Martha Hinton

AMPK and FoxO1 regulate catalase expression in hypoxic pulmonary arterial smooth muscle

Hypoxia and reactive oxygen species (ROS) including H2O2 play major roles in triggering and progression of pulmonary vascular remodeling in persistent pulmonary hypertension. Catalase (CAT), the major endogenous enzyme scavenging H2O2, is regulated in a tissue‐ and context‐specific manner.

Assessment of P-glycoprotein Activity in the Blood-Brain Barrier (BBB) Using Near Infrared Fluorescence (NIRF) Imaging Techniques

ABSTRACTPurposeTo examine functional activity of P-glycoprotein (P-gp) in the blood-brain barrier (BBB) using near infrared fluorescence (NIRF) imaging techniques.MethodsCellular accumulation and bi-directional permeability of the NIRF probe, rhodamine 800 (R800) was determined in MDCKMDR1 and MDCKwt monolayers under normal conditions and following P-gp inhibition with GF120918. Functional P-gp activity was also assessed in mice following administration of R800 alone and with GF230918. Quantitative analysis of R800 fluorescence in brain tissue and blood was measured ex-vivo using Odyssey Near Infrared imaging.ResultsR800 accumulation was reduced in MDCKMDR1 compared to MDCKwt monolayers. Addition of GF120918, resulted in increased R800 accumulation in MDCKMDR1 monolayers. Permeability of R800 in MDCKMDR1 monolayers was significantly enhanced (4-fold) in the basolateral to apical direction under control conditions and was abolished following treatment with GF120918. With the exception of the choriod plexus, there was very little penetration of R800 into the brain under control conditions. Treatment of mice with GF120918 resulted in a nearly 4-fold increase in R800 fluorescence in the brain. In contrast, GF120918 had no effect on brain penetration of a vascular permeability marker.ConclusionsIn vitro studies demonstrate the P-gp transporter properties of the NIRF probe R800. Preliminary in vivo studies confirm the P-gp transporter liabilities of R800 and suggest this probe may be useful as a molecular imaging agent for examining P-gp activity in the BBB.

Evaluation of drug efflux transporter liabilities of darifenacin in cell culture models of the blood–brain and blood–ocular barriers†‡

The objective of the present study was to evaluate drug efflux transporter interactions of darifenacin and examine the impact of such transporter interactions on darifenacin permeability in an in vitro model of the blood–brain barrier (BBB) and blood–ocular barrier (BOB).

Hypoxia and nitric oxide exposure promote apoptotic signaling in contractile pulmonary arterial smooth muscle but not in pulmonary epithelium.

Neonatal pulmonary hypertension is characterized by hypoxia, abnormal vascular remodeling, and impaired alveolarization. Nitric oxide (NO) regulates cell replication and activation of apoptosis. Our objective was to examine cell phenotype‐specific effects of hypoxia and NO exposure on cumulative apoptotic signal in neonatal pulmonary epithelial cells and arterial smooth muscle.

Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324.

Dysregulation of the thromboxane A2 (TP) receptor, resulting in agonist hypersensitivity and hyper‐responsiveness, contributes to exaggerated vasoconstriction in the hypoxic pulmonary artery in neonatal persistent pulmonary hypertension. We previously reported that hypoxia inhibits TP receptor phosphorylation, causing desensitization. Hence, we examined the role of PKA‐accessible serine residues in determining TP receptor affinity, using site‐directed mutational analysis.

Palmitoylation of Gαq Determines Its Association with the Thromboxane Receptor in Hypoxic Pulmonary Hypertension

Pulmonary arterial vasoconstriction is a hallmark of persistent pulmonary hypertension of the newborn (PPHN). We reported increased calcium responses to thromboxane and selectively increased thromboxane prostanoid (TP) association with Gαq in hypoxic pulmonary artery. Palmitoylation of Gαq is important for efficient receptor-Gαq-phospholipase-C interactions. TPα receptor is not itself amenable to palmitoylation. We studied the role of Gαq palmitoylation in constriction of hypoxic pulmonary artery using pharmacological palmitoylation inhibition, the effects of hypoxia on palmitoylation, and the effects of site-specific cysteine substitution mutations of Gαq on Gαq membrane targeting, TPα association, and calcium dose-response curve to a TP agonist. PPHN pulmonary artery and HEK293T cells expressing TPα were exposed to irreversible palmitoylation inhibitor 2-bromopalmitate before challenge with TP agonist U46619. Palmitate uptake was studied in hypoxic and normoxic myocytes. Wild-type Gαq and Gαq cysteine-to-alanine mutants C9A, C10A, and C9A/C10A were transiently coexpressed in HEK293T cells stably expressing TPα. We examined membrane localization of Gαq, TP receptor-Gαq association by coimmunoprecipitation, and Ca(2+) responses to U46619 in hypoxic and normoxic cells. Gαq palmitoylation is essential for the Ca(2+) response to TPα stimulation. Inhibition of palmitoylation reduces contractile force to thromboxane in PPHN but not in control pulmonary artery. Hypoxia increases palmitoylation of Gαq; the hypoxic. but not the normoxic, response to thromboxane is palmitoylation sensitive. Palmitoylation of one N-terminal cysteine is required for physical association of Gαq with the TPα receptor. Palmitoylation of both cysteines is required for Gαq membrane localization and Ca(2+) mobilization. Depalmitoylation of any one Gαq cysteine reduces the hypoxic response to thromboxane challenge to equal that of normoxic cells.

Characterization of GPCR signaling in hypoxia

G protein-coupled receptors (GPCRs) signal in response to various external stimuli including stress. GPCR signaling has been shown to play a critical role in the adaptation of cell response to limited oxygen supply. Hypoxia has been implicated in cardiovascular diseases, human pulmonary arterial responses, and persistent pulmonary hypertension in newborns. One of the key GPCRs implicated in hypoxia is the prostanoid receptor, thromboxane A2 receptor (TP). Hypoxia can affect TP localization, stability, and activity both in vivo and in vitro. To elucidate hypoxia-mediated GPCR signaling in vitro, we lay out a general strategy to perform hypoxic experiments using both primary pulmonary artery smooth muscle cells and TP expressed in HEK293T cells. We describe assay for measuring moderate tissue hypoxia using static cell cultures, monitoring pericellular media oxygen content, and signaling of TP.