decay of a uniform pulse with distance and values of the
MEMBRANE PROPERTIES OF RAT ANOCOCCYGEUS 53 length constant (A) ranged from 2-14 to 3-63 mm (mean + S.D. = 2-73 + 0-44, n = 17). For hyperpolarizing current pulses, the amplitude of the electrotonic potential was linearly proportional to the intensity of the applied current (Fig. 2). For small depolarizing currents the relationship was also linear so that with the recording electrode adjacent to the stimulating plate depolarizations of 20 mV were obtained. For larger currents rectification appeared and the amplitude of the electrotonic potential did not exceed 40-50 mV. There was usually no evidence of an active response to small depolarizing currents although sometimes active transient depolarizations were seen superimposed on the early part of the electrotonic potential. These were not seen in the presence of phentolamine (1 X 10-6 M) and were therefore probably nervous in origin. For large current pulses the time course of the depolarizing electrotonic potential was often faster than for the hyperpolarizing potential and may indicate a conductance change in the membrane. An approximate value of the time constant of the membrane (Tm) was obtained from the time course of the electrotonic potentials recorded close to the stimulating plate. The electrotonic potential increased with time in the manner described by an error function and values of 135--260 msec were obtained for Tm. Estimates were also obtained from the relation between the time to reach a half of the steady potential and the distance from the plate since: Slope = Tm/2A. The values obtained were variable and ranged between 126 and 221 msec (178 + 32 msec, n = 13). These results suggest that the cable equations can be applied to the rat anococcygeus. Dependence of resistance on membrane potential In an attempt to determine whether brief changes in membrane potential are associated with changes in membrane resistance, 10 msec intracellular current pulses at a frequency of 10 Hz were applied during the electrotonic potential produced by passage of current between the plates. The amplitude of the recorded pulses, which is directly proportional to the input resistance, remained constant during hyperpolarization to - 120 mV and, in the absence of an active response, was unchanged with depolarization up to -20 mV (Fig. 3). When the extracellular depolarizing current evoked an active response, the amplitude of the pulses was reduced by up to 18 % at the peak, but returned to the control level before the end of the 1 see extracellular current pulse. A significant reduction in amplitude also occurred during the active response produced by stimulation of the excitatory nerves (Creed et al. 1975). This suggests that the
54 A12721TE E. CREED input resistance does not vary with passive displacements of the membrane potential in the range - 120 to -20 mV. Owing to the complex relationship between input resistance and membrane resistance, it cannot be concluded that the latter is also unchanged but if a change does occur it will be small compared with that associated with the active response.
en ~~~~A
mV j ~~~~~50
_1
1 sec Fig. 3. The effect of passive displacements of the membrane potential on the input resistance of the rat anococcygeus. The membrane potential was depolarized (top) or hyperpolarized by 1 sec pulses shown in the bottom trace. The input resistance is proportional to the amplitude of the pulse recorded in response to application of 1 msec current pulses through the recording electrode. An active response is superimposed on the larger passive depolarization (top right).
With more prolonged displacements of the membrane potential, it is possible to measure changes in the membrane resistance by a more direct method. If the properties of the muscle can be explained in terms of the cable equations, the membrane resistance (rm) is related to the amplitude of the electrotonic potential (P) by the equation: P = mJ(rmri) e(-x1A)
(1)
Therefore, if the internal resistance (ri) remains constant and records are taken close to the stimulating electrode (i.e. the distance (x) is small compared with the length constant (A)), a relative change in resistance can be expressed as: rm
4=,
P2
_~ ~50m~V
MEMBRANE PROPERTIES OF RAT ANOCOCCYGEUS 55 The membrane potential was- displaced by passage of current between the two plates for about 10 sec. A second step of 1 see duration and uniform intensity was applied between the two plates about 8 sec after the beginning of the displacement. The amplitude of the electrotonic potential produced by the second step and measured within 0-1 mm of the plate remained constant when the membrane was hyperpolarized, or when it was depolarized to -25 to -20 mV (Fig. 4). With larger displacements the amplitude and time course of the electrotonic potential were reduced. This therefore suggests that the membrane resistance is independent of membrane potential up to this value in the resting state.
64 mV
47 mV
35 mV
16 mV I sec
Fig. 4. Electrotonic potentials recorded from a cell 0.1 mm from the stimulating plate at four levels of membrane potential in the rat anococcygeus. The membrane potential (middle trace) was displaced by passage of current between the plates for several seconds beforehand. A second pulse of 1 sec duration and uniform intensity (shown in the bottom trace) was then applied. The top trace is a reference potential.
Field stimulation The motor nerves in the anococcygeus are adrenergic and contraction, induced by either nerve stimulation or noradrenaline, is associated with depolarization of the membrane (Creed et al. 1975). Experiments were carried out to investigate the nature of this potential change from the alteration in passive properties during activity. (a) Transmembrane potential. Stimulation of the nerves within the muscle with trains of pulses at 30 Hz and 1 msec pulse duration produced
.KAXTE E. CBEED depolarization of the membrane, the maximum amplitude of which ranged between 35 and 51 mV. This represented a mean transmembrane potential at the peak of the response of - 21-2 + 4-7 mV (n = 148) compared with a mean resting potential of - 62 07 + 7*83 mV (n = 201) in this series of experiments. With prolonged periods of stimulation the amplitude was not increased further although the duration of the response 56
was increased (Fig. 5), the mean duration of the response at 50 % amplitude being 549-4 + 93 3 msec (n = 87) in response to 6 pulses at 30 Hz
0
50 mV
100 I
I
I sec
Fig. 5. Depolarization produced by stimulation of motor nerves with 4, 7 and 10 pulses at 30 Hz in the rat anococcygeus. The top trace is a reference potential, the middle is the membrane potential and the bottom trace is the stimulus. With increasing length of train the duration but not the amplitude of the response was increased.
MEMBRANE PROPERTIES OF RAT ANOCOCCYGEUS 57 and 627*0 msec in response to 12 pulses, a difference statistically significant at the 0.1 % level (Student's paired t test). Application of intracellular current pulses during the depolarization produced by field stimulation indicated that the input resistance was significantly reduced at the peak of the response (Creed et al. 1974). This therefore suggests that the action of the transmitter may be to open ionic channels in the membrane so that the potential tends towards an equilibrium potential (E). In this situation the value of the membrane potential at the peak of the response (V') is given by
VI = V-
g+O
(v-E),
(2)
where V and G are the potential and conductance in the absence of transmitter and g is the increase in conductance (Ginsborg, 1967). The depolarization (V - V') should therefore vary linearly with the displacement of the membrane from the equilibrium potential (V - E) when the ionic channels are fully opened and no depolarization should occur when V = E.
*
*] i OV *3 0
15OrnV
1sec the effect of of 6. The membrane displacement potential on the Fig. depolarization produced in response to nerve stimulation. The two records on the left show responses to nerve stimulation at the normal membrane potential. In the other records two levels of depolarization (top) or hyperpolarization were produced by 1 sec current pulses shown in the bottom trace. Two superimposed traces of the membrane potential (middle) are shown one with, the other without nerve stimulation applied shortly after the beginning of the membrane displacement. The top traces are reference potentials. Neither depolarization nor hyperpolarization altered the absolute value of the potential at the peak of the response.
KATE E. CREED The membrane potential was displaced by passage of extracellular current between two plates for 1 sec. Field stimulationwas applied 200 msec after the beginning of the current pulse and was sufficient to produce a response of maximum amplitude. This was to ensure that the conductance change was large (i.e. g/(g + G) 1). For displacements of the membrane potential to between -20 and - 120 mV the transmembrane potential at the peak of the response remained constant throughout the range for any cell and varied from -18 to -24 mV (Fig. 6). There was an approximately linear relationship between the amplitude of depolarization pro-
58
-
-120
-
x
x
-100
7;~~
,-80-xx _
-60
/
-
0
E=-22 mV C -
-40
-
-20
0 20 40 60 80 100 Amplitude of depolarization (mV) (V-V') Fig. 7. The relationship between the amplitude of the response to nerve stimulation (V-V') and the level of the displaced membrane potential (V) in the rat anococcygeus. By extrapolation no depolarization would occur when the membrane potential was -22 mV, suggesting that this is the equilibrium potential. *, Normal membrane potential; x, membrane potential displaced by 1 sec current pulses passed between two stimulating plates.
duced in response to field stimulation (V - V') and the membrane potential (V) (Fig. 7). The intercept of the line on the ordinate indicated that when (V - V') = 0, the value of V, and therefore of the equilibrium potential (E), was between - 17 and 26 mV in different cells (- 21-6 + 30 mV, n = 8). Because of the rectifying property of the membrane, it was not possible to displace the membrane potential to less than -20 mV even
MEMBRANE PROPERTIES OF RAT ANOCOCCYGEUS 59 with large currents recorded close to the stimulating plate. However, in some cells it was possible to abolish the response, so that supramaximal field stimulation did not affect the potential change produced by current passage (i.e. V-V' = 0). (b) Decay of the response. At the end of a train of stimulation the membrane repolarized to the resting level. The time course of the decay was approximately exponential with the time constant (i.e. the time to reach 37 % of the peak amplitude) increasing from a mean of 184-6 + 26-5 msec (n = 18) for bursts of less than 4 stimuli to over 300 msec for 7 or more stimuli. Following long periods of stimulation (over 1 sec) rhythmic oscillations in potential at about 1 Hz appeared during the repolarization. The decay of the electrotonic potential to 37 % took 100-165 msec. Even for brief trains, therefore, the decay was longer than for the passive response of the membrane to extracellular current pulses when no transmitter was present.
Effect of noradrenaline Addition of noradrenaline, to give a concentration of 3 x 10-5 M, produced depolarization of the membrane. In some cells the potential showed rhythmic depolarizations at about -20 mV. In the absence of such oscillations the minimum value of the transmembrane potential was -15 mV. In order to study the resistance changes that occurred during the action of noradrenaline extracellular current pulses were applied. Since rm G+g rm G' from eqn. (1) g4%a= il()2,
substituting into equation (2),
VI
=
V-(-(
(V -E).
Thus if the effect of noradrenaline is to increase membrane conductance, the level of depolarization (V - V') produced by noradrenaline will be proportional to (1- (PI/P)2) and as (PI/P)2 approaches zero, V' approaches the equilibrium potential (E). This equation, which was discussed in detail by Bolton (1972), assumes that records are taken close to the stimulating plate and that the conductance change (g) is produced solely by the action of noradrenaline and is independent of the voltage. The membrane potential was recorded continuously from a cell within 0'5 mm (usually at about 0 1 mm) of the stimulating partition and hyperpolarizing current pulse of constant amplitude was applied every 10 sec.
60 KATE E. CREED Noradrenaline was added to the bath so that the concentration increased to about 3 x 10-5 M, a supramaximal dose, over several minutes. Following the addition of the drug, depolarization occurred and a reduction in amplitude of the electrotonic potential confirmed that there was an increase in membrane conductance. A linear relation was found between the membrane potential and the expression (1_ (PI/P)2) (Fig. 8). In two experiments extrapolation indicated that when this expression equalled 1, V' (and therefore presumably the equilibrium potential) gave values of -20 and -22 mV. A forty-fold change in conductance took the membrane potential to within about 1 mV of the equilibrium values. -60
-
E=-20 8 mV
-40
x
4
20 .0 0. m
x
-60\ E=-22O0 mV
E
E -40
-2nl 0
0-25
0-5
075
1.0
Fig. 8. The relationship between the membrane potential (V') and the relative conductance (1- (P'/P)2) in two cells of the rat anococcygeus in the presence of noradrenaline. The equilibrium potential (E) is the value of V' when the relative conductance is 1. DISCUSSION
The lack of spontaneous activity in the rat anococcygeus muscle and the inability to stimulate it directly (Creed et al. 1975) suggest a multiunit organization, with little interaction between cells. However, application of current pulses with extracellular electrodes showed that the muscle has cable properties, since electrotonic potentials could be recorded 7 mm from the stimulating plate and decayed exponentially with distance. The absence of propagated activity within the muscle may therefore result
MEMBRANE PROPERTIES OF RAT ANOCOCCYCEUS 61 directly from active properties of the membrane, which do not favour spike generation, together with the electrical inexcitability of the muscle. The value of the length constant (A) at 2-8 mm was higher than in intestinal smooth muscles (I0-16 mm), but comparable values have been obtained from guinea-pig vas deferens (2.1 mm) (Tomita, 1967), guinea-pig ureter (2.5 mm) (Kuriyama, Osa & Toida, 1967) and fowl mesenteric artery (2-8 mm) (Bolton & Nishihara, 1970), all of which lack spontaneous activity. Since A2 = r./ri, this could indicate that the resistance of the intercellular connexions is relatively low, and probably certainly reflects a relatively high membrane resistance (rm) associated with the stable, fairly high membrane potential. This is supported by the value of the time constant of the membrane (Tm) which was also higher than in intestinal muscle, since Tm = Rm. Cm. The input resistance was of the same order of magnitude as in other smooth muscles but the relationship between this and the membrane resistance is complex and depends on the extent of intercellular current spread. The time course of the electrotonic potential, approximated to an exponential, was less than the decay of the active response to very short trains of stimuli at 30 Hz. The decay is therefore not entirely passive. The large separation of nerves from smooth muscle cells (mean 2600 A) as seen in the electron microscope suggests that transmitter application is diffuse so that the time course of its action will be comparatively long (Bennett, 1972). Even with short trains some residual effect on the membrane during repolarization can be expected. With long bursts recovery was in some cases prolonged for several seconds. The transmembrane potential at the peak of the response to supramaximal field stimulation or to high concentrations of noradrenaline was about -20 mV, and it was suggested that this might represent an equilibrium potential for noradrenaline (Creed et al. 1975). The observation that the amplitude of the response to field stimulation was proportional to the level of the displaced membrane potential is consistent with this theory and indicated that the equilibrium potential was - 21-6 mV. Furthermore the peak of the response to field stimulation was associated with a decrease in input resistance (Creed et al. 1975) and the transmitter noradrenaline also reduced the membrane resistance, whereas only minor changes occurred when the membrane potential was passively displaced to -20 mV. It can therefore be concluded that the action of the transmitter, noradrenaline, is to increase membrane permeability so that the membrane potential moves towards an equilibrium potential of about -21 mV. It is not yet possible to say which ions are involved in the permeability increase in the anococcygeus and no equilibrium potentials have been reported in other tissues in the presence of noradrenaline. In the guinea-pig
KATE E. CREED ileum Bolton (1972) demonstrated that during excitation by carbachol the membrane potential moved towards an equilibrium potential of -9 mV and concluded that this resulted from increased sodium and potassium ion permeability. Acetylcholine was also shown to produce depolarization by increased permeability to sodium and potassium ions in oestrogendominated guinea-pig myometrium (Szurszewski & Bulbring, 1973) but noradrenaline, which is also excitatory, was believed to act in addition by increasing chloride ion conductance in this tissue and also in rabbit common carotid artery (Mekata & Niu, 1972). In the rat anococcygeus muscle preliminary experiments have indicated that intracellular concentrations of sodium, potassium and chloride ions are 88, 127 and 79 mM with corresponding equilibrium potentials of + 13, -81 and -13 mV (K. E. Creed & D. Pollock, unpublished). Increase in permeability of chloride or sodium could therefore depolarize the muscle from the resting membrane potential of -62 mV. 62
This work was carried out with financial assistance from the Medical Research Council and from medical research funds of Glasgow University. REFERENCES ABE, Y. & TOMITA, T. (1968). Cable properties of smooth muscle. J. Physiol. 196, 87-100. BENNETT, M. R. (1972). Autonomic Neuromuscular Transmission. Cambridge: University Press. BOLTON, T. B. (1972). The depolarizing action of acetylcholine or carbachol in intestinal smooth muscle. J. Physiol. 220, 647-671. BOLTON, T. B. & NISHIHARA, H. (1970). The fine structure of avian vascular muscle and its electrical constants obtained with intracellular microelectrodes. J. Physiol. 208, 20-21 P. BOZLER, E. (1948). Conduction, automaticity and tonus of visceral muscles. Experientia 4, 213-229. BtLBRING, E. & TOMITA, T. (1969). Increase of membrane conductance by adrenaline in the smooth muscle of guinea-pig taenia coil. Proc. R. Soc. B 172, 89-102. CREED, K. E. & GILLEsPIE, J. S. (1974). The effect of excitatory or inhibitory nerve stimulation on the membrane potential of the rat anococcygeus muscle. J. Physiol. 237, 47-48P. CREED, K. E., GILLESPIE, J. S. & Mun, T. C. (1975). The electrical basis of excitation and inhibition in the rat anococcygeus muscle. J. Physiol. 245, 33-47. GILLESPIE, J. S. (1972). The rat anococcygeus muscle and its response to nerve stimulation and to some drugs. Br. J. Pharmac. 45, 404-416. GINSBORG, B. L. (1967). Ion movements in junctional transmission. Pharmac. Rev. 19, 289-316. KU~rYAMA, H., OSA, T. & TOIDA, N. (1967). Membrane properties of the smooth muscle of guinea-pig ureter. J. Physiol. 191, 225-238. MEKATA, F. & Niu, H. (1972). Biophysical effects of adrenaline on the smooth muscle of the rabbit common carotid artery. J. gen. Physiol. 59, 92-102. SzU szEwsKi, J. H. & BULBRING, E. (1973). The stimulant action of acetylcholine and catecholamines on the uterus. Phil. Trans. R. Soc. B 265, 149-156. TOMITA, T. (1967). Current spread in the smooth muscle of the guinea-pig vas deferens. J. Physiol. 189, 163-176.
J. Physiol. (1975), 245, pp. 49-62 With 8 text-figureB Printed in Great Britain
MEMBRANE PROPERTIES OF THE SMOOTH MUSCLE CELLS OF THE RAT ANOCOCCYGEUS MUSCLE
BY KATE E. CREED* From the Department of Pharmacology, University of Glasgow, Glasgow G12 8QQ
(Received 10 January 1974) SUMMARY
1. The membrane properties of the rat anococcygeus muscle, during rest and activity, were investigated with micro-electrodes and partition stimulation. 2. Intercellular current spread occurred within the muscle and the mean length constant was 2-7 mm. The membrane showed rectification to depolarizing pulses. 3. The mean resting potential was - 62-1 mV and the input resistance was 23-0 MQ. Stimulation of intramural nerves produced depolarization to -21 mV and a 10 % reduction in input resistance. Displacement of the membrane potential indicated that the transmembrane potential at the peak of the response was independent of the membrane potential. 4. Noradrenaline also produced depolarization and this was accompanied by a decrease in membrane resistance as indicated by a reduction in amplitude of the electrotonic potential. 5. It was concluded that the muscle possesses cable properties and that the action of the transmitter, noradrenaline, is to increase membrane permeability so that the membrane potential moves towards an equilibrium potential. INTRODUCTION
Experiments with micro-electrodes have indicated that the anococcygeus muscle of the rat has no spontaneous electrical activity (Creed, Gillespie & Muir, 1975). Direct electrical stimulation of the muscle is not normally possible but contraction can be evoked by noradrenaline and other drugs (Gillespie, 1972). Field stimulation with pulses of 1 msec duration also produces contraction which is blocked by phentolamine, suggesting that noradrenaline released by nerve activity is responsible for excitation of the muscle and that myogenic activity and conduction *
M.R.C. Research Fellow.
KATE E. CREED within the muscle does not occur. This corresponds to Bozler's description of a multiunit muscle (1948). Maximal field stimulation of the motor nerves or noradrenaline produces depolarization of the membrane to the same value of about -20 mV. In other smooth muscles the ax-action of noradrenaline is to increase membrane ionic permeability (guinea-pig taenia coli, Bulbring & Tomita, 1969; rabbit common carotid artery, Mekata & Niu, 1972; oestrogen-dominated guinea-pig uterus, Szurszewski & BUlbring, 1973). The present experiments were in part intended to determine whether the a-action of noradrenaline in the anococcygeus involves a similar increase in ionic permeability. In addition the passive properties of the muscle at rest and during activity were examined. In spite of indirect evidence of a multi unit organization, it was found that intercellular current spread can occur. It was confirmed that excitation is associated with a decrease in membrane resistance and a noradrenaline equilibrium potential of about -21 mV was indicated. Some of these results have been presented to the Physiological Society (Creed & Gillespie, 1974). 50
METHODS
Male Wistar rats weighing 230-270 g were stunned and bled. The anococcygeus muscle was removed and set up as previously described (Creed et at. 1975). The muscle was stimulated by passage of current between Ag-AgCl plates, 10 mm apart, through which the caudal end of the muscle passed (partition stimulating method Abe & Tomita, 1968). This method was also used to displace the membrane potential in order to study the effect of this on the response to field stimulation. Intracellular stimulation was carried out through the recording microelectrode with the WPI current injection pre-amplifier (Model M4A). The distance of the micro-electrode from the nearest stimulating plate was measured through a binocular microscope. In order to reduce movement the muscle was progressively stretched until contraction to repetitive stimulation at 30 Hz, seen in the binocular microscope, was
just abolished. RESULTS
Passive electrical properties Passage of current across the cell membrane was used to study the passive properties of single cells. Intracellular current pulses of 10 msec duration were applied through the same electrode that was used for measurement of the transmembrane potential. Micro-electrodes were chosen so that the pulse recorded in response to passage of current was approximately square when the tip of the electrode was extracellular. It was then assumed that, when the tip was intracellular, that part of the voltage with a slow time course of onset was due to the voltage drop across the membrane and the balance adjusted accordingly. The amplitude of the recorded pulse was linearly proportional to the intensity of the applied
MEMBRANE PROPERTIES OF RAT ANOCOCCYGEUS 51 current although slight rectification to depolarizing currents was seen in a few cells. No active response could be evoked in response to depolarizing currents. From the relationship between the intensity of the applied current and the amplitude of the recorded pulse, the input resistance at
21 mm
50 mV
3-7 mm
61 mm
-I
I
I sec
40 1=243 mm
20 1-
E
-o
10
E.
E
4
2
I
I
1
2
L
I
3 4 Distance (mm)
I
5
6
7
Fig. 1. Electrotonic potentials recorded in the rat anococcygeus muscle at four distances from the stimulating plate are shown in the top four panels. In each record the middle trace is the membrane potential. The bottom trace indicates the passage of current between the two stimulating plates and in the record at 6-1 mm the gain was reduced by half. The steadystate amplitude of the electrotonic potential has been plotted against distance (below) and indicates that the decay was exponential, with a length constant (A) of 2-43 mm in this preparation.
KATE E. CBEED 52 rest was 18-28 MQ (mean + S.D. = 23-0 + 3-6 mQ, n = 12). The rise time of the potential was approximately exponential and gave a time constant of about 2-5 msec. In order to study the spread of current within the tissue, square pulses of 1 see duration were applied to the tissue through extracellular plate electrodes and recorded at various distances from the stimulating partition. Although the smooth muscle cells are orientated in a longitudinal direction and bundles of two to eight cells can be seen in the electron 40
008 Current (V/cm)
-20 E ._ 0 c-
0~ -40
Fig. 2. The relationship between the amplitude of the electrotonic potential recorded from the rat anococcygeus at four distances from the stimulating plate and the intensity of the applied current, expressed as a voltage gradient. For hyperpolarizing currents the relationship was linear but some rectification occurred to depolarizing currents.
microscope, these bundles are not obvious in the dissecting microscope. Therefore to ensure, as far as possible, that recordings were from functionally related cells, the electrode was inserted into cells lying approximately the same distance in from the side of the muscle for each series of measurements. Electrotonic potentials could be recorded up to 7 mm from the stimulating plate and were found to decay exponentially with distance (Fig. 1). There was some variation from one preparation to another in the decay of a uniform pulse with distance and values of the
MEMBRANE PROPERTIES OF RAT ANOCOCCYGEUS 53 length constant (A) ranged from 2-14 to 3-63 mm (mean + S.D. = 2-73 + 0-44, n = 17). For hyperpolarizing current pulses, the amplitude of the electrotonic potential was linearly proportional to the intensity of the applied current (Fig. 2). For small depolarizing currents the relationship was also linear so that with the recording electrode adjacent to the stimulating plate depolarizations of 20 mV were obtained. For larger currents rectification appeared and the amplitude of the electrotonic potential did not exceed 40-50 mV. There was usually no evidence of an active response to small depolarizing currents although sometimes active transient depolarizations were seen superimposed on the early part of the electrotonic potential. These were not seen in the presence of phentolamine (1 X 10-6 M) and were therefore probably nervous in origin. For large current pulses the time course of the depolarizing electrotonic potential was often faster than for the hyperpolarizing potential and may indicate a conductance change in the membrane. An approximate value of the time constant of the membrane (Tm) was obtained from the time course of the electrotonic potentials recorded close to the stimulating plate. The electrotonic potential increased with time in the manner described by an error function and values of 135--260 msec were obtained for Tm. Estimates were also obtained from the relation between the time to reach a half of the steady potential and the distance from the plate since: Slope = Tm/2A. The values obtained were variable and ranged between 126 and 221 msec (178 + 32 msec, n = 13). These results suggest that the cable equations can be applied to the rat anococcygeus. Dependence of resistance on membrane potential In an attempt to determine whether brief changes in membrane potential are associated with changes in membrane resistance, 10 msec intracellular current pulses at a frequency of 10 Hz were applied during the electrotonic potential produced by passage of current between the plates. The amplitude of the recorded pulses, which is directly proportional to the input resistance, remained constant during hyperpolarization to - 120 mV and, in the absence of an active response, was unchanged with depolarization up to -20 mV (Fig. 3). When the extracellular depolarizing current evoked an active response, the amplitude of the pulses was reduced by up to 18 % at the peak, but returned to the control level before the end of the 1 see extracellular current pulse. A significant reduction in amplitude also occurred during the active response produced by stimulation of the excitatory nerves (Creed et al. 1975). This suggests that the
54 A12721TE E. CREED input resistance does not vary with passive displacements of the membrane potential in the range - 120 to -20 mV. Owing to the complex relationship between input resistance and membrane resistance, it cannot be concluded that the latter is also unchanged but if a change does occur it will be small compared with that associated with the active response.
en ~~~~A
mV j ~~~~~50
_1
1 sec Fig. 3. The effect of passive displacements of the membrane potential on the input resistance of the rat anococcygeus. The membrane potential was depolarized (top) or hyperpolarized by 1 sec pulses shown in the bottom trace. The input resistance is proportional to the amplitude of the pulse recorded in response to application of 1 msec current pulses through the recording electrode. An active response is superimposed on the larger passive depolarization (top right).
With more prolonged displacements of the membrane potential, it is possible to measure changes in the membrane resistance by a more direct method. If the properties of the muscle can be explained in terms of the cable equations, the membrane resistance (rm) is related to the amplitude of the electrotonic potential (P) by the equation: P = mJ(rmri) e(-x1A)
(1)
Therefore, if the internal resistance (ri) remains constant and records are taken close to the stimulating electrode (i.e. the distance (x) is small compared with the length constant (A)), a relative change in resistance can be expressed as: rm
4=,
P2
_~ ~50m~V
MEMBRANE PROPERTIES OF RAT ANOCOCCYGEUS 55 The membrane potential was- displaced by passage of current between the two plates for about 10 sec. A second step of 1 see duration and uniform intensity was applied between the two plates about 8 sec after the beginning of the displacement. The amplitude of the electrotonic potential produced by the second step and measured within 0-1 mm of the plate remained constant when the membrane was hyperpolarized, or when it was depolarized to -25 to -20 mV (Fig. 4). With larger displacements the amplitude and time course of the electrotonic potential were reduced. This therefore suggests that the membrane resistance is independent of membrane potential up to this value in the resting state.
64 mV
47 mV
35 mV
16 mV I sec
Fig. 4. Electrotonic potentials recorded from a cell 0.1 mm from the stimulating plate at four levels of membrane potential in the rat anococcygeus. The membrane potential (middle trace) was displaced by passage of current between the plates for several seconds beforehand. A second pulse of 1 sec duration and uniform intensity (shown in the bottom trace) was then applied. The top trace is a reference potential.
Field stimulation The motor nerves in the anococcygeus are adrenergic and contraction, induced by either nerve stimulation or noradrenaline, is associated with depolarization of the membrane (Creed et al. 1975). Experiments were carried out to investigate the nature of this potential change from the alteration in passive properties during activity. (a) Transmembrane potential. Stimulation of the nerves within the muscle with trains of pulses at 30 Hz and 1 msec pulse duration produced
.KAXTE E. CBEED depolarization of the membrane, the maximum amplitude of which ranged between 35 and 51 mV. This represented a mean transmembrane potential at the peak of the response of - 21-2 + 4-7 mV (n = 148) compared with a mean resting potential of - 62 07 + 7*83 mV (n = 201) in this series of experiments. With prolonged periods of stimulation the amplitude was not increased further although the duration of the response 56
was increased (Fig. 5), the mean duration of the response at 50 % amplitude being 549-4 + 93 3 msec (n = 87) in response to 6 pulses at 30 Hz
0
50 mV
100 I
I
I sec
Fig. 5. Depolarization produced by stimulation of motor nerves with 4, 7 and 10 pulses at 30 Hz in the rat anococcygeus. The top trace is a reference potential, the middle is the membrane potential and the bottom trace is the stimulus. With increasing length of train the duration but not the amplitude of the response was increased.
MEMBRANE PROPERTIES OF RAT ANOCOCCYGEUS 57 and 627*0 msec in response to 12 pulses, a difference statistically significant at the 0.1 % level (Student's paired t test). Application of intracellular current pulses during the depolarization produced by field stimulation indicated that the input resistance was significantly reduced at the peak of the response (Creed et al. 1974). This therefore suggests that the action of the transmitter may be to open ionic channels in the membrane so that the potential tends towards an equilibrium potential (E). In this situation the value of the membrane potential at the peak of the response (V') is given by
VI = V-
g+O
(v-E),
(2)
where V and G are the potential and conductance in the absence of transmitter and g is the increase in conductance (Ginsborg, 1967). The depolarization (V - V') should therefore vary linearly with the displacement of the membrane from the equilibrium potential (V - E) when the ionic channels are fully opened and no depolarization should occur when V = E.
*
*] i OV *3 0
15OrnV
1sec the effect of of 6. The membrane displacement potential on the Fig. depolarization produced in response to nerve stimulation. The two records on the left show responses to nerve stimulation at the normal membrane potential. In the other records two levels of depolarization (top) or hyperpolarization were produced by 1 sec current pulses shown in the bottom trace. Two superimposed traces of the membrane potential (middle) are shown one with, the other without nerve stimulation applied shortly after the beginning of the membrane displacement. The top traces are reference potentials. Neither depolarization nor hyperpolarization altered the absolute value of the potential at the peak of the response.
KATE E. CREED The membrane potential was displaced by passage of extracellular current between two plates for 1 sec. Field stimulationwas applied 200 msec after the beginning of the current pulse and was sufficient to produce a response of maximum amplitude. This was to ensure that the conductance change was large (i.e. g/(g + G) 1). For displacements of the membrane potential to between -20 and - 120 mV the transmembrane potential at the peak of the response remained constant throughout the range for any cell and varied from -18 to -24 mV (Fig. 6). There was an approximately linear relationship between the amplitude of depolarization pro-
58
-
-120
-
x
x
-100
7;~~
,-80-xx _
-60
/
-
0
E=-22 mV C -
-40
-
-20
0 20 40 60 80 100 Amplitude of depolarization (mV) (V-V') Fig. 7. The relationship between the amplitude of the response to nerve stimulation (V-V') and the level of the displaced membrane potential (V) in the rat anococcygeus. By extrapolation no depolarization would occur when the membrane potential was -22 mV, suggesting that this is the equilibrium potential. *, Normal membrane potential; x, membrane potential displaced by 1 sec current pulses passed between two stimulating plates.
duced in response to field stimulation (V - V') and the membrane potential (V) (Fig. 7). The intercept of the line on the ordinate indicated that when (V - V') = 0, the value of V, and therefore of the equilibrium potential (E), was between - 17 and 26 mV in different cells (- 21-6 + 30 mV, n = 8). Because of the rectifying property of the membrane, it was not possible to displace the membrane potential to less than -20 mV even
MEMBRANE PROPERTIES OF RAT ANOCOCCYGEUS 59 with large currents recorded close to the stimulating plate. However, in some cells it was possible to abolish the response, so that supramaximal field stimulation did not affect the potential change produced by current passage (i.e. V-V' = 0). (b) Decay of the response. At the end of a train of stimulation the membrane repolarized to the resting level. The time course of the decay was approximately exponential with the time constant (i.e. the time to reach 37 % of the peak amplitude) increasing from a mean of 184-6 + 26-5 msec (n = 18) for bursts of less than 4 stimuli to over 300 msec for 7 or more stimuli. Following long periods of stimulation (over 1 sec) rhythmic oscillations in potential at about 1 Hz appeared during the repolarization. The decay of the electrotonic potential to 37 % took 100-165 msec. Even for brief trains, therefore, the decay was longer than for the passive response of the membrane to extracellular current pulses when no transmitter was present.
Effect of noradrenaline Addition of noradrenaline, to give a concentration of 3 x 10-5 M, produced depolarization of the membrane. In some cells the potential showed rhythmic depolarizations at about -20 mV. In the absence of such oscillations the minimum value of the transmembrane potential was -15 mV. In order to study the resistance changes that occurred during the action of noradrenaline extracellular current pulses were applied. Since rm G+g rm G' from eqn. (1) g4%a= il()2,
substituting into equation (2),
VI
=
V-(-(
(V -E).
Thus if the effect of noradrenaline is to increase membrane conductance, the level of depolarization (V - V') produced by noradrenaline will be proportional to (1- (PI/P)2) and as (PI/P)2 approaches zero, V' approaches the equilibrium potential (E). This equation, which was discussed in detail by Bolton (1972), assumes that records are taken close to the stimulating plate and that the conductance change (g) is produced solely by the action of noradrenaline and is independent of the voltage. The membrane potential was recorded continuously from a cell within 0'5 mm (usually at about 0 1 mm) of the stimulating partition and hyperpolarizing current pulse of constant amplitude was applied every 10 sec.
60 KATE E. CREED Noradrenaline was added to the bath so that the concentration increased to about 3 x 10-5 M, a supramaximal dose, over several minutes. Following the addition of the drug, depolarization occurred and a reduction in amplitude of the electrotonic potential confirmed that there was an increase in membrane conductance. A linear relation was found between the membrane potential and the expression (1_ (PI/P)2) (Fig. 8). In two experiments extrapolation indicated that when this expression equalled 1, V' (and therefore presumably the equilibrium potential) gave values of -20 and -22 mV. A forty-fold change in conductance took the membrane potential to within about 1 mV of the equilibrium values. -60
-
E=-20 8 mV
-40
x
4
20 .0 0. m
x
-60\ E=-22O0 mV
E
E -40
-2nl 0
0-25
0-5
075
1.0
Fig. 8. The relationship between the membrane potential (V') and the relative conductance (1- (P'/P)2) in two cells of the rat anococcygeus in the presence of noradrenaline. The equilibrium potential (E) is the value of V' when the relative conductance is 1. DISCUSSION
The lack of spontaneous activity in the rat anococcygeus muscle and the inability to stimulate it directly (Creed et al. 1975) suggest a multiunit organization, with little interaction between cells. However, application of current pulses with extracellular electrodes showed that the muscle has cable properties, since electrotonic potentials could be recorded 7 mm from the stimulating plate and decayed exponentially with distance. The absence of propagated activity within the muscle may therefore result
MEMBRANE PROPERTIES OF RAT ANOCOCCYCEUS 61 directly from active properties of the membrane, which do not favour spike generation, together with the electrical inexcitability of the muscle. The value of the length constant (A) at 2-8 mm was higher than in intestinal smooth muscles (I0-16 mm), but comparable values have been obtained from guinea-pig vas deferens (2.1 mm) (Tomita, 1967), guinea-pig ureter (2.5 mm) (Kuriyama, Osa & Toida, 1967) and fowl mesenteric artery (2-8 mm) (Bolton & Nishihara, 1970), all of which lack spontaneous activity. Since A2 = r./ri, this could indicate that the resistance of the intercellular connexions is relatively low, and probably certainly reflects a relatively high membrane resistance (rm) associated with the stable, fairly high membrane potential. This is supported by the value of the time constant of the membrane (Tm) which was also higher than in intestinal muscle, since Tm = Rm. Cm. The input resistance was of the same order of magnitude as in other smooth muscles but the relationship between this and the membrane resistance is complex and depends on the extent of intercellular current spread. The time course of the electrotonic potential, approximated to an exponential, was less than the decay of the active response to very short trains of stimuli at 30 Hz. The decay is therefore not entirely passive. The large separation of nerves from smooth muscle cells (mean 2600 A) as seen in the electron microscope suggests that transmitter application is diffuse so that the time course of its action will be comparatively long (Bennett, 1972). Even with short trains some residual effect on the membrane during repolarization can be expected. With long bursts recovery was in some cases prolonged for several seconds. The transmembrane potential at the peak of the response to supramaximal field stimulation or to high concentrations of noradrenaline was about -20 mV, and it was suggested that this might represent an equilibrium potential for noradrenaline (Creed et al. 1975). The observation that the amplitude of the response to field stimulation was proportional to the level of the displaced membrane potential is consistent with this theory and indicated that the equilibrium potential was - 21-6 mV. Furthermore the peak of the response to field stimulation was associated with a decrease in input resistance (Creed et al. 1975) and the transmitter noradrenaline also reduced the membrane resistance, whereas only minor changes occurred when the membrane potential was passively displaced to -20 mV. It can therefore be concluded that the action of the transmitter, noradrenaline, is to increase membrane permeability so that the membrane potential moves towards an equilibrium potential of about -21 mV. It is not yet possible to say which ions are involved in the permeability increase in the anococcygeus and no equilibrium potentials have been reported in other tissues in the presence of noradrenaline. In the guinea-pig
KATE E. CREED ileum Bolton (1972) demonstrated that during excitation by carbachol the membrane potential moved towards an equilibrium potential of -9 mV and concluded that this resulted from increased sodium and potassium ion permeability. Acetylcholine was also shown to produce depolarization by increased permeability to sodium and potassium ions in oestrogendominated guinea-pig myometrium (Szurszewski & Bulbring, 1973) but noradrenaline, which is also excitatory, was believed to act in addition by increasing chloride ion conductance in this tissue and also in rabbit common carotid artery (Mekata & Niu, 1972). In the rat anococcygeus muscle preliminary experiments have indicated that intracellular concentrations of sodium, potassium and chloride ions are 88, 127 and 79 mM with corresponding equilibrium potentials of + 13, -81 and -13 mV (K. E. Creed & D. Pollock, unpublished). Increase in permeability of chloride or sodium could therefore depolarize the muscle from the resting membrane potential of -62 mV. 62
This work was carried out with financial assistance from the Medical Research Council and from medical research funds of Glasgow University. REFERENCES ABE, Y. & TOMITA, T. (1968). Cable properties of smooth muscle. J. Physiol. 196, 87-100. BENNETT, M. R. (1972). Autonomic Neuromuscular Transmission. Cambridge: University Press. BOLTON, T. B. (1972). The depolarizing action of acetylcholine or carbachol in intestinal smooth muscle. J. Physiol. 220, 647-671. BOLTON, T. B. & NISHIHARA, H. (1970). The fine structure of avian vascular muscle and its electrical constants obtained with intracellular microelectrodes. J. Physiol. 208, 20-21 P. BOZLER, E. (1948). Conduction, automaticity and tonus of visceral muscles. Experientia 4, 213-229. BtLBRING, E. & TOMITA, T. (1969). Increase of membrane conductance by adrenaline in the smooth muscle of guinea-pig taenia coil. Proc. R. Soc. B 172, 89-102. CREED, K. E. & GILLEsPIE, J. S. (1974). The effect of excitatory or inhibitory nerve stimulation on the membrane potential of the rat anococcygeus muscle. J. Physiol. 237, 47-48P. CREED, K. E., GILLESPIE, J. S. & Mun, T. C. (1975). The electrical basis of excitation and inhibition in the rat anococcygeus muscle. J. Physiol. 245, 33-47. GILLESPIE, J. S. (1972). The rat anococcygeus muscle and its response to nerve stimulation and to some drugs. Br. J. Pharmac. 45, 404-416. GINSBORG, B. L. (1967). Ion movements in junctional transmission. Pharmac. Rev. 19, 289-316. KU~rYAMA, H., OSA, T. & TOIDA, N. (1967). Membrane properties of the smooth muscle of guinea-pig ureter. J. Physiol. 191, 225-238. MEKATA, F. & Niu, H. (1972). Biophysical effects of adrenaline on the smooth muscle of the rabbit common carotid artery. J. gen. Physiol. 59, 92-102. SzU szEwsKi, J. H. & BULBRING, E. (1973). The stimulant action of acetylcholine and catecholamines on the uterus. Phil. Trans. R. Soc. B 265, 149-156. TOMITA, T. (1967). Current spread in the smooth muscle of the guinea-pig vas deferens. J. Physiol. 189, 163-176.
