Introduction

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Primary pulmonary hypertension (PPH) is an uncommon disease of unknown cause (1). Familial studies suggest a genetic susceptibility (2). There is also an inherited platelet storage-pool disease in humans in which the uptake and storage of serotonin (5-hydroxytryptamine [5-HT]) and other biogenic amines by the platelets is reduced, which is associated with pulmonary hypertension (3). A role for 5-HT is suspected in PPH, as its circulating plasma levels are high in these patients (4). Also, use of anorectic drugs, such as dexfenfluramine, has been associated with an increased risk of PPH (5, 6). These agents impair platelet storage of 5-HT and elevate plasma levels. Such anorectics also downregulate the expression of the 5-HT transporter (5-HTT) (7).

The fawn-hooded rat (FHR) is an animal model used to study systemic hypertension and chronic renal failure (8, 9). It has an inherited platelet storage disorder, not seen in other strains such as Wistar, with both reduced 5-HT uptake and storage (10, 11). Uniquely, the FHR develops pulmonary hypertension when it is exposed to mild hypoxia (12).

A number of cells store and take up 5-HT, including neurons, endothelial cells, and platelets. The principal route of uptake is the 5-HTT, encoded by a single gene in humans and murine species (13, 14). Storage is a separate function, release from which is effected by an exocytotic process involving potassium (K⁺) and calcium (Ca²⁺) channels (15). This can be triggered by lithium (18). Dexfenfluramine both inhibits the 5-HTT (19) and increases exocytotic release of 5-HT (15). Physiologically, this anorectic mimics the platelet defect of the FHR. It was therefore considered important to study details of the 5-HTT in the FHR as a strategy to learn more of the mechanisms leading to the development of pulmonary hypertension.

	Materials and Methods

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Animals

Male Wistar rats (University of Sheffield) and male FHRs were studied at 9-10 wk of age. The FHRs were obtained from an inbred colony from Dr. Baurier Margrain (Center d'elevage, Las Jeunes St. Isle, Paris, France). All animals were maintained on standard rat chow diet and allowed free access to food and water; regular 12-h diurnal cycles of light were maintained. Rats were killed by administration of pentobarbitone anesthetic (Sagatal, 60 mg/kg) followed by exsanguination. Ten milliliters of citrated blood were obtained from each rat. Samples of the lung (cardiac lobe) were dissected and rapidly frozen in liquid nitrogen until assay.

Preparation of Platelet RNA

Platelets were pooled from three animals in each experiment. They were separated by centrifugation at 210 × g for 10 min at room temperature. The platelet-rich plasma was recentrifuged at 2,500 × g for a further 10 min. The pellet was resuspended in 1 ml of guanidine thiocyanate-phenol-chloroform (RNAzol B; Biogenesis Ltd., Bournemouth, UK), and total RNA was isolated. After phenol/chloroform extraction and isopropanol precipitation, the RNA was washed in 70% ethanol and quantified at 260 nm using an ATI Unicam Spectrometer (Cambridge, UK). Purity was assessed by the ratio of absorption at 260/280 nm and the integrity was confirmed by the appearance of clear bands for 18S and 28S after electrophoresis through 1% agarose gels containing 2.2 M formaldehyde (20).

Platelet Function

Serotonin uptake and release by the platelets was measured in both Wistar rats and FHRs using a method modified from Steinberg and Das (21), which measures the uptake of 5-hydroxy [side chain-2-¹⁴C] tryptamine creatinine sulfate at 37°C into a suspension of 3 × 10¹¹ platelets/liter. ¹⁴C uptake was measured using an LKB/Pharmacia Scintillation Counter (Cambridge, UK).

Reverse Transcription and Polymerase Chain Reaction Amplification

All reagents used were obtained from GIBCO, BRL (Paisley, UK) unless otherwise stated. Reverse transcription (RT) of 1 µg of total RNA from Wistar and FHR platelets or lung tissue was used for cDNA synthesis. Tissue from at least six animals was used. cDNA synthesis was performed using 300 U avian myeloblastosis virus reverse transcriptase, 1 mM dNTPs, 25 ng/µl random hexamer primers or oligodT (0.5 µg/µg RNA), 1 U/µl of ribonuclease inhibitor, and reaction buffer for 1 h at 42°C and 5 min at 99°C. Samples of these reactions (5 µl) were used in polymerase chain reaction (PCR) amplification using overlapping oligonucleotide primers specific for the 5-HTT gene (Figure 1).

View larger version (21K): [in this window] [in a new window]   Figure 1.   Specific primer pairs for the coding region of the 5-HTT used for PCR amplification of overlapping segments of the gene. cDNA was synthesized from RNA isolated from platelets and lung. The open reading frame for the protein coding region is indicated as a bar.

Negative controls contained all components except total RNA. All PCR reagents were obtained from Promega (Southampton, UK). PCR amplification was performed at 95°C for 5 min, 35 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min, and a final extension of 72°C for 10 min. Each reaction contained 2.5 U Taq DNA polymerase, 0.4 µM specific primers, 2 mM MgCl₂, 200 µM dNTPs, and 1× PCR buffer (as supplied) in a final volume of 50 µl. Five microliters of the RT-PCR products were analyzed by 1% agarose gel electrophoresis and visualized under ultraviolet light in the presence of ethidium bromide. The remainder of the reactions were purified on Qiaquick columns (Qiagen Inc., Crawley, UK) according to manufacturer's protocol for sequencing. Pools of triplicate reactions were used to counter the effect of any misincorporation events early in the amplification.

Sequence Analysis

PCR products were sequenced by automated dideoxy chain termination. Both DNA strands of each PCR product were sequenced using an Applied Biosystems Model 373A automated sequencer (Warrington, UK) in all cases.

	Results

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We amplified the serotonin transporter cDNA (5-HTT) using three sets of specific primers for the coding region in two strains of ratthe Wistar and the FHR (Rattus norvegicus). We submitted the cDNA sequence for the FHR to the EMBL database with accession number Y11024. We found significant reduction in the platelet uptake of 5-HT in the FHR compared with the Wistar rat, and greater release (Table 1). No difference in the predicted primary structure of the 5-HTT from platelet or lung was observed between the Wistar and the FHR strains (Figure 2, top).

View larger version (81K): [in this window] [in a new window]   View larger version (9K): [in this window] [in a new window]   Figure 2.   (Top panel ) Amino acid sequence alignment reported here for the FH rat and the Wistar (W/ FHR; Rattus norvegicus), cell lines derived from R. norvegicus (Hoffman; Reference 24), R. rattus (Blakely; Reference 23), and Homo sapiens (Hs sert; Reference 22). EMBL accession numbers are W/FHR, Y11024; Hoffman, M79450; Blakely, X63253; and Hs sert, X70697. (Bottom panel ) The frameshift from two nucleotide inserts (see text) results in a truncated primary structure of 630 aa, differing from Hoffman, but with 100% homology to that of the brown rat (R. rattus; Reference 23) and 92% homology to that of Homo sapiens (22).

The cDNA sequence predicts a 630-amino acid protein of 70.1 kD with 92% homology with that of humans and 100% with that of the brown rat (Rattus rattus) (22, 23). Our results show some differences in the cDNA sequence by Hoffman (24) (Figure 2, top) obtained from rat basophilic leukemia cell lines (RBL 2H3), and from R. norvegicus, to which our nucleotide identifications correspond (EMBL accession no. M79450). However, we found single base changes at position 1451 (C instead of G) and at position 2049 (C instead of G). The former would result in an amino acid changeAla 415 rather than Glywhereas the latter does not predict an amino acid change (CGG to CGC, both Arg). Furthermore, we found an additional two nucleotides at position 1828 (CC) and a further insert of one nucleotide at position 1835. The resultant frame shift in this region (Figure 2, bottom) predicts an amino acid sequence identical to that of the common brown rat (R. rattus) and more closely resembles that of humans (Homo sapiens) (22, 23).

	Discussion

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We have confirmed a 5-HT platelet storage-pool defect of the FHR (11, 12) by measurement of platelet uptake of [¹⁴C]5-HT. Platelet uptake of 5-HT was reduced and leakage increased in the FHR compared with the Wistar rat strain. Our data suggest that this is not associated with an alteration in the primary structure of the serotonin transporter (5-HTT). We found no difference in the predicted amino acid sequence of the 5-HTT, studied in both the lung and the platelets, compared with the Wistar rat.

The gene for the 5-HTT has been localized to chromosome 17q11.1-q12 in humans and has been mapped to a locus close to D17S58 and D17S73 by family linkage analysis (25). The homologous mouse gene has been located to chromosome 11 (14). The complete organization of the gene in humans and the cDNA sequence in the rat has been published (13, 23). It is expressed in platelets, placenta, brain, epithelium of the gastrointestinal tract, and endothelial cells of the lung (26).

The FHR was initially bred for behavioral disturbances linked with a disturbed 5-HT metabolism, notably a depressive disorder and alcohol abuse (29, 30). In humans, depression and unipolar disorder are associated with a variable number of tandem repeats in intron 2 of the 5-HTT gene (31). This does not alter the primary structure of the transporter, but it could alter expression. A similar effect could account for the functional defect in the FHR. Expression studies for the transporter in the FH and Wistar rats, using semiquantitative RT-PCR (32) and Northern blot hybridization, are presently underway in our laboratory.

The distinctive coloring and coat pattern of the FHR has been shown in segregational analysis to be linked to the gene for the red-eye dilution (r) found on chromosome 1 (8). The same also appears to be true for the platelet storage-pool defect, which appears part of a generalized impairment of biogenic amine storage defect, not confined to the platelets but also seen in the endothelium (33). The FHR is of special interest as three further genes, linked with diseases, have also been localized to chromosome 1 and associated with (r). Two are independently associated with the development of renal impairment, and one, Bpfh-1, is responsible for 26% of the genetic variation of systemic blood pressure (8). This strain of rat is also susceptible to the development of pulmonary hypertension (PH) when exposed to mild hypoxia (10, 34), which is associated with characteristic pulmonary artery remodeling and increased expression of the peptide mitogen/vasoconstrictor endothelin-1 (35).

The association between reduced 5-HT storage and the susceptibility to PH in the FHR parallels some forms of human PPH. The two forms of PPH, sporadic and familial, are similar in their natural history and pathology. A gene for familial PPH has been recently located to chromosome 2 (2), but this form of the disease only accounts for 6% of PPH cases. We have chosen a rat model, the FHR, because its reduced 5-HT uptake and increased leakage in the platelets mimics the platelet storage deficiency observed in some patients suffering from PPH (3). FHR also has an inherited susceptibility to the development of PH with mild hypoxia (12) and therefore offers a model for secondary PH.

A link between PPH and disturbed 5-HT metabolism (3, 4) and the risk of PPH by use of a 5-HTT inhibitor, dexfenfluramine (6), highlight an important role of 5-HT in its development. Our observation of a normal primary structure of the 5-HTT in the FHR emphasizes the potential importance of the storage mechanisms for 5-HT. For example, a 90-kD glycoprotein in the platelets of the FHR differs from the "normal" glycoprotein (36). This is thought to be integral in the membrane of the dense storage granules of the platelets.

In summary, the reduced uptake of serotonin in our strain of the FHR is not associated with an alteration of the primary structure of the serotonin transporter. Other mechanisms for this defect should be sought.