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Sodium-lithium countertransport activity and its sensitivity to inhibitors with erythrocyte ageing in man
CIinica Chimica Acta, 178 (1988) 51-58 Elsevier
51
CCA 04293
Sodium-lit~um countertransport activity and its sensitivity to inhibitors with erythrocyte ageing in man S. Carr, T.H. Thomas and R. Wilkinson department of
University of Neweastfe upon Tyne and department of ~ephrolo~, Freeman Hospital, Newcastle upon Tvne {UK)
Medicine,
(Received 27 February 1988; revision received 26 July 1988; accepted 3 August 1988) Key words: Ageing; Amiloride; Erythrocyte; Phloretin; Sodium transport
Summary
Sodium-lithium countertransport is believed to depend on a specific protein and since such proteins usually decline with erythrocyte age it would be expected to be reduced in older cells. In fact, so~um-lit~um countertransport increased with ageing, whereas the rate constant of the sodium pump decreased. The increase in sodium-lithium countertransport with erythrocyte ageing was due to a phloretin insensitive component that was not present in young erythrocytes. Raised sodiumlithium countertransport in patients with essential hypertension was due mainly to an increased phloretin sensitive component but the phloretin insensitive component was also higher in middle aged erythrocytes. Amiloride had no effect upon sodiumlithium counter-transport or unidirectional sodium influx in cells of any age. This suggests that sodium-lithium countertransport is not a mode of action of the sodium-proton exchanger in the erythrocyte.
In some patients with essential hypertension there is an increase in the rate of sodium-lithium countertransport in erythrocytes [1,2]. The same membrane ion transporter under physiological conditions is responsible for a 1: 1, sodium-sodium exchange but this does not alter intracellular electrolyte balance and its role is difficult to explain [3]. In order to form a hypothesis of an aetiological link between
Correspondence and requests for reprints to: Dr. S. Carr, Department of Medicine and Nephrology, Freeman Hospital, Freeman Road, Newcastle upon Tyne NE7 7DN, UK. ~9-8981/88/~03.50
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
52
raised sodium-lithium countertransport and essential hypertension the same membrane transporter has been suggested to be synonymous with sodium-hydrogen exchange activity [4,5]. A generalized increase in sodium-hydrogen exchange could increase sodium reabsorption in the renal proximal tubule and predispose to proliferation in vascular smooth muscle [4-61. The membrane sodium-hydrogen exchanger has been well characterized and is defined by its sensitivity to amiloride which competes with sodium at an external site [4]. This transporter gives net sodium influx, which is normally reversed by the sodium-potassium ATPase pump but would accumulate when the pump was inhibited by ouabain [7]. Reports in the literature do not support the hypothesis that sodium-lithium and sodium hydrogen countertransport activities are synonymous [8]. However, although sodium-lithium countertransport is characteristically inhibited by phloretin [8] its lack of sensitivity to amiloride remains unconfirmed. Erythrocytes in the circulation are ageing and unable to replace damaged or degraded proteins. There is loss of membrane and sodium-potassium ATPase activity during this process. Therefore, with erythrocyte ageing other membrane ion transporters may lose activity or have qualitative changes such as a change in sensitivity to inhibitors. In addition, sensitivity to inhibitors of sodium-lithium countertransport and changes in activity with erythrocyte ageing may be different in a group of patients, such as essential hypertensives, where this ion transporter is known to be abnormal. We therefore studied sodium-lithium countertransport and the effects of phloretin and amiloride in erythrocytes of different ages from both normal subjects and essential hypertensive patients known to have a raised sodium-lithium countertransport activity. Methods Eight normal individuals (age 53.6, SD 1.4 years; systolic blood pressure (mm Hg) 128, SD 3.2; diastolic blood pressure 77.6, SD 1.9) were used to study the effects of amiloride on sodium-lithium countertransport and ouabain-sensitive sodium flux. Nine normal individuals and 7 patients with essential hypertension (age 46.0, SD 1.4 years; systolic blood pressure (mm Hg) 142.5, SD 38.6; diastolic blood pressure 93.8, SD 4.78) known to have raised sodium-lithium countertransport were used to study the effects of phloretin on sodium-lithium countertransport in cells of different ages. Four patients in this group were receiving treatment with antihypertensive agents including beta blockers and angiotensin-converting enzyme inhibitors. Erythrocyte sodium potassium and water contents Venous blood was collected into tubes containing lithium heparin. Blood was centrifuged at 2500 x g at 4” C for 5 min and the plasma removed. Erythrocytes were then washed three times with isotonic MgCl, solution (290 mosm/kg) to remove plasma contamination. The washed erythrocytes were centrifuged in 6 X 45 mm polythene tubes at 20000 X g for 30 min at 15” C, similar to the method of
53
Murphy [9] but using a swingout head. Young cells were obtained by slicing the top of the erythrocyte column, old cells from the bottom, and cells from the centre of the tube were take to be of intermediate age. Each slice contained 5-7.5s of the erythrocyte column. For measurement of sodium and potassium, erythrocytes were dispersed in 1.5 mmol/l CsCl and directly aspirated into an IL9483 flame photometer. Water and dry cell content were measured by weighing cell slices before and after drying to constant weight at 37’ C for 24 h. Ouabain sensitive sodium jlux and its rate constant Whole blood samples were incubated with either ouabain (lop4 mmol/l) or ouabain (10e4 mmol/l) and amiloride (lop3 mmol/l) for 2 h at 37°C and the erythrocyte sodium was determined as described above. Ouabain sensitive sodium flux ( fO) was calculated as the increase in erythrocyte sodium in one hour and when ouabain alone was used the rate constant (k,) was determined from the equation as described by Cumberbatch and Morgan [lo]. Amiloride was a fo = k0 x ]Nal.ac gift from Merck, Sharp and Dohme Research Laboratories, Hertfordshire, UK. Sodium-lithium countertransport The method used was similar to that described by Clegg et al. [ll]. Erythrocytes (2 ml) were suspended in lithium loading solution (8 ml; 150 mmol/l LiCl, 10 mmol/l Tris acetate, 10 mmol/l glucose) for 3 h at 37°C. The erythrocytes were then washed twice with isotonic MgCl, and once with sodium free incubation 85 mmol/l sucrose, 10 mmol/l Tris acetate, 10 medium (75 mmol/l MgCl,, mmol/l glucose, lop4 mol/l ouabain, pH 7.4, 290 mosm/kg). Erythrocytes (0.6 ml) were then incubated in 3 ml of either sodium free medium or sodium medium (150 mmol/l NaCl, 10 mmol/l Tris acetate, 10 mmol/l glucose, 1O-4 mol/l ouabain, pH 7.4, 290 mosm/kg) for 1 h. Erythrocytes were also incubated in media containing either amiloride (lop3 mol/l) or phloretin (lop4 mol/l). Phloretin was obtained from the Sigma Chemical Co. Erythrocyte lithium content was measured by flame photometry after lithium loading and after incubation in each medium and the erythrocytes were prepared by centrifugation in small polythene tubes as described for sodium and potassium determination. Sodium-lithium countertransport was determined from the difference in lithium efflux from erythrocytes in the sodium containing and sodium free media. Statistics The effect of the erythocyte age fraction or raised total sodium-lithium countertransport on other results was assessed using analysis of variance. The significance of differences between treatments and between erythrocytes fractions was assessed using the paired t test. Results Erythrocyte fractionation The age related fractionation of erythrocytes haemoglobin (HbA,) [12] and cell water content.
was verified using glycosylated Between young and old erythro-
54 TABLE
I
The effect of erythrocyte age on sodium-lithium countertransport, passive lithium effhtx in 8 normal subjects (mean f SEM)
sodium
pump
rate
constant
age
Sodium-lithium countertransport (mm01 Li/h per kg cells)
(h-‘)
Passive lithium efflux (mm01 Li/h per kg cells)
Young Middle Old
0.21 f 0.02 * * 0.30 f 0.06 0.48 + 0.04 * * *
0.27 + 0.01 0.28 + 0.01 0.24 k 0.02 *
0.22 * 0.03 0.21+ 0.04 0.26 f 0.05
Erythrocyte
* p < 0.01, **p
***
Sodium-pump rate constant
p < 0.001 compared
with the middle
erythrocyte
and
fraction.
cytes HbA, increased by 44% and cell water decreased by 5%. Measurements of cell water showed that the age related fractionation did not change when erythrocytes were incubated in the solutions used for the measurement of flux rates.
Sodium-lithium countertransport and the rate constant of the sodium pump with erythrocyte ageing The rate constant of the sodium pump (k,) decreased with erythrocyte age (Table I) which indicated the loss of active sodium pumps from the erythrocyte membrane. Most of the ageing change occurred between the middle and old age fractions. There was also increased variance in k, in the older fractions which presumably reflects, variability in the ageing process. Sodium-lithium countertransport increased with erythrocyte age (Table I) in contrast to the change in k,, but as with k,, most of the change occurred between
TABLE
II
The effect of erythrocyte age on total, phloretin sensitive and phloretin countertransport in 9 normal subjects and 7 essential hypertensive patients raised countertransport activity (mean f SEM) Subjects
Erythrocyte
age
Total a,b
Sodium-lithium countertransport (mmol Li/h per kg cells) Phloretin insensitive
Normal
controls
Essential hypertensives
insensitive sodium-lithium who were known to have
a.C
Phloretin sensitive b
Young Middle Old
0.16 f 0.04 0.27 f 0.03 0.62 f 0.09
0.03 f 0.02 0.09 * 0.02 0.47 + 0.09
0.12 f 0.04 0.20 f 0.04 0.17rtO.06
Young Middle Old
0.38 f 0.03 0.50 f 0.03 0.70 f 0.04
0.06 + 0.03 0.23 f 0.04 0.35 f 0.05
0.31+ 0.02 0.27 f 0.04 0.35 f 0.07
age on the activity; b p < 0.01 for an Analysis of variance showed: a p i 0.01 for an effect of erythrocyte effect of hypertension on the activity; and ’ p -z 0.05 for an interaction between erythrocyte age and hypertension on the activity.
55
the middle and old cell fractions. There was also increased variance in the older cell fractions. The changes in sodium-lithium countertransport and ouabain sensitive sodium flux with erythrocyte age were similar and the converse of the changes in sodium pump rate constant. However, none of these changes were related between individuals, suggesting that they were due to independent ageing changes in the erythrocyte membrane. Passive lithium efflux did not change significantly with erythrocyte ageing (Table I). Analysis of variance showed very significant effect of erythrocyte age ( f = 43.2, p < 0.01) and essential hypertension (f = 31.7, p < 0.01) on sodium-lithium countertransport but there was no significant interaction between the effect of essential hypertension and the erythrocyte age (Table XI). However, although the two groups had significantly different total sodium-lit~um countertransport activity in the young (p < 0.005) as well as the middle cells, there was no difference between the old cells. Effect of phloretin on sodium-lithium countertransport
In normal subjects phloretin completely inhibited sodium-lithium countertransport in young erythrocytes but in the middle fraction of cells 35% was phloretin insensitive and in old cells this had increased to 75% of sodium-~t~urn countertransport that was phloretin sensitive. The phloretin sensitive component of sodium-lithium countertransport did not change during the erythrocyte lifespan. In patients with essential hypertension and raised total sodium-lithium countertransport in middle aged erythrocytes, analysis of variance showed that the phloretin sensitive component was raised (f= 11.62, p < 0.01) and was also not affected by erythrocyte age. The overall increase in sodium-lithium countertransport was due to a marked increase in the phloretin insensitive component (f = 27.5, p -C0.01). However, the pattern of change in this component with erythrocyte ageing was significantly different (f = 6.27, p < 0.05) in the essential hypertensives (Table II). Effect of amiloride on sodium-lithium co~ntertran~port and ouabain sensitiue sodium fr UX
Amiloride, the definitive inhibitor of sodium-hydrogen countertransport, had no effect on sodium-lithium countertransport in erythrocytes of any age (Table III).
TABLE III The effect of amilorideon sodium-lithium countertransportand ouabain sensitive sodium flux in young, middle and old aged erythrocytes in 8 normal subjects (Mean f SEM)
Young Middle Old
Sodium-lithium countertransport (mm01 Li/h per kg cells)
Ouabain-sensitive sodium flux mm01 Na/h per kg celts
Control
Amiloride
ControI
Amiloride
0.25 * 0.03 0.34 * 0.03 0.51 & 0.06
0.27 f 0.03 0.34 * 0.03 0.47 f 0.05
1.761 f0.02 1.806 * 0.07 2.142*0.11
1.742 f 0.03 1.892kO.03 2.035 k 0.07
56
Amiloride also had no effect on ouabain sensitive sodium flux ( fO), which is primarily a measure of sodium influx and tended to increase with erythrocyte age.
Discussion There has recently been interest in erythrocyte sodium-lithium countertransport because it has been reported to be increased in some patients with essential hypertension [1,2]. It is well known that the sodium-proton exchanger, which is present in all cells, will exchange lithium in place of a proton [6]. Therefore, it has been suggested that the raised erythrocyte sodium-lithium countertransport in essential hypertension may reflect a raised level of sodium-proton exchange [6,7]. This could form a pathophysiological link to some of the observed changes in essential hypertension. Otherwise, the raised sodium-lithium countertransport may simple be a marker of a particular group of hypertensives without itself mediating the rise in blood pressure. Since most specific proteins, notably enzymes, are reduced in activity with erythrocyte age, young, middle and old aged erythrocytes were studied. The age fractions were assessed from their increasing haemoglobin A, and decreasing cell water with increasing cell age. As expected there was a decline in active sodium pumps with erythrocyte age as shown by the fall in rate constant (k,) [13,14]. This occurred mainly in the latter half of the erythrocyte life span. However, the rate of sodium efflux achieved by the sodium pumps, the ouabain sensitive sodium flux ( fO), increased with erythrocyte age (Tables I and III). This was most significant in the early half of the life span and was achieved by increasing steady state intracellular sodium concentration. There were very significant increases in the sodium-lithium countertransport during erythrocyte ageing that were similar in pattern to the increase in f0 and decrease in k, for the whole group but not related to them (Tables I and III) since the changes were not related between individuals. The cell age related increase in total sodium-lithium counter-transport activity was similar in normal subjects and essential hypertensive patients with raised activity in the middle erythrocyte fraction. However, the distinction between the two groups was poor in the old cells with no significant difference between the groups of subjects. This cell ageing related increase in sodium-lithium countertransport is difficult to explain if sodium-lithium countertransport is due to a specific membrane protein, as there is no facility to synthesize new protein units after cells have left the bone marrow. However, it may be possible to assemble subunits as can occur with the sodium pump [15]. Alternatively the general degradation and loss of proteins during erythrocyte ageing could result in the loss of a suppressing control function before the countertransport activity is lost. This possibility was supported by the differential effects of phloretin on ageing erythrocytes (Table II). This has been reported to inhibit sodium-lithium counteralthough low intracellular lithium transport in mixed erythrocyte populations, concentrations were used [5].
The effect of phloretin was not the same in erythrocytes of all ages and sodium-lithium countertransport became less sensitive to this inhibitor as the erythrocytes aged. This supports the concept that a control function of sodiumlithium countertransport was degrading during erythrocyte ageing and allowing greater activity of the remaining countertransporter. It is interesting to note, however, that the phloretin sensitive component was constant during erythrocyte ageing and the appearance of a second phloretin insensitive pathway with membrane ageing changes cannot be excluded. The increase in phloretin sensitive sodium-lithium countertransport in the essential hypertensive patients remained constant with erythrocyte ageing; however, there was a differential ageing effect in the phloretin insensitive component (Table II). This suggested that the phloretin insensitive component increased earlier with erythrocyte ageing in the essential hypertensive patients. If this component does arise from loss of inhibitory control it could indicate that patients with raised sodium-lithium countertransport have a less stable control of this membrane function. Although the old cells were smaller and therefore contained more membrane in unit mass, it is unlikely that a reduction in cell volume from 95 to 85 fl could account for the 120% increase in sodium-lithium countertransport between young and old cells. In addition, there was no difference in passive lithium efflux with erythrocyte age. The lithium content of all cell fractions was above saturating concentration [16] for sodium-lithium countertransport although older erythrocytes had a greater lithium loading. It therefore appears most likely that the control of sodium-lithium countertransport activity was affected during erythrocyte ageing. However, amiloride, the definitive inhibitor of sodium-proton countertransport had no effect on sodiumlithium countertransport in young, middle or old aged erythrocytes. Erythrocyte sodium-lithium countertransport is therefore very unlikely to be a function of the sodium-proton countertransporter unless there is a rapid loss of amiloride sensitivity during reticulocyte maturation which continues as a further change of inhibitory sensitivity in the mature erythrocytes. Sodium-proton exchange is normally a pathway of net sodium influx. This is reversed by the sodium pump and is therefore a part of the ouabain sensitive sodium flux (to). However, amiloride had no effect on f. at any cell age. This suggests that either sodium-proton exchange in erythrocytes has lost amiloride sensitivity or that sodium-proton exchange does not contribute to net sodium influx in erythrocytes. Thus, there is no support for a direct pathogenetic link between raised sodiumlithium countertransport in erythrocytes and hypertension although it may be a marker for a subgroup of hypertensives. The increased levels of activity could be due either to more carrier or more rapid loss of inhibitory control observed in hypertension. References 1 Canessa M, Adragna N, Solomon HS, Connolly TM, Tosteson counter-transport 1980;302:772-776.
in
red
cells
of
patients
with
essential
DC. Increased sodium-lithium hypertension. New Engl J Med
58 2 Morgan DB, Stewart AD, Davidson C. Relations between erythrocyte lithium efflux, blood pressure and family histories of hypertension and cardiovascular disease: studies in a factory workforce and hypertension clinic. J Hypertension 1986;4:609-615. 3 Duhm J, Becker BF. Studies on lithium transport across the red cell membrane. V. On the nature of the Na-dependent Li countertransport system of mammalian erythrocytes. J Membr Biol 1979;51:263-286. 4 Mahnensmith RL, Aronson PS. The plasma membrane sodium sodium-hydrogen exchanger and its role in physiological and pathophysiological processes. Circ Res 1985;56:773-788. 5 Funder J, Wieth JO, Jensen HE, Ibsen KK. The sodium/lithium exchange mechanism in essential hypertension. Is it a sodium/proton exchanger? In: Villareal H, Sambhi MP, eds. Topics in pathophysiology of hypertension. Boston: Martinus Nijhoff, 1984;147-161. 6 Weder AB. Red-cell lithium-sodium countertransport and renal lithium clearance in hypertension. New Engl J Med 1986;314:198-201. 7 Escobales N, Canessa M. Amiloride-sensitive Na+ transport in human red cells: Evidence for a Na/H exchange system. J Membr Biol 1986;90:21-28. 8 Pandey GN, Sarkadi B, Haas M, Gunn RB, Davis JM, Tosteson DC. Lithium transport pathways in human red blood cells. J Gen Phys 1978;72:233-247. 9 Murphy J. Influence of temperature and method of centrifugation on the separation of erythrocytes. J Lab Clin Med 19733;82:334-341. 10 Cumberbatch M, Morgan DB. A simple technique for the measurement of ouabain-sensitive sodium transport in red cells. Clin Chim Acta 1978;89:221-230. 11 Clegg G, Morgan DB, Davidson C. The heterogeneity of essential hypertension: relation between lithium efflux and sodium content of erythrocytes and a family history of hypertension. Lancet 1982;ii:891-894. 12 Fitzgibbons JF, Koler RD, Jones RT. Red cell age related changes of haemoglobins Alath and A,, in normal and diabetic subjects. J Clin Invest 1976;58:820-824. 13 Hentschel WM, Wu LL, Tobin Go, Anstall HB, Smith JB, Williams RR, Ash KO. Erythrocyte cation transport activities as a function of cell age. Clin Chim Acta 1986;157:33-43. 14 Kamber E, Poyiagi A, Deliconstantinos G. Modifications in the activities of membrane-bound enzymes during in vivo ageing of human and rabbit erythrocytes. Comp Biochem Physiol 1984;77B:95-99. 15 Rubython EJ, Morgan DB. Effect of hypokalaemia on the ouabain-sensitive sodium transport and the ouabain binding capacity in human erythrocytes. Clin Sci 1983;64:177-182. 16 Ibsen KK, Jensen HE, Wieth JO, Funder J. Essential hypertension: sodium-lithium countertransport in erythrocytes from patients and from children having one hypertensive parent. Hypertension 1982;4:703-709.
51
CCA 04293
Sodium-lit~um countertransport activity and its sensitivity to inhibitors with erythrocyte ageing in man S. Carr, T.H. Thomas and R. Wilkinson department of
University of Neweastfe upon Tyne and department of ~ephrolo~, Freeman Hospital, Newcastle upon Tvne {UK)
Medicine,
(Received 27 February 1988; revision received 26 July 1988; accepted 3 August 1988) Key words: Ageing; Amiloride; Erythrocyte; Phloretin; Sodium transport
Summary
Sodium-lithium countertransport is believed to depend on a specific protein and since such proteins usually decline with erythrocyte age it would be expected to be reduced in older cells. In fact, so~um-lit~um countertransport increased with ageing, whereas the rate constant of the sodium pump decreased. The increase in sodium-lithium countertransport with erythrocyte ageing was due to a phloretin insensitive component that was not present in young erythrocytes. Raised sodiumlithium countertransport in patients with essential hypertension was due mainly to an increased phloretin sensitive component but the phloretin insensitive component was also higher in middle aged erythrocytes. Amiloride had no effect upon sodiumlithium counter-transport or unidirectional sodium influx in cells of any age. This suggests that sodium-lithium countertransport is not a mode of action of the sodium-proton exchanger in the erythrocyte.
In some patients with essential hypertension there is an increase in the rate of sodium-lithium countertransport in erythrocytes [1,2]. The same membrane ion transporter under physiological conditions is responsible for a 1: 1, sodium-sodium exchange but this does not alter intracellular electrolyte balance and its role is difficult to explain [3]. In order to form a hypothesis of an aetiological link between
Correspondence and requests for reprints to: Dr. S. Carr, Department of Medicine and Nephrology, Freeman Hospital, Freeman Road, Newcastle upon Tyne NE7 7DN, UK. ~9-8981/88/~03.50
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
52
raised sodium-lithium countertransport and essential hypertension the same membrane transporter has been suggested to be synonymous with sodium-hydrogen exchange activity [4,5]. A generalized increase in sodium-hydrogen exchange could increase sodium reabsorption in the renal proximal tubule and predispose to proliferation in vascular smooth muscle [4-61. The membrane sodium-hydrogen exchanger has been well characterized and is defined by its sensitivity to amiloride which competes with sodium at an external site [4]. This transporter gives net sodium influx, which is normally reversed by the sodium-potassium ATPase pump but would accumulate when the pump was inhibited by ouabain [7]. Reports in the literature do not support the hypothesis that sodium-lithium and sodium hydrogen countertransport activities are synonymous [8]. However, although sodium-lithium countertransport is characteristically inhibited by phloretin [8] its lack of sensitivity to amiloride remains unconfirmed. Erythrocytes in the circulation are ageing and unable to replace damaged or degraded proteins. There is loss of membrane and sodium-potassium ATPase activity during this process. Therefore, with erythrocyte ageing other membrane ion transporters may lose activity or have qualitative changes such as a change in sensitivity to inhibitors. In addition, sensitivity to inhibitors of sodium-lithium countertransport and changes in activity with erythrocyte ageing may be different in a group of patients, such as essential hypertensives, where this ion transporter is known to be abnormal. We therefore studied sodium-lithium countertransport and the effects of phloretin and amiloride in erythrocytes of different ages from both normal subjects and essential hypertensive patients known to have a raised sodium-lithium countertransport activity. Methods Eight normal individuals (age 53.6, SD 1.4 years; systolic blood pressure (mm Hg) 128, SD 3.2; diastolic blood pressure 77.6, SD 1.9) were used to study the effects of amiloride on sodium-lithium countertransport and ouabain-sensitive sodium flux. Nine normal individuals and 7 patients with essential hypertension (age 46.0, SD 1.4 years; systolic blood pressure (mm Hg) 142.5, SD 38.6; diastolic blood pressure 93.8, SD 4.78) known to have raised sodium-lithium countertransport were used to study the effects of phloretin on sodium-lithium countertransport in cells of different ages. Four patients in this group were receiving treatment with antihypertensive agents including beta blockers and angiotensin-converting enzyme inhibitors. Erythrocyte sodium potassium and water contents Venous blood was collected into tubes containing lithium heparin. Blood was centrifuged at 2500 x g at 4” C for 5 min and the plasma removed. Erythrocytes were then washed three times with isotonic MgCl, solution (290 mosm/kg) to remove plasma contamination. The washed erythrocytes were centrifuged in 6 X 45 mm polythene tubes at 20000 X g for 30 min at 15” C, similar to the method of
53
Murphy [9] but using a swingout head. Young cells were obtained by slicing the top of the erythrocyte column, old cells from the bottom, and cells from the centre of the tube were take to be of intermediate age. Each slice contained 5-7.5s of the erythrocyte column. For measurement of sodium and potassium, erythrocytes were dispersed in 1.5 mmol/l CsCl and directly aspirated into an IL9483 flame photometer. Water and dry cell content were measured by weighing cell slices before and after drying to constant weight at 37’ C for 24 h. Ouabain sensitive sodium jlux and its rate constant Whole blood samples were incubated with either ouabain (lop4 mmol/l) or ouabain (10e4 mmol/l) and amiloride (lop3 mmol/l) for 2 h at 37°C and the erythrocyte sodium was determined as described above. Ouabain sensitive sodium flux ( fO) was calculated as the increase in erythrocyte sodium in one hour and when ouabain alone was used the rate constant (k,) was determined from the equation as described by Cumberbatch and Morgan [lo]. Amiloride was a fo = k0 x ]Nal.ac gift from Merck, Sharp and Dohme Research Laboratories, Hertfordshire, UK. Sodium-lithium countertransport The method used was similar to that described by Clegg et al. [ll]. Erythrocytes (2 ml) were suspended in lithium loading solution (8 ml; 150 mmol/l LiCl, 10 mmol/l Tris acetate, 10 mmol/l glucose) for 3 h at 37°C. The erythrocytes were then washed twice with isotonic MgCl, and once with sodium free incubation 85 mmol/l sucrose, 10 mmol/l Tris acetate, 10 medium (75 mmol/l MgCl,, mmol/l glucose, lop4 mol/l ouabain, pH 7.4, 290 mosm/kg). Erythrocytes (0.6 ml) were then incubated in 3 ml of either sodium free medium or sodium medium (150 mmol/l NaCl, 10 mmol/l Tris acetate, 10 mmol/l glucose, 1O-4 mol/l ouabain, pH 7.4, 290 mosm/kg) for 1 h. Erythrocytes were also incubated in media containing either amiloride (lop3 mol/l) or phloretin (lop4 mol/l). Phloretin was obtained from the Sigma Chemical Co. Erythrocyte lithium content was measured by flame photometry after lithium loading and after incubation in each medium and the erythrocytes were prepared by centrifugation in small polythene tubes as described for sodium and potassium determination. Sodium-lithium countertransport was determined from the difference in lithium efflux from erythrocytes in the sodium containing and sodium free media. Statistics The effect of the erythocyte age fraction or raised total sodium-lithium countertransport on other results was assessed using analysis of variance. The significance of differences between treatments and between erythrocytes fractions was assessed using the paired t test. Results Erythrocyte fractionation The age related fractionation of erythrocytes haemoglobin (HbA,) [12] and cell water content.
was verified using glycosylated Between young and old erythro-
54 TABLE
I
The effect of erythrocyte age on sodium-lithium countertransport, passive lithium effhtx in 8 normal subjects (mean f SEM)
sodium
pump
rate
constant
age
Sodium-lithium countertransport (mm01 Li/h per kg cells)
(h-‘)
Passive lithium efflux (mm01 Li/h per kg cells)
Young Middle Old
0.21 f 0.02 * * 0.30 f 0.06 0.48 + 0.04 * * *
0.27 + 0.01 0.28 + 0.01 0.24 k 0.02 *
0.22 * 0.03 0.21+ 0.04 0.26 f 0.05
Erythrocyte
* p < 0.01, **p
***
Sodium-pump rate constant
p < 0.001 compared
with the middle
erythrocyte
and
fraction.
cytes HbA, increased by 44% and cell water decreased by 5%. Measurements of cell water showed that the age related fractionation did not change when erythrocytes were incubated in the solutions used for the measurement of flux rates.
Sodium-lithium countertransport and the rate constant of the sodium pump with erythrocyte ageing The rate constant of the sodium pump (k,) decreased with erythrocyte age (Table I) which indicated the loss of active sodium pumps from the erythrocyte membrane. Most of the ageing change occurred between the middle and old age fractions. There was also increased variance in k, in the older fractions which presumably reflects, variability in the ageing process. Sodium-lithium countertransport increased with erythrocyte age (Table I) in contrast to the change in k,, but as with k,, most of the change occurred between
TABLE
II
The effect of erythrocyte age on total, phloretin sensitive and phloretin countertransport in 9 normal subjects and 7 essential hypertensive patients raised countertransport activity (mean f SEM) Subjects
Erythrocyte
age
Total a,b
Sodium-lithium countertransport (mmol Li/h per kg cells) Phloretin insensitive
Normal
controls
Essential hypertensives
insensitive sodium-lithium who were known to have
a.C
Phloretin sensitive b
Young Middle Old
0.16 f 0.04 0.27 f 0.03 0.62 f 0.09
0.03 f 0.02 0.09 * 0.02 0.47 + 0.09
0.12 f 0.04 0.20 f 0.04 0.17rtO.06
Young Middle Old
0.38 f 0.03 0.50 f 0.03 0.70 f 0.04
0.06 + 0.03 0.23 f 0.04 0.35 f 0.05
0.31+ 0.02 0.27 f 0.04 0.35 f 0.07
age on the activity; b p < 0.01 for an Analysis of variance showed: a p i 0.01 for an effect of erythrocyte effect of hypertension on the activity; and ’ p -z 0.05 for an interaction between erythrocyte age and hypertension on the activity.
55
the middle and old cell fractions. There was also increased variance in the older cell fractions. The changes in sodium-lithium countertransport and ouabain sensitive sodium flux with erythrocyte age were similar and the converse of the changes in sodium pump rate constant. However, none of these changes were related between individuals, suggesting that they were due to independent ageing changes in the erythrocyte membrane. Passive lithium efflux did not change significantly with erythrocyte ageing (Table I). Analysis of variance showed very significant effect of erythrocyte age ( f = 43.2, p < 0.01) and essential hypertension (f = 31.7, p < 0.01) on sodium-lithium countertransport but there was no significant interaction between the effect of essential hypertension and the erythrocyte age (Table XI). However, although the two groups had significantly different total sodium-lit~um countertransport activity in the young (p < 0.005) as well as the middle cells, there was no difference between the old cells. Effect of phloretin on sodium-lithium countertransport
In normal subjects phloretin completely inhibited sodium-lithium countertransport in young erythrocytes but in the middle fraction of cells 35% was phloretin insensitive and in old cells this had increased to 75% of sodium-~t~urn countertransport that was phloretin sensitive. The phloretin sensitive component of sodium-lithium countertransport did not change during the erythrocyte lifespan. In patients with essential hypertension and raised total sodium-lithium countertransport in middle aged erythrocytes, analysis of variance showed that the phloretin sensitive component was raised (f= 11.62, p < 0.01) and was also not affected by erythrocyte age. The overall increase in sodium-lithium countertransport was due to a marked increase in the phloretin insensitive component (f = 27.5, p -C0.01). However, the pattern of change in this component with erythrocyte ageing was significantly different (f = 6.27, p < 0.05) in the essential hypertensives (Table II). Effect of amiloride on sodium-lithium co~ntertran~port and ouabain sensitiue sodium fr UX
Amiloride, the definitive inhibitor of sodium-hydrogen countertransport, had no effect on sodium-lithium countertransport in erythrocytes of any age (Table III).
TABLE III The effect of amilorideon sodium-lithium countertransportand ouabain sensitive sodium flux in young, middle and old aged erythrocytes in 8 normal subjects (Mean f SEM)
Young Middle Old
Sodium-lithium countertransport (mm01 Li/h per kg cells)
Ouabain-sensitive sodium flux mm01 Na/h per kg celts
Control
Amiloride
ControI
Amiloride
0.25 * 0.03 0.34 * 0.03 0.51 & 0.06
0.27 f 0.03 0.34 * 0.03 0.47 f 0.05
1.761 f0.02 1.806 * 0.07 2.142*0.11
1.742 f 0.03 1.892kO.03 2.035 k 0.07
56
Amiloride also had no effect on ouabain sensitive sodium flux ( fO), which is primarily a measure of sodium influx and tended to increase with erythrocyte age.
Discussion There has recently been interest in erythrocyte sodium-lithium countertransport because it has been reported to be increased in some patients with essential hypertension [1,2]. It is well known that the sodium-proton exchanger, which is present in all cells, will exchange lithium in place of a proton [6]. Therefore, it has been suggested that the raised erythrocyte sodium-lithium countertransport in essential hypertension may reflect a raised level of sodium-proton exchange [6,7]. This could form a pathophysiological link to some of the observed changes in essential hypertension. Otherwise, the raised sodium-lithium countertransport may simple be a marker of a particular group of hypertensives without itself mediating the rise in blood pressure. Since most specific proteins, notably enzymes, are reduced in activity with erythrocyte age, young, middle and old aged erythrocytes were studied. The age fractions were assessed from their increasing haemoglobin A, and decreasing cell water with increasing cell age. As expected there was a decline in active sodium pumps with erythrocyte age as shown by the fall in rate constant (k,) [13,14]. This occurred mainly in the latter half of the erythrocyte life span. However, the rate of sodium efflux achieved by the sodium pumps, the ouabain sensitive sodium flux ( fO), increased with erythrocyte age (Tables I and III). This was most significant in the early half of the life span and was achieved by increasing steady state intracellular sodium concentration. There were very significant increases in the sodium-lithium countertransport during erythrocyte ageing that were similar in pattern to the increase in f0 and decrease in k, for the whole group but not related to them (Tables I and III) since the changes were not related between individuals. The cell age related increase in total sodium-lithium counter-transport activity was similar in normal subjects and essential hypertensive patients with raised activity in the middle erythrocyte fraction. However, the distinction between the two groups was poor in the old cells with no significant difference between the groups of subjects. This cell ageing related increase in sodium-lithium countertransport is difficult to explain if sodium-lithium countertransport is due to a specific membrane protein, as there is no facility to synthesize new protein units after cells have left the bone marrow. However, it may be possible to assemble subunits as can occur with the sodium pump [15]. Alternatively the general degradation and loss of proteins during erythrocyte ageing could result in the loss of a suppressing control function before the countertransport activity is lost. This possibility was supported by the differential effects of phloretin on ageing erythrocytes (Table II). This has been reported to inhibit sodium-lithium counteralthough low intracellular lithium transport in mixed erythrocyte populations, concentrations were used [5].
The effect of phloretin was not the same in erythrocytes of all ages and sodium-lithium countertransport became less sensitive to this inhibitor as the erythrocytes aged. This supports the concept that a control function of sodiumlithium countertransport was degrading during erythrocyte ageing and allowing greater activity of the remaining countertransporter. It is interesting to note, however, that the phloretin sensitive component was constant during erythrocyte ageing and the appearance of a second phloretin insensitive pathway with membrane ageing changes cannot be excluded. The increase in phloretin sensitive sodium-lithium countertransport in the essential hypertensive patients remained constant with erythrocyte ageing; however, there was a differential ageing effect in the phloretin insensitive component (Table II). This suggested that the phloretin insensitive component increased earlier with erythrocyte ageing in the essential hypertensive patients. If this component does arise from loss of inhibitory control it could indicate that patients with raised sodium-lithium countertransport have a less stable control of this membrane function. Although the old cells were smaller and therefore contained more membrane in unit mass, it is unlikely that a reduction in cell volume from 95 to 85 fl could account for the 120% increase in sodium-lithium countertransport between young and old cells. In addition, there was no difference in passive lithium efflux with erythrocyte age. The lithium content of all cell fractions was above saturating concentration [16] for sodium-lithium countertransport although older erythrocytes had a greater lithium loading. It therefore appears most likely that the control of sodium-lithium countertransport activity was affected during erythrocyte ageing. However, amiloride, the definitive inhibitor of sodium-proton countertransport had no effect on sodiumlithium countertransport in young, middle or old aged erythrocytes. Erythrocyte sodium-lithium countertransport is therefore very unlikely to be a function of the sodium-proton countertransporter unless there is a rapid loss of amiloride sensitivity during reticulocyte maturation which continues as a further change of inhibitory sensitivity in the mature erythrocytes. Sodium-proton exchange is normally a pathway of net sodium influx. This is reversed by the sodium pump and is therefore a part of the ouabain sensitive sodium flux (to). However, amiloride had no effect on f. at any cell age. This suggests that either sodium-proton exchange in erythrocytes has lost amiloride sensitivity or that sodium-proton exchange does not contribute to net sodium influx in erythrocytes. Thus, there is no support for a direct pathogenetic link between raised sodiumlithium countertransport in erythrocytes and hypertension although it may be a marker for a subgroup of hypertensives. The increased levels of activity could be due either to more carrier or more rapid loss of inhibitory control observed in hypertension. References 1 Canessa M, Adragna N, Solomon HS, Connolly TM, Tosteson counter-transport 1980;302:772-776.
in
red
cells
of
patients
with
essential
DC. Increased sodium-lithium hypertension. New Engl J Med
58 2 Morgan DB, Stewart AD, Davidson C. Relations between erythrocyte lithium efflux, blood pressure and family histories of hypertension and cardiovascular disease: studies in a factory workforce and hypertension clinic. J Hypertension 1986;4:609-615. 3 Duhm J, Becker BF. Studies on lithium transport across the red cell membrane. V. On the nature of the Na-dependent Li countertransport system of mammalian erythrocytes. J Membr Biol 1979;51:263-286. 4 Mahnensmith RL, Aronson PS. The plasma membrane sodium sodium-hydrogen exchanger and its role in physiological and pathophysiological processes. Circ Res 1985;56:773-788. 5 Funder J, Wieth JO, Jensen HE, Ibsen KK. The sodium/lithium exchange mechanism in essential hypertension. Is it a sodium/proton exchanger? In: Villareal H, Sambhi MP, eds. Topics in pathophysiology of hypertension. Boston: Martinus Nijhoff, 1984;147-161. 6 Weder AB. Red-cell lithium-sodium countertransport and renal lithium clearance in hypertension. New Engl J Med 1986;314:198-201. 7 Escobales N, Canessa M. Amiloride-sensitive Na+ transport in human red cells: Evidence for a Na/H exchange system. J Membr Biol 1986;90:21-28. 8 Pandey GN, Sarkadi B, Haas M, Gunn RB, Davis JM, Tosteson DC. Lithium transport pathways in human red blood cells. J Gen Phys 1978;72:233-247. 9 Murphy J. Influence of temperature and method of centrifugation on the separation of erythrocytes. J Lab Clin Med 19733;82:334-341. 10 Cumberbatch M, Morgan DB. A simple technique for the measurement of ouabain-sensitive sodium transport in red cells. Clin Chim Acta 1978;89:221-230. 11 Clegg G, Morgan DB, Davidson C. The heterogeneity of essential hypertension: relation between lithium efflux and sodium content of erythrocytes and a family history of hypertension. Lancet 1982;ii:891-894. 12 Fitzgibbons JF, Koler RD, Jones RT. Red cell age related changes of haemoglobins Alath and A,, in normal and diabetic subjects. J Clin Invest 1976;58:820-824. 13 Hentschel WM, Wu LL, Tobin Go, Anstall HB, Smith JB, Williams RR, Ash KO. Erythrocyte cation transport activities as a function of cell age. Clin Chim Acta 1986;157:33-43. 14 Kamber E, Poyiagi A, Deliconstantinos G. Modifications in the activities of membrane-bound enzymes during in vivo ageing of human and rabbit erythrocytes. Comp Biochem Physiol 1984;77B:95-99. 15 Rubython EJ, Morgan DB. Effect of hypokalaemia on the ouabain-sensitive sodium transport and the ouabain binding capacity in human erythrocytes. Clin Sci 1983;64:177-182. 16 Ibsen KK, Jensen HE, Wieth JO, Funder J. Essential hypertension: sodium-lithium countertransport in erythrocytes from patients and from children having one hypertensive parent. Hypertension 1982;4:703-709.