Dysfunction of Golgi tethers, SNAREs, and SNAPs in monocrotaline-induced pulmonary hypertension

2007 ◽  
Vol 292 (6) ◽  
pp. L1526-L1542 ◽  
Author(s):  
Pravin B. Sehgal ◽  
Somshuvra Mukhopadhyay ◽  
Fang Xu ◽  
Kirit Patel ◽  
Mehul Shah
Keyword(s):  
Cell Culture ◽  
Pulmonary Hypertension ◽  
Membrane Fusion ◽  
Vesicular Trafficking ◽  
Lung Epithelial Cells ◽  
Cell Fractionation ◽  
Golgi Membranes ◽  
Protein Receptors ◽  
Attachment Proteins ◽  
Endothelial Nitric Oxide

Monocrotaline (MCT)-induced pulmonary hypertension (PH) in the rat is a widely used experimental model. We have previously shown that MCT pyrrole (MCTP) produces loss of caveolin-1 (cav-1) and endothelial nitric oxide synthase from plasma membrane raft microdomains in pulmonary arterial endothelial cells (PAEC) with the trapping of these proteins in the Golgi organelle (the Golgi blockade hypothesis). In the present study, we investigated the mechanisms underlying this intracellular trafficking block in experiments in cell culture and in the MCT-treated rat. In cell culture, PAEC showed trapping of cav-1 in Golgi membranes as early as 6 h after exposure to MCTP. Phenotypic megalocytosis and a reduction in anterograde trafficking (assayed in terms of the secretion of horseradish peroxidase derived from exogenously transfected expression constructs) were evident within 12 h after MCTP. Cell fractionation and immunofluorescence techniques revealed the marked accumulation of diverse Golgi tethers, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), and soluble NSF attachment proteins (SNAPs), which mediate membrane fusion during vesicular trafficking (GM130, p115, giantin, golgin 84, clathrin heavy chain, syntaxin-4, -6, Vti1a, Vti1b, GS15, GS27, GS28, SNAP23, and α-SNAP) in the enlarged/circumnuclear Golgi in MCTP-treated PAEC and A549 lung epithelial cells. Moreover, NSF, an ATPase required for the “disassembly” of SNARE complexes subsequent to membrane fusion, was increasingly sequestered in non-Golgi membranes. Immunofluorescence studies of lung tissue from MCT-treated rats confirmed enlargement of perinuclear Golgi elements in lung arterial endothelial and parenchymal cells as early as 4 days after MCT. Thus MCT-induced PH represents a disease state characterized by dysfunction of Golgi tethers, SNAREs, and SNAPs and of intracellular vesicular trafficking.

scholarly journals Depletion of the ATPase NSF from Golgi membranes with hypo-S-nitrosylation of vasorelevant proteins in endothelial cells exposed to monocrotaline pyrrole

2008 ◽  
Vol 295 (5) ◽  
pp. H1943-H1955 ◽  
Author(s):  
Somshuvra Mukhopadhyay ◽  
Jason Lee ◽  
Pravin B. Sehgal
Keyword(s):  
Endothelial Cells ◽  
Caveolin 1 ◽  
Golgi Membranes ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Pulmonary Arterial ◽  
Attachment Proteins ◽  
Biotin Switch ◽  
Monocrotaline Pyrrole ◽  
Arterial Endothelial Cells

Investigations of regulated S-nitrosylation and denitrosylation of vasorelevant proteins are a newly emergent area in vascular biology. We previously showed that monocrotaline pyrrole (MCTP)-induced megalocytosis of pulmonary arterial endothelial cells (PAECs), which underlies the development of pulmonary arterial hypertension, was associated with a Golgi blockade characterized by the trapping of diverse vesicle tethers, soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs), and soluble NSF-attachment proteins (SNAPs) in the Golgi; reduced trafficking of caveolin-1 (cav-1) and endotheial nitric oxide (NO) synthase (eNOS) from the Golgi to the plasma membrane; and decreased caveolar NO. We have investigated whether NSF, the ATPase involved in all SNARE disassembly, might be the upstream target of MCTP and whether MCTP might regulate NSF by S-nitrosylation. Immunofluorescence microscopy and Golgi purification techniques revealed the discordant decrease of NSF by ∼50% in Golgi membranes after MCTP despite increases in α-SNAP, cav-1, eNOS, and syntaxin-6. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide failed to affect the initiation or progression of MCTP megalocytosis despite a reduction of 4,5-diaminofluorescein diacetate fluorescence and inhibition of S-nitrosylation of eNOS as assayed using the biotin-switch method. Moreover, the latter assay not only revealed constitutive S-nitrosylation of NSF, eNOS, cav-1, and clathrin heavy chain (CHC) in PAECs but also a dramatic 70–95% decrease in the S-nitrosylation of NSF, eNOS, cav-1, and CHC after MCTP. These data point to depletion of NSF from Golgi membranes as a mechanism for Golgi blockade after MCTP and to denitrosylation of vasorelevant proteins as critical to the development of endothelial cell megalocytosis.

scholarly journals Exogenous Ghrelin Attenuates the Progression of Chronic Hypoxia-Induced Pulmonary Hypertension in Conscious Rats

Endocrinology ◽  
2007 ◽  
Vol 149 (1) ◽  
pp. 237-244 ◽  
Author(s):  
Daryl O. Schwenke ◽  
Takeshi Tokudome ◽  
Mikiyasu Shirai ◽  
Hiroshi Hosoda ◽  
Takeshi Horio ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Pulmonary Hypertension ◽  
Right Ventricular ◽  
Endothelial Nitric Oxide Synthase ◽  
Ventricular Hypertrophy ◽  
Chronic Hypoxia ◽  
Right Ventricular Hypertrophy ◽  
Pulmonary Arterial ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Chronic exposure to hypoxia, a common adverse consequence of most pulmonary disorders, can lead to a sustained increase in pulmonary arterial pressure (PAP), right ventricular hypertrophy, and is, therefore, closely associated with heart failure and increased mortality. Ghrelin, originally identified as an endogenous GH secretagogue, has recently been shown to possess potent vasodilator properties, likely involving modulation of the vascular endothelium and its associated vasoactive peptides. In this study we hypothesized that ghrelin would impede the pathogenesis of pulmonary arterial hypertension during chronic hypoxia (CH). PAP was continuously measured using radiotelemetry, in conscious male Sprague Dawley rats, in normoxia and during 2-wk CH (10% O2). During this hypoxic period, rats received a daily sc injection of either saline or ghrelin (150 μg/kg). Subsequently, heart and lung samples were collected for morphological, histological, and molecular analyses. CH significantly elevated PAP in saline-treated rats, increased wall thickness of peripheral pulmonary arteries, and, consequently, induced right ventricular hypertrophy. In these rats, CH also led to the overexpression of endothelial nitric oxide synthase mRNA and protein, as well as endothelin-1 mRNA within the lung. Exogenous ghrelin administration attenuated the CH-induced overexpression of endothelial nitric oxide synthase mRNA and protein, as well as endothelin-1 mRNA. Consequently, ghrelin significantly attenuated the development of pulmonary arterial hypertension, pulmonary vascular remodeling, and right ventricular hypertrophy. These results demonstrate the therapeutic benefits of ghrelin for impeding the pathogenesis of pulmonary hypertension and right ventricular hypertrophy, particularly in subjects prone to CH (e.g. pulmonary disorders).

Estradiol-induced attenuation of pulmonary hypertension is not associated with altered eNOS expression

2001 ◽  
Vol 280 (1) ◽  
pp. L88-L97 ◽  
Author(s):  
Thomas C. Resta ◽  
Nancy L. Kanagy ◽  
Benjimen R. Walker
Keyword(s):  
Pulmonary Hypertension ◽  
Chronic Hypoxia ◽  
Vascular Tissue ◽  
Female Rats ◽  
Ovariectomized Rats ◽  
Inhibitory Influence ◽  
Enos Expression ◽  
17Β Estradiol ◽  
Spermine Nonoate ◽  
Endothelial Nitric Oxide

Female rats develop less severe pulmonary hypertension (PH) in response to chronic hypoxia compared with males, thus implicating a potential role for ovarian hormones in mediating this gender difference. Considering that estrogen upregulates endothelial nitric oxide (NO) synthase (eNOS) in systemic vascular tissue, we hypothesized that estrogen inhibits hypoxic PH by increasing eNOS expression and activity. To test this hypothesis, we examined responses to the endothelium-derived NO-dependent dilator ionomycin and the NO donors S-nitroso- N-acetylpenicillamine and spermine NONOate in U-46619-constricted, isolated, saline-perfused lungs from the following groups: 1) normoxic rats with intact ovaries, 2) chronic hypoxic (CH) rats with intact ovaries, 3) CH ovariectomized rats given 17β-estradiol (E2β), and 4) CH ovariectomized rats given vehicle. Additional experiments assessed pulmonary eNOS levels in each group by Western blotting. Our findings indicate that E2β attenuated chronic hypoxia-induced right ventricular hypertrophy, pulmonary arterial remodeling, and polycythemia. Furthermore, although CH augmented vasodilatory responsiveness to ionomycin and increased pulmonary eNOS expression, these responses were not potentiated by E2β. Finally, responses to S-nitroso- N-acetylpenicillamine and spermine NONOate were similarly attenuated in all CH groups compared with normoxic control groups. We conclude that the inhibitory influence of E2β on chronic hypoxia-induced PH is not associated with increased eNOS expression or activity.

scholarly journals Protein complex formation with heat shock protein 90 in chronic hypoxia-induced pulmonary hypertension in newborn piglets

2010 ◽  
Vol 299 (4) ◽  
pp. H1190-H1204 ◽  
Author(s):  
Candice D. Fike ◽  
Sandra L. Pfister ◽  
James C. Slaughter ◽  
Mark R. Kaplowitz ◽  
Yongmei Zhang ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Chronic Hypoxia ◽  
Heat Shock Protein 90 ◽  
Resistance Arteries ◽  
Newborn Piglets ◽  
Protein Complex Formation ◽  
Endothelial Nitric Oxide ◽  
The Impact

Aberrant interactions between heat shock protein (Hsp)90 and its client proteins could contribute to pulmonary hypertension. We tested the hypotheses that 1) the interaction between Hsp90 and its known client protein, endothelial nitric oxide synthase (eNOS), is impaired in pulmonary resistance arteries (PRAs) from piglets with pulmonary hypertension caused by exposure to 3 or 10 days of hypoxia and 2) Hsp90 interacts with the prostanoid pathway proteins prostacyclin synthase (PGIS) and/or thromboxane synthase (TXAS). We also determined whether Hsp90 antagonism with geldanamycin alters the agonist-induced synthesis of prostacyclin and thromboxane or alters PRA responses to these prostaglandin metabolites. Compared with normoxic piglets, less eNOS coimmunoprecipitated with Hsp90 in PRAs from hypoxic piglets. Despite reduced Hsp90-eNOS interactions, dilation to ACh was enhanced in geldanamycin-treated PRAs from hypoxic, but not normoxic, piglets. In PRAs from all groups of piglets, PGIS and TXAS coimmunoprecipitated with Hsp90. Geldanamycin reduced the ACh-induced synthesis of prostacyclin and thromboxane and altered responses to the thromboxane mimetic U-46619 in PRAs from all groups. Although geldanamycin enhanced responses to prostacyclin in PRAs from both groups of hypoxic piglets, geldanamycin had no effect on prostacyclin responses in PRAs from either group of normoxic piglets. Our findings indicate that Hsp90 influences both prostanoid and eNOS signaling in the pulmonary circulation of newborn piglets and that the impact of pharmacological inhibition of Hsp90 on these signaling pathways is altered during exposure to chronic hypoxia.

scholarly journals Lipid Rafts: Keys to Sperm Maturation, Fertilization, and Early Embryogenesis

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Natsuko Kawano ◽  
Kaoru Yoshida ◽  
Kenji Miyado ◽  
Manabu Yoshida
Keyword(s):  
Cell Membrane ◽  
Cell Signaling ◽  
Membrane Fusion ◽  
Lipid Rafts ◽  
Gene Transcription ◽  
Early Embryogenesis ◽  
Sperm Maturation ◽  
Protein Receptors ◽  
Structure And Functions

Cell membranes are composed of many different lipids and protein receptors, which are important for regulating intracellular functions and cell signaling. To orchestrate these activities, the cell membrane is compartmentalized into microdomains that are stably or transiently formed. These compartments are called “lipid rafts”. In gamete cells that lack gene transcription, distribution of lipids and proteins on these lipid rafts is focused during changes in their structure and functions such as starting flagella movement and membrane fusion. In this paper, we describe the role of lipid rafts in gamete maturation, fertilization, and early embryogenesis.

Endothelial nitric oxide synthase (NOS) is upregulated in rapid progressive pulmonary hypertension of the newborn

2003 ◽  
Vol 29 (10) ◽  
pp. 1757-1762 ◽  
Author(s):  
Thomas Hoehn ◽  
Anthony A. Preston ◽  
Allan R. McPhaden ◽  
Brigitte Stiller ◽  
Martin Vogel ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Pulmonary Hypertension ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide
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