Increased Expression Of 5-HT Transporter In Monocrotaline-induced Pulmonary Hypertension In Rat

INTRODUCTIONPulmonary hypertension (PHT) is characterized by abnormal proliferation of vascular endothelial and smooth muscle cells, leading to occlusion of pulmonary arterioles, pulmonary hypertension, right ventricular failure, and even death. Several studies have suggested a role for serotonin (5 - HT) in both remodeling of the pulmonary circulation and in increased pulmonary vascular tone, which implicated involvement of serotonin in the etiology of PHT. Serotonin is one of the vasoactive substances in serum, which is produced mainly in the en-terochromaffin cells of the intestine and also locally released from pulmonary neuroendocrine cells and neuroepithelial bodies distributed throughout the airway. Large amounts of serotonin from these cells in response to airway hypoxia directly contribute to secondary pulmonary arterial hypertension. Serotonin in the micromolar range is a known mitogen for smooth muscle cells (SMCs) isolated from bovine, porcine, and rat aorta as well as rat and bovine pulmonary arteries. In the systemic circulation, the 5 - HT_2A receptor mediates arterial vaso-constriction to 5 - HT. However, in PHT rats, 5 - HT - induced pulmonary vas-oconstriction is mediated by the 5 - HT_1B receptor. Several studies have concluded that 5 - hydroxytryptamine transporter (5 - HTT) plays a key role in the mitogenic effect of 5 - HT on hypoxic pulmonary artery smooth muscle cells (PASMCs). More recently, Eddahibi et al. reported that PASMCs from patients with primary pulmonary hypertension grow faster than the cells from control subjects due to increased expression of 5 - HTT. In the model of " inflammatory" PHT, monocrotaline (MCT) given to rats results in cardiopulmonary pathophysi-ologic sequelae similar to the forms of chronic pulmonary hypertension in man. The toxicology of MCT is complex. It causes lung injury, pulmonary hypertension , and right heart enlargement. High levels of plasma 5 - HT and remodelingof pulmonary arteries have been observed in MCT - induced PHT rats, which indicates an underlying cause may exist between the two phenomena. Increasing recognition of the importance of remodeling to the pathogenesis of PHT suggests new therapeutic possibilities. So, it may be of importance to clarify the role of 5 -HTT in remodeling of pulmonary arteries (PAs) in MCT rats. The present study, therefore, was designed to compare the expression of 5 - HTT gene in PAs from normal and chronic " inflammatory" PHT rats.MATERIALS AND METHODSMonocrotaline model of chronic "inflammatory" PHT in rats In this study, 24 Wistar rats from Laboratory Animal Center, China Medical University , were randomly divided into two groups. The test rats were treated with a single , intraperitoneal injection of monocrotaline 60 mg/kg, while the control rats were treated with an equal volume of its vehicle, then these rats were fed solid food and water ad lib in an alternating 12 h light/dark cycle under controlled temperature (18℃ -22℃ ) and humidity (50 % -70 %) for 3 weeks.In vivo studies The rats were anesthetized by intraperitoneal injection of 10 % chloral hydrate (4 ml/kg) on d 21 after MCT or its vehicle administration (8 and 12 rats respectively) and placed on a heating pad to maintain body temperature at 37℃ to 38℃ throughout the study. A polyethylene catheter (PE - 50) was inserted into the right carotid artery to measure heart rate and arterial pressure. A PV - 1 catheter was inserted through the right jugular vein into the pulmonary artery for measurement of pulmonary arterial pressure. These hemody-namic variables were measured with a pressure transducer connected to a polygraph and recorded with a thermal recorder. Then, vein and artery blood was got. PaO₂ and PaCO₂ were measured.After completion of the above measurements, the right ventricle (RV) , septum and the left ventricle ( S + LV) were dissected and the ratio of RV weight to S + LV weight was calculated as index of the right ventricular hypertrophy.In vitro studies The rat main pulmonary arteries were dissected from con-nective tissues and cut into cylindrical segments (2 - 3 mm in width). Then the PA rings were threaded onto two stainless steel hooks and mounted on isometric wire - myographs. Vessels were bathed in 10 ml Krebs - buffer solution (composition in mmol/L: NaCl 119; KCl 4. 7; MgSO₄ 0. 6; KH₂PO₄ 1. 2; CaCl₂ 2.5; NaHCO₃ 25 and Glucose 11.1, pH 7.4) bubbled constantly with 95 % O₂ - 5 % CO₂ gas mixture at 37℃ and connected to a force - displacement transducter under optimal resting tension (1.0 g) and were equilibrated for 1 h before the beginning of each experiment, and rinsed with buffer every 15 min.Following equilibration, the response to KCl with a final concentration of 50 mmol/L to get the maximum contraction was determined, which was then used through the remainder of experiments and provide a reference contraction against which subsequent responses could be normalized. The contractile responses to 5- HT were expressed as percentage of the contractile response to 50 mmol/L KCl. The cumulative concentration — contractile response curves of pulmonary artery rings to 5 - HT were established. The maximum contractile response (E_max) and the potency of the 5 - HT (pEC₅₀) were calculated.Morphometric analysis of pulmonary arteries Paraffin sections with 5 μm thick each were obtained from rats PAs and stained with hematoxylin and eo-sin (HE stain) for examination by light microscopy. Statistical analysis of the medial wall thickness of the pulmonary arteries was performed. The external diameter and the medial wall thickness were measured in 20 muscular arteries (ranging in size from 70 to 100 m in external diameter). For each artery, 8 values of medial wall thickness were obtained by every 45 angle, and then the mean value was got (5 sections per PA).RT - PCR Pulmonary arteries (endothelium - denuded) were dissected free from peripheral tissues and immediately stored at - 70℃. Total RNA was extracted with TRIzol Reagent as describe in its directions. The concentration and purification of the total RNA was estimated by optical density measurement at 260 nm with ultraviolet spectrophotometer.RT - PCR to identify mRNA expression of 5 - HT transporter in rat PAs was performed with RNA PCR Kit (AMV) Ver. 2. 1. 5 - HT transporter and β- actin primers were synthesized as follows: 5 - HT transporter a: 5 ' - GGATCC CTG CTC ACA CTG-3' (nucleotides 1541 -1558) , b: 5' - TTA CAC AGC ATT CAT GCG - 3' ( nucleotides 2008 - 1991) ; (3 - actin a: 5 ' - ATC ATG TTT GAG ACC TTC AAC A -3' , b: 5' - CAT CTC TTG GTC GAA GTC CA-3'.Total RNA (1 μg /sample) was reverse - transcribed in the volume of 20 μL and amplified in the volume of 100 μL using 5 U AMV Reverse Tran-scriptase, 10 mmol/L dNTP and 2.5 U TaKaRa Taq DNA polymerase. The reaction conditions were as follows: reverse transcription of RNAs at 42℃ for 30 min, then inactive AMV at 99℃ for 5 min. 35 cycle amplifications were performed at 94℃ for 1 min, 56℃ for 30 s, 72℃ for 1min. PCR products from the 5 - HTT mRNA and the (3 - actin mRNA contained 484 and 308 base pairs, respectively. They were electrophoresed in 2 % agarose gel and stained with ethidium bromide (5 mg/L). Then, the gel was scanned and semi - quantified using a gel analyzer. The gene expression of 5 — HTT was represented by the relative yield to the β - actin gene.Statistical methods All data in this text and illustrations are expressed as mean ± S. An unpaired Student's t test was used to detect significant differences when 2 groups were compared. A value of P < 0.05 was considered statistically significant. Correlation between thickness of pulmonary vascular medial walls and 5 - HTT mRNA expression of pulmonary arteries was accessed by linear regression analysis.RESULTSMCT - induced pulmonary hypertension in rats Chronic PHT model in rats induced by MCT was successfully established at end of 3 weeks and confirmed by a significant increase of mean pulmonary arterial pressure (16. 7 ±3. 3 mmHg, n = 8 vs control 10.7 ±1.7 mmHg, n = 12, P < 0.01) and right ventricular hypertrophy index (0.46 ±0.07, n = 8 vs control 0.32 ±0.03, n = 12, P < 0.01). PaO₂ in artery blood was decreased(70.7 ±5.7, n = 8 vs control 84. 6±5.7,n = 12, P <0.05). The thickness of pulmonary vascular medial walls was increased in MCT rats ( 17. 3 ±2. 6 μm vs 3. 9 ±0. 5 μm, P< 0.01).Effect of MCT on concentration - contractile response to 5 - HT Themaximum contraction to 5 - HT was enhanced in MCT rat vs control, with an E_maxvalue increased from 68.2 % ±16.9 % ( response to 50 mmol/L KCl, n = 12) to 105.9 % ±15.2 % (n = 8, P < 0.01) and the pEC₅₀ value were 3. 53 ±0.28 for control, and 3.42 ±0.31 for MCT rats.Effect of MCT on 5 - HTT mRNA expression of pulmonary arteries RT - PCR was performed to detect mRNA of 5 - HTT in PAs from rats using specific primers. β - actin gene, used as a control, was detected in all preparations. PCR products obtained were consistent with the estimated bases. 5 - HTT mRNA levels measured by RT - PCR in PAs from control and from MCT rats were significantly difference. The ratio of the PCR products of 5 - HTT gene to those of β - actin gene was much higher in MCT rats than in control rats (1. 73 ±0.32, n = 8 vs control 1.32 ±0.39, n = 12, P < 0.05). 5-HTT mRNA expression of pulmonary arteries correlated with the thickness of pulmonary vascular medial walls (r = 0.741, P < 0.01).DISCUSSIONPulmonary hypertension is a common complication of cardiopulmonary diseases , for which the underlying cause is not quite clear. Yet several vasoactive factors, such as 5 - HT, endothelin - 1 ( ET - 1) , thromboxane A₂ (TXA₂) , platelet -derived growth factor (PDGF) , epidermal growth factor (EGF) , an-giotensin â…¡ (Ag â…¡ ) etc, were certain to be involved in pulmonary hypertension. More and more data demonstrated that 5 - HT is one of the most important factors in PHT, for which either directly, or via a synergism with other vasoactive factors to cause pulmonary arterial vasoconstraction, pulmonary vascular SMC proliferation, and local microthrombosis in patients and animal models. Further more, 5 - HT has been shown to exert mitogenic and comitogenic effects on PASMCs. Several studies have shown that pulmonary arterial responses to 5 - HT is mediated by 5 - HT_1B receptor in the human isolated small muscular pulmonary arteries and in MCT rat pulmonary arteries. The mechanism by which5 - HT causes vascular smooth muscular cell proliferation varies with species and cell types.Several studies have concluded that the mitogenic action of 5 - HT was resulted from an energy - dependent transport of 5 - HT into the cells. 5 - HT transporter in pulmonary vascular SMCs has many attributes, suggesting that it may be a key determinant in this process. In addition to contributing to the uptake and subsequent inactivation of 5 - HT passing through the lung, 5 - HTT mediates the proliferation of pulmonary vascular SMCs through its ability to internalize indoleamine. Lee and Eddahibi observed that bovine and rat PASMC proliferation induced by 5 - HT was dose - dependently inhibited by highly selective inhibitors of 5 - HT transporter such as paroxetine and fluoxetine. The level of 5 - HTT expression appeared to be much greater in human lungs than in the brain, suggesting that altered 5 - HTT expression may have direct consequences on PASMCs function. Moreover, it was recently observed that mice with targeted 5 - HTT gene disruption developed mild PHT by hypoxic challenge than wild -type controls.In this study, monocrotaline - induced pulmonary hypertension in the rat was successfully established. It was found that MCT - induced pulmonary vascular remodeling was accompanied with an increase in contractile response of pulmonary artery rings to 5 - HT. Further more, 5 - HTT mRNA expression in the PAs was increased and the levels of 5 — HTT mRNA expression was correlated with the thickness of pulmonary vascular medial walls. Taken these evidences from hypoxia - and MCT - induced pulmonary hypertensive animal models together , we considered that 5 - HTT plays a key role in the pathophysiological processes of pulmonary hypertension and this may provide a potential therapeutic target for this disease.In summary, the pulmonary vascular remodeling and increased vascular contractile responsiveness to 5 - HT were accompanied with the increased expression of 5 - HTT, and there existed correlation between the wall thickness of pulmonary arteries and 5 - HTT mRNA expression in MCT - induced pulmonary hypertensive rats. Therefore, we concluded that 5 - HTT plays an important role in pulmonary hypertension.