Plasma serotonin levels and the platelet serotonin transporter

Abstract

Serotonin (5HT) is a platelet-stored vasoconstrictor. Altered concentrations of circulating 5HT are implicated in several pathologic conditions, including hypertension. The actions of 5HT are mediated by different types of receptors and terminated by a single 5HT transporter (SERT). Therefore, SERT is a major mechanism that regulates plasma 5HT levels to prevent vasoconstriction and thereby secure a stable blood flow. In this study, the response of platelet SERT to the plasma 5HT levels was examined within two models: (i) in subjects with chronic hypertension or normotension; (ii) on platelets isolated from normotensive subjects and pretreated with 5HT at various concentrations. The platelet 5HT uptake rates were lower during hypertension due to a decrease in Vmax with a similar Km; also, the decrease in Vmax was primarily due to a decrease in the density of SERT on the platelet membrane, with no change in whole cell expression. Additionally, while the platelet 5HT content decreased 33%, the plasma 5HT content increased 33%. Furthermore, exogenous 5HT altered the 5HT uptake rates by changing the density of SERT molecules on the plasma membrane in a biphasic manner. Therefore, we hypothesize that in a hypertensive state, the elevated plasma 5HT levels induces a loss in 5HT uptake function in platelets via a decrease in the density of SERT molecules on the plasma membrane. Through the feedback effect of this proposed mechanism, plasma 5HT controls its own concentration levels by modulating the uptake properties of platelet SERT.

Fig. 1

Serotonin (5HT) contents of platelet and platelet-poor plasma (PPP) in hypertensive and normotensive states. Using a competitive ELISA technique, platelet and PPP 5HT concentrations from 16 subjects with hypertension were measured and compared with the values from normotension following the manufacturer’s instructions. Samples were read at 405 nm on an ELISA plate reader. 5HT amounts were quantified using standards supplied by the manufacturer, and analyzed using Origin software. The (*) represents the results of a two-tailed Student’s t-test with p < 0.001 (compared with normotensive subjects).

Fig. 2

Serotonin (5HT) uptake rates of platelet single 5HT transporter (SERT) from hypertensive and normotensive subjects. (a) Platelets from the blood samples of seven hypertensive and seven normotensive subjects were prepared and assayed for [³H]-5HT uptake at 1, 2.5, 10, 15, 17.5, 20, 30, 50 and 60 min as described in Materials and methods. Background accumulation of [³H]-5HT was measured in the same experiment by treating platelets with 0.1 µmol/L high-affinity cocaine analog, 2β-carbomethoxy-3-tropane (β-CIT) and subtracted from each experimental value. The difference between the 5HT uptake rates of hypertensive and normotensive samples was most pronounced at the earliest time points, 10 min. (b) 5HT uptake rates of platelets prepared from 1 mL blood samples of seven hypertensive and seven normotensive subjects were measured by incubating with [³H]-5HT for 10 min as described in Materials and methods. The (*) represents the results of a two-tailed Student’s t-test with p < 0.001 (compared with normotensive subjects).

Fig. 3

Substrate dependence of platelet hSERT from hypertensive and normotensive subjects. Platelets prepared from 7 mL blood samples of seven hypertensive and seven normotensive subjects were prepared as described under Materials and methods. Initial rates of 5HT uptake in platelets from hypertensive subjects (a) and normotensive subjects (b) were measured over the indicated range of 5HT concentrations using 20 nmol/L ³H-5HT with unlabeled 5HT added to the final concentration. K_m and V_max values were determined by fitting the rate versus concentration data. The inset shows an Eadie–Hofstee plot of the data with lines drawn from the derived kinetic constants. See Table 2 for a summary of the calculated data. Background SERT-independent accumulation of ³H-5HT was measured in the same experiment by treating platelets with 0.1 µmol/L high-affinity cocaine analog, 2β-carbomethoxy-3-tropane (β-CIT), and subtracted from each experimental value. Cocaine (0.1 mmol/L) totally inactivates the 5HT uptake function of SERT (Kilic and Rudnick 2000; Kilic et al. 2003; Kocabas et al. 2003; Ozaslan et al. 2003). The 5HT transporter uptake rates were calculated as means of standard deviation values from three independent experiments performed in triplicate.

Fig. 4

Analysis of single 5HT transporter (SERT) expression in platelets from hypertensive and normotensive subjects. For whole-cell expression, platelets from 2 mL blood samples of seven hypertensive and seven normotensive subjects with (lanes 1 and 2, respectively) were lysed, and proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by western blot analysis with anti-SERT-Ab (a). The integrated density values were determined by densitometry (b). For cell surface expression, platelets from the subjects with hypertension and normotension (lanes 3 and 4, respectively) were biotinylated, and the labeled cell surface proteins were precipitated with streptavidin beads and then separated and visualized as above. The results of western blot analysis are shown above a summary of combined data from four densitometric scans. The (*) represents the results of a two-tailed Student’s t-test with p < 0.001 (compared with normotensive subjects).

Fig. 5

Serotonin (5HT) uptake rates of single 5HT transporter (SERT) on isolated platelets pretreated with 5HT at different concentrations. Platelets prepared from 5 mL blood samples of three normotensive subjects were first pre-treated with 5HT over a wide concentration range (0–2.5 nmol/L) for 30 min. Then the [³H]-5HT uptake rates were measured in intact platelet. Rate of uptake is expressed as the means and SD values of triplicate determinations from three independent experiments. The (*) and (**) represent the results of a two-tailed Student’s t-test with both p < 0.001 (compared with untreated platelet uptake rates).

Fig. 6

Expression of single 5HT transporter (SERT) on isolated platelets with and without 5HT pre-treatment. Platelets prepared from 5 mL blood samples of 3 normotensive subjects were first pretreated with 0, 7.5, 1.5 nmol/L 5HT for 30 min. Then, they were biotinylated, and the labeled cell surface proteins were precipitated with streptavidin beads and then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by western blot analysis using anti-SERT antibody followed by HRP-conjugated rabbit IgG (a). The integrated density values were determined by densitometry (b). The results of western blot analysis are shown in figure is the summary of combined data from three densitometric scans (Table 3). Actin was used as the loading control (inset in Figure 45 kD). The (* and **) represents the two-tailed Student’s t-test with p < 0.001 (compared with 0 nmol/L 5HT-matched samples).

Fig. 7

Model for Serotonin (5HT)-dependent plasma membrane density of platelet single 5HT transporter (SERT). In the presence of high (>1 nmol/L) plasma 5HT levels, the density and the 5HT uptake rates of platelet SERT decrease in platelets samples of hypertensive subjects and 5HT-pre-treated platelets obtained from normotensive subjects. Therefore, we hypothesize that the low 5HT uptake capacity of platelet SERT results from a feedback effect of increased plasma 5HT levels. Individuals with hypertension might have an impaired 5HT uptake mechanism, which abates as the hypertensive state improves.