A study of the specific membrane properties in different types of smooth muscle

The primary question which I have attempted to answer by the experiments described in this thesis was: What are the electrical properties of the smooth muscle membrane?
Intracellular stimulation
The first approach consisted of a study of the responses to intracellularly applied currents recorded intracellularly, with the same microelectrode using the Wheatstone bridge method.
Effect of intracellular current application on single cells.
The experiments were carried out on the spontaneously active taenia coli of the guinea-pig. The spontaneous discharge consisted of a slow and a fast (spike) component. They were clearly related. The slow component usually preceded the spike, often triggered it but not always, and persisted as a depolarization delaying the polarisation of the membrane following the spike.
When the membrane was depolarized by the internal current application the spike component became smaller and its rate of rise decreased while, when the membrane was hyperpolarised, the spike component became larger and its rate of rise increased. With very strong hyperpolarization the spike component was blocked. This change of the spike amplitude and its rate of rise was just as expected from our knowledge of the effect of the membrane potential on the sodium carrying system in other excitable tissues. However, the slow component was scarcely affected by the membrane polarisation and no frequency modulation of the spontaneous discharge was observed. Even when the spike component was blocked by strong hyperpolarization, the size and frequency of the slow component remained normal.
The effective membrane resistance, calculated from the current-voltage relationship, was about 40 MΩ. The time constant of the electro tonic potential was 2-4 msec.
In the majority of the cells, no active response was observed in response to intracellular stimulation, even though a normal resting potential and spontaneous spikes with overshoot were recorded with the same micro-electrode which was used for stimulation. Only occasionally a graded action potential was produced. The amplitude of the evoked spike was increased by conditioning hyperpolarization exactly like that of the spontaneously generated spike.
Electrical interaction between cells.
For these experiments, two independent Wheatstone bridge circuits were used, one for each micro-electrode. Since the spontaneous electrical activity of two cells within 50 μ separation was well synchronised, it was expected that there would be electro-tonic spread between cells. However, intracellular current application to one cell had almost no effect on the membrane potential of adjacent cells. Even if the stimulating current evoked a spike, no effect was observed in a neighbouring cell.
From the results of these experiments it is difficult to explain how the spike is evoked, what the slow component is, or how the spike propagates from cell to cell. The possibility suggested itself that the cell membrane is not homogeneous but composed of different areas with different properties and, furthermore, that a single cell is not the unit of excitation. If so, external field stimulation might produce more information than intracellular stimulation.
Therefore, the second approach in the investigation of the membrane properties of the smooth muscle was made by external current application.
External Field stimulation in normal Krebs solution
Large stimulating electrodes were used, the arrangement of which was varied according to the experimental requirements.
Polarization of the membrane by long current pulses.
When current was applied externally, the spontaneous spike frequency was modulated. In contrast to intracellular stimulation, depolarization increased the frequency of the spike discharge while hyperpolarization decreased it. Moreover, depolarizing current easily triggered a spike in every cell.
These phenomena might involve the effect of nerve stimulation because the tissue has intrinsic nerve fibres. In fact, it has been shown that a short current pulse (<1 msec) applied externally causes a transient hyperpolarization ("inhibitory potential") of about 1 sec duration and a latency of 100-200 msec. Therefore it was necessary to discriminate the direct response of the muscle membrane to external stimulation from the indirect (nerve mediated) response.
The inhibitory potential
When two stimulating electrodes were placed, one at each end of the tissue, which was therefore exposed to a uniform longitudinal potential field, the inhibitory potential behaved quite differently from the response of the muscle membrane to polarisation.
Though the muscle membrane was depolarised near the cathode, hyperpolarized near the anode and not polarized in the centre of the tissue (about 3 cm length), the inhibitory potential was recorded everywhere with the same amplitude in response to both polarities of the stimulating current if its parameters were kept constant. If the length of the tissue was reduced to about 1 mm, the spike became graded and threshold current intensity was increased, while the inhibitory potential was produced with little change in size and threshold current intensity.
The strength duration curves for the inhibitory potential and for the spike were obtained. The chronaxie for the inhibitory potential was about 1 msec and that for the spike was about 30 msec. A short current pulse of less than 1 msec produced only the inhibitory potential but not the spike. Tetrodotoxin (10-7 g/ml) abolished the inhibitory potential without affecting the resting potential nor the spontaneous and evoked spike potential of the muscle fibre.
From the results, the inhibitory potential can be explained as a response to stimulation of nerve fibres and can easily be discriminated from the direct response of the muscle fibres.
External field stimulation
Hypertonic solution (Krebs solution containing 10% sucrose) abolishes the spontaneous spike activity. A normal spike can still be evoked but there is no mechanical response. The inhibitory potential is abolished within 20 min. Therefore, the membrane properties can be studied using external current application without interference from spontaneous activity, or movements, or the inhibitory potential.
Electrotonic potential
Interpolar polarisation was studied with preparations of more than 10 mm length placed between two stimulating electrodes, one at each end. It was found that cells near the cathode were depolarized, those near the anode were hyperpolarized and those in the middle part of the tissue were not polarized.
When an insulating partition was placed between toe stimulating site and the recording site, the electrotonic potential not only decreased in size roughly exponentially with the distance from the partition, but also in its rate of rise and fall. It was also found that the electrotonic potentials had a time course similar to that predicted from the cable theory applied to a nerve fibre. The space constant of the electrotonic potential was 1.6 mm (range 1.4-1.9 mm); the time constant was 60-100 msec.
Spike generation and propagation
When the membrane was depolarized by about 25mV, a spike was evoked in every cell. The minimum longitudinal potential gradient to produce the spike was 2-3 mV/100 μ. Since the spike was propagated over the whole tissue (conduction velocity: 7 cm/sec) and the electrotonic potential decayed with a space constant of 1.6 mm, the apparent firing level was very low far away from the stimulating electrode.
There were some qualitative differences between the responses to long end short stimuli. When the stimulating current was short (less than 10 msec), the spike was produced in a graded manner with a short latency. Spikes thus produced did not show a clear slow component. When the stimulating current was long (more than 20 msec) the spike appeared in an all-or-none manner and its latency was very long (up to 1 sec) at threshold intensity.
During external conditioning hyperpolarization the spike amplitude was decreased stepwise and notches or humps appeared on the rising or falling phases of the spike.
These phenomena may be related to the different mechanisms and sites involved in the generation of the slow component and the spike. On the basis of the results obtained by intracellular and external current applications, the following two hypotheses are presented. One is that the smooth muscle cell membrane is not homogeneous but composed of areas with different electrical properties. The second is that many cells are electrically interconnected forming a functional bundle.
Evidence for different areas in a single cell membrane
Since the properties of the electrotonic potential produced by field stimulation (See V) are direct evidence that cells are electrically interconnected and act as a functional syncytium, there must be some membrane areas which act as a current pathway between cells.
Spontaneous electrical activity consists of two clearly different components, the slow and spike components. Intracellularly applied current affects only the spike component. Frequency modulation is observed only by external but not by intracellular current application. These results indicate that the slow component is generated in a different area from that in which the spike is produced, and that this area which generates the slow component must be near the area which acts as current pathway between cells.
In smooth muscle the spike amplitude is small and its rate of rise is slow compared with most other excitable membranes. The spike is insensitive to the external Na concentration, and it is very difficult to trigger a spike by intracellular current application. These facts suggest that the smooth muscle membrane is lees excitable probably due to a poorly developed sodium carrying system.
The hypothesis is as follows (Terms A, B and C are introduced for convenience):
Areas A may be the nexuses i.e. patches of membrane very close to neighbouring cells. When a external current is applied longitudinally to the tissue, most of the current flows through these areas from cell to cell.
Areas B may surround A. They would produce the slow component and have a long time constant. Current passing through area A might affect area B. If this is depolarized, a local potential, the slow component, is initiated. If it Is hyperpolarized, the slow component is suppressed. Hence the frequency modulation.
Area C may be the remaining part of the membrane. It would produce the spike when the slow component has reached threshold level. It would have a shorter time constant than area B and might be electrically less excitable. Daring intracellular stimulation, most of the current would flow through area C.
On the basis of the hypothesis, when a long current pulse is applied externally, area B which has a long tine constant is excited and a slow component is produced which then generates an all-or-none spike. However, when a short current pulse is applied through an external electrode, this only stimulates area C which has a short time constant and produces a spike in graded manner.
Evidence for electrical Interconnections between cells
Although the electrotonic potential produced by intracellular current application is not detected in the neighbouring cell, the fact a that the space constant (1.6 mm) of the electrotonic potential, produced by external current application, is such longer than the cell length (100 μ), and that interpolar polarisation is the same as that expected from a long core conductor, are direct evidence for the electrical interconnections between muscle fibres.
Furthermore, from the minimum longitudinal potential gradient necessary to produce the spike (2-3 mV/100 μ) and the critical firing level of the cell membrane (about 25 mV), it must be assumed that the fibres are interconnected, because the potential gradient per cell length (100 μ) is too small to produce a depolarization of 25 mV.
The failure to obtain evidence for cell-to-cell interaction by intracellular current application is not necessarily a contradiction to the idea of the presence of interconnections between cells, because the spatial decay of the intracellularly applied current would be very sharp due to the three-dimensional current spread in a muscle bundle composed of many aggregated cells.
This situation was simulated using analogous electrical model circuits and the results obtained with these models agreed with the above prediction, i.e. the spatial decay of the current distribution becomes very sharp if there are interconnections between cells.
Deduction of the membrane parameters from direct and indirect evidence.
The membrane resistance could not be measured directly because, when current was applied intracellularly, there was no accurate method of calculation due to the three-dimensional current spread, and because, when current was applied externally, the intensity of the current which passed through the membrane could not be measured with the present method. However, it may be possible to estimate the membrane resistance if the membrane capacity of the taenia is assumed to be the same as that of the slow skeletal muscle fibre (about 2 μF/cm2) because both muscle types have no sarcoplasmic tubular system which causes the high membrane capacity in the twitch fibre. From the capacity of 2 μF/cm2 and the time constant of 60 - 100 msec, the membrane resistance of the muscle fibre is calculated to be 30 - 50 KΩ cm2 which is of the same order as that of the slow skeletal muscle fibre (28 KΩ cm2). From the data of K42 efflux, a high membrane resistance (7 - 27 KΩ cm2) is also predicted. The low K permeability may be responsible for the high membrane resistance and also for the high ratio of Na permeability to K permeability compared with other excitable membrane. The smooth muscle fibres are aggregated in functional bundles of about 30 μ diameter. The capacity of a bundle would then be 12 μF/cm2 (because of the large surface area) and the membrane resistance of the bundle would be 5 - 8 KΩ cm2.
Final conclusion
In spontaneously active muscle (guinea-pig taenia coli), intracellular stimulation causes no frequency modulation; the time constant of the electrotonic potential is 3 msec; a spike is triggered only occasionally; it has no slow component; and no electrotonic spread is detected in adjacent cells. On the other hand, external current application modulates spike frequency; a spike with a slow component is evoked in all cells; and the electrotonic potential fits the cable theory. The space constant is 1.6 mm and the time constant is 60 - 100 msec.
The difference between intracellular and external stimulation could be explained by differences in current distribution. If many fibres with three-dimensional inter-connections were aggregated to form functional bundles, and the possibility that a single muscle fibre membrane is composed of areas with different properties. Participation of many muscle fibres appears to be necessary for spike production and propagation, the fundamental mechanism being electrotonic spread, as in the ephaptic junction between neurones of low animals. A single muscle fibre of the taenia has several such junctions, and the electrotonic spread from many surrounding cells must summate to generate first an active slow component, which then triggers the spike.