Contribution of the 5-HT1B Receptor to Hypoxia-Induced Pulmonary Hypertension
Converging Evidence Using 5-HT1B-Receptor Knockout Mice and the 5-HT1B/1D-Receptor Antagonist GR127935
5-Hydroxytryptamine (5-HT)1B receptors mediate contraction in human pulmonary arteries, and 5-HT1B receptor-mediated contraction is enhanced in pulmonary arteries from hypoxic rats. Here we further examine the role of this receptor in the development of pulmonary hypertension (PHT) by examining (1) the effects of a 5-HT1B/1D-receptor antagonist (GR127935) on hypoxia-induced PHT (CHPHT) in rats and (2) CHPHT in 5-HT1B-receptor knockout mice. In rats, hypoxia increased right ventricular pressure and right ventricular hypertrophy and induced pulmonary vascular remodeling associated with an increase in pulmonary arterial wall thickness. GR127935 (3 mg · kg−1 · d−1) reduced all of these indices. 5-HT1-mediated contraction was enhanced in pulmonary arteries of the CHPHT rats. The effects of GR127935 on PHT indices were associated with an attenuation of the enhanced contractile responses to 5-HT and the 5-HT1-receptor agonist, 5-carboxamidotryptamine (5-CT), in isolated pulmonary arteries. In wild-type mice, hypoxia increased right ventricular hypertrophy, which was absent in 5-HT1B-receptor knockout mice. Hypoxia increased pulmonary vascular remodeling in wild-type mice, and this was reduced in the 5-HT1B-receptor knockout mice. Hypoxia increased 5-HT1-mediated contraction in pulmonary arteries from the wild-type mice and this was attenuated in the 5-HT1B-receptor knockout mice. In conclusion, the 5-HT1B receptor plays a role in the development of CHPHT. One possible mechanism may be via enhanced 5-HT1 receptor-mediated contraction of the pulmonary arterial circulation.
Acute hypoxia causes pulmonary arteriolar vasoconstriction and increased pulmonary arterial pressure. Chronic hypoxia induces a sustained increase in pulmonary arterial pressure and pulmonary vascular smooth muscle cell proliferation.1
5-HT is a potent pulmonary vasoconstrictor and co-mitogen and studies suggest a role for 5-HT in both remodeling of the pulmonary circulation and in increased pulmonary vascular tone, associated with exposure to hypoxia.2 The chronic hypoxic pulmonary hypertensive (CHPHT) rat shows a marked right ventricular hypertrophy, an index of pulmonary hypertension (PHT), on exposure to hypoxia.3 Treatment with 5-HT potentiates the development of PHT in this model,4 and enhanced responses to 5-HT have been demonstrated at all levels of the pulmonary arterial circulation in the CHPHT rat.5 Plasma and platelet levels of 5-HT are also increased in primary PHT6 and pulmonary arteries isolated from primary PHT patients demonstrate enhanced contractile responses to 5-HT.7 Recently, a link has been found between PHT and the use of anorectic agents that can promote the release, and inhibit the re-uptake, of 5-HT.8
In the systemic circulation, the 5-HT2A receptor mediates arterial vasoconstriction to 5-HT.9 In the human pulmonary circulation, the 5-HT1B/1D receptor mediates vasoconstriction of large and small pulmonary arteries.10,11 In nonhypoxic rats, pulmonary vasoconstriction to 5-HT is mediated predominantly by the 5-HT2A receptor. However, in CHPHT rats, 5-HT-induced vasoconstriction is mediated by both the 5-HT2A and 5-HT1B receptor.5 This increased 5-HT1B receptor activity may be due to pharmacological synergism between 5-HT1B receptor-mediated Gi-protein coupling and increased Gq-protein activation.12 In addition, levels of mRNA for the 5-HT1B receptor are increased in pulmonary arteries from the CHPHT rat.13
Taken together, both rat and human studies suggest that 5-HT1B receptors play a role in the pathobiology of PHT. To further test this hypothesis, we have adopted two approaches, examining (1) the effect of treatment with GR127935 (selective 5-HT1D/1B-receptor antagonist14) on the development of PHT secondary to hypoxic exposure in rats and (2) the development of hypoxia-induced PHT in a 5-HT1B-receptor knockout mouse. We also studied the sensitivity of isolated pulmonary arteries, to 5-HT and the 5-HT1-receptor agonist 5-carboxamidotryptamine (5-CT), comparing responses in rat larger elastic pulmonary arteries with those in the small muscular pulmonary arteries, as we have reported regional receptor heterogeneity in the rat pulmonary arterial circulation, both for 5-HT-induced responses5 and endothelin (ET)-1-induced responses.15
Materials and Methods
The investigation conforms with the United Kingdom Animal Procedures Act, 1986 and with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).
Male Wistar rats (33 days old; Harlan UK) or male mice (strain 129.SV; 90 days old) were placed in a hypobaric chamber. This was depressurized over the course of 2 days to 550 mbar (equivalent to 10% O2). Temperature was maintained at 21 to 22°C and the chamber was ventilated with air at approximately 45 L · min−1. The duration of hypoxia was 14 days. Rat groups were treated orally with GR127935 ([N-[4-methoxy-3(4-methyl-1-piperazinyl)phenyl]2′methyl-4′(5-methyl-1,2,4-oxadiazol-3-yl)1,1′biphenyl4-carboxamide] 3 mg · kg−1 · d−1 in distilled H2O) or H2O alone, starting 2 days before hypoxic exposure. Aged-matched control groups were maintained in room air and received GR127935 treatment or H2O alone. Mice were either wild-type (5-HT1B+/+; Harlan UK) or 5-HT1B knockout (5-HT1B−/−; Columbia University) and appropriate aged-matched control groups were also employed.
Assessment of PHT
Measurement of Right VentricularPressure and Right Ventricular Hypertrophy
Right ventricular pressure was measured in rats as previously described for rabbits.16 Right ventricular hypertrophy was assessed by measuring the right ventricular free wall (RV) and left ventricle together with the septum (LV+S) separately. Total ventricular weight (TV) was calculated as RV+(LV+S) and the ratio RV/TV calculated.
One sagittal section was obtained from the left lungs sample populations. Sections were stained with Elastica-Van Gieson (EVG) stain and microscopically assessed in a blinded fashion. Pulmonary arteries (25 to 100 μm external diameter) associated with an airway distal to the respiratory bronchiole were counted. The arteries were considered muscularized if they possessed a distinct double-elastic lamina visible for at least half the diameter in the vessel cross-section. The percentage of vessels containing double-elastic lamina was calculated as number of muscularized vessels/total number of vessels counted×100. Vessels (46±3) were counted per mouse lung section (n=5 lungs studied per group), and 72±6 vessels were counted per rat lung section (n=5 lungs studied per group). Fully muscularized vessels with an external diameter <80 μm were analyzed using computer-assisted imaging. The vessel diameters were calculated and the percent wall thickness calculated as external elastic lamina diameter minus the internal lamina diameter divided by the diameter of the external elastic lamina. Three vessels were analyzed per lung section and the results meaned (n=5 lungs studied per group).
In Vitro Studies
Animals were euthanized by intraperitoneal injection of sodium pentobarbitone.
Rat Extralobar Pulmonary Arteries
The rat first branch pulmonary artery (2 to 3 mm ID) was dissected out and set up in 5 mL organ baths under optimal resting tension (1.5 g) and vessels set up under conditions previously described.5,15
Small Muscular Pulmonary Arteries
Two 2-mm long segments of pulmonary arteries (≈200 to 250 μm ID) were threaded onto 40 μm stainless steel wire and mounted on isometric wire-myographs (J-P Trading). Vessels were bathed in Krebs-buffer solution with a constant supply of 16% O2/5% CO2 (balance N2), as previously described.5,15 Tension was applied to give transmural pressures equivalent of ≈12 to 14 mm Hg for controls and 30 to 33 mm Hg for hypoxic animals. These pressures are similar to those experienced by the vessels in vivo in rats.17
In Vitro Experimental Protocols
Following equilibration, the response to 50 mmol/L KCl was determined, the concentration which produced maximal contraction. Endothelial-dependent vasodilation was assessed by preconstricting vessels with 1 μmol/L phenylephrine and relaxing vessels with 1 μmol/L acetylcholine. Cumulative concentration-response curves (CCRCs) to 5-HT or 5-CT were constructed. To examine whether residual 5-HT1 receptor-mediated response in the 5-HT1B knockout mice was mediated by the 5-HT1D receptor, in 5-HT1B−/− mice vessels, the CCRCs to 5-CT were repeated in the presence of the 5-HT1D-receptor antagonist BRL1557218 (500 nmol/L, 1 hour preincubation).
Responses to 5-HT or 5-CT were expressed as a percentage of the initial response to KCl to calculate the maximum contraction (Emax). pEC50 values were calculated from individual CCRCs by graphical interpolation (Graphpad Prism). Statistical comparisons were made by one-way analysis of variance. When significance was attained (P<0.05), differences were established using the Newman-Keuls multiple comparison test. Data are expressed as mean±SEM.
CHPHT Rat Study
Effect of GR127935 Pretreatment on Right Ventricular Pressure
Hypoxia resulted in a ≈50% elevation in RVP in vehicle-treated rats. In GR127935-treated CHPHT rats, the elevation in RVP with hypoxia was reduced to ≈41% (Figure 1A). Mean systemic arterial pressure did not differ between groups, the values being 85.6±1.6 mm Hg (n=6), 87.1±1.3 mm Hg (n=5), 88.2±0.8 mm Hg (n=6), and 86.8±4.4 mm Hg (n=5) for vehicle-treated, GR127935-treated, vehicle-treated CHPHT, and GR127935-treated CHPHT rats, respectively. Similarly, heart rate showed no significant difference between groups, being 396.6±14.6 bpm for the vehicle-treated controls, 401.6±13.4 bpm for GR127935-treated controls, 370.0±6.9 bpm for vehicle-treated CHPHT rats, and 407.6±17.4 bpm for GR127935-treated CHPHT rats.
Effect of GR127935 Pretreatment on RV/TV Ratio
Hypoxia caused a ≈59% increase in the RV/TV weight ratio in vehicle-treated CHPHT rats compared with vehicle-treated controls (Figure 1B). GR127935 had no effect on the RV/TV weight ratio in control rats whereas the development of right ventricular hypertrophy was partly offset by treating CHPHT rats with GR127935, such that the RV/TV weight ratio was increased by ≈40% (Figure 1B).
Effect of GR127935 Pretreatment on Pulmonary Vascular Remodeling
There was a 10-fold increase in the percentage of vessels showing double elastic lamina in vehicle-treated CHPHT in comparison to vehicle-treated controls (Figure 1C). GR127935 did not alter the percent of remodeled vessels in control rats. In CHPHT rats, GR127935, however, significantly decreased the percentage of remodeled vessels. In fully muscularized small vessels, the percent wall thickness was markedly increased in both CHPHT rat groups (Table 1, Figure 2). Percent wall thickness was not affected by GR127935 in control rats but GR127935 significantly reduced the percent wall thickness in CHPHT rats (Table 1). There was no other change in lung morphology after drug treatment.
Responses to KCl and Acetylcholine-Induced Vasodilation in First Branch Pulmonary Arteries
GR127935 had no effect on acetylcholine-induced vasodilation. In control vehicle-treated rats this was 54±5% (n=8) of phenylephrine-induced preconstriction; in control GR127935-treated rats this was 55±5% (n=8). In vehicle-treated hypoxic rats the percent relaxation was 43±4% (n=15), and in the hypoxic GR127935-treated rats it was 45±3% (n=15). The degree of preconstriction with 1 μmol/L phenylephrine was not significantly different between groups being (percent of contraction to 50 mmol/L KCl in each vessel): 139±9% (control, vehicle-treated), 138±8% (control, GR127935-treated), 135±10% (hypoxic, vehicle-treated), and 119±5% (hypoxic, GR127935-treated). Likewise the response to 50 mmol/L KCl was equal, being (in mN) 5.0±0.6, 4.7±0.6, 5.0±0.9, and 5.8±0.8, respectively. This also demonstrates that GR127935 had no effect on responses to 1 μmol/L phenylephrine or 50 mmol/L KCl.
Responses to 5-HT in First Branch Pulmonary Arteries
The maximum contraction to 5-HT was enhanced by ≈100% in vehicle-treated rats with CHPHT (Figure 3A). The Emax value increased from 78.8±11.3% (response to 50 mmol/L KCl) to 157.6±12.2% (n=6, P<0.05). Treatment with GR127935 did not alter the Emax value for 5-HT in control rats. In sharp contrast, pretreatment of CHPHT rats with GR127935 attenuated the Emax to 5-HT (93.3±15.6% (n=7); P<0.05 versus vehicle-treated CHPHT rats). Neither hypoxia nor GR127935 affected sensitivity to 5-HT as shown by pEC50 values, which were 5.74±0.29 (n=6), 5.45±0.37 (n=7), 6.04±0.11 (n=6), and 5.80±0.16 (n=7) for vehicle-treated controls, GR127935-treated controls, vehicle-treated CHPHT, and GR127935-treated CHPHT rats, respectively.
Responses to 5-CT in First Branch Pulmonary Arteries
5-CT only elicited a contractile response in rats exposed to hypoxia (Figure 3B). GR127935 significantly (P<0.05) reduced Emax to 5-CT from 138.8±23.1% (n=9) to 64.5±17.0% (n=8). Sensitivity to 5-CT did not differ between groups, the pEC50 values being 5.88±0.15 and 5.57±0.15 for vehicle-treated and GR127935-treated CHPHT groups, respectively.
Acetylcholine-Induced Vasodilation in Rat Small Muscular Pulmonary Arteries
GR127935 had no effect on acetylcholine-induced vasodilation in either control or CHPHT rats. In control vehicle-treated rats this was 45±5% (n=8) of phenylephrine-induced preconstriction; in control GR127935-treated rats this was 47±5% (n=10). In vehicle-treated hypoxic rats the % relaxation was 39±5% (n=9) and in the hypoxic GR127935-treated rats it was 47±6% (n=9). The degree of preconstriction with 1 μmol/L phenylephrine was not different between control and GR127935-treated groups, being (percent of contraction to 50 mmol/L KCl in each vessel) 15±2% for control, vehicle-treated rats and 20±3% for control, GR127935-treated rats. Likewise the degree of preconstriction was the same in the 2 chronic hypoxic rat groups, being 34±6% for vehicle-treated rats and 26±4% for the GR127935-treated rats. The response to 50 mmol/L KCl was equal, being (mN) 3.4±0.7, 4.1±0.5, 4.1±0.5, and 3.0±0.5, respectively. This also demonstrates that GR127935 had no effect on the response to 1 μmol/L phenylephrine or 50 mmol/L KCl although responses to phenylephrine were elevated in the chronic hypoxic rat vessels (P<0.05).
Responses to 5-HT in Rat Small Muscular Pulmonary Arteries In Vitro
Control vessels (234±5 μm ID, n=20) were set up at equivalent pressures of 13.8±0.4 mm Hg. Vessels from CHPHT rats (231±10 μm ID, n=18) were set up at equivalent pressures of 33.1±0.5 mm Hg. Contractile responses to 5-HT were markedly (P<0.001) increased in vehicle-treated CHPHT rats compared with vehicle-treated controls, the Emax values being 84.9±5.6% (n=11) and 27.6±5.9% (n=9), respectively (Figure 4A). The maximum contractile response was not significantly changed in GR127935-treated control rats. GR127935 partially reversed the enhanced response to 5-HT in CHPHT vessels such that Emax (55.4±8.2%, n=11) was reduced (P<0.01) compared with vehicle-treated CHPHT levels, although it was still elevated (P<0.05) in comparison to vehicle-treated controls. Hypoxia increased (P<0.001) the sensitivity to 5-HT in vehicle-treated CHPHT rats (pEC50: 6.8±0.1, n=8) compared with vehicle-treated controls (pEC50: 5.9±0.1, n=9). There was a significant (P<0.001) increase in sensitivity to 5-HT in GR127935-treated controls (pEC50: 6.7±0.1) compared with their vehicle-treated counterparts. GR127935 reduced sensitivity to 5-HT (pEC50: 6.4±0.1; P<0.05 versus vehicle-treated CHPHT rats and GR127935-treated controls) although it was still elevated compared with vehicle-treated controls (P<0.01).
Responses to 5-CT in Rat Small Muscular Pulmonary Arteries In Vitro
Control vessels (224±9 μm ID, n=13) were set up at equivalent pressures of 13.6±0.4 mm Hg. Vessels from CHPHT rats (228±7 μm ID, n=14) were set up at equivalent pressures of 32.4±0.6 mm Hg. 5-CT-induced contractions in control vessels were extremely small and not significant until relatively high concentrations >0.3 μmol/L (Figure 4B). There was an increased response to 5-CT in vehicle-treated CHPHT rats compared with in vehicle-treated controls such that significant contractions were evident at 30 nmol/L, and the Emax was increased from 17.2±6.5%, n=7 to 57.0±14.6%, n=7, P<0.05. Treatment with GR127935 for the duration of hypoxia prevented this increase so that the Emax value (24.7±3.7%, n=7) did not differ from vehicle-treated control levels (23.6±5.9%, n=6). Sensitivity to 5-CT was increased in vehicle-treated CHPHT rats (pEC50: 6.4±0.3) compared with vehicle-treated controls (pEC50: 5.2±0.2; P<0.01). GR127935 altered sensitivity to 5-CT in CHPHT rats, so that the pEC50 value (pEC50: 5.7±0.2) lay between the vehicle-treated control and CHPHT levels (not differing significantly from either). Similarly, in control rats GR127935 changed the sensitivity of the vessel to 5-CT, so that the pEC50 value (5.8±0.1) no longer differed significantly from vehicle-treated CHPHT levels.
KO Mouse Study
RV/TV Ratio and Body Weight
The RV/TV weight ratio was increased ≈41% by hypoxia in 5-HT1B+/+ compared with 5-HT1B+/+ controls (Figure 5A). 5-HT1B-receptor knockout itself had no effect on the RV/TV weight ratio in that the RV/TV weight ratio was the same in the control 5-HT1B−/− versus 5-HT1B+/+ mice. Right ventricular hypertrophy was not observed in 5-HT1B−/− mice (Figure 5A). Body weights at euthanasia were not different between groups, being 5-HT1B+/+ controls, 27±1 g (n=23); 5-HT1B+/+ hypoxic, 25±1 g (n=19); 5-HT1B−/− controls, 28±1 g (n=21); 5-HT1B−/− hypoxic, 27±1 g (n=18).
Pulmonary Vascular Remodeling
Whereas there were very few vessels exhibiting a double elastic lamina in 5-HT1B+/+ controls, 5-HT1B+/+ mice exposed to hypoxia exhibited marked pulmonary vascular remodeling (Figure 5B). 5-HT1B-receptor knockout itself had no effect on the percentage of remodeled vessels in control mice. There was an increase in remodeling in the CHPHT 5-HT1B−/− mice although this was less than that observed in the CHPHT 5-HT1B+/+ mice (Figure 5B). In fully muscularized vessels, the percent wall thickness was markedly increased in both CHPHT mice groups (Table 1, Figure 2). Percent wall thickness was not affected by 5-HT1B-receptor knockout itself but was significantly reduced in CHPHT 5-HT1B−/− mice compared with 5-HT1B+/+ CHPHT mice (Table 2). There was no other change in lung morphology in the 5-HT1B−/− mice.
Responses to 5-HT in Pulmonary Arteries
Control vessels (221±11 μm ID, n=13) were set up at equivalent pressures of 13.7±0.4 mm Hg; vessels from CHPHT mice (229±9 μm ID, n=17) were set up at equivalent pressures of 33.1±0.5 mm Hg. 5-HT induced a marked vasoconstrictor response in the mice arteries (Figure 6A). The maximum response in the wild-type mice was 182.2±16.1% (n=6) compared with the response in the rat small muscular pulmonary arteries of ≈85% (Figure 4A). The maximum contractile response to 5-HT was markedly reduced (≈70%) in control 5-HT1B−/− mice (Emax: 107.0±17.2%, n=7) compared with wild-types (P<0.05, Figure 6A). Hypoxia did not significantly alter the maximum response to 5-HT in vessels from the 5-HT1B+/+ mice. However, hypoxia increased the maximum response to 5-HT in 5-HT1B−/− mice (Emax: 169.5±13.1%, P<0.05). The sensitivity to 5-HT did not differ significantly between groups, the pEC50 values being 7.14±0.2 and 7.17±0.1 for 5-HT1B+/+ and 5-HT1B−/− controls and 7.35±0.09 and 7.21±0.14 for CHPHT 5-HT1B+/+ and CHPHT 5-HT1B−/−, respectively. No acetylcholine-mediated vasodilator responses were observed in the mice pulmonary arteries.
Responses to 5-CT in Small Muscular Pulmonary Arteries
Control vessels (231±6 μm ID n=15) were set up at equivalent pressures of 13.1±0.3 mm Hg; vessels from CHPHT mice (234±6 μm ID n=15) were set up at equivalent pressures of 32.2±0.6 mm Hg. As shown in Figure 6B, hypoxia had no influence on the contractile responses to 5-CT in the 5-HT1B−/− vessels but significantly increased the maximum contractile response to 5-CT in the 5-HT1B+/+ vessels from 111.4±8.2% (n=7) to 175.4±18.9% (n=8, P<0.05). Hypoxia increased the sensitivity to 5-CT in 5-HT1B+/+ mice (pEC50: 6.14±0.07) compared with both 5-HT1B+/+ (pEC50: 5.73±0.14; P<0.05) and 5-HT1B−/− (pEC50: 5.45±0.11; P<0.01) controls, respectively. The pEC50 value in hypoxic 5-HT1B−/− mice was 5.57±0.11, which was not significantly different from control levels. The response to 5-CT in the 5-HT1B−/− mice vessels was markedly inhibited by the 5-HT1D-receptor antagonist BRL15572 (Figure 6B).
The results confirm previous reports that hypoxia-induced PHT is associated with increases in right ventricular pressure and hypertrophy associated with extensive pulmonary vascular remodeling in both mice3,19 and rats.4 We observed that remodeling was associated with increased wall thickness. The wall thickness values measured, in control and hypoxic groups, were similar to those previously reported in rats and mice.20,21
In CHPHT rats, GR127935 attenuated right ventricular pressure, right ventricular remodeling, and vascular remodeling although GR127935 had no effect on heart rate or systemic blood pressure. This suggests that the 5-HT1B receptor is involved in the development of PHT and the consequent development of pulmonary vascular remodeling. In support of this, hypoxia did not induce right ventricular hypertrophy, and vascular remodeling was significantly reduced in the 5-HT1B knockout mice. The 5-HT1B knockout mice did not differ from the wild-type in terms of body weight, and although they have been reported to have lower heart rates than wild-type mice,22 there is no published evidence for other cardiovascular abnormalities.
Contractile responses to 5-HT were markedly enhanced in the CHPHT rat arteries, the maximum response being increased 3-fold in the small muscular arteries. We have previously studied the contractile response to other vasoconstrictors in these arteries in this model and reported that maximum responses to ET-1 are increased 1.5-fold,23 responses to noradrenaline are increased 2.3-fold,24 and responses to KCl are unaffected.23,24 Indeed we show here that responses to phenylephrine were increased 2-fold. It is clear, therefore, that receptor systems of many pulmonary vascular mediators are affected by exposure to hypoxia and the net rise in pulmonary arterial pressure will be the result of many interacting influences. Perhaps what makes the enhanced response to 5-HT of particular interest is the extent of the increase in 5-HT-mediated responses and that, in humans, the 5-HT receptors mediating contraction in the pulmonary circulation (5-HT1B) are different to those mediating contraction in the systemic circulation (5-HT2A) and may offer a pulmonary selective therapeutic target.
GR127935 reversed the enhanced response to 5-HT in the larger elastic arteries and reduced responses to 5-HT in the small arteries. GR127935 had no effect on contractile responses to phenylephrine or KCl in either large or small arteries suggesting that its effects were selective for responses to 5-HT. Curiously, GR127935 increased the sensitivity to 5-HT in small muscular arteries from normoxic control rats. The 5-HT1B or 5-HT1D receptors have been shown to mediate nitric oxide-dependent vasodilator responses.25,26 We have, however, been unable to demonstrate substantial 5-HT- or 5-CT-induced vasodilation in rat elastic pulmonary arteries (unpublished observation) making this explanation unlikely. In addition, we show GR127935 has no effect on acetylcholine-induced vasodilation, implying that GR127935 does not impair receptor-mediated nitric oxide synthesis. Another explanation may be that GR127935 has moderate 5-HT1B-, Gi-mediated partial agonist activity both in vivo and in vitro.27 This would synergize with Gq-mediated 5-HT signaling pathways such that the potency of 5-HT is increased.12 This may explain why this effect was only observed in the smaller arteries and only to 5-HT, where the response to 5-HT is entirely dependent on the 5-HT2A receptor, which is Gq-coupled.5 The effects of GR127935 on indices of PHT, and the interpretation that this is due to 5-HT1B-receptor blockade, are supported by the results obtained from the 5-HT1B knockout mouse where the changes can definitely be attributed to selective ablation of the 5-HT1B receptor.
The results highlight regional and species differences in the responses of pulmonary arteries to 5-HT. As previously observed, 5-CT did not constrict control rat elastic branch pulmonary arteries, only those removed from the CHPHT rats.5 In the small muscular arteries of control rats, small 5-CT-induced vasoconstrictor responses were evident although only at high concentrations. We have previously observed such heterogeneous distribution of ET with ETA receptors being evident in large pulmonary arteries and only ETB receptors being present in smaller pulmonary arteries.15
In the mouse small pulmonary arteries, the maximum response to 5-HT was >180% of the response to 50 mmol/L KCl, being magnitudes greater that that observed in equivalent vessels from the rat (this study, ≈30%) and humans11 (≈110%). The pEC50 for 5-HT in the mouse (≈7.0) is the same as that observed in human vessels,11 but 5-HT is much more potent than in the rat where the pEC50 is ≈5.9 (this study). This may explain why hypoxic exposure did not enhance the response to 5-HT in the 5-HT1B+/+ mice, as the response to 5-HT was already maximal.
The maximum responses to 5-HT were markedly reduced in the 5-HT1B−/− mice by ≈40%. This indicates that ≈40% of the contractile response to 5-HT may normally be mediated by the 5-HT1B receptor. The residual response is likely to be carried by the other contraction-mediating receptors, the 5-HT1D and/or the 5-HT2A receptor. Indeed there is evidence for the presence of the 5-HT1D receptor as the 5-CT-induced contractile response in the 5-HT1B−/− mice was markedly inhibited by the 5-HT1D-receptor antagonist BRL15572. This is consistent with previous studies in the 5-HT1B−/− mice that demonstrate that 5-CT does not activate the 5-HT1B receptor in some mouse strains.28 The contractile response to 5-HT in the 5-HT1B−/− mice was potentiated by hypoxia. Hypoxia did not enhance the 5-HT1D-mediated response to 5-CT observed in the 5-HT1B−/− mice. Hypoxia did, however, enhance the response to 5-CT in the wild-type mice. This suggests that both 5-HT1B- and 5-HT2A-mediated responses are enhanced by hypoxia in the mice small pulmonary arteries.
5-HT has been shown to induce proliferation of rat pulmonary arterial smooth muscle cells,29 depending on its active transport into the cell. Proliferation is inhibited by selective inhibitors of 5-HT transport but not 5-HT-receptor antagonism.2 Hypoxia induces the expression of the 5-HT transporter in rat pulmonary smooth muscle cells and this contributes to cellular proliferation.30 Indeed there is attenuated CHPHT in mice lacking the 5-HT transporter gene.19 Thus, evidence suggests that 5-HT-induced vascular remodeling is dependent on cellular internalization rather than receptor activation. In some cellular systems, however, the 5-HT2A receptor does induce pulmonary proliferation. Indeed, we have examined pulmonary fibroblast proliferation in response to 5-HT in our CHPHT and control rats and shown that this is inhibited by the 5-HT2A-receptor antagonist ketanserin but not the 5-HT1B/1D-receptor antagonist GR55562.31 This indicates that proliferation in the CHPHT rat can be mediated via the 5-HT2A receptor but not the 5-HT1B or 5-HT1D receptor. The effect of the GR127935 and 5-HT1B knockout on pulmonary vascular remodeling observed in the present study is likely, therefore, to be secondary to the reductions in pulmonary vascular tone.
In conclusion, there is converging evidence using 5-HT1B knockout mice and the 5-HT1B/1D antagonist GR127935 that the 5-HT1B receptor is involved in the development of hypoxia-induced PHT. This study suggests that one possible mechanism is via enhanced 5-HT1 receptor-mediated contraction of the pulmonary arterial circulation.
This work was funded by The Wellcome Trust. We thank Glaxo Wellcome, UK, for the donation of GR127935, Smith Kline Beecham, PA, for the donation of BRL15572, and Columbia University for the supply of 5-HT1B knockout mice.
Original received June 11, 2001; revision received September 5, 2001; accepted October 10, 2001.
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