![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CARDIOVASCULAR
Services de Biochimie et d'Hématologie Biologique, Hôpital Lariboisière, Assistance Publique-Hopitaux de Paris, Paris, France (J.C., K.P., C.T., L.D., J.M.L.); Institut Federatif de Recherche 139, Paris, France (J.C., K.P., C.T., L.D., J.M.L.); Institut de Génétique et Biologie Moléculaire et Cellulaire, Illkirch, France (J.M.E., L.M.); Institut National de la Santé et de la Recherche Médicale U536, Illkirch, France (J.M.E., L.M.); Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7104, Illkirch, France (J.M.E., L.M.); University of Strasbourg, Illkirch, France (J.M.E., L.M.); Laboratoire de Chirurgie Expérimentale Unité Propre de Recherche et de l'Enseignement Supérieur, Equipe d'Accueil, Paris South University, Hôpital Marie Lannelongue, Le Plessis-Robinson, France (P.H.); Institut National de la Santé et de la Recherche Médicale U616, Paris, France (L.M.); Hopital Pitié-Salpetrière, Paris, France (L.M.); and University Pierre et Marie Curie, Paris, France (L.M.)
Received November 7, 2005; accepted February 2, 2006.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). Several studies have suggested a role for serotonin [5-hydroxytryptamine (5-HT)] in pulmonary hypertension (for review, see Egermayer et al., 1999). A correlation between high plasma serotonin levels and total pulmonary resistance, first observed in a patient with a defect in platelet serotonin storage capacity (Hervé et al., 1990), was confirmed in more than 80% of pulmonary hypertensive patients (Hosoda, 1994; Hervé et al., 1995; Kéreveur et al., 2000). In rats, roles for serotonin in the initiation and progression of monocrotaline-induced pulmonary hypertension have been suggested since plasma serotonin levels are raised, and pulmonary arteries show hyper-reactivity to serotonin. In these experiments, treatments with 5-HT2R antagonists inhibited the development of pulmonary hypertension along with a decrease in the plasma serotonin concentrations and the number of proliferative cells, substantiating the pivotal role of plasma serotonin in the development of monocrotaline- or interleukin-6-induced pulmonary hypertension (Miyata et al., 2001).
The main peripheral sources of serotonin are as a neurotransmitter and local hormone in the gastrointestinal tract enterochromaffin cells and pulmonary neuroepithelial bodies. Blood serotonin, which is concentrated in the platelet-dense granules (>99%), is almost absent from plasma (nanomolar range) (Da Prada and Picotti, 1979). Defective platelet uptake (Awabdy et al., 2003) or release from activated platelets could generate increased plasma serotonin as in platelet storage diseases (Hervé et al., 1990). Furthermore, lungs have been reported to remove efficiently "free" serotonin from plasma (Gillis and Pitt, 1982). High levels of plasma serotonin could, thus, result from an impaired endothelial metabolism that would decrease 5-hydroxyindole-acetic acid (5-HIAA) levels and/or serotonin uptake. As well as being a pulmonary vasoconstrictor, plasma serotonin may exert a comitogenic influence on pulmonary vascular smooth muscle cells (Liu and Fanburg, 2005) and contribute to both hypoxia-induced acute vasoconstriction and chronic vascular remodeling through various serotonin receptors (5-HTRs) and the serotonin transporter (SERT).
When exposed to a few weeks of hypoxia (10% O2), wild-type mice develop pulmonary hypertension and lung vascular remodeling recapitulating at least in part the human pathology. Smooth muscle and endothelial cells from human and rodent pulmonary arteries express mRNAs for 5-HT1B, 5-HT2A, 5-HT7, and 5-HT2BRs and SERT (Ullmer et al., 1995). Mice deficient for 5-HT1BRs or for SERT are still responsive to chronic hypoxia though the increase in pulmonary artery blood pressure and pulmonary vascular remodeling are reduced when compared with control mice. Using the chronic hypoxic mouse model of pulmonary hypertension, we previously reported that the hypoxia-dependent increases in pulmonary blood pressure and lung remodeling are totally abolished in mice with genetically or pharmacologically inactive 5-HT2BRs (Launay et al., 2002). An interference of the 5-HT2B-/- mice congenital cardiomyopathy to the lung hypoxic response has been ruled out since the cardiac phenotype of 5-HT2B-/- mice is characterized by left ventricular dysfunctions, but no apparent right ventricular alterations; the basal pulmonary pressure in 5-HT2B-/- mice is indistinguishable from +/+ mice; and in +/+ mice exposed to chronic hypoxia in presence of RS-127445, one of the most selective 5-HT2BR antagonists, all tested lung parameters are not significantly different from untreated values, clearly demonstrating that the activation of 5-HT2BRs is required for hypoxia-induced pathology independently of any prior cardiac phenotype. In both humans and mice, pulmonary hypertension is associated with a substantial increase in 5-HT2BR, 5-HT1BR, but not 5-HT2AR expression in pulmonary arteries (Launay et al., 2002). Recently, a heterozygous mutation causing premature truncation of the 5-HT2BR protein was found in one patient with fenfluramine-associated pulmonary hypertension (Blanpain et al., 2003) that clearly increases proliferative index of this mutant receptor (Deraet et al., 2005). However, the link between 5-HT2BRs and pulmonary hypertension pathophysiology remains to be discovered.
Since a correlation between plasma serotonin levels and total pulmonary resistance was consistently observed in pulmonary hypertensive patients, we asked whether serotonin receptors could participate in the control of plasma serotonin levels. In the present study, we are reporting that 5-HT2BRs are involved in a carrier-dependent control of plasma serotonin levels in mice.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Hypoxia. The 5-HT2BR mutant mice (-/-) were generated in the 129PAS pure genetic background that was used as controls (+/+) (Nebigil et al., 2000). Mice 6 weeks of age (20-25 g) were randomly divided into groups of 50% male and 50% female mice, maintained either in room air (21% O2) or in normobaric chambers (21-23°C) with hypoxic air (10% O2, 90% N2, 2 or 5 weeks; Launay et al., 2002). Normoxic mice were kept in the similar room with the same 12-/12-h light/dark cycle. Classically, groups of 10 mice were treated with vehicle, a highly selective 5-HT2BR (full neutral) antagonist RS-127445 at 1 mg/kg/day or dexfenfluramine at the "therapeutic" dose of 2.5 mg/kg/day delivered by miniosmotic pumps (ALZET; DURECT Corporation, Cupertino, CA) from the beginning of the hypoxic treatments. All animal care and procedures were in accordance with institutional guidelines and European regulations.
Cardiovascular Evaluations. Mice were anesthetized with i.p. injection of ketamine hydrochloride (60 mg/kg) and xylazine (8 mg/kg). Right ventricular systolic pressure (RVSP) was measured by insertion into the right ventricle of a 26-gauge needle connected to a pressure transducer. After a blood sample was collected by cardiac puncture for measurements, the pulmonary artery was cannulated through an incision in the right ventricle and perfused with Earle's balanced salt solution (37°C, 20 cm of H2O pressure).
Lung Parameters. Lung vascular bed around pulmonary arteries was collected and prepared for culture (Launay et al., 2002). The culture was maintained in Dulbecco's modified Eagle's medium supplemented with serotonin-depleted serum (10%) for 24 h, washed with Hanks' balanced salt solution, and grown for 24 h in serum-free medium [Dulbecco's modified Eagle's medium/F-12 (1:1) with 5 µg/ml insulin, 5 µg/ml transferrin, 30 nM selenium, 20 nM progesterone, and 100 µM putrescine] before serotonin uptake was performed.
To measure serotonin uptake, after incubation for 1 min with 25, 50, and 100 nM [3H]serotonin binoxalate, cells were lysed by addition of 0.1 N NaOH and radioactivity was counted using liquid scintillation spectrometry. Specific serotonin uptake was assessed as the difference between total uptake and uptake in the presence of 1 µM paroxetine. Due to a lack of materials, only three concentrations of [3H]serotonin were used, leading only to an estimate of the initial rates (1 min uptake) of specific serotonin uptake versus serotonin concentration. After a nonlinear regression analysis according to a hyperbolic model (EZ-FIT program; Department of Medical Products, du Pont de Nemours, Glenolden, PA), we determined the Michaelis-Menten constant (Km) and initial rate (Vi) of serotonin uptake into lung vascular cells.
Dosage of the 5-HT2BR expression was performed by binding experiments using a selective tritiated radioligand (LY-266097). Briefly, cell membranes were prepared by four cycles of homogenization (Brinkmann P10 disrupter; Brinkmann Instruments, Westbury, NY) and centrifugation (48,000g for 15 min). The assay was established to achieve steady-state conditions and to optimize specific binding (Kellermann et al., 1996). Fifty micrograms of membrane proteins was incubated with 1 nM [3H]LY-266097 at 4°C for 60 min. Nonspecific binding was determined using 1 µM RS-127445. Assays were terminated by vacuum filtration through glass fiber filters (GF/B), which had been pretreated with 0.1% polyethylenimine. Total and bound radioactivity was determined by liquid scintillation counting. Greater than 80% specific binding was achieved in these assays.
For elastolytic activities, conditioned media (380 µl) were incubated at 37°C with 200 µg [3H]elastin produced by radiolabeling of purified insoluble elastin from bovine nuchal ligament (Elastin Products Company, Inc., Owensville, MO) with [3H]NaBH4 as previously described (Takahashi et al., 1973). After 24 h, the radioactivity in 250 µl of the supernatant was determined by liquid scintillation spectrometry as a measure of elastolysis. To control for nonenzymatic degradation, radiolabeled elastin was incubated with medium from tissue-free cultures. Elastase activity was related to a standard curve generated with human leukocyte elastase (0.075 to 5.0 ng) (Elastin Products Company, Inc.).
Blood Parameters. Plasma 3,4-dihydroxyphenylglycol (DHPG) and 5-HIAA levels were measured by high-performance liquid chromatography, and serotonin in both plasma and whole blood was measured by radioenzymology (Berlin et al., 1995; Kéreveur et al., 2000).
Acute Injections. Acute i.p. injections of the preferential 5-HT2BR agonist BW-723C86 (10 mg/kg), selective 5-HT2BR antagonist RS-127445 (1 mg/kg), dexfenfluramine (10 mg/kg), or nordexfenfluramine (10 mg/kg) were performed on 5-HT2BR+/+ and -/- mice before blood was collected by cardiac puncture for measurements over a 30-min time period. Pretreatment with paroxetine was performed by i.p. injection twice a day of the selective SERT blocker paroxetine (1 mg/kg) over 2 days, reducing platelet serotonin uptake by more than 90%.
Statistics. The reported data represent the mean of individual values ± S.E.M. (n = number of individuals at the end of treatments as indicated in the text). Comparisons were performed using the nonparametric Kruskal-Wallis test. Significance was set at P < 0.0001. This level of significance also applies to correlation between individual values, assessed by Kendall rank coefficients. For the group comparisons, statistical comparisons were made by one-way analysis of variance. Difference between groups was established using the Bonferroni-Dunn multiple comparison tests (P < 0.0001).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). A progressive increase in RVSP was detected in +/+ mice treated from 2 to 5 weeks under hypoxia (10% O2) (Fig. 1A). We tested serotonin levels in blood samples of the hypoxia-treated mice after acute or 2 or 5 weeks of hypoxia. Strikingly, as the expression of 5-HT2BRs in lungs (Fig. 1B), plasma serotonin levels (Fig. 1C) were significantly increased in +/+ mice after exposure to 2 weeks of chronic hypoxia, before significant changes in remodeling factors could be evidenced (e.g., elastase activity: Fig. 1D) but not in hypoxic 5-HT2BR-/- mice. No change in whole-blood serotonin could be evidenced.
|
|
The Hypoxia-Dependent Increase in Plasma Serotonin Levels Is Correlated with RVSP and 5-HT2BR Expression. When comparing the plasma serotonin levels and the others parameters affected by hypoxia, we observed significant (P < 0.0001) correlations between all the individual plasma serotonin values and RVSP values (Fig. 3A), lung 5-HT2BR expression site numbers (Fig. 3B), or plasma 5-HIAA values (Fig. 3C). Moreover, the ratios of serotonin/5-HIAA were also significantly correlated with plasma DHPG values, confirming that these ratios reflect monoamine oxidase A (MAOA) activity (Fig. 3E). However, the correlation was not significant between individual plasma and whole-blood serotonin levels (Fig. 3D). These results further substantiate the evidence that 5-HT2BRs are required for the regulation of plasma serotonin levels, independently of platelets, which store most of the whole-blood serotonin.
|
|
Direct in Vivo Activation of 5-HT2BRs Results in an Increase of Plasma Serotonin Levels. To test whether normoxic mice respond to activation of 5-HT2BRs, we acutely injected 5-HT2BR agonists and assessed the serotonin plasma levels. Over a 30-min time period, plasma levels were extremely stable after vehicle injection in mice. A significant increase in plasma serotonin levels over this control value was observed at 10 min after i.p. injection of the 5-HT2BR preferential agonist BW-723C86 (10 mg/kg) (76 ± 12% over basal, n = 6) in +/+ mice (Fig. 4A). In contrast, in 5-HT2BR-/- mice (Fig. 4B) or in 5-HT2BR antagonist, RS-127445-treated (1 mg/kg, n = 4) +/+ mice (Fig. 4C), this increase could not be detected. Interestingly, acute i.p. injection of nordexfenfluramine (10 mg/kg, n = 6) fully mimicked the BW-723C86-dependent increase in plasma serotonin levels in timing (peak at 10 min) and magnitude (82 ± 17% over basal) in +/+ mice, with again no effect on 5-HT2BR inactive mice. However, acute i.p. dexfenfluramine injection (10 mg/kg, n = 6) produced a faster (5 min) and sharper increase in plasma serotonin, which was observed in both +/+ and 5-HT2BR inactive mice. The dexfenfluramine effect was clearly distinct from that of nordexfenfluramine since the nordexfenfluramine as the BW-723C86 effects on peripheral serotonin release required 5-HT2BRs. No variation in whole-blood serotonin could be detected in this time window in either genotype after similar injections (not illustrated). Activation of 5-HT2BRs can thus increase plasma serotonin levels.
|
The in Vivo 5-HT2BR-Dependent Increase in Plasma Serotonin Levels Is SERT-Dependent. We tested the effect of SERT inhibition using a pretreatment by the highly selective SERT blocker paroxetine or SERT-/- on dexfenfluramine, BW-723C86, or nordexfenfluramine in vivo acute i.p. injection. Strikingly, no change in plasma serotonin levels could be detected after acute injection of nordexfenfluramine or BW-723C86 when the mice were pretreated by paroxetine (1 mg/kg, n = 6) twice a day for 2 days in +/+, 5-HT2BR-/-, and SERT-/- mice (not illustrated). In contrast, a lower but significant increase in plasma serotonin levels over the control value was still observed at 5 min after dexfenfluramine injection in +/+ mice (62 ± 4% of plasma level without paroxetine) (Fig. 5A) and in 5-HT2BR-/- mice after paroxetine treatment (64 ± 8% of plasma level without paroxetine) (Fig. 5B). No variation in total blood serotonin could be detected in this time window in either genotype after similar injections (not illustrated). The BW-723C86- and nordexfenfluramine-dependent increases in plasma serotonin levels appear entirely 5-HT2BR- and SERT-dependent, whereas that of dexfenfluramine appears 5-HT2BR-independent and only partially SERT-dependent.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
An interference of the 5-HT2B-/- mice congenital defects has been ruled out since treatment of +/+ mice by RS-127445, the most selective 5-HT2BR antagonist, reproduces the results obtained with 5-HT2B-/- mice. The serotonergic anorectic agent and amphetamine derivative, dexfenfluramine, increases the risk of developing pulmonary hypertension (Rich et al., 2000). In vitro, dexfenfluramine and its main metabolite nordexfenfluramine have been shown to inhibit serotonin reuptake and to stimulate its release from brain synaptosomes (Mennini et al., 1981). Moreover, both compounds bind to 5-HT2Rs with appreciable affinity. In fact, dexfenfluramine binds weakly to rodent and human 5-HT2A,B,CRs, but nordexfenfluramine behaves as a high-affinity agonist for 5-HT2B and 5-HT2CRs and more moderately for 5-HT2ARs (Porter et al., 1999; Fitzgerald et al., 2000; Rothman and Baumann, 2002; Setola et al., 2003; Supplementary table). In mice, chronic exposure to dexfenfluramine 2.5 mg/kg/day leads to complete conversion into nordexfenfluramine with a final plasma concentration of about 500 nM (Caccia et al., 1985; Launay et al., 2002), fully stimulating 5-HT2BRs but not 5-HT2ARs (Supplementary table), 5-HT2CRs being below detection level in the periphery. Chronic effects of dexfenfluramine therefore appeared nordexfenfluramine-mediated.
The mechanisms leading to hypoxia-induced plasma serotonin elevation might be a consequence of platelet defects or of a slower serotonin uptake by platelets due to a kinetic change in SERT activity (Awabdy et al., 2003). We did not observe any significant variation in whole-blood serotonin content (>99% of which is represented by platelets) in hypoxic mice (Fig. 2B), thus eliminating major changes in serotonin synthesis, platelet number, or their serotonin uptake capacity. Furthermore, the absence of correlation between individual values of plasma and blood serotonin (Fig. 3D) indicates that mechanisms controlling serotonin platelet uptake and plasma levels are not coordinately regulated by hypoxia. Moreover, mature platelets express 5-HT2ARs but not 5-HT2BRs, and ketanserin, a selective 5-HT2AR antagonist, does not significantly change pulmonary artery pressure in hypoxic mice (Marcos et al., 2003). Recently, platelets serotonin uptake inhibition by both dexfenfluramine and nordexfenfluramine (IC50, dexfenfluramine approximately 3 µM; nordexfenfluramine approximately 10 µM) has been reported, but no serotonin efflux occurs at these concentrations (Johnson et al., 2003). Together with our chronic in vivo results, these findings indicate that regulation of platelets serotonin content and that of plasma serotonin are under different molecular controls in hypoxic mice.
A change in metabolism might also explain the increase in plasma serotonin levels in hypoxic pulmonary hypertension. Lung endothelial cells control serotonin clearance by catabolism into 5-HIAA by MAOA, and lung is known as the main site of serotonin removal from plasma (Gillis and Pitt, 1982). Low oxygen levels are expected to directly decrease MAO activity and thus serotonin metabolism. The nonsignificant reduction in serotonin degradation observed in hypoxic 5-HT2B-/- or +/+ mice (Fig. 3, C-E) indicates that 5-HT2BRs have no major contribution to the MAOA activity under hypoxia, and the increase in plasma serotonin cannot be explained solely by changes in MAOA activity. In conclusion, a generalized deficit of the serotonin metabolism could not explain the increase in plasma serotonin encountered during hypoxic pulmonary hypertension with no changes in whole-blood serotonin.
Since the lung endothelium is the major site of carrier-mediated peripheral serotonin fast removal (Gillis and Pitt, 1982), reduced SERT uptake activity in lungs might also raise plasma serotonin levels. Recent evidence obtained by positron emission tomography scan of SERT ligands in healthy volunteers confirmed that lung is the main fast high-uptake organs in the body (Suhara et al., 1998; Takano et al., 2002). Our result showing a reduced rate of lung serotonin uptake upon chronic hypoxia, which is not observed when the 5-HT2BR are inactivated (Table 1), agrees with previous reports in lung of hypoxic rats and mice (Jeffery et al., 2000). In perfused rat lung, SERT can mediate an efflux of serotonin (James and Bryan-Lluka, 1997), and the serotonin efflux caused by SERT substrates (such as amphetamine derivatives) is carrier-mediated (Hilber et al., 2005). Together, the reduced lung serotonin uptake and the increased plasma serotonin levels in hypoxic mice suggest that hypoxia-dependent 5-HT2BR activation can trigger the increase in plasma serotonin, probably via a negative regulation of SERT activity. Whether this regulation is restricted to lungs remains to be determined.
Another site known to express 5-HT2BRs is the gut, which is the main site of peripheral serotonin synthesis by enterochromaffin cells. The recent observation that, in perfused rat ileum, dexfenfluramine (EC50 around 100 µM) or nordexfenfluramine (EC50 around 10 µM) can increase serotonin levels in the venous effluent strongly supports their ability to increase its release from the small intestine (Rezaie-Majd et al., 2004). Given the plasma concentrations of dexfenfluramine and nordexfenfluramine in submicromolar range (Caccia et al., 1985; Launay et al., 2002) and the nanomolar affinity of nordexfenfluramine for 5-HT2Rs (Supplementary table), a direct action at 5-HT2Rs to the regulation of serotonin release from intestine seems unlikely but cannot be totally excluded.
In a different experimental in vivo setup, our data show that acute injection of the 5-HT2BR preferential agonist BW-723C86, as nordexfenfluramine (another preferential 5-HT2B/2CR agonist), significantly increases plasma serotonin levels with identical kinetic and 5-HT2BR dependence, whereas dexfenfluramine acts with a faster kinetic and generates a serotonin increase that is independent of 5-HT2BRs (Fig. 4). Evidence that the mechanism by which nordexfenfluramine induces serotonin efflux is different from that underlying dexfenfluramine-induced release had been documented previously; work in synaptosomes showed that nordexfenfluramine acts, at least in part, on the release of a serotonin cytoplasmic pool (Mennini et al., 1981), whereas dexfenfluramine can induce a Ca2+-dependent serotonin release from a vesicular pool (Gobbi et al., 1998); independent work using microdialysis showed also that dexfenfluramine, but not nordexfenfluramine, uses calcium to increase extracellular serotonin (Puig de Parada et al., 1995).
Although evaluated in the periphery, our work substantiates a dual mode of action for these compounds in modulating plasma serotonin levels. Our observations that acute injection of dexfenfluramine triggers serotonin releasing effects that are rapid, mainly paroxetine-insensitive and independent of 5-HT2BRs, support a rapid mechanism of serotonin release by dexfenfluramine. The modulation by BW-723C86 or nordexfenfluramine of serotonin plasma levels is blocked by pharmacological (paroxetine at 1 mg/kg, Fig. 5) or genetic (mutant mice, not illustrated) SERT inhibition. In humans, similar paroxetine treatment leads to plasma concentration less than 500 nM (Lindsay De Vane, 1999). Thus, assuming a similar bioavailability in mice, it is unlikely that this concentration could affect 5-HT2A, 5-HT2C, or 5-HT2BRs (Supplementary table). The elimination of both BW-723C86- and nordexfenfluramine-induced plasma serotonin increases in 5-HT2BR-/- mice, in 5-HT2BR antagonist-treated +/+ mice, by selective SERT blocker and in SERT-/- mice, strongly supports the hypothesis that serotonin release is controlled by 5-HT2BRs via a regulation of SERT uptake activity. Reports showing that both dexfenfluramine and nordexfenfluramine at micromolar per liter concentrations generated no efflux of serotonin from platelets (Johnson et al., 2003) and that in ileum, both compounds increased serotonin levels but at concentrations over 10 µM (Rezaie-Majd et al., 2004) support the implication of other organs such as the lungs in this serotonin release.
A 5-HT2BR-dependent SERT phosphorylation (Launay et al., 1998) in vascular endothelium could be an essential component of the 5-HT2BR-dependent regulation of plasma serotonin independently of platelets. However, the molecular pathway leading to the control of SERT activity by 5-HT2BRs remains to be molecularly detailed. Our data support the hypothesis that the 5-HT2BR is an essential partner in signaling pathways regulating the plasma serotonin levels via a control of SERT activity. The previous observations that, in response to hypoxia, increases in RVSP and in lung vascular remodeling are reduced in mice deficient for SERT, together with our finding that paroxetine blocks 5-HT2BR efflux effects, may explain the paradoxical reports showing that some SERT inhibitors have putative beneficial effects in pulmonary hypertension (Marcos et al., 2003), whereas injection of serotonin potentiates the development of pulmonary hypertension in rats exposed to chronic hypoxia (Eddahibi et al., 1997). Nevertheless, our work demonstrates that 5-HT2BR can control plasma serotonin levels in mice and further suggests that such a control may participate in other types of pulmonary hypertension in other organisms.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindole-acetic acid; 5-HTR, serotonin receptor; SERT, serotonin transporter; RS-127445, 2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine; LY-266097, 1-(2-chloro-3,4-dimethoxybenzyl)-6-methyl-1,2,3,4-tetrahydro-9Hpyrido[3,4-b]indole hydrochloride; BW-723C86, 1-[5-(2-thienylmethoxy)-1H-3-indolyl]propan-2-amine HCl; RVSP, right ventricular systolic pressure; DHPG, 3,4-dihydroxyphenylglycol; MAOA, monoamine oxidase A.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
1 Current affiliation: Laboratoire de Biochimie, Unité de Formation et de Recherche de Pharmacie, Reims, France.
Address correspondence to: Dr. Luc Maroteaux, Institut National de la Santé et de la Recherche Médicale, U616, Hopital Pitié-Salpetrière, Université Pierre et Marie Curie Paris, Bat Pédiatrie, 47 Boulevard de l'Hopital, 75013 Paris, France. E-mail: luc.maroteaux{at}chups.jussieu.fr
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Awabdy D, Bryan-Lluka LJ, and Wanstall JC (2003) 5-Hydroxytryptamine and platelets: uptake and aggregation in hypoxic pulmonary hypertensive rats. Eur J Pharmacol 459: 1-7.[CrossRef][Medline]
Berlin I, Said S, Spreux-Varoquaux O, Launay JM, Olivares R, Millet V, Lecrubier Y, and Puech AJ (1995) A reversible monoamine oxidase A inhibitor (moclobemide) facilitates smoking cessation and abstinence in heavy, dependent smokers. Clin Pharmacol Ther 58: 444-452.[CrossRef][Medline]
Blanpain C, Le Poul E, Parma J, Knoop C, Detheux M, Parmentier M, Vassart G, and Abramowicz MJ (2003) Serotonin 5-HT2B receptor loss of function mutation in a patient with fenfluramine-associated primary pulmonary hypertension. Cardiovasc Res 60: 518-528.
Caccia S, Conforti I, Duchier J, and Garattini S (1985) Pharmacokinetics of fenfluramine and norfenfluramine in volunteers given D- and DL-fenfluramine for 15 days. Eur J Clin Pharmacol 29: 221-224.[CrossRef][Medline]
Da Prada M and Picotti GB (1979) Content and subcellular localization of catecholamines and 5-hydroxytryptamine in human and animal blood platelets: monoamine distribution between platelets and plasma. Br J Pharmacol 65: 653-662.[Medline]
Deraet M, Manivet P, Janoshazi A, Callebert J, Guenther S, Drouet L, Launay JM, and Maroteaux L (2005) The natural mutation encoding a C terminus-truncated 5-hydroxytryptamine 2B receptor is a gain of proliferative functions. Mol Pharmacol 67: 983-991.
Eddahibi S, Raffestin B, Pham I, Launay JM, Aegerter P, Sitbon M, and Adnot S (1997) Treatment with 5-HT potentiates development of pulmonary hypertension in chronically hypoxic rats. Am J Physiol 272: H1173-H1181.[Medline]
Egermayer P, Town GI, and Peacock AJ (1999) Role of serotonin in the pathogenesis of acute and chronic pulmonary hypertension. Thorax 54: 161-168.
Fitzgerald LW, Burn TC, Brown BS, Patterson JP, Corjay MH, Valentine PA, Sun J-H, Link JR, Abbaszade I, Hollis JM, et al. (2000) Possible role of valvular serotonin 5-HT2B receptors in the cardiopathy associated with fenfluramine. Mol Pharmacol 57: 75-81.
Gillis CN and Pitt BR (1982) The fate of circulating amines within the pulmonary circulation. Annu Rev Physiol 44: 269-281.[CrossRef][Medline]
Gobbi M, Parazzoli A, and Mennini T (1998) In vitro studies on the mechanism by which (+)-norfenfluramine induces serotonin and dopamine release from the vesicular storage pool. Naunyn-Schmiedeberg's Arch Pharmacol 358: 323-327.[CrossRef][Medline]
Hervé P, Drouet L, Dosquet C, Launay JM, Rain B, Simonneau G, Caen J, and Duroux P (1990) Primary pulmonary hypertension in a patient with a familial platelet storage pool disease: role of serotonin. Am J Med 89: 117-120.[CrossRef][Medline]
Hervé P, Launay JM, Scrobohaci ML, Brenot F, Simonneau G, Petitpretz P, Poubeau P, Cerrina J, Duroux P, and Drouet L (1995) Increased plasma serotonin in primary pulmonary hypertension. Am J Med 99: 249-254.[CrossRef][Medline]
Hilber B, Scholze P, Dorostkar MM, Sandtner W, Holy M, Boehm S, Singer EA, and Sitte HH (2005) Serotonin-transporter mediated efflux: a pharmacological analysis of amphetamines and non-amphetamines. Neuropharmacology 49: 811-819.[CrossRef][Medline]
Hosoda Y (1994) Pathology of pulmonary hypertension: a human and experimental study. Pathol Int 44: 241-267.[Medline]
James KM and Bryan-Lluka LJ (1997) Efflux studies allow further characterisation of the noradrenaline and 5-hydroxytryptamine transporters in rat lungs. Naunyn-Schmiedeberg's Arch Pharmacol 356: 126-133.[CrossRef][Medline]
Jeffery TK, Bryan-Lluka LJ, and Wanstall JC (2000) Specific uptake of 5-hydroxytryptamine is reduced in lungs from hypoxic pulmonary hypertensive rats. Eur J Pharmacol 396: 137-140.[CrossRef][Medline]
Johnson GJ, Leis LA, Dunlop PC, and Weir EK (2003) The effect of the anorectic agent, D-fenfluramine and its primary metabolite, D-norfenfluramine, on intact human platelet serotonin uptake and efflux. J Thromb Haemost 1: 2663-2668.[CrossRef][Medline]
Kellermann O, Loric S, Maroteaux L, and Launay J-M (1996) Sequential onset of three 5-HT receptors during the 5-hydroxytryptaminergic differentiation of the murine 1C11 cell line. Br J Pharmacol 118: 1161-1170.[Medline]
Kéreveur A, Callebert J, Humbert M, Hervé P, Simonneau G, Launay JM, and Drouet L (2000) High plasma serotonin levels in primary pulmonary hypertension: effect of long-term epoprostenol (Prostacyclin) therapy. Arterioscler Thromb Vasc Biol 20: 2233-2239.
Launay JM, Hervé P, Peoc'h K, Tournois C, Callebert J, Nebigil C, Etienne N, Drouet L, Humbert M, Simonneau G, et al. (2002) Function of the serotonin 5-hydroxytryptamine 2B receptor in pulmonary hypertension. Nat Med 8: 1129-1135.[CrossRef][Medline]
Launay JM, Loric S, Mutel V, and Kellermann O (1998) The 5-HT2B receptor controls the overall 5-HT transport system in the 1C11 serotonergic cell line. Ann NY Acad Sci 861: 247.
Lindsay De Vane C (1999) Metabolism and pharmacokinetics of selective serotonin reuptake inhibitors. Cell Mol Neurobiol 19: 443-466.[CrossRef][Medline]
Liu Y and Fanburg BL (2006) Serotonin-induced growth of pulmonary artery smooth muscle requires activation of PI3K/Akt/S6K1. Am J Respir Cell Mol Biol 34: 182-191
Loscalzo J (2001) Genetic clues to the cause of primary pulmonary hypertension. N Engl J Med 345: 367-371.
Marcos E, Adnot S, Pham MH, Nosjean A, Raffestin B, Hamon M, and Eddahibi S (2003) Serotonin transporter inhibitors protect against hypoxic pulmonary hypertension. Am J Respir Crit Care Med 168: 487-493.
Mennini T, Borroni E, Samanin R, and Garattini S (1981) Evidence of the existence of two different intraneuronal pools from which pharmacological agents can release serotonin. Neurochem Int 3: 289-294.[CrossRef]
Miyata M, Ito M, Sasajima T, Ohira H, and Kasukawa R (2001) Effect of a serotonin receptor antagonist on interleukin-6-induced pulmonary hypertension in rats. Chest 119: 554-561.[CrossRef][Medline]
Nebigil CG, Choi D-S, Dierich A, Hickel P, Le Meur M, Messaddeq N, Launay J-M, and Maroteaux L (2000) Serotonin 2B receptor is required for heart development. Proc Natl Acad Sci USA 97: 9508-9513.
Porter RH, Benwell KR, Lamb H, Malcolm CS, Allen NH, Revell DF, Adams DR, and Sheardown MJ (1999) Functional characterization of agonists at recombinant human 5-HT2A, 5-HT2B and 5-HT2C receptors in CHO-K1 cells. Br J Pharmacol 128: 13-20.[CrossRef][Medline]
Puig de Parada M, Parada MA, Pothos E, and Hoebel BG (1995) D-Fenfluramine, but not D-norfenfluramine, uses calcium to increase extracellular serotonin. Life Sci 56: L415-L420.
Rezaie-Majd S, Murar J, Nelson DP, Kelly RF, Hong Z, Lang IM, Varghese A, and Weir EK (2004) Increased release of serotonin from rat ileum due to dexfenfluramine. Am J Physiol 287: R1209-R1213.[CrossRef]
Rich S, Rubin L, Walker AM, Schneeweiss S, and Abenhaim L (2000) Anorexigens and pulmonary hypertension in the United States: results from the surveillance of North American pulmonary hypertension. Chest 117: 870-874.[CrossRef][Medline]
Rothman RB and Baumann MH (2002) Therapeutic and adverse actions of serotonin transporter substrates. Pharmacol Ther 95: 73-88.[CrossRef][Medline]
Setola V, Hufeisen SJ, Grande-Allen KJ, Vesely I, Glennon RA, Blough B, Rothman RB, and Roth BL (2003) 3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") induces fenfluramine-like proliferative actions on human cardiac valvular interstitial cells in vitro. Mol Pharmacol 63: 1223-1229.
Suhara T, Sudo Y, Yoshida K, Okubo Y, Fukuda H, Obata T, Yoshikawa K, Suzuki K, and Sasaki Y (1998) Lung as reservoir for antidepressants in pharmacokinetic drug interactions. Lancet 351: 332-335.[CrossRef][Medline]
Takahashi S, Seifter S, and Yang FC (1973) A new radioactive assay for enzymes with elastolytic activity using reduced tritiated elastin: the effect of sodium dodecyl sulfate on elastolysis. Biochim Biophys Acta 327: 138-145.[Medline]
Takano A, Suhara T, Sudo Y, Inoue M, Hashimoto K, Zhang MR, Ichimiya T, Yasuno F, and Suzuki K (2002) Comparative evaluation of two serotonin transporter ligands in the human brain: [11C](+)McN5652 and [11C]cyanoimipramine. Eur J Nucl Med Mol Imaging 29: 1289-1297.[CrossRef][Medline]
Ullmer C, Schmuck K, Kalkman HO, and Lübbert H (1995) Expression of serotonin receptor mRNA in blood vessels. FEBS Lett 370: 215-221.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  C. Fligny, Y. Fromes, P. Bonnin, M. Darmon, E. Bayard, J.-M. Launay, F. Cote, J. Mallet, and G. Vodjdani Maternal serotonin influences cardiac function in adult offspring FASEB J, July 1, 2008; 22(7): 2340 - 2349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Doly, E. Valjent, V. Setola, J. Callebert, D. Herve, J.-M. Launay, and L. Maroteaux Serotonin 5-HT2B Receptors Are Required for 3,4-Methylenedioxymethamphetamine-Induced Hyperlocomotion and 5-HT Release In Vivo and In Vitro J. Neurosci., March 12, 2008; 28(11): 2933 - 2940. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Collet, C. Schiltz, V. Geoffroy, L. Maroteaux, J.-M. Launay, and M.-C. de Vernejoul The serotonin 5-HT2B receptor controls bone mass via osteoblast recruitment and proliferation FASEB J, February 1, 2008; 22(2): 418 - 427. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Papageorgiou and C. Denef Stimulation of Growth Hormone Release by 5-Hydroxytryptamine (5-HT) in Cultured Rat Anterior Pituitary Cell Aggregates: Evidence for Mediation by 5-HT2B, 5-HT7, 5-HT1B, and Ketanserin-Sensitive Receptors Endocrinology, September 1, 2007; 148(9): 4509 - 4522. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Li, Y. Liu, P. Dutt, B. L. Fanburg, and D. Toksoz Inhibition of serotonin-induced mitogenesis, migration, and ERK MAPK nuclear translocation in vascular smooth muscle cells by atorvastatin Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293(2): L463 - L471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Papageorgiou and C. Denef Estradiol Induces Expression of 5-Hydroxytryptamine (5-HT) 4, 5-HT5, and 5-HT6 Receptor Messenger Ribonucleic Acid in Rat Anterior Pituitary Cell Aggregates and Allows Prolactin Release via the 5-HT4 Receptor Endocrinology, March 1, 2007; 148(3): 1384 - 1395. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Morecroft, Y. Dempsie, M. Bader, D. J. Walther, K. Kotnik, L. Loughlin, M. Nilsen, and M. R. MacLean Effect of Tryptophan Hydroxylase 1 Deficiency on the Development of Hypoxia-Induced Pulmonary Hypertension Hypertension, January 1, 2007; 49(1): 232 - 236. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Ni, K. Lookingland, and S. W. Watts Arterial 5-Hydroxytryptamine Transporter Function Is Impaired in Deoxycorticosterone Acetate and N{omega}-Nitro-L-Arginine But Not Spontaneously Hypertensive Rats Hypertension, July 1, 2006; 48(1): 134 - 140. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
, any statistical difference from chronic hypoxia values (P < 0.0001).
