Lisa A. Palmer

Hypoxia induces type II NOS gene expression in pulmonary artery endothelial cells via HIF-1.

Type II nitric oxide synthase (NOS) is upregulated in the pulmonary vasculature in a chronic hypoxia model of pulmonary hypertension. In situ hybridization analysis demonstrates that type II NOS RNA is increased in the endothelium as well as in the vascular smooth muscle in the lung. The current studies examine the role of hypoxia-inducible factor (HIF)-1 in regulating type II NOS gene expression in response to hypoxia in pulmonary artery endothelial cells. Northern blot analyses demonstrate a twofold increase in HIF-1α but not in HIF-1β RNA with hypoxia in vivo and in vitro. Electrophoretic mobility shift assays show the induction of specific DNA binding activity when endothelial cells were subjected to hypoxia. This DNA binding complex was identified as HIF-1 using antibodies directed against HIF-1α and HIF-1β. Transient transfection of endothelial cells resulted in a 2.7-fold increase in type II NOS promoter activity in response to hypoxia compared with nonhypoxic controls. Mutation or deletion of the HIF-1 site eliminated the response to hypoxia. These results demonstrate that HIF-1 is essential for the hypoxic regulation of type II NOS gene transcription in pulmonary endothelium.Type II nitric oxide synthase (NOS) is upregulated in the pulmonary vasculature in a chronic hypoxia model of pulmonary hypertension. In situ hybridization analysis demonstrates that type II NOS RNA is increased in the endothelium as well as in the vascular smooth muscle in the lung. The current studies examine the role of hypoxia-inducible factor (HIF)-1 in regulating type II NOS gene expression in response to hypoxia in pulmonary artery endothelial cells. Northern blot analyses demonstrate a two fold increase in HIF-1 alpha but not in HIF-1 beta RNA with hypoxia in vivo and in vitro. Electrophoretic mobility shift assays show the induction of specific DNA binding activity when endothelial cells were subjected to hypoxia. This DNA binding complex was identified as HIF-1 using antibodies directed against HIF-1 alpha and HIF-1 beta. Transient transfection of endothelial cells resulted in a 2.7-fold increase in type II NOS promoter activity in response to hypoxia compared with nonhypoxic controls. Mutation or deletion of the HIF-1 site eliminated the response to hypoxia. These results demonstrate that HIF-1 is essential for the hypoxic regulation of type II NOS gene transcription in pulmonary endothelium.

Hypoxic Regulation of Inducible Nitric Oxide Synthase via Hypoxia Inducible Factor-1 in Cardiac Myocytes

The relationship between hypoxia and regulation of nitric oxide synthase (NOS) in myocardial tissue is not well understood. We investigated the role of hypoxia inducible factor-1 (HIF-1) on expression of the inducible NOS (iNOS) in myocardial cells in vivo and in vitro. In situ hybridization in myocardial tissue from rats exposed to hypoxia for 3 weeks demonstrated increased iNOS mRNA expression. Northern analysis of RNA from hearts of those animals and from cells exposed to hypoxia for 12 hours in vitro demonstrated an increase of HIF-1 RNA expression. Electrophoretic mobility shift assays using oligonucleotides containing the iNOS HIF-1 DNA binding site and nuclear extracts from cardiac myocytes showed induction of specific DNA binding in cells subjected to hypoxia. Transient transfection of cardiac myocytes using the murine iNOS promoter resulted in a 3.43-fold increase in promoter activity under hypoxia compared with normoxia. Mutation or deletion of the HIF-1 site eliminated the hypoxic response. As cytokines have been shown to regulate iNOS expression in myocardial cells, cultured neonatal cardiac myocytes were stimulated with interleukin-1beta causing a dramatic induction of iNOS protein expression under normoxia, with further augmentation under hypoxia. Transient transfection of cells stimulated with interleukin-1beta showed an increased iNOS promoter activity under normoxic conditions compared with unstimulated cells, with a further increase in response to hypoxia, which was dependent on HIF-1. These results demonstrate that hypoxia causes an increase in iNOS expression in cardiac myocytes and that HIF-1 is essential for the hypoxic regulation of iNOS gene expression.

S-Nitrosothiols signal hypoxia-mimetic vascular pathology

NO transfer reactions between protein and peptide cysteines have been proposed to represent regulated signaling processes. We used the pharmaceutical antioxidant N-acetylcysteine (NAC) as a bait reactant to measure NO transfer reactions in blood and to study the vascular effects of these reactions in vivo. NAC was converted to S-nitroso-N-acetylcysteine (SNOAC), decreasing erythrocytic S-nitrosothiol content, both during whole-blood deoxygenation ex vivo and during a 3-week protocol in which mice received high-dose NAC in vivo. Strikingly, the NAC-treated mice developed pulmonary arterial hypertension (PAH) that mimicked the effects of chronic hypoxia. Moreover, systemic SNOAC administration recapitulated effects of both NAC and hypoxia. eNOS-deficient mice were protected from the effects of NAC but not SNOAC, suggesting that conversion of NAC to SNOAC was necessary for the development of PAH. These data reveal an unanticipated adverse effect of chronic NAC administration and introduce a new animal model of PAH. Moreover, evidence that conversion of NAC to SNOAC during blood deoxygenation is necessary for the development of PAH in this model challenges conventional views of oxygen sensing and of NO signaling.

Compartmentalized Connexin 43 S-Nitrosylation/Denitrosylation Regulates Heterocellular Communication in the Vessel Wall

Objective—To determine whether S-nitrosylation of connexins (Cxs) modulates gap junction communication between endothelium and smooth muscle. Methods and Results—Heterocellular communication is essential for endothelium control of smooth muscle constriction; however, the exact mechanism governing this action remains unknown. Cxs and NO have been implicated in regulating heterocellular communication in the vessel wall. The myoendothelial junction serves as a conduit to facilitate gap junction communication between endothelial cells and vascular smooth muscle cells within the resistance vasculature. By using isolated vessels and a vascular cell coculture, we found that Cx43 is constitutively S-nitrosylated on cysteine 271 because of active endothelial NO synthase compartmentalized at the myoendothelial junction. Conversely, we found that stimulation of smooth muscle cells with the constrictor phenylephrine caused Cx43 to become denitrosylated because of compartmentalized S-nitrosoglutathione reductase, which attenuated channel permeability. We measured S-nitrosoglutathione breakdown and NOx concentrations at the myoendothelial junction and found S-nitrosoglutathione reductase activity to precede NO release. Conclusion—This study provides evidence for compartmentalized S-nitrosylation/denitrosylation in the regulation of smooth muscle cell to endothelial cell communication.

S-Nitrosylating Agents: A Novel Class of Compounds That Increase Cystic Fibrosis Transmembrane Conductance Regulator Expression and Maturation in Epithelial Cells

The endogenous bronchodilator, S-nitrosoglutathione (GSNO), increases expression, maturation, and function of both the wild-type and the ΔF508 mutant of the cystic fibrosis transmembrane conductance regulatory protein (CFTR). Though transcriptional mechanisms of action have been identified, GSNO seems also to have post-transcriptional effects on CFTR maturation. Here, we report that 1) GSNO is only one of a class of S-nitrosylating agents that, at low micromolar concentrations, increase ΔF508 and wild-type CFTR expression and maturation; 2) NO itself (at these concentrations) and 8-bromocyclic GMP are minimally active on CFTR; 3) a novel agent, S-nitrosoglutathione diethyl ester, bypasses the need for GSNO bioactivation by γ-glutamyl transpeptidase to increase CFTR maturation; 4) surprisingly, expression—but not S-nitrosylation—of cysteine string proteins (Csp) 1 and 2 is increased by GSNO; 5) the effect of GSNO to increase full maturation of wild-type CFTR is inhibited by Csp silencing (si)RNA; 6) proteins relevant to CFTR trafficking are SNO-modified, and SNO proteins traffic through the endoplasmic reticulum (ER) and Golgi after GSNO exposure; and 7) GSNO alters the interactions of ΔF508 CFTR with Csp and Hsc70 in the ER and Golgi. These data suggest that GSNO is one of a class of S-nitrosylating agents that act independently of the classic NO radical/cyclic GMP pathway to increase CFTR expression and maturation. They also suggest that the effect of GSNO is dependent on Csp and on intracellular SNO trafficking. We speculate that these data will be of relevance to the development of NO donor-based therapies for CF.

Jun Blockade of Erythropoiesis: Role for Repression of GATA-1 by HERP2

ABSTRACT Although Jun upregulation and activation have been established as critical to oncogenesis, the relevant downstream pathways remain incompletely characterized. In this study, we found that c-Jun blocks erythroid differentiation in primary human hematopoietic progenitors and, correspondingly, that Jun factors block transcriptional activation by GATA-1, the central regulator of erythroid differentiation. Mutagenesis of c-Jun suggested that its repression of GATA-1 occurs through a transcriptional mechanism involving activation of downstream genes. We identified the hairy-enhancer-of-split-related factor HERP2 as a novel gene upregulated by c-Jun. HERP2 showed physical interaction with GATA-1 and repressed GATA-1 transcriptional activation. Furthermore, transduction of HERP2 into primary human hematopoietic progenitors inhibited erythroid differentiation. These results thus define a novel regulatory pathway linking the transcription factors c-Jun, HERP2, and GATA-1. Furthermore, these results establish a connection between the Notch signaling pathway, of which the HERP factors are a critical component, and the GATA family, which participates in programming of cellular differentiation.

Hemoglobin α/eNOS Coupling at Myoendothelial Junctions Is Required for Nitric Oxide Scavenging During Vasoconstriction

Objective— Hemoglobin &agr; (Hb &agr;) and endothelial nitric oxide synthase (eNOS) form a macromolecular complex at myoendothelial junctions; the functional role of this interaction remains undefined. To test if coupling of eNOS and Hb &agr; regulates nitric oxide signaling, vascular reactivity, and blood pressure using a mimetic peptide of Hb &agr; to disrupt this interaction. Approach and Results— In silico modeling of Hb &agr; and eNOS identified a conserved sequence of interaction. By mutating portions of Hb &agr;, we identified a specific sequence that binds eNOS. A mimetic peptide of the Hb &agr; sequence (Hb &agr; X) was generated to disrupt this complex. Using in vitro binding assays with purified Hb &agr; and eNOS and ex vivo proximity ligation assays on resistance arteries, we have demonstrated that Hb &agr; X significantly decreased interaction between eNOS and Hb &agr;. Fluorescein isothiocyanate labeling of Hb &agr; X revealed localization to holes in the internal elastic lamina (ie, myoendothelial junctions). To test the functional effects of Hb &agr; X, we measured cyclic guanosine monophosphate and vascular reactivity. Our results reveal augmented cyclic guanosine monophosphate production and altered vasoconstriction with Hb &agr; X. To test the in vivo effects of these peptides on blood pressure, normotensive and hypertensive mice were injected with Hb &agr; X, which caused a significant decrease in blood pressure; injection of Hb &agr; X into eNOS-/- mice had no effect. Conclusions— These results identify a novel sequence on Hb &agr; that is important for Hb &agr;/eNOS complex formation and is critical for nitric oxide signaling at myoendothelial junctions.

Loss of Collectrin, an Angiotensin-Converting Enzyme 2 Homolog, Uncouples Endothelial Nitric Oxide Synthase and Causes Hypertension and Vascular Dysfunction

Background— Collectrin is an orphan member of the renin-angiotensin system and is a homolog of angiotensin-converting enzyme 2, sharing ≈50% sequence identity. Unlike angiotensin-converting enzyme 2, collectrin lacks any catalytic domain. Collectrin has been shown to function as a chaperone of amino acid transporters. In rodents, the renal expression of collectrin is increased after subtotal nephrectomy and during high-salt feeding, raising the question of whether collectrin has any direct role in blood pressure regulation. Methods and Results— Using a susceptible genetic background, we demonstrate that deletion of collectrin results in hypertension, exaggerated salt sensitivity, and impaired pressure natriuresis. Collectrin knockout mice display impaired endothelium-dependent vasorelaxation that is associated with vascular remodeling, endothelial nitric oxide synthase uncoupling, decreased nitric oxide production, and increased superoxide generation. Treatment with Tempol, a superoxide scavenger, attenuates the augmented sodium sensitivity in collectrin knockout mice. We report for the first time that collectrin is expressed in endothelial cells. Furthermore, collectrin directly regulates L-arginine uptake and plasma membrane levels of CAT1 and y+LAT1 amino acid transporters in endothelial cells. Treatment with L-arginine modestly lowers blood pressure of collectrin knockout mice. Conclusions— Collectrin is a consequential link between the transport of L-arginine and endothelial nitric oxide synthase uncoupling in hypertension.

Loss of Collectrin, an ACE2 Homologue, Uncouples Endothelial Nitric Oxide Synthase and Causes Hypertension and Vascular Dysfunction

Background— Collectrin is an orphan member of the renin-angiotensin system and is a homolog of angiotensin-converting enzyme 2, sharing ≈50% sequence identity. Unlike angiotensin-converting enzyme 2, collectrin lacks any catalytic domain. Collectrin has been shown to function as a chaperone of amino acid transporters. In rodents, the renal expression of collectrin is increased after subtotal nephrectomy and during high-salt feeding, raising the question of whether collectrin has any direct role in blood pressure regulation. Methods and Results— Using a susceptible genetic background, we demonstrate that deletion of collectrin results in hypertension, exaggerated salt sensitivity, and impaired pressure natriuresis. Collectrin knockout mice display impaired endothelium-dependent vasorelaxation that is associated with vascular remodeling, endothelial nitric oxide synthase uncoupling, decreased nitric oxide production, and increased superoxide generation. Treatment with Tempol, a superoxide scavenger, attenuates the augmented sodium sensitivity in collectrin knockout mice. We report for the first time that collectrin is expressed in endothelial cells. Furthermore, collectrin directly regulates L-arginine uptake and plasma membrane levels of CAT1 and y+LAT1 amino acid transporters in endothelial cells. Treatment with L-arginine modestly lowers blood pressure of collectrin knockout mice. Conclusions— Collectrin is a consequential link between the transport of L-arginine and endothelial nitric oxide synthase uncoupling in hypertension.

Hypoxia-induced ventilatory responses in conscious mice: gender differences in ventilatory roll-off and facilitation.

The aim of this study was to compare the ventilatory responses of C57BL6 female and male mice during a 15 min exposure to a hypoxic-hypercapnic (H-H) or a hypoxic (10% O(2), 90% N(2)) challenge and subsequent return to room air. The ventilatory responses to H-H were similar in males and females whereas there were pronounced gender differences in the ventilatory responses during and following hypoxic challenge. In males, the hypoxic response included initial increases in minute volume via increases in tidal volume and frequency of breathing. These responses declined substantially (roll-off) during hypoxic exposure. Upon return to room-air, relatively sustained increases in these ventilatory parameters (short-term potentiation) were observed. In females, the initial responses to hypoxia were similar to those in males whereas roll-off was greater and post-hypoxia facilitation was smaller than in males. The marked differences in ventilatory roll-off and post-hypoxia facilitation between female and male C57BL6 mice provide evidence that gender is of vital importance to ventilatory control.