Ian Morecroft

Serotonin Increases Susceptibility to Pulmonary Hypertension in BMPR2-Deficient Mice

Heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II (BMPR-II) receptor underlie the majority (>70%) of cases of familial pulmonary arterial hypertension (FPAH), and dysfunction of BMP signaling has been implicated in other forms of PAH. The reduced disease gene penetrance in FPAH indicates that other genetic and/or environmental factors may also be required for the clinical manifestation of disease. Of these, the serotonin pathway has been implicated as a major factor in PAH pathogenesis. We investigated the pulmonary circulation of mice deficient in BMPR-II (BMPR2+/− mice) and show that pulmonary hemodynamics and vascular morphometry of BMPR2+/− mice were similar to wild-type littermate controls under normoxic or chronic hypoxic (2- to 3-week) conditions. However, chronic infusion of serotonin caused increased pulmonary artery systolic pressure, right ventricular hypertrophy, and pulmonary artery remodeling in BMPR2+/− mice compared with wild-type littermates, an effect that was exaggerated under hypoxic conditions. In addition, pulmonary, but not systemic, resistance arteries from BMPR2+/− mice exhibited increased contractile responses to serotonin mediated by both 5-HT2 and 5-HT1 receptors. Furthermore, pulmonary artery smooth muscle cells from BMPR2+/− mice exhibited a heightened DNA synthesis and activation of extracellular signal-regulated kinase 1/2 in response to serotonin compared with wild-type cells. In vitro and in vivo experiments suggested that serotonin inhibits BMP signaling via Smad proteins and the expression of BMP responsive genes. These findings provide the first evidence for an interaction between BMPR-II-mediated signaling and the serotonin pathway, perturbation of which may be critical to the pathogenesis of PAH.

Overexpression of the 5-Hydroxytryptamine Transporter Gene Effect on Pulmonary Hemodynamics and Hypoxia-Induced Pulmonary Hypertension

Background—Increased serotonin (5-hydroxytryptamine, 5-HT) transporter activity has been observed in human familial pulmonary hypertension. Methods and Results—We investigated pulmonary hemodynamics and the development of hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling in mice overexpressing the gene for the 5-HT transporter (5-HTT+ mice). Right ventricular pressure was elevated 3-fold in normoxic 5-HTT+ mice compared with their wild-type controls. Hypoxia-induced increases in right ventricular hypertrophy and pulmonary vascular remodeling were also potentiated in the 5-HTT+ mice. 5-HTT–like immunoreactivity, protein, and binding sites were markedly increased in the lungs from the 5-HTT+ mice. Hypoxia, however, decreased 5-HT transporter immunoreactivity, mRNA transcription, protein, and binding sites in both wild-type and 5-HTT+ mice. Conclusions—Increased 5-HT transporter expression causes elevated right ventricular pressures, and this occurs before the onset of right ventricular hypertrophy or pulmonary arterial remodeling. Hypoxia-induced remodeling is, however, increased in 5-HTT+ mice, whereas hypoxia inhibits 5-HTT expression. This provides a unique model that demonstrates differential mechanisms for familial pulmonary arterial hypertension and pulmonary arterial hypertension with hypoxemia.

Contractile responses to human urotensin-II in rat and human pulmonary arteries: effect of endothelial factors and chronic hypoxia in the rat.

Responses to human urotensin‐II (hU‐II) were investigated in human and rat pulmonary arteries. Rat pulmonary arteries: hU‐II was a potent vasoconstrictor of main pulmonary arteries (2–3 mm i.d.) (pEC50, 8.55±0.08, n=21) and was ∼4 fold more potent than endothelin‐1 [ET‐1] (P<0.01), although its Emax was considerably less (∼2.5 fold, P<0.001). The potency of hU‐II increased 2.5 fold with endothelium removal (P<0.05) and after raising vascular tone with ET‐1 (P<0.01). Emax was enhanced ∼1.5 fold in the presence of Nω‐nitro‐L‐arginine methylester (L‐NAME, 100 μM, P<0.01) and ∼2 fold in vessels from pulmonary hypertensive rats exposed to 2 weeks chronic hypoxia (P<0.05). hU‐II did not constrict smaller pulmonary arteries. Human pulmonary arteries (∼250 μm i.d.): in the presence of L‐NAME, 3 out of 10 vessels contracted to hU‐II and this contraction was highly variable. hU‐II is, therefore, a potent vasoconstrictor of rat main pulmonary arteries and this response is increased by endothelial factors, vascular tone and onset of pulmonary hypertension. Inhibition of nitric oxide synthase uncovers contractile responses to hU‐II in human pulmonary arteries.

5-Hydroxytryptamine receptors mediating vasoconstriction in pulmonary arteries from control and pulmonary hypertensive rats.

1 We investigated 5‐hydroxytryptamine (5‐HT)‐receptor mediated vasoconstriction in the main, first branch and resistance pulmonary arteries removed from control and pulmonary hypertensive rats. Contractile responses to 5‐HT, 5‐carboxamidotryptamine (5‐CT, non‐selective 5‐HT1 agonist), and sumatriptan (5‐HT1D‐like receptor agonist) were studied. The effects of methiothepin (non‐selective 5‐HT1+2‐receptor antagonist) and ketanserin (5‐HT2A receptor antagonist) and GR55562 (a novel selective 5‐HT1D receptor antagonist) on 5‐HT‐mediated responses were also studied. Basal levels of adenosine 3′:5′‐cyclic monophosphate ([cyclic AMP]i) and guanosine 3′:5′‐cyclic monophosphate ([cyclic GMP]i) were determined and we assessed the degree of inherent tone in the vessels under study. 2 5‐HT was most potent in the resistance arteries. pEC50 values were 5.6 ± 0.1, 5.3 ± 0.1, 5.0 ± 0.2 in the resistance arteries, pulmonary branch and main pulmonary artery, respectively (n = at least 5 from 5 animals). The sensitivity to, and maximum response of, 5‐HT was increased in all the arteries removed from the chronic hypoxic (CH) rats. In CH rats the pEC50 values were 5.9 ± 0.2, 6.3 ± 0.2, 6.4 ± 0.2 and the increase in the maximum response was 35%, 51% and 41% in the resistance arteries, pulmonary branch and main pulmonary artery, respectively. Sumatriptan did not contract any vessel from the control rats whilst 5‐CT did contract the resistance arteries. In the CH rats, however, they both contracted the resistance arteries (responses to sumatriptan were small) (pEC50: 5‐CT; 5.4 ± 0.2) and the pulmonary artery branches (pEC50: sumatriptan, 5.4 ± 0.2; 5‐CT, 5.4 ± 0.2). 5‐CT also caused a contraction in the main pulmonary artery (pEC50: 6.0 ± 0.3). 3 Ketanserin (1 nM‐1 μm) caused a competitive antagonism of the 5‐HT response in all vessels tested. In control rats, the estimated pKb values for ketanserin in resistance arteries, pulmonary branches and main pulmonary artery were 8.3, 7.8 and 9.2, respectively. Methiothepin (1 nM‐1 μm) inhibited responses to 5‐HT in the first branch (estimated pKb value: 7.8) and main pulmonary artery. In CH rats, the estimated pKb values for ketanserin in resistance arteries, pulmonary branches and main pulmonary artery were 7.7, 8.3 and 9.6, respectively. Methiothepin also inhibited contractions to 5‐HT in the pulmonary artery branch and main pulmonary artery with estimated pKb values of 7 and 9.5, respectively. In control animals, GR55562 had no effect on responses to 5‐HT in any of the vessels tested. In the CH rats the estimated pKb values for GR55562 were 6.5, 7.8 and 7.0 in the pulmonary resistance arteries, first branch and main pulmonary artery, respectively. 4 Large pulmonary arteries from controls demonstrated inherent tone and this was increased three fold in the CH rats. The resistance arteries from controls demonstrated little inherent tone though this was enhanced in those from the CH rats. 5 [Cyclic AMP]i was 259 ± 23 pmol mg−1 protein in the pulmonary artery branches removed from control rats and decreased to 192 ± 11 pml mg−1 protein in the CH rats (P < 0.01, n = 8). [Cyclic GMP]i also decreased in the pulmonary artery branches (from 550 ± 15, control to 462 ± 31 pmol mg−1 protein in CH vessels, n = 8, P < 0.01) and in the main pulmonary arteries (from 566 ± 33, control to 370 ± 25 pmol mg−1 protein in CH vessels, n = 8, P < 0.001). No changes in either [cyclic AMP]i or [cyclic GMP]i were observed in the resistance arteries. 6 The results suggest that the increased vasoconstrictor response to 5‐HT in CH rat pulmonary arteries is due to an increase in 5‐HT2A‐receptor mediated contraction combined with an increase in r5‐HT1B‐like receptor‐mediated contraction. An increase in vascular tone and decreased levels of [cyclic GMP]i in the large pulmonary arteries may contribute to the observed increase in activity of r5‐HT1B‐like receptors.

5-Hydroxytryptamine receptors mediating contraction in human small muscular pulmonary arteries : importance of the 5-HT1B receptor

The 5‐hydroxytryptamine (5‐HT) receptors mediating vasoconstriction in isolated human small muscular pulmonary arteries (SMPAs) were determined using techniques of wire myography and reverse transcription‐polymerase chain reaction (RT–PCR). The agonists 5‐HT, 5‐carboxamidotryptamine (5‐CT, unselective for 5‐HT1 receptors) and sumatriptan (selective for 5‐HT1B/D receptors) all caused contraction and were equipotent (pEC50s: 7.0±0.2, 7.1±0.3 and 6.7±0.1, respectively) suggesting the presence of a 5‐HT1 receptor. Ketanserin (5‐HT2A‐selective antagonist, 0.1 μM) inhibited 5‐HT‐induced contractions only at non‐physiological/pathological concentrations of 5‐HT (>0.1 μM) whilst GR55562 (5‐HT1B/1D‐selective antagonist, 1 μM) inhibited 5‐HT‐induced contractions at all concentrations of 5‐HT (estimated pKB=7.7±0.2). SB‐224289 (5‐HT1B‐selective antagonist, 0.2 μM) inhibited sumatriptan‐induced contractions (estimated pKB=8.4±0.1) whilst these were unaffected by the 5‐HT1D‐selective antagonist BRL15572 (0.5 μM) suggesting that the 5‐HT1B receptor mediates vasoconstriction in this vessel. RT–PCR confirmed the presence of substantial amounts of mRNA for the 5‐HT2A and 5‐HT1B receptor subtypes in these arteries whilst only trace amounts of 5‐HT1D receptor message were evident. These findings suggest that a heterogeneous population of 5‐HT2A and 5‐HT1B receptors co‐exist in human small muscular pulmonary arteries but that the 5‐HT1B receptor mediates 5‐HT‐induced vasoconstriction at physiological and pathophysiological concentrations of 5‐HT. These results have important implications for the treatment of pulmonary hypertension in which the 5‐HT1B receptor may provide a novel and potentially important therapeutic target.

Effect of Tryptophan Hydroxylase 1 Deficiency on the Development of Hypoxia-Induced Pulmonary Hypertension

Tryptophan hydroxylase 1 catalyzes the rate-limiting step in the synthesis of serotonin in the periphery. Recently, it has been shown that expression of the tryptophan hydroxylase 1 gene is increased in lungs and pulmonary endothelial cells from patients with idiopathic pulmonary arterial hypertension. Here we investigated the effect of genetic deletion of tryptophan hydroxylase 1 on hypoxia-induced pulmonary arterial hypertension in mice by measuring pulmonary hemodynamics and pulmonary vascular remodeling before and after 2 weeks of hypoxia. In wild-type mice, hypoxia increased right ventricular pressure and pulmonary vascular remodeling. These effects of hypoxia were attenuated in the tryptophan hydroxylase 1−/−mice. Hypoxia increased right ventricular hypertrophy in both wild-type and tryptophan hydroxylase 1−/−mice suggesting that in vivo peripheral serotonin has a differential effect on the pulmonary vasculature and right ventricular hypertrophy. Contractile responses to serotonin were increased in pulmonary arteries from tryptophan hydroxylase 1−/−mice. Hypoxia increased serotonin-mediated contraction in vessels from the wild-type mice, but this was not further increased by hypoxia in the tryptophan hydroxylase 1−/−mice. In conclusion, these results indicate that tryptophan hydroxylase 1 and peripheral serotonin play an essential role in the development of hypoxia-induced elevations in pulmonary pressures and hypoxia-induced pulmonary vascular remodeling. In addition, the results suggest that, in mice, serotonin has differential effects on the pulmonary vasculature and right ventricular hypertrophy.

EndothelinB receptor-mediated contraction in human pulmonary resistance arteries.

1 Using wire myography, we have examined the endothelin (ET) receptor subtypes mediating vasoconstriction to ET peptides in human pulmonary resistance arteries (150–200 μm, i.d.). 2 Cumulative concentration‐response curves to ET‐1, sarafotoxin 6c (SX6c) and ET‐3 were constructed in the presence and absence of the selective antagonists FR 139317 (ETA‐selective), BMS 182874 (ETA‐selective) and BQ‐788 (ETB‐selective). 3 All agonists induced concentration‐dependent contractions. However, the response curves to ET‐1 were biphasic in nature. The first component demonstrated a shallow slope up to 1 nM ET‐1. Above 1 nM ET‐1 the response curve was markedly steeper. Maximum responses to ET‐3 and SX6c were the same as those to 1 nM ET‐1 and 30% of those to 0.1 μm ET‐1. The order of potency, taking 0.3 μm as a maximum concentration was SX6c > > ET‐3 > ET‐1 (pEC50 values of: 10.75 ± 0.27, 9.05 ± 0.19, 8.32 ± 0.08 respectively). Taking 1 nM ET‐1 as a maximum, the EC50 for ET‐1 was 10.08 ± 0.13 and therefore ET‐1 was equipotent to ET‐3 and SX6c over the first component of the response curve. 4 Responses to ET‐1 up to 1 nM were resistant to the effects of the ETA receptor antagonists, FR 139317 and BMS 182874 but were inhibited by the ETB receptor antagonist, BQ‐788. Conversely, responses to ET‐1 over 1 nM were inhibited by the ETA receptor antagonists, FR 139317 and BMS 182874 but unaffected by the ETB receptor antagonist, BQ‐788. 5 The results suggest that at concentrations up to 1 nM, responses to ET‐1 are mediated via the ETB receptor, whilst the responses to higher concentrations are mediated by ETA receptors.

Evidence for 5-HT1-like receptor-mediated vasoconstriction in human pulmonary artery

1 The 5‐hydroxytryptamine (5‐HT) receptors mediating contraction of human isolated pulmonary artery rings were investigated. Responses to the agonists 5‐carboximidotryptamine (5‐CT, non‐selective 5‐HT1 agonist), sumatriptan (5‐HT1D‐like receptor agonist), 5‐HT and 8‐hydroxy‐2‐(di‐n‐propylamino)‐tetralin (8‐OH‐DPAT, 5‐HT1A receptor agonist) were studied. Responses to 5‐HT and sumatriptan in the presence of the antagonists, methiothepin (non‐selective 5‐HT1+2‐receptor antagonist), ketanserin (5‐HT2A receptor antagonist) and the novel antagonist, GR55562 (5‐HT1D receptor antagonist) were also studied. 2 All agonists contracted human pulmonary artery ring preparations in the following order of potency 5‐CT > 5‐HT = sumatriptan > 8‐OH‐DPAT. Maximum responses to 5‐HT, 5‐CT and sumatriptan were not significantly different. 3 Methiothepin 1 nM and 10 nM, but not 0.1 nM reduced the maximum contractile responses to 5‐HT but did not alter tissue sensitivity to 5‐HT. Methiothepin 0.1 nM, 1 nM and 10 nM had a similar effect on responses to sumatriptan. 4 The 5‐HT2A receptor antagonist ketanserin (10 nM, 100 nM and 1 μm) also reduced the maximum contractile response to both 5‐HT and sumatriptan without affecting tissue sensitivity to these agonists. 5 The novel 5‐HT1D receptor antagonist, GR55562, inhibited responses to 5‐HT and sumatriptan in a true competitive fashion. 6 The results suggest that the human pulmonary artery has a functional population of 5‐HT1D‐like receptors which are involved in the contractile response to 5‐HT.

Is the Pregnancy Hormone Relaxin Also a Vasodilator Peptide Secreted by the Heart

Background—It has been shown recently that the pregnancy and parturition hormone, relaxin, is secreted by the heart. This study examined the effects of relaxin in small human resistance arteries from the systemic and pulmonary circulations. Methods and Results—Arteries were obtained from gluteal biopsies and resected lung tissue and studied with the use of wire myography. Cumulative concentration relaxation curves were constructed in systemic arteries with substance P, epoprostenol, atrial natriuretic peptide, and relaxin (concentration range 10−13 -10−7M). The maximal responses were 88(±5)%, 67(±10)%, 52(±16)% and 66(±16)%, respectively. Endothelium removal virtually abolished the action of relaxin. Relaxin had no vasodilator effect in pulmonary arteries. Conclusions—Relaxin is a powerful dilator of systemic resistance arteries secreted by the heart that may contribute to cardiovascular regulation.

Converging evidence in support of the serotonin hypothesis of dexfenfluramine-induced pulmonary hypertension with novel transgenic mice.

Background— The incidence of pulmonary arterial hypertension secondary to the use of indirect serotinergic agonists such as aminorex and dexfenfluramine led to the “serotonin hypothesis” of pulmonary arterial hypertension; however, the role of serotonin in dexfenfluramine-induced pulmonary arterial hypertension remains controversial. Here, we used novel transgenic mice lacking peripheral serotonin (deficient in tryptophan hydroxylase-1; Tph1−/− mice) or overexpressing the gene for the human serotonin transporter (SERT; SERT+ mice) to investigate this further. Methods and Results— Dexfenfluramine administration (5 mg · kg−1 · d−1 PO for 28 days) increased systolic right ventricular pressure and pulmonary vascular remodeling in wild-type mice but not in Tph1−/− mice, which suggests that dexfenfluramine-induced pulmonary arterial hypertension is dependent on serotonin synthesis. Dexfenfluramine was also administered to normoxic SERT+ mice and SERT+ mice exposed to chronic hypoxia. Dexfenfluramine and SERT overexpression had additive effects in increasing pulmonary vascular remodeling; however, in hypoxic SERT+ mice, dexfenfluramine reduced both systolic right ventricular pressure and pulmonary vascular remodeling. Pulmonary arterial fibroblasts from SERT+ mice, but not wild-type mice, proliferated in response to hypoxia. Dexfenfluramine inhibited hypoxia-induced proliferation of pulmonary arterial fibroblasts derived from SERT+ mice in a manner dependent on SERT activity. Dexfenfluramine also inhibited the hypoxia-mediated increase in phosphorylation of p38 mitogen-activated protein kinase in SERT+ pulmonary arterial fibroblasts. Conclusions— The results suggest that peripheral serotonin is critical for the development of dexfenfluramine-induced pulmonary arterial hypertension and that dexfenfluramine and SERT overexpression have additive effects on pulmonary vascular remodeling. We propose that dexfenfluramine can also inhibit hypoxia-induced pulmonary vascular remodeling via SERT activity and inhibition of hypoxia-induced p38 mitogen-activated protein kinase.