again leaves and K enters against the gradients (Harris, 1941). It has been

Transcription

1 59 J. Physiol. (I95I) II2, I2.III.1 FACTORS IN THE ACTIVE TRANSPORT OF CATIONS BY MONTAGUE MAIZELS* From the Department of Pathology, University College Hospital Medical School, London (Received 26 January 195) In fresh human blood cells K is high and Na low, while in plasma K is low and Na high. On cold-storage or on incubation in the absence of glucose, cations flow with the concentration gradients, cell K falling and Na rising (Maizels, 1943, 1949). On incubation in the presence of glucose, Na in cold-stored cells again leaves and K enters against the gradients (Harris, 1941). It has been shown that output of Na is active and uptake of K secondary and passive, and that total base (Na + K) and cell volume increase above the cold-storage values in the absence of glucose and fall below the cold-storage values in the presence of glucose (Maizels, 1949; Flynn & Maizels, 1949); it was concluded that output of Na subserves the function of checking invasion of the cells by Na from the Na-rich external medium, so maintaining constancy of cell composition and volume. In the energizing of these cation movements during incubation glucose is normally an essential factor: the present paper is concerned with (a) the correlation of glycolysis with active transport, (b) the effects on active transport of substances which affect one aspect or another of cell metabolism, and (c) the replacement of glucose as a source of energy by other substances. METHODS These involve: (1) The preparation of packed fresh erythrocytes as a reference sample for volume changes, etc. (2) Cold-storage for 6-8 days of the fresh blood mixed with one-ninth of its volume of trisodium citrate (3 %) and glucose or other sugars. (3) Incubation, usually for hr., of 1 ml. portions of the cold-stored blood with KCI (-15 N) and NaCl (-15 N), together with the addition of the appropriate amounts of alkali to give a series of final ph values between 7-2 and 6-8. When during incubation the bloods were not rocked, 7 ml. KCI and 2 ml. NaCl were added; with rocked bloods the amounts were 4 and 1 ml. (4) Treatment of cold-stored blood as in the preceding item but with the addition of inhibiting agents, etc., and enough acid or alkali to give a final ph of about 7. Thus where no inhibition of glycolysis is expected alkali is added to ph 7-6 or 7-8, and after incubation the value sinks to about 7; where inhibition occurs little or no alkali is added and the ph shifts from about 7-1 to 7 during incubation. It will be realized that the ph values recorded in the tables are measured at the end of incubation. (5) Treatment of the * In receipt of a grant for technical assistance from the Medical Research Council.

2 6 MONTAGUE MAIZELS stored blood as in item 3 above, but with sodium fluoride added to 3 mm. thus giving as a base-line for cell glucose the value found when glycolysis has been completely inhibited throughout incubation; this is called the cell glucose control sample. (6) Reagents were Seitz-filtered or autoclaved. The actual methods used are the same as those described elsewhere (Maizels, 1943, 1949). The phosphate method carried an error of up to ± 2 % and hence the results for easily hydrolysable P (a difference value) have a considerable possible error which may reach an absolute value of ±-3 mg. % when cell phosphate is high and of -15 mg. when this is low. RESULTS In the present investigation of stored blood before and after incubation, tables show cell volume and ph; they also show cell K, Na and P (total acid soluble, hydrolysable and inorganic), all these being corrected for cell shrinkage or swelling by reference to the original cell volume. In addition, cell glucose after incubation is shown and may be compared with values in the incubated cell when glycolysis has been entirely inhibited with fluoride. Before considering results in detail, several points require elucidation. (1) When using inhibiting agents, these may themselves be so acid or alkaline (e.g. cyanide) as to inhibit active transport non-specifically and it is essential so to adjust systems that the ph lies between 7-6 and 6-8 for the greater part of the incubation period. (2) Liberation of inorganic phosphate: this leads to an increase in cell inorganic P but the true rise is not fully apparent since inorganic phosphate, unlike the phosphoric esters, is able to penetrate the cell membrane and so is lost in the plasma. The correct amount of inorganic P liberated during incubation thus equals the observed change in cell inorganic P plus cell phosphate lost by diffusion, the latter equalling the difference in total cell P before and after incubation. Thus if cell inorganic P rises from 6 to 1 mg. % and total acid soluble P falls from 5 to 4 mg. %, then inorganic P liberated is 14 mg. % and it is this corrected value for inorganic P which is shown in the tables. In what follows it is thus assumed that differences in the amounts of inorganic P liberated in the presence of various agents depend on the direct effects of these agents on enzymatic processes, but it is of course possible that any agent by increasing cell permeability to inorganic phosphate would lead to a greater loss of inorganic phosphate, disturbing the equilibrium between inorganic and organic phosphate within the cells, and hence leading indirectly to increased phosphorolysis. That most agents do indeed act directly on phosphorolysis is shown by the fact that usually total inorganic P liberated varies directly with cell inorganic P, whereas if liberation of inorganic P depended mainly on increased permeability, total inorganic P liberated and inorganic P remaining in the cells would vary inversely: in fact, it is only when cell depletion is very marked that the inverse relation tends to appear. (3) With regard to the values for cell glucose: the figure recorded is the resultant of glucose removed by metabolic activity within the cells and of glucose diffusing in from the glucose-rich plasma between the settled cells; the findings are

3 FACTORS IN ACTIVE TRANSPORT OF CATIONS 61 compared with those in the cells of a similar system where glycolysis during incubation is inhibited by fluoride (cell glucose control sample), and they thus afford a mere index of metabolic activity and not an absolute figure. An absolute figure may be obtained by rocking in an incubating chamber and estimating total glucose disappearing from the whole system in a given time. This was not done as a rule because of the necessity for concentrating the cells suspended by rocking, because rocking and concentration accentuate any tendency to cell lysis and chiefly because loss of inorganic phosphate is much greater from rocked than from settled cells and this in turn accelerates the breakdown of phosphoric esters. The loss of cell phosphate leads to disturbance of the physico-chemical system with fall in content and concentration of cell base, while depletion of phosphoric esters may sometimes be so marked as to lead to premature breakdown of the glycolytic system. Finally, one can correlate glucose metabolized with the apparent output of Na under standard conditions but not with real output, since the latter is complicated by passive diffusion of Na inwards and this value is unknown and will vary from system to system with cell permeability. Further, if conditions are not standardized correlation between glucose metabolized and Na actively transported becomes obscured. This is discussed in the next section. (4) Acid liberated during incubation in the presence of a given agent may be gauged by comparing alkali added at the beginning of incubation and ph achieved at the end with the corresponding findings in poison-free control systems. Active transport and glycolysis In the experiments of this section blood suspensions in bottles were rocked in an incubating room and the absolute amount of glucose metabolized was measured. Effects of glucose content of the system. Variations in this had little effect on gain of Na and loss of K in the cold (Table 1) while during incubation, provided that glucose sufficed for glycolysis throughout the period, output of Na was little affected by the actual glucose level, though perhaps rather more glycolysis accompanied a given Na output in Na-rich systems. Effecs of ph. At ph 6*6, glycolysis is slight and active movements small. At ph 7-1, 65 mole of glucose disappear for 1 of Na expelled (Table 1, Exp. If, i compared with g, j), and at ph 7 3, 83 mole. In Exp. 2, the figures are a little different but it is clear that the relation of glycolysis to active transport varies with ph, and if the system be sufficiently alkaline, glycolysis may be very marked yet no active transport be apparent at all. Effects of time. At 6 hr. 45 m-glucose have disappeared for 1 M-Na expelled; at hr. the figure is 63 (Exps. id-f and 2e, g and h); hence, between 6 and hr. the figure is 1-1 M-glucose per 1 M-Na. Clearly then, the single hr. figure has little absolute significance even in rocked systems, though it may be

4 62 MONTAGUE MAIZELS used as an index of correlation between glycolysis and Na transport at a given ph. If one had a measure of passive diffusion and so of real active transport, it might indeed be desirable to have a 4- or 5-point curve for each experiment and so correlate rate of glycolysis with rate of transport. Even so, the difficulties would be considerable: thus when investigating the effects of cyanide one would need two cyanide samples and two cyanide free controls each at ph 6-9 and 7d1, and each of these four would have to be divided into four or five lots TABLE 1. Effects of time and ph on glycolysis and sodium output of incubated erythrocytes (rocked). (Seven days cold-storage followed by variable incubation; approximate external concentrations, K 33 and Na 12 m.equiv./l.) NaOH Glucose Glucose added to Incuba- in cell used cell tion K content Na content suspen- per litre suspension period ph of cells of cells sion of cells Exp. (m.equiv./l.)* (hr.) at 2 (m.equiv./l.)t (m.equiv./l.)t (mg. %) (mm.) Ia b c 4-2 7* d 2-4 7* e 2* if A i j a b c d e f lh * At beginning of incubation. f Referred to original cell volume. incubated for varying times, giving twenty lots in all, each lot needing a large number of different analyses done in duplicate. In the following experiments, therefore, time curves were not usually done, but glucose levels, etc., were observed at hr., sometimes in whole blood but more often in the incubated cells alone. Effects of metabolic poisons Poison-free controls. The higher the ph, the greater is glycolysis and the slower is phosphorolysis (see observations b and c and sometimes d in all experiments). Easily hydrolysable P lay as a rule between 3 and 5 mg. % at the end of cold-storage and fell by about 1 mg. during incubation. Optimum active transport corresponded to a final ph between 7-2 and 6-8. In view of normal variations of findings with ph, controls at several ph levels are given, and the

5 FACTORS IN ACTIVE TRANSPORT OF CATIONS 63 final ph of poisoned systems are planned to fall in this range. Deviations from the normal controls are evident on simple inspection, but where three control values are available curves may be drawn and results in poisoned systems compared with values at the corresponding control ph. Sodium fluoride. When the usual amounts of alkali were added to blood incubated with fluoride, high alkalinity resulted; without alkali, ph was about the same as in incubated fluoride-free systems with added alkali, showing that glycolysis and hence liberation of acid were inhibited. This was confarmed by sugar estimations which showed that with an overall fluoride content of 1 mm./l. glycolysis was almost completely inhibited (Table 2, Exp. 1): for Exp. Inc. (hr.) la b c d e f 9 h i j 2a b c d e f g h i 1 m TABLE 2. Effects of fluoride and mono-iodoacetate on active transport in incubated erythrocytes of unrocked blood (Approximate external concentrations, K 33 and Na 12 m.equiv./l.) Overall concentrations of additions in cell+plasm water (mm./l.) (unincubated) NaOH 4-2 NaOH 2-4 NaOH, NaF 1 NaOH, NaF 1, Na Py 2 NaOH 1-, NaF 4 NaOH 1-, NaF 4, Na Py 2 NaOH 2-6, NaF 1-5 NaOH 2-6, Na? 1-5, Na Py 2 Cell glucose control, NaF 3 (unincubated) NaOH 6-2 NaOH 4-5 NaOH 2-8 NaOH -6, MIA -8 NaOH -6, MIA -8, Na Py 2 NaOH 1-, MIA -2 NaOH 1-, MIA -2, Na Py 2 NaOH 2-2, MIA -5 NaOH 2-2, MIA -5, Na Py 2 NaOH 2-8, MIA -2 NaOH 2-8, MIA -2, Na Py 2 Cell glucose control H v i Contents referred to original cell volume (m.equiv./l.) (mg. %) ph2 'N rp ER? CeIL G at 2 K Na TP EHP IPL* (mg.%) Inc. =incubated. H =haemolysis, + =slight; ± =trace. V =percentage of original volume of fresh cells. TP =total acid soluble phosphorus. EHP=easfly hydrolysable phosphorus. IPL* =inorganic phosphorus liberated during incubation; at the beginning of incubation this equals. Cell G=cell glucose. Na Py=Na pyruvate. MIA=monoiodoacetic acid. Cell glucose control =cell glucose in blood incubated with NaF (-3 x) to give&complete inhibition of glycolysis. this reason cells incubated with NaF (.3 M) were used in other experiments as controls to give the cell glucose level in the absence of glycolysis and the difference between this value and that found after incubation with a given agent was a measure of the agent's effect on glycolysis

6 64 MONTAGUE MAIZELS NaF also largely inhibited the breakdown of phosphoric esters, except for hydrolysable phosphate which fell to a very low level; as a result of conservation of phosphoric esters, total cell P fell little during incubation. Active cation transport diminished and with the higher concentrations of fluoride, Na so far from leaving the cell increased there by passive diffusion while the level of K fell. The net result was a rise of cell total-base and volume, though the increase was sometimes less than would have been expected from the failure of active transport at high fluoride levels. There was also some lysis not explained by cell swelling and therefore attributable to damage of the cell membrane-possibly resulting from interference with nutrition. Addition of pyruvate to X2 M had the following effects on these findings: haemolysis was consistently and definitely less than when pyruvate was absent and passive diffusion of Na inwards and of K out was usually slightly decreased. In fourteen paired observations with pyruvate-fluoride and simple fluoride systems cell Na averaged 2*5 + 1'5 m.equiv./l. less in the former: in two instances it was slightly greater in the pyruvate-fluoride system, in three it was the same, and in nine observations from 1 to 4 m. equiv./l. less. These findings were thought to arise from the effects of pyruvate on permeability rather than on metabolism. They are also shown in the rocked system of Table 3 (Exp. 1), where absolute changes in glucose levels are also shown. Monoiodoacetic acid. The effects of this agent were similar to those of fluoride except that phosphorolysis was hastened and loss of total P from the cells marked (Table 2, Exp. 2). In conformity with this, glycolysis was inhibited and cell Na was higher and K lower than in the controls, while total base (Na + K) and cell volume were also greater than in the absence of iodoacetate. Increase in base and volume were greater than with fluoride. The addition of pyruvate had the same effect as with fluoride: haemolysis was less and cell Na tended to be about 2 m.equiv./l. lower than when pyruvate was absent. Cyanide. When cyanide was added along with the usual amounts of alkali, incubated systems were very alkaline and haemolysis was marked; even without added alkali the alkalinity of the cyanide gave a ph which was still high at the end of incubation and active transport was poor (Table 4, Exp. 1 e). The following gave satisfactory results: cyanide solution was Seitz-filtered and added along with a little HCI to stored blood in a vessel so small as to be almost filled. Under these circumstances glycolysis and phosphorolysis were more active than in cyanide-free controls, while active transport was well marked even when the cyanide concentration approached M/1. Carbon monoxide. Here, 4 ml. cell suspension were placed in a 34 ml. bottle which was closed by a rubber washer and perforated cap. Long and short hollow needles were pushed into the bottles and washed CO passed through and over the blood for 3 min., after which the needles were withdrawn and the punctures sealed. In this way the cell suspension was saturated with CO

7 FACTORS IN ACTIVE TRANSPORT OF CATIONS 65 and in constant contact with a large volume of oxygen-free CO gas. Table 4, Exp. 2, shows that haemolysis was rather more marked than usual, presumably because of the agitation produced by bubbling; otherwise CO systems were indistinguishable from CO-free controls, though glycolysis was rather more marked. TABLE 3. Effects of various poisons on active transport in incubated erythrocytes of rocked blood (Approximate external concentrations, K 28 and Na 125 m.equiv./l.) Contents referred to original cell volume A A Glucose Glucose Overall concentrations of (m.equiv./l.) (mg. %) in cell used by Inc. additions in cell +plasma ph suspension cells Exp. (hr.) water (mx./l.) at 2 K Na TP EHP IPL* (mg. %) (mm./l.) la (unincubated) b c NaOH 4-2 NaOH d NaOH, NaF e NaOH, NaF 8, Na Py f NaOH -6, NaF NaOH -6, NaF 4, Na Py a (unincubated) b NaOH c NaOH d NaOH e HCl 3-, NaCN f NaOH 2-2, NaCN NaOH, NaCN h NaOH 2-2, NaCN a (unincubated) b NaOH c NaOH d NaOH e NaOH, NaN f NaOH 2-2, NaN NaOH 5-7, NaN { h i NaOH 9-4, DNP -9 NaOH 6-2, DNP Inc. =incubated. TP =total acid soluble phosphorus. EHP =easily hydrolysable phosphorus. IPL* =inorganic phosphorus liberated during incubation; in the unincubated samples this equals. Na Py =Na pyruvate. DNP= dinitrophenol. In the suspensions of Exps. 1-3 cells respectively occupied 1-6, 16- anid 16-7 % by volume. Sodium azide (Table 4, Exp. 3). Active transport was practically uninhibited by azide (Seitz-filtered) up to -8 M and was evident in concentrations up to -16 M. Glycolysis and phosphorolysis were rather more marked than in the controls, but this effect was not quite so constant as with cyanide. All the preceding three agents, then, caused some increase in glycolysis as compared with the simple controls (a property shared by dinitro-phenol), but there was no evidence that this was accompanied by a corresponding increase of active transport, for Na was the same as, or rather less than, in the controls. This is also seen in Table 3 (Exps. 2 and 3), where in rocking experiments, cyanide, azide and dinitro-phenol caused an increased consumption of glucose PH. CXII. 5

8 66 MONTAGUE MAIZELS but without an increase in active transport. It was further thought possible that changes in active transport might be obscured after hr. incubation when glycolysis in the controls, though less than with cyanide, was still well marked and that the changes might be evident after only 6 hr. incubation when glycolysis was less advanced. In fact, glycolysis was relatively more marked with cyanide at this time, but active transport was not increased. The results present no special features and have not been tabulated. TABLE 4. Effects of cyanide, carbon monoxide and azide on active transport in incubated erythrocytes of unrocked blood (Approximate external concentrations, K 33 and Na 12 m.equiv./l.) Contents referred to original cell volume Overall concentrations of (m.equiv./l.) (mg. %) Inc. additions in cell +plasma ph, Cel G Exp. (hr.) water (mm./l.) H V at 2 K Na TP EHP IPL* (mg. %) la (unincubated) 17 7* b NaOH c NaOH d NaOH e NaOH, NaCN f HCl 4-5, NaCN NaOH 1-7, NaCN * h NaOH 3-4, NaCN v i Cell glucose control, NaF 3 _ a (unincubated) * b c NaOH 6-2 NaOH d NaOH 4.5, CO saturated ± e Cell glucose control, NaF a (unincubated) b c NaOH 6-2 NaOH d NaOH 4, NaN e NaOH 5., NaN f NaOH 5-, NaN Cell glucose control, NaF Inc. =incubation. V =percentage of original volume of fresh cells. H =haemolysis. + =slight. ± =trace. TP=total acid soluble phosphorus. EHP=easily hydrolysable phosphorus. IPL*=inorganic phosphorus liberated during incubation; at the beginning of incubation this equals. Cell G =cell glucose. Cell glucose control=cell glucose in blood incubated with NaF (-3 M.) to give complete inhibition of glycolysis. Mepacrine. This was not a satisfactory agent to use, for at moderate concentrations it induced lysis, and even when more dilute caused erythrocytes to adhere in a slimy mass, indicating that the cell exterior was certainly and permeability possibly altered. Glycolysis and phosphorolysis were not obviously affected, but there was apparent partial inhibition of Na output with marked uptake of K against the concentration gradient. Cell total base and volume increased. The cation changes with mepacrine suggest that there is a marked uptake of K with only a small corresponding output of Na a finding which is contrary to earlier conclusions (Flynn & Maizels, 1949). The position is clarified if

9 FACTORS IN ACTIVE TRANSPORT OF CATIONS 67 observations are made after 4 as well as at hr. incubation (Table 5, Exp. 2). The normal control shows a progressive fall of cell Na and a smaller rise of K thoughout incubation, and this is apparent after only 4 hr. when cell ph is still fairly high; thereafter, between 4 and hr. cell Na falls by 16 m.equiv./l. (from 31 to 15), while K rises by 11 m.equiv. (Exp. 2b, c). With mepacrine, cell Na rises between and 4 hr. (from 39 to 46 m.equiv./l.), and it is only when cell ph approaches neutrality that output of Na becomes really active. However, even here cell Na falls by 16 m.equiv./l. (from 46 to 3) between 4 and hr. and this exceeds the corresponding rise in K of 12 m.equiv. (Exp. 2d, e). Presumably, mepacrine increases the permeability of erythrocytes to cations TABLE 5. Exp. la b c d e f 9 2a b c d e 3a b c d e f Inc. (hr.) 4 4 Effects of mepacrine and arsenite on active transport in incubated erythrocytes of unrocked blood (Approximate extemal concentrations, K 33 and Na 12 m.equiv./l.) Contents referred to original cell volume Overall concentrations of additions in cell +plasma water (mm./i.) (unincubated) NaOH 6-2 NaOH 4-5 NaOH 3- NaOH 4'5, mepacrine 5 NaOH 4 5, mepacrine X25 Cell glucose control, NaF 3 (unincubated) NaOH 4-5 NaOH 4-5 NaOH 4-5, mepacrine -5 NaOH 4-5, mepacrine '5 (unincubated) NaOH 5-7 NaOH 4-5 NaOH 4-, Na arsenite 2-8 NaOH 5, Na arsenite 1-4 NaOH 5-, Na arsenite '7 H + V ph at ' ' ' (m.equiv./l.) (mg. %) K Na TP EHP IPL* ' Cell G (mg. %) 9 Cell glucose control, NaF 3 Inc. =incubation. V =percentage of original volume of fresh cells. H =haemolysis. + =slight. ± =trace. TP=total acid soluble phosphorus. EHP=easily hydrolysable phosphorus. IPL*=inorganic phosphorus liberated during incubation; at the beginning of incubation this equals. Cell G =cell glucose. Cell glucose control=cell glucose in blood incubated with NaF (-3 m) to give complete inhibition of glycolysis. at moderately high ph, and this will increase passive entry of the large hydrated Na ions early in incubation with normal output in the later, less alkaline stages. The smaller K ions in any case penetrate red cells relatively quickly, and their diffusion outwards with the concentration gradient (normally restrained by the equilibrium requirements of non-penetrating anions within the cells) will be less affected by increased permeability than the corresponding diffusion of Na inwards. If this is so, then early in incubation there will be a large passive gain of Na with little or no loss of K (as in Table 5, Exp. 2 d) and later in

10 68 MONTAGUE MAIZELS incubation, the usual output of Na with compensatory gain of K (Exp. 2e). In this way may be explained the seemingly anomalous transport of cations observed after hr. incubation with mepacrine, although, as we have seen, the actual active output of Na between 4 and hr. is, in fact, considerable and exceeds the corresponding uptake of K. Similar cation changes are observed when erythrocytes are incubated with a small volume of very alkaline hypertonic saline instead of with a large volume of slightly alkaline isotonic solution. Sodium ar8senite (Table 5). This agent caused a little inhibition of glycolysis with no obvious effect on phosphorolysis. In the concentrations used, there was some inhibition of Na output as compared with the controls, uptake of K against the gradient being less affected. As with mepacrine, this effect was ascribed to an increase in permeability: a view supported by the constant slight haemolysis observed with this substance. TABic 6. Effects of dinitrophenol, methylene blue and malonate on active transport in incubated erythrocytes of unrocked blood (Approximate external concentrations, K 33 and Na 12 m.equiv./l.) Contents referred to original cell volume (m.equiv./l.) (mg. %) Inc. Additions per litre of ph,,, Cell G Exp. (hr.) cell+plasma water H V at 2 K Na TP EHP IPL* (mg.%) la (unincubated) b NaOH 6.2 m.equiv * c NaOH 4.5 m.equiv. 16 6X d NaOH 2X8 m.equiv X NaOH 7 3 m.equiv., DNP 2.4 mm. ± f NaOH 7.3 m.equiv., DNP 1.2 mx NaOH 6*7 m.equiv., DNP -6 mx NaOH 6-7 m.equiv., DNP -2 mx. 12 6X ' i NaOH 6*7 m.equiv., MB 12 mg. i j NaOH 6-7 m.equiv., MB 3 mg k Cell glucose control, NaF 3 mx a (unincubated) b NaOH 6-2 m.equiv c NaOH 4-5 m.equiv d NaOH 2-8 m.equiv c NaOH 4-5 m.equiv., Malon lo mx. 1 7* f NaOH 4*5 m.equiv., Malon 5 mx Inc. =incubation. V =percentage of original volume of fresh cells. H =haemolysis. + =slight; + =trace. TP =total acid soluble phosphorus. EHP =easily hydrolysable phosphorus. IPL* =organic phosphorus liberated during incubation; at the beginning of incubation this equals. Cell G=cell glucose. DNP =dinitrophenol. MB =methylene blue. Malon =Na malonate. Cell glucose control =cell glucose in blood incubated with NaF (.3 x) to give complete inhibition of glycolysis. Dinitrophenol (Tables 3 and 6). This substance in concentrations higher than 2*4 mm./l. caused slight haemolysis associated with decreased output of Na. Below this level active transport was practically unaffected, but phosphorolysis and glycolysis were both increased and ph was lower than in the corresponding controls (Table 3, Exp. 3, h compared with b); it is possible that a similar ph

11 FACTORS IN ACTIVE TRANSPORT OF CATIONS 69 effect might be observed with cyanide and azide were it not obscured by the natural alkalinity of these agents. Methylene blue. This increased the amount of sugar used and hence the acidity developed in the system during incubation (Table 6, Exp. 1 i and j compared with b; Table 8, Exp. 2 e and f compared with b); but this did not raise the activity of cation transport. Phosphorolysis was inhibited by methylene blue. Malonate (Table 6). Na malonate up to -1 M had no effect on active transport. Sulphanilamide. This up to 2 mm./i. was without effect on active transport. Glucose and other sugars as sources of energy When blood is cold-stored with or without glucose, cation distribution after 6 days is similar (Tables 7-1; unincubated levels); presumably, the store of natural glucose originally present in fresh blood glycolyses so slowly at low temperatures that a remnant persists even after a week. But during subsequent incubation in the absence of added sugar, cell glucose quickly disappears-more especially in unrocked systems where the settled cell mass is out of contact with the main volume of plasma-and cations continue to run with the gradient and not against it, so that cell Na, total base and volume rise above the cold-storage level while total and hydrolysable phosphate fall and inorganic phosphate liberated rises. As might be expected, little acid is set free during incubation of blood without added glucose and a very small addition of alkali suffices to keep the ph at about 7. Glucose, fructose, mannose and galactose all penetrate the erythrocyte readily: the first three are all capable of energizing active transport; galactose is not (Table 7). The effects of glucose and mannose are very similar. The effects of fructose are peculiar: when this sugar is present in high concentration, as in Table 7, fructolysis and liberation of acid is marked, cation transport is active and hydrolysable phosphate which usually decreases when erythrocytes are incubated with other sugars, actually increases above the cold-storage level in the presence of fructose-usually at the expense of inorganic phosphate liberated. When, on the other hand, blood is incubated with low concentrations of fructose as in the shaking experiments of Table 8, fructolysis is small compared with glycolysis in the glucose systems and acid liberation is slight, so that when alkali is added to both systems to -12 N, cell ph after incubation with glucose is 6-93 and with fructose So, too, output of Na in low glucose systems is about 2 m.equiv./l. and in low fructose systems only 8. With systems containing galactose little acid is liberated and active transport is not apparent. Arabinose and xylose (Table 9). Little acid is liberated in the presence of xylose and arabinose and any ph shift may well be ascribed to glycolysis of residual natural glucose. Both these substances appear to give rise to very small Na outputs, and the effect is more significant wheh compared with the

12 7 MONTAGUE MAIZELS Inc. Exp. (hr.) la b c d TABLE 7. Monosaccharides and pyruvate as activators of cation transport in the incubated erythrocytes of unrocked blood (Approximate external concentrations, K 35 and Na 115 m.equiv./l.) Contents referred to original cell volume Additions,A_ A, 5(m.equiv./l.) (mg. %) NaOH ph I CellS Sugar, etc. (m.equiv./l.) H V at 2 K Na TP EHP IPL* (mg.%) Glucose (unincubated) Glucose Glucose 4* Glucose 3* e f Galactose (unincubated) g Galactose h Cell galactose control i Fructose (unincubated) Fructose Cell fructose control 1 Mannose (unincubated) m Mannose n Cell mannose control o No sugar (unincubated) p No sugar q No sugar, pyruvate + r Sugar-free control Cell glucose control ± ± ? ± ? ?1 Inc. =incubated. H =haemolysis; + =slight, ± =trace. V =percentage of original volume of fresh cells. TP =total acid soluble phosphorus. EHP =easily hydrolysable phosphorus. IPL* =inorganic phosphorus liberated during incubation; at the beginning of incubation this equals. Cell S =cell sugar. Pyruvate + =pyruvate added to 2 m.equiv./l. Cell-sugar controls =cell sugar in blood incubated with NaF (43 i) to give complete inhibition of glycolysis. Inc. Exp. (hr.) la b c d e f 9 h i 2a b c d c f TABLE 8. Effects of monosaccharides and methylene blue on active transport in the incubated erythrocytes of rocked blood (Approximate external concentrations, K 28 and Na 125 m.equiv./l.) Contents referred to original cell volume Sugar A Additions in cell Sugar (m.equiv./l.) (mg. %) suspen- used by NaOH ph,- sion cells Sugar, etc. (m.equiv./l.) at 2 K Na TP EHP IPL* (mg. %) (mm./l.) Glucose (unincubated) Glucose Glucose * Galactose (unincubated) Galactose Fructose (unincubated) Fructose Mannose (unincubated) Mannose Glucose (unincubated) Glucose Glucose Glucose Glucose, MB 12 mg. % Glucose, MB 3 mg. % Inc. =incubated. TP =total acid soluble phosphorus. EHP =easily hydrolysable phosphorus. IPL* =inorganic phosphorus liberated during incubation. MB =methylene blue.

13 FACTORS IN ACTIVE TRANSPORT OF CATIONS 71 findings in bloods incubated without any sugar being added (Table 9, cf. Exp. 1 f and g, i and j, 1 andm). This matter is considered later. TABLE 9. Pentoses and cation transport in the incubated erythrocytes of unrocked blood (Approximate external concentrations, K 33 and Na 115 m.equiv./l.) Contents referred to original cell volume Additions (m.equiv./l.) (mg. %) Inc. NaOH ph, A CellS Exp. (hr.) Sugar, etc. (m.equiv./l.) H V at 2' K Na TP EHP IPL* (mg.%) la Glucose (unincubated) b Glucose c Glucose d Glucose e Cel glucose control f Arabinose (unincubated) Arabinose h Cell arabinosecontrol i Xylose (unincubated) j Xylose 1-2 ± k Cell ylose control No sugar (unincubated) m No sugar 1- ± 115 7* *7 3? n Sugar-free control <1 Inc.=incubated. H=haemolysis. +=slight. ±=trace. V =percentage of original volume of fresh cells. TP =total acid soluble phosphorus. EHP =easily hydrolysable phosphorus. IPL* =inorganic phosphorus liberated during incubation. CellS =cell sugar. Cell-sugar controls =cell sugar in blood incubated with NaF (-3M) to give complete inhibition of glycolysis. TABLE 1. Disaccharides and cation transport in the incubated erythrocytes of unrocked blood (Approximate external concentrations, K 35 and Na 115 m.equiv./l.) Contents referred to original cell volume Additions,r A- (m.equiv./l.) (mg. %) Inc. Sugar, etc. NaOH ph, --- Cell S Exp. (hr.) (m.equiv./l.) H V at 2' K Na TP EHP IPL* (m.%) la Glucose (unincubated) b Glucose c Glucose d Cell glucose control e Lactose (unincubated) f Lactose Cel lactose control h Maltose (unincubated) - 1 7{ i Maltose j Cell maltose control k Sucrose (unincubated) Sucrose 1-2 ± m Cell sucrose control n No sugar (unincubated) o No sugar 1- ± ? p Sugar-free control - 14 Inc. -incubated. H =haemolysis, + =slight; ±- =trace. V =percentage of original volume of fresh cells. TP =total acid soluble phosphorus. EHP =easily hydrolysable phosphorus. IPL* =inorganic phosphorus liberated during incubation. Cell S =sugar. Cell-sugar controls =cell sugar in blood incubated with NaF (-3 ms) to give complete inhibition of glycolysis. Lactose, maltose and sucrose (Table 1). These sugars also liberate little or no acid during incubation, and in the case of sucrose active transport is slight or

14 72 MONTAGUE MAIZELS absent. Lactose is apparently associated with a small amount of active cation transport; with maltose, the effect is slightly greater. Pyruvate (Table 7). This substance probably does not supply energy for cation transport, and in the absence of added glucose cell Na, total base and volume all increase. DISCUSSION According to Rothstein (1948) yeast cells probably have some of their enzyme systems situated on the cell surface, but in the case of the red cell (which is not concerned with non-diffusible external substrates) the glycolysing and phosphorylating systems are probably situated in the substance of the cell membrane or on its inner face (Maizels, 1949). Hence, when assessing the potency of an inhibiting agent it is necessary to be sure that the agent is able to penetrate the cell and if so, that enough of it remains after combination with the external cell face and internal proteins (haemoglobin) to exert specific effects, if any, on the cell-enzyme systems. Of the substances investigated, those which exert a clear inhibitory effect present no difficulties: such are fluoride and iodoacetate. In the case of cyanide, CO and azide simplicity of structure renders penetration certain and, in fact, cyanide and iodoacetate have been shown to penetrate the red-cell membrane rapidly (Maizels, 1934), while penetration by azide and CO is made evident by changes in the colour of cell haemoglobin. With regard to other agents: those which are surface active also penetrate cell membranes readily. But if the agent is strongly adsorbed at the outer cell face, the effective concentration of the agent within the system will be far below the initial concentration and it will be difficult to assess its effects on the enzyme systems of the inner cell face. However, in the present investigation adsorption is not sufficiently powerful to affect the bulk concentration significantly. Thus, even with mepacrine which is strongly adsorbed, the actual concentration in the system was only 8 % less than the theoretical value when the latter was 5 mm./l. and the volume of the external phase nine times that of the cells. It is therefore probable that the actual effective intracellular concentration of mepacrine is not much below the theoretical overall value. The same applies with more force to dinitrophenol which is less strongly adsorbed than mepacrine; here, moreover, evidence in favour of free penetration is afforded by analogy to nitrobenzoate which enters human red cells rapidly (Maizels, 1934). With methylene blue loss of colour in the extracellular fluid was more marked, either from adsorption or reduction: however, it is clear that methylene blue accelerates breakdown of sugar within the cell and so adequate penetration may be presumed; further, it is known that methylene blue stains nuclear granules in nucleated red cells. Hexoses, pentoses and pyruvate all penetrate erythrocytes adequatelv under the present experimental conditions: the disaccharides, little if at all. It is therefore not possible to ascribe the inactivity of the disaccharides to their

15 FACTORS IN ACTIVE TRANSPORT OF CATIONS 73 inability to participate in the cell metabolic cycle. In the case of arsenite alone, there is doubt about the amount of penetration, if any, which occurs. Multivalent inorganic anions like phosphate and presumably arsenite, penetrate the erythrocyte membrane slowly at low temperatures, though at 37 phosphate certainly and arsenite possibly, penetrate fairly rapidly; in support of the latter possibility is the inhibitory effect exerted by arsenite on glycolysis (Table 5). Action of m4tabolic poisons It is generally accepted that metabolism in human erythrocytes follows the well-known Embden-Meyerhof-Parnas cycle: thus, the red cells show reciprocity between glycolysis and phosphorylation, and the effects of ph on these activities are characteristic, as are the inhibitory actions of fluoride and iodoacetate. Further, all the ingredients of the cycle are present, including glucose, adenosinetriphosphate (whose P accounts for 2% of the total acid soluble P), di- and tri-phosphopyridine nucleotide, co-carboxylase and phosphoric esters. However, with regard to the latter, the human erythrocyte differs from many cells in that half the total acid soluble P is contributed by 2, 3-diphosphoglycerate, a stable ester to be distinguished from the labile 1, 3-ester which appears to be a normal intermediary in glycolysis (Guest & Rapoport, 1941). It is also important to remember that metabolism in the human red cell is normally conducted through channels of aerobic glycolysisa route shared by many cells where aerobic glycolysis is active though accompanied by a greater or lesser amount of respiration; among -such cells are spermatozoa, retina, pus and others. Fluoride and iodoacetate. It has been seen that active cation movements require the presence of glucose (Harris, 1941; Maizels, 1949), and the fact that fluoride and iodoacetate interfere with cation transport suggests that the energy needed is acquired during normal processes of glycolysis or respiration, and presumably the former, since respiration in the red cell is very slight. The detailed effects of these inhibitors present features of interest. Thus, in the case of fluoride, breakdown of phosphoric esters is much decreased (Table 2, Exp. 1, and Table 3, Exp. 1) presumably because of the inhibition of enzyme systems acting between the phosphoglycerate-phosphopyruvate stages. As a result, the balance between dephosphorylation and rephosphorylation of adenylic acid fails and hydrolysable phosphate is much decreased; at the same time glycolysis is inhibited and the blood system liberates very little acid during incubation. Iodoacetic acid likewise inhibits glycolysis and acid production and here (Table 2, Exp. 2) alkali was added at the beginning of incubation to neutralize the acidity of iodoacetic acid added as such and not to neutralize any acid metabolites that might be produced during incubation. Monoiodoacetate differs from fluoride, however, in that it leads to a considerable liberation of inorganic phosphate presumably because it stops the cycle between 3-phospho-

16 74 MONTAGUE MAIZELS glyceraldehyde and 1, 3-diphosphoglycerate and so disturbs the balance between de- and re-phosphorylation of 2, 3-diphosphoglycerate: progressive breakdown ofthe latter then providing most of the inorganic phosphate liberated under the influence of iodoacetate. This inorganic phosphate set free should suffice to maintain the level of hydrolysable phosphate; its failure to do so suggests that iodoacetate interferes with the rephosphorylation of adenylic acid. Cyanide, azide and carbon monoxide. It has already been suggested that in view of the slight respiration of non-nucleated erythrocytes, it is unlikely that respiratory mechanisms could contribute to cation transport. This view is confirmed by the failure of cyanide, azide and CO to inhibit output of Na and uptake of K (Table 3, Exps. 2 and 3, and Table 4). These substances inhibit cytochrome oxidase, and hence the usual oxidative routes may be excluded as energizers. There remain oxidative systems not involving cytochrome: how far these function under normal conditions is by no means clear; but in the CO experiments where gas was bubbled through blood for half an hour, it is unlikely that any respiratory process could function. It therefore seems clear that active cation movements are energized by glycolysis whether be present or not. One other point is of interest here: all these agents, cyanide, azide, CO and also dinitrophenol, increase the rate of aerobic glycolysis so that this exceeds (though not greatly) glycolysis observed in untreated controls; this is an example of inhibition of the Pasteur effect observed in the case of erythrocytes and many other tissues (for references, see Burk, 1939). Nevertheless, the increased glycolysis is not accompanied by increased active transport, possibly because all these agents while increasing glycolysis, tend to inhibit phosphorylation. It is likely that high energy phosphate bonds mediate in active transport and this may account for the absence of increased transport, in spite of the more rapid glycolysis. It follows that the rate of glycolysis is not necessarily an index of metabolic efficiency. 4, 6-dinitrophenol. Many observations have been made on the action of this substance which appears to be twofold: it stimulates glycolysis and also respiration (Dodds & Greville, 1934). The extent to which each is affected varies with the tissue or cell and substrate (Pickett & Clifton, 1941). This absolute increase of metabolic activity appears to be due to a diversion of substances normally incorporated into the cell substance to glycolytic or respiratory breakdown or, in other words, to a failure of synthesis. It may be noted that when increased respiration occurs with dinitrophenol, it is cyanide sensitive (Bodine & Boell, 1938) thus differing from the respiration stimulated by methylene blue, which is cyanide insensitive. In the erythrocytes of the goose, dinitrophenol causes a marked increase of glycolysis and respiration (de Meio & Barron, 1935), but with the non-nucleated mature human erythrocyte increased respiration does not occur, nor is it to be expected, since dinitrophenol,

17 FACI'ORS IN ACTIVE TRANSPORT OF CATIONS 75 unlike methylene blue, is not a H acceptor. But as increase of glycolysis does in fact occur and can hardly be due to a block in synthesis, it must be ascribed to an increase in the rate of glycolysis and not to an absolute increase in the amount of substance glycolysable. The extra glycolysis induced by dinitrophenol seems to follow the usual cycle since it is inhibited by iodoacetate (Ronzoni & Ehrenfest, 1936), and so the absence of increased active transport in the presence of this agent may perhaps be attributed to interference with phosphorylation. This interference is illustrated in Table 3, Exp. 3, and Table 6, Exp. 1: it has been noted previously by Loomis & Lippman (1948) and by Judah & Williams-Ashman (1949). It may be observed that the optimum-stimulating dose of dinitrophenol varies with the system examined and perhaps with the amount of protein in the system. In the case of human red cells the optimum appears to lie between -2 and 1-2 mlm./l.; with higher concentrations slight haemolysis occurs and active transport fails. Mepacrine. According to Haas (1944) mepacrine inhibits respiration by competing with riboflavine phosphate for cytochrome reductase, and it also prevents the conversion of glucose-6-phosphate to phosphohexonic acid. Hence, mepacrine should be without action on active cation transport which is energized by glycolysis. This is in fact the case, though output of Na is obscured-by effects of mepacrine on permeability. That active output does occur, however, is made clear by reference to a time curve (Table 5, Exp. 2 a, d and e); the matter is discussed more fully in an earlier section. Arsenite. This agent acts mainly on the aerobic phase of metabolism, inhibiting the oxidation of pyruvate (Peters, Sinclair & Thompson, 1946). Hence it should leave cation transport unaffected. But, in fact, fall in cell Na is less than in arsenite-free controls, while uptake of K against the concentration gradient is little affected (Table 5, Exp. 3). The findings resemble those obtained with mepacrine, and may similarly be due to permeability changes giving a marked rise of cell Na early in incubation followed by a fall of normal extent which leaves cell Na at the end of incubation higher than in the controls. Arsenite in the concentrations used always caused slight haemolysis, and this may be associated with changes in permeability. Arsenite also had some inhibitory effect on glycolysis, presumably exerted directly on the glycolytic cycle. Malonate. This substance inhibits respiration by competing for succinoxidase. It should therefore have no action on active transport. Table 6, Exp. 2, shows that this is the case. Methylene blue. Nucleated red cells and reticulocytes have co-existing respiratory and glycolytic cycles, while glycolysis predominates in adult cells and respiration is negligible. But addition of methylene blue, which is a H acceptor raises consumption to the levels found in nucleated cells (Harrop & Barron, 1928). This form of respiration is cyanide resistant, and so differs from

18 76 MONTAGUE MAIZELS the usual respiratory process. The presence of methylene blue in amounts sufficient to increase greatly the disappearance of glucose appears to leave active transport unaffected. There are two possibilities: (a) cyanide-resistant respiration is associated with partial inhibition of glycolysis but by contributing to active transport maintains the usual output of Na and uptake of K; (b) glycolysis goes on side by side with, and uninhibited by, the cyanide-resistant respiration induced by methylene blue and since cation movements are not increased by the dye, they must be motivated solely by glycolysis. Preliminary investigations (Judah and Maizels) show that the addition of fluoride may be so adjusted as to inhibit glycolysis and to leave cyanide-resistant respiration practically unaffected; under these conditions, active transport fails. Hence, the second possibility indicated above is probably correct. In this connexion it may be noted that barley roots contrast with erythrocytes in that they need to respire in order to effect active transport, but the induction of cyanide-resistant respiration with methylene blue, so far from increasing salt accumulation, inhibits it (Hoagland & Broyer, 1942). Inhibition of phosphorolysis by methylene blue remains unexplained. Glucose and other possible sources of energy for activation cation transport Glucose, mannose, fructose and galactose. Of these, the first three energize active transport; galactose does not and its failure to figure in the glycolysis cycle is associated with the loss of very little sugar from the system and with the rapid hydrolysis of easily hydrolysable and other phosphoric esters (Tables 7 and 8). Fructose is peculiar in that with high fructose bloods (as in the unrocked system of Table 7, Exp. l i-k) there is marked active transport and phosphorolysis is no greater and may be less than in glucose systems; indeed, there is an actual synthesis of easily hydrolysable phosphate. In low fructose bloods, on the other hand (as in the rocked systems of Table 8, Exp. 1) loss of fructose from the incubated blood is less than loss of mannose or glucose from corresponding systems. Cation transport is less effective and no synthesis of hydrolysable phosphate is seen. The findings suggest that the reaction rates of the several parts of the glycolytic cycle vary with the kind and amount of sugar participating; they cannot be explained by variations in the penetration rates of these sugars, for though such variations may well exist, all penetrate freely during cold-storage and incubation under the experimental conditions set up. The findings correspond with those of Meyerhof & Geliazkowa (1947) who show, in the case of brain and sarcoma slices, that glucose and mannose give active glycolysis, that galactose is always inactive and that fructose is relatively inactive in 2 % solution but glycolyses as rapidly as glucose in 2% solution. These findings also occur in homogenates and are not due to failure of the sugar to penetrate tissue cells. The writers attribute the findings to different affinities of the sugar for hexokinase so that at low concentrations the affinity

19 FACTORS IN ACTIVE TRANSPORT OF CATIONS 77 of fructose appro2ximates to that of galactose and at high levels to glucose and mannose. In the case of red cells too, it has been found that hexokinase phosphorylates glucose, mannose and fructose, but not galactose (Christensen, Plimpton, Calvin & Ball, 1949), while according to Spicer & Clarke (1949) erythrocytes of dog, cat and rabbit metabolize glucose, mannose and fructose readily and galactose only slightly. So too, glucose, mannose and fructose, but not galactose, serve to maintain motility in spermatozoa (Mann, 1949). Arabinose, xylose, maltose and latose (Tables 9 and 1). These are associated with the liberation of very little acid during incubation as compared with the glucose containing controls, and cation transport is slight. Nevertheless, passive increase of cell Na, evident in the simple glucose-free systems (Table 9, Exp. 14, m, and Table 1, Esxp. 1 n, o) is absent and a small output of Na occurs. This may be explained in several ways: (a) The sugar added is metabolized; this is unlikely on general grounds and more especially because dephosphorylation of esters, including those that are easily hydrolysable, is no less in the presence of arabinose, xylose, lactose and maltose than it is in the cells of glucose-free bloods; further, the disappearance of reducing substances during the incubation of these systems is very small. Finally, though pentoses penetrate human erythrocytes readily, it is known that disaccharides do not (Kozawa, 1914); Table 1 shows that after incubation with lactose or maltose, about 1 mg. % of the sugar are associated with the cells, but preliminary investigations suggest that this is adsorbed on the cell surface and does not penetrate. (b) The natural glucose present in blood persists dluring coldstorage and promotes a little active transport during incubation; against this is the observation that no Na output occurs in glucose-free samples. (c) Glucose present as an impurity in the sugar added is the cause of the output. In fact, however, pentoses and disaccharides were shown to be free of glucose, except for maltose which evolved a trace of gas on fermentation with yeast. (d) It will be shown later that only a small part of the energy set free by glycolysis is available for active transport and it is possible that sugars, themselves not glycolysed might in some way divert energy from other cell activities to transport, the source of energy being natural blood glucose surviving after coldstorage. (e) These sugars by decreasing the permeability of the cell membrane would check simple diffusion of K and Na and so favour whatever cation transport might be possible. However this may be, it is clear that output of Na in the presence of arabinose, xylose, lactose and maltose is very small, that the sugars themselves probably do not participate in any chemical cycle and that their effects compared with those of glucose, fructose and mannose, are insignificant. Saccharose. This sugar, which does not penetrate erythrocytes, was incapable of supporting active transport.

20 78 MONTAGUE MAIZELS Pyruvate. Energy can only be derived from pyruvate (or lactate) by respiratory processes, and since cation transport in human red cells depends on glycolysis, it is not to be expected that pyruvate or lactate will act as energizers and experimentally this is found to be the case (Table 7, Exp. 1 q). Nor is it to be expected that the addition of pyruvate to systems incubated with'fluoride or iodoacetate would help to originate active transport, for though tissues poisoned with these agents may respire when pyruvate or lactate is added, they will not manifest glycolysis on which, in red cells at least, active transport depends. Nevertheless, the addition of pyruvate to blood incubated with fluoride or monoiodoacetate does as a rule lower the Na level by 2 or 3 m.equiv./l. as compared with'the pyruvate-free system (Tables 2 and 3). Further, it has been seen that fluoride causes cell haemolysis, not due to osmotic swelling and possibly metabolic in origin; this lysis is also inhibited by pyruvate. The findings 'are paralleled by those of Wilbrandt (194) which show that the permeability of red cells increases in the presence of fluoride and that the increase is abolished by the addition of pyruvate. Presumably, the lytic effect of fluoride has a metabolic basis; the cause of inhibition by pyruvate is obscure. Phosphoric esters. As these do not penetrate the erythrocyte membrane (Hevesy & Aten, 1939) their capacity to energize cation transport cannot be investigated. Types of cation transport It has been seen that as a result of active transport in human erythrocytes incubated after cold-storage, there is a marked output of Na, with a lesser compensatory uptake of K and a net loss of total base (Flynn & Maizels, 1949). The transport tends to keep cell base, water and volume constant and so prevent cell swelling and rupture by physical forces: this mechanism may well be common to all individual cells unprotected by an internal or external resistant structure. The process is inhibited by fluoride and iodoacetate, but not by cyanide, CO or azide, and depends not on respiration but on glycolysis. With regard to energy available (Table 1), it has been seen that with external Na rising from 12 to 122 m.equiv./l., cell Na falls from 52 to 32 m.equiv. after 6 hr. incubation with a conversion of 9 mm. glucose to lactic acid and the liberation of energy equivalent to about 5 calories; this has to provide for a total output of Na which exceeds the apparent output (2 m.equiv./l.) by an amount equal to Na diffusing into the cells passively. E. J. Harris (personal communication) calculates that apparent output requires 4-8 cal./hr. and the total about 7'2 cal./hr.; this is less than 1 % of the 8 cal./hr. liberated in the first 6 hr. incubation. In contrast to this form of active transport in and out of cells for their individual purposes, is active transport through or across a membrane of cells. An example of this is the passage of Na through the frog's skin from a lowexternal to a high-internal level (Huf, 1935; Ussing, 1949); but this process is

21 FACTORS IN ACTIVE TRANSPORT OF CATIONS inhibited by cyanide even at the optimum ph of 8 (Ussing, 1949) and also by monobromacetate, though with the latter some transport may be restored by adding pyruvate (Huf, 1935). It follows that respiration and not glycolysis is responsible for transport across the frog's skin. The activity serves to replace Na loss by renal excretion; it is conceivable that this could be accomplished by an anaerobic mechanism, but possibly anaerobic conditions do not provide enough energy for the rapid transport of sodium ions from the low level of pond water to the high level of plasma. A respiratory mechanism also seems to account for the expulsion of Na by the distal convoluted tubule of the frog's kidney, for here also output is inhibited by cyanide (Conway, Fitzgerald & MacDougald, 1946). With regard to frog's muscle, Conway (1947) states that the metabolism of the resting muscle does not provide enough energy for expelling Na, and he holds that the high K and low Na levels are maintained almost entirely by physical means-a view which he supports by the observation that the anomalous cation distribution is unaffected by the presence of cyanide. But as we have seen, absence of respiration is no bar to some forms of transport and since, according to Harris (195), active expulsion of Na from frog's muscle only requires about 15% of the total resting energy available, the existence of active transport in frog's muscle seems probable. Another example of active transport is furnished by barley roots (Lundegardh, 1945) which take up and concentrate ions preferentially from dilute solutions; the process is poisoned by cyanide, and the energy needed is apparently derived from glucose by way of respiration. It is noteworthy that this form of active transport has as its object not only the selective removal of ions, but also the formation of a sap with a pressure of several atmospheres. We have thus considered three types of active transport, one in red cells leading mainly to cation exchange with little osmotic work and the others, in frog's skin and barley roots, requiring much osmotic work. The former involves cells as individual units, is based on glycolysis and is presumably phylogenetically older than transport across a film or community of cells where respiration provides the energy. How far this finding is general, is a matter for further investigation. Mechanisms for active transport Most workers are agreed that simple mechanical models cannot explain selective transport. Thus, following Teorell's model (1935), continuous production of acid based on glycolysis in erythrocytes during incubation would lead to escape of mobile H ions to the exterior, the interior of the cell becoming negatively charged and attracting external K and Na. Since Na is presumed to penetrate more slowly than K, the latter might be expected to accumulate. However, this device cannot explain passage against the gradient of Na already in the cell, more especially when Na expelled exceeds K taken up. 79

22 8 MONTAGUE MAIZELS Conway's views (1947) on K accumulation in muscle are also not applicable to the human erythrocyte. He suggests that amino-acids and inorganic phosphate can diffuse in and out of muscle cells, but during rest these substances on entering the cells are converted into large protein and phosphoric ester molecules, which are then unable to diffuse out of the fibres. If permeability of the muscle fibre to Na is slight and K considerable, K will enter greatly in excess of Na to combine with the new-formed non-diffusible anions; during activity these changes will be reversed. Such mechanisms, however, are not attributable to the human erythrocyte, first because the cell gains K during incubation at a time when total cell phosphate is falling (examples will be found in all the tables); secondly, even though Na were to penetrate red cells with extreme slowness, this would not explain why Na already in the cell is expelled against a gradient during incubation: rather the reverse. Conway states that the erythrocyte is 'essentially a moribund cell'; Krogh's (1946) views are at variance with this and it would seem more correct to regard the red cell as an adult structure which has lost its nucleus in specialization for carriage, and in view of the fact that it has a life span of up to 3 months, has an active and complex metabolic cycle and is capable of expelling 2 m.equiv./l. Na in 6 hr. against a considerable contrary gradient, the cell can in no sense be considered as senescent. The view is preferred (Maizels, 1949) that expulsion of Na is a primary activity and uptake of K secondary, the object being maintenance of constancy of cell composition and volume in the face of the erythrocyte's high content of non-penetrating anion and of its permeability to inorganic anions and cations. With regard to possible mechanisms for the actual expulsion of Na from cells, Conway (1947) supports a pore theory. He suggests that expulsion may be accomplished by a dehydrating device (within the cells) resulting on the one hand in a high external concentration of slowly penetrating hydrated Na ions and a low internal level, and on the other hand in a high internal concentration of rapidly penetrating dehydrated Na ions with a low external concentration. The view implies that normally in a watery medium a small proportion of Na ions are not hydrated. It is difficult to imagine any way in which the cell might bring about this active dehydration, but in any case it could only work in a two-phase system consisting of the dehydrating cell and the hydrating exterior. It could not work in the three-phase red-cell system with a dehydrating membrane separating external and internal hydrating phases, for in this case hydrated Na ions received from the exterior and dehydrated by the cell membrane, would still diffuse to the phase where the concentration of dehydrated Na ions was lower, that is to the cell interior. In fact, although uptake of K secondary to Na output can readily be understood, there is no clear explanation of the immediate method of Na output. There must clearly be an initial selective binding of Na on the inside of the cell membrane, transmission to the external face and release there. It is commonly

23 FACTORS IN ACTIVE TRANSPORT OF CATIONS 81 assumed that K is more strongly adsorbed than Na, but Hodgkin (1947) remarks that 'the tendency of sodium to combine with a lipoid soluble carrier might conceivably be greater than that of potassium'. Expulsion of Na would then follow 'if there were a continuous production and leakage of the carrier or if the carrier remained in the cell membrane, but was altered chemically in a cyclical manner as a result of metabolic activity'. Hodgkin thus offers alternatives of which the second is more probable, since it is unlikely that the adult red cell has the power of continually producing lipoid carriers. Lundegardh (1945) suggests that random rotation of surface molecules may suffice to carry ions to the cell exterior provided that a detaching mechanism co-exists; he further suggests that some types of active transport might be mediated by a cytochrome system. This may well be so, though the mechanism envisaged is not primarily selective nor is it applicable to human erythrocytes which do not respire. That the cell membrane is a dynamic structure is supported by the observations of Pulvertaft (1949) who, with the phase-contrast microscope, found the red-cell membrane to be in continuous shimmering movementa phenomenon which decreased with time and was inhibited by fluoride. Finally, these theories are merely intended to indicate possible modes of action in an obscure phenomenon and to suggest future lines of research. Our actual knowledge is limited to the obvious facts that Na adheres to the inside of the cell surface, is carried to the outside and detached there. SUMMARY 1. When human blood is cold-stored cations flow with the concentration gradient, Na rising and K falling in the cells. On incubating the cold-stored blood without added glucose these movements are all accelerated, but on incubation in the presence of extra glucose the cation flow is reversed so that cell Na falls and K rises, in each case against the respective concentration gradients. 2. Glucose, mannose and fructose all energize cation transport. Mannose is as effective as glucose. Fructose is as effective as glucose when present in high concentration, but when the fructose concentration is low active transport is much less than in systems containing a correspondingly low level of glucose. 3. Galactose, arabinose, xylose, the disaccharides, pyruvate and lactate do not energize active cation transport. 4. The energy for active transport in human erythrocytes is an accompaniment of glycolysis and not of respiration. 5. When glucose or mannose are present in incubated blood, the cells liberate acids with a decrease in sugar and in phosphoric esters, including hydrolysable phosphate. Similar findings occur with fructose systems, except that hydrolysable phosphate increases above the cold-storage level. In the absence of glucose, mannose or fructose erythrocytes liberate little acid during incubation, PH. CXII. 6

24 82 MONTAGUE MAIZELS phosphorolysis is hastened and hydrolysable phosphate practically disappears within hr. 6. Fluoride and monoiodoacetate tend to inhibit glycolysis and also activ.e transport. The disappearance of hydrolysable phosphate is hastened by both agents, but apart from this ester, phosphorolysis is hastened by iodoacetate and delayed by fluoride. 7. Inhibitors of cytochrome systems-cyanide, azide and CO-do not inhibit active transport, unless their concentration is very high and then the effect is probably non-specific. 8. Other inhibitors of respiratory processes are also without significant effect on active transport (unless their concentrations are relatively high). Such agents are dinitrophenol, malonate, mepacrine and arsenite, though the last two may cause an apparent decrease in transport through their effects on permeability. 9. Cyanide, azide and dirnitrophenol hasten aerobic glycolysis and also phosphorolysis, but increased consumption of glucose is not accompanied by increase in active cation transport. 1. Methylene blue hastens the disappearance of glucose but inhibits phosphorolysis; active transport is not accelerated by the dye. REFERENCES Bodine, J. H. & Boell, E. J. (1938). J. ceu. comp. Physiol. 11, 41. Burk, D. (1939). Cold Spr. Harb. Symp. quant. Biol. 7, 42. Chrisnsen, W. R., Plimpton, C. H., Calvin, H. & Ball, E. G. (1949). J. biol. Chem., 791. Conway, E. J. (1947). Irish J. med. Sci. 262, 593. Conway, E. J., Fitzgerald,. & MacDougald, T. C. (1946). J. gen. Phys8. 29, 35. Dodds, E. C. & Greville, G. D. (1934). Lancet, i, 398. Flynn, F. & Maizels, M. (1949). J. Physiol. 11, 31. Guest, G. M. & Rapoport, S. (1941). Physiol. Rev. 21, 41. Haas, E. (1944). J. biol. Chem. 155, 321. Harris, E. J. (195). Trans. Faraday Soc. (in the Press). Harris, J. E. (1941). J. biol. Chem. 141, 579. Harrop, G. A. & Barron, E. S. G. (1928). J. exp. Med. 48, 27. Hevesy, G. & Aten, A. H. W. (1939). K. danske vidensk. Sel8k. Biol. Medd. 14, 5. Hoagland, D. R. & Broyer, T. C. (1942). J. gen. Physiol. 25, 865. Hodgkin, A. L. (1947). J. Physiol. 16, 319. Huf, E. (1935). Pflug. Arch. gee. Physiol. 235, 655. Judah, J. D. & Williams-Ashman, H. G. (1949). Biochem. J. 44, xl. Kozawa, S. (1914). Biochem. Z. 6, 231. Krogh, A. (1946). Proc. Roy. Soc. B, 133, 14. Loomis, W. F. & Lippman, F. (1948). J. biol. Chem. 173, 87. Lundegardh, H. (1945). Ark. Bot. 32A, no. 12. Maizels, M. (1934). Biochem. J. 28, 337. Maizels, M. (1943). Quart. J. exp. Physiol. 32, 143. Maizels, M. (1949). J. Physio. 18, 247. Mann, T. (1949). Advanc. in Enzymol. 9, 329. de Meio, R. H. & Barron, E. S. G. (1935). Proc. Soc. exp. Biol., N.Y., 32, 36.