Commentary

A role for the serotonin transporter in hypoxia-induced pulmonary hypertension

Barry L. Fanburg and Sheu-Ling Lee

Pulmonary and Critical Care Division, New England Medical Center, Boston, Massachusetts, USA

Address correspondence to: Barry L. Fanburg, Pulmonary and Critical Care Division, New England Medical Center, 750 Washington Street, #257, Boston, Massachusetts 02111, USA. Phone: (617) 636-5871; Fax: (617) 636-5953; E-mail: bfanburg@lifespan.org.

Serotonin (5-hydroxytryptamine; 5-HT) is one of many vasoactive substances postulated to participate in the development of hypoxia-induced pulmonary hypertension. Pulmonary vasoactive responses to hypoxia are intensified by 5-HT (1), but attempts to block hypoxia-induced pulmonary hypertension with 5-HT receptor antagonists have met with mixed success. Furthermore, it has been difficult to establish a causal relationship between 5-HT and the physiological response to hypoxia. Lacking in much of this work is a clear distinction between two classes of molecules, 5-HT transporters and 5-HT receptors, either or both of which may participate in the response of pulmonary vascular smooth muscle cells to 5-HT.

High levels of 5-HT have been associated with pulmonary hypertension in several systems. Herve et al. (2) described a patient with pulmonary hypertension who had high levels of circulating 5-HT due to a platelet storage disease, and other individuals have been described with primary pulmonary hypertension associated with elevated serum 5-HT levels (3). In the fawn-hooded rat, an animal model for platelet storage disease that exhibits high circulating levels of 5-HT, mild hypoxia also leads to pulmonary hypertension (4, 5). Finally, pulmonary hypertension is also associated with appetite suppressants, such as fenfluramine or dexfenfluramine, which block reuptake of 5-HT.

Transduction of 5-HT signals through the serotonin transporter and the 5-HT receptors

Several subtypes of signal-transducing 5-HT receptors have been characterized pharmacologically and cloned (see review in ref. 6). Depending on their subtype, these receptors may act on G-proteins and thereby activate phospholipase C or adenylate cyclase (6). By analogy with other signaling molecules, it is generally assumed that these receptors operate at the cell surface, without necessarily mediating the uptake of 5-HT. In addition, 5-HT may be internalized into a variety of cell types, including platelets, neurons, mast cells, endothelial cells, and smooth muscle cells, through an active transport mechanism that is powered by a transmembrane Na⁺/Cl^– gradient (7). Transporter genes have been cloned in rat, human, mouse, bovine, and Drosophilia (8–13). The serotonin transporter (SERT) belongs to a large family of sodium chloride–dependent γ-aminobutyric acid/norepinephrine transporters. The precise relationship between 5-HT receptors and the transporter has never been adequately defined. Some cells appear to contain receptors, others transporters, and some both.

Both bovine pulmonary artery endothelial and smooth muscle cells actively transport 5-HT (14, 15), and this transport is enhanced by exposure of cells to hypoxia (15, 16). Hypoxia also activates the mitogen-activated protein (MAP) kinase (17), thereby inducing c-fos transcription, which leads to activation of hypoxia-inducible factor-1 (HIF-1) (18). In the bovine pulmonary artery smooth muscle cell, only one 5-HT receptor has been identified, which resembles the 5-HT1A or 5-HT4 receptor (19, 20). Stimulation of this receptor in the presence of a phosphodiesterase inhibitor elevates cAMP and inhibits cellular proliferation (21). On the other hand, in the absence of phosphodiesterase inhibition, cellular cAMP does not rise, and 5-HT transport predominates over transmembrane signaling, resulting in cellular proliferation and hypertrophy at concentrations of 5-HT in the micromolar range (22). Monoamine oxidase inhibitors, which block the intracellular degradation of 5-HT, augment this proliferative response. Thus, in this case, it appears that the intracellular accumulation of 5-HT drives the proliferative process, since it is doubtful that monoamine oxidase affects 5-HT metabolism at the cell surface.

5-HT signaling through SERT also acts through a variety of other signaling molecules — tyrosine phosphorylation of GTPase-activating protein, Ras activation, activation of NAD(P)H oxidase to produce reactive oxygen species (ROSs), and activation of ERK1/ERK2 (extracellular signal-regulated kinase-1/-2) MAP kinase — to induce smooth muscle cell hyperplasia or hypertrophy (Figure 1) (23–25). These signals occur only in intact cells, not in cellular homogenates (23). Although bovine pulmonary artery endothelial cells actively transport 5-HT, they neither proliferate nor induce protein tyrosine phosphorylation in response to this stimulus (23). We previously demonstrated that Chinese hamster lung fibroblasts respond to 5-HT through both SERT and a 5-HT2 receptor, leading to both transmembrane signaling and proliferation (25). Similarly, in rat mesangial cells, 5-HT activates a 5-HT2A receptor through a Gq-protein–coupled pathway, leading to activation of phospholipase C, NAD(P)H oxidase, and ERK MAP kinase (26, 27).

Figure 1

Stimulation begins with contact between 5-HT and either a transporter or receptor, which may lead to activation of Ras or Rac or both. The signal then activates a NAD(P)H oxidase that has not been fully characterized, leading to release of reactive oxygen species (ROS) and the activation of the ERK1 or ERK2 MAP kinases. The consequent changes in gene expression culminate in both cellular hyperplasia and hypertrophy.

In Chinese ovary fibroblasts expressing an exogenous 5-HT1A receptor, 5-HT also activates NAD(P)H oxidase, ROS formation, and the ERK MAP kinase through a Gi-protein–mediated pathway (28). Humblot et al. reported that, in PC12 cells, 5-HT induces tyrosine phosphorylation and subsequently activates TIS8/egr-1 and c-fos expression (29). Furthermore, Nebigil and colleagues recently showed that the 5-HT2B receptor stimulates cyclin-dependent kinase activities by at least two mechanisms: it acts through c-Src and the receptor tyrosine kinase platelet-derived growth factor receptor (PDGFR) to stimulate cyclin D1/cdk4 activity, and it acts through c-Src alone to stimulate cyclin E/cdk2 (30). However, since SERT and 5-HT receptors may coexist in a single cell type, it remains possible that some of these effects require SERT rather than the 5-HT receptor. Nevertheless, it appears that activation of either SERT or receptor by 5-HT, depending upon cell type, can initiate a signaling process that activates cell proliferation (Figure 1).

SERT in the pulmonary response to hypoxia

These observations raise fundamental questions about the functions of 5-HT receptors and transporters: Do 5-HT receptors internalize 5-HT? Do receptors and transporters expressed in the same cell interact, and, if so, how? Resolution of these issues is complicated by the long-standing assumption in the literature that 5-HT receptors, rather than transporters, stimulate proliferation (6). The article by Eddahibi and associates in this issue of the JCI (31) follows closely on their recent work (32) showing that hypoxia in rats upregulates SERT mRNA. Now these authors find that mutant mice lacking this transporter are protected from developing pulmonary hypertension. After excluding the possibility that hypoxic pulmonary hypertension is reduced in mutant mice because of attenuation of acute pressure changes, Eddahibi et al. argue that the 5-HT transporter is required directly in the development of this condition (31). The present data are consistent with the thesis that remodeling of the pulmonary circulation, either as a result of or contributor to increased pulmonary vascular pressure, depends on the presence of SERT. They also fit with in vitro data showing that 5-HT causes vascular smooth muscle cell proliferation and that hypoxia enhances the uptake of 5-HT (15, 21, 22). 5-HT in the circulation, probably released from platelets, likely provides the substrate for the 5-HT transporter. This hypothesis can also account for dexfenfluramine-associated pulmonary hypertension, since this drug stimulates release of 5-HT from platelets and neurons, inhibits 5-HT reuptake by platelets (33), and acts through similar signaling pathways as 5-HT to stimulate smooth muscle cell proliferation directly (S.-L. Lee et al., unpublished data).

A better conceptual framework is still needed regarding the structural identities of or relationships between the 5-HT receptor and the 5-HT transporter. More complete information is needed about the precise cellular signaling pathways of 5-HT. Perhaps most importantly, the oxidase that serves as an intermediate in this pathway and the specific ROS it generates must be characterized more fully. Finally, as a corollary, it should be determined whether any of the components of the signaling pathways could be blocked to prevent or reverse pulmonary hypertension for therapeutic benefit.

Acknowledgments

Studies of the authors have been supported by a research grant from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL-32723).

References

1. Eddahibi, S, et al. Treatment with 5-HT potentiates development of pulmonary hypertension in chronically hypoxic rats. Am J Physiol 1997. 272:H1173-H1181.
   View this article via: PubMed
2. Herve, P, et al. Primary pulmonary hypertension in a patient with a familial platelet storage pool disease: role of serotonin. Am J Med 1990. 89:117-120.
   View this article via: PubMed CrossRef
3. Herve, P, et al. Increased plasma serotonin in primary pulmonary hypertension. Am J Med 1995. 99:249-254.
   View this article via: PubMed CrossRef
4. Tschopp, TB, Zucker, MB. Hereditary defect in platelet function in rats. Blood 1972. 40:217-226.
   View this article via: PubMed
5. Sato, K, et al. Factors influencing the idiopathic development of pulmonary hypertension in the fawn hooded rats. Am Rev Respir Dis 1992. 145:793-797.
   View this article via: PubMed
6. Fanburg, BL, Lee, SL. A new role for an old molecule: serotonin as a mitogen. Am J Physiol 1997. 272:L795-L806.
   View this article via: PubMed
7. Junod, AF. Uptake metabolism and efflux of ¹⁴C-5-hydroxytryptamine in isolated perfused rat lungs. J Pharmacol Exp Ther 1972. 183:341-355.
   View this article via: PubMed
8. Blakely, RD, et al. Cloning and expression of a functional serotonin transporter from rat brain. Nature 1991. 354:66-70.
   View this article via: PubMed CrossRef
9. Corey, JL, Quick, MW, Davidson, N, Lester, HA, Guastella, J. A cocaine-sensitive Drosophila serotonin transporter: cloning, expression, and electrophysiological characterization. Proc Natl Acad Sci USA 1994. 91:1188-1192.
   View this article via: PubMed CrossRef
10. Demchyshyn, LL, et al. Cloning expression and localization of a chloride-facilitated cocaine-sensitive serotonin transporter from Drosophila melanogaster. Proc Natl Acad Sci USA 1994. 91:5158-5162.
    View this article via: PubMed CrossRef
11. Hoffman, BJ, Mezey, E, Brownstein, MJ. Cloning of a serotonin transporter affected by anti-depressants. Science 1991. 254:579-580.
    View this article via: PubMed CrossRef
12. Mortensen, OV, Kristensen, AS, Rudnick, G, Wiborg, O. Molecular cloning, expression and characterization of a bovine serotonin transporter. Brain Res Mol Brain Res 1999. 71:120-126.
    View this article via: PubMed CrossRef
13. Ramamoorthy, S, et al. Antidepressant- and cocaine-sensitive human serotonin transporter: molecular cloning, expression and chromosomal localization. Proc Natl Acad Sci USA 1993. 90:2542-2546.
    View this article via: PubMed CrossRef
14. Lee, SL, Fanburg, BL. Serotonin uptake by bovine pulmonary artery endothelial cells in culture. I. Characterization. Am J Physiol 1986. 250:C761-C765.
    View this article via: PubMed
15. Lee, SL, Dunn, J, Yu, FS, Fanburg, BL. Serotonin uptake and configuration change of bovine pulmonary artery smooth muscle cells in culture. J Cell Physiol 1989. 138:145-153.
    View this article via: PubMed CrossRef
16. Lee, SL, Fanburg, BL. Serotonin uptake by bovine pulmonary artery endothelial cells in culture. II. Stimulation by hypoxia. Am J Physiol 1986. 250:C766-C770.
    View this article via: PubMed
17. Muller, JM, Krauss, B, Kaltschmidt, C, Baeuerle, PA, Ruper, RA. Hypoxia induces c-fos transcription via a mitogen-activated protein kinase-dependent pathway. J Biol Chem 1997. 272:23435-23439.
    View this article via: PubMed CrossRef
18. Minet, E, et al. ERK activation upon hypoxia: involvement in HIF-1 activation. FEBS Lett 2000. 18:53-58.
    View this article via: PubMed CrossRef
19. Lee, SL, Fanburg, BL. Serotonin produces a configurational change of cultured smooth muscle cells that is associated with elevation of intracellular cAMP. J Cell Physiol 1992. 150:396-405.
    View this article via: PubMed CrossRef
20. Becker, BN, et al. 8-hydroxy-2-(di-n-propylamino)tetralin-responsive 5-hydroxytryptamine4-like receptor expressed in bovine pulmonary artery smooth muscle cells. Mol Pharmacol 1992. 42:817-825.
    View this article via: PubMed
21. Lee, SL, Wang, WW, Moore, BJ, Fanburg, BL. Dual effect of serotonin on growth of bovine pulmonary artery smooth muscle cells in culture. Circ Res 1991. 68:1362-1368.
    View this article via: PubMed
22. Lee, SL, Wang, WW, Lanzillo, JJ, Fanburg, BL. Serotonin produces both hyperplasia and hypertrophy of bovine pulmonary artery smooth muscle cells in culture. Am J Physiol 1994. 266:L46-L52.
    View this article via: PubMed
23. Lee, SL, Wang, WW, Fanburg, BL. Association of Tyr phosphorylation of GTPase-activating protein with mitogenic action of serotonin. Am J Physiol 1997. 272:C223-C230.
    View this article via: PubMed
24. Lee, SL, Wang, WW, Fanburg, BL. Superoxide as an intermediate signal for serotonin-induced mitogenesis. Free Radic Biol Med 1998. 24:855-858.
    View this article via: PubMed CrossRef
25. Lee, SL, Wang, WW, Finlay, GA, Fanburg, BL. Serotonin stimulates mitogen-activated protein kinase activity through the formation of superoxide anion. Am J Physiol 1999. 277:L282-L291.
    View this article via: PubMed
26. Greene, EL, et al. 5-HT(2A) receptors stimulate mitogen-activated protein kinase via H₂O₂ generation in rat renal mesangial cells. Am J Physiol Renal Physiol 2000. 278:F650-F658.
    View this article via: PubMed
27. Watts, SW. Activation of the mitogen-activated protein kinase pathway via the 5-HT2A receptor. Ann NY Acad Sci 1998. 861:162-168.
    View this article via: PubMed CrossRef
28. Mukhin, YV, et al. 5-hydroxytryptamine 1A receptor/Gi betagamma stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of Src in Chinese hamster ovary fibroblasts. Biochem J 2000. 347:61-67.
    View this article via: PubMed CrossRef
29. Humblot, N, Esteve, L, Burgun, C, Aunis, D, Zwiller, J. 5-hydroxytryptamine induces TIS8/erg-1 and c-fos expression in PC12 cells. Involvement of tyrosine protein phosphorylation. Eur J Neurosci 1997. 9:84-92.
    View this article via: PubMed CrossRef
30. Nebigil, CG, Launay, JM, Hickel, P, Tournois, C, Maroteaux, L. 5-hydroxytryptamine 2B receptor regulates cell-cycle progression: cross-talk with tyrosine kinase pathways. Proc Natl Acad Sci USA 2000. 97:2591-2596.
    View this article via: PubMed CrossRef
31. Eddahibi, S, et al. Attenuated hypoxic pulmonary hypertension in mice lacking the 5-hydroxytryptamine transporter gene. J Clin Invest 2000. 105:1555-1562.
    View this article via: JCI.org PubMed CrossRef
32. Eddahibi, S, et al. Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells: relationship with the mitogenic action of serotonin. Circ Res 1999. 84:329-336.
    View this article via: PubMed
33. Buczko, W, De Gaetano, G, Garattini, S. Effect of fenfluramine on 5-hydroxytryptamine uptake and release by rat blood platelets. Br J Pharmacol 1975. 53:563-568.
    View this article via: PubMed