Site-Directed Mutations and the Polymorphic Variant Ala160Thr in the Human Thromboxane Receptor Uncover a Structural Role for Transmembrane Helix 4

The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic (nsSNP) variants. Interestingly, A160T is a novel nsSNP variant with unknown structure and function. Additionally, within this helix in TP, Ala1604.53 is highly conserved as is Gly1644.57. Here we target Ala1604.53 and Gly1644.57 in the TP for detailed structure-function analysis. Amino acid replacements with smaller residues, A160S and G164A mutants, were tolerated, while bulkier beta-branched replacements, A160T and A160V showed a significant decrease in receptor expression (Bmax). The nsSNP variant A160T displayed significant agonist-independent activity (constitutive activity). Guided by molecular modeling, a series of compensatory mutations were made on TM3, in order to accommodate the bulkier replacements on TM4. The A160V/F115A double mutant showed a moderate increase in expression level compared to either A160V or F115A single mutants. Thermal activity assays showed decrease in receptor stability in the order, wild type>A160S>A160V>A160T>G164A, with G164A being the least stable. Our study reveals that Ala1604.53 and Gly1644.57 in the TP play critical structural roles in packing of TM3 and TM4 helices. Naturally occurring mutations in conjunction with site-directed replacements can serve as powerful tools in assessing the importance of regional helix-helix interactions

Serial serum brain-type natriuretic peptide (BNP) identifies compromised blood flow in infants with hemodynamically significant patent ductus arteriosus

Background: The physiological correlates of elevated serum brain-type natriuretic peptide (BNP) in hemodynamically significant patent ductus arteriosus (HSPDA) are unclear. Objective: To determine if serial BNP measured at 48-72 hours of age, before and after non-steroidal anti-inflammatory drugs (NSAID) treatment of HSPDA reflect compromised blood flow indices, in infants < 31 weeks of gestational age (GA). Design/methods: In a prospective blinded study, 70 infants < 31 weeks GA, admitted to Winnipeg NICUs from August 2010 to September 2011, had serum BNPs and echocardiograms at 48-72 hours of age, before and after medical treatment of HSPDA. All BNP and logarithm of BNP (logBNP) were correlated by linear regression with contemporaneous blood flow indices for: 1) systemic hemodynamic indices (corrected left and right ventricular outputs [LVO, RVO], RVO/LVO ratio, superior vena cava flow [SVCF], SVCF/LVO); 2) regional blood flow indices (middle cerebral artery flow [MCAF], MCAF/LVO ratio, middle cerebral artery resistive index [MCARI], middle cerebral artery pulsatility index [MCAPI]; celiac artery flow [CAF], CAF/LVO ratio, celiac artery resistive index [CARI], and celiac artery pulsatility index [CAPI]). Results: Twenty-six of 70 infants developed HSPDA at 6 ± 2 days. Both BNP and logBNP had similar correlations with all indices, but logBNP showed better goodness of fit. The best correlation was at 48-72 hours of life. Analyzing systemic hemodynamics, logBNP best correlated with SVCF (β -0.49, R2 0.24, p < 0.0001), SVCF/LVO (β -0.55, R2 0.31, p < 0.0001), RVO/LVO (β -0.59, R2 0.35, p < 0.0001), LVO (β 0.4, R2 0.16, p < 0.0001), and RVO (β -0.35, R2 0.12, p < 0.0001). For regional blood flow, logBNP best correlated with MCARI (β 0.6, R2 0.35, p < 0.0001), MCAPI (β 0.5, R2 0.29, p < 0.001), and MCAF (β -0.34, R2 0.12, p < 0.0001). Conclusions: BNP correlates with blood flow indices in preterm infants with HSPDA mainly at 48-72 hours reflecting the value of pre-symptomatic physiologic prediction by BNP

Lipid metabolites as regulators of airway smooth muscle function

Compelling evidence identifies airway smooth muscle (ASM) not only as a target but also a cellular source for a diverse range of mediators underlying the processes of airway narrowing and airway hyperresponsiveness in diseases such as asthma. These include the growing family of plasma membrane phospholipid-derived polyunsaturated fatty acids broadly characterised by the prostaglandins, leukotrienes, lipoxins, isoprostanes and lysophospholipids. In this review, we describe the enzymatic and nonenzymatic biosynthetic pathways of these lipid mediators and how these are influenced by drug treatment, oxidative stress and airways disease. Additionally, we outline their cognate receptors, many of which are expressed by ASM. We describe potential deleterious and protective roles for these lipid mediators in airway inflammatory and remodelling processes by describing their effects on diverse functions of ASM in asthma that have the potential to contribute to asthma pathogenesis and symptoms. These functions include contractile tone development cytokine and extracellular matrix production, and cellular proliferation and migration. (C) 2008 Elsevier Ltd. All rights reserved

Pre-symptomatic prediction of morbitidies in preterm infants with patent ductus arteriosus by targeted neonatal echocardiography and brain-type natriuretic peptide

Objectives: Our objective was to compare patent ductus arteriosus (PDA) diameter, PDA score and brain-type natriuretic peptide (BNP) measurements at 48-72 hours of life, for prediction of neonatal morbidities. We hypothesized that use of a PDA score with BNP, may improve pre-symptomatic prediction of PDAs associated with adverse outcomes. Method: Infants < 31 weeks GA were prospectively studied by targeted neonatal echocardiogram (TNE) at 48-72 hours age, composite PDA score and serum BNP assay; the clinical team remained blinded. PDA was independently diagnosed by echocardiography at time of clinical suspicion (6 ± 2 days), and treated at discretion of the clinical team. Primary outcome was survival with one or more of adverse outcomes (intraventricular hemorrhage [IVH], bronchopulmonary dysplasia [BPD], retinopathy of prematurity [ROP], necrotizing enterocolitis [NEC]). Results: A PDA was present in 56 of 70 infants studied at 48-72 hours; 30 were eventually diagnosed with PDA but never required treatment, 19 required medical treatment, 7 surgical ligation. After adjustment for gestation, PDA diameter did not predict any adverse outcome, PDA score was associated with increased risk of any adverse outcome and high BNP was associated with IVH, BPD, or survival with any adverse outcome. Conclusions: Comprehensive PDA evaluation at 48-72 hours of age may predict the subsequent occurrence of adverse outcomes and may be useful to define the PDA treatment threshold

Inverse Agonism of SQ 29,548 and Ramatroban on Thromboxane A2 Receptor

<div><p>G protein-coupled receptors (GPCRs) show some level of basal activity even in the absence of an agonist, a phenomenon referred to as constitutive activity. Such constitutive activity in GPCRs is known to have important pathophysiological roles in human disease. The thromboxane A2 receptor (TP) is a GPCR that promotes thrombosis in response to binding of the prostanoid, thromboxane A2. TP dysfunction is widely implicated in pathophysiological conditions such as bleeding disorders, hypertension and cardiovascular disease. Recently, we reported the characterization of a few constitutively active mutants (CAMs) in TP, including a genetic variant A160T. Using these CAMs as reporters, we now test the inverse agonist properties of known antagonists of TP, SQ 29,548, Ramatroban, L-670596 and Diclofenac, in HEK293T cells. Interestingly, SQ 29,548 reduced the basal activity of both, WT-TP and the CAMs while Ramatroban was able to reduce the basal activity of only the CAMs. Diclofenac and L-670596 showed no statistically significant reduction in basal activity of WT-TP or CAMs. To investigate the role of these compounds on human platelet function, we tested their effects on human megakaryocyte based system for platelet activation. Both SQ 29,548 and Ramatroban reduced the platelet hyperactivity of the A160T genetic variant. Taken together, our results suggest that SQ 29,548 and Ramatroban are inverse agonists for TP, whereas, L-670596 and Diclofenac are neutral antagonists. Our findings have important therapeutic applications in the treatment of TP mediated pathophysiological conditions.</p></div