University of Bristol, Bristol BS8 1TD

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1 J. Physiol. (1976), 255, pp With 11 text-ft ure Prined in Great Britain THE EFFECT OF CARBON DIOXIDE ON THE INTRACELLULAR ph AND BUFFERING POWER OF SNAIL NEURONES BY R. C. THOMAS From the Department of Physiology, Medical School, University of Bristol, Bristol BS8 1TD (Received 18 August 1975) SUMMARY 1. Intracellular ph (phi) was measured using ph-sensitive glass microelectrodes. The effects on phi of CO2 applied externally and HCO3-, H+ and NH4+ injected iontophoretically, were investigated. 2. The transport numbers for iontophoretic injection into aqueous micro-droplets were found by potentiometric titration to be 0 3 for HCO3- and 0 94 for H+. 3. Exposure to Ringer, ph 7 5, equilibrated with 2-2 % CO2 caused a rapid, but only transient, fall in phi. Within 1 or 2 min phi began to return exponentially to normal, with a time constant of about 5 min. 4. When external CO2 was removed, phi rapidly increased, and then slowly returned to normal. The phi changes with CO2 application or removal gave a calculated intracellular buffer value of about 30 m-equiv H+/pH unit per litre. 5. Injection of HCO3- caused a rise in phi very similar to that seen on removal of external CO2. 6. The phi responses to CO2 application, CO2 removal and HCO3- injection were slowed by the carbonic anhydrase inhibitor acetazolamide. 7. H+ injection caused a transient fall i phi. In CO2 Ringer ph1 fell less and recovered faster than in C02-free Ringer. Calculation of the internal buffer value from the phi responses to H+ and HCO3- injection gave very similar values. 8. The internal buffer value (measured by H+ injection) was greatly increased by exposure to CO2 Ringer. Acetazolamide reduced this effect of CO2, suggesting that the function of intracellular carbonic anhydrase may be to maximize the internal buffering power in CO2. 9. It was concluded that the internal HCO3- was determined primarily by the CO2 level and phi, that internal HC03- made a large contribution to the buffering power, and that after internal acidification phi was

2 716 R. C. THOMAS restored to normal by active transport of H+, OH- or HC03- across the cell membrane. The active transport was much faster in C02 than in C02- free Ringer. INTRODUCTION It is now established that H+ ions are not passively distributed across nerve cell membranes, but little is known about the way that intracellular ph (ph,) is regulated. A constant phi is desirable because of the high chemical reactivity of the H+ ion, especially with substances such as proteins. The two most important factors in the control of phi are the active transport of H+ (or OH- or HCO3-) ions across the cell membrane and the intracellular buffering power. (The buffering power or buffer value of a solution is a measure of its ability to resist ph changes in response to added acid or base. The higher the buffer value the less will the ph change.) The intracellular buffering power will determine the immediate ph1 response to the addition of acid or base, while active transport will determine the longer-term response (see Woodbury, 1965). Carbon dioxide may be related to phi in two ways, directly and indirectly. Directly, through its effect on carbonic acid, and indirectly through its possible role in intracellular buffering. In solution, C02 readily crosses cell membranes (Jacobs, 1940) so that an alteration in extracellular C02 will rapidly alter internal C02. In the absence of carbonic anhydrase, the alteration in internal C02 will then slowly change internal H2CO3 and hence ph1 and internal HCO3-. If snail neurones do contain carbonic anhydrase, then the ph1 change will be faster. The size of the phi change will be related to, and may be used to estimate, the intracellular buffering power. It is well known that extracellularly the C02-bicarbonate system is an important physiological buffer. Intracellularly, however, its role is uncertain, but it could be very important in conditions in which the external C02 is constant, especially if the internal HC03- concentration is determined principally by the C02 and ph1 (as shown in skeletal muscle by Khuri, Bogharian & Agulian, 1974). If C02 is able to cross the cell membrane and react with water sufficiently rapidly, the internal H2CO3 level will be fixed by the external C02. In this situation, internal HC03- will have four times the normal molar buffer value (see Woodbury, 1965) and could well contribute over half the total intracellular buffering power. Intracellular buffer values at constant external C02 levels have not been measured previously, so the extent to which HC03- may contribute is unknown. In an earlier study of the phi of snail neurones (Thomas, 1974a) the

3 C02 AND INTRACELLULAR ph 717 snail Ringer used had a normal ph of 8-0. This ph was too alkaline for stable C02-bicarbonate solutions, and in fact Burton (1969) has found that the blood of aestivating snails has a ph of about 7 5, and a bicarbonate level close to 20 mm. In the present experiments the ph of the Ringer was therefore changed to 7-5, so that it has been possible to investigate the effects of C02 at a constant extracellular ph. The internal buffering power was measured both by changing the external C02 and by iontophoretic injection of KHCO3 and HCl. The transport numbers for KHCO3 and HCl iontophoresis were therefore determined, using a specially developed microtitration method. The results show that the cell membrane must contain a H+-pump, or its equivalent, which can be very active, especially when the cell is in CObicarbonate Ringer. When exposed to C02, the cell accumulates HCO3- ions and the buffering power is greatly increased. Acetazolamide, an inhibitor of carbonic anhydrase, slows down the phi responses to C02 addition or removal, and reduces the internal buffer value in cells exposed to CO2. Preliminary reports of some of this work have been published (Thomas, 1974b, 1975c). METHODS These were generally the same as previously described (Thomas, 1974a) except that the ph of the normal snail Ringer was changed from 8-0 to 7-5. Details will be given only where they differ from the earlier descriptions. General. Most of the experiments were done on the large nerve cell 1 of Helix aspersa (Neild & Thomas, 1974) located at the rear of the right pallial ganglion. Special care was taken to expose the cell as completely as possible. At least 1 hr was allowed for the preparation to equilibrate before the experiment, with about half of this time being allowed for electrode equilibration outside the cell. The cell diameter was measured at two angles using an eyepiece micrometer in the dissecting microscope, magnification 80 x. The ph-sensitive micro-electrode was inserted first, then the reference (or membrane potential) micro-electrode, and finally the current-passing electrodes, if required. The ph electrode was inserted perpendicularly, but the other electrodes were usually inserted sideways, that is each electrode was lowered vertically, to make a small dimple in the cell surface, and then carefully moved horizontally until it suddenly penetrated the cell. It was then moved horizontally back to its original position. This technique, rather surprisingly, usually caused less damage than direct penetration. Results were rejected if, on removal from the cell, the voltage of the membranepotential or ph-sensitive electrode was more than 3 mv different from its value at the beginning of the experiment. In practice, the ph-sensitive electrodes were far more stable than the conventional micro-electrodes. Solutions. The normal (nominally C02-free) Ringer had a ph of 7-5, was in equilibrium with air, and had the following composition (in mm): KC1, 4; NaCl, 80; CaC12, 7; MgCl2, 5; Tris maleate or Na salt of HEPES, 2-N-2-hydroxyethyl piperaxine-n'-yl ethanesulphonic acid, 20. The CO2 Ringer solutions were saturated with 0-9, 2-2 or 4-4 % CO2 in 02, and had

4 718 R. C. THOMAS the same composition as the normal Ringer except that they contained NaHCO3 instead of Tris or HEPES, and contained 0 1 mm phosphate to prevent precipitation of bicarbonates (Burton, 1975). NaHCO3 was added as follows: 0 9 % Co2 had 8-4 mm; 2-2 % had 20mM; and 4-4% had 40 mm. The NaCl content was adjusted to keep the total NaCl + NaHCO3 to 100 mm. Conventional micro-electrodes. Potential-recording electrodes were filled first with distilled water, as previously described, and then with boiled 2-5 m-kc1, buffered to about ph 8-5 with 10 mm glycyl-glycine. Current-passing electrodes were filled directly with a 2 m solution of the required salt, and tested for their ability to carry a 100 na current in either direction. Electrode resistances were between 20 and 40 Mn. c ec HO Injection current dt (KHCO)Fl a e orkhco3 i. 10m n ph KCI~~~~~~~- (reference) Ag Droplet j6.5 Ag~~ 7Thait 7.5 UL 'LI L_...U A m la0 i- Droplet 10smm- Droplet 10 mm- HCO; diameter HCO- diameter HCO; 3.50 pm 290 pm Fig. 1. Interbarrel iontophoresis of KHCO.. A, diagram of the experimental arrangement for ph measurement during the injection of KHCO3 into an aqueous droplet mounted on a silver wire under liquid paraffin. B, penrecording of part of an experiment to measure KHCO3 output. The ph changes during KHCO injection into two droplets are shown: between each injection the ph and reference electrodes were placed in a droplet containing 10 mm-hc03e. The arrowheads indicate the point at which the KHCO3 micro-electrode was placed in the droplet: some leakage of KHCO3 then occurred before the current was switched on. ph-senlitive micro-electrodes. These were made as previously described, except that the exposed length of ph glass aimed at was about 25am, and the electrodes were filled and aged in one operation by gentle boiling in distilled water for 2-3 hr. The water was then sucked out and replaced with the final filling solution. (For a recent detailed account of the construction and properties of the recessed-tip ph-sensitive micro-electrode see Thomas, 1975b.) Electrical arrangements. These were the same as before. The circuit of the floating current clamp has now been published (Thomas 1975a). -wr Estimation of intracellular HCO3- concentration. Graphs of ph against HC03 constructed by titration. The ph of an initially HC03 -free CO2 Ringer solution,

5 CO2 AND INTRACELLULAR ph 719 bubbled with 0-9, 2-2 or 4-4% CO. in 0,, was followed while measured aliquots of solid NaHCO were added and dissolved. Intracellular HCO3- concentrations were than taken from these graphs. Measurement of transport numbers for HCO3- and H+ iontophoresi8. The quantity of HCl or KHCO, released by interbarrel iontophoresis was determined by titration, using an un-insulated ph-sensitive micro-electrode to follow changes in the ph of droplets of aqueous solution under liquid paraffin. The current-passing and reference micro-electrodes were the same as used for snail neurones; the arrangement is illustrated in Fig. 1A. Droplets mm in diameter were made on the ends of earthed PTFE-coated silver wires. The solution was ejected from siliconized micropipettes on to the wires by air pressure from a hand-operated syringe. Droplet diameters were measured by the method described above for snail neurones. For HCO3- determinations the experimental droplets contained 100 mm-kcl and 2 mm-nacl, and the reference droplets 100 mm-kcl and 10 mm-nahco. Since HCO,- solutions are only stable in the presence of a fixed level of CO, the liquid paraffin was continuously bubbled with 2-2% CO2 in 02. The titrations were done by measuring the ph of an experimental droplet while injecting HCO3- ions with a constant current, as shown in Fig. I B. The end-point was the ph equal to that of the reference droplets, which remained constant for many hr. In this way the charge required to inject HCO,- to a concentration of 10 mm into a droplet of known diameter was measured. For the H+ determinations the experimental droplets contained 100 mm-kcl and 20 mm-nahco3 or NaOH. The liquid paraffin was in equilibrium with air. The reference droplets contained citrate buffer, adjusted to ph 3-8, the ph of the titration end-point. Since there were only short periods between injections when the injection electrodes were in a droplet, no attempts were made to stop leakage from the electrodes by the use of backing-off currents. RESULTS Transport number for HCO3- and H+ iontophoresis If during an iontophoretic injection all the current flowing through a KHCO3-filled micro-electrode is carried by HCO3- ions leaving the electrode, and no other HC03- movement occurs, the transport number will be 1-0. The equivalents of HCO3- ejected will be given by the charge in coulombs divided by 96,500. If, however, some of the current is carried by cations entering the electrode, the transport number will be less than unity. The results obtained when HC03- or H+ was injected into aqueous droplets are shown in Fig. 2. The amount of HC03- determined from the ph change was much less than expected from the charge passed, if the transport number had been unity. The mean ratio of HCO3- calculated from the charge to that calculated from the titration was With HC1 iontophoresis, however, there was much better agreement between HCl calculated from the charge and from the titration: the mean ratio was Thus when calculating the quantity of HC03- or H+ injected into snail

6 720 R. C. THOMAS neurones in the experiments described below, the transport number for ECO3- was taken to be 0 3, and that for He to be The effect of 2*2 % C02 on the internal ph (Thomas, 1974a) C02 applied externally rapidly de- As shown earlier creases the ph, of snail neurones. This occurs through the well-known reaction inside the cell: C02 + H2O=H2CO3=H+ + HCO3-. The H+ produced intracellularly is, of course, the direct cause of the fall in ph. In these earlier experiments the normal Ringer ph 8-0 was too high to enable significant levels of C02 to be applied without changing the external ph. In the present experiments, however, it was possible to make, stable solutions in equilibrium with C02 levels up to 4-4% at the normal ph of o 600 HCI 0 -w Calculated from charge (p-equiv) Fig. 2. Relationship between calculated and measured quantities of HC1 or KHCOO injected iontophoretically from three different HCI-filled and five different KHCO3-filled micro-electrodes into aqueous droplets. Vertical axis shows the quantity found by titration, horizontal axis shows that calculated from the charge, assuming a transport number of one. The continuous line has a slope of 1P0, the dashed line a slope of 0*3. A complete, although unusually brief experiment, in which 2-2 % C02 was applied for 18 min and then for three short periods is shown in Fig. 3. The record starts with both electrodes outside the cell. Then the ph-

7 C02 AND INTRACELLULAR ph 721 sensitive micro-electrode was inserted. Inside the cell it recorded the sum of the membrane potential and the equilibrium potential for H+ ions, giving a negative deflexion of about 20 mv. Next the reference microelectrode was inserted into the same cell. This caused a deflexion of about 40 mv downwards on the upper trace, and a similar simultaneous upward deflexion of the lower trace as the membrane potential was subtracted from the ph-electrode output. From this point the ph trace had no membrane potential component. 10 > 20 E E Em min p I 8 0l-02-2 % CO2, U 1 I U U ph 6-S ph 7 5 CO2 CO2 CO2 Fig. 3. Pen-recording of an experiment to measure the effects of C02 application and removal on the internal ph, phi, of a snail neurone. The voltage recorded by the KCl-filled membrane potential electrode (Em) is shown at the top, and the voltage recorded by the ph-sensitive microelectrode is shown at the bottom. Except where indicated in this and later Figs., the preparation was superfused by a nominally C02-free Ringer of ph 7.5. The long time constant of the pen-recorder has reduced the size of the spontaneously-occurring action potentials to only a few mv. At the beginning and end of the record illustrated, both micro-electrodes were outside the cell. The ph response was checked at the end of the experiment. The initial ph1 was 7-2. It slowly rose until, about 12 min after the begging of the experiment, the solution flowing through the bath was changed from normal snail Ringer, ph 7-5, to one equilibrated with 2-2 % C02 at the same ph. As the C02 reached the cell, phi fell rapidly by a total of 0-17 units. But this fall was only transient; within 2 min ph1 began to increase towards a higher value than before the CO2 application. A similar increase in ph during a long exposure to C02 has recently been observed in squid giant axons (Boron & de Weer, 1975) and crab muscle fibres (Aickin & Thomas, 1975), but the time course of the phi changes

8 722 R. C. THOMAS was very much slower. In the experiment illustrated in Fig. 3 the ph, increase was roughly exponential, with a time constant of about 2-9 mi, although this was unusually fast (cf. Figs. 4, 5, 6, 7 and 11). The membrane potential was also increased during the exposure to C02, but without any initial fall. The high permeability of the cell membrane to C02, as shown by the rapid phi responses, suggests that after some time in C02 Ringer the internal C02 would be virtually the same as the external C02. The speed of the phi response to C02 also suggests that the internal HCO3- concentration would be determined primarily by phi and the C02 level. Assuming that the relevant equilibrium constants are the same inside and outside the cell, the internal HCO3- concentration after 15 min in C02 was about 15 mm, the internal ph being When the solution was changed back to the normal C02-free Ringer, phi rapidly increased, reaching a peak value of 7-84 within 2 min. Presumably, removal of external C02 caused C02 to leave the cell interior, and upset the equilibrium between internal C02 and HC03-. Thus internal HCO3- took up H+ ions (causing the increase in ph1) and left the cell as C02. Three further applications of C02 were made before the end of the experiment. The lower the ph, at which the C02 was applied, the smaller the ph change observed. The first brief C02 application caused a ph decrease of 0-48 units, the second 0-36, and the third At the end of the experiment both electrodes were withdrawn from the cell and their response to ph 7-5 Ringer was tested. Buffering power. If HCO3-, H+ and OH- do not cross the cell membrane actively or passively, the size of the rapid ph, response to CO2 application will depend only on the initial ph, and buffering power. Intracellular hydration of CO2 will produce equal numbers of H+ and HCO3 ions. It can be assumed that when ph, stops decreasing, there will be an approximate equilibrium between the ph1 and the internal CO2 and HC03-. Thus the HC03- produced can be calculated from the minimum ph, reached in C02, assuming that the carbonic acid dissociation constant and C02 concentration are the same inside as outside the cell. The estimated internal buffering power is then the HCO3- produced divided by the ph change. For the first CO2 application shown in Fig. 3, the buffering power calculated in this way was 40-6 m-equiv H+/pH unit per litre, and for the other three applications it was 27-6, 25-0 and The effect of different levels of CO2 on the internal ph In most of the experiments described in this paper, C02 was applied at 2-2 % in 02, but some experiments were done with more or less C02, as illustrated in Fig. 4. It is clear that the higher the level of C02 the larger were the ph changes observed. In all three exposures to C02, the internal

9 CO2 AND INTRACELLULAR ph 723 acidification was only transient. During the period in C02, phi returned roughly exponentially towards the normal value of about 7 4, with time constants for the three CO2 levels of 4-6, 7-3 and 8-9 min respectively. E E 40 E ~~~~~ ~~~~~~~~~~30. min 7-0 ' % C2 2-2 % CO2 4.4 % CO2 Fig. 4. Pen-recording of part of an experiment to show the effects on the membrane potential, Ems and internal ph, phi, of exposures to 0-9, 2-2 and 4-4% Co2. The external ph was maintained at 7-5 throughout. Internal buffering power by extrapolation In estimating the buffering power from the results illustrated in Fig. 3, it was assumed that there was no movement of H+, OH- or HCO3- across the cell membrane. For any but the shortest of exposures to C02, this is clearly wrong; the transient nature of the phi changes during a long exposure to C02 suggests that there was active transport of H+, OH- or HC03-. Thus to obtain a more accurate estimate of the phi change on application or removal of C02, the ph trace was extrapolated on each side of the minimum or maximum point to give a sharp peak. This was done for all phi changes occurring at the beginning or end of long exposures to C02. The buffering powers calculated from the three C02 exposures shown in Fig. 4 were in reasonable agreement: for 0-9 % C02 they were 28-9 m- equiv H+/pH unit per litre for the C02 application and 25-3 for the C02 removal; for 2-2% they were 26-9 and 32-4; and for 4-4% they were 26-1 and Similar measurements were made with 2-2% C02 on fourteen cells. From fifteen applications of C02 the mean buffering power was (S.E. of mean) while from fifteen removals of C02 it was There have been few previous measurements of the intracellular buffer value of nerve cells, and none in which allowance has been made for active transport. Using the DMO technique to measure the phi of cat brain,

10 724 R. C. THOMAS Roos (1965) obtained a value for the internal buffering power of 36-7 m- equiv H+/pH unit per litre, but this may well be too high. With the snail neurones, if phi had been measured only before and perhaps 30 min after changing the external CO2, active transport would have made the buffer value appear very large indeed min > 20 - E_ w 40 _ m X 7 phi e vacetazolamide (10 #H) % CO2 22% CO2 Fig. 5. Pen-recording of part. of an experiment to show the effects of 10-6 m acetazolamide on the response to CO2 application and removal. The effect of acetazolamide on the response to C02 The speed of the response of phi to C02 application or removal suggests that the hydration of C02 and dehydration of H2CO3 was being catalysed by carbonic anhydrase. Perhaps the simplest way of testing this is to apply an inhibitor such as acetazolamide (Diamox; Lederle), well known to specifically block carbonic anhydrase activity. Fig. 5 shows that acetazolamide did indeed considerably slow the phi response to both C02 addition and removal. It appears to have little effect on the change in phi during C02, but greatly slows the return to normal after C02 removal. The slowing of the ph, increase following CO2 removal is somewhat surprising: the uncatalysed dehydration of H2CO3 is much faster than the reverse reaction (Woodbury, 1965). To check that acetazolamide was not reducing the membrane permeability to dissolved gases, the experiment shown in Fig. 6 was done. The result shows that the ph response to injected NH4+ ions was not changed by acetazolamide. The fall in ph following NH4+ injection is caused by the intracellular conversion of NH4+ to H+ and NH3, the latter then leaving the cell. The ph1 responses to both C02 removal and HC03- injection were considerably slowed. In some preliminary experiments communicated to the Physiological Society

11 CO2 AND INTRACELLULAR ph 725 (Thomas, 1974b) 1 used NH,+4 injections to measure the intracellular buffering power, with the assumption that the transport number was 10. Later, however, when making H+ and NH4+ injections into the same cell, I found that NH4+ gave a higher apparent buffering power than with H+ injection. Possibly the transport number for NH4+ is significantly lower than that for H+. I have found the ph of aqueous droplets containing NH4+ too unstable to allow me to measure the transport number for NH4+. 'E 20 l- _ > NH4HCO E 30 uj Injection current thco- I % CO ph >E 20 r E 30 _ Em Lu40.,..- _ 50 ] oo ph i o - 10-SM 2-2 % CO,. ph Fig. 6. Pen-recording of a complete experiment to compare the effects of acetazolamide on the response to C02 application and removal with the responses to NH4+ or HCO3- injection. The bottom half of the Fig. is a continuation of the top half. The two arrows above the top record indicate the points at which the two current-carrying micro-electrodes were inserted. In each half of the figure the first trace is the membrane potential, E., the second is the injection current, and the third is the internal ph, phi. Comparison of HCO3- injection and CO2 removal If the ph1 increase seen on removal of CO2 from outside a cell is caused by internal HCO3- taking up H+ ions and leaving the cell as C02, it should be possible to mimic the phi response by injecting HC03- ions. Fig. 7 illustrates an experiment in which three injections of HC03- were made, followed by a 20 min exposure to 2-2 % CO2. The results show that the

12 726 R. C. THOMAS more HCO3- is injected, the bigger is the rise in phi, and that the time courses of the responses are similar to those seen on removal of external C02. In making quantitative comparisons a major problem is the need to know the cell volume to calculate the HCO3- concentrations resulting from the injections. In this paper cell volumes have been estimated by measuring the apparent cell diameter and then calculating the volume of the equivalent sphere. When this is done, injected HCO3- appears to be 30 min 10 _ KHCO. electrode 5~ out E, 320 E Infection current (KHCOJ ph1 [18 6] C* 7.8 8* % C02, ph 7-5 Fig. 7. Pen-recording of part of an experiment to compare the effects of HCO3- injection with the effects of C02 removal. The arrow above the top trace indicates the point at which the KHCO3-filled micro-electrode was withdrawn from the cell to prevent any leakage of KHCO3 into the cell. Before that, leakage was minimized by the passage of a 5 na back-off current, in the reverse direction to that used for HCO3- injection. The numbers in square brackets are the calculated HCO3- concentrations. more effective than HC03- accumulated in C02. The results from a total of five experiments similar to that of Fig. 7 are plotted in Fig. 8. It is clear that injected HC03- always causes a larger phi change than would be expected from the apparently equivalent amount of HCO3- accumulated during exposure to C02. Thus the buffering power calculated from the sixteen HC03- injections was m-equiv H+/pH unit per litre, while that calculated from the seven responses to C02 removal was

13 CO2 AND INTTRACELLULAR ph * (The discrepancy between these values is considered fully in the Discussion.) Measurement of buffering power by HCl injection The most direct way of measuring the buffering power of a solution is to add acid or alkali and measure the ph change. So far I have been unable to inject KOH, but HC1 is one of the easiest compounds I have ever tried to inject iontophoretically. Thus I have used HC1 to measure the buffering power with and without CO2 outside the cell, the cell volume being estimated from the apparent cell diameter, as described above El*05 ~ /4 -/ El 0 o 00O4 0J 0 bo r U injection 03 Ca; removal / Calculated intracellular HCO-3 (mm) Fig. 8. The relationship between the intracellular ph change and internal HC03-: the HCO3- being either injected (circles) or accumulated during exposure to CO2 (triangles). The two lines are drawn to fit the average buffering power calculated from each set of points: 10-7 for the injections, and 30-7 for the C02 withdrawal. A transport number of 0-3 was used in calculating the quantity of HCO3- injected, and the cell contents were assumed to be essentially the same as the external environment, except for ph, in calculating the internal HCO3- in CO2. Buffering power in the absence of CO2 Fig. 9 shows the effect on phi of three equal injections of HCl, and also illustrates some of the procedures used and problems encountered. The ph-sensitive and KCl-filled reference micro-electrodes were inserted first. Next a second KCl-filled micro-electrode was inserted, causing little change in the internal ph but a transient fall in the membrane potential. Then the HCl-filled micro-electrode was pushed into the cell, causing

14 728 R. C. THOMAS another transient depolarization. As HCl began to leak out of the microelectrode into the cell, phi began to fall. Then a 5 na braking current was switched on between the two current-passing electrodes, and phi began to recover. 10 L ~20 E~ ~~~E -_ Injection current ph1 %% 7.3 II C.7.5 % min Fig. 9. Pen-recording of the beginning and end of an experiment to measure the intracellular buffering power by the injection of HCl. The two arrows indicate the points at which the two current-passing electrodes (filled with KCl and HCl) were inserted. The central portion of the experiment, about 110 min long, is not shown. (During this section the HC1 micro-electrode was removed and replaced with one of a higher resistance to reduce leakage, the cell was exposed to C02 and acetazolamide, and a total of four further injections of HCl were made.) The injection current was reversed 10 min later and increased to 35 na for 60 sec, and then returned to its previous value. If the cell had had no buffering capacity, this injection would have increased the internal H+ concentration by 5-75 mm (and ph, would have decreased by over 3 units): in fact ph, changed by 0-43 units, giving a buffering power of 13-4 m- equiv H+/pH unit per litre. The recovery of the ph after this injection was rather slow, suggesting that HCl was again leaking from the electrode, in spite of the 5 na braking current. Thus, in the part of the experiment not illustrated, the HCl micro-electrode was withdrawn and replaced with one having a higher resistance. In the last part of the experiment two more injections of HOl

15 C02 AND INTRACELLULAR ph 729 were made, giving calculated buffering powers of 15-0 and The three HOl injections covered the ph range , suggesting that the buffering power did not vary near the normal internal ph. (See also Fig. 5 of Thomas, 1975c.) For the last two injections one of the current-passing micro-electrodes was incompletely inserted, or partially blocked, so that the membrane potential was slightly affected by the injection current. This happened relatively infrequently, but when it did the only reliable remedy was replacement of the relevant micro-electrode. The normal lack of effect of the injection current on the membrane potential can be seen in Figs. 6, 7 and 11, and the earlier injections of Fig. 10. The mean calculated buffering power from eighteen HCl injections into seven cells in normal Ringer was '7, which agrees well with the figure of obtained from the HCO3- injections. The effect of C02 on the internal buffering power The molar buffering power of a bicarbonate solution in equilibrium with a fixed level of C02, and hence having a constant H2CO3 concentration, is 2x3. This is four times higher than that of a normal buffer (Woodbury, 1965). If C02 is able to cross the cell membrane sufficiently rapidly, the HCO3- accumulated intracellularly during exposure to CO2 should greatly increase the cell's buffering power. If the internal buffer value in C02-free Ringer was 30, it would be more than doubled in C02 Ringer if the internal HCO3- was 15 mm. Fig. 10 shows the effects of HCl injection into a cell during and after exposure to C02. The buffering power was much higher in the 2-2 % C02 Ringer than in the nominally C02-free Ringer. For example, the second and fifth injections were of the same quantity of HCl; phi changed by 021 units in C02 Ringer, but by 0 57 units in normal Ringer. For a total of fourteen HOl injections into seven cells exposed for some time to 2-2 % C02 Ringer the average buffering power was 31' , while for the same cells in C02-free Ringer it was only The recovery of the ph after an HCl injection was faster in C02 Ringer than in normal Ringer. In the normal Ringer the recovery time constant was about 12 min, but for C02 Ringer it was only about 5 min (calculated from the experiment shown in Fig. 10). The effect of acetazolamide If rapid dehydration of H2CO3 is essential to enable C02 to reduce the ph response to injected acid, carbonic anhydrase inhibition should reduce

16 730 R. C. THOMAS the effectiveness of C02. Fig. 11 shows that acetazolamide treatment does lower the internal buffering power from its high value in C02 Ringer. (The transient increase in phi when the acetazolamide was applied was probably due to loss of C02 by the solution in the tubing.) min I.., 30 EM< 76 (24r7r(270 ecio (32.3) 80 22%CO2. ph75 Fig. 10. Pen-recording of part of an experiment to show the effect of CO2 on 10-2the internal buffering power. Between the first and second injections of HCl the H~l-ifilled micro-electrode was moved so that its tip was less close to the ph-sensitive electrode. The numbers in parentheses under the ph record are the calculated buffering powers for each injection. 10 r 30 min E 20 E > ;~ ~ Injection current (HCI) 307m3 0 ecf 80 Fig. 11. Pen-recording of part of an experiment to show the effect on the intracellular buffering power of 002 and 002 plus acetazolamide. The numbers in parentheses under the ph record are the calculated buffering powers for ech injection.

17 CO2 AND INTRACELLULAR ph7 731 For three cells treated with acetazolamide the buffering power in C02 fell from 34-6 m-equiv H+/pH unit per litre to a mean of Acetazolamide had no effect on the buffering power in C02-free Ringer. TABLE 1. Intracellular buffering power of snail neurones (m-equiv H+/pH unit per litre) External solution CO2 + C02-free 2.2 %CO2 acetazolamide Method used (m-equiv) (m-equiv) (m-equiv) Application of 2-2 % CO Application of 0 9 % CO Removal of 2.2 % CO2 28-9±1 0 Removal of 0 9 % CO KHCO3 injection, volume not 10-7 ± corrected* HCl injection, volume not 10-9 ± ± corrected* HCl injection, volume (29 5) correctedt Results are shown as mean + s.e. of mean. * Volume of the cell assumed to be the same as that of a sphere of the same diameter as the cell. v t Volume corrected by assuming true internal buffering power is DISCUSSION Perhaps the most interesting conclusions to be drawn from the above results concern the part played by carbonic anhydrase in increasing the internal buffer value in C02, and the high activity of the 'H+-pump' (a convenient term for H+, OH- or HCO3- active transport across the cell membrane) especially in C02-Ringer. Before discussing these points, however, I will consider the large difference between buffer values as measured by C02 application and by HOl injection, and the accumulation of HCO3- by cells exposed to C02. The various values for internal buffering power are collected together in Table 1. The internal buffering power from CO2 application or removal As mentioned in the Results section, the accuracy of the estimation of the buffering power by the C02 method depends on the following assumptions: the C02 levels and H2CO3 dissociation constant were the same inside and outside the cell; before and soon after a period of exposure to C02 the

18 732 R. C. THOMAS internal C02 and thus the internal HCOG3- were negligible; there was no significant passive entry or exit of HC03- ions; and active transport of H+, OH- or HCO3- ions could be allowed for by extrapolation of the phi trace. The first three assumptions seem reasonable, so that the only likely source of error is the allowance made for the H+-pump. The time course of the recovery of phi after displacement from normal appears to be exponential, at least for ph, decreases. Thus linear extrapolation will tend to underestimate the phi change, and, in the case of C02 addition, over-estimate the HCO3- produced. These errors will both cause overestimation of the internal buffer value. The over-estimation is unlikely to be large, however; errors of extrapolation will be smaller with small ph changes, and the buffering powers calculated from the ph changes with 0 9 % C02 are not much less than with 2 2 % CO2. For C02 applications the average value for 0 9 % C02 was 27*1 m-equiv H+/pH unit per litre, as against 30-1 for 2-2 0C02, and for C02 removal the value for 0-9 % C02 was 24-7 as against It is perhaps worth noting that in earlier experiments when the external C02 and ph were changed simultaneously, and there was no need for extrapolation, the internal buffering power was estimated to be at least 25 (Thomas, 1974a). Internal buffering power from HCl injection There is a large difference between the buffering power measured by C02 and measured by iontophoretic injection. The simplest explanation for this is that the intracellular volume is much smaller than that of a sphere having the same apparent diameter as the cell. A large fraction of the sphere volume is probably, by analogy with Aplysia neurones, occupied by extracellular space in membrane infoldings (Coggeshall, 1967) and many subcellular organelles are likely to be relatively impermeable to H+ or other ions. If the buffer value of the contents of the 'HCl accessible space' is that given by the responses to C02, then its volume can be estimated by multiplying the sphere volume by a correction factor. This correction factor will be the ratio of buffer value measured by injection to that measured by C02. Taking the latter figure as 29-5, the mean correction factor is Thus the HCl accessible space is on average only 36% of the volume of a sphere of the same diameter as the cell. If this average correction factor is used to correct the buffer values determined by HCl injection into cells in 2-2 % C02, the very high value of 86 is obtained. Such a high buffer value cannot be accounted for even if all the accumulated HCO3- makes its maximum possible contribution, which would give at most a mean internal buffer value of Presumably, because C02 can readily cross the membrane of subcellular

19 C02 AND INTRACELLULAR ph 733 organelles (cf. Elder & Lehlninger, 1973), the effective intracellular volume will be larger in C02. It seems probable, then, that CO2 increases the internal buffering power not only by enabling internal HC03- in the HCl-accessible space to make a large contribution, but also by enabling the buffers inside subcellular organelles to participate. The internal HCO3- in a cell exposed to C(2 Cell membranes are highly permeable to C02, so that, except when it is being taken up or released very rapidly, the internal and external CO2 levels will be the same. The speed of the initial response of the snail neurone phi to CO2 addition or removal shows that internal CO2 is rapidly hydrated or dehydrated, so that the internal HCO3- concentration will be determined simply by phi and the internal CO2. (The bicarbonate equilibrium potential will be the same as that for H+ ions, so that any net passive movement of HC03- ions across the cell membrane will be outwards. Such movements are unlikely to be large enough to reduce internal HCO3- concentration significantly.) If the HC03- accumulated by a cell in C02 is estimated by comparing the ph1 changes seen with C02 removal with those caused by HC03- injection, the values obtained are much lower than estimated from the phi and external CO2. But the internal buffering power determined by KHCO3 injection is very similar to that determined by HCl, so the internal space accessible to injected KHCO3 is probably the same as the HCl assessible space. Correcting for this gives good agreement between the two ways of estimating internal HC03- concentration. Thus in snail neurones, as well as in frog and rat skeletal muscle (Khuri et al. 1974) the internal bicarbonate is determined by the CO2 and phi, not the membrane potential and the external HC03-. The role of intracellulakr carbonic anhydrase Since acetazolamide decreases the buffering power measured by HCl injection into cells exposed to C02, carbonic anhydrase activity must be required to enable C02 and intracellular HC03- to contribute fully. The rapidity of the ph, responses to C02 addition and removal also appears to depend on carbonic anhydrase, but it is hard to see any physiological advantage in this. The function of intracellular carbonic anhydrase in many cells is unknown (Carter, 1972): perhaps its primary role, at least in nerve cells, is to maximize internal buffering in cells exposed to a constant level of C PHY 255

20 734 R. C. THOMAS Activity of the H+-pump One of the more striking aspects of the response of phi to CO2 application is the speed at which phi recovers from the initial fall. There must be some active process involved in this ph change: the extrusion of H+ ions, or uptake of OH- ions, or uptake of HCO3- ions would all be against their electrochemical gradients. In the experiment shown in Fig. 3, phi had recovered to 7-36 after 10 min in C02, and the cell should have accumulated over 14 mm internal HCO3-. Thus the H+-pump must have effectively extruded at least an equivalent number of H+ ions in increasing phi to this value. That the H+ ions must have been extruded rather than sequestered is perhaps clearer from the HCl injection experiments. Considerable quantities of HCl could be injected repeatedly into the same cell, and phi always recovered, as shown in Figs. 9, 10 and 11. The HCl experiments also show clearly that the H+-pump is much more active in CO2 Ringer than in C02-free; not only does phi recover more rapidly, but the higher buffering power in CO2 means that more H+ ions must be extruded for a similar phi change. It would be fruitless to speculate much at this stage on the mechanism of the H+-pump. There is a striking similarity, however, between the ph1 responses to HCl injection and the intracellular Na+ concentration, [Na+]i, responses to Na+ injection (see Fig. 12 of Thomas, 1972). The time courses of the exponential recoveries of ph1 or [Na+]i after an injection are very similar, with time constants of around 5 min. These points suggest that the H+-pump may work in much the same way as the Na+-pump. Finally, it is worth pointing out that the snail neurones as well as having a higher buffering power, were less susceptible to damage when in the CO2 Ringer. This suggests that for Ringer solutions C02-bicarbonate buffers are physiologically the best. Note added in proof. W. F. Boron & P. de Weer (J. gen. Phy8iol., in the press) have shown that the increase in ph, in squid axon during a long exposure to CO2 must be due to a H+ pump or its equivalent. More recentiy (Nature, Lond. in the press) they have also found that this H+ pump is stimulated by CO2 and HCO3. I wish to thank Mrs Vicky Martin for technical assistance, Miss Lilian Patterson for analysing gas mixtures, and Miss Pat Cragg for lending un-insulated microelectrodes. I am grateful to Professor A. J. Buller and Dr C. C. Ashley for reading the manuscript, and to the Medical Research Council for money.

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