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Title: A study of the specific membrane properties in different types of smooth muscle
Author: Tomita, Tadao
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1966
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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?

  1. 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.

    1. 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.

    2. 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.

  2. External Field stimulation in normal Krebs solution Large stimulating electrodes were used, the arrangement of which was varied according to the experimental requirements.

    1. 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.

    2. 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.

  3. 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.

    1. 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.

    2. 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.

Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available