MEMBRANE ATPase ACTIVITIES AND ERYTHROCYTE ION CONTENT

Transcription

1 Br. J. clin. Pharmac. (1978), 5, THE EFFECT OF LITHIUM TREATMENT ON ERYTHROCYTE MEMBRANE ATPase ACTIVITIES AND ERYTHROCYTE ION CONTENT J.E. HESKETH*, J.B. LOUDON, H.W. READING & A.I.M. GLEN M.R.C. Brain Metabolism Unit, University Department of Pharmacology, 1, George Square, Edinburgh, Scotland I ATPase activities were studied in erythrocyte membranes prepared from blood of suffering from affective disorders. 2 Long-term (9-12 months) administration of lithium led to an increase in the erythrocyte membrane Na/K ATPase (54%) when studied on an age and sex matched basis or when the were studied before and after treatment. The Mg ATPase was also increased (38%) but there was no consistent effect of lithium treatment on Ca stimulated ATPase in the membranes. It is suggested that the effect of lithium treatment on Na/K ATPase was due to recovery of the rather than an effect of the drug. 3 MgATPase increased regardless of clinical condition. Short-term lithium treatment (2-4 weeks) led to increased Mg ATPase (43%). These results suggest that lithium treatment itself causes an increase in Mg ATPase and that this effect is not dependent on changes in protein synthesis. 4 Lithium treatment (long-term) increased the erythrocyte sodium content by 15%. No effect on plasma sodium, magnesium, potassium or erythrocyte magnesium and potassium was observed. Introduction Lithium salts are widely used for the treatment of affective disorders but their mode of action is unknown (Johnson, 1975). Lithium treatment has been shown to be associated with an increase in Na/K ATPase (adenosine triphosphatase (EC )) (Dick, Naylor, Dick & Moody, 1974) and ATPases have been suggested as important in the mode of action of lithium (Glen & Reading, 1973). The present paper describes studies of various ATPase activities during lithium treatment and also attempts to answer certain specific questions concerning the observed effects. Since depression has been shown to be associated with reduced Na/K ATPase (Hesketh, Glen & Reading, 1977a) and recovery from depression to be associated with increased (Naylor et al., 1973) the observed increase in membrane Na/K ATPase during lithium treatment (Dick et al., 1974) could be due to an effect of recovery rather than one of the drug. We have attempted to investigate this problem. The work on Na/K ATPase was extended to investigate the effects of lithium on Mg and Ca dependent ATPases present in erythrocyte membranes and to examine whether observed changes were due to effects on protein synthesis. Erythrocyte ion content * Present address: Centre de Neurochimie du CNRS, 11 rue Humann, Strasbourg, France. was measured in some so as to investigate if ATPase changes were related to ion s. Some of this work has been presented previously (Hesketh, 1976a). Methods Fresh heparinised venous blood was used throughout. Erythrocyte membranes were prepared by hypotonic haemolysis in 10 mm Tris-HCl buffer following the method of Dick, Dick & Tosteson (1969) except that a temperature of 9 C was used for all post-lysis centrifugations. ATPase was measured by the liberation of inorganic phosphate following incubation with 3mM disodium ATP (Boehringer Ltd). Incubation was at 370C in 30mM Tris-HC1 buffer, ph 7.4, 0.1mM EDTA with either; (a) 100mM NaCl, 5mM KCI and 6mM MgCl2. (b) 6mM MgCl2 alone or, (c) 6mM MgCl2 and 0.15 CaCl2. The in medium B was defined as the MgATPase, the difference in in A to that in B as the Na/K stimulated (Na/K ATPase), the in C as the Ca + Mg ATPase and the difference between C and B as the Ca stimulated ATPase (Ca ATPase). Inorganic phosphate

2 324 J.E. HESKETH, J.B. LOUDON, H.W. READING & A.I.M. GLEN was estimated by the method of Stoward as described by Naylor, Dick, Dick, Le Poidevin & Whyte (1973). For ion estimations, erythrocytes were separated from plasma and washed following the methods of Smith & Samuel (1970) and Glen & Bellinger (1973). Fresh, heparinised blood (5-10 ml) was centrifuged by bringing momentarily to 16,000g and the plasma removed. This and subsequent centrifugations were done at 10 C. The erythrocyte pellet was resuspended in 285mosm sucrose and the centrifugation repeated. After three such washes the buffy coat and top layer of cells were removed and an aliquot of the erythrocyte pellet lysed in distilled, deionised water (1:10). All ion measurements were by atomic absorption spectroscopy using a Perkin-Elmer 360 atomic absorptiometer. Samples were diluted in 0.1% (w/v) lanthanum chloride for analysis. The studied took only a light breakfast on the morning of sampling but otherwise there was no dietary control. Those receiving lithium carbonate took no medication on the morning of sampling. All untreated were in- at the Metabolic Ward, Craig House, Royal Edinburgh Hospital while of the lithium-treated some were in- in the same ward but the majority were out-. All studied were suffering from or had suffered from a despressive illness. Patients were diagnosed by two independent psychiatrists, the refering clinical psychiatrist and a research psychiatrist. Criteria for the diagnosis of depression were those recommended by the M.R.C. Clinical Psychiatry Committee (1965). Patients receiving lithium were assessed as to whether they were ill or recovered. Results The first part of this study consisted of a comparison between a group of untreated, depressed and a group of (including some ill and some recovered) who had received lithium treatment for at least one year. The groups were not age and sex matched but their composition in terms of age and sex was not significantly different. Comparison of untreated with lithium-treated showed a significant increase (P< 0.05) in the Mg ATPase in the lithium-failed group. The increase in the lithium-recovered group was not statistically significant. There was also a significant decrease (P< 0.01) in the Ca ATPase in the lithium-recovered group but no change in the Ca + Mg ATPase was observed. Neither of the lithiumtreated groups showed any significant change in Na/K ATPase when compared to untreated. The lithium-recovered group showed an increased Na/K ATPase compared to the lithium-failed group but the difference was not statistically significant. The results in Table 1 show considerable variance. In an attempt to reduce such variance untreated, ill and lithium-treated were compared on an age and sex matched basis. The results (Table 2) showed both an increased Na/K ATPase and an increased Mg ATPase in the lithiumtreated group (P< 0.025, P <0.05 respectively). There were no differences in the Ca+ Mg ATPase or Ca ATPase activities between the two groups. A further study was carried out in which samples were taken from some while ill and untreated and from same after they had received lithium treatment for between 9 months and 1 year. All these except one had recovered and had no episode of illness since starting lithium. The results from this experiment (Table 3) show an increased of all the ATPases after lithium treatment, although the increase in Mg ATPase is barely statistically significant. ATPase activities were measured in membranes prepared from blood of a group of depressed while ill and untreated and again after 2-4 weeks of receiving lithium therapy. The results are shown in Table 4. They show an increased Mg ATPase specific (P< 0.05) after lithium treatment but there was no difference in the activities of the other ATPases. Two of the studied had also received electroconvulsive shock treatment during the period between sampling. Plasma and erythrocyte ion s were measured in a group of ill, untreated and a group of who had received lithium treatment for at least 1 year. The results are shown in Table 5. The only significant difference between the two groups was the increased intracellular sodium in erythrocytes from lithium-treated. Discussion In each group of studied there was a large variance in the activities of all the four ATPase activities measured. Examination of weekly serial samples from control subjects (Hesketh 1976b; Hesketh & Reading, unpublished observations) showed considerable variation in erythrocyte membrane ATPase activities from week to week. This large intra-individual variation was not due to methodological error in the ATPase assay or to quantitative changes in the membrane preparations. However qualitative membrane changes or the presence of modulators of enzyme could not be ruled out as causative factors. Such intra-individual variation may account for the high variance of the data in the present paper, in particular the variance of data on studied before and after treatment. The large variation in ATPase activities found within an individual makes it difficult to allot with confidence values for the erythrocyte membrane ATPase

3 LITHIUM AND ERYTHROCYTE MEMBRANE ATPases 325 Table 1 Specific activities of ATPases in erythrocyte membranes prepared from blood of a group of ill, untreated depressive and a group of who had received lithium treatment for at least 1 year. The lithium-treated group was divided into those who had recovered and those who were ill (lithiumfailed). Activities are expressed as nmol Pi h-' mg-1 protein. Values given are means + s.d. with the number of studied in parentheses. Groups were compared using an analysis of variance, P<0.05; **P<0.01 compared to the untreated group. Age, sex and diagnostic composition of the groups are also shown. 111, untreated Lithium-recovered Lithium-failed Na/K ATPase Mg ATPase Ca + Mg ATPase Ca ATPase 217±137(21) 279±107 (21) 894± 280 (19) 589± 189 (19) 248 ± 95 (9) 373 ± 130 (9) 768 ±298 (9) 359 ± 116 (9)** 179 ± 65 (5) 384 ± 66* (5) 975 ± 183 (5) 591 ± 161 (5) Age (years) Sex Diagnosis 46± 15 (21) 8 male 13 female 11 bipolar 10 unipolar 47± 17 (9) 3 male 6 female 7 bipolar 2 unipolar 54± 11 (5) 3 male 2 female 4 bipolar 1 unipolar Table 2 Specific activities of ATPases in erythrocyte membranes prepared from blood of a group of ill, untreated depressive and an age and sex matched group of who had received lithium treatment for at least one year. Seven of the lithium-treated group had recovered whilst two were ill despite lithium treatment. Activities are expressed as nmol Pi h-1 mg-1 protein. Values are given as means + s.d. with the number of studied in parentheses. Groups were compared using a paired t-test, *P<0.05; *P < Na/K ATPase Mg ATPase Ca+ Mg ATPase Ca ATPase Diagnosis 131 ± 43 (9) 238 ± 79 (9) 768±240 (9) 530± 247 (9) 3 bipolar 5 unipolar 1 mixed affective Lithium-treated 208 ± 65 (9)** 343 ± 130 (9)* 851 ±225 (9) 507 ±244 (9) 6 bipolar 3 unipolar Table 3 Specific activities of ATPases in erythrocyte membranes prepared from blood of a group of nine depressive while ill and untreated and again after 9-12 months lithium treatment. All but one of the had recovered following lithium treatment. Activities are expressed as nmol PI h-' mg-' protein. Values given are means + s.d. Groups were compared using a paired t-test, *P<0.1; **P< Na/K ATPase Mg ATPase Ca+ Mg ATPase Ca ATPase 185± ± ± ±175 Diagnosis: 5 were unipolar depressives, 4 were bipolar. Lithium-treated 273 ± 80** 439 ± 203* 1191 ±292* 751 ± 183**

4 326 J.E. HESKETH, J.B. LOUDON, H.W. READING & A.I.M. GLEN activities in a given person. Thus analysis of changes following drug treatment is problematical. However in the present experiments certain ATPase activities were found to increase after lithium treatment when were compared on more than one basis. Such changes may be real. Since unipolar have been shown to differ from bipolar depressives in their erythrocyte membrane Na/K ATPase (Hesketh et al., 1977a) the diagnostic composition of the groups in the present work is also of importance for analysis of the data. For example the groups compared in Table 2 differed in the ratio of unipolar to bipolar and this difference between the two groups might explain the observed increase in Na/K ATPase in the lithium-treated group. However Mg ATPase does not seem to differ between unipolar and bipolar depressive (Hesketh et al., 1977a). Comparison of untreated on an age and sex matched basis with who had received lithium treatment for at least one year (Table 2) showed the lithium-treated group to have a greater erythrocyte Table 4 Specific activities of ATPases in erythrocyte membranes prepared from blood of a group of depressive while ill and untreated and again after 2-4 weeks of lithium therapy. Two were judged to need additional antidepressant therapy and they (marked ECT in the table) also received electroconvulsive shock treatment. Five of the were bipolar depressives, one was unipolar. Activities are expressed as nmol Pi h-1 mg-1 protein. Individual values, before and after treatment, are given together with mean ±s.d. Groups were compared using a paired t test, *P<0.05. Na/KATPase MgATPase Ca + Mg ATPase Ca ATPase ECT ECT Mean+s.d ±86* ± ± Table 5 Plasma and erythrocyte ion s for a group of ill, untreated depressive and a group of who had received lithium treatment for at least 1 year. Plasma ion s are expressed as mm, erythrocyte ion s as mmol/kg wet cells. Values given are mean ± s.d. with the number of subjects in parentheses. Groups were compared using a Students t-test, P<0.05. Plasma magnesium Plasma sodium Plasma potassium Plasma lithium Erythrocyte magnesium Erythrocyte sodium Erythrocyte potassium Erythrocyte lithium ± ± 1.66* Lithium-treated ± ± * ±0.13

5 LITHIUM AND ERYTHROCYTE MEMBRANE ATPases 327 membrane Na/K ATPase but this effect, as discussed above, could have been due to diagnostic differences in the two groups. Patients studied both untreated and after 9-12 months lithium treatment also showed increased Na/K ATPase after lithium treatment. Both these lithium-treated groups consisted largely of who had recovered so that the increased enzyme might have been due to change in clinical state. This problem was investigated by splitting the data from a group of lithium-treated into those from who had recovered and those from who were ill despite receiving lithium therapy (Table 1). There was no significant difference between the values of the Na/K ATPase for the untreated lithiumrecovered and lithium-failed groups. All these values were less than that found for non-depressive control subjects (342 nmol Pi h-' mg-' protein-hesketh et al., 1977a). The results in Table 1 show however a trend towards increased Na/K ATPase in those lithium-treated who had recovered compared to those who were ill. The small changes are difficult to interpret in view of the large variance in the data but they suggest that lithium treatment only causes an increase in Na/K ATPase when there is an associated recovery of the. This is supported by the finding that amelioration of mood is correlated with increased Na/K ATPase (Naylor et al., 1973). Lithium itself may not cause any change in erythrocyte membrane Na/K ATPase. However our results confirm the previous finding that lithium-treated show an increased erythrocyte membrane Na/K ATPase (Dick et al., 1974). Lithium-treated showed an increased intracellular erythrocyte sodium compared to untreated. The majority of the lithium-treated group had recovered and so it was not possible to separate drug and recovery effects in this case. Erythrocyte sodium has been shown to decrease following recovery from depressive illness not associated with lithium treatment (Naylor, McNamee & Moody, 1971, Naylor et al., 1973) whereas recovery from depression following lithium treatment has been found to be associated with an increase in erythrocyte sodium (Mendels, Frazer, Secunda & Stoken, 1971; Mendels & Frazer, 1974-however this was not confirmed by Mendels, Frazer & Secunda, 1972). Our results show lithium treatment to be associated with an increase in both the erythrocyte sodium and erythrocyte membrane Na/K ATPase. It is possible that this apparent anomaly was caused by intracellular lithium increasing the intracellular sodium by inhibiting active efflux of sodium. Such inhibition of the sodium-pump at the sodiumsensitive side has been suggested previously (Glen & Reading, 1973; Hesketh, 1977). Both short-term (2-4 weeks) and long-term (9-12 months) lithium treatment caused an increase in the erythrocyte membrane Mg ATPase although in one experiment the increase was barely significant. The effect of lithium was observed with short-term treatment where recovery was at most only partial and with long-term treatment regardless of clinical state and this suggests that the increased is due to an action of lithium and not due to a change in clinical state of the. Diagnostic composition of the groups is unlikely to have been the cause of the observed effect. It has been reported that people with high reticulocyte counts (10-30%) have an increased specific of erythrocyte membrane Mg ATPase compared to control subjects (Feig & Guidotti, 1974). This raised the possibility that the observed effect of lithium-treatment on Mg ATPase was due to an effect on erythrocyte production. However such an explanation of the observed effect is unlikely since lithium-treated have reticulocyte counts within the normal range (Bille, Jensen, Kaalund- Jensen & Paulson, 1975). Since the lifespan of the -human erythrocyte is 120 days (Harris & Kellermeyer, 1970) the increase in Mg ATPase observed after only 2-4 weeks of lithium treatment suggests that the effect of lithium on specific enzyme was not dependent on the production of new cells. As the mature human erythrocyte does not synthesize protein (Harris & Kellermeyer, 1970) this in turn suggests that the effect of lithium on Mg ATPase was not dependent on the synthesis of more enzyme. No lithium could be detected in membranes prepared from erythrocytes of lithium-treated (Hesketh, 1976a) and therefore it is unlikely that the increased ATPase was due to in vitro activation by lithium present in the membrane preparations. The effect of lithium was most likely due to activation of the Mg ATPase by some unknown factor. In this context the weekly variation in membrane ATPase activities (control subjects) suggests activation of the enzyme may be possible even in an extensively washed preparation. Lithium treatment had no consistent effect on Ca ATPase or Ca+ Mg ATPase activities. Such calcium dependent ATPase is thought to reflect, in part at least, the active calcium transport system. Calcium is of such importance to many aspects of cell function (Cuthbert, 1970) that the varied nature of our results suggests further study may be of value. There have been several conflicting reports concerning the effects of lithium administration to humans on plasma magnesium s. Lithium has been found to increase (Bunney, Goodwin, Davis & Fawcett, 1968; Aronoff, Evans & Durell, 1971), decrease (Frizel, Coppen & Marks, 1968) and have no effect upon (Dunner, Meltzer, Schreiner & Feigelson, 1975) plasma magnesium. The present results show no effect of lithium treatment (9-12 months) on plasma magnesium.

6 328 J.E. HESKETH, J.B. LOUDON, H.W. READING & A.I.M. GLEN In agreement with previous workers (Dunner et al., 1975) our results show no effect of lithium treatment on human erythrocyte magnesium. The lack of effect of lithium treatment on human plasma and erythrocyte magnesium s compared to its effect of increasing erythrocyte membrane Mg ATPase suggests there is no relationship between magnesium s and Mg ATPase. It has been suggested that lithium might exert its action on magnesium dependent enzymes through its effects on magnesium distribution (Glen, 1976). This does not seem to be the case for the Mg ATPase of the erythrocyte membrane. In summary, these results suggest lithium treatment to increase Mg ATPase in the erythrocyte membrane probably due to activation by some unknown factor. The physiological significance of the increased Mg ATPase is obscure since the function of this enzyme in the erythrocyte membrane is unknown. However the observed increase is of interest since chronic lithium administration to rats increases synaptic membrane Mg ATPase (Hesketh, 1976a; Hesketh, Kinloch & Reading, 1977b). In contrast although lithium-treated showed increased erythrocyte membrane Na/K ATPase this increase may have been a result of change in the clinical state of the. The results do not provide unequivocable evidence that lithium itself increases erythrocyte membrane Na/K ATPase. Although acute lithium application is known to affect Na/K ATPase and sodium transport (Glen, Bradbury & Wilson, 1972; Ploeger, 1974; Tobin, Akera, Han & Brody, 1974, Hesketh, 1977) chronic diet administration of lithium to rats showed no effect on synaptic membrane Na/K ATPase (Hesketh et al., 1977b). It may be that chronic lithium treatment does not affect the measured Na/K ATPase, that is the activation or amount of the enzyme as judged from its in in vitro preparations in the absence of lithium. We thank Mr A. McGovern and Miss A. Oliver for technical assistance and Mrs B. Hulme for much help in obtaining clinical samples. J.E.H. thanks the M.R.C. for a scholarship. References ARONOFF, M.S., EVANS, R.C. & DURELL, J. (1971). Effect of lithium salts on electrolyte metabolism. J. Psychiat. Res., 8, BILLE, P.E., JENSEN, M.K., KAALUND-JENSEN, J.P. & PAULSEN, J.C. (1975). Studies on the haematologic and cytogenetic effect of lithium. Acta Med. Scand., 198, BUNNEY, W.E., GOODWIN, F.K., DAVIS, J.M. & FAWCE1T, J.A. (1968). A behavioural-biochemical study of lithium treatment. Am. J. Psychisat., 125, CUTHBERT, A.W. (1970). Calcium and Cell Function. London: Macmillan Press. DICK, D.A.T., DICK, E.G. & TOSTESON, D.C. (1969). Inhibition of adenosine triphosphatase in sheep red cell membranes by oxidised glutathione. J. gen. Physiol., 54, DICK, D.A.T., NAYLOR, G.J., DICK, E.G. & MOODY, J. (1974). Erythrocyte sodium-plus-potassium ion activated adenosine triphosphatase in depressive. Biochem. Soc. 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