Roles for Nox4 in the contractile response of bovine pulmonary arteries to hypoxia

Hypoxia appears to promote contraction [hypoxic pulmonary vasoconstriction (HPV)] of bovine pulmonary arteries (BPA) through removal of a peroxide-mediated relaxation. This study examines the roles of BPA Nox oxidases and mitochondria in the HPV response. Inhibitors of Nox2 (0.1 mM apocynin and 50 μM gp91-dstat) and mitochondrial electron transport (10 μM antimycin and rotenone) decreased superoxide generation in BPA without affecting contraction to 25 mM KCl or the HPV response. Transfection of BPA with small inhibitory RNA (siRNA) for Nox2 and Nox4 decreased Nox2 and Nox4 protein expression, respectively, associated with an attenuation of superoxide detection, without affecting 25 mM KCl contraction. However, Nox4 siRNA, but not Nox2, attenuated HPV in BPA. A Nox4 inhibitor plumbagin (10 μM) increased basal force, decreased superoxide detection and peroxide release, and caused BPA to relax under hypoxia. Although acute removal of peroxide with 0.1 mM ebselen increased 25 mM KCl contraction and decreased hypoxic contraction, prolonged treatment with ebselen only decreased hypoxic contraction without affecting 25 mM KCl contraction, suggesting basal peroxide levels also maintain a contractile mechanism not removed by acute hypoxia. Organ culture of BPA with transforming growth factor (TGF)-β1 (4 nM) increased Nox4 expression, superoxide, peroxide, and the HPV response. Thus Nox2 and mitochondria are sources for superoxide generation in BPA, which do not appear to influence the HPV response. However, peroxide derived from superoxide generated by Nox4 appears to maintain a basal relaxation in BPA under normoxic conditions, which is removed under hypoxia leading to HPV. Peroxide generated by Nox4 may also function to maintain a contractile mechanism, which is not reversed by acute hypoxia.

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Inhibition of hypoxic pulmonary vasoconstriction by antagonists of store-operated Ca2+ and nonselective cation channels

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00044.2005 ◽

2005 ◽

Vol 289 (1) ◽

pp. L5-L13 ◽

Cited By ~ 87

Author(s):

Letitia Weigand ◽

Joshua Foxson ◽

Jian Wang ◽

Larissa A. Shimoda ◽

J. T. Sylvester

Keyword(s):

Hypoxic Pulmonary Vasoconstriction ◽

Acute Hypoxia ◽

Pulmonary Arteries ◽

Cation Channels ◽

Pulmonary Vasoconstriction ◽

Nonselective Cation Channels ◽

Pressor Responses ◽

Intracellular Ca ◽

Pulmonary Arterial

Previous studies indicated that acute hypoxia increased intracellular Ca2+ concentration ([Ca2+]i), Ca2+ influx, and capacitative Ca2+ entry (CCE) through store-operated Ca2+ channels (SOCC) in smooth muscle cells from distal pulmonary arteries (PASMC), which are thought to be a major locus of hypoxic pulmonary vasoconstriction (HPV). Moreover, these effects were blocked by Ca2+-free conditions and antagonists of SOCC and nonselective cation channels (NSCC). To test the hypothesis that in vivo HPV requires CCE, we measured the effects of SOCC/NSCC antagonists (SKF-96365, NiCl2, and LaCl3) on pulmonary arterial pressor responses to 2% O2 and high-KCl concentrations in isolated rat lungs. At concentrations that blocked CCE and [Ca2+]i responses to hypoxia in PASMC, SKF-96365 and NiCl2 prevented and reversed HPV but did not alter pressor responses to KCl. At 10 μM, LaCl3 had similar effects, but higher concentrations (30 and 100 μM) caused vasoconstriction during normoxia and potentiated HPV, indicating actions other than SOCC blockade. Ca2+-free perfusate and the voltage-operated Ca2+ channel (VOCC) antagonist nifedipine were potent inhibitors of pressor responses to both hypoxia and KCl. We conclude that HPV required influx of Ca2+ through both SOCC and VOCC. This dual requirement and virtual abolition of HPV by either SOCC or VOCC antagonists suggests that neither channel provided enough Ca2+ on its own to trigger PASMC contraction and/or that during hypoxia, SOCC-dependent depolarization caused secondary activation of VOCC.

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Modulatory role of EDRF in hypoxic contraction of isolated porcine pulmonary arteries

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1992.262.3.h691 ◽

1992 ◽

Vol 262 (3) ◽

pp. H691-H697 ◽

Cited By ~ 6

Author(s):

M. Ogata ◽

M. Ohe ◽

D. Katayose ◽

T. Takishima

Keyword(s):

Contractile Response ◽

Isometric Force ◽

Hypoxic Pulmonary Vasoconstriction ◽

Pulmonary Arteries ◽

Pulmonary Vasoconstriction ◽

Basal Release ◽

Endothelium Derived Relaxing Factor ◽

Endothelium Dependent Relaxation ◽

Porcine Pulmonary Artery

To examine the hypothesis that suppression of basal release of endothelium-derived relaxing factor (EDRF) by hypoxia might be related to the mechanism of hypoxic pulmonary vasoconstriction, rings of porcine pulmonary artery (PA, 2 mm OD) were suspended in organ chambers and changes in isometric force were measured. Hypoxia significantly reduced endothelium-dependent relaxation induced by acetylcholine and augmented contractile response to phenylephrine. This augmentation by hypoxia was not seen in rings without endothelium. Contractile response to phenylephrine was also enhanced by removal of endothelium. With 15 min of hypoxia, PA contracted and guanosine 3',5'-cyclic monophosphate content decreased. Pretreatment with 10(-6) M methylene blue, 3 x 10(-7) M oxyhemoglobin, and 9.6 x 10(-5) M NG-monomethyl-L-arginine significantly enhanced hypoxic contraction. Furthermore, removal of endothelium also enhanced hypoxic contraction. These results suggest that suppression of basally released EDRF by hypoxia was not the cause of the contractile response to hypoxia and that EDRF modulates the hypoxic contraction of porcine PA in basal conditions at this diameter.

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The Contractile Response of Isolated Small Pulmonary Arteries Induced by Activated Macrophages

Physiological Research ◽

10.33549/physiolres.932698 ◽

2014 ◽

pp. 267-270

Author(s):

M. ŽALOUDÍKOVÁ ◽

J. HERGET ◽

M. VÍZEK

Keyword(s):

Contractile Response ◽

Hypoxic Pulmonary Vasoconstriction ◽

Pulmonary Arteries ◽

Activated Macrophages ◽

Pulmonary Vasoconstriction ◽

In Vitro Response ◽

Ros Scavenger ◽

Membrane Bound ◽

Intravascular Pressure

To test whether macrophages can play any role in hypoxic pulmonary vasoconstriction, we tested the in vitro response of rings from small pulmonary arteries to the activation of macrophages by FMLP, a substance stimulating predominantly membrane-bound NADPH oxidase. A small vessel myograph was used to measure the responses of rings from small pulmonary arteries (300-400 μm) isolated from rat lungs. Rings from 5 rats were placed into both chambers of the myograph. The vessels were stabilized for 40 min and then normalized by automatic stretching to a wall tension equivalent to the intravascular pressure 30 mm Hg. At the start of each experiment, vessels were exposed to 80 mM K+ to obtain maximal contractile response, which was used to normalize subsequent contractile responses. 2x106 viable macrophages, obtained by peritoneal lavage, were added into one chamber, then 5 μM FMLP was administrated to both chambers and the tension measurement was started. The hydrogen peroxide concentration produced by stimulated macrophages was measured luminometrically. The concentrations of H2O2 in specimens from chambers containing activated macrophages rose from 3.5±1.5 nM to 110±28 nM within 25 min of stimulation, while FMLP itself didn’t increase the H2O2 concentration from the baseline value (4.5±3 nM) in samples from control chambers. After FMLP administration, the tension of the vessel rings in the presence of macrophages reached 0.23±0.07 of maximal contractile response, it did not change in controls. The addition of ROS scavenger 4-hydroxy-TEMPO blocked the contractile response to the activation of macrophages. We conclude that the activation of macrophages stimulates the contraction of small pulmonary arteries and that this contraction is probably mediated by reactive oxygen species.

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Precursors and inhibitors of hydrogen sulfide synthesis affect acute hypoxic pulmonary vasoconstriction in the intact lung

Journal of Applied Physiology ◽

10.1152/japplphysiol.01049.2011 ◽

2012 ◽

Vol 112 (3) ◽

pp. 411-418 ◽

Cited By ~ 38

Author(s):

Jane A. Madden ◽

Susan B. Ahlf ◽

Mark W. Dantuma ◽

Kenneth R. Olson ◽

David L. Roerig

Keyword(s):

Hydrogen Sulfide ◽

Arterial Pressure ◽

Lung Tissue ◽

Hypoxic Pulmonary Vasoconstriction ◽

Acute Hypoxia ◽

Pulmonary Arteries ◽

Tissue Homogenate ◽

Rat Lung ◽

Western Blots ◽

Pulmonary Vasoconstriction

The effects of hydrogen sulfide (H2S) and acute hypoxia are similar in isolated pulmonary arteries from various species. However, the involvement of H2S in hypoxic pulmonary vasoconstriction (HPV) has not been studied in the intact lung. The present study used an intact, isolated, perfused rat lung preparation to examine whether adding compounds essential to H2S synthesis or to its inhibition would result in a corresponding increase or decrease in the magnitude of HPV. Western blots performed in lung tissue identified the presence of the H2S-synthesizing enzymes, cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST), but not cystathionine β-synthase (CBS). Adding three H2S synthesis precursors, cysteine and oxidized or reduced glutathione, to the perfusate significantly increased peak arterial pressure during hypoxia compared with control ( P < 0.05). Adding α-ketoglutarate to enhance the 3-MST enzyme pathway also resulted in an increase ( P < 0.05). Both aspartate, which inhibits the 3-MST synthesis pathway, and propargylglycine (PPG), which inhibits the CSE pathway, significantly reduced the increases in arterial pressure during hypoxia. Diethylmaleate (DEM), which conjugates sulfhydryls, also reduced the peak hypoxic arterial pressure at concentrations >2 mM. Finally, H2S concentrations as measured with a specially designed polarographic electrode decreased markedly in lung tissue homogenate and in small pulmonary arteries when air was added to the hypoxic environment of the measurement chamber. The results of this study provide evidence that the rate of H2S synthesis plays a role in the magnitude of acute HPV in the isolated perfused rat lung.

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Acute hypoxia increases intracellular [Ca2+] in pulmonary arterial smooth muscle by enhancing capacitative Ca2+ entry

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00448.2004 ◽

2005 ◽

Vol 288 (6) ◽

pp. L1059-L1069 ◽

Cited By ~ 96

Author(s):

Jian Wang ◽

Larissa A. Shimoda ◽

Letitia Weigand ◽

Wenqian Wang ◽

Dejun Sun ◽

...

Keyword(s):

Smooth Muscle ◽

Hypoxic Pulmonary Vasoconstriction ◽

Acute Hypoxia ◽

Primary Cultures ◽

Fluorescent Microscopy ◽

Pulmonary Arteries ◽

Arterial Smooth Muscle ◽

Fura 2 ◽

Pulmonary Arterial ◽

Pulmonary Arterial Smooth Muscle

Hypoxic pulmonary vasoconstriction (HPV) requires influx of extracellular Ca2+ in pulmonary arterial smooth muscle cells (PASMCs). To determine whether capacitative Ca2+ entry (CCE) through store-operated Ca2+ channels (SOCCs) contributes to this influx, we used fluorescent microscopy and the Ca2+-sensitive dye fura-2 to measure effects of 4% O2 on intracellular [Ca2+] ([Ca2+]i) and CCE in primary cultures of PASMCs from rat distal pulmonary arteries. In PASMCs perfused with Ca2+-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca2+ stores in sarcoplasmic reticulum and nifedipine to prevent Ca2+ entry through L-type voltage-operated Ca2+ channels (VOCCs), hypoxia markedly enhanced both the increase in [Ca2+]i caused by restoration of extracellular [Ca2+] and the rate at which extracellular Mn2+ quenched fura-2 fluorescence. These effects, as well as the increased [Ca2+]i caused by hypoxia in PASMCs perfused with normal salt solutions, were blocked by the SOCC antagonists SKF-96365, NiCl2, and LaCl3 at concentrations that inhibited CCE >80% but did not alter [Ca2+]i responses to 60 mM KCl. In contrast, the VOCC antagonist nifedipine inhibited [Ca2+]i responses to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca2+]i caused by hypoxia was completely reversed by perfusion with Ca2+-free KRBS. LaCl3 increased basal [Ca2+]i during normoxia, indicating effects other than inhibition of SOCCs. Our results suggest that acute hypoxia enhances CCE through SOCCs in distal PASMCs, leading to depolarization, secondary activation of VOCCs, and increased [Ca2+]i. SOCCs and CCE may play important roles in HPV.

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Abstract 276: Heme Oxygenase-1 Induction Modulates Hypoxic Pulmonary Vasoconstriction

Circulation ◽

10.1161/circ.116.suppl_16.ii_35-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Mansoor Ahmad ◽

Nader G Abraham ◽

Michael S Wolin

Keyword(s):

Organ Culture ◽

Heme Oxygenase ◽

Cobalt Chloride ◽

Hypoxic Pulmonary Vasoconstriction ◽

Pulmonary Arteries ◽

Heme Oxygenase 1 ◽

Force Development ◽

Pulmonary Vasoconstriction ◽

Copper Chelator ◽

Relaxing Effect

Endothelium removed Bovine pulmonary arteries (BPA) contract to hypoxia through a mechanism potentially involving lowering of superoxide-derived hydrogen peroxide and removing its basal relaxing effect. Induction of heme oxygenase-1 (HO-1) in BPA by 24 hr organ culture with 0.1mM cobalt chloride was accompanied by a decrease in 5μM lucigenin-detectable superoxide and an increase in horseradish peroxidase-luminol detectable peroxide levels. Force development to 20mM KCl in BPA was not affected by HO-1, but hypoxic pulmonary vasoconstriction (HPV) was significantly reduced. Organ culture with a HO-1 inhibitor (10μM chromium mesoporphyrin) reversed the effects of HO-1 on HPV and peroxide. Pretreatment of BPA with a copper chelator 10mM diethyldithiocarbamate (DETCA) to inactivate Cu,Zn-SOD, prevented the conversion of superoxide to peroxide, and attenuated HPV. DETCA treatment increased superoxide and decreased peroxide to similar levels in control and HO-1 induced BPA. Peroxide scavenging with 0.1mM ebselen increased force development to 20mM KCl and partially reversed the decrease in HPV seen on induction of HO-1. Thus HO-1 induction in BPA causes an increase in superoxide scavenging by Cu,Zn-SOD resulting in increased levels of peroxide, leading to an attenuation of HPV. The generation of superoxide in BPA is not affected by HO-1 induction as DETCA treated control and HO-1 BPA show similar levels of superoxide. Thus, HO-1 induction appears to attenuate HPV in BPA by increasing the conversion of superoxide to peroxide, leading to peroxide levels which may not be adequately lowered by hypoxia.

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Hypoxic induction of Ca2+-dependent action potentials in small pulmonary arteries of the cat

Journal of Applied Physiology ◽

10.1152/jappl.1985.59.5.1389 ◽

1985 ◽

Vol 59 (5) ◽

pp. 1389-1393 ◽

Cited By ~ 73

Author(s):

D. R. Harder ◽

J. A. Madden ◽

C. Dawson

Keyword(s):

Action Potentials ◽

Hypoxic Pulmonary Vasoconstriction ◽

Pulmonary Arteries ◽

Input Resistance ◽

Potential Generation ◽

Applied Current ◽

Pulmonary Vasoconstriction ◽

Ionic Conductances ◽

The Relationship ◽

K Permeability

Small pulmonary arteries (less than 300 micron) from cats were mounted in myographs to record mechanical and electrical responses to hypoxia. When these preparations were exposed to a PO2 of 30–50 Torr after equilibration at 300 Torr they consistently developed active force, which increased or decreased in amplitude as [Ca2+] was raised or lowered, respectively, and was blocked on addition of verapamil. Intracellular electrical recording with glass microelectrodes demonstrated membrane depolarization and action potential generation when PO2 was lowered. Steady-state voltage vs. applied current curves obtained before and during hypoxia showed a significant reduction in input resistance. The relationship between membrane potential and extracellular K+ was not different during hypoxia compared with control, suggesting that there were not marked changes in K+ permeability under this condition. In the presence of verapamil to block Ca2+ inward current the hypoxia-induced action potentials were abolished concomitant with partial membrane repolarization. The results of these studies suggest that in certain isolated pulmonary arteries hypoxia induces contraction by a mechanism involving an increased Ca2+ conductance. These data suggest that the sensor involved in hypoxic pulmonary vasoconstriction may lie within the vessel wall and somehow mediates changes in smooth muscle ionic conductances.

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Development of the pulmonary vasculature in newborn lambs: structure-function relationships

Journal of Applied Physiology ◽

10.1152/jappl.1991.70.3.1255 ◽

1991 ◽

Vol 70 (3) ◽

pp. 1255-1264 ◽

Cited By ~ 18

Author(s):

R. P. Michel ◽

J. B. Gordon ◽

K. Chu

Keyword(s):

Light Microscopy ◽

Hypoxic Pulmonary Vasoconstriction ◽

Venous Pressure ◽

Pulmonary Arteries ◽

Muscle Thickness ◽

Pulmonary Vasculature ◽

Pressure Gradients ◽

Middle Segment ◽

Pulmonary Vasoconstriction ◽

Quantitative Light Microscopy

Our objectives were 1) to describe the quantitative light microscopy and ultrastructure of newborn lamb lungs and 2) to correlate hemodynamic changes during normoxia and hypoxia with the morphology. By light microscopy, we measured the percent muscle thickness (%MT) and peripheral muscularization of pulmonary arteries and veins from 25 lambs aged less than 24 h, 2-4 days, 2 wk, and 1 mo. At the same ages, lungs were isolated and perfused in situ and, after cyclooxygenase blockade with indomethacin, total, arterial (delta Pa), middle (delta Pm), and venous pressure gradients at inspired O2 fractions of 0.28 (mild hyperoxia) and 0.04 (hypoxia) were determined with inflow-outflow occlusion. During mild hyperoxia, delta Pa and delta Pm fell significantly between 2-4 days and 2 wk, whereas during hypoxia, only delta Pm fell. The %MT of all arteries (less than 50 to greater than 1,000 microns diam) decreased, and peripheral muscularization of less than 100-microns-diam arteries fell between less than 4 days and greater than 2 wk. Our data suggest that 1) the %MT of arteries determines normoxic pulmonary vascular resistance, because only arterial and middle segment resistance fell, 2) peripheral muscularization is a major determinant of hypoxic pulmonary vasoconstriction, because we observed a fall with age in peripheral muscularization of less than 100-micron-diam arteries and in delta Pm with hypoxia, and 3) the arterial limit of the middle segment defined by inflow-outflow occlusion lies in 100- to 1,000-microns-diam arteries.

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Responses of isolated guinea pig pulmonary venules to hypoxia and anoxia

Journal of Applied Physiology ◽

10.1152/jappl.1989.67.5.2147 ◽

1989 ◽

Vol 67 (5) ◽

pp. 2147-2153 ◽

Cited By ~ 8

Author(s):

W. R. Tracey ◽

J. T. Hamilton ◽

I. D. Craig ◽

N. A. Paterson

Keyword(s):

Guinea Pig ◽

Hypoxic Pulmonary Vasoconstriction ◽

Pulmonary Arteries ◽

Major Site ◽

Sustained Contraction ◽

Pulmonary Vasoconstriction ◽

Ph Dependent

Because small pulmonary arteries are believed to be the major site of hypoxic pulmonary vasoconstriction (HPV), pulmonary venular responses to hypoxia have received little attention. Therefore the responses of isolated guinea pig pulmonary venules to hypoxia (bath PO2, 25 Torr) and anoxia (bath PO2, 0 Torr) were characterized. Pulmonary venules [effective lumen radius (ELR), 116 +/- 2 microns] with an adherent layer of parenchyma responded to hypoxia and anoxia with a graded sustained contraction (hypoxia, 0.03 +/- 0.01; anoxia, 0.26 +/- 0.03 mN/mm), whereas paired femoral venules (ELR, 184 +/- 7 microns) contracted to anoxia only (0.05 +/- 0.02 mN/mm). Repeated challenges with hypoxia and anoxia continued to elicit sustained pulmonary venular contractions; femoral venule contractions to anoxia were not repeatable. Hypoxia- and anoxia-induced pulmonary venular contractions were calcium and pH dependent. Dissection of the parenchyma from pulmonary venules did not alter contractions to decreased PO2. Anoxic contractions of pulmonary venules were variably reduced by replacement of the bath fluid; however, the release of a contractile mediator(s) from pulmonary venules during hypoxia or anoxia was not demonstrated. Pulmonary venular responses to hypoxia and anoxia are similar to those induced by hypoxia in vivo, and results obtained from this model may be useful in predicting mechanisms of HPV.

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