Transmission from perivascular inhibitory nerves to the smooth muscle of the guinea-pig taenia coli

1. Membrane potential changes of the smooth muscle cells of the taenia coli were recorded during stimulation of the perivascular inhibitory nerves.2. Some spontaneous action potentials were preceded by a slow pacemaker-like potential. Others began at or near the maximum level of the membrane potential and were not preceded by pacemaker-like potentials.3. There were no changes in the membrane potential of smooth muscle cells when the inhibitory nerves were stimulated with a single pulse. Stimulation at frequencies greater than 5 pulses/sec caused a hyperpolarization of the smooth muscle membrane. This resulted in a decrease in spike frequency and relaxation.4. When the frequency of stimulation of the inhibitory nerves was increased there was an increase in the amplitude and rate of rise of the hyperpolarization and a decrease of the latency. The latency varied from 150 to 300 msec, and the largest hyperpolarization recorded was 16 mV.5. The effect of the hyperpolarization due to nerve stimulation in cells showing pacemaker-like activity was to increase the level of the membrane potential at which the action potentials began and to increase the membrane potential to which the action potentials repolarized. Action potentials which occurred during hyperpolarizations of the membrane had greater rates of rise and fall and larger amplitudes than did the action potentials which occurred before hyperpolarization.6. The effect of the hyperpolarization due to nerve stimulation in cells which did not show pacemaker-like activity depended on the amplitude of the hyperpolarization. Small hyperpolarizations exposed small depolarizations of the membrane which occurred when an action potential would normally have been expected. Large hyperpolarizations blocked the action potentials entirely.7. Action potentials did not begin firing again at the normal rate immediately after stimulation ceased. The time taken for the rate of firing of action potentials to return to normal increased with increasing frequency of stimulation.8. The hyperpolarization in response to perivascular inhibitory nerve stimulation was blocked by guanethidine and bretylium.     

Transmission from intramural inhibitory nerves to the smooth muscle of the guinea-pig taenia coli

1. Membrane potential changes of smooth muscle cells were recorded during stimulation of the intramural inhibitory nerves to the taenia coli.2. Stimulation across the taenia coli with single pulses of 200 musec duration excites the intramural nerves and not the muscle directly.3. The membrane potential changes due to stimulation of the intramural inhibitory nerves were different from those produced by perivascular inhibitory nerve stimulation in the following ways: hyperpolarizations (i.j.p.'s) of up to 25 mV were produced in response to single pulses; the latency, i.e. the time taken for the membrane to hyperpolarize after a stimulus of maximal strength, was as short as 80 msec; when the nerves were stimulated repetitively the membrane was hyperpolarized by up to 35 mV and all spontaneous activity was abolished; the mean hyperpolarization due to repetitive stimulation increased with the frequency of stimulation up to 10 pulses/sec and then remained constant; the hyperpolarization due to stimulation at frequencies greater than 5 pulses/sec was not maintained but decreased after 3-5 sec of stimulation; and finally when stimulation had ceased action potentials commenced firing at frequencies greater than normal.4. The amplitude and rate of hyperpolarization of the i.j.p. increased with increasing strength of stimulation until a maximum amplitude and rate of hyperpolarization was reached. The recovery or depolarizing phase of the i.j.p. was exponential with a time constant which varied from about 250 msec to 500 msec and could not therefore be due to the discharge of the membrane capacitance. In some cases there was an inflexion on this depolarizing phase and in these cases recovery led directly into an action potential.5. Spontaneous hyperpolarizations of the membrane were seen in some cells, and these hyperpolarizations were similar to those recorded on submaximal stimulation of the intramural nerves.6. There were no changes in the characteristics of the i.j.p. in the presence of guanethidine or bretylium.     

Transmission from intramural excitatory nerves to the smooth muscle cells of the guinea-pig taenia coli

1. A study has been made of transmission from intramural excitatory nerves to the smooth muscle cells of the guinea-pig taenia coli.2. Only ten cells out of eighty gave depolarizing (i.e. excitatory junction potentials, E.J.P.S) on stimulating the intramural nerves, the remaining cells gave hyperpolarizing responses (i.e. inhibitory junction potentials, I.J.P.S). E.J.P.S were recorded in cells which were less than 1 mm away from cells which gave I.J.P.S.3. In some cells stimulation of the intramural nerves with single pulses of maximal strength gave E.J.P.S of about 20 mV amplitude after a latency of 100-200 msec. In quiescent cells these E.J.P.S gave rise to action potentials. Repetitive stimulation above 1 c/s depolarized the membrane for less than about 1 sec, and during the remainder of the stimulation no action potentials fired, even in spontaneous cells.4. In some cells stimulation of the intramural nerves gave an I.J.P. The largest sized I.J.P.S were generally only about half the size of the I.J.P.S recorded in atropinized preparations. The decreased amplitude of the I.J.P.S enabled rebound action potentials to be fired by successive I.J.P.S when the intramural nerves were stimulated at about 1 c/s. At higher frequencies all spontaneous activity was suppressed.5. The effect of neostigmine (10(-9)-10(-7) g/ml.) on the transmission was studied. There was no detectable increase in the number of cells giving E.J.P. responses in the presence of neostigmine.6. The electrophysiological characteristics of intramural excitatory and inhibitory nerve transmission are discussed.     

Ionic currents in the guinea-pig taenia coli.

Short segments of portions of taenia coli of the guinea-pig averaging 54 mum X 219 mum X ca. 200 mum have been studied by a double sucrose-gap voltage-clamp technique. 2. The average total capacitance was 0-4 muF, corresponding to approximately 10(4) cells, if a specific membrane capacitance of 3 muF/cm2 were assumed. 3. A significant resistance, averaging 11-4omega, was in series with the membrane, and seriously limited the accuracy of the voltage control possible. 4. On depolarization, an early transient inward current was followed by a late maintained outwary current. 5. The late current was carried mainly by K+, because its direction could be reversed if the preparation were first depolarized in isotonic K2SO4 and held back to the original resting potential. 6. After appropriate corrections for residual capacitative and leakage currents, a reversal potential for the late current (Eb) was determined to be 15-20 mV more negative than the natural resting potential. It was not affected by the amplitude or the duration of the activating voltage step, but could be changed by prolonged applications of holding current. 7. At rest, the ratio of PNa:PK was 0-16:1; for Eb it was 0-05:1. 8. The reversal potential for the transient early inward current (Ea) averaged 22 mV in Krebs-bicarbonate solution, but was shifted to about 35 mV when the late current was first suppressed with tetraethylammonium ion. The shift suggested that there was some overlap of the early and late currents. 9. Reduction of [Na+]o to 50% of normal, or replacement of all Na+ with dimethyldiethanol ammonium ion and choline ion, failed to cause any significant shifts in the reversal potential of the early current or reduce the magnitude of the early current. 10. Reduction of [Ca2+]o to 0-25 or 0-1 of the normal caused shifts of the Ea toward the negative and reductions in the early current. These changes can occur without changes in the maximum chord conductance of the early current, such as might happen in ordinary Krebs-bicarbonate solution, or in preparations which had been depolarized by prior treatment with isotonic K2SO4 and then held back to the original membrane voltage. 11. Increase of [Ca2+]o to 5 times normal increased the early inward current, and the maximum chord conductances of the early and late currents, but did not shift the Ea. 12. In preparations pretreated with TEA, increasing [Ca2+]o to 5 times normal shifted Ea toward 45 mV. 13. The various observations are interpreted to mean that the early current in the taenia coli is carried principally by influx of Ca2+, and not by Na+.     

Rebound excitation of the smooth muscle cells of the guinea-pig taenia coli after stimulation of intramural inhibitory nerves

1. A study has been made of the increase in the rate of action potential firing in spontaneously active cells and of the initiation of action potential firing in quiescent cells of the taenia coli after stimulation of the intramural inhibitory nerves.2. In the majority of cells which fired action potentials spontaneously at intervals of about 1 sec, stimulation of the intramural inhibitory nerves with single pulses gave an inhibitory junction potential (I.J.P.) which was followed by action potentials which occurred at intervals as small as 0.5 sec. The increased rate of firing lasted up to 30 sec.3. A small number of cells were either not spontaneously active or only fired action potentials at intervals greater than 5 sec. After stimulation of the intramural inhibitory nerves with either single or repetitive pulses, the quiescent cells gave I.J.P.S which were followed by either a single action potential or a burst of action potentials.4. The rate of firing of action potentials after an I.J.P., and the duration of this enhanced rate of firing increased with an increase in the mean amplitude of the hyperpolarization during the I.J.P. As the amplitude of the I.J.P. increases with an increase in frequency of stimulation of the nerves, the rebound excitation increases with an increase in the frequency of stimulation of the inhibitory nerves.     

Mechanical characteristics of chemically skinned guinea-pig taenia coli

Strips of intact and chemically skinned (Triton X-100) taenia coli were mounted for isometric and quick-release experiments at 23°C. Active force increased in repeated high-K⁺ induced contractures in the intact muscle. Stable maximal force was 313±24 mN/mm² (n=6). The skinned preparations activated by Ca²⁺, at 2 mM Mg²⁺, 3.2 mM MgATP and ionic strength 0.085 M, gave half maximal force atpCa=5.62±0.4 and a maximal force (63±8 mN/mm²) atpCa=4.5 (20–25 of the control K⁺-responses prior to skinning but about 60% of the first K⁺-response). Force-velocity relations were obtained from intact muscles and from the same muscles chemically skinned and activated at optimal Ca²⁺. Maximal shortening velocity (V _max) was unaltered in the skinned preparation compared to the intact muscle (0.138±0.011 vs 0.140±0.006 L/s) indicating similar kinetics of actomyosin interaction. In the intact muscle a decrease inV _max was found when the Ca²⁺ concentration was reduced. Calmodulin (1M) increased Ca²⁺ sensitivity (by about 0.6 log units) of the skinned preparation but at optimal Ca²⁺ caused no alteration in isometric force orV _max Apreliminary report of some of the results presented here was given at the Scandinavian Physiology Society Meeting in Århus, November 1981. Arner A, Hellstrand P (1982) Acta Physiol Scand (Abstract) 114: 38 A.     

Time series analysis of guinea-pig taenia coli spontaneous activity

Summary Spontaneous tension changes of isolated strips of guinea-pig taenia coli were studied in using the time series analysis (evaluation of autocorrelation functions and power spectra). The results indicated three typical power spectral maxima with frequencies of 0.02–0.03 Hz, 0.06–0.07 Hz, and 0.1–0.14 Hz. These smooth muscle oscillation components were compared with the vascular autooscillations which previously were measured in the isolated rat kidney.This work was supported by Grant TAG-229 of Turkish Scientific and Technical Research Council (T.B.T.A.K.).     

Spike propagation in the smooth muscle of the guinea-pig taenia coli

1. Spike activity was produced by external field stimulation of the guinea-pig taenia coli. Spikes were evoked by depolarization of the muscle membrane. Though the activity was usually also observed during hyperpolarization, this was shown to be conducted activity from the depolarized region of the tissue. Spike conduction was blocked when hyperpolarization exceeded 10 mV.2. The shape of the conducted spike was influenced by membrane polarization. Sometimes a notch appeared on the spike and sometimes the spike was split into two by hyperpolarization. This is probably due to the fact that functional bundles form a network and that the branches between bundles are more susceptible to the membrane polarization.3. There was a critical spike amplitude for normal propagation. Therefore, different spike amplitudes were observed near the stimulating electrode, but only spikes of nearly full size amplitude were recorded far from the stimulating electrode, i.e. at a distance of more than 5 mm.4. When repetitive stimulation was applied, the spike amplitude decreased with increasing frequency of stimulation. No steady level was reached, however, but the spike amplitude fluctuated at about 0.3 c/s.     

Diffusion and distribution of dimethyl sulphoxide in the isolated guinea-pig taenia coli

1. The diffusion of the cryoprotective non-electrolyte dimethyl sulphoxide (DMSO) in the isolated guinea-pig taenia coli at 37, 25 and 0 degrees C has been studied using [(35)S]DMSO.2. Within 1 hr after immersion at 37 degrees C in Krebs solution containing 20% (w/v) DMSO and trace amounts of [(35)S]DMSO, the non-electrolyte was distributed uniformly throughout a volume equivalent to the total initial water content of the muscle.3. The kinetics of efflux of [(35)S]DMSO from muscles at constant volume were analysed on the basis of two models: one incorporated radial diffusion in extracellular fluid with simultaneous permeation into the cells, the other involved only radial diffusion in homogeneous cylinders of tissue having no internal barriers to diffusion; the former was found to give a better representation of the efflux kinetics.4. If it was assumed that the rate of diffusion of DMSO in the extracellular space of taenia coli was the same as that in the bathing medium, the values of the extracellular space and the permeability of smooth muscle to DMSO, obtained from the analysis of the efflux kinetics, were 454 +/- 19 ml./kg and 2.36 +/- 0.05 x 10(-6) cm sec(-1) at 37 degrees C.5. The activation energy for the transfer of DMSO across the surface of the cell was estimated to be 6.0 kcal/mole at 37 degrees C, 6.6 kcal/mole at 25 degrees C and 11.6 kcal/mole at 0 degrees C, indicating either that the equivalent pore radius of the cells decreased with temperature or that the cell permeability represented the sum of two fluxes, one through the aqueous pores of the cell and the other through the lipid phase of the cell membrane, each with a different energy of activation.6. A net flux of water across the surface of the cells, superimposed on the efflux of DMSO, markedly affected the rate of diffusion of the non-electrolyte out of the whole tissue; however, it was considered that an analysis of the efflux kinetics was not possible under these conditions.7. These results provide a basis for methods which will be used to investigate the possibility of preserving tissue in unfrozen aqueous media at sub-zero temperatures.     

Conductance change during the inhibitory potential in the guinea-pig taenia coli

1. Effects of membrane polarization and of reduction in external K and Cl concentration on the inhibitory potential were investigated in the guinea-pig taenia coli.2. Depolarization of the membrane increased the inhibitory potential while hyperpolarization decreased it. The relationship between the degree of membrane polarization and the amplitude of inhibitory potential was linear. The inhibitory potential was abolished or slightly reversed in polarity, when the membrane was hyperpolarized by 25-40 mV in different preparations.3. Removal of external K ion depolarized the membrane for about 5 min and increased the inhibitory potential more than could be accounted for by the depolarization. Readmission of K transiently hyperpolarized the membrane, probably due to an activation of the Na-K pump, and reduced the inhibitory potential, but no reversal of polarity in the inhibitory potential was observed during this hyperpolarizing phase.4. The membrane was transiently depolarized when the external Cl concentration was reduced by substituting with isethionate. Hyperpolarization was produced by restoring the external Cl concentration to normal. Changes in the amplitude of inhibitory potentials during alterations in Cl concentration occurred as expected from the shift of the membrane potential.5. From the results, it is concluded that the membrane conductance is increased during the inhibitory potential, and that an increase in the K permeability is the main factor for hyperpolarization of the membrane.     

Oxygen consumption by the isolated smooth muscle of guinea-pig taenia coli

1. An apparatus is described for simultaneous measurement of oxygen consumption and electrical and mechanical activity of isolated smooth muscle preparations.

2. The mean oxygen uptake by the isolated taenia coli of the guinea-pig was 10-20 μl./g/min.

3. In spontaneously active preparations, adrenaline (10^-8-10^-7 g/ml.) caused, with the inhibition of electrical and mechanical activity, a reduction in oxygen uptake.

4. After prolonged exposure to substrate free solution spontaneous activity ceased periodically. Adrenaline, when applied during a silent period, had no detectable effect on resting oxygen consumption, while readmission of substrate, either glucose or β-hydroxybutyrate, increased oxygen uptake.

5. Adrenaline did not modify the increased oxygen uptake during the initial recovery period when it was given simultaneously with the substrate. However, adrenaline shortened the time interval which elapsed from the addition of substrate until spontaneous activity was resumed, indicating an acceleration of the recovery process.

     

The calcium content of the smooth muscle of the guinea-pig taenia coli

1. The in vitro calcium content of the smooth muscle of the guinea-pig taenia coli was 3.0 m-mole Ca/kg wet wt. when phosphate was omitted from the bathing medium, and was almost independent of pH changes in the range 6.7-7.6.2. The calcium content was not changed when 1 mM phosphate was included in the medium, if the pH was 6.7 or 7.0. However, when the pH was 7.6, the calcium content increased by 1.5 m-mole Ca/kg wet wt. in the presence of phosphate.3. The calcium content rose by 1.1 m-mole Ca/kg wet wt. when NaCl in the bathing medium was replaced by isotonic sucrose, and rose by 0.7 m-mole Ca/kg wet wt. when MgCl(2) in the bathing medium was replaced. These increases may reflect a competition between Ca(2+) and other cations for fixed negative sites in the tissue.4. The initial rapid phase of (42)K exchange corresponded to an ;extra-cellular (42)K-space' of 470 ml./kg fresh wt. in normal solution, rising to 560 ml./kg. fresh wt. in low-sodium solution and to 760 ml./kg fresh wt. in calcium-free low-sodium solution. In this last medium the extra-cellular [(14)C]sorbitol space was only 390 ml./kg fresh wt., so that there was a large excess of rapidly-exchanging potassium which may have been competing at fixed negative sites.     

The extracellular space of the smooth muscle of the guinea-pig taenia coli

1. The measured extracellular space of the taenia coli is large when small ions or molecules are used for the determination, and small when large molecules are used, even with identical experimental procedures.

2. Extracellular hyaluronic acid has been detected histologically. It is apparently reduced by hyaluronidase.

3. The extracellular inulin space increases after the tissue has been pretreated with hyaluronidase, although the ionic composition and wet weight are unchanged.

4. It is suggested that the hyaluronic acid prevents the free entry of macromolecules such as inulin into the extracellular space by steric hindrance. Monatomic ions such as Na⁺, Li⁺, Cl^-, Ca²⁺ therefore have a larger extracellular space available than is calculated on the basis of the inulin space.

5. A slight shrinkage of the muscle cells can be detected when the incubation period is prolonged.

     

Impedance components in longitudinal direction in the guinea-pig taenia coli.

1. When the tissue impedance of the guinea-pig taenia coli was measured across a 2 mm sucrose gap in a longitudinal direction, the impedance locus could be fitted by two different circular arcs. Their characteristic frequencies were about 0-6 and 240 Hz after 60 min superfusion with sucrose solution. From the effects of changing the width of sucrose gap and of transection of tissue, and also from taking the difference between impedances measured at two distances, it was concluded that the low-frequency locus corresponds to the transverse impedance of the plasma membrane and the high-frequency locus to the longitudinal tissue impedance. 2. A change in the longitudinal tissue impedance was measured during superfusion with sucrose solution, using a frequency range between 5 Hz and 10 kHz. The admittance decreased with time of superfusion and this time course could be expressed by the sum of three exponential terms. The fastest component, having a time constant of 1-3 min at 10 kHz, was interpreted to correspond to a process of wash-out of extracellular medium. 3. Admittances at zero and infinite frequencies were obtained from the impedance locus. The decrease in these admittances with the time was analysed and the values at the start of washing were obtained by extrapolating the admittance change to zero time. 4. From these values it was estimated that the myoplasmic resistance was 214 omega cm, the junctional resistance 372 omega cm, and the junctional capacity 3-1 muF/cm at 25 degrees C. In these calculations the equivalent circuit of tissue was assumed to be expressed by two components in series: one for the myoplasmic resistance and the other for the junction which has the junctional resistance and capacity in parallel. 5. After 90 min superfusion with sucrose solution, the total tissue impedance measured at zero frequency was increased from 586 to 3034 omega cm. In the total impedance the myoplasmic resistance was increased from 214 to 914 omega cm and the junctional resistance from 372 to 2120 omega cm. Thus, the change in junctional resistance was greater than that in myoplasmic resistance during superfusion of sucrose solution.