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Intracellular Ca2+ in Helix neurons: Effect of intracellular azide
Comp. Biochent Physiol., Vol. 66C, pp. 261 to 263
0306-4492/80/0701-02~1502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
INTRACELLULAR C a 2+ IN H E L I X NEURONS" EFFECT OF INTRACELLULAR AZIDE L. SIMONSEN University of Copenhagen, Zoophysiological Laboratory B, August Krogh Institute, 13, Universitetsparken DK-2100 Copenhagen O, Denmark (Received 17 October 1979) Abstract--1. The effect of extracellularly applied azide on membrane potential and intracellular calcium activity was examined in neurons from Helix pomatia at low extracellular pH and normal versus low extracellular calcium activity. 2. The effect of intracellularly applied azide at low and normal extracellular pH was followed. 3. It was found that the source of Ca 2 + involved in enhancement of intracellular activity at exposure to azide at low pH is of extracellular origin, and further that the primary site of action of azide is at the outside of the cell membrane.
INTRODUCTION The presence of extracellular azide in concentrations of 2-10 m M at pH 5.5 leads to a 2-10 times reversible increment of intracellular calcium activity in nerve cells from Helix pomatia after few seconds of exposure (Simonsen & Christoffersen, 1979). The fact that the rise of activity begins immediately after the exposure of N ~ indicates that N~ affects the Ca 2 + transport system directly as opposed to e.g. the C N - induced activity increase in squid axons (Blanstein & Hodgkin, 1969). Hence this effect has presently been object for further investigation. Azide may exert its effect from outside the cell or it may penetrate the membrane at the low pH where leakage conductance is enhanced (Brown et al., 1970). In an attempt to elucidate these possibilities intracellular Ca-activity (ale,) and membrane potential (EM) was followed during intracellular ionophoretic injection of azide at normal and low extracellular pH and during extracellular exposure to azide at normal (2.0 x 1 0 - 3 M ) a n d low (.13 x 1 0 - 6 M ) extracellular Ca 2 + activity. MATERIALS AND METHODS
Cells The measurements were carried out on the big mediorostral cells of the suboesophageal ganglia from the snail Helix pomatia. Salt solutions The preparation which was mounted in a continuous flow chamber was bathed in the salt solution of Kerkut &
Thomas (1965). Deviations from this solution are listed in Table 1. The solution with no Ca 2+ added was found to have a pCa of 5.20 (Simonsen & Christoffersen, 1979). When 1 mM of EGTA was added to this solution (C, Table 1) the resulting pCa at pH 5.5 was calculated to 5.90 according to Caldwell (1970). Using Kc,.EGr^ = 1011 which is in agreement with calcium electrode measurements (Owen, 1976) measurements of pCa in solution C with the calcium electrode gave pCa 5.85. pH was buffered with Tris maleate. 7he electrodes A KCl-filled micropipettc was used for measuring Eu, tip resistance was between 5--10MfJ. The Ca 2+ sensitive microelectrode used differed from the one described earlier (Christoffcrsen & Simonsen 1977): In order to reduce tip resistance and make the penetration easier, the micropipettes were bevelled after they had been exposed to a saturated atmosphere of dichlor--dimethyi silan for approx 90 sea The micropipettes were siliconized before bevelling to avoid introduction of the 10% ethanol, used for cooling during bevelling into the tip of the pipettes. The electrode bevelling was modified from Brown & Flaming (1974): Cromoxid grind-paper CMoyco" Flexi-i grid) with medium grain size of 0.5 :tin was used as grinding surface. The micropipettes were bevelled to an o.d. of 2.4 #m. The calcium-carrier used was the same as described by Amman et al. (1975). The carrier was filled into the siliconized and bevelled micropipettes by dipping the tip into the carrier mixture for 10-15 min. The shaft of the electrode was filled with 10 mM CaClz. Electrode resistance for an electrode with an o.d. of approx 3/xm was 109 f~ which is approximately two orders
Table 1. Composition of the salt-solutions used Solution (mM)
NaCl
KCI
CaCI2
MgCI2
TM*
EGTA
NaN3
pH
"Normal" A
80 80
4 4
7 7
5 5
10 10
0 0
0 0
B C
75 75
4 4
7 0
5 0
10 46
0 1
5 5
7.8 5.5 5.5
* TM--Tris-malate buffer; also used as substitute for CaCI2 and MgCI2 in solution C. 261
5.5
262
L. SIMONSEN
CF A~A~
Ei'c° CRO EM CRO
)v~ Fig. 1. Experimental set up: E1 the KCl-filled electrode is connected to A1 a high impedance preamplifier (NF-1Bioelectric) and to the negative input of A2, a high impedance differential voltage probe (input impedance approx 10tsD). E 2 is the Ca2+-sensitive microelectrode connected to the non-inverting input of A2. Membranepotential (EM) and "Calcium potential" (E~.c~) are monitored on a pen recorder and an oscilloscope. E3 the double barrelled injection electrode can, through contact B, be connected to either the "floating" current generator (CF) or the "grounded" current generator (C~): where 1/i is monitoring the current sent through the electrode and Vr is monitoring the voltage needed to send the current. In this way it was possible to test the injection electrode before use, and to measure membrane resistance.
of magnitude lower than the previously used calcium microelectrode base on Ca-DOPP/DOPP, in PVC. Calibration of the electrode was carried out as described earlier (Christoffersen & Simonsen, 1977). Electrophoretic intracellular injection was carried out as described by Eccles & Eccles (1964) by using a double barrelled electrode pulled from "R. & D. Optical System" style 1 Theta tubing glass (2.0 mm o.d.) filled with 1 M solutions of NaN3 and KCI, or NaCI.
sible fall (Fig. 2B). This fall was in agreement with previously observed changes in intracellular Ca 2 + at low extracellular calcium activity with no azide applied (Simonsen & Christoffersen, 1979, Fig. 1C).
Membrane potential and intracellular Ca-activity at intracellular addition of NaN3 and KN3 When NaNa and KN3 were injected into the cells at low concentrations (1-5 mM) they had no influence on intracellular calcium activity (the amount of injected chemicals was calculated by estimating cellvolume and measuring duration and strength of the injection current. When KN3 was injected into the cell in high concentration (up to 80 mM) (Fig. 3), the membrane potential hyperpolarized in agreement with reported transport number of that cell (Tk0.8) Christoffersen (1973). The simultaneous change of Eca indicated a rise in ai.ca, but this change failed to appear when NaN3 was injected in same amount. It therefore was concluded that the observed change in Eca was due to the intracellular rise in potassium. An assumption in agreement with reported selectivity constants of the used Ca-carrier Kc~,K = 6.03 × 10 -6 (Amman et al., 1979). When NaC1 was injected, it led to a brief hyperpolarisation of the membrane potential (Fig. 3) probably due to the electrogenic property of the N a - K - p u m p (Thomas, 1969).
A 5mM
N~
pHo= S./.S
Experimental set up The ionophoretic injection of chemicals into the cells was carried out with a floating constant current generator (Fig. 1). To ensure that the double barrelled electrode was inside the cell a contact (B, Fig. 1) could switch the electrode to a current generator passing current over the membrane. The following change of EM was measured. Measurements were only carried out when membrane resistance was greater than 1 M f~, and Eu more negative than - 4 0 mV. The same equipment as described earlier (Christoffersen & Simonsen, 1979) was used for registration of EM and Ei.c~ (see Fig. 1).
5 rain B
m M N~ 0rnMCQ2° 1mMEGTA pHo=5£5
RESULTS
Membrane potential and intracellular Ca-activity at extracellular poisoning with azide at pH 5.5 and pCao 2.7 and 5.9 It has been reported previously that extracellularly applied azide at low pH leads to a rise in intraceUular Ca 2+ (Simonsen & Christoffersen, 1979). Poisoning with azide has now been carried out at normal (2 x I0 -a M) and low (1.3 x 10 -6 M) extracellular calcium activity (Fig. 2). At normal calcium activity the previously observed intracellular calcium rise was seen (Fig. 2A). At low extracellular calcium activity the intracellular Ca2+-rise was changed to a rever-
5 rain
J
Fig. 2. Effect of extracellular N~ at normal and low extracellular Ca z+ . A is a penrecording of the effect of azide on EM and E~.ca at pH5.5 and normal Ca-activity (2mM). The membrane potential is reversibly reduced with 30 mV accompanied by a reversible change in E~.ca of 10 inV. At 2b the extracellular Ca-activity is reduced to 0.0013 mM simultaneously with the exposure of N~. This again leads to a reduction in EM but a fall in E~.ca of approx 14 mV.
Intracellular Ca 2 + in Helix neurons -90 nA
*90 nA
10 min Fig. 3. Pen recording of Eu and E~.ca at ionophoretic injection of KN3 and NaCI. The sign of the injection current, indicated at the top of the figure, is with respect to the NaNa filled barrel of the injection electrode. At - 9 0 nA KN3 is injected into the cell to a final concentration of approx 80 mM. At + 90 nA NaCI is injected into the cell to a final concentration of approx 160 mM provided no active transport or diffusion. Notice the different sensitivity of the two tracks. DISCUSSION The increase in intracellular calcium activity taking place at addition of N ~ at low pH fails to appear when extracellular Ca 2 ÷ is lowered corresponding to a change of ao, ca/ai, ca from 5000 to 3.5. This indicates that the source of Ca 2 + leading to the intraceilular rise is of extracellular origin. This in connection with the previously reported observation: that the azideinduced calcium activity rise is not enhanced by removal of extracellular sodium (Simonsen & Christoffersen, 1979) supports the conclusion that the Na/Caexchange mechanism described in squid axons (Baker, 1975) is of m i n o r importance in snail neurons (Simonsen & Christoffersen, 1979). A supposition for this conclusion was a Ca 2 + source of extracellular origin. N o intracellular calcium rise is seen when azide is applied intracellularly at concentrations up to 10 m M (observations for approx 3 0 m i n ) although the changes of EM indicate that azide is actually injected into the cells. It therefore can be concluded that the major effect of azide on at, ca is exerted from the outside of the membrane. The effect of pH lowering may be a demasking of azide-sensitive Ca-transport site. Acknowledoement--The used Ca 2 + carrier was provided by Dr W. Simon Schweitzerland.
263 REFERENCES
AMMAN D., Gi.)GGI M., PRETSCH E. & SIMON W. (1975) Improved calcium ion-sensitive electrodes based on a neutral carrier. Analyt. Lett. 8, 709-720. AMMAN D., Gi)GGI M., PRETSCH E. & SIMON W, (1979) Design and use of Ca 2 + of selective microelectrodes. In Detection and Measurements of Free Ca 2 + Ions in Cells (Edited by ASHLEY C. C. & CAMPBELL A. K.). North Holland, Amsterdam. BAKER P. F. (1975) Transport and metabolism of calcium ions in nerve cells. In Calcium Movements in Excitable Cells. Pergamon Press, Oxford. BLANSTEIN M. P. & HODGKIN A. L. (1969) The effect of cyanide on the efflux of calcium from squid axons. J. Physiol. Lond. 200, 497-527. BLANSTEIN M. P., RATZLOFF R. W. t~ SCHWEITZER E. S. (1978) Calcium buffering in presynaptic nerve terminals. J. gen. Physiol. 72, 43-66. BROWN K. T. ,~¢ FLAMING D. G. (1974) Bevelling of fine micropipette electrodes by a rapid precision method. Science, N. Y 185, 693-695. BROWN A. M., WALKER I. L. t~ SUTTON R. B. (1970) Increased chloride conductance as the proximate cause of hydrogen ion concentration effects in Aplysia neurons J. gen. Physiol. 56, 559-582. CLADWELL P. C. (1970) Calcium chelation and buffers. In Calcium and Cellular Function, pp. 10-16. Macmillan, New York. CHRISTOFFERSEN G. R. J. (1973) Calcium concentration and chloride conductance. Comp. Biochem. Physiol. 46A, 371-389. CHRISTOEFERSEN G. R..J. & SIMONSEN L. (1977) Ca 2÷sensitive microelectrode: intracellular steady-state measurements in nerve cells. Acta physiol, scand. 101, 492~494. CHRISTOFFERSEN G. R. J. & SIMONSEN L. (1979) Ca 2+sensitive microelectrode: Intracellular use in nerve cells. Detection and Measurements of Free Ca Ions in Cells (Edited by ASHLEYC. C. & CAMPBELLA. K.) North Holland, Amsterdam. KERKUT G. A. & THOMASR. C. (1965) An electrogenic sodium pump in small neurons. Comp. Biochem. Physiol. 14, 167-183. OWEN J. D. (1976) The determination of the stability constants for calcium-EGTA. Biochem. biophys. Acta 451, 321-325. SIMONSEN L. & CHRISTOFEERSEN G. R. J. (1979) Intracellular Ca 2 ÷-activity in Helix neurons: Effects of extracellular Ca 2÷, H +, Na ÷ and N~-. Comp. Biochem. Physiol. 63A, p. 615-618. THOMAS R. C. (1969) Membrane current and intracellular sodium in a snail neuron during excursion of injected sodium. J. Physiol, Lond. 201, 495-514.
0306-4492/80/0701-02~1502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
INTRACELLULAR C a 2+ IN H E L I X NEURONS" EFFECT OF INTRACELLULAR AZIDE L. SIMONSEN University of Copenhagen, Zoophysiological Laboratory B, August Krogh Institute, 13, Universitetsparken DK-2100 Copenhagen O, Denmark (Received 17 October 1979) Abstract--1. The effect of extracellularly applied azide on membrane potential and intracellular calcium activity was examined in neurons from Helix pomatia at low extracellular pH and normal versus low extracellular calcium activity. 2. The effect of intracellularly applied azide at low and normal extracellular pH was followed. 3. It was found that the source of Ca 2 + involved in enhancement of intracellular activity at exposure to azide at low pH is of extracellular origin, and further that the primary site of action of azide is at the outside of the cell membrane.
INTRODUCTION The presence of extracellular azide in concentrations of 2-10 m M at pH 5.5 leads to a 2-10 times reversible increment of intracellular calcium activity in nerve cells from Helix pomatia after few seconds of exposure (Simonsen & Christoffersen, 1979). The fact that the rise of activity begins immediately after the exposure of N ~ indicates that N~ affects the Ca 2 + transport system directly as opposed to e.g. the C N - induced activity increase in squid axons (Blanstein & Hodgkin, 1969). Hence this effect has presently been object for further investigation. Azide may exert its effect from outside the cell or it may penetrate the membrane at the low pH where leakage conductance is enhanced (Brown et al., 1970). In an attempt to elucidate these possibilities intracellular Ca-activity (ale,) and membrane potential (EM) was followed during intracellular ionophoretic injection of azide at normal and low extracellular pH and during extracellular exposure to azide at normal (2.0 x 1 0 - 3 M ) a n d low (.13 x 1 0 - 6 M ) extracellular Ca 2 + activity. MATERIALS AND METHODS
Cells The measurements were carried out on the big mediorostral cells of the suboesophageal ganglia from the snail Helix pomatia. Salt solutions The preparation which was mounted in a continuous flow chamber was bathed in the salt solution of Kerkut &
Thomas (1965). Deviations from this solution are listed in Table 1. The solution with no Ca 2+ added was found to have a pCa of 5.20 (Simonsen & Christoffersen, 1979). When 1 mM of EGTA was added to this solution (C, Table 1) the resulting pCa at pH 5.5 was calculated to 5.90 according to Caldwell (1970). Using Kc,.EGr^ = 1011 which is in agreement with calcium electrode measurements (Owen, 1976) measurements of pCa in solution C with the calcium electrode gave pCa 5.85. pH was buffered with Tris maleate. 7he electrodes A KCl-filled micropipettc was used for measuring Eu, tip resistance was between 5--10MfJ. The Ca 2+ sensitive microelectrode used differed from the one described earlier (Christoffcrsen & Simonsen 1977): In order to reduce tip resistance and make the penetration easier, the micropipettes were bevelled after they had been exposed to a saturated atmosphere of dichlor--dimethyi silan for approx 90 sea The micropipettes were siliconized before bevelling to avoid introduction of the 10% ethanol, used for cooling during bevelling into the tip of the pipettes. The electrode bevelling was modified from Brown & Flaming (1974): Cromoxid grind-paper CMoyco" Flexi-i grid) with medium grain size of 0.5 :tin was used as grinding surface. The micropipettes were bevelled to an o.d. of 2.4 #m. The calcium-carrier used was the same as described by Amman et al. (1975). The carrier was filled into the siliconized and bevelled micropipettes by dipping the tip into the carrier mixture for 10-15 min. The shaft of the electrode was filled with 10 mM CaClz. Electrode resistance for an electrode with an o.d. of approx 3/xm was 109 f~ which is approximately two orders
Table 1. Composition of the salt-solutions used Solution (mM)
NaCl
KCI
CaCI2
MgCI2
TM*
EGTA
NaN3
pH
"Normal" A
80 80
4 4
7 7
5 5
10 10
0 0
0 0
B C
75 75
4 4
7 0
5 0
10 46
0 1
5 5
7.8 5.5 5.5
* TM--Tris-malate buffer; also used as substitute for CaCI2 and MgCI2 in solution C. 261
5.5
262
L. SIMONSEN
CF A~A~
Ei'c° CRO EM CRO
)v~ Fig. 1. Experimental set up: E1 the KCl-filled electrode is connected to A1 a high impedance preamplifier (NF-1Bioelectric) and to the negative input of A2, a high impedance differential voltage probe (input impedance approx 10tsD). E 2 is the Ca2+-sensitive microelectrode connected to the non-inverting input of A2. Membranepotential (EM) and "Calcium potential" (E~.c~) are monitored on a pen recorder and an oscilloscope. E3 the double barrelled injection electrode can, through contact B, be connected to either the "floating" current generator (CF) or the "grounded" current generator (C~): where 1/i is monitoring the current sent through the electrode and Vr is monitoring the voltage needed to send the current. In this way it was possible to test the injection electrode before use, and to measure membrane resistance.
of magnitude lower than the previously used calcium microelectrode base on Ca-DOPP/DOPP, in PVC. Calibration of the electrode was carried out as described earlier (Christoffersen & Simonsen, 1977). Electrophoretic intracellular injection was carried out as described by Eccles & Eccles (1964) by using a double barrelled electrode pulled from "R. & D. Optical System" style 1 Theta tubing glass (2.0 mm o.d.) filled with 1 M solutions of NaN3 and KCI, or NaCI.
sible fall (Fig. 2B). This fall was in agreement with previously observed changes in intracellular Ca 2 + at low extracellular calcium activity with no azide applied (Simonsen & Christoffersen, 1979, Fig. 1C).
Membrane potential and intracellular Ca-activity at intracellular addition of NaN3 and KN3 When NaNa and KN3 were injected into the cells at low concentrations (1-5 mM) they had no influence on intracellular calcium activity (the amount of injected chemicals was calculated by estimating cellvolume and measuring duration and strength of the injection current. When KN3 was injected into the cell in high concentration (up to 80 mM) (Fig. 3), the membrane potential hyperpolarized in agreement with reported transport number of that cell (Tk0.8) Christoffersen (1973). The simultaneous change of Eca indicated a rise in ai.ca, but this change failed to appear when NaN3 was injected in same amount. It therefore was concluded that the observed change in Eca was due to the intracellular rise in potassium. An assumption in agreement with reported selectivity constants of the used Ca-carrier Kc~,K = 6.03 × 10 -6 (Amman et al., 1979). When NaC1 was injected, it led to a brief hyperpolarisation of the membrane potential (Fig. 3) probably due to the electrogenic property of the N a - K - p u m p (Thomas, 1969).
A 5mM
N~
pHo= S./.S
Experimental set up The ionophoretic injection of chemicals into the cells was carried out with a floating constant current generator (Fig. 1). To ensure that the double barrelled electrode was inside the cell a contact (B, Fig. 1) could switch the electrode to a current generator passing current over the membrane. The following change of EM was measured. Measurements were only carried out when membrane resistance was greater than 1 M f~, and Eu more negative than - 4 0 mV. The same equipment as described earlier (Christoffersen & Simonsen, 1979) was used for registration of EM and Ei.c~ (see Fig. 1).
5 rain B
m M N~ 0rnMCQ2° 1mMEGTA pHo=5£5
RESULTS
Membrane potential and intracellular Ca-activity at extracellular poisoning with azide at pH 5.5 and pCao 2.7 and 5.9 It has been reported previously that extracellularly applied azide at low pH leads to a rise in intraceUular Ca 2+ (Simonsen & Christoffersen, 1979). Poisoning with azide has now been carried out at normal (2 x I0 -a M) and low (1.3 x 10 -6 M) extracellular calcium activity (Fig. 2). At normal calcium activity the previously observed intracellular calcium rise was seen (Fig. 2A). At low extracellular calcium activity the intracellular Ca2+-rise was changed to a rever-
5 rain
J
Fig. 2. Effect of extracellular N~ at normal and low extracellular Ca z+ . A is a penrecording of the effect of azide on EM and E~.ca at pH5.5 and normal Ca-activity (2mM). The membrane potential is reversibly reduced with 30 mV accompanied by a reversible change in E~.ca of 10 inV. At 2b the extracellular Ca-activity is reduced to 0.0013 mM simultaneously with the exposure of N~. This again leads to a reduction in EM but a fall in E~.ca of approx 14 mV.
Intracellular Ca 2 + in Helix neurons -90 nA
*90 nA
10 min Fig. 3. Pen recording of Eu and E~.ca at ionophoretic injection of KN3 and NaCI. The sign of the injection current, indicated at the top of the figure, is with respect to the NaNa filled barrel of the injection electrode. At - 9 0 nA KN3 is injected into the cell to a final concentration of approx 80 mM. At + 90 nA NaCI is injected into the cell to a final concentration of approx 160 mM provided no active transport or diffusion. Notice the different sensitivity of the two tracks. DISCUSSION The increase in intracellular calcium activity taking place at addition of N ~ at low pH fails to appear when extracellular Ca 2 ÷ is lowered corresponding to a change of ao, ca/ai, ca from 5000 to 3.5. This indicates that the source of Ca 2 + leading to the intraceilular rise is of extracellular origin. This in connection with the previously reported observation: that the azideinduced calcium activity rise is not enhanced by removal of extracellular sodium (Simonsen & Christoffersen, 1979) supports the conclusion that the Na/Caexchange mechanism described in squid axons (Baker, 1975) is of m i n o r importance in snail neurons (Simonsen & Christoffersen, 1979). A supposition for this conclusion was a Ca 2 + source of extracellular origin. N o intracellular calcium rise is seen when azide is applied intracellularly at concentrations up to 10 m M (observations for approx 3 0 m i n ) although the changes of EM indicate that azide is actually injected into the cells. It therefore can be concluded that the major effect of azide on at, ca is exerted from the outside of the membrane. The effect of pH lowering may be a demasking of azide-sensitive Ca-transport site. Acknowledoement--The used Ca 2 + carrier was provided by Dr W. Simon Schweitzerland.
263 REFERENCES
AMMAN D., Gi.)GGI M., PRETSCH E. & SIMON W. (1975) Improved calcium ion-sensitive electrodes based on a neutral carrier. Analyt. Lett. 8, 709-720. AMMAN D., Gi)GGI M., PRETSCH E. & SIMON W, (1979) Design and use of Ca 2 + of selective microelectrodes. In Detection and Measurements of Free Ca 2 + Ions in Cells (Edited by ASHLEY C. C. & CAMPBELL A. K.). North Holland, Amsterdam. BAKER P. F. (1975) Transport and metabolism of calcium ions in nerve cells. In Calcium Movements in Excitable Cells. Pergamon Press, Oxford. BLANSTEIN M. P. & HODGKIN A. L. (1969) The effect of cyanide on the efflux of calcium from squid axons. J. Physiol. Lond. 200, 497-527. BLANSTEIN M. P., RATZLOFF R. W. t~ SCHWEITZER E. S. (1978) Calcium buffering in presynaptic nerve terminals. J. gen. Physiol. 72, 43-66. BROWN K. T. ,~¢ FLAMING D. G. (1974) Bevelling of fine micropipette electrodes by a rapid precision method. Science, N. Y 185, 693-695. BROWN A. M., WALKER I. L. t~ SUTTON R. B. (1970) Increased chloride conductance as the proximate cause of hydrogen ion concentration effects in Aplysia neurons J. gen. Physiol. 56, 559-582. CLADWELL P. C. (1970) Calcium chelation and buffers. In Calcium and Cellular Function, pp. 10-16. Macmillan, New York. CHRISTOFFERSEN G. R. J. (1973) Calcium concentration and chloride conductance. Comp. Biochem. Physiol. 46A, 371-389. CHRISTOEFERSEN G. R..J. & SIMONSEN L. (1977) Ca 2÷sensitive microelectrode: intracellular steady-state measurements in nerve cells. Acta physiol, scand. 101, 492~494. CHRISTOFFERSEN G. R. J. & SIMONSEN L. (1979) Ca 2+sensitive microelectrode: Intracellular use in nerve cells. Detection and Measurements of Free Ca Ions in Cells (Edited by ASHLEYC. C. & CAMPBELLA. K.) North Holland, Amsterdam. KERKUT G. A. & THOMASR. C. (1965) An electrogenic sodium pump in small neurons. Comp. Biochem. Physiol. 14, 167-183. OWEN J. D. (1976) The determination of the stability constants for calcium-EGTA. Biochem. biophys. Acta 451, 321-325. SIMONSEN L. & CHRISTOFEERSEN G. R. J. (1979) Intracellular Ca 2 ÷-activity in Helix neurons: Effects of extracellular Ca 2÷, H +, Na ÷ and N~-. Comp. Biochem. Physiol. 63A, p. 615-618. THOMAS R. C. (1969) Membrane current and intracellular sodium in a snail neuron during excursion of injected sodium. J. Physiol, Lond. 201, 495-514.