J. Phy8iol. (1975), 250, pp. 231-245 With 11 text-fitgurem Printed in Great Britain
231
EFFECT OF INTRACELLULAR INJECTION OF CALCIUM AND STRONTIUM ON CELL COMMUNICATION IN HEART
BY WALMOR C. DE MELLO From the Department of Pharmacology, Medical Sciences Campus, U.P.R. San Juan, P.R. 00963, U.S.A.
(Received 1 September 1974) SUMMARY
1. The influence of Ca and Sr on the electrical coupling of canine Purkinje cells was investigated by injecting the ions electrophoretically into the cytoplasm. 2. It was found that the intracellular injection produced electrical uncoupling which was spontaneously reversed. 3. No change in resting potential of the cell adjacent to the injection site was found except in those fibres not completely healed. 4. The input resistance of the injected cell increased concomitantly with the establishment of the electrical uncoupling. 5. Caffeine (6 mM), added to the extracellular fluid, reduced the rate of spontaneous recoupling. Reduction of temperature of the Tyrode solution had the same effect. 6. The abolition of cell communication produced by Ca injection seems to indicate that the ion plays an important role in the control of junctional conductance in heart fibres. INTRODUCTION
Electron microscopic studies have shown that cardiac cells are not organized as an anatomical syncytium but are completely isolated. The areas of cell to cell contact are, however, characterized by the presence of differentiated structures - the so-called intercalated disks (Spiro & Sonnenblick, 1964). Electrophysiological observation led to the conclusion that heart cells are electrically connected. The studies of Woodbury & Crill (1961), in rat atrium, indicated that the propagation of action potentials, in this tissue, requires an extremely low resistance of the intercellular junctions. On the other hand, the low longitudinal core resistance found in Purkinje fibres (Weidmann, 1952) and the evidence that radiopotassium can diffuse across the intercalated disks (Weidmann, 1966) support the view that heart cells are, indeed, connected through low resistance pathways.
232 232 ~~WALMOR C. DE MELLO It is known that Ca is essential for the maintenance of the integrity of the intercellular 'cement' in many tissues (Chambers, 1940). In heart muscle, calcium ions also play an important role in maintaining the integrity of the intercellular junctions (De Mello, Motta & Chapeau, 1969) and are essential for the quick recovery of resting potential which occurs after damage (healing-over; De'leze, 1965; De Mello et al. 1969). Indirect evidence has been described which indicates that the influence of calcium on the healing-over process of cardiac muscle could be related to its effect on junctional conductance (De Mello et al. 1969; iDe Mello & Dexter, 1970, iDe Mello, 1972 a). Searching for more information on this area, we thought important to investigate the role of calcium and strontium ions on the electrical coupling of heart cells by injecting the ions electrophoretically into the cell. The detailed results of this study, previously reported in abstracts (De Mello, 1972b, 1974), are described in this paper. METHODS
Dogs were anaesthetized with sodium pentobarbitone and the heart was immnediately removed and immersed in cooled Tyrode solution. Strands of Purkinje fibres (about 15 mmn in length) were excised from the left ventricle and transferred to a transparent Plexiglass chamber through which Tyrode solution at 340 C, saturated with a mixture of 95 %02+5% C02, was flowed continuously. The fibres were pinned on a thin layer of silicone rubber to the bottom of the chamber. The preparations were transilluminated and observed under a stereoscopic microscope. In some experiments, single Purkinje cells were visualized by treating the preparation with toluidine blue (10 mg/I.) until the nucleus became visible. In these experiments a water immersion objective was used and the micro-electrodes were impaled horizontally at the edge of the bundle. This procedure, kindly suggested to us by Professor Weidmann, made it possible to record the voltage change and to inject ions near the nucleus of contiguous cells. Solution. The Tyrode solution had the following composition (mm): NaCl 137; KCl 5-4; NaHCO, 12; MgCl2 0-5; NaH2PO4 3-6; dextrose, 5-5; CaCl2 2-7. In some experiments, the Ca was replaced by Sr isosmotically. When caffeine was used a stock solution of the drug was prepared immediately before the experiment and added to Tyrode solution to give the final concentration. All the solutions were prepared with demineralized distilled water which was used also to wash the glassware. pH of all solutions was 7. Electrical recording. Measurements of membrane potential were made with conventional KCl micro-electrodes. After inserting two micro-electrodes in adjacent Purkinje cells visualized by the method described above, the degree of electrical coupling was determined by injecting rectangular current pulses (40-100 msec in duration) delivered by an electronic stimulator and isolation unit through one of the micropipettes and recording the resulting voltage drop with the other micro-electrode (Fatt & Katz, 1951). The voltage recording micro-electrode was connected to a standard cathode follower and DC amplifier and displayed simultaneously with voltage on the second beam of the oscilloscope. Measurements of the input resistance of single Purkinje cells were made with a single 3 m-KCl micro-electrode connected to a balance bridge circuit used to pass
Ca INJECTION AND CELL UNCOUPLING IN HEART 233 current and record the voltage drop. On these experiments, a precision electrometer (Model M-4) from W-P Instruments, Inc. was used. Experimental procedure of balancing circuit. While the micro-electrode tip was in contact with the Tyrode solution rectangular current pulses of 40 msec duration and of reasonable intensity were sent to the bridge. A trial and error method was used to balance the circuit so that no square-wave deflexion could be detected on the oscilloscope. At the end of each experiment the micro-electrode was withdrawn and the balance was checked again; if the circuit was unbalanced the experiment was rejected. The electrometer used provided direct reading of electrode resistance from the balanced control when the balance was fully achieved. Ca injection. Ca was injected electrophoretically into the cell using the techniques of Nastuk (1953) and del Castillo & Katz (1955). Micro-electrodes were filled with 0*15 M-CaCl2 or with a mixture of 0*15 m-KCl and 0 15 M-CaCl2. To control the precise moment of Ca injection and to prevent the diffusion of Ca from the micropipette tip a steady and constant negative bias was applied to the interior of the micropipette. The ejection of Ca was then triggered by applying outward current pulses (40 msec duration, 4 cls) in which a positive coulomb quantity of calcium was moved from the pipette to the cell interior. The outward current pulses were monitored on the second beam of the oscilloscope. To control the position of the micro-electrode with respect to cell interior, inward current pulses were applied before and during impalement. -65
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-95
-65 > -75
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-
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> -75 _ -95 -
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Fig. 1. Electrical uncoupling and hyperpolarization produced by intracellular Ca injection on incompletely healed fibres. Top traces in A, B and C, show reduction in size of electrotonic potentials due to intracellular calcium injection into a neighbouring cell. Tracesaresequentialrecordsfrom the same preparation. Voltage scale at the left edge indicates values of resting potential. Current pulses in A, B and C, 1-8 x 10-7 A. Temperature, 340 C. IO
P HY 250
WALMOR C. DE MELLO
234
RESULTS
Influence of intracellular calcium injection on electrical coupling The electrical coupling between adjacent Purkinje cells immersed in Tyrode solution at 360 C was largely decreased or even suppressed by injecting calcium intracellularly. As is shown in Fig. 1, injection of calcium gradually reduced the size of the electronic potentials recorded from an adjacent cell, leading to complete uncoupling. The time required for total interruption of cell communication was variable. In some experiments, as Ca"'injection 100
MOn
Off
~80 bO ~-60
0
*
40
LU
20 0L
0
20
40
60 80 100 120 Time (sec) Fig. 2. Partial uncoupling of heart cells caused by intracellular Ca injection and spontaneous re-establishment of cell communication after interruption of injection. Coupling of adjacent cells before Ca injection was taken as 100%. Temperature, 340 C.
in that illustrated in Fig. 1, about 5 min were necessary for complete abolition of cell communication while in others a shorter (2-3 min) or a longer time (8-12 min) was required. This discrepancy is probably explained by the variable location of the tip of the Ca micropipette in the cytoplasm (see Discussion). In many experiments the uncoupling produced by Ca injection was substantial but incomplete as is shown in Fig. 2 (average from four experiments). As can be seen in Fig. 1 the injection of Ca also raised the resting membrane potential of the neighbouring cell. This increment in membrane polarization was usually seen when the ion was injected between the cut
Ca INJECTION AND CELL UNCOUPLING IN HEART 235 end and the recording site in fibres that were partially depolarized (75 MV or less) by incomplete healing after the dissection procedure. In those fibres with good resting potentials (85-90 mV) the injection of Ca reduced the electrical coupling without increasing the resting potential and in some experiments a slight depolarization was found (Fig. 3). A 85
B
82 mV
80 mV
Fig. 3. Electrical uncoupling caused by intracellular Ca injection after complete healing. A, control; between A and B injection was started and almost complete uncoupling is shown in C. Resting potential is indicated at
left. Vertical calibration line, 5 mV; horizontal calibration, 120 msec. Inward current pulses (7 x 10-8 A), 40 msec duration. Temperature, 340 C.
Experiments performed with a single Ca micro-electrode connected to balance bridge circuit showed that, concomitantly with the fall in size of the electronic potentials recorded from cells adjacent to the site of Ca injection, the input resistance of the injected cell increased appreciably, as can be seen in Fig. 4. These results indicate that the decrease in amplitude of the electronic potentials recorded from cells located nearby the injection
a
IO-2
236 WALMOR C. DE MELLO site is not related to a fall in non-junctional membrane resistance of the injected cell. On the other hand, measurements of the input resistance from single Purkinje cells made with a single KCl micro-electrode showed that variations of the external calcium concentration from 12 mm to free Ca solutions for periods of 2-10 min had no effect on input resistance or on electrical coupling (Fig. 5A and B). Similar observations made by Loewenstein, Nakas & Socolar (1967) in salivary glands of Chironomus, indicated that the cell uncoupling produced by intracellular Ca injection was not related to a change in conductance of the non-junctional membrane. 8 AA A A
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Fig. 4. Increase of input membrane resistance (V/I) recorded from the same Purkinje cell to which Ca was injected intracellularly. Temperature, 340 C.
The impairment or total failure of cell communication caused by intracellular Ca injection was largely reversible. As can be seen in Figs. 2 and 6 (average from four experiments) as soon as the release of Ca from the micropipette was interrupted, the size of the electrotonic potentials, recorded from a nearby cell, gradually increasedin amplitude and cell communication was completely re-established. In those instances in which Ca injection reduced the electrical coupling by only 50-70 % (Fig. 2), the time required for complete recovery of cell communication was shorter (about 80-120 sec) than when total uncoupling was achieved. In this case, 10 min or more were usually necessary for complete recovery of electrical coupling (Fig. 6).
UNCOUPLING IN HEART
Ca INJECTION AND CELL 4
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238
WALMOR C. DE MELLO
Fig. 7. Influence of intracellular Sr injection on electrical coupling of Purkinje cells. A, control; between A and B injection was started. C, after 70 sec of Sr injection. Vertical calibration 8 mV; horizontal calibration, 300 msec. Inward current pulses 10-7 A in A. In B and C the intensity of current was slightly increased. Temperature, 340 C.
Influence of intracellular injection of strontium and magnesium on the electrical coupling It is known that Sr replaces Ca on the processes of healing-over (Escobar & De Mello, 1971) and cardiac contraction (Niedergerke & Harris, 1957). The effects of Ca injections on cell communication described above led us
Ca INJECTION AND CELL UNCOUPLING IN HEART 239 to investigate the influence of intracellular strontium injection on electrical coupling of Purkinje cells. The electrical interaction between normal cells was measured as described above before and after injection of Sr. Fig. 7 shows the influence of intracellular injection of Sr on cell communication. As can be seen the electrical coupling between adjacent Purkinje cells was substantially reduced by increasing the intracellular Sr concentration. As with Ca, the reduction of cell communication produced by injection of strontium was reversible (Fig. 8A) and the time course and rate of the recovery process SR2+ injection 100
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120
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140
Fig. 8. A, spontaneous recovery of cell communication previously depressed by intracellular Sr injection. B, lack of action of variation of the extracellular Sr concentration on input resistance of a single Purkinje cell. Preparation was exposed to Sr free solution. for 2-5 min. Similar results were obtained with 10 min of incubation. Temperature, 340 C.
were similar to that found after intracellular Ca injection. Control experiments in which the extracellular Sr concentration was changed from 12 to 0 (for 10 min) produced no variation in input resistance (Fig. 8B) which strongly suggests that the conductance of the non-junctional membrane did not vary. Magnesium, which does not replace Ca on the healing-over process of cardiac muscle (De Mello et al. 1969), had no influence on junctional conductance as judged from the lack of action of an intracellular injection of it on electrical coupling (Fig. 9).
WALMOR C. DE MELLO
240 1l00
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I
160
200
240
Fig. 9. Lack of action of intracellular Mg injection on electrical coupling of Purkinje cells. Inward current pulses used to inject Mg had the same intensity and duration described in Fig. 3. Temperature, 340 C.
On the nature of the reversibility of the electrical uncoupling The re-establishment of cell communication found after interruption of calcium injection is probably due to the operation of mechanisms which keep a low concentration of Ca in the cytoplasm namely (a) an active extrusion of Ca through the surface cell membrane; (b) the uptake of Ca by elements of the sarcoplasmic reticulum or by mitochondria. The possible contribution of sarcoplasmic reticulum to the re-establishment of cell communication previously abolished by Ca injection was investigated by exposing the preparation to caffeine, a drug which releases Ca from terminal cysternae and also inhibits its uptake by the same structures (Herz & Weber, 1965). In these experiments Ca was injected into the cell before and after treatment with caffeine (6 mM) and the time required for complete recovery of cell communication, after stopping the Ca injection, was measured in both situations. Since the time required for total re-establishment of electrical coupling was variable at different sites of injection, measurements of the recovery process were made keeping the micro-electrodes impaled into the same cells throughout the experiment. This was not an easy task considering the relatively long time required for such measurements. Fig. 10 is the average of four highly successful experiments. As is shown on this figure, the rate of recovery of cell communication, after interruption of Ca injection, was markedly reduced by caffeine. Control measurements showed that the drug has no influence on electrical coupling, at least under the conditions used on these experiments. It is also important to mention that in the presence of caffeine, suppression of cell communication by Ca injection was more easily obtained than in absence of the drug, which is probably related to the inhibitory action of the drug on the uptake of Ca by the sarcoplasmic reticulum and the accumulation of Ca in the cytoplasm.
Ca INJECTION AND CELL UNCOUPLING IN HEART 241 In view of the probable mechanisms involved in the re-establishment of cell communication after Ca injection, it seemed pertinent to investigate the influence of temperature on the rate of electrical re-coupling. For this, measurements of cell communication were made at 360 C and then the temperature was dropped to 200 C while keeping the electrodes inside the cell. After a few minutes of equilibration at 200 C the degree of coupling was again determined and Ca injected into the cell until suppression of cell communication achieved. The injection of Ca was then interrupted and the rate of recovery of electrical coupling measured. A 100
O.-
-
-
Control 80 -~~~~
Caffeine 60
LU
O
~~~~ 500 -Control ~~2 20
/10
0 0
200
Caffeine (6 mm)
6
10
A-.ATime (min)
400 600 Time (sec)
800
1000
Fig. 10. A, influence of caffeine (6 mm) added to the extracellular fluid on the rate of recovery of cell communication previously abolished by intracellular Ca injection. B, lack of action of the drug on the electrical coupling of adjacent Purkinje cells in absence of Ca injection. Temperature, 340 C.
The results obtained indicated that the reduction of temperature not only decreased the resting potential by about 8 mV but also reduced the amplitude of the electrotonic potentials. Control measurements of the input resistance performed with single micro-electrodes indicated that the drop in temperature caused an increase of input resistance (about 17 %) which is probably explained by the well known reduction in potassium conductance of the surface cell membrane (Carmeliet, 1961). Although previous studies of Coraboeuf & Weidmann (1954) in Purkinje fibres and of De Mello (1972c) in toad's ventricle indicated no apparent effect of temperature on electrical coupling, the observations described above suggest that the fall in amplitude of the electrotonic potentials
242 WALMOR C. DE MELLO found at 200 C could be related to a decrease in junctional conductance. Experiments made on salivary glands of Chironomus (Politoff, Socolar & Loewenstein, 1969) indicated, indeed, that cell communication in this tissue is largely dependent on temperature. The rate of recovery of cell communication previously blocked by Ca injection was also reduced at 200 C (Fig. 11). On these experiments the muscles were immersed in Tyrode solution at 200 C before Ca injection and kept at this temperature throughout the experiment. The 100 % value used in Fig. 11 indicates maximal coupling between adjacent Purkinje cells at 200 C. 200 C Ca2+ injections 100 'olo in Out
80
0
.E60 U0
40
-
~20
/
\/
0 0
200
400
600
800 1000 Time (sec)
1200
1400
1600
Fig. 11. Effect of low temperature (200 C) on the rate of spontaneous cell recoupling. Control measurements at 340 C (not included) indicated complete recoupling in about 10 min. DISCUSSION
The present results indicate that calcium plays an important role in cell communication in heart. The complete recovery of electrical coupling which occurs after interruption of calcium injection is probably related to the re-establishment of the low concentration of ionized calcium in the cytoplasm. Evidence has been provided, indeed, that Ca is actively taken by elements of the sarcoplasmic reticulum and that caffeine inhibits this uptake (Herz & Weber, 1965). The decrease on the rate of re-establishment of electrical coupling caused by caffeine could indicate that the uptake of Ca by the sarcoplasmic reticulum is involved in the recovery of electrical coupling found after interruption of Ca injection. The observation that
Ca INJECTION AND CELL UNCOUPLING IN HEART 243 uncoupling is more easily obtained by injecting calcium in preparations exposed to caffeine seems to support this view. The decrease in junctional conductance produced by Ca injection can be permanent if the calcium concentration is kept high at one side of the intercellular junction, as is the case when the fibre is damaged (De Mello et al. 1969). Similar conclusions were obtained from experiments performed in damaged salivary glands (Oliveira-Castro & Loewenstein, 1971). Under normal conditions, however, when the sarcoplasmic reticulum is working properly or calcium is being actively extruded through the surface cell membrane, the increase in concentration of ionized calcium in the cytoplasm is transient and long lasting changes in junctional conductance, in cardiac cells, will not occur. The decreased rate of recoupling found at 200 C is probably related to some impairment of the active Ca uptake by the sarcoplasmic reticulum. The variable delay between the initiation of Ca injection and the moment the electrical coupling starts to change was probably related to several factors. It is known, for instance, that the diffusion of Ca in the sarcoplasm is slower that in free solution (Niedergerke, 1957; Kushmerick & Podolsky, 1969). Slight differences of distance between the site of injection and the intercellular junction could produce large variations in the time delay. The binding of calcium by troponin or the presence of elements of the sarcoplasmic reticulum in the vicinity of the micro-electrode tip may also be important. These variables make it difficult to estimate the effective concentration of calcium that reaches the intercellular junctions. Since no change in transparency or a large fall on resting potential were produced by Ca injection, the possibility of an injurious effect of the injection seems unlikely. It is important to mention that in salivary glands of Chironomus, uncoupling can be produced by depolarizing the surface cell membrane (Socolar & Politoff, 1971) and the proposed mechanism for the uncoupling was the internal release of Ca due to the fall of membrane polarization. In this preparation the uncoupling produced by depolarization can be reversed by repolarizing the cell membrane (Rose & Loewenstein, 1971). Preliminary experiments performed in cardiac fibres, however, indicated that the injection of outward current pulses using a conventional KCl micro-electrode did not alter coupling (W. C. De Mello, unpublished). Similarly, it is known that electrical communication in heart is not abolished even in preparations completely depolarized by isotonic K2SO4 solutions. The evidence presented above that calcium is largely involved in the control of junctional conductance in heart fibres provides support of the hypothesis that the healing-over process is related to a change in junctional resistance mainly due to an increase in Ca concentration at one side of the junction (De Mello et al. 1969).
244
WALMOR C. DE MELLO
We wish to thank Professors S. Weidmann and D. Noble for reading the manuscript and for helpful discussion. This work was supported by a grant from National Heart and Lung Institute (HL-10897), Bethesda, Maryland; from the Puerto Rico Heart Association and in part by Grant no. NS-07464 from the NINDS. REFERENCES
CARMELIET, E. E. (1961). Chloride and potassium permeability in cardiac Purkinje fibres. Arscia Uitgaven Brussel, pp. 117 118. CHAMBERS, R. (1940). Relation of extraneous coats to the organization and permeability of cellular membranes. Cold Spring Harb. Symp. quant. Biol. 8, 144-168. CORABOEUF, E. & WEIDMANN, S. (1954). Temperature effects on the electrical activity of Purkinje fibers. Helv. physiol. pharmac. Acta 12, 32-41. DEL CASTILLO, J. & KATZ, B. (1955). On the localization of acetylcholine receptors. J. Physiol. 128, 157-181. DE'LtZE, J. (1965). Calcium ions and the healing-over of heart fibres. In Electrophysiology of the Heart, ed. TACCARDI, B. & MARCHETTI, G., pp. 147-148. DE MELLO, W. C. (1972a). The healing-over process in cardiac and other muscle fibers. In Electrical Phenomena of the Heart, ed. DE MELLO, W. C., pp. 323-351. New York: Academic Press. DE MELLO, W. C. (1972b). Electrical uncoupling of heart cells produced by intracellular injection of calcium and its relation to healing-over. In Fifth International Congress on Pharmacology Abstracts, pp. 55. DE MELLO, W. C. (1972c). Influence of temperature on myocardial healing over. Experientia 28, 832-833. DE MELLO, W. C. (1974). Intracellular calcium injection and cell communication in heart. Fedn Proc. 33, 445. DE MELLO, W. C. & DEXTER, D. (1970). Increased rate of sealing in beating heart muscle of the frog. Circulation Res. 26, 481-498. DE MELLO, W. C., MOTTA, G. & CHAPEAU, M. (1969). A study on the healing-over of myocardial cells of toads. Circulation Res. 24, 475-487. ESCOBAR, I. & DE MELLO, W. C. (1971). Correlation between healing-over and muscle contractions in heart. Fedn Proc. 30, 326. FATT, P. & KATZ, B. (1951). An analysis of the end-plate potential recorded with an intracellular micro-electrode. J. Physiol. 115, 320-370. HERZ, R. & WEBER, A. (1965). Caffeine inhibition of Ca uptake by muscle reticulum. Fedn Proc. 24, 208. KUSHMERICK, M. J. & PODOLSKY, R. J. (1969). Ionic mobility in muscle cells. Science, N.Y. 166, 1297. LOEWENSTEIN, W. R., NAKAS, M. & SOCOLAR, S. J. (1967). Junctional membrane uncoupling. Permeability transformation at the cell membrane junction. J. gen. Physiol. 50, 1865-1891. NASTUK, W. L. (1953). Membrane potential changes at a single muscle end plate produced by transitory application of acetyleholine with an electrically controlled microjet. Fedn Proc. 12, 102. NIEDERGERKE, R. (1957). The rate of action of calcium ions on the contraction of the heart. J. Physiol. 138, 506-515. NIEDERGERKE, R. & HARRIS, E. J. (1957). Accumulation of calcium (or strontium) under conditions of increasing contractility. Nature, Lond. 179, 1068-1069. OLIVEIRA-CASTRO, G. & LOEWENSTEIN, W. (1971). Junctional membrane permeability. Effects of divalent nations. J. membrane Biol. 5, 51-77.
Ca INJECTION AND CELL UNCOUPLING IN HEART 245 POLITOFF, A. L., SOCOLAR, S. J. & LOEWENSTEIN, W. R. (1969). Permeability of a cell membrane junction. Dependence on energy metabolism. J. gen. Physiol. 53, 498-515. ROSE, B. & LOEWEINSTEIN, W. R. (1971). Junctional membrane permeability. Depression by substitution of Li for extracellular Na, and by long-term lack of Ca and Mg; restoration by cell repolarization. J. membrane Biol. 5, 20-50. SOCOLAR, S. J. & POLITOFF, A. L. (1971). Uncoupling a cell junction in a glandular epithelium by depolarizing current. Science, N.Y. 172, 492. SPIRO, D. & SONNENBLICK, E. H. (1964). Comparison of the ultrastructural basis of the contractile process in heart and skeletal muscle. Circulation Res. 15, suppl. 2, 14-37. WEIDMANN, S. (1952). The electrical constants of Purkinje fibres. J. Physiol. 118, 348-360. WEIDMANN, S. (1966). The diffusion of radiopotassium across intercalated disks of mammalian cardiac muscle. J. Physiol. 187, 323-342. WOODBURY, J. W. & CRILL, W. E. (1961). On the problem of impulse conduction in the atrium. In Nervous Inhibition, pp. 124-135. Oxford: Pergamon Press.
231
EFFECT OF INTRACELLULAR INJECTION OF CALCIUM AND STRONTIUM ON CELL COMMUNICATION IN HEART
BY WALMOR C. DE MELLO From the Department of Pharmacology, Medical Sciences Campus, U.P.R. San Juan, P.R. 00963, U.S.A.
(Received 1 September 1974) SUMMARY
1. The influence of Ca and Sr on the electrical coupling of canine Purkinje cells was investigated by injecting the ions electrophoretically into the cytoplasm. 2. It was found that the intracellular injection produced electrical uncoupling which was spontaneously reversed. 3. No change in resting potential of the cell adjacent to the injection site was found except in those fibres not completely healed. 4. The input resistance of the injected cell increased concomitantly with the establishment of the electrical uncoupling. 5. Caffeine (6 mM), added to the extracellular fluid, reduced the rate of spontaneous recoupling. Reduction of temperature of the Tyrode solution had the same effect. 6. The abolition of cell communication produced by Ca injection seems to indicate that the ion plays an important role in the control of junctional conductance in heart fibres. INTRODUCTION
Electron microscopic studies have shown that cardiac cells are not organized as an anatomical syncytium but are completely isolated. The areas of cell to cell contact are, however, characterized by the presence of differentiated structures - the so-called intercalated disks (Spiro & Sonnenblick, 1964). Electrophysiological observation led to the conclusion that heart cells are electrically connected. The studies of Woodbury & Crill (1961), in rat atrium, indicated that the propagation of action potentials, in this tissue, requires an extremely low resistance of the intercellular junctions. On the other hand, the low longitudinal core resistance found in Purkinje fibres (Weidmann, 1952) and the evidence that radiopotassium can diffuse across the intercalated disks (Weidmann, 1966) support the view that heart cells are, indeed, connected through low resistance pathways.
232 232 ~~WALMOR C. DE MELLO It is known that Ca is essential for the maintenance of the integrity of the intercellular 'cement' in many tissues (Chambers, 1940). In heart muscle, calcium ions also play an important role in maintaining the integrity of the intercellular junctions (De Mello, Motta & Chapeau, 1969) and are essential for the quick recovery of resting potential which occurs after damage (healing-over; De'leze, 1965; De Mello et al. 1969). Indirect evidence has been described which indicates that the influence of calcium on the healing-over process of cardiac muscle could be related to its effect on junctional conductance (De Mello et al. 1969; iDe Mello & Dexter, 1970, iDe Mello, 1972 a). Searching for more information on this area, we thought important to investigate the role of calcium and strontium ions on the electrical coupling of heart cells by injecting the ions electrophoretically into the cell. The detailed results of this study, previously reported in abstracts (De Mello, 1972b, 1974), are described in this paper. METHODS
Dogs were anaesthetized with sodium pentobarbitone and the heart was immnediately removed and immersed in cooled Tyrode solution. Strands of Purkinje fibres (about 15 mmn in length) were excised from the left ventricle and transferred to a transparent Plexiglass chamber through which Tyrode solution at 340 C, saturated with a mixture of 95 %02+5% C02, was flowed continuously. The fibres were pinned on a thin layer of silicone rubber to the bottom of the chamber. The preparations were transilluminated and observed under a stereoscopic microscope. In some experiments, single Purkinje cells were visualized by treating the preparation with toluidine blue (10 mg/I.) until the nucleus became visible. In these experiments a water immersion objective was used and the micro-electrodes were impaled horizontally at the edge of the bundle. This procedure, kindly suggested to us by Professor Weidmann, made it possible to record the voltage change and to inject ions near the nucleus of contiguous cells. Solution. The Tyrode solution had the following composition (mm): NaCl 137; KCl 5-4; NaHCO, 12; MgCl2 0-5; NaH2PO4 3-6; dextrose, 5-5; CaCl2 2-7. In some experiments, the Ca was replaced by Sr isosmotically. When caffeine was used a stock solution of the drug was prepared immediately before the experiment and added to Tyrode solution to give the final concentration. All the solutions were prepared with demineralized distilled water which was used also to wash the glassware. pH of all solutions was 7. Electrical recording. Measurements of membrane potential were made with conventional KCl micro-electrodes. After inserting two micro-electrodes in adjacent Purkinje cells visualized by the method described above, the degree of electrical coupling was determined by injecting rectangular current pulses (40-100 msec in duration) delivered by an electronic stimulator and isolation unit through one of the micropipettes and recording the resulting voltage drop with the other micro-electrode (Fatt & Katz, 1951). The voltage recording micro-electrode was connected to a standard cathode follower and DC amplifier and displayed simultaneously with voltage on the second beam of the oscilloscope. Measurements of the input resistance of single Purkinje cells were made with a single 3 m-KCl micro-electrode connected to a balance bridge circuit used to pass
Ca INJECTION AND CELL UNCOUPLING IN HEART 233 current and record the voltage drop. On these experiments, a precision electrometer (Model M-4) from W-P Instruments, Inc. was used. Experimental procedure of balancing circuit. While the micro-electrode tip was in contact with the Tyrode solution rectangular current pulses of 40 msec duration and of reasonable intensity were sent to the bridge. A trial and error method was used to balance the circuit so that no square-wave deflexion could be detected on the oscilloscope. At the end of each experiment the micro-electrode was withdrawn and the balance was checked again; if the circuit was unbalanced the experiment was rejected. The electrometer used provided direct reading of electrode resistance from the balanced control when the balance was fully achieved. Ca injection. Ca was injected electrophoretically into the cell using the techniques of Nastuk (1953) and del Castillo & Katz (1955). Micro-electrodes were filled with 0*15 M-CaCl2 or with a mixture of 0*15 m-KCl and 0 15 M-CaCl2. To control the precise moment of Ca injection and to prevent the diffusion of Ca from the micropipette tip a steady and constant negative bias was applied to the interior of the micropipette. The ejection of Ca was then triggered by applying outward current pulses (40 msec duration, 4 cls) in which a positive coulomb quantity of calcium was moved from the pipette to the cell interior. The outward current pulses were monitored on the second beam of the oscilloscope. To control the position of the micro-electrode with respect to cell interior, inward current pulses were applied before and during impalement. -65
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Fig. 1. Electrical uncoupling and hyperpolarization produced by intracellular Ca injection on incompletely healed fibres. Top traces in A, B and C, show reduction in size of electrotonic potentials due to intracellular calcium injection into a neighbouring cell. Tracesaresequentialrecordsfrom the same preparation. Voltage scale at the left edge indicates values of resting potential. Current pulses in A, B and C, 1-8 x 10-7 A. Temperature, 340 C. IO
P HY 250
WALMOR C. DE MELLO
234
RESULTS
Influence of intracellular calcium injection on electrical coupling The electrical coupling between adjacent Purkinje cells immersed in Tyrode solution at 360 C was largely decreased or even suppressed by injecting calcium intracellularly. As is shown in Fig. 1, injection of calcium gradually reduced the size of the electronic potentials recorded from an adjacent cell, leading to complete uncoupling. The time required for total interruption of cell communication was variable. In some experiments, as Ca"'injection 100
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0
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40
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20 0L
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20
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60 80 100 120 Time (sec) Fig. 2. Partial uncoupling of heart cells caused by intracellular Ca injection and spontaneous re-establishment of cell communication after interruption of injection. Coupling of adjacent cells before Ca injection was taken as 100%. Temperature, 340 C.
in that illustrated in Fig. 1, about 5 min were necessary for complete abolition of cell communication while in others a shorter (2-3 min) or a longer time (8-12 min) was required. This discrepancy is probably explained by the variable location of the tip of the Ca micropipette in the cytoplasm (see Discussion). In many experiments the uncoupling produced by Ca injection was substantial but incomplete as is shown in Fig. 2 (average from four experiments). As can be seen in Fig. 1 the injection of Ca also raised the resting membrane potential of the neighbouring cell. This increment in membrane polarization was usually seen when the ion was injected between the cut
Ca INJECTION AND CELL UNCOUPLING IN HEART 235 end and the recording site in fibres that were partially depolarized (75 MV or less) by incomplete healing after the dissection procedure. In those fibres with good resting potentials (85-90 mV) the injection of Ca reduced the electrical coupling without increasing the resting potential and in some experiments a slight depolarization was found (Fig. 3). A 85
B
82 mV
80 mV
Fig. 3. Electrical uncoupling caused by intracellular Ca injection after complete healing. A, control; between A and B injection was started and almost complete uncoupling is shown in C. Resting potential is indicated at
left. Vertical calibration line, 5 mV; horizontal calibration, 120 msec. Inward current pulses (7 x 10-8 A), 40 msec duration. Temperature, 340 C.
Experiments performed with a single Ca micro-electrode connected to balance bridge circuit showed that, concomitantly with the fall in size of the electronic potentials recorded from cells adjacent to the site of Ca injection, the input resistance of the injected cell increased appreciably, as can be seen in Fig. 4. These results indicate that the decrease in amplitude of the electronic potentials recorded from cells located nearby the injection
a
IO-2
236 WALMOR C. DE MELLO site is not related to a fall in non-junctional membrane resistance of the injected cell. On the other hand, measurements of the input resistance from single Purkinje cells made with a single KCl micro-electrode showed that variations of the external calcium concentration from 12 mm to free Ca solutions for periods of 2-10 min had no effect on input resistance or on electrical coupling (Fig. 5A and B). Similar observations made by Loewenstein, Nakas & Socolar (1967) in salivary glands of Chironomus, indicated that the cell uncoupling produced by intracellular Ca injection was not related to a change in conductance of the non-junctional membrane. 8 AA A A
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Fig. 4. Increase of input membrane resistance (V/I) recorded from the same Purkinje cell to which Ca was injected intracellularly. Temperature, 340 C.
The impairment or total failure of cell communication caused by intracellular Ca injection was largely reversible. As can be seen in Figs. 2 and 6 (average from four experiments) as soon as the release of Ca from the micropipette was interrupted, the size of the electrotonic potentials, recorded from a nearby cell, gradually increasedin amplitude and cell communication was completely re-established. In those instances in which Ca injection reduced the electrical coupling by only 50-70 % (Fig. 2), the time required for complete recovery of cell communication was shorter (about 80-120 sec) than when total uncoupling was achieved. In this case, 10 min or more were usually necessary for complete recovery of electrical coupling (Fig. 6).
UNCOUPLING IN HEART
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WALMOR C. DE MELLO
Fig. 7. Influence of intracellular Sr injection on electrical coupling of Purkinje cells. A, control; between A and B injection was started. C, after 70 sec of Sr injection. Vertical calibration 8 mV; horizontal calibration, 300 msec. Inward current pulses 10-7 A in A. In B and C the intensity of current was slightly increased. Temperature, 340 C.
Influence of intracellular injection of strontium and magnesium on the electrical coupling It is known that Sr replaces Ca on the processes of healing-over (Escobar & De Mello, 1971) and cardiac contraction (Niedergerke & Harris, 1957). The effects of Ca injections on cell communication described above led us
Ca INJECTION AND CELL UNCOUPLING IN HEART 239 to investigate the influence of intracellular strontium injection on electrical coupling of Purkinje cells. The electrical interaction between normal cells was measured as described above before and after injection of Sr. Fig. 7 shows the influence of intracellular injection of Sr on cell communication. As can be seen the electrical coupling between adjacent Purkinje cells was substantially reduced by increasing the intracellular Sr concentration. As with Ca, the reduction of cell communication produced by injection of strontium was reversible (Fig. 8A) and the time course and rate of the recovery process SR2+ injection 100
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Fig. 8. A, spontaneous recovery of cell communication previously depressed by intracellular Sr injection. B, lack of action of variation of the extracellular Sr concentration on input resistance of a single Purkinje cell. Preparation was exposed to Sr free solution. for 2-5 min. Similar results were obtained with 10 min of incubation. Temperature, 340 C.
were similar to that found after intracellular Ca injection. Control experiments in which the extracellular Sr concentration was changed from 12 to 0 (for 10 min) produced no variation in input resistance (Fig. 8B) which strongly suggests that the conductance of the non-junctional membrane did not vary. Magnesium, which does not replace Ca on the healing-over process of cardiac muscle (De Mello et al. 1969), had no influence on junctional conductance as judged from the lack of action of an intracellular injection of it on electrical coupling (Fig. 9).
WALMOR C. DE MELLO
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I
160
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Fig. 9. Lack of action of intracellular Mg injection on electrical coupling of Purkinje cells. Inward current pulses used to inject Mg had the same intensity and duration described in Fig. 3. Temperature, 340 C.
On the nature of the reversibility of the electrical uncoupling The re-establishment of cell communication found after interruption of calcium injection is probably due to the operation of mechanisms which keep a low concentration of Ca in the cytoplasm namely (a) an active extrusion of Ca through the surface cell membrane; (b) the uptake of Ca by elements of the sarcoplasmic reticulum or by mitochondria. The possible contribution of sarcoplasmic reticulum to the re-establishment of cell communication previously abolished by Ca injection was investigated by exposing the preparation to caffeine, a drug which releases Ca from terminal cysternae and also inhibits its uptake by the same structures (Herz & Weber, 1965). In these experiments Ca was injected into the cell before and after treatment with caffeine (6 mM) and the time required for complete recovery of cell communication, after stopping the Ca injection, was measured in both situations. Since the time required for total re-establishment of electrical coupling was variable at different sites of injection, measurements of the recovery process were made keeping the micro-electrodes impaled into the same cells throughout the experiment. This was not an easy task considering the relatively long time required for such measurements. Fig. 10 is the average of four highly successful experiments. As is shown on this figure, the rate of recovery of cell communication, after interruption of Ca injection, was markedly reduced by caffeine. Control measurements showed that the drug has no influence on electrical coupling, at least under the conditions used on these experiments. It is also important to mention that in the presence of caffeine, suppression of cell communication by Ca injection was more easily obtained than in absence of the drug, which is probably related to the inhibitory action of the drug on the uptake of Ca by the sarcoplasmic reticulum and the accumulation of Ca in the cytoplasm.
Ca INJECTION AND CELL UNCOUPLING IN HEART 241 In view of the probable mechanisms involved in the re-establishment of cell communication after Ca injection, it seemed pertinent to investigate the influence of temperature on the rate of electrical re-coupling. For this, measurements of cell communication were made at 360 C and then the temperature was dropped to 200 C while keeping the electrodes inside the cell. After a few minutes of equilibration at 200 C the degree of coupling was again determined and Ca injected into the cell until suppression of cell communication achieved. The injection of Ca was then interrupted and the rate of recovery of electrical coupling measured. A 100
O.-
-
-
Control 80 -~~~~
Caffeine 60
LU
O
~~~~ 500 -Control ~~2 20
/10
0 0
200
Caffeine (6 mm)
6
10
A-.ATime (min)
400 600 Time (sec)
800
1000
Fig. 10. A, influence of caffeine (6 mm) added to the extracellular fluid on the rate of recovery of cell communication previously abolished by intracellular Ca injection. B, lack of action of the drug on the electrical coupling of adjacent Purkinje cells in absence of Ca injection. Temperature, 340 C.
The results obtained indicated that the reduction of temperature not only decreased the resting potential by about 8 mV but also reduced the amplitude of the electrotonic potentials. Control measurements of the input resistance performed with single micro-electrodes indicated that the drop in temperature caused an increase of input resistance (about 17 %) which is probably explained by the well known reduction in potassium conductance of the surface cell membrane (Carmeliet, 1961). Although previous studies of Coraboeuf & Weidmann (1954) in Purkinje fibres and of De Mello (1972c) in toad's ventricle indicated no apparent effect of temperature on electrical coupling, the observations described above suggest that the fall in amplitude of the electrotonic potentials
242 WALMOR C. DE MELLO found at 200 C could be related to a decrease in junctional conductance. Experiments made on salivary glands of Chironomus (Politoff, Socolar & Loewenstein, 1969) indicated, indeed, that cell communication in this tissue is largely dependent on temperature. The rate of recovery of cell communication previously blocked by Ca injection was also reduced at 200 C (Fig. 11). On these experiments the muscles were immersed in Tyrode solution at 200 C before Ca injection and kept at this temperature throughout the experiment. The 100 % value used in Fig. 11 indicates maximal coupling between adjacent Purkinje cells at 200 C. 200 C Ca2+ injections 100 'olo in Out
80
0
.E60 U0
40
-
~20
/
\/
0 0
200
400
600
800 1000 Time (sec)
1200
1400
1600
Fig. 11. Effect of low temperature (200 C) on the rate of spontaneous cell recoupling. Control measurements at 340 C (not included) indicated complete recoupling in about 10 min. DISCUSSION
The present results indicate that calcium plays an important role in cell communication in heart. The complete recovery of electrical coupling which occurs after interruption of calcium injection is probably related to the re-establishment of the low concentration of ionized calcium in the cytoplasm. Evidence has been provided, indeed, that Ca is actively taken by elements of the sarcoplasmic reticulum and that caffeine inhibits this uptake (Herz & Weber, 1965). The decrease on the rate of re-establishment of electrical coupling caused by caffeine could indicate that the uptake of Ca by the sarcoplasmic reticulum is involved in the recovery of electrical coupling found after interruption of Ca injection. The observation that
Ca INJECTION AND CELL UNCOUPLING IN HEART 243 uncoupling is more easily obtained by injecting calcium in preparations exposed to caffeine seems to support this view. The decrease in junctional conductance produced by Ca injection can be permanent if the calcium concentration is kept high at one side of the intercellular junction, as is the case when the fibre is damaged (De Mello et al. 1969). Similar conclusions were obtained from experiments performed in damaged salivary glands (Oliveira-Castro & Loewenstein, 1971). Under normal conditions, however, when the sarcoplasmic reticulum is working properly or calcium is being actively extruded through the surface cell membrane, the increase in concentration of ionized calcium in the cytoplasm is transient and long lasting changes in junctional conductance, in cardiac cells, will not occur. The decreased rate of recoupling found at 200 C is probably related to some impairment of the active Ca uptake by the sarcoplasmic reticulum. The variable delay between the initiation of Ca injection and the moment the electrical coupling starts to change was probably related to several factors. It is known, for instance, that the diffusion of Ca in the sarcoplasm is slower that in free solution (Niedergerke, 1957; Kushmerick & Podolsky, 1969). Slight differences of distance between the site of injection and the intercellular junction could produce large variations in the time delay. The binding of calcium by troponin or the presence of elements of the sarcoplasmic reticulum in the vicinity of the micro-electrode tip may also be important. These variables make it difficult to estimate the effective concentration of calcium that reaches the intercellular junctions. Since no change in transparency or a large fall on resting potential were produced by Ca injection, the possibility of an injurious effect of the injection seems unlikely. It is important to mention that in salivary glands of Chironomus, uncoupling can be produced by depolarizing the surface cell membrane (Socolar & Politoff, 1971) and the proposed mechanism for the uncoupling was the internal release of Ca due to the fall of membrane polarization. In this preparation the uncoupling produced by depolarization can be reversed by repolarizing the cell membrane (Rose & Loewenstein, 1971). Preliminary experiments performed in cardiac fibres, however, indicated that the injection of outward current pulses using a conventional KCl micro-electrode did not alter coupling (W. C. De Mello, unpublished). Similarly, it is known that electrical communication in heart is not abolished even in preparations completely depolarized by isotonic K2SO4 solutions. The evidence presented above that calcium is largely involved in the control of junctional conductance in heart fibres provides support of the hypothesis that the healing-over process is related to a change in junctional resistance mainly due to an increase in Ca concentration at one side of the junction (De Mello et al. 1969).
244
WALMOR C. DE MELLO
We wish to thank Professors S. Weidmann and D. Noble for reading the manuscript and for helpful discussion. This work was supported by a grant from National Heart and Lung Institute (HL-10897), Bethesda, Maryland; from the Puerto Rico Heart Association and in part by Grant no. NS-07464 from the NINDS. REFERENCES
CARMELIET, E. E. (1961). Chloride and potassium permeability in cardiac Purkinje fibres. Arscia Uitgaven Brussel, pp. 117 118. CHAMBERS, R. (1940). Relation of extraneous coats to the organization and permeability of cellular membranes. Cold Spring Harb. Symp. quant. Biol. 8, 144-168. CORABOEUF, E. & WEIDMANN, S. (1954). Temperature effects on the electrical activity of Purkinje fibers. Helv. physiol. pharmac. Acta 12, 32-41. DEL CASTILLO, J. & KATZ, B. (1955). On the localization of acetylcholine receptors. J. Physiol. 128, 157-181. DE'LtZE, J. (1965). Calcium ions and the healing-over of heart fibres. In Electrophysiology of the Heart, ed. TACCARDI, B. & MARCHETTI, G., pp. 147-148. DE MELLO, W. C. (1972a). The healing-over process in cardiac and other muscle fibers. In Electrical Phenomena of the Heart, ed. DE MELLO, W. C., pp. 323-351. New York: Academic Press. DE MELLO, W. C. (1972b). Electrical uncoupling of heart cells produced by intracellular injection of calcium and its relation to healing-over. In Fifth International Congress on Pharmacology Abstracts, pp. 55. DE MELLO, W. C. (1972c). Influence of temperature on myocardial healing over. Experientia 28, 832-833. DE MELLO, W. C. (1974). Intracellular calcium injection and cell communication in heart. Fedn Proc. 33, 445. DE MELLO, W. C. & DEXTER, D. (1970). Increased rate of sealing in beating heart muscle of the frog. Circulation Res. 26, 481-498. DE MELLO, W. C., MOTTA, G. & CHAPEAU, M. (1969). A study on the healing-over of myocardial cells of toads. Circulation Res. 24, 475-487. ESCOBAR, I. & DE MELLO, W. C. (1971). Correlation between healing-over and muscle contractions in heart. Fedn Proc. 30, 326. FATT, P. & KATZ, B. (1951). An analysis of the end-plate potential recorded with an intracellular micro-electrode. J. Physiol. 115, 320-370. HERZ, R. & WEBER, A. (1965). Caffeine inhibition of Ca uptake by muscle reticulum. Fedn Proc. 24, 208. KUSHMERICK, M. J. & PODOLSKY, R. J. (1969). Ionic mobility in muscle cells. Science, N.Y. 166, 1297. LOEWENSTEIN, W. R., NAKAS, M. & SOCOLAR, S. J. (1967). Junctional membrane uncoupling. Permeability transformation at the cell membrane junction. J. gen. Physiol. 50, 1865-1891. NASTUK, W. L. (1953). Membrane potential changes at a single muscle end plate produced by transitory application of acetyleholine with an electrically controlled microjet. Fedn Proc. 12, 102. NIEDERGERKE, R. (1957). The rate of action of calcium ions on the contraction of the heart. J. Physiol. 138, 506-515. NIEDERGERKE, R. & HARRIS, E. J. (1957). Accumulation of calcium (or strontium) under conditions of increasing contractility. Nature, Lond. 179, 1068-1069. OLIVEIRA-CASTRO, G. & LOEWENSTEIN, W. (1971). Junctional membrane permeability. Effects of divalent nations. J. membrane Biol. 5, 51-77.
Ca INJECTION AND CELL UNCOUPLING IN HEART 245 POLITOFF, A. L., SOCOLAR, S. J. & LOEWENSTEIN, W. R. (1969). Permeability of a cell membrane junction. Dependence on energy metabolism. J. gen. Physiol. 53, 498-515. ROSE, B. & LOEWEINSTEIN, W. R. (1971). Junctional membrane permeability. Depression by substitution of Li for extracellular Na, and by long-term lack of Ca and Mg; restoration by cell repolarization. J. membrane Biol. 5, 20-50. SOCOLAR, S. J. & POLITOFF, A. L. (1971). Uncoupling a cell junction in a glandular epithelium by depolarizing current. Science, N.Y. 172, 492. SPIRO, D. & SONNENBLICK, E. H. (1964). Comparison of the ultrastructural basis of the contractile process in heart and skeletal muscle. Circulation Res. 15, suppl. 2, 14-37. WEIDMANN, S. (1952). The electrical constants of Purkinje fibres. J. Physiol. 118, 348-360. WEIDMANN, S. (1966). The diffusion of radiopotassium across intercalated disks of mammalian cardiac muscle. J. Physiol. 187, 323-342. WOODBURY, J. W. & CRILL, W. E. (1961). On the problem of impulse conduction in the atrium. In Nervous Inhibition, pp. 124-135. Oxford: Pergamon Press.
