Only a small amount of cations is lost from the cells. A considerable. (Received 5 September 1967)

Transcription

1 J. Physiol. (1968), 195, pp With 5 text-figures Printed in Great Britain THE EFFECT OF HYPO- AND HYPERTONIC SOLUTIONS ON VOLUME AND ION DISTRIBUTION OF SMOOTH MUSCLE OF GUINEA-PIG TAENIA COLI BY ALISON F. BRADING AND J. SETEKLEIV* From the Department of Pharmacology, University of Oxford (Received 5 September 1967) SUMMARY 1. The intra- and extracellular spaces and ionic content of the taenia coli of the guinea-pig have been measured in a series of bathing solutions in which the tonicity varied from 0.5 to 3 times the tonicity of the normal Krebs solution. 2. Equilibrium of the tissue in the experimental solution is reached within about 30 min. 3. The absolute values of the parameters measured have been shown to depend on the blotting technique used. These differences were eliminated by expressing the results as a percentage of the values found in the normal Krebs solution. 4. In hypertonic solutions the cell behaves as a perfect osmometer, the cell volume changing in proportion to the tonicity of the bathing medium. Only a small amount of cations is lost from the cells. A considerable amount of chloride is lost, making the postulation of its replacement by some other anions necessary in order to maintain electroneutrality of the intracellular solution. 5. In hypotonic solutions the cells do not behave as predicted for a perfect osmometer. In 0 5 hypotonic solution an actual decrease in cell volume was observed associated with an increase of the extracellular space probably due to penetration of [14C]sorbitol into the cell. The intracellular ionic concentration was decreased. These findings suggest damage of the cell membrane. 6. The observed hyperpolarization of the membrane in hypertonic solution can be explained by the increased intracellular potassium concentration. * Wellcome Trust Research Fellow. Present address: Department of Pharmacology, University of Oslo, Blindern, Oslo, Norway.

2 108 ALISON F. BRADING AND J. SETEKLEIV INTRODUCTION The guinea-pig taenia coli muscle exhibits rhythmic electrical and mechanical activity under normal experimental conditions (Biulbring, 1955). Sustained intracellular recordings of the membrane potential under these conditions are difficult, because the mechanical activity tends to dislodge the electrode. Tomita (1966) has found that a Krebs solution made twice hypertonic with sucrose hyperpolarizes the membrane below threshold for spike activity, and also reduces the mechanical response of the muscle to applied electrical stimuli, although the membrane properties appear to be almost unchanged and normal spike activity can be restored by depolarization with applied current or high potassium. In striated muscle where effects of tonicity have been studied in some detail (Hodgkin & Horowicz, 1957; Howarth, 1958; Dydynska & Wilkie, 1963; Blinks, 1965), hypertonic solutions are known to produce large changes in volume and mechanical properties of the cells. In smooth muscle of frog stomach various osmotic phenomena have been investigated by Bozler (1959, 1961, 1962, 1965), but these experiments were carried out under abnormal ionic conditions. Barr, Dewey & Evans (1965) investigated the effect of hypertonic solution on the nexus and spike propagation in guinea-pig taenia coli. No systematic study has been made of the simultaneous changes in tissue volume, extracellular and intracellular spaces and electrolyte distribution in a range of different tonicities. Such a study has been the object of the present investigation, which was undertaken in the hope that the results would help to elucidate the effect of hypertonic solution on the electrical and mechanical activity in the taenia coli of the guinea-pig. METHODS Preparation. White guinea-pigs were stunned and bled, and eight pieces of taenia coli between 10 and 20 mg wt. were dissected quickly from the caecum, weighed on a torsion balance to determine the fresh weight and mounted on an adjustable stainless steel holder. In order to ensure that pieces were under similar tension, the length was adjusted to give a ratio of weight (mg)/length (mm) of about 1. After dissection, all pieces were left to equilibrate in normal solution at 350 C for at least 1 hr. Solutions. The normal Krebs solution used in all experiments was modified Krebs solution prepared from isotonic stock solutions. It contained (mm) Na , K+ 5-9, Ca2+ 2-5, Mg2+ 1-2, HC , H2P04-1-2, Cl and glucose 11-5, equilibrated with a gas mixture of 3 % CO2 and 97 % 02. Hypertonic solutions were made by adding solid sucrose to the normal solution. A solution of 10 g sucrose/100 ml. is isosmotic to the normal solution: 2-5, 5, 10, and 20 g of sucrose were added to 100 ml. of the normal Krebs to make solutions of approximately 1-25, 1-5, 2 and 3 times normal tonicity. The addition of sucrose leads to some dilution of the Krebs solution especially at the higher tonicities, and this has been corrected in our calculations (10 g/100 ml. increases the volume by about 6 %/'). Hypotonic solutions were made by diluting the normal Krebs with distilled water to which had been

3 OSMOTIC PROPERTIES OF SMOOTH MUSCLE 109 added the appropriate amount of CaC12, to maintain the normal calcium concentration, since calcium is known to be important in maintaining membrane permeability. Experimental procedure. After equilibration in normal Krebs solution, pieces of taenia coli were transferred at intervals to the experimental solutions. At least one piece from each guinea-pigremained in the normal solution as control. The fast phase of uptake of sorbitol by the tissue in normal solution is completed within 10 min (Goodford & Leach, 1966). Experiments with sorbitol uptake in other tonicities have confirmed that the phase is completed within this time over the range of solutions used, and so, to estimate the extracellular space, each piece was transferred to an ionically identical solution containing [14C]sorbitol for the last 10 min of exposure to the experimental solution. The tissues were removed from the holders, blotted and reweighed to determine the wet weight. For extracellular space analysis the radioactivity was extracted in distilled water for 12 hr, and a small sample taken for the estimation of 14C by liquid scintillating counting. The ionic content was determined by flame photometry, using the techniques described by Casteels & Kuriyama (1965). From the results the wet weight and extracellular space were calculated as a percentage of the fresh weight of the tissue. By subtraction, the intracellular space was determined, and the ionic contents expressed with relation to any of these three parameters, as appropriate. Estimations of the cell water in these particular samples have not been made, but can easily be calculated, since the dry weight was consistently found to be between 16 % and 17 % of the fresh weight. Blotting techniques. Two methods of blotting were used in these experiments: one by placing the muscle on filter paper, gently blotting with another piece of paper, moving the muscle and repeating the blotting; and the other was by dragging the muscle across black Perspex until a wet trail was no longer seen. In one series, two experiments were performed in which the procedures were identical except for the blotting technique applied, and the comparison of these two methods is described in the results. RESULTS Time taken to reach equilibrium. Figure 1 shows results of an experiment carried out in two times hypertonic Krebs solution. Pieces were left in the experimental solution for a range of times up to 2 hr. The blotting was done by the filter-paper method. The ionic content of the whole tissue is expressed as m-moles/kg fresh wt., to show the actual loss of ions from the tissue, and also as m-moles/kg wet wt., which gives an indication of the changes in intracellular concentration of the ions. Figure 1A shows that the tissue achieved a constant weight in under 30 min, and Figs. 1B, C and D show that a constant ionic content was also achieved within this time. Other experiments carried out in solutions of different tonicity confirmed that equilibrium was reached within half an hour. Presentation of results. Table 1 shows the results of two experiments performed under identical conditions except for the blotting techniques employed. It can be seen that there are very considerable differences between the results obtained with the two methods of blotting, although the calculated intracellular spaces are comparable. However, if the results are expressed as a percentage of the control muscle (from the same guineapig), a reasonably reliable estimation of the changes that take place under experimental conditions can be made with either technique. For this reason

4 110 ALISON F. BRADING AND J. SETEKLEIV -4. >0 to ;t k A - -i- - I i C B Ill _ne-a liii -B _ I..,I_ - -AIl IIl ti I III Minutes I I I 1 I I I I I I Minutes C 90 _ D C; _~~~ is/ T C5 0 u i a Minutes Minutes Fig. 1. Time course of equilibration in Krebs solution made 2 times hypertonic with added sucrose. A, Change in weight of tissue; B, change in sodium concentration; C, change in potassium concentration; D, change in chloride concentration. *, A, *: ion concentration, expressed as m-moles/kg fresh wt. of tissue. 0, EO, ion concentration, expressed as m-moles/kg wet wt. of tissue. Blotting Normal Krebs Stroking Normal Krebs Blotting 2 x tonicity Stroking 2 x tonicity Blotting 2 x tonicity Stroking 2 x tonicity TABLE 1. Comparison of blotting technique % fresh weight Extra- Intra- m-m4 ileslkg fresh wt. cellular cellular e.c.s. Wet wt. space space i.c.s. C- Na+ K Results expressed as % normal Krebs C) c ?-45

5 OSMOTIC PROPERTIES OF SMOOTH MUSCLE 111 subsequent results are expressed as a percentage of the value in normal Krebs solution, and the mean value obtained in the normal solution is given with each graph + standard error, with the number of experiments 110 A ; 90_ g 4-80 ~~~~~ I I I I I I I I Tonicity B 120 { 110 _ 10 o > I I I I I I I I l I I Q Tonicity Fig. 2. Change in weight (A) and extracellular space (B) of tissue in hypo- and hypertonic solutions. Ordinate: values expressed as a percentage of the value in normal Krebs solution. Abscissae: tonicity expressed with the normal Krebs solution taken as 1. Mean normal wet weight: (25) % fresh weight. Mean normal extracellular space: (9) % fresh weight. in parentheses. As a result, the graphs illustrate the relative magnitude of changes induced by exposure to various tonicities, rather than absolute values. Effect of tonicity on wet weight. Since the tissue reaches a steady state within half an hour of exposure to hypertonic solutions (Fig. 1), this period was used throughout. Figure 2 A shows the results of several experi-

6 112 ALISON F. BRADING AND J. SETEKLEIV ments. The wet weight has been plotted as a percentage of the fresh weight in normal solution. In hypertonic solution up to 2 times tonicity the tissue weight decreased. In 3 times hypertonic solution the tissue appeared to be slightly heavier, but that may be due to the weight of sucrose in the extracellular space, which has not been corrected for since \ _ ~~~~~10\ i8~~~~~ Tonicity Fig. 3. The influence of tonicity on the intracellular space. The points plotted are values obtained by subtraction of the extracellular space and weight of sucrose from the wet weight of the tissue. The continuous line is drawn from Ponder's equation for a perfect osmometer: V = W[(1/T) - 1)]+ 100, where V = intracellular space, T = tonicity and W = cell water (taken as 74 % of the fresh weight). Ordinate: intracellular space expressed as a percentage of the value in normal solution. Abscissa: tonicity expressed with the normal Krebs solution taken as 1. Mean normal intracellular space (9) % fresh weight. the extracellular space was not always measured in these experiments. Surprisingly little increase in tissue weight was observed in hypotonic solution. Effects of tonicity on extracellular space. The extracellular space decreased slightly when the tissue was placed in 1x25 hypertonic solution but showed little further change on increasing tonicity (Fig. 2B). A decrease was also produced in hypotonic solution by reducing the tonicity to 075. Further decrease in tonicity resulted in a large increase in the measured extracellular space, but the scatter of these values was great, indicating that the tissue was damaged, allowing penetration of [14C]sorbital into the cells. Effects of tonicity on intracellular space. The smooth-muscle cells shrank in hypertonic solution in proportion to the tonicity of the bathing solution (Fig. 3). In hypotonic solution the cells swelled when the tonicity was

7 OSMOTIC PROPERTIES OF SMOOTH MUSCLE 113 lowered to 075. At half tonicity the values obtained for the intracellular space appeared to be less. This is again probably largely due to damage of the cell membrane. The continuous line in Fig. 3 (which shows the changes which would be expected if the cells behaved as simple osmotic bags filled with solutions of a fixed amount of ideal solute) is calculated from Ponder's equation 100 o / Tonicity Fig. 4. The influence of tonicity on tissue ionic content. Ordinate: concentrations expressed as a percentage of the concentrations found in normal Krebs. Abscissa: tonicity expressed with that of normal Krebs taken as 1. The mean values for tissue ionic concentrations in normal solution were: sodium, *, (9) m-moles/kg fresh wt.; potassium, A, (9) m-moles/kg fresh wt.; chloride, *, (9) m-moles/kg fresh wt. V = W[(1/T) - 1] + 100, where V = cell volume (intracellular space)(100 when T = 1), W = cell water (74 % cell volume) and T = tonicity. There is good agreement between the experimental values and those calculated from Ponder's equation. Effect of tonicity on ionic concentration. The total tissue concentrations of sodium, potassium and chloride ions (m-moles/kg fresh wt.) in different tonicities of bathing solution is shown in Fig. 4. Any change in tonicity was accompanied by a decrease in total tissue ions: the greatest change occurred in chloride and the least in potassium. Since these changes also include ions gained or lost in the extracellular space, the values have been recalculated to show the concentration in the cells (Fig. 5). The continuous lines indicate the expected concentrations that would be found if no ions were lost from the cells. In hypotonic solution a small amount of potassium and chloride was 8 Physiol. I95

8 114 ALISON F. BRADING AND J. SETEKLEIV lost at 0 75 tonicity, but at 0-5 tonicity a considerable amount of all three ions was lost, indicating an increased permeability of the cell membrane. Hypertonicity caused an increase in the intracellular concentration of sodium and potassium although at higher tonicities the increase was c-., A,. A A j _. U * _ h Tonicity Fig. 5. Figure showing the expected intracellular concentration of ions, assuming no loss from the cells (continuous line), and the observed concentrations in hypoand hypertonic solutions. A, Potassium concentration m-moles/kg intracellular space; *, chloride concentration m-moles/kg intracellular space; 0, sodium concentration m-moles/kg intracellular space. smaller than expected, indicating again a loss of ions from the intracellular space. The chloride concentration, however, always decreased when the tonicity was changed. At 2 times normal tonicity the loss of sodium + potassium from the intracellular space was 24 m-moles/kg while the loss of chloride was 44 m-moles/kg intracellular space.

9 OSMOTIC PROPERTIES OF SMOOTH MUSCLE 115 DISCUSSION Volume changes. The result of the experiments measuring the intracellular volume of the smooth muscle cells indicates that over a wide range of tonicity they behave in a manner similar to that expected by a perfect osmometer. A similar conclusion has also been reached by Dydynska & Wilkie (1963) and Blinks (1965) for amphibian skeletal muscle which has been shown to exhibit ideal osmotic behaviour over a very wide range of osmotic strengths. In most cell systems in which osmotic behaviour has been studied, when the cell volume is plotted against 1/1T (r being the osmotic pressure of the bathing solution) a straight-line relationship is observed and the intercept, where 1/pr = 0, gives a value for the non-solvent volume of the cell (Dick, 1965). It has usually been foundthat this volume is larger than that measured directly by comparison with wet and dry weights, and this discrepancy has been expressed by Ponder as a ratio which he designates by the letter R (Ponder, 1948). In taenia coli the osmotically inactive fraction of the intracellular space appears to be equal to the dry weight, and thus the value for R is approximately unity. This figure assumes the whole dry weight to be derived from the cells, but if there is a significant amount of extracellular dry weight the figure is too high, and therefore the R value will be less than unity. When the tonicity of the bathing solution is reduced to three-quarters of the original level, the cells of the taenia coli swell, but not quite to the extent predicted by Ponder's equation. This may be due to inextensibility of the cell membrane. In solutions of half the tonicity, however, there is an actual loss of cell water, which may be explained by damage to the cell membrane associated with loss of salts. Bozler (1965) examined the osmotic properties of amphibian sartorius, heart and stomach muscle and obtained similar results. He found that, in solutions made hypertonic with added NaCl, all three muscles shrank in proportion to the tonicity of the solution, but in hypotonic solutions the stomach and heart muscles swelled less than would be expected for an osmometer, and also suffered a certain loss of intracellular electrolyte. Ionic distribution. Although the behaviour of the tissue indicates that, in hypertonic solutions, water is lost according to simple osmotic laws, yet a study of the sodium potassium and chloride content of the tissue brings to light some additional interesting phenomena. Measurements of the ionic content of the whole tissue indicate a loss of all three ions (see Fig. 4). Some of the ion loss can be accounted for by the reduction of the extracellular space, but a comparison of the intracellular ion concentration with the expected concentration (Fig. 5) shows that there is still a loss of sodium 8-2

10 116 ALISON F. BRADING AND J. SETEKLEIV and potassium from the cells at the higher tonicities. This might be caused to some extent by the dilution of the ionic concentration of the Krebs solution that occurs on addition of solid sucrose. The chloride concentration of the intracellular space is, however, considerably less than predicted, indicating a significant loss of this ion. At 2 times tonicity, the loss of sodium + potassium from the cells is 24 m-moles, while the loss of chloride is 44 m-moles/kg intracellular space. The loss of chloride in the taenia must presumably be compensated for, in order to maintain electroneutrality in the intracellular fluid, and this would imply either the loss of some other cation or gain of some intracellular anion. A similar problem was encountered by Casteels (1965), who found that in solutions in which chloride was replaced by isotonic ethanesulphonate in the external medium the cells shrank by 10 % and lost chloride, but there was no loss of intracellular cations. The penetration of ethanesulphonate into the cell was insufficient to compensate for the chloride lost. This author postulates that the chloride is replaced by an anion, probably bicarbonate, produced by intracellular metabolism. Exchanges of chloride with intracellular bicarbonate are known to occur in red blood cells associated with cell volume changes and ph changes (Roughton, 1935). It has recently been established (Vijai & Foster, 1967) that in bovine plasma albumin about 40 % of the carboxylate groups on the protein are masked in the normal condition. Changes in ph or ionic strength can unmask these groups, and thus effectively increase the negative charges on the protein. If similar masked groups occur in the muscle cell proteins, increase in intracellular ionic strength might unmask these groups and increase the intracellular fixed anions. This in turn could lead to a loss of intracellular chloride to maintain electroneutrality. ph changes have also been observed to occur in smooth muscle cells during contraction (Dubuisson 1939). Figure 1 B-D shows the time course of change in ionic concentration in 2 times tonicity. Because the change in extracellular space is relatively small, the concentrations of ions expressed as m-moles/kg wet wt. reflect mainly the change in intracellular concentration. Initially the sodium, potassium and chloride concentrations all increase, presumably because water is lost from the cells but, whereas the sodium and potassium concentrations soon reach a steady level, the chloride concentration shows a decrease as if redistribution of chloride is taking place separately from the water movement. This redistribution could be due to unmasking of anionic sites on intracellular protein, resulting in an excess of anions. Some chloride loss might also be expected from the hyperpolarization that is known to occur in hypertonic solution, if the distribution of chlorides is determined by the electrical potential across the membranes.

11 OSMOTIC PROPERTIES OF SMOOTH MUSCLE 117 Electrical and mechanical behaviour. The hyperpolarization of the cell membrane can be explained by the increase in intracellular potassium concentration. In normal solution the cell water takes up about 74 % of the intracellular space, and at 2 times tonicity about 59 %. If the potassium is all in the cell water, then (using the values shown in Fig. 5) the concentration of potassium in the cell water will be 150 mm at normal tonicity and 278 mm at 2 times tonicity. The EK values will be 85*7 and 102 mv respectively. The actual hyperpolarization observed is about 12 mv (51-63 mv) (Tomita, 1966). The extracellular space of the taenia coli was found to shrink in hypoas well as in hypertonic solution. Similar findings were made in frog smooth muscle by Bozler (1965). The fact that the extracellular space shrinks in hypertonic solution indicates that there is little or no fixed arrangement of the cells in the tissue. In hypertonic solution the tissue shrinks and becomes increasingly rigid and inextensible. This phenomenon has also been observed in skeletal muscle (Howarth, 1958). Contractile proteins are known to be sensitive to ionic strength, and this is used in procedures for their extraction (Portzehl, 1951). Actin, for instance, can be extracted with water from muscle in which the myosin has previously been removed. The actin solution undergoes a considerable increase in viscosity when the ph is decreased or ionic strength increased (Davson, 1964). It may well be that the increased intracellular ionic strength observed in the present experiments with taenia coli in hypertonic solution could in some way affect the contractile proteins and account for both the decreased extensibility and the reduced contractile activity observed on stimulating the muscle. We would like to thank Dr Edith Bulbring for her help and encouragement, Professor W. D. M. Paton for his hospitality in the laboratory, and the Medical Research Council for a grant for one of us. REFERENCES BARR, L., DEWEY, M. M. & EVANS, H. (1965). The role of the nexus in the propagation of action potentials of cardiac and smooth muscle. Fedn Proc. 24, 142. BLINKS, J. R. (1965). Influence of osmotic strength on cross section and volume of isolated single muscle fibres. J. Physiol. 177, BOZLER, E. (1959). Osmotic effects and diffusion of nonelectrolytes in muscle. Am. J. Physiol. 197, BOZLER, E. (1961). Electrolytes and osmotic balance of muscle in solutions ofnonelectrolytes. Am. J. Physiol. 200, BOZLER, E. (1962). Osmotic phenomena in smooth muscle. Am. J. Physiol. 203, BOZLER, E. (1965). Osmotic properties of amphibian muscles. J. gen. Physiol BULBRING, E. (1955). The correlation between the membrane potential, spike discharge and tension in smooth muscle. J. Physiol. 128, CASTEELS, R. (1965). The relation between the membrane potential and the ion content of smooth muscle cells. Ph.D. Thesis, Oxford.

12 118 ALISON F. BRADING AND J. SETEKLEIV CASTEELS, R. & KURIYAMA, H. (1965). Membrane potential and ionic content in pregnant and nonpregnant rat myometrium. J. Physiol. 177, DAVSON, H. (1964). A Text Book of General Physiology, 3rd edn., p London: Churchill. DICK, D. A. T. (1965). Cell Water, pp London: Butterworths. DuBuIssoN, M. (1939). Studies on the chemical processes which occur in muscle before, during and after contraction. J. Physiol. 94, DYDYNSKA, MARIA & WILKIE, D. R. (1963). The osmotic properties of striated muscle fibres in hypertonic solution. J. Physiol. 169, GOODFORD, P. J. & LEACH, E. H. (1966). The extracellular space of the smooth muscle of the guinea-pig taenia coli. J. Physiol. 186, HODGKIN, A. L. & HoRoWICZ, P. (1957). The differential action ofhypertonic solutions on the twitch and action potential of a muscle fibre. J. Physiol. 136, 17-18P. HOWARTH, J. V. (1958). The behaviour of frog muscle in hypertonic solutions. J. Physiol. 144, PONDER, E. (1948). Hemolysis and Related Phenomena. London: Churchill. PORTZEHL, H. (1951). Muskelkontraktion and modellkontraktion. II. Z. Naturf. 6b, ROUGHTON, F. J. W. (1935). Recent work on carbon dioxide transport by the blood. Physiol. Rev. 15, TOMITA, T. (1966). Electrical responses of smooth muscle to external stimulation in hypertonic solution. J. Physiol. 183, VIJAI, K. K. & FOSTER, J. F. (1967). The amphoteric behaviour of bovine plasma albumin. Evidence for masked carboxylate groups in the native protein. Biochemistry, N.Y. 6,