[3H]serotonin uptake in human blood platelets is reduced by ouabain and endogenous digitalis-like inhibitors of Na+,K+-ATPase

European Journal of Pharmacologr', 92 (1983) 167-168

167

Elsevier

Rapid communication [ 3 H ] S E R O T O N I N UPTAKE IN H U M A N B L O O D P L A T E L E T S IS REDUCED BY OUABAIN AND E N D O G E N O U S DIGITALIS-LIKE I N H I B I T O R S OF Na+,K +-ATPase L.A. K A M A L *, J.F. CLOIX, M.A. D E V Y N C K and P. M E Y E R

Department of Pharmacology, 1NSERM U7, Hopital Necker, 161 rue de Sbvres, 75015 Paris, France Received 5 July 1983, accepted 11 July 1983

The uptake of serotonin in blood platelets is a carrier-mediated sodium-dependent transport process (Sneddon, 1969). The intracellular sodium concentration is kept relatively low by means of a sodium pump linked to the activity of sodium- and potassium-stimulated adenosine triphosphatase (Na+,K+-ATPase). Inhibitors of Na+,K+-ATPase have been reported in experimental animals and man undergoing extracellular volume expansion (see review De Wardener and Clarkson, 1982). The precise origin and characteristics of these inhibitors are as yet unknown. However, inhibitors of Na+,K+-ATPase having digitalis-like properties have been isolated from plasma, urine, brain, kidney and heart (see review, De Wardener and Clarkson, 1982). The uptake of [3H]serotonin in human blood platelets was studied in the presence of a cardiac glycoside, ouabain, specific inhibitor of Na+,K +ATPase and in the presence of endogenous inhibitor-enriched fractions of Na+,K+-ATPase. These heat stable, low molecular weight inhibitors were isolated from human plasma by gel filtration and separated by their negative charge by anion exchange chromatography as previously described by Cloix et al. (1983). Forty ml of venous blood was withdrawn from healthy normotensive subjects and centrifuged at 300 g for 10 min at 15°C. The supernatant containing platelet rich plasma was removed and aliquots containing 1-2 × 108 platelets were preincubated at 37°C for 50 min with varying concentrations of either ouabain (10 -1° to 10 -5 M)

* To whom all correspondence should be addressed. 0014-2999/83/$03.00 '~ 1983 Elsevier Science Publishers B.V.

(Sigma), increasing amounts of the Na+,K +ATPase inhibitor enriched fractions, inactive fractions derived in the same manner or with saline buffer. The uptake of [3H]serotonin (Amersham, 10.8 Ci/mmol) was initiated by the addition of [3H]serotonin, 4.4. × 10 - 7 M (final concentration), at 37°C. After 30 s of incubation, uptake was terminated by filtration on Millipore filters (0.45 /zm). The filters were rinsed with 10 ml of buffer, dried and counted. The uptake of [3H]serotonin in platelets determined by Lineweaver-Burk plots was previously found to be linear, suggesting simple MichaelisMenten uptake kinetics (Kamal et al., unpublished observations). In the presence of ouabain, the uptake of [3H]serotonin was inhibited in a concentration-dependent manner (fig. 1). Similarly, in the presence of the Na÷,K+-ATPase inhibitor-enriched fractions, the uptake of [3H]serotonin was reduced and depended upon the amount of each fraction (fig. 1). These results are in agreement with the activity of these fractions in inhibiting Na+,K+-ATPase from dog kidney and [3H]ouabain binding in human erythrocytes (Cloix et al., 1983). The present findings showing a similar effect of ouabain and these endogenous inhibitors on [ 3H]serotonin uptake represent an additional argument favoring their action on Na+,K+-ATPase inhibition. These results also suggest that in an intact cell preparation there is a possible influence of circulating factors which can act locally to modulate cell functions. Circulating inhibitors of Na+,K + pumps have been proposed as playing a role in the development of hypertension. It has been demonstrated

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that a correlation exists between the levels of a plasma inhibitor of Na+,K+-ATPase activity and mean arterial pressure (Hamlyn et al., 1982). In addition, the uptake of [3H]serotonin and endogenous serotonin content is reduced in subjects with essential hypertension (Kamal et al., unpublished observations). The decrease in uptake of [3H]serotonin may thus be linked to the presence of these circulating factors. This is the first report that partially purified circulating plasma inhibitors of Na+,K+-ATPase inhibit the uptake of [3H]serotonin in platelets. The uptake of [3H]serotonin in blood platelets adds to the biological assays available to test the effects of these endogenous Na+,K+-ATPase inhibitors.

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EQUIVALENTPLASMACONCENTRATION Fig. 1. Effect of ouabain and endogenous inhibitors of Na+,K+-ATPase (B,C, D). Ordinate." percent inhibition of [3H]serotonin uptake in l0 s platelets at a concentration of 4.4 × 10 -7 M serotonin in the presence of ouabain or endogenous inhibitors. Abscissa: molar concentrations of ouabain or volume of plasma inhibitors, determined from the original plasma volume before purification. Values shown are the means of 5-10 experiments.

Cloix, J.F., M.A. Devynck, J.L. Elghozi, L.A. Kamal, L. Lacerda-Jacomini, P, Meyer, M.G. Pernollet, J.B. Rosenfeld and H. De The, Plasma endogenous sodium pump inhibitor in essential hypertension, Hypertension (in press). De Wardener, H.E. and E.M. Clarkson, 1982, The natriuretic hormone: recent developments, Clin. Sci. 63, 415. Hamlyn, J.M., R. Ringel, Schaeffer, P.D. Levinson, B.P. Hamilton, A.A. Kowarski and M.P. Blaustein, 1982, A circulating inhibitor of (Na + + K +) ATPase associated with essential hypertension, Nature 300, 650. Sneddon, J.M., 1969, Sodium-dependent accumulation of 5-hydroxytryptamine by rat blood platelets, Br. J. Pharmacol. 37, 680.