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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.
Serotonin (5HT) is an intermediate product of tryptophan metabolism located primarily in the enterochromaffin cells of the intestines, serotonergic neurons of the brain, and blood platelets, and is a well established neurotransmitter in the central nervous system. Although altered concentrations of circulating 5HT are implicated in several pathologic conditions, including hypertension, the mechanism underlying involvement of 5HT remains elusive. 5HT has potent vasoconstrictor activity (Watts 2005) and also enhances the hypertensive effects of known vasoconstrictors (Golino et al. 1991;Gujrati et al. 1994; Azzadin et al. 1995; Yildiz et al. 1996; Watts 2005). In hypertension and atherosclerosis (Golino et al. 1991; Yildiz et al. 1996), the blood vessels become more sensitive to the vasoconstrictor effects of 5HT, and this effect is further amplified by subcontractile levels of other vasoconstrictors such as angiotensin and endothelin (Yildiz et al. 1998;Watts 2005). Furthermore, 5HT levels and turnover are increased in primary hypertension and certain types of secondary hypertension, such as pregnancy, erythropoietin, and cyclosporine-induced hypertension (Gujrati et al. 1994; Azzadin et al. 1995; Watts 2005). The actions of 5HT are mediated by different types of receptors, but are terminated by a single 5HT transporter (SERT) (Blakely et al. 1991;Hoffman et al. 1991; Lesch et al. 1993; Ramamoorthy et al. 1993b). The uptake mechanism of platelet SERT regulates plasma 5HT levels and secures stable blood flow by decreasing the possibility of platelet-activation, which produces clots in blood vessels, resulting in platelet aggregation, and subsequently, hypertension (Pettinger et al. 1973; Shulman et al. 1989; Lichtman et al. 2002; Blann et al. 2003; Stoddard et al. 2003; Pidgeon et al. 2004). The relation between the plasma 5HT level and the uptake ability of SERT has been demonstrated using SERT knock-out mice (Bengel et al. 1998), polymorphisms in the SERT-linked promoter region, and SERT inhibitors (Heils et al. 1996; Lesch et al. 1996; Serebruany et al. 2001; Johnson et al. 2003). Overall, these studies emphasize that the 5HT uptake efficiency of SERT depends on the plasma 5HT level and the number of transporters on the plasma membrane (Ramamoorthy and Blakely 1999). However, neither the contributions of these phenomena nor the order of events in the hypertensive state are known.
In the present study, we first measured 5HT levels in platelets and platelet-poor plasma (PPP). We then characterized the platelet 5HT uptake rates, the catalytic properties (Vmax), and the substrate-dependence (Km) of SERT in platelets from subjects with chronic hypertension or normotension. The platelet 5HT uptake rate during hypertension was lower due to a decrease in Vmax with no change in Km for the platelet SERT. To understand the mechanism for the reduction in 5HT uptake, the expressions of SERT on the plasma membrane and in the intracellular pools were measured. The findings indicated that the decrease in Vmax was due to a decrease in the amount of SERT molecules expressed on the platelet membrane but not in whole cell expression. Furthermore, we studied the interaction between the plasma 5HT levels and the density of SERT molecules on the platelet membrane. The effects of exogenous 5HT on the uptake rates and plasma membrane densities of SERT were measured in isolated platelets after pre-treating them with various concentrations of 5HT.
Together, our findings demonstrated that exogenous 5HT affects the uptake capacity of SERT by controlling the density of transporter molecules on the plasma membrane in a biphasic manner. Therefore, we hypothesize that in a hypertensive state, the elevated plasma 5HT levels induce a loss in the uptake function of platelets via a decrease in the density of SERT molecules on the plasma membrane. Through the feedback effect, plasma 5HT controls its own concentration levels by modulating the kinetic properties of platelet SERT.
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- Materials and methods
5HT was first isolated 55 years ago and identified as a vasoconstrictor compound in serum (Freyburger et al. 1952; Willerson 1995; Reis et al. 2000). Today the impact of 5HT on the cardiovascular system is well-established. Specifically, in hypertension and atherosclerosis (Golino et al. 1991; Yildiz et al. 1996), there is a very high sensitivity of blood vessels to the vasoconstrictive effects of 5HT, and these effects are amplified further by even subcontractile concentrations of endogenous hypertensive mediators (Watts 2005). Thus, 5HT, which markedly induces vasoconstriction, also enhances the hypertensive effects of known vasoconstrictors.
Altered 5HT plasma level is one of the multiple mechanisms associated with certain types of hypertension (Woittiez et al. 1986; Dabire et al. 1987; Egan et al. 1988; Wocial et al. 1990; Gujrati et al. 1994; Azzadin et al. 1995; Yildiz et al. 1998;Brass 1999; Reis et al. 2000; Watts 2005). Plasma 5HT plasma levels are regulated by SERT in the platelet membranes (Pettinger et al. 1973; Shulman et al. 1989; Lichtman et al. 2002; Blann et al. 2003; Doggrell 2003); Stoddard et al. 2003; Pidgeon et al. 2004; Ni et al., 2006). Therefore, the uptake efficiency of platelet SERT is one of the major factors in controlling blood pressure levels by regulating plasma 5HT concentration. In the present study, we first demonstrated that plasma 5HT levels of hypertensive subjects are increased while the amounts of 5HT in their platelets are decreased. These findings are consistent with previous reports (Jafri et al., 1992; Watts 2005). Then, we compared the biochemical characteristics of platelet SERT from hypertensive with the normotensive subjects to determine whether platelet SERT undergoes hypertension-associated alterations. We performed our studies in two model systems (i) the platelets prepared from hypertensive and normotensive subjects; and (ii) the platelets isolated from normotensive subjects and pretreated with various concentrations of 5HT. The main conclusions from our findings are that the 5HT uptake rate of SERT is lower in platelets from hypertensive subjects than those from normotensive subjects due to the decrease in Vmax and the decreased number of SERT molecules on the platelet membrane. Several independent lines of evidence support these conclusions. First, we differentiated the expression levels of transporter protein on the platelet membrane and in whole cells by biotinylation of membrane proteins. The density of SERT on the plasma membrane of platelets from hypertensive subjects was less than that of the normotensive subjects. Second, the decreased Vmax of SERT during the hypertensive state could be due to the decreased surface expression of SERT. Third, we confirmed these findings in an in vitro model system, isolated platelets pretreated with 5HT at various concentrations (Fig. 7). Our data demonstrated that exogenous 5HT at lower concentrations (0.75 nmol/L ≤ [5HT] ≤ 1.5 nmol/L) increases the uptake rates and surface density of platelet SERT by 32% and 35%, respectively. However, at higher concentrations ([5HT] ≥ 1.5 nmol/L) 5HT decreases the uptake rates and surface density of platelet SERT by 31% and 30%, respectively (Table 3). These findings nicely agreed with the data obtained from the platelets of hypertensive subjects when the plasma 5HT levels were around 1 nmol/L, the 5HT uptake rates and the surface density of platelet SERT decreases 45% and 35%, respectively. The overall data show that in serum during the formation of hypertension plasma 5HT levels are important and have more impact than the one we obtained with the in vitro system. This is a reasonably expected outcome.
Figure 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.
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Table 3. Effect of 5HT pretreatment on platelet single 5HT transporter (SERT)
|% Change of SERT in 5HT pretreated platelets compare to the untreated ones||5HT pre-treatment|
|0.75 nmol/L||1.5 nmol/L|
|5HT uptake rate||32% increase||31% decrease|
|Surface expression||35%* increase||30%** decrease|
|Intracellular expression||29% decrease||20% increase|
The regulatory roles of substrates and inhibitors on their transporters are well established (Wuller et al. 2004). Glutamate increases the surface expression of EAAT1 (Duan et al. 1999) and GABA increases GAT1 activity, through an increase in cell surface expression, which occurs within minutes (Bernstein and Quick 1999;Quick 2003). Dopamine transporter function and plasma membrane expression are regulated by dopamine, amphetamine, and cocaine (Kahlig and Galli 2003). Protein kinase C activators decrease the numbers of SERT molecules; 5HT pretreatment altered the density of SERT on the plasma membrane by interfering with SERT phosphorylation and internalization (Ramamoorthy and Blakely 1999). Cyclic adenosine monophosphate activators, however, increase SERT density in cell membranes (Ramamoorthy et al. 1993a). These studies confirm that the functional efficiency of SERT depends on the density of functionally active transporters on the plasma membrane (Ramamoorthy et al. 1993a; Ramamoorthy and Blakely 1999; Koldzic-Zivanovic et al. 2004). Therefore, we then examined whether plasma 5HT levels play a role in regulating the density of SERT molecules on the plasma membrane. Our findings indicate that at lower concentrations (≤1 nmol/L) 5HT-pre-treatment promotes the plasma membrane expression of SERT which subsequently increases the rate of 5HT uptake (Table 3). Pre-treatment with 1.5 nmol/L 5HT decreased the 5HT uptake rate and the density of SERT on the plasma membrane. On the basis of these findings, we hypothesize that during hypertension, the 5HT uptake rate of the platelet SERT system decreases due to the number of available SERT molecules on the platelet membrane; therefore, the average rate of transport per cell surface SERT molecule is decreased (Fig. 7). Our findings, together with those of others, suggest that plasma 5HT regulates its own concentration via altering the density of SERT molecules on the platelet membrane. Elucidating the molecular basis of communication between 5HT and the SERT protein will lead to the identification of the intracellular activities of platelets in hypertension.
During hypertension, plasma 5HT levels are higher than during normotension. Our current findings indicate that the 5HT uptake rates of SERT undergo hypertension-associated alterations. This is an important finding establishing that SERT on the platelet membrane can be a major regulator of plasma 5HT levels. More specifically, we demonstrated that the 5HT uptake rate of SERT decreases due to a decrease in Vmax and the amount of SERT molecules on the platelet membrane. These findings support the idea that SERT is an attractive candidate for studies of the control of hypertension via regulating the plasma 5HT levels. On the basis of these findings, without further investigation, it would be speculative to relate the plasma 5HT levels directly with the uptake capacity of platelet SERT.