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Nicholas W Morrell

Publications and source records attributed to Nicholas W Morrell.

27 records · Page 2Linked to original sources
PubMed2004

Mechanism of cicaprost-induced desensitization in rat pulmonary artery smooth muscle cells involves a PKA-mediated inhibition of adenylyl cyclase.

Long-term infusion of prostacyclin, or its analogs, is an effective treatment for severe pulmonary arterial hypertension. However, dose escalation is often required to maintain efficacy. The aim of this study was to investigate the mechanisms of prostacyclin receptor desensitization using the prostacyclin analog cicaprost in rat pulmonary artery smooth muscle cells (PASMCs). Desensitization of the cAMP response occurred in 63 nM cicaprost after a 6-h preincubation with agonist. This desensitization was reversed 12 h after agonist removal, and resensitization was inhibited by 10 microg/ml of cycloheximide. Desensitization was heterologous since desensitization to other G(s)alpha-adenylyl cyclase (AC)-coupled agonists, isoproterenol (1 microM), adrenomedullin (100 nM), or bradykinin (1 microM), was also reduced by preincubation with cicaprost. The reduced cAMP response to prolonged cicaprost exposure appeared to be due to inhibition of AC activity since the responses to the directly acting AC agonist forskolin (3 microM) and the selective AC5 activator NKH-477 were similarly reduced. Expression of AC2 and AC5/6 protein levels transiently decreased after 1 h of cicaprost exposure. The PKA inhibitor H-89 (1 microM) added 1 h before cicaprost preincubation (6 h, 63 nM) completely reversed cicaprost-induced desensitization, whereas the PKC inhibitor bisindolylmaleimide (100 nM) was only partly effective. Desensitization was not prevented by the G(i) inhibitor pertussis toxin. In conclusion, chronic treatment of PASMCs with cicaprost induced heterologous, reversible desensitization by inhibition of AC activity. Our data suggest that heterologous G(s)alpha desensitization by cicaprost is mediated predominantly by a PKA-inhibitable isoform of AC, most likely AC5/6.

Adenylyl Cyclases↗
PubMed2004

Primary pulmonary hypertension: molecular basis and potential for therapy.

Primary pulmonary hypertension (PPH) is defined clinically by sustained elevation of pulmonary arterial pressure without a demonstrable cause, and is a progressive, often-fatal disease. PPH can be associated with ingestion of appetite suppressants, human immunodeficiency virus infection and certain autoimmune diseases. Familial PPH is known to account for 6% of all cases. Mutations in the gene encoding the bone morphogenetic protein (BMP) type II receptor have been identified in 72% of affected families and 26% of apparently sporadic cases. BMPs are members of the transforming growth factor b superfamily and affect intracellular signalling via Smads and mitogen-activated protein kinases. Evidence supports a 'two-hit' hypothesis in which PPH is triggered by accumulation of genetic and environmental insults in a susceptible individual. Elucidation of the precise molecular and cellular mechanisms underlying PPH will provide a powerful basis for the development of novel therapeutic strategies in the treatment of this devastating condition.

Bone Morphogenetic Protein Receptors, Type II↗
PubMed2003

Functional interaction between BMPR-II and Tctex-1, a light chain of Dynein, is isoform-specific and disrupted by mutations underlying primary pulmonary hypertension.

Diverse heterozygous mutations of bone morphogenetic receptor type II (BMPR-II) underlie the inherited form of the vascular disorder primary pulmonary hypertension (PPH). As yet, the molecular detail of how such defects contribute to the pathogenesis of PPH remains unclear. BMPR-II is a member of the transforming growth factor-beta cell signalling superfamily. Ligand binding induces cell surface receptor complex formation and activates a cascade of phosphorylation events of intracellular intermediaries termed Smads, which initiate transcriptional regulation. Some 30% of PPH-causing mutations localize to exon 12, which may be spliced out forming an isoform depleted of the unusually long BMPR-II cytoplasmic tail. To further elucidate the consequences of BMPR2 mutation, we sought to characterize aspects of the cytoplasmic domain function by seeking intracellular binding partners. We now report that Tctex-1, a light chain of the motor complex dynein, interacts with the cytoplasmic domain of BMPR-II and demonstrate that Tctex-1 is phosphorylated by BMPR-II, a function disrupted by PPH disease causing mutations within exon 12. Finally we show that BMPR-II and Tctex-1 co-localize to endothelium and smooth muscle within the media of pulmonary arterioles, key sites of vascular remodelling in PPH. Taken together, these data demonstrate a discrete function for the cytoplasmic domain of BMPR-II and justify further investigation of whether the interaction with and phosphorylation of Tctex-1 contributes to the pathogenesis of PPH.

Bone Morphogenetic Protein Receptors, Type II↗
PubMed2002

Characterization of high-altitude pulmonary hypertension in the Kyrgyz: association with angiotensin-converting enzyme genotype.

Previous studies have suggested a genetic component in susceptibility to hypoxia-induced pulmonary hypertension. We therefore estimated the prevalence of high-altitude pulmonary hypertension (HAPH) in a Kyrgyz population and whether the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene associates with HAPH. An electrocardiographic survey of 741 highlanders demonstrated electrocardiogram signs of cor pulmonale in 14% of subjects. Pulmonary artery hemodynamics measured in an independent group of 136 male highlanders with symptoms of dyspnea at altitude revealed established pulmonary hypertension (mean pulmonary artery pressure [MPAP] > or = 25 mm Hg) in 20%. However, 26% of the normal subjects demonstrated an exaggerated response (twofold or greater increase in MPAP) to inhalation of 11% oxygen, and were classified as hyperresponsive. Ten-year follow-up of this group revealed increases in the MPAP, but not in normal subjects. Comparison of ACE I/D genotypes in the catheterized group revealed a threefold higher frequency of the I/I genotype in highlanders with HAPH, compared with normal highlanders (chi2 = 11.59, p = 0.003). In addition, MPAP was higher in highlanders with the I/I genotype (26.9 +/- 4.0 mm Hg) compared with the I/D genotype (20.6 +/- 1.2 mm Hg) or the D/D genotype (18.3 +/- 0.9 mm Hg) (p < 0.05). We conclude that HAPH is associated with ACE I/D genotype among Kyrgyz highlanders and the development of HAPH in this population and may be predicted by hyperresponsiveness to acute hypoxia.

Adolescent↗
PubMed2002

Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension.

A wide range of mutations in the type II receptor for bone morphogenetic protein (BMPR-II) have been shown to underlie primary pulmonary hypertension. To determine the mechanism of altered BMPR-II function, we employed transient transfection studies in cell lines and primary cultures of pulmonary vascular smooth muscle cells using green fluorescent protein (GFP)-tagged wild-type and mutant BMPR2 constructs and confocal microscopy to localize receptors. Substitution of cysteine residues in the ligand binding or kinase domain prevented trafficking of BMPR-II to the cell surface, and reduced binding of (125)I-BMP4. In addition, transfection of cysteine-substituted BMPR-II markedly reduced basal and BMP4-stimulated transcriptional activity of a BMP/Smad responsive luciferase reporter gene (3GC2wt-Lux), compared with wild-type BMPR-II, suggesting a dominant-negative effect of these mutants on Smad signalling. In contrast, BMPR-II containing non-cysteine substitutions in the kinase domain were localized to the cell membrane, although these also suppressed the activity of 3GC2wt-Lux. Interestingly, BMPR-II mutations within the cytoplasmic tail trafficked to the cell surface, but retained the ability to activate 3GC2wt-Lux. Transfection of mutant, but not wild-type, constructs into a mouse epithelial cell line (NMuMG cells) led to activation of p38(MAPK) and increased serum-induced proliferation compared with the wild-type receptor, which was partly p38(MAPK)-dependent. We conclude that mutations in BMPR-II heterogeneously inhibit BMP/Smad-mediated signalling by diverse molecular mechanisms. However, all mutants studied demonstrate a gain of function involving upregulation of p38(MAPK)-dependent proproliferative pathways.

Animals↗
PubMed2002

Primary pulmonary hypertension is associated with reduced pulmonary vascular expression of type II bone morphogenetic protein receptor.

BACKGROUND: Mutations in the type II receptor for bone morphogenetic protein (BMPR-II), a receptor member of the transforming growth factor-beta (TGF-beta) superfamily, underlie many familial and sporadic cases of primary pulmonary hypertension (PPH). METHODS AND RESULTS: Because the sites of expression of BMPR-II in the normal and hypertensive lung are unknown, we studied the cellular localization of BMPR-II and the related type I and II receptors for TGF-beta by immunohistochemistry in lung sections from patients undergoing heart-lung transplantation for PPH (n=11, including 3 familial cases) or secondary pulmonary hypertension (n=6) and from unused donor lungs (n=4). In situ hybridization was performed for BMPR-II mRNA. Patients were screened for the presence of mutations in BMPR2. In normal lungs, BMPR-II expression was prominent on vascular endothelium, with minimal expression in airway and arterial smooth muscle. In pulmonary hypertension cases, the intensity of BMPR-II immunostaining varied between lesions but involved endothelial and myofibroblast components. Image analysis confirmed that expression of BMPR-II was markedly reduced in the peripheral lung of PPH patients, especially in those harboring heterozygous BMPR2 mutations. A less marked reduction was also observed in patients with secondary pulmonary hypertension. In contrast, there was no difference in level of staining for TGF-betaRII or the endothelial marker CD31. CONCLUSIONS: The cellular localization of BMPR-II is consistent with a role in the formation of pulmonary vascular lesions in PPH, and reduced BMPR-II expression may contribute to the process of vascular obliteration in severe pulmonary hypertension.

Activin Receptors, Type I↗
PubMed2002

ET(A) and ET(B) receptors modulate the proliferation of human pulmonary artery smooth muscle cells.

We determined the distribution of ET(A) and ET(B) receptors in pulmonary arteries from pulmonary hypertensive patients and control subjects, using in vitro autoradiography, and investigated their role in mediating the proliferative effects of endothelin-1 (ET-1) on distal human pulmonary artery smooth muscle cells (PASMCs). Distal arteries possessed more medial [(125)I]-ET-1 binding sites (105 +/- 10 versus 45 +/- 6 amol/mm(2); p < 0.001) and a greater proportion of ET(B) receptors than proximal arteries (36 +/- 3% versus 3 +/- 1%; p < 0.001). Receptor density in distal arteries and lung parenchyma was twofold greater (p < 0.05) in pulmonary hypertensive patients than in control subjects. ET-1 (10(-9)-10(-7) mol/L) stimulated DNA synthesis (147 +/- 10% of control subjects; p < 0.05) and attenuated the antiproliferative action of cicaprost and forskolin on PASMCs, these effects being mediated via ET(A) and ET(B) receptors. Serum-stimulated proliferation was attenuated by inhibiting either endogenous ET-1 release with phosphoramidon (10(-5) mol/L) or its action with PD145065 (10(-5) mol/L). Cicaprost (10(-10)-10(-7) mol/L) inhibited ET-1 release from PASMCs (49 +/- 16% of control after 24 h; p < 0.001) and increased intracellular cAMP levels, whereas ET(B) receptor stimulation selectively reduced cAMP levels. In conclusion, ET(A) and ET(B) receptors are differentially distributed in human pulmonary arteries. Both receptors promote the proliferation of PASMCs in vitro and may contribute to vascular remodeling in pulmonary hypertension.

Adult↗
PubMed2002

Hypoxic induction of cox-2 regulates proliferation of human pulmonary artery smooth muscle cells.

Chronic hypoxia-induced pulmonary hypertension results partly from proliferation of smooth muscle cells in small peripheral pulmonary arteries. Therefore, we examined the effect of hypoxia on growth of pulmonary artery smooth muscle cells (PASMCs) from human distal pulmonary arteries. Initial studies identified that serum-induced proliferation of explant-derived PASMCs was inhibited under hypoxic conditions (3-4 kPa in medium). However, selection of hypoxia-stimulated cells was achieved by culturing cells at low density under conditions of prolonged hypoxia (1-2 wk). In hypoxia-inhibited and -stimulated cells, Western blotting revealed hypoxic induction of cyclooxygenase (COX)-2, which was dependent on the activation of p38(MAPK), but not COX-1, inducible nitric oxide synthase (iNOS), or hemoxygenase-1 (HO-1). Hypoxic induction of COX-2 was also observed in the media of pulmonary arteries in lung organ culture. Hypoxia induced a 4- to 5-fold increase (P < 0.001) in prostaglandin (PG)E(2), PGD(2), PGF(2alpha), and 6-keto-PGF(1alpha) release from PASMCs. Hypoxic inhibition of proliferation was attenuated by incubation with indomethacin (10 micro M), or the COX-2 antagonist, NS398 (10 micro M), but not by the COX-1 antagonist, valeryl salicylate (0.5 mM). In conclusion, we have isolated cells from human peripheral pulmonary arteries that are either inhibited or stimulated by culture under hypoxic conditions. In both cell types hypoxia modulates cell proliferation by induction of COX-2 and production of antiproliferative prostaglandins. Induction of COX-2 may contribute to the inhibition of hypoxia-induced pulmonary vascular remodeling.

6-Ketoprostaglandin F1 alpha↗