Life Sciences Vol . 22, pp . 157-164 Printed in the U .S .A .

Pergamon Press

TRANSFER OF SODIUM IONS ACROSS THE ERYTHROCYTE MEMBRANE IN MANIC DEPRESSIVE ILLNESS : TREATMENT WITH LITHIUM CARBONATE Z Alan Frazer, Joseph Mendels, David Brunswick, and T . Alan Ramsey Veterans Administration Hospital and Departments of Psychiatry and Pharmacology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104 (Received in final form November 21, 1977) Summary The efflux of 22Na from erythrocytes was measured in vitro under experimental conditions such that rate constants due to efflux either by active transport, passive diffusion, or exchange diffusion could be calculated . No significant differences were found in the rate constants for 22 Na efflux between seven male bipolar depressed pabents and eight male control subjects, who had no personal or family history of psychiatric illness . Treatment of patients with lithium carbonate either for less than one week or for 4-5 weeks produced no changes in the rate constants describing the efflux of 22 Na from red cells . Also, addition of 1mM LiCl _in vitro did not alter the active transport of 22Na from erythrocytes These data provide no evidence for either an abnormality in 22Na transfer across the red cell membrane of bipolar depressives or an effect of the lithium ion upon such transfer . In spite of twenty years of extensive research, the biological factors associated with affective disorders remain unclear . While much research has centered around the role of biogenic amines in these disorders (1,2), interest has been focused more recently on possible abnormalities in either electrolyte metabolism (2,3) or cell membrane properties regulating cation transport (2,4, 5,6) . In part, this is the result of the successful use of the lithium ion (Li+) in the treatment of mania, in long term maintenance treatment of primary affective disorders, and as an antidepressant in a subgroup of depressed patients . In the past few years, several reports have appeared in which the properties of the cation transport syatem(s) of erythrocytes have been examined in patients with affective disorders . The human erythrocyte is the most popular cell for the study of ion transport processes because of the considerable experimental advantages it affords. Furthermore, there are qualitative similarities in the processes involved in the active transport of sodium ions (Na+) and potassium ions (R+) across the erythrocyte membrane and the membrane of excitable cells like nerve (see 6) . Nevertheless, extrapolation of results obtained with human red cells to more complex systems like nerve must be made only with great caution . The present study was undertaken in an attempt to confirm and extend previous reports (7-10) of a defect in the active transport of canons across the 1Supported by Research Funds from the Veterans Administration and by NIMH Grant SRO-1MH-25433 . 157

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Lithium and 22Na Transfer

Vol . 22, No . 2,

1978

erythrocyte membrane in patienta with affective disorders ; furthermore, the effects of the lithium ion on these transport parameters was examined both _in vitro and in vivo . Methods S Materials Male patients diagnosed as having a primary affective disorder according to the criteria of Feighner et al . (11), and who were hospitalized on a psychiatric clinical research ward were utilized in this study . The diagnosis of bi polar illness was made on the basis of a history of at least one previous manic or hypomanic episode . However, the patients utilized in this study were clinically depressed, the severity of which was evaluated by the use of the Hamilton Depression Scale (12) . The actual selection of the patient for the study was made after the patient had spent ten days on the ward . Patients with any significant medical illness, including organic brain disorder or previous psychiatric illness other than mania or depression were excluded from the study. All subjects were drug free for at least two weeks prior to the determination of 22Na transport from their erythrocytes . Patients were selected for treatment with lithium carbonate based on clinThe initial dose of lithium carical criteria which have been published (13) . bonate was 900-1200mg/day ; this was adjusted, if indicated, to produce plasma levels of the cation between 0 .6-1 .2mM . All patients signed an informed consent form voluntarily agreeing to participate in the study after it had been fully explained to them . Control subjects studied were male employees of the Veterans Administration Hospital, who were paid for their services . Control subjects had no significant medical or psychiatric illness, no family history of affective ill ness, nor were they taking any medications for at least two weeks prior to the drawing of blood . 22Na Efflux The efflux of 22 Na from erythrocytes was measured essentially as described Red cells were isolated from heparinized blood and by Sacha and Welt (.14) . washed twice with iso-osmotic MgCl2 . The washed red cells were loaded with 22Na by incubation for 2 hra at 37° in an iso-osmotic medium containing 22NaC1 0.15uC/ml medium ; NaCl 38mM ; CaC12 1mM; Na2HP04 1 .2mM ; MgC12 53mM ; glucose lOmM ; glycylglycine 33mM, adjusted to pH 7 .4 with Tris . The 22 Na- loaded cells The cells were then were washed four times with ice-cold iso-osmotic MgCl2 . placed in one of three different efflux media maintained at 37° in a reciprocating water-bath . The hematocrit o£ the incubation media was approximately lOZ . The following media were used to measure the different components of transfer o£ 22 Na from the red cell : 1) total efflux ; NaCl 140mM; R2HPOy 2mM ; CaC12 1mM; MgSOq 1mM ; glucose lOmM ; pH 7 .4 with Tris . 2) "passive" and "exchange diffusion" efflux : ouabain (O .1mM) was added to the above buffer . 3) "passive" efflux : the NaCl concentration of the first media was replaced by an iso osmotic amount of MgC1 2 and ouabain (O .1mM) was added. Aliquots of the cell suspension were taken at 0 and 60 min for estimation of cell and supernatant 22 Na content . Preliminary experiments showed that over the time course of the experiment, efflux of 22Na from the loaded cells was first order . All incubations were done in duplicate . From the results obtained for the efflux of 22 Na in the different incuba-

Vol . 22, No . 2, 1978

Lithitmi and 22Na Transfer

15 9

tion media, movement of sodium ions due to either active transport, passive diffusion, or exchange diffusion can be calculated . When 22 Na efflux from red cells was determined on patients being treated with lithium carbonate, the buffer used to load the cells with the radioisotope was the same as that described above but, in addition, contained LiCl, 1mM . To measure the effect of Li+ of 22 Na efflux _in vitro , LiCl (1mM) was added to each of the three incubation media previously described . Influx Experiments The influx of 22Na into erythrocytes was measured essentially as described by Dunn (15) . Erythrocytes were incubated at 37° in a reciprocating water bath at a hematocrit of 8 percent in a buffer of the following composition : NaCl 145mM ; RC1 12mM ; MgSO 4 1mM; CaC1 2 1mM; ouabain O.1mM, adjusted to pH 7.4 with Approximately O.luC Tris . If LiCl was present, its concentration was 1mM . 22Na/ml medium was added to each incubation . Aliquots of the cell suspension The cells were washed were removed at 0 and 30 min . and centrifuged at 4° . four times with ice cold iso-osmotic MgC1 2 and the washed cells and the incubation buffer were analysed for radioactivity. In all experiments, 22 Na activity was estimated in a well-type gamma counMeasurements of ter (Intertechnique CG 30 automatic gamma spectrometer) . plasma, supernatant fluid, or erythrocyte concentrations of L1+ were done by Carrier free 22 NaC1 was obtained atomic absorption spectrophotometry (16) . Nuclear Corp . (Boston, Mass .) . Ouabain was obtained from from New England Sigma Chemical Co . (St . Louis, Mo .) . Statistical evaluations were done by Student's or paired t-teats (17) . Only a P0 .4) . significant correlation between age and the magnitude of the rate constants for Na+ transfer ; for example, the correlation between age and tr act was 0 .021 . As there was no significant difference in the cell Na+ concentration between the group of bipolar patients (6 .8 t 0 .36 meq/1 cell) and that of the control population (6 .8 t 0 .53 meq/1 cell ; P>0 .5), the results presented in Table 1 would be essentially the same if expressed as the flux of sodium ions from the erythrocyte. It should be noted that the rate constants for efflux of 22Na presented in Table 1 are in excellent agreement with values in the literature (18) . As there have been reports that treatment of patients with Li+ alters either the active transport of Na+ (19) or the activity of Na, K-activated adenosine triphosphatase (ATPase) (20), we examined the effect of Li+ treatment on sodium ion transport parameters . Six patients were examined prior to treatment and again 5-7 days af~er treatment was started. For this group of patients, the mean plasma L1 concentration was 0.56 t .03mM, and the concentra-

Lithium and 22Na Transfer

160

Vol . 22, No . 2, 1978

TABLE 1 Rate Constants for the Efflux of 22 Na from Erythrocytes of Control and Bipolar Depressed Male Subjects ~~1) -

hr-1)

Subjects

(h=t1)

Controls (g)b

0 .25 ± .013a

0 .06 ± .008

0 .05 ± .007

Bipolar-Dep (7) b

0 .28 t .012

0 .04 ± .006

0 .06 ± .008

aR±SEM b number of subjects tion in the red cells was 0 .27 ± .05meq/1 cells . In five patients, measurements were repeated between 28-35 days after initiation of treatment with lithium carbonate . The mean plasma and red cell concentrations of L1+ in this group of subjects were 0 .89 t .16mM and 0 .59 ± .llmeq/1 cells respectively . The results obtained are shown in Table 2 . TABLE 2 Effect of Treatment with Lithium on 22 Na Efflux from Erythrocytes fact (hr -1 )

Treatment

~as (hY 1 )

kex (hi 1 )

None (6) b

0 .27

± .02a

0 .03

± .007

0 .05

± .01,

Li+ " 5-7 days (6)~

0 .27

± .04

0 .04

t .005

0 .05

± .O1

None (5) b

0 .26

± .O1

0 .03

± .005

0 .05

± .004

Li+ 28-35 days (5)~

0 .28

± .02

0 .04

± .007

0 .04

± .007

aXtSEM b number of subjects Neither short-term nor longer term treatment of patients with lithium carbonate altered significantly the rate constants for either active transport, or passive or exchange diffusion of Na+ . It should be noted that when cells were obtained from patients treated with lithium carbonate, loading with 22 Na occurred in the presence of 1mM LiCl . These cells, then, contained Li+ during the time period when efflux of 22Na was measured, the mean cell concentration of Li+ being 0 .37 ± .06meq/1 cells . Neither treatment with lithium carbonate

Vol . 22, No . 2, 1978

Lithitms sad 22Na Transfer

nor the physical presence of Li+ in the cells altered the rate constants for efflux of 22Na from the erythrocytes . However, when 1mM LiCl was added to media in which 22Na efflux was occurring, the rate of efflux of 22 Na from the cells was affected (Table 3) . In particular, there was a significant increase in the rate constant for passive diffusion of Na+, whereas the apparent rate constant for exchange diffusion decreased . Addition of Li+ _in vitro had no effect on the rate constant for active sodium transport . In three experiments, when NaCl (1mM) was added to the media used to measure kpas, it did not mimic the effect of 1mM LiCl, i.e ., no effect of lmèt NaCl on kpas was noted. TABLE 3 Effect of Li (].mM) In Vitro on 22Na Efflux Rate Constants

Addition

N

(hr~l)

(hi$1 )

(hr-1 )

None

9

0 .28 ± .02a

0 .029 t .005

0 .047 t .006

Li+ (1mM)

9

0 .27 ± .O1

0 .040 t .007**

0 .035 ± .004*

If Li + is truly increasing , then it should do so regardless of whethkpa er kp s is estimated by measuring t~e efflux of 22 Na from cells previously loade~ with the radioisotope or by determining the uptake of 22 Na into erythro cytes incubated in media containing ouabain (O .1mM) . In four paired experiments, LiCl (1mM) had no significant effect on the uptake on Na+ into the cell ; the flux of Na+ into the cell was 2 .0 ± 0 .05mM/1 cells/hr t SEM) in the absence of the lithium ion and was 2.3 ± 0 .19 when Li+ was present (P>0 .4, paired t-test) . Thin result implies that Li+ is not really increasing ~pas for Na+ but rather is promoting lose of 22 Na from the cell by some other mechanism.

(x

Discussion There were no significant differences in the rate constants for the transfer of 22 Na across the erythrocyte membrane between male control subjects and male bipolar depressives . Furthermore, neither short nor longer-term treatment of patients with lithium carbonate affected the rate of transfer of 22 Na across the red blood cell membrane . Addition of 1mM LiCl _in vitro did not alter significantly the active transport of 22 Na . Rather there was an apparent increase in the passive efflux of Na+ from the erythrocyte . In light of the fact that this concentration of Li+ did not increase the passive uptake of Na+ into the erythrocyte, it is unl~kely that the lithium ion is truly affecting the passive permeability of Na . Rather, under the conditions in which the "passive" efflux of Na+ is determined, this low concentration of Li+ may be promoting the efflux of Na from the erythrocyte by activation of a recently described L1 + -Na+ countertransport or exchange diffusion system (21-23) . As 1mM NaCl did not mimic the effect of 1mM LiCl in this s~atem, the affinity of the external site of the countertransport system for Li is apparently greater than the affinity for Na+. Further work is needed, though, to establish this .

16 2

Lithium and 22Na Transfer

Vol . 22, No . 2, 1978

Nevertheless, if Li + does promote the efflux of the sodium ion in this way, it is probably not relevant clinically, due to the presence of high concentrations of the sodium ion in plasma . The Li+ -induced decrease in the rate constant for exchange diffusion of Na+ (Table 3) is also more apparent than real . It occurs because efflux of 22Na increased in the media containing ouabain and no added NaCl but did not increase in the media containing both ouabain and sodium ions ; the rate constant for exchange diffusion is calculated from the difference in 22Na efflux occurring in these media . There have been reports claiming that the transport of the sodium ion from the red cell is different either between controls and depressed patients or between different types of depressives . In an initial report, Naylor and his associates (7) reported that the active transport of Na from erythrocytes obtained from psychotic depressives was about 17 percent lower than such transport in erythrocytes obtained from neurotic depressive patients ; this difference was statistically significant . No difference in the active transport of Na+ was noted between red cells obtained from the patients, all of whom were female, and four female "control" subjects, all of whom had subnormal intelligence . There are several obvious differences between this study and our own, such as the sex of the study subjects and the type of "control" subjects . Furthermore, the criteria used by Naylor et al . for diagnosing patients appear to have been quite different to those used by us . Thus, it is difficult to compare the two studies . In a subsequent study by this group of investigators (8), no significant difference was noted in the activity of erythrocyte Na, K-activated ATPase between psychotic and neurotic depressives . This result would appear to be somewhat inconsistent with their previous report . More recently, Naylor et al . (10) have reported no difference in either erythrocyte Na, R-activated ATPase activity or ouabain-sensitive K+ flux between manic patients and controls . In summary, it seems that the data of Naylor and associates provide no convincing evidence for an abnormality in erythrocyte cation transport or Na, K-activated ATPase activity in depressed or manic patients . Recently, Hokin-Neaveraon et al . (9) utilized an interesting strategy to explore active sodium ion transport in erythrocytes of bipolar patients during the manic phase of their illness . These investigators loaded red cells with sodium ions, by incubating erythrocytes for two days in the cold, and then measured active Na+ efflux . They found that under these conditions, erythrocytes obtained from bipolar patients had about a 14 percent decrease in active Na+ efflux as compared to that measured in red cells obtained from age- and sex-matched controls . This study differs from ours in two important aspect~-patients were studied when manic rather than while depressed and red cells were loaded with sodium ions -- either of which may contribute to the difference in the results . Loading the cells with the Na+ increases the rate of active Na+ transport (24) and this may reveal a defect not apparent at normal cell Na+ concentrations . In general agreement with the results of Hokin-Neaverson et al . (9), Choi and his associates (25) have reported recently low Na, R-activated ATPase activity in erythrocytes obtained from primary depressives . However, the con trol subjects studied by Choi et al . were not sex-matched with the patient population . Furthermore, Cam stimulated ATPase activity was also decreased in erythrocytes obtained from the patients, which raises questions about the specificity of the reported defect . Thus, the data reported here together with the comments on some of the

Vol . 22, No . 2, 1978

Lithium and 22Na TraaefeT. .

16 3

limits of previous studies, suggests that abnormalities in cation transport in erythrocytes of patients with affective disorders have not yet been demonstrated conclusively . This does not rule out the possibility of such abnormalities in other cells, such se neurone. Of course, it offers no evidence in support of such an idea . We have examined also the question of whether the lithium ion alters the activity of the sodium pump . Our data provide no support for this . Neither treatment of patients with the lithium ion nor addition of a "therapeutic" con centration of Li+ _in vitro altered significantly the rate constant for active Na+efflux from the erythrocyte . Other investigators have found effects of Li+ either on the active transport of Na+ or the activity of Na, R-activated ATPase . Glen et al . (26) reported that 3mM LiCl stimulated the total efflux of 22 Na from red cells when the incubation was carried out in the presence of 2mM RC1 but not if 6mM RC1 was present . This result is not unexpected as Li+ has been shown to have affinity for the potassium ion site of the Na, R-activated ATPase (27) and stimulation of the sodium pump by Li+ would be anticipated at suboptimal concentrations of RC1 like 2mM. Thus, this finding cannot be extrapolated to the clinical situation, where the plasma concentration of the potassium ion is between 4-SmM. In our experiments, the concentration of R+ in the incubation medium was 4mM and no effect of Li+ on either the active or total efflux of 22Na from the erythrocyte was observed . More recently, Hokin-Neaverson et al . (19) found that treatment of patients with the lithium ion was associated with a significant increase in the active The red cells used in this study had efflux of 22Na from the erythrocyte . undergone a prior loading with NaCl, such that erythrocyte Na+ was raised to a concentration of about 35meq/1 cells. This procedure may reveal some effect of Li+ on 22Na efflux that is not apparent under our experimental conditions of "normal" cell Na+ . In addition to measuring sodium-pump activity, HokinSubjects Neaveraon et al . (19) determined ouâbain-sensitive ATPase activity . being treated with lithium carbonate for varying periods of time were found to have a significant (17X) increase in ouabain-sensitive ATPase activity over that measured in erythrocytes obtained from a mixed group of subjects not receiving Li+. Naylor et al . (20) also found lithium ion treatment of manic depressive patients to be associated with a significant increase in red cell Na, R-activated ATPase activity but, somewhat inconsistently, no effect oa oubain sensitive R+ influx was noted. In subjects with no history of psychiatric illness, Naylor et al . (28) found two weeks of Li+ treatment to produce results different from those found in manic-depresaive .patiente, namely no change in erythrocyte Na, R-activated ATPase activity but a significant deFurthermore, Li treatment of rats did crease in ouabain-sensitive R influx . not effect erythrocyte Na, R-activated ATPase activity . In summary, then, it seems to us that consistent effects of the lithium ion on sodium-pump activity or Na, R-activated ATPase activity have not been observed under clinically relevant conditions . While effects of Li+ on cation transport and Na, R-activated ATPase activity have been either inferred (29,30) or observed (31,32) in nervous tissue of animals, the relevance of these observations to the therapeutic effects of Li+ in affective disorders remains unknown at this time . Acknowledgements The excellent technical assistance of Mr . Charles Cortez and Mr . Michael Bane is gratefully acknowledged .

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Lithium and 22Na Traaefer

Vol . 22, No . 2, 1978

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