867
J. Phy8iol. (1977), 265, pp. 867-879 With 6 text-figurem Printed in Great Britain
THE EFFECT OF CALCIUM INJECTION ON THE INTRACELLULAR SODIUM AND pH OF SNAIL NEURONES
By R. W. MEECH AND R. C. THOMAS From the A.R.C. Unit of Invertebrate Chemistry and Physiology, Department of Zoology, Downing Street, Cambridge CB2 3EJ, and the Department of Physiology, University of Bristol, Bristol B88 lTD
(Received 22 September 1976) SUMMARY
1. Ion-sensitive glass micro-electrodes were used to measure the intracellular pH (pH,) and the intracellular sodium ion concentration, [Na+]i, in identified Helix aspersa neurones. 2. The injection of small volumes of 01 M-CaC12, which increased the membrane potential by 10-15 mV for 1-2 min, had little or no effect on [Na+]i. Increases of up to 1 mM in [Na+]i could be reversibly induced by larger injections. 3. Calcium injection caused an immediate decrease in pH,, which appeared to be directly proportional to the amount of calcium injected. Injections causing hyperpolarizations of 10-20 mV which recovered in 2-5 min caused pH, decreases of 0-04-0 15 units. After each of these injections both pHi and the membrane potential recovered exponentially but with different time constants. 4. The injection of calcium at a low rate could decrease pHi without affecting the membrane potential. 5. Neither membrane potential nor pH1 were- affected by the injection of small volumes of 0.1 M-MgCl2. Injection of CoCl2 produced a large transient decrease in pHi but no significant change in membrane potential. 6. Exposure of the cell to saline equilibrated with 2-5 % CO2 greatly reduced the pHi decrease caused by calcium injection but had only small effects on the membrane potential response. 7. It is concluded that most of the injected calcium is exchanged for protons inside the cell. INTRODUCTION Injection of calcium into nerve cells causes an increase in potassium permeability which can be ascribed to a direct effect of calcium on the
intracellular surface of the membrane (Meech, 1972, 1974; Krnjevic & 30-2
R~. W. MEECH AND R. C. THOMAS Lisiewicz, 1972). A large proportion of the injected calcium appears to be rapidly removed from the cytoplasm. It has been assumed that much of this calcium is taken up into intracellular organelles such as mitochondria and pumped from the cell in exchange for sodium ions (see Baker, 1972). The uptake of cations by isolated mitochondria is associated with the production of hydrogen ions (Bartley & Amoore, 1958; Chappell, Greville & Bicknell, 1962). The possibility that a rise in intracellular calcium may lead to a decrease in pH1, the intracellular pH, although discussed by Baker (1972), has not yet been investigated. We have now used Na+- -and pH-sensitive glass micro-electrodes (Thomas, 1972, 1974) to show that calcium injection into snail neurones causes only small increases in internal sodium [Na+]i but relatively large changes in pH1. 868868
METHODS
The suboesophageal ganglion of the snail, Helix a8per8a, waa dissected as described previously (Thomas, 1974). A single identified neurone, cell A, was used in each preparation (Meech, 1974). The normal (nominally CO.-free) saline had a pH of 7*5, was in equilibrium with air and had the following composition (in mm): KCl, 4; NaCl, 90; CaCl2, 7; MgCl2, 5; HEPES (2-N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid), 10. The CO2 saline was saturated with 2-5 % CO02in 02, and had the same composition and pH as the C02-free saline except that it contained 23 mmNaHCO3 instead of HEPES, only 80 mm-NaCl and 0- I mm phosphate. All experiments were done at room temperature, 20-25o C. Conventional glass micropipettes filled with 2-5 m-KCl were used to measure the membrane potential and to pass current. The Na+. sensitive and pH-sensitive micro-electrodes were prepared as previously described (Thomas, 1972, 1974, 1976a,b). The membrane potential micropipette was connected to a unity gain current amplifier, the output of which was led to an oscilloscope, pen recorder and, via a low pasa filter, to the low impedance input of a vibrating capacitor electrometer. The ion-sensitive micro-electrode was connected to the high impedance input of the electrometer, the output of which was recorded on another channel of the penrecorder. Calcium chloride was pressure-injected as previously described (Meech, 1972, 1974) from glass micropipettea prepared with tip diameters of 1-2 ,sm. The injection pipette normally contained 100 mMMCaCl2 mixed with 100 mm-KCI but in certain experiments the dye Fast Green FCF was also present (1 % w.v.). A photon-coupled floating current-clamp was used to pass a 5-40 nA current between the injection pipette and an intracellular KCl-filled micropipette. This was necessary to reduce the risk of the calcium filled micropipette becoming blocked. RESULTS
The effect of calcium injection on intracellular Bodium Calcium efflux from squid axons, and cardiac tissue is reduced by removing external sodium (Baker, Blaustein, Hodgkin & Steinhardt, 1969; Reuter & Seitz, 1968). This suggests a coupling between calcium efflux
Ca INJECPION AND INTRACELLULAR pH 869 and some component of sodium influx. If reversible exchange of calcium and sodium ions occurs across the outer membrane of Helix neurones there should be an appreciable increase in [Na+]1 following the injection of small volumes of 100 mm-CaCl2. Fig. 1 shows recordings from an experiment in which a Na+-sensitive micro-electrode was used to measure [Na+]i in cell A. The upper trace shows the membrane potential of the neurone together with CaCI2
[ KI
100
CaCI2
5 min
CaCI2
Ca21 injections
[Nai] 2
Fig. 1. Pen-recording of an experiment to measure the effect of CaCl2 injection on internal sodium, [Na+],. The voltage (Em) recorded by an intracellularKCI-filled micropipette is at the top, and [Nae]1, recorded by a sodiumsensitive micro-electrode, is at the bottom. The sodium electrode was inserted into the cell first, as shown by the large deflexion near the beginning of the lower trace. Next, the KCl-filled Em-recording micropipette was serted, as indicated by the simultaneous deflexions of the E. and [Na+]i records. Then a second KCI-ifilled micropipette was inserted as indicated above the E. record. A 5 nA current passed between this KOL-ifilled pipette and an external CaCl2.filled injection pipette decreased Em while the CaCl2ifilled pipette was extracellular. When inserted, the CaCl2-filled pipette was found to be blocked. It was removed and replaced as indicated. During this exchange [Na+], was not recorded. Pressure-injection of CaCl2 is indicated below Em. The cell was in nominally C02-free saline throughout. attenuated spontaneous action potentials. The lower trace gives [Na+]i. At the first arrow a third micropipette was inserted into the cell. Current passed between this KCl-filled micropipette and a calcium injection pipette positioned outside the cell caused a temporary depolarization of the membrane until the calcium pipette penetrated the neurone (second arrow). The calcium pipette was found to be blocked. It was removed (third arrow) and at the fourth arrow replaced with a fresh calcium-filled
~R. W. MEECH AND R. C. THOMAS micropipette. Injection of calcium then produced an immediate hyperpolarization of the membrane but no change in [Na+]1. A second calcium injection lasted 15 sec and produced a recorded increase in [Na+]i of approximately 0-1 mmw. However, 75 sec later the third calcium injection, which lasted 25 sec, increased [Na+], from 4-9 to 6 1 mm. The significance of these findings depends on an estimate of the amount of calcium injected and on the Na+: Cal+ coupling ratio of the exchange mechanism. The ratio appears to depend on the ATP level (Baker & McNaughton, 1976c) and on the intracellular concentration of ionized calcium (Mullins & Brinley, 1975). It may be as high as 3Na+: Wea2 in dialysed squid axons (Mullins & Brinley, 1975). A method of calculating the amount of calcium 'Injected is discussed later but our best estimate is that a 15 sec injection like that in Fig. 1 should raise the intracellular calcium level by 3-5 mm. Consequently exchange of intracellular calcium for sodium appears to play only a minor role in regulating cytoplasmic calcium after injection. It may be more significant under conditions of increased calcium influx when the ionized calcium at the internal surface may reach high levels. 870870
The effect of calcium injection on1 intracelluiar pH Injection of calcium into cell A produces a decrease in pH,, as shown in Fig. 2. This experiment was similar to that shown in Fig. 1 except that the Na+-sensitive electrode was replaced by one sensitive to pH. On this occasion a dye, Fast Green FCF, was included in the injection mixture. Following the insertion of the pH-sensitive micro-electrode and two KCI-filled micropipettes a depolarizing current was injected into the cell as before. The membrane potential (upper trace) returned to its resting state when the calcium injection pipette penetrated the cell. These manipulations had very little effect on pH1. The effect of calcium injection on the membrane potential depends on both the quantity of calcium injected and its rate of entry (Meech, 1976). Fig. 2 shows four separate injections of CaCl2. Typical hyperpolarizing responses followed the first two injections. The injection pipette appeared to become blocked at this point, however, because a third injection failed to hyperpolarize the membrane. This is not an altogether unknown phenomenon in this type of experiment (see Fig. 1) and generally removal of the injection pipette from the cell confirms that normal injection pressures (up to 30 lb./in. ) are insufficient to eject fluid from the tip. On this occasion however entry of green dye into the cell indicated that the blockage was only partial. Increased pressure (fourth injection) was sufficient to generate a prolonged hyperpolarizing response associated with an immediate decrease in pH1 of close to 0-3 unit. The first two injections also generated immediate pH1
Ca INJECTION AND INTRACELLULAR pH 871 changes. The slow fall in pH, during the third injection presumably therefore results from the slow rate of entry of calcium. The absence of a hyperpolarizing response indicates that the increase in ionized calcium is restricted to the region of cytoplasm close to the injection electrode (see also Podolsky & Costantin, 1964; Rose & Loewenstein, 1975). The implication of this experiment is that some of the effects seen in cells iontophoretically injected with calcium ions may be secondary and mediated by changes in pH1. O -- |
r
minI ~~~~~~~~~~5
E40 LU
60 Ca2' injections
80 0
7-0~~~~~
o
-
0
_
8-00
Fig. 2. Pen-recording of an experiment to measure the effect of CaCl2 injection on pH,. The membrane potential (Em) recorded by a KCl-filled micropipette is at the top and pHi, recorded by a pH-sensitive micro-electrode, is at the bottom. The pH-electrode was inserted into the cell first followed by the Em-recording micropipette. A second KCl-filled micropipette was then inserted and used to pass 5 nA current to a CaCl2-filled injection micropipette outside the cell. This current depolarized Em until the CaCl2 pipette penetrated the cell. Pressure injection of CaCl2 is indicated below Em. The cell was in nominally C02-free saline throughout.
The effect of calcium injection on both intracellular sodium and intracellular pH A more direct comparison of the effect of calcium injection on [Na+]i and pH, was achieved by taking measurements first with a Na+-sensitive electrode as described above and then replacing it with one sensitive to pH. A series of 30 sec calcium injections produced little or no change in [Na+]1 but, during a subsequent series of 30 sec injections in which comparable membrane hyperpolarizations were recorded, pHi fell by approximately 0 3 pH unit. (Fig. 3 is taken from the latter half of this experiment.)
JB. W. MEECH AND R. C. THOMAS The intracellular buffering power of snail neurones under the conditions of the experiment (nominally C02-free saline) is 29-5 m-equiv H+/pH unit per litre (Thomas, 1976a). This means that almost 9 m-equiv H+/L. were released into the cytoplasm following each calcium injection. Under the same conditions [Na+]1 increased by less than 041 mm. If much of the 872 872
min ~~~~~~~~~~~~~10
I
20 El
>
Ca2' injections
7.0
Fig. 3. Part of an experiment to test the effect of the duration of CaCl2 injection on the change in E. (top trace) and pH, (bottom trace). CaCI2 Was injected (as indicated below E.) at the same pressure for periods of 15, 5, 10 and 20 sec respectively. The cell was bathed in nominally C02-free saline during this part of the experiment. An earlier part of the experiment (referred to in the text) showed that a similar injection of 30 sec produced insignificant changes in [Na+]i.
injected calcium is rapidly accumulated by intracellular organelles, the concentration of ionized calcium at the internal surface of the cell membrane may be maintained at a low level in spite of relatively large calcium injections. Thus although the experiment indicated that much of the injected calcium is exchanged for protons it does not exclude the possibility that a large proportion of the calcium at the membrane may be exchanged for sodium. The effect of injection duration on the change in pHi In the absence of Fast Green FCF each of a series of similar calcium injections would in many cases give consistent changes of pHi and membrane potential. Figure 3 shows a record of such a series in which the pressure of each injection was constant and the induced pH1 change was approximately proportional to the duration of the injection. This is shown in Fig. 4, which also shows that after each injection the membrane potential and the pH, change recovered in a simple exponential fashion but with different time constants.
Ca INJECTION AND INTRACELLULAR pH
873
The effect of CO2 on the calcium induced pHi change The intracellular buffering power of snail neurones can be greatly increased by exposing the preparation to C02 saline without changing the external pH (Thomas, 1976a). A saline with about 20 mM-NaHCO3 and equilibrated with 2-5 % C02 gives a reasonable approximation to the in vivo condition (Burton, 1969). As Fig. 5 shows, exposure to the C02 A
0 10
B
.
0
E10
-J
I E 8
.to _Jo
0-03LI0
6
0.16 r
3 -
9 6 Time (min)
12
0 12
4
E
W406 Ca2+ injections
80
0
a
7-0 ~
8S5
~
0
0
0
H
25 % CO2, pH75
Fig. 5. Part of an experiment to show the effect of HCO -CO2, buffered saline on the response of Em (top trace) and pH, (bottom trace) to CaCl2 injection. The CaCl2 injections indicated below Em were made using the same pressure and duration. For the period indicated the cell was exposed to saline equilibrated with 2-5 % C02, adjusted to pH 7-5 by the addition of 23 mM-NaHCO3. At other times the cell was in nominally C02-free saline.
gives a value of 3-8 m-equiv/l. for the released protons. A possible reason for the difference between the two figures is that the injected calcium may be accumulated more rapidly in C02-containing saline (see Fig. 5). Recovery of pH,, which occurred in an exponential fashion, can be attributed to the activity of a proton pump (Thomas, 1976a). Thus the recorded change in pH, will give an underestimate of the Ca2+/H+ exchange. Extrapolation of a semi-log. plot of pHi recovery against time to the end of the injection period should compensate for most of the extruded protons. In C02-free saline this extrapolated value was approximately 0 15 pH unit giving 4.4 m-equiv H+/l. as the concentration of released protons. This figure is in reasonable agreement with the value in C02 saline which increased to 5-3 m-equiv/l. This experiment shows that small changes in pH, may be expected with calcium injection even in cells bathed in HCO3-CO2 buffer in conditions closer to those believed to exist in vivo.
Ca INJECTION AND INTRACELLULAR pH
875
Injection of magnesium chloride Small quantities of 100 mM-MgCl2 and Fast Green FCF were injected into cell A as a control. Volumes of the mixture comparable to those used during the calcium injection experiments could be injected by comparing the colour of the injected cells. Injection of magnesium under these conditions had little or no effect on the membrane potential or on pH1. or
E
E
10 min
CoCI, KCI~
>20 L
LLJ~EEM! 40t 60 L 40
Co0
I
d
injections
705 8-0
L
Fig. 6. Pen-recording of an experiment to show the effect of cobalt chloride injection on Em and pHi. Em measured byaKCl-Ifilledmicropipetteis shown on the top trace andpH, at the bottom. Cobalt chloride was pressure injected as indicated below Em.
Cobalt injection The large decrease in pH, following calcium injection is not responsible for the hyperpolarization observed since injection of HCl produces, if anything, a small depolarization of the membrane potential (Thomas, 1976a). Injection of cobalt chloride also has little effect on the membrane potential but produces a large decrease in pHi as shown in Fig. 6. The effect of lanthanum injection A preliminary experiment showed that injection of a solution containing both calcium chloride and lanthanum chloride generated an unusually small fall in pH1. The recovery of pHi was also considerably faster than the recovery observed following injection of calcium chloride alone. The calcium-induced membrane hyperpolarization, on the other hand, considerably outlasted the pH change. DISCUSSION
The intracellular concentration of ionized calcium in invertebrate nerves probably lies between 10-8 and 10-7 M when the external calcium concentration is 10-2 M (Baker, Hodgkin & Ridgway, 1971; Meech & Standen, 1975; Dipolo, Requena, Brinley, Mullins, Scarpa & Tiffert, 1976).
8. W. MEECH AND R. C. THOMAS This low level is increased by depolarization, by exposure to sodium-free solution and by metabolic poisons. Regulation of intracellular calcium levels appears to involve a calcium extrusion system in the plasma membrane and intracellular sequestration by macromolecules and organelles within the cytoplasm (see Baker, 1972). 876
Intracellular sequestration of calcium Mitochondria are known to take up cations in exchange for protons (Bartley & Amoore, 1958). The large and rapid changes in the pHi of snail neurones during calcium injection suggest that much of the injected calcium is accumulated in this way. The suggestion receives some support from the effect of lanthanum since the uptake of calcium into mitochondria is blocked by low levels of lanthanum ions (Mela, 1968). The active binding of calcium by mitochondria has been extensively studied since it was first reported by Vasington & Murphy (1961). Experiments using pH electrodes in suspensions of isolated mitochondria show that in the presence of chloride ions each calcium taken up is associated with the release of one proton (Chappell et al. 1962) although in the presence of phosphate the H+/Ca2+ ratio is closer to 07 (Chance, 1965). If each calcium is exchanged for one proton in Helix neurones a calcium injection which released 4-5 m-equiv H+/l. (for example, see Fig. 5) must have increased the intracellular calcium concentration by at least 4-5 mm. Yet at the cell membrane the local ionized calcium concentration probably increased to a value a thousand times less than this (Meech, 1974). The rate of uptake or release of protons at a fixed site will tend to be increased when the pH-buffering capacity of the medium is increased. The presence of pH buffer facilitates the diffusion of protons (Engasser & Horvath, 1974) and reduces the possibility of large localized changes in pH in the region of the site of calcium/hydrogen exchange. The intracellular buffering power of snail neurones is about three times greater in C02 saline than it is in the absence of C02 (Thomas, 1976a) and C02 saline reduces the delay in the recovery of the cell following calcium injection. In addition HC00-002 buffer can replace phosphate as a source of counter ion for the accumulation of calcium by respiring mitochondria (Elder & Lehninger, 1973). This may also stimulate uptake but there is no evidence for an associated change in the Ca2+: H+ ratio.
Calcium exchange at the plasma membrane A large proportion of the calcium efflux from squid axons is activated by, but apparently not exchanged for, external calcium (Baker & McNaughton, 1976a, b). It is possible that this process may contribute to the changes in pHi reported above, either as a result of ATP hydrolysis or
Ca INJECTION AND INTRACELLULAR pH 877 by direct exchange with extracellular protons. The sensitivity of this sodium-independent calcium efflux, like that of mitochondrial calcium/ hydrogen exchange, to low concentrations of lanthanum make its contribution difficult to assess and a contribution of ATP hydrolysis to the calcium-induced fall in pH, remains a possibility. Calcium injection produced little change in [Na+]i unless the injection was prolonged. But the situation niay be different during depolarization of the cell membrane since under conditions of enhanced calcium influx the concentration of calcium at the inner membrane surface may rise to high levels. On the other hand at least part of the observed increase in [Na+]i may be a secondary result of the calcium-induced decrease in pH,. Injection of HCl into Helix neurones also produces a small increase in [Na+]i (R. C. Thomas, unpublished).
Effect of pHi on membrane properties The relationship between the fall in pHi and pCa, caused by calcium injection depends to a large extent on the rate at which calcium enters the cytoplasm (see Fig. 2). A low rate such as that produced by iontophoretic injection may generate a large fall in pHi but little or no change in pCal. It is important therefore to distinguish between direct effects of internal calcium and secondary effects of decreased pHi. In snail neurones a fall in pHi caused by exposing the cells to pH 7*1 CO2 saline reduces the potassium current recorded under voltage clamp (G. V. Lees and R. W. Meech, unpublished). A recent report that calcium injection also depresses these currents (Heyer & Lux, 1976) can perhaps also be attributed to the effects of reduced pHi, since the immediate effect of calcium injection is to increase the potassium conductance (Meech, 1972, 1976). An effect of C02 saline on potassium conductance has previously been reported in barnacle photoreceptors (Brown & Meech, 1976). This is associated with a reduction in light sensitivity of the receptor and with a fall in pHi of 0-2-0-3 unit. Iontophoretic injection of calcium into Limulus photoreceptors over a period of 2 min also progressively reduces the sensitivity of the receptor to light (Lisman & Brown, 1972). It seems possible that this effect also is a secondary phenomenon associated with an induced fall in pHi (Meech & Brown, 1976).
878
R. W. MEECH AND R. C. THOMAS REFERENCES
BAKER, P. F. (1972). Transport and metabolism of calcium ions in nerve. Prog. Biophy8. moles. Biol. 24, 177-223. BAKER, P. F., BLAUSTEIN, M. P., HODGKIN, A. L. & STEINHARDT, R. A. (1969). The influence of calcium on sodium efflux in squid axons. J. Phy8iol. 200, 431-458. BAKER, P. F., HODGKIN, A. L. & RIDGWAY, E. B. (1 97 1). Depolarization and calcium entry in squid giant axons. J. Phy8iol. 218, 709-755. BAKER, P. F. & MCNAUGHTON, P. A. (1976a). Calcium-dependent calcium efflux from intact squid axons: Ca-Ca exchange or net extrusion? J. Physiol. 258, 97-98P. BAKER, P. F. & MCNAUGHTON, P. A. (1976b). Kinetics and energetic of calcium efflux from intact giant squid axons. J. Physiol. 259, 103-144. BAKER, P. F. & MCNAUGHTON, P. A. (1976c). The effect of membrane potential on the calcium transport systems in squid axons. J. Phy8iol; 260, 24-25P. BARTLEY, W. & AMOORE, J. E. (1958). The effects of manganese on the solute content of rat-liver mitochondria. Biochem. J. 69, 348-360. BROWN, H. M. & MEECH, R. W. (1976). Intracellular pH and light adaptation in barnacle photoreceptors. J. Physiol. 263, 218P. BURTON, R. F. (1969). Buffers in the blood of the snail. Helix pomatia L. Comp. Biochem. Phyriol. 29, 919-930. CHANCE, B. (1965). The energy-linked reaction of calcium with mitochondria. J. biol. Chem. 240, 2729-2748. CHAPPELL, J. B., GREVILLE, G. D. & BICKNELL, K. E. (1962). Stimulation of respiration of isolated mitochondria by manganese ions. Biochem. J. 84, 61 P. DIPOLO, R., REQUENA, J., BRINLEY, F.J., JR, MULLINS, L. J., SCARPA, A. & TIFFERT, T. (1976). Ionized calcium concentrations in squid axons. J. gen. Physiol. 67, 433-467. ELDER, J. A. & LEHNINGER, A. L. (1973). Respiration-dependent transport of carbon dioxide into rat liver mitochondria. Biochemistry, N.Y. 12, 976-982. ENGASSER, J.-M. & HORVATH, C. (1974). Buffer-facilitated proton transport. pH profile of bound enzymes. Biochim. biophy8. Acta 358, 178-192. HEYER, C. B. & Lux, H. D. (1976). Control of the delayed outward potassium currents in bursting pace-maker neurones of the snail Helix pomatia. J. Physiol. 262, 349-382. KRNJEVI6, K. & LISIEWICZ, A. (1972). Injections of calcium ions into spinal motoneurones. J. Physiol. 225, 363-390. LISMAN, J. E. & BROWN, J. E. (1972). The effects of intracellular iontophoretic injection of calcium and sodium ions on the light response of Limulus ventral photoreceptors. J. gen. Physiol. 59, 701-719. MEECH, R. W. (1972). Intracellular calcium injection causes increased potassium conductance in Aply8ia nerve cells. Comp. Biochem. Physiol. 42A, 493-499. MEECH, R. W. (1974). The sensitivity of Helix aspersa neurones to injected calcium ions. J. Physiol. 237, 259-277. MEECH, R. W. (1976). Intracellular calcium and the control of membrane permeability. Symp. Soc. exp. Biol. 30, 161-191. MEECH, R. W. & BROWN, H. M. (1976). Invertebrate photoreceptors: a survey of recent experiments on photoreceptors from Balanue and Litnulus. In Perspectives in Experimental Biology, vol. 1, ed. SPENCER DAVIES, P., pp. 331-351. Oxford and New York: Pergamon Press. MEECH, R. W. & STANDEN, N: B. (1975). Potassium activation in Helix aspersa neurones under voltage clamp: a component mediated by calcium influx. J. Physiol. 249. 211-239.
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MELA, L. (1968). Interactions of La3+ and local anesthetic drugs with mitochondrial Ca++ and Mn++ uptake. Archs Biochem. Biophy8. 123, 286-293. MULLINS, L. J. & BRINLEY, F. J., JR (1975). Sensitivity of calcium efflux from squid axons to changes in membrane potential. J. gen. Physiol. 65, 135-152. PODOLSKY, R. J. & COSTANTIN, L. L. (1964). Regulation by calcium of the contraction and relaxation of muscle fibers. Fedn Proc. 23, 933-939. REUTER, H. & SEITZ, N. (1968). The dependence of calcium efflux from cardiac muscle on temperature and external ion composition. J. Physiol. 195, 451-470. Rosk, B. & LOEWENSTEIN, W. R. (1975). Permeability of cell junction depends on local cytoplasmic calcium activity. Nature, Lond. 254, 250-252. THOMAS, R. C. (1972). Intracellular sodium activity and the sodium pump in snail neurons. J. Physiol. 220, 55-71. THOMAS, R. C. (1974). Intracellular pH of snail neurones measured with a new pH-sensitive glass micro-electrode. J. Physiol. 238, 159-180. THOMAS, R. C. (1976a). The effect of carbon dioxide on the intracellular pH and buffering power of snail neurones. J. Physiol. 255, 715-735. THOMAS, R. C. (1976b). Construction and properties of recessed-tip micro-electrodes for sodium and chloride ions and pH. In Ion and Enzyme Electrodes in Biology and Medicine, ed. KESSLER, M., CLARK, L. C., LUBBERS, D. W., SILVER, I. A. & SIMON, W., pp. 141-148. Munich: Urban and Schwarzenberg. VASINGTON, F. D. & MURPHY, J. V. (1961). Active binding of calcium by mitochondria. Fedn Proc. 20, 146.
J. Phy8iol. (1977), 265, pp. 867-879 With 6 text-figurem Printed in Great Britain
THE EFFECT OF CALCIUM INJECTION ON THE INTRACELLULAR SODIUM AND pH OF SNAIL NEURONES
By R. W. MEECH AND R. C. THOMAS From the A.R.C. Unit of Invertebrate Chemistry and Physiology, Department of Zoology, Downing Street, Cambridge CB2 3EJ, and the Department of Physiology, University of Bristol, Bristol B88 lTD
(Received 22 September 1976) SUMMARY
1. Ion-sensitive glass micro-electrodes were used to measure the intracellular pH (pH,) and the intracellular sodium ion concentration, [Na+]i, in identified Helix aspersa neurones. 2. The injection of small volumes of 01 M-CaC12, which increased the membrane potential by 10-15 mV for 1-2 min, had little or no effect on [Na+]i. Increases of up to 1 mM in [Na+]i could be reversibly induced by larger injections. 3. Calcium injection caused an immediate decrease in pH,, which appeared to be directly proportional to the amount of calcium injected. Injections causing hyperpolarizations of 10-20 mV which recovered in 2-5 min caused pH, decreases of 0-04-0 15 units. After each of these injections both pHi and the membrane potential recovered exponentially but with different time constants. 4. The injection of calcium at a low rate could decrease pHi without affecting the membrane potential. 5. Neither membrane potential nor pH1 were- affected by the injection of small volumes of 0.1 M-MgCl2. Injection of CoCl2 produced a large transient decrease in pHi but no significant change in membrane potential. 6. Exposure of the cell to saline equilibrated with 2-5 % CO2 greatly reduced the pHi decrease caused by calcium injection but had only small effects on the membrane potential response. 7. It is concluded that most of the injected calcium is exchanged for protons inside the cell. INTRODUCTION Injection of calcium into nerve cells causes an increase in potassium permeability which can be ascribed to a direct effect of calcium on the
intracellular surface of the membrane (Meech, 1972, 1974; Krnjevic & 30-2
R~. W. MEECH AND R. C. THOMAS Lisiewicz, 1972). A large proportion of the injected calcium appears to be rapidly removed from the cytoplasm. It has been assumed that much of this calcium is taken up into intracellular organelles such as mitochondria and pumped from the cell in exchange for sodium ions (see Baker, 1972). The uptake of cations by isolated mitochondria is associated with the production of hydrogen ions (Bartley & Amoore, 1958; Chappell, Greville & Bicknell, 1962). The possibility that a rise in intracellular calcium may lead to a decrease in pH1, the intracellular pH, although discussed by Baker (1972), has not yet been investigated. We have now used Na+- -and pH-sensitive glass micro-electrodes (Thomas, 1972, 1974) to show that calcium injection into snail neurones causes only small increases in internal sodium [Na+]i but relatively large changes in pH1. 868868
METHODS
The suboesophageal ganglion of the snail, Helix a8per8a, waa dissected as described previously (Thomas, 1974). A single identified neurone, cell A, was used in each preparation (Meech, 1974). The normal (nominally CO.-free) saline had a pH of 7*5, was in equilibrium with air and had the following composition (in mm): KCl, 4; NaCl, 90; CaCl2, 7; MgCl2, 5; HEPES (2-N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid), 10. The CO2 saline was saturated with 2-5 % CO02in 02, and had the same composition and pH as the C02-free saline except that it contained 23 mmNaHCO3 instead of HEPES, only 80 mm-NaCl and 0- I mm phosphate. All experiments were done at room temperature, 20-25o C. Conventional glass micropipettes filled with 2-5 m-KCl were used to measure the membrane potential and to pass current. The Na+. sensitive and pH-sensitive micro-electrodes were prepared as previously described (Thomas, 1972, 1974, 1976a,b). The membrane potential micropipette was connected to a unity gain current amplifier, the output of which was led to an oscilloscope, pen recorder and, via a low pasa filter, to the low impedance input of a vibrating capacitor electrometer. The ion-sensitive micro-electrode was connected to the high impedance input of the electrometer, the output of which was recorded on another channel of the penrecorder. Calcium chloride was pressure-injected as previously described (Meech, 1972, 1974) from glass micropipettea prepared with tip diameters of 1-2 ,sm. The injection pipette normally contained 100 mMMCaCl2 mixed with 100 mm-KCI but in certain experiments the dye Fast Green FCF was also present (1 % w.v.). A photon-coupled floating current-clamp was used to pass a 5-40 nA current between the injection pipette and an intracellular KCl-filled micropipette. This was necessary to reduce the risk of the calcium filled micropipette becoming blocked. RESULTS
The effect of calcium injection on intracellular Bodium Calcium efflux from squid axons, and cardiac tissue is reduced by removing external sodium (Baker, Blaustein, Hodgkin & Steinhardt, 1969; Reuter & Seitz, 1968). This suggests a coupling between calcium efflux
Ca INJECPION AND INTRACELLULAR pH 869 and some component of sodium influx. If reversible exchange of calcium and sodium ions occurs across the outer membrane of Helix neurones there should be an appreciable increase in [Na+]1 following the injection of small volumes of 100 mm-CaCl2. Fig. 1 shows recordings from an experiment in which a Na+-sensitive micro-electrode was used to measure [Na+]i in cell A. The upper trace shows the membrane potential of the neurone together with CaCI2
[ KI
100
CaCI2
5 min
CaCI2
Ca21 injections
[Nai] 2
Fig. 1. Pen-recording of an experiment to measure the effect of CaCl2 injection on internal sodium, [Na+],. The voltage (Em) recorded by an intracellularKCI-filled micropipette is at the top, and [Nae]1, recorded by a sodiumsensitive micro-electrode, is at the bottom. The sodium electrode was inserted into the cell first, as shown by the large deflexion near the beginning of the lower trace. Next, the KCl-filled Em-recording micropipette was serted, as indicated by the simultaneous deflexions of the E. and [Na+]i records. Then a second KCI-ifilled micropipette was inserted as indicated above the E. record. A 5 nA current passed between this KOL-ifilled pipette and an external CaCl2.filled injection pipette decreased Em while the CaCl2ifilled pipette was extracellular. When inserted, the CaCl2-filled pipette was found to be blocked. It was removed and replaced as indicated. During this exchange [Na+], was not recorded. Pressure-injection of CaCl2 is indicated below Em. The cell was in nominally C02-free saline throughout. attenuated spontaneous action potentials. The lower trace gives [Na+]i. At the first arrow a third micropipette was inserted into the cell. Current passed between this KCl-filled micropipette and a calcium injection pipette positioned outside the cell caused a temporary depolarization of the membrane until the calcium pipette penetrated the neurone (second arrow). The calcium pipette was found to be blocked. It was removed (third arrow) and at the fourth arrow replaced with a fresh calcium-filled
~R. W. MEECH AND R. C. THOMAS micropipette. Injection of calcium then produced an immediate hyperpolarization of the membrane but no change in [Na+]1. A second calcium injection lasted 15 sec and produced a recorded increase in [Na+]i of approximately 0-1 mmw. However, 75 sec later the third calcium injection, which lasted 25 sec, increased [Na+], from 4-9 to 6 1 mm. The significance of these findings depends on an estimate of the amount of calcium injected and on the Na+: Cal+ coupling ratio of the exchange mechanism. The ratio appears to depend on the ATP level (Baker & McNaughton, 1976c) and on the intracellular concentration of ionized calcium (Mullins & Brinley, 1975). It may be as high as 3Na+: Wea2 in dialysed squid axons (Mullins & Brinley, 1975). A method of calculating the amount of calcium 'Injected is discussed later but our best estimate is that a 15 sec injection like that in Fig. 1 should raise the intracellular calcium level by 3-5 mm. Consequently exchange of intracellular calcium for sodium appears to play only a minor role in regulating cytoplasmic calcium after injection. It may be more significant under conditions of increased calcium influx when the ionized calcium at the internal surface may reach high levels. 870870
The effect of calcium injection on1 intracelluiar pH Injection of calcium into cell A produces a decrease in pH,, as shown in Fig. 2. This experiment was similar to that shown in Fig. 1 except that the Na+-sensitive electrode was replaced by one sensitive to pH. On this occasion a dye, Fast Green FCF, was included in the injection mixture. Following the insertion of the pH-sensitive micro-electrode and two KCI-filled micropipettes a depolarizing current was injected into the cell as before. The membrane potential (upper trace) returned to its resting state when the calcium injection pipette penetrated the cell. These manipulations had very little effect on pH1. The effect of calcium injection on the membrane potential depends on both the quantity of calcium injected and its rate of entry (Meech, 1976). Fig. 2 shows four separate injections of CaCl2. Typical hyperpolarizing responses followed the first two injections. The injection pipette appeared to become blocked at this point, however, because a third injection failed to hyperpolarize the membrane. This is not an altogether unknown phenomenon in this type of experiment (see Fig. 1) and generally removal of the injection pipette from the cell confirms that normal injection pressures (up to 30 lb./in. ) are insufficient to eject fluid from the tip. On this occasion however entry of green dye into the cell indicated that the blockage was only partial. Increased pressure (fourth injection) was sufficient to generate a prolonged hyperpolarizing response associated with an immediate decrease in pH1 of close to 0-3 unit. The first two injections also generated immediate pH1
Ca INJECTION AND INTRACELLULAR pH 871 changes. The slow fall in pH, during the third injection presumably therefore results from the slow rate of entry of calcium. The absence of a hyperpolarizing response indicates that the increase in ionized calcium is restricted to the region of cytoplasm close to the injection electrode (see also Podolsky & Costantin, 1964; Rose & Loewenstein, 1975). The implication of this experiment is that some of the effects seen in cells iontophoretically injected with calcium ions may be secondary and mediated by changes in pH1. O -- |
r
minI ~~~~~~~~~~5
E40 LU
60 Ca2' injections
80 0
7-0~~~~~
o
-
0
_
8-00
Fig. 2. Pen-recording of an experiment to measure the effect of CaCl2 injection on pH,. The membrane potential (Em) recorded by a KCl-filled micropipette is at the top and pHi, recorded by a pH-sensitive micro-electrode, is at the bottom. The pH-electrode was inserted into the cell first followed by the Em-recording micropipette. A second KCl-filled micropipette was then inserted and used to pass 5 nA current to a CaCl2-filled injection micropipette outside the cell. This current depolarized Em until the CaCl2 pipette penetrated the cell. Pressure injection of CaCl2 is indicated below Em. The cell was in nominally C02-free saline throughout.
The effect of calcium injection on both intracellular sodium and intracellular pH A more direct comparison of the effect of calcium injection on [Na+]i and pH, was achieved by taking measurements first with a Na+-sensitive electrode as described above and then replacing it with one sensitive to pH. A series of 30 sec calcium injections produced little or no change in [Na+]1 but, during a subsequent series of 30 sec injections in which comparable membrane hyperpolarizations were recorded, pHi fell by approximately 0 3 pH unit. (Fig. 3 is taken from the latter half of this experiment.)
JB. W. MEECH AND R. C. THOMAS The intracellular buffering power of snail neurones under the conditions of the experiment (nominally C02-free saline) is 29-5 m-equiv H+/pH unit per litre (Thomas, 1976a). This means that almost 9 m-equiv H+/L. were released into the cytoplasm following each calcium injection. Under the same conditions [Na+]1 increased by less than 041 mm. If much of the 872 872
min ~~~~~~~~~~~~~10
I
20 El
>
Ca2' injections
7.0
Fig. 3. Part of an experiment to test the effect of the duration of CaCl2 injection on the change in E. (top trace) and pH, (bottom trace). CaCI2 Was injected (as indicated below E.) at the same pressure for periods of 15, 5, 10 and 20 sec respectively. The cell was bathed in nominally C02-free saline during this part of the experiment. An earlier part of the experiment (referred to in the text) showed that a similar injection of 30 sec produced insignificant changes in [Na+]i.
injected calcium is rapidly accumulated by intracellular organelles, the concentration of ionized calcium at the internal surface of the cell membrane may be maintained at a low level in spite of relatively large calcium injections. Thus although the experiment indicated that much of the injected calcium is exchanged for protons it does not exclude the possibility that a large proportion of the calcium at the membrane may be exchanged for sodium. The effect of injection duration on the change in pHi In the absence of Fast Green FCF each of a series of similar calcium injections would in many cases give consistent changes of pHi and membrane potential. Figure 3 shows a record of such a series in which the pressure of each injection was constant and the induced pH1 change was approximately proportional to the duration of the injection. This is shown in Fig. 4, which also shows that after each injection the membrane potential and the pH, change recovered in a simple exponential fashion but with different time constants.
Ca INJECTION AND INTRACELLULAR pH
873
The effect of CO2 on the calcium induced pHi change The intracellular buffering power of snail neurones can be greatly increased by exposing the preparation to C02 saline without changing the external pH (Thomas, 1976a). A saline with about 20 mM-NaHCO3 and equilibrated with 2-5 % C02 gives a reasonable approximation to the in vivo condition (Burton, 1969). As Fig. 5 shows, exposure to the C02 A
0 10
B
.
0
E10
-J
I E 8
.to _Jo
0-03LI0
6
0.16 r
3 -
9 6 Time (min)
12
0 12
4
E
W406 Ca2+ injections
80
0
a
7-0 ~
8S5
~
0
0
0
H
25 % CO2, pH75
Fig. 5. Part of an experiment to show the effect of HCO -CO2, buffered saline on the response of Em (top trace) and pH, (bottom trace) to CaCl2 injection. The CaCl2 injections indicated below Em were made using the same pressure and duration. For the period indicated the cell was exposed to saline equilibrated with 2-5 % C02, adjusted to pH 7-5 by the addition of 23 mM-NaHCO3. At other times the cell was in nominally C02-free saline.
gives a value of 3-8 m-equiv/l. for the released protons. A possible reason for the difference between the two figures is that the injected calcium may be accumulated more rapidly in C02-containing saline (see Fig. 5). Recovery of pH,, which occurred in an exponential fashion, can be attributed to the activity of a proton pump (Thomas, 1976a). Thus the recorded change in pH, will give an underestimate of the Ca2+/H+ exchange. Extrapolation of a semi-log. plot of pHi recovery against time to the end of the injection period should compensate for most of the extruded protons. In C02-free saline this extrapolated value was approximately 0 15 pH unit giving 4.4 m-equiv H+/l. as the concentration of released protons. This figure is in reasonable agreement with the value in C02 saline which increased to 5-3 m-equiv/l. This experiment shows that small changes in pH, may be expected with calcium injection even in cells bathed in HCO3-CO2 buffer in conditions closer to those believed to exist in vivo.
Ca INJECTION AND INTRACELLULAR pH
875
Injection of magnesium chloride Small quantities of 100 mM-MgCl2 and Fast Green FCF were injected into cell A as a control. Volumes of the mixture comparable to those used during the calcium injection experiments could be injected by comparing the colour of the injected cells. Injection of magnesium under these conditions had little or no effect on the membrane potential or on pH1. or
E
E
10 min
CoCI, KCI~
>20 L
LLJ~EEM! 40t 60 L 40
Co0
I
d
injections
705 8-0
L
Fig. 6. Pen-recording of an experiment to show the effect of cobalt chloride injection on Em and pHi. Em measured byaKCl-Ifilledmicropipetteis shown on the top trace andpH, at the bottom. Cobalt chloride was pressure injected as indicated below Em.
Cobalt injection The large decrease in pH, following calcium injection is not responsible for the hyperpolarization observed since injection of HCl produces, if anything, a small depolarization of the membrane potential (Thomas, 1976a). Injection of cobalt chloride also has little effect on the membrane potential but produces a large decrease in pHi as shown in Fig. 6. The effect of lanthanum injection A preliminary experiment showed that injection of a solution containing both calcium chloride and lanthanum chloride generated an unusually small fall in pH1. The recovery of pHi was also considerably faster than the recovery observed following injection of calcium chloride alone. The calcium-induced membrane hyperpolarization, on the other hand, considerably outlasted the pH change. DISCUSSION
The intracellular concentration of ionized calcium in invertebrate nerves probably lies between 10-8 and 10-7 M when the external calcium concentration is 10-2 M (Baker, Hodgkin & Ridgway, 1971; Meech & Standen, 1975; Dipolo, Requena, Brinley, Mullins, Scarpa & Tiffert, 1976).
8. W. MEECH AND R. C. THOMAS This low level is increased by depolarization, by exposure to sodium-free solution and by metabolic poisons. Regulation of intracellular calcium levels appears to involve a calcium extrusion system in the plasma membrane and intracellular sequestration by macromolecules and organelles within the cytoplasm (see Baker, 1972). 876
Intracellular sequestration of calcium Mitochondria are known to take up cations in exchange for protons (Bartley & Amoore, 1958). The large and rapid changes in the pHi of snail neurones during calcium injection suggest that much of the injected calcium is accumulated in this way. The suggestion receives some support from the effect of lanthanum since the uptake of calcium into mitochondria is blocked by low levels of lanthanum ions (Mela, 1968). The active binding of calcium by mitochondria has been extensively studied since it was first reported by Vasington & Murphy (1961). Experiments using pH electrodes in suspensions of isolated mitochondria show that in the presence of chloride ions each calcium taken up is associated with the release of one proton (Chappell et al. 1962) although in the presence of phosphate the H+/Ca2+ ratio is closer to 07 (Chance, 1965). If each calcium is exchanged for one proton in Helix neurones a calcium injection which released 4-5 m-equiv H+/l. (for example, see Fig. 5) must have increased the intracellular calcium concentration by at least 4-5 mm. Yet at the cell membrane the local ionized calcium concentration probably increased to a value a thousand times less than this (Meech, 1974). The rate of uptake or release of protons at a fixed site will tend to be increased when the pH-buffering capacity of the medium is increased. The presence of pH buffer facilitates the diffusion of protons (Engasser & Horvath, 1974) and reduces the possibility of large localized changes in pH in the region of the site of calcium/hydrogen exchange. The intracellular buffering power of snail neurones is about three times greater in C02 saline than it is in the absence of C02 (Thomas, 1976a) and C02 saline reduces the delay in the recovery of the cell following calcium injection. In addition HC00-002 buffer can replace phosphate as a source of counter ion for the accumulation of calcium by respiring mitochondria (Elder & Lehninger, 1973). This may also stimulate uptake but there is no evidence for an associated change in the Ca2+: H+ ratio.
Calcium exchange at the plasma membrane A large proportion of the calcium efflux from squid axons is activated by, but apparently not exchanged for, external calcium (Baker & McNaughton, 1976a, b). It is possible that this process may contribute to the changes in pHi reported above, either as a result of ATP hydrolysis or
Ca INJECTION AND INTRACELLULAR pH 877 by direct exchange with extracellular protons. The sensitivity of this sodium-independent calcium efflux, like that of mitochondrial calcium/ hydrogen exchange, to low concentrations of lanthanum make its contribution difficult to assess and a contribution of ATP hydrolysis to the calcium-induced fall in pH, remains a possibility. Calcium injection produced little change in [Na+]i unless the injection was prolonged. But the situation niay be different during depolarization of the cell membrane since under conditions of enhanced calcium influx the concentration of calcium at the inner membrane surface may rise to high levels. On the other hand at least part of the observed increase in [Na+]i may be a secondary result of the calcium-induced decrease in pH,. Injection of HCl into Helix neurones also produces a small increase in [Na+]i (R. C. Thomas, unpublished).
Effect of pHi on membrane properties The relationship between the fall in pHi and pCa, caused by calcium injection depends to a large extent on the rate at which calcium enters the cytoplasm (see Fig. 2). A low rate such as that produced by iontophoretic injection may generate a large fall in pHi but little or no change in pCal. It is important therefore to distinguish between direct effects of internal calcium and secondary effects of decreased pHi. In snail neurones a fall in pHi caused by exposing the cells to pH 7*1 CO2 saline reduces the potassium current recorded under voltage clamp (G. V. Lees and R. W. Meech, unpublished). A recent report that calcium injection also depresses these currents (Heyer & Lux, 1976) can perhaps also be attributed to the effects of reduced pHi, since the immediate effect of calcium injection is to increase the potassium conductance (Meech, 1972, 1976). An effect of C02 saline on potassium conductance has previously been reported in barnacle photoreceptors (Brown & Meech, 1976). This is associated with a reduction in light sensitivity of the receptor and with a fall in pHi of 0-2-0-3 unit. Iontophoretic injection of calcium into Limulus photoreceptors over a period of 2 min also progressively reduces the sensitivity of the receptor to light (Lisman & Brown, 1972). It seems possible that this effect also is a secondary phenomenon associated with an induced fall in pHi (Meech & Brown, 1976).
878
R. W. MEECH AND R. C. THOMAS REFERENCES
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