Rubidium Uptake of Mononuclear Leukocytes From Normotensive and Borderline Hypertensive First Degree Relatives to

Patients With Essential Hypertension

Torben Johansenl, Jens Rokkedal Nielsenz, Lars Poulsgardl Torben Knudsenl, Knud E. Pedersen3 and Niels A. Klitgaardb

Departments of 1Pharmacology, 2Clinical Physiology, 3Internal Medicine and k l i n i c a l Chemistry, Odense University, Odense, DENMARK

Summary Uptake of 86Rubidium of mononuclear leukocytes (MNL) was used as a measure of cellular sodium-potassium pump activity. 86Rb-uptake was deter- mined w i th the pump stimulated mainly f rom inside the cells by sodium as wel l as wi th a combined stimulation f rom inside by sodium and f rom outside by Rb. I n the f i r s t case there was an increased pump act iv i ty in MNL f rom borderline hypertensive offspring of hypertensive patients (BHO), and th is may be related to an increased number of pump sites observed previously (10). Estimation of maximal pump act iv i ty (Vmax) of MNL suggested that Vmax of each pump site in MNL from BHO may be decreased compared t o control value of MNL from healthy normotensive subjects.

Introduction There i s an increasing evidence of disturbances in intracellular sodium- potassium regulation associated w i th essential hypertension. Tobian and Binion (1) reported in 1952 an increase i n the sodium content of renal arteries, and i n 1960 Losse e t al. ( 2 ) reported abnormalities in red cells. Since then various changes in sodium-flux and potassium-flux of red and white cells f rom patients w i th essential hypertension have been demonstrated (3, 4, 5, 6).

Whereas the physiological role of the sodium-sodium countertransport ( 4 ) and the sodium-potassium cotransport (5, 7) for cellular electrolyte regulation are not clarified, the sodium-potassium pump (8) i s considered t o be of major importance. In the present investigation we have measured sodium-potassium pump act iv i ty in mononuclear leukocytes (MNL) f rom normotensive and borderline hypertensive f i rst degree relatives to patients wi th essential hyper- tension.

Subjects, Methods and Materials Subjects

Normotensive (NTO) and borderline hypertensive (BHO) f i r s t degree relatives to hypertensive patients, borderline hypertensive subjects (BH) and normoten- sive controls (N) (Table I) were investigated. Only one of the parents o f NTO and BHO had hypertension.

Methods Isolation of leukocytes

MNL were isolated from whole blood by centrifugation on a discontinuous density gradient of Percol l as described by Boon e t al. (9). Brief ly, whole venous blood, anticoagulated with 1% EDTA, was diluted w i th phosphate- buffered saline solution, mixed w i th dextran (1.2% w/v) and l e f t a t room temperature for 30 min to allow the erythrocytes t o sediment. The superna- tant was removed, and the cellular content was washed w i th phosphate- buffered saline. A suspension of pure MNL was then prepared by dif ferential centrifugation of the mixed cel l suspension through a Percol l gradient consist- ing of two layers (1.06 and 1.08 g/ml). MNL were concentrated between the two layers. The majori ty of the platelets did not penetrate the upper layer

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This work was supported by Car l og El len Hertz's Legat and funds obtained from P. Car l Petersens Foundation (Nos. 61294, 61356).

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and polymorphnuclear leukocytes and remaining erythrocytes moved through both layers. MNL were harvested by aspiration into a syringe and then washed in potassium-free Ringer solution. Cel l number was counted in a hemocyto- meter, and purity of the cel l suspension was determined by inspection of a stained smear (Diff-Quick, Harleco, Belgium). Ce l l purity: 97.9% mononuclear cells.

In f ive control experiments the mononuclear fract ion contained 79% lymphocytes and 19% monocytes.

Incubation

The MNL suspension was divided into aliquots w i th the same ce l l density in a final volume of 0.6 ml. The cel l density i n the experiments varied f rom 2.4~106 to 8.0~106 cells per ml. The cel l suspensions i n potassium-free Ringer solution were temperature-equilibrated by 10 min preincubation a t 37OC. Then the cells were incubated with f ive dif ferent concentrations of 86Rb (1.56- 16.67 mmol/L) for 2.5, 5, 7.5 and 10 min or wi th 86Rb 8.3 umol /L for 60 min. The f inal concentration of NaCl in the incubation medium was adjusted t o compensate for presence of the dif ferent concentrations of 86Rb. A f te r the incubation the reaction was stopped by adding a 15.7 times higher volume of ice-chilled potassium-free Ringer solution to the cell suspensions. Then the cells were washed three times. The cellular content of 86Rb was measured in a Mark Ill l iquid scintillation spectrometer (Nulear Chicago) using the preset isotop program for 32P. Counting efficiency was 90%.

Materials

Bovine albumin and dextran were supplied by Sigma Chemical Company (St. Louis, USA). Percol l was supplied by Pharmacia Fine Chemicals (Sweden), and 86Rb by Amersham, Buckinghamshire, England. A l l other chemicals were o f analytical grade.

Phosphate-buffered saline solution contained (in mmol/L): NaCl 125, Na2HP04 13.8, NaH2P04 3.2, EDTA 0.34, glucose 1 mg/ml, albumin 1 mg/ml.

Potassium-free Rinqer solution contained ( in mmol/L): NaCl 125, CaC12 1.3, MgS04 1.3, Na2HP04 13.8, NaH2P04 3.2, glucose 1 mg/ml, albumin 1 mg/ml.

Resulta

Ef fec t of Rb 8 umol/L

The uptake of 86Rb in presence o f a very low concentration of extracellular Rb was significantly higher i n MNL from BHO compared to the uptake of MNL from N (P < 0.05 by the Mann-Whitney Test). However, there was no difference between the uptake in MNL from NTO or B H compared to control value (fig. 1).

Ef fec t of Rb 4 mmol/L

I n presence of Rb 4 mmol/L there was a linear relat ion between cellular uptake of 86Rb and t ime of incubation. The rate of Rb-uptake (pmol/106 cells/min) of MNL from BHO and NTO were not significantly dif ferent f rom that observed with MNL from N (fig. 2).

Estimation of maximal act ivi ty of the Na+-K+-pump

There was a linear relation between cellular uptake of 86Rb and t ime of incubation wi th a l l f ive concentrations of Rb. The rates of Rb-uptake were calculated and a Lineweaver-Burk plot (reciprocal of Rb concentration versus the reciprocal of the rate of Rb-uptake) was used to estimate the maximal rate of Rb-uptake (Vmax). As shown i n fig. 3, there was no significant difference in maximal Rb-uptake by MNL from BHO or NTO compared t o the values observed w i th MNL from N.

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Discussion

I n this investigation two types o f experiments were performed i n order t o characterize MNL sodium-potassium pump activity. Firstly, the pump was mainly stimulated by a high intracellular concentration of sodium. Secondly, the pump was stimulated by both high inside concentration o f sodium and high outside concentration of Rb. The high inside concentration was caused by use of potassium-free solutions for the isolation procedure which lasted 3-4 hours. This is known to make the cel l gain sodium i n exchange for potassium (10). The increased 86Rb-uptake of MNL f rom BHO in the f i rst type of experiment confirmed our previous observation (11). Thus, i n MNL f rom BHO inside stimulation of the pump with sodium caused the pump activi ty to increase to a greater extent than that i n MNL f rom NTO and BH. This may be related to our previous observation of an increased number of pump sites o f lymphocytes from BHO (11). However, there was a normal maximal pump act iv i ty (Vmax) of MNL from BHO. Apparently, Vmax of each pump site in MNL from these subjects are lower than control value from N. This i s supported by the normal pump act iv i ty in MNL from BHO in presence of Rb 4 mmol/L.

I t may be observed that Vmax of MNL f rom 5 subjects o f a total of 26 subjects w i th disposition for hypertension were higher than the upper 95% confidence l imit for control value o f Vmax. The prevalence of hypertension of f i r s t degree relatives to parents w i th one having essential hypertension is 28% (12). One may speculate that these 5 subjects may have an increased risk of developing hypertension.

References

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2.

3.

4.

5.

6.

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8.

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10.

11.

12.

Tobian, L., Binion, J.T.: Tissue cations and water in arterial hypertension. Circulat ion 5:754-758, 1952. Losse, H., Wehmeyer, H., Wessel, F.: The water and electrolyte content o f erythrocytes in arterial hypertension. Klin Wochenschr 38:393-395, 1960. Postnov, Y.V., Orlov, S.N., Shevchenko, A., Adler, A.M.: Altered sodium permeability, calcium binding and Na-K-ATPase act iv i ty in the red blood cel l membrane in essential hypertension. Pflugers Arch 371963-269, 1977. Canessa, M., Adragna, N., Solomon, H.S., Connolly, T., Tosteson, D.C.: Increased sodium-lithium countertransport in red cells of patients w i th essential hypertension. New Engl J Med 302:772-776, 1980. Garay, R.P., Elghozi, J.-L., Dagher, G., Meyer, P.: Laboratory distinction between essential and secondary hypertension by measurement of erythro- cyte cation fluxes. New Engl J Med 302:769-771, 1980. Thomas, R.D., Edmondson, R.P.S., Hilton, P.J., Jones, N.F.: Abnormal sodium transport in leucocytes f rom patients w i th essential hypertension and the ef fect of treatment. C l in Sci Mo l Med 48:169~-170s, 1975. Garay, R.P, Meyer, P.: A new test showing abnormal net Na+ and K+ fluxes i n erythrocytes of essential hypertensive patients. Lancet 349-353, 1979. Skou, J.C.: Enzymatic basis for active transport of Na+ and K+ across cel l membrane. Physiol Rev 45:596-617, 1965. Boon, N.A., Oh, V.M.S., Taylor, E.A., Johansen, T., Aronson, J.K., Grahame-Smith, D.G.: Measurement of specific (3H)-ouabain binding to different types of human leucocytes. Br J Cl in Pharmac 18:153-161, 1984. Simchowitz, L., Spilberg, I., De Weer, P.: Sodium and potassium fluxes and membrane potential of human neutrophils. Evidence for an electrogenic sodium pump. J Gen Physiol 79:453-479, 1982. Nielsen, J.R., Pedersen, K.E., Johansen, T., Klitgaard, N.A.: Ouabain- binding and %ubidiurn-uptake in lymphocytes of normal and borderline hypertensive subjects. Scand J C l in Invest 43:393-399, 1983. Ayman, D.: Heridity in arteriolar (essential) hypertension. A clinical study of the blood pressure of 1524 members of 227 families. Arch Intern Med 54:792-802, 1934.

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1ow-

-

ow - -

6W -

LW -

200 -

-

0-

EFFECT OF RUBIDIUM 83pM unstimulated pump activity

EFFECT OF RUBIDIUM 4 m M stimulated pump activity

Rb uptoke pmolllo' ~ ~ 1 1 ~ 1 6 0 mn 60

50

LO

30

20

10

0

600

500

600

300

20E

101

. U

0 .

0

0 0

. 8 c

I : i

m 0 . .

0

N BH NTO BHO I N BH

Table I

NTO BHO

MAXIMAL PUMP ACTIVITY (Vmax)

Rb uptake pmolI1o'cet lhm

0 N NTO BHO BH

B

No. of subjects 26 24 16 11

Age 2725 2524 2625 2727

Systolic blood 12928 122+11 1 4 2 1 5 e 139214 pressure (mm Hg)

Diastolic blood 8828 8226 97210a 9328 pressure (mm Hg)

Mean blood 10127 9527 11Z2Ba 10B29b pressure (mm Hg)

0 .

0 . m

c..

0

0 . . 8

N BH

a: p < 0.01 (N/BHO); b: p c 0.02 (N/BH)

The table shows mean values end standard deviations

NTO BHO