Ca-activated K channels by the intracellular redox potential in
pulmonary and ear arterial smooth muscle cells of the rabbit.
Eur } Physiol 1995; 430:308-14
26 Yuan X-J, Tod M, Rubin L, et al. Deoxyglucose and reduced
glutathione mimic effects of hypoxia on K+ and Ca2+ con¬
ductances in pulmonaiy artery cells. Am J Physiol 1994;
2994:L52-63
27 Archer S, Huang J, Henry T, et al. A redox-based 02 sensor
in rat pulmonary vasculature. Circ Res 1993; 73:1100-12
28 Archer S, Nelson D, Weir E. Simultaneous measurement of
02 radicals and pulmonary vascular reactivity in rat lung.
J Appl Physiol 1989; 67:1903-11
Paky A, Michael }, Burke-Wolin T, et al. Endogenous pro¬
duction of superoxide by rabbit lungs: effects of hypoxia or
metabolic inhibitors. J Appl Physiol 1993; 74:2868-74
Mohazzab HK, Wolin M. Properties of a superoxide aniongenerating microsomal NADH oxidoreductase, a potential pul¬
monary arteiy Po2 sensor. Am J Physiol 1994; 267:L823-31
Marshall C, Mamary A, Verhoeven A, et al. Pulmonaiy artery
NADPH-oxidase is activated in hypoxic pulmonaiy vasocon¬
striction. Am J Respir Cell Moi Biol 1996; 15:633-44
29
30
31
32 Mohazzab HK, Fayngersh R, Kaminski P. Potential role of
NADPH oxidoreductase-derived reactive 02 species in calf
pulmonary arterial Po2-elicited responses. Am J Physiol 1995;
269:L637-44
Gatley S, Sherratt H. The effects of diphenyleneiodonium on
mitochondrial reactions. Biochem J 1976; 158:307-15
34 Stuehr D, Fasehun O, Kwon N, et al. Inhibition of macro¬
phage and endothelial cell nitric oxide synthase by diphenyle¬
neiodonium and its analogs. FASEB } 1991; 5:98-103
35 Cross A, Henderson L, Jones O, et al. Involvement of an
NAD(P)H oxidase as a Po2 sensor protein in the rat carotid
body. Biochem J 1990; 272:743-47
36 Thomas H III, Carson R, Fried E, et al. Inhibition of hypoxic
33
pulmonary
by diphenyleneiodonium. Bio¬
chem Pharmacol 1991; 42:R9-12
37 Youngson C, Nurse C, Yeger H, et al. Oxygen sensing in
airway chemoreceptors. Nature 1993; 365:153-55
38 Grimminger F, Weissmann N, Spriestersbach R, et al. Effects
of NADPH oxidase inhibitors on hypoxic vasoconstriction in
buffer-perfused rabbit lungs. Lung Cell Moi Physiol 1995;
12:L747-52
39 Weir E, Wyatt C, Reeve H, et al. Diphenyleneiodonium
inhibits both potassium and calcium currents in isolated
pulmonary artery smooth muscle cells. J Appl Physiol 1994;
vasoconstriction
76:2611-15
40 Acker
H, Xue D. Mechanisms of 02 sensing
in the carotid
46 Rounds
S, McMurtry I. Inhibitors of oxidative ATP pro¬
duction cause transient vasoconstriction and block subse¬
quent pressor responses in rat lungs. Circ Res 1981;
48:393-400
47 Gorlach A, Holtermann G,
48
49
50
51
Jelkmann W, et al. Photometric
characteristics of haem proteins in erythropoielin-producing
hepatoma cells (HepG2). Biochem J 1993; 290:771-76
McCormack T, McCormack K. Shaker K+ channel B subunits belong to an NAD(P)H-dependent oxidoreductase su¬
perfamily. Cell 1994; 79:1133-35
Kummer W, Acker H. Immunohistochemical demonstration
of four subunits of neutrophil NAD(P)H oxidase in type I
cells of carotid body. ] Appl Physiol 1995; 78:1904-09
Gleadle J, Ebert B, Ratcliffe P. Diphenylene iodonium
inhibits the induction of erythropoietin and other mammalian
genes by hypoxia: implications for the mechanism of oxygen
sensing. Eur J Biochem 1995; 234:92-99
Meier B, Jesaitis A, Emmendorffer A, et al. The cytochrome
b-558 molecules involved in the fibroblast and polymorphonuclear leucocyte superoxide-generating NADPH oxidase
systems are structurally and genetically distinct. Biochem J
1993; 289:481-86
52 McMurtry I, Davidson B, Reeves
J,
et
al. Inhibition of
hypoxic pulmonary vasoconstriction by calcium antagonists in
isolated rat lungs. Circ Res 1976; 38:99-104
53 Franco-Obregon A, Lopez-Barneo J. Differential oxygen
sensitivity of calcium channels in rabbit smooth muscle cells
of conduit and resistance pulmonary arteries. J Physiol 1996;
491:511-18
54 Archer S, Huang J, Reeve H, et al. Differential distribution of
electrophysiologically distinct myocytes in conduit and resis¬
tance arteries determines their response to nitric oxide and
hypoxia. Circ Res 1996; 78:431-42
Huang J, et al. Anorexic agents aminorex,
fenfluramine, and dexfenfluramine inhibit potassium current
55 Weir E, Reeve H,
pulmonary
in rat
vascular smooth muscle and cause
nary vasoconstriction. Circulation 1996; 94:2216-20
pulmo¬
Oxidant Stress Stimulates Na/K
Pump Activity in Bovine
Pulmonary Arterial Endothelial
Cells*
body in comparison with other 02-sensing cells. News Physiol
Sci
41
1995; 10:211-16
Wang D, Youngson C, Wong V, et al. NADPH-oxidase and a
hydrogen peroxide-sensitive K+ channel may function as an
oxygen sensor complex in airway chemoreceptors and small
cell lung carcinoma cell lines. Proc Natl Acad Sci USA 1996;
93:13182-87
42 Weir E, Archer S. The mechanism of acute hypoxic pulmo¬
vasoconstriction: the tale of two
nary
channels. FASEB
1995; 9:183-89
43
J
Meury J, Robin A. Glutathione-gated K+ channels of Esche¬
richia coli carry out K+ efflux controlled by the redox state of
the cell. Arch Microbiol 1990; 154:475-82
44 Kuo S, Saad A, Koong A, et al. Potassium-channel activation
in response to low doses of irradiation involves reactive
oxygen intermediates in nonexcitatory cells. Proc Natl Acad
Sci USA 1993; 90:908-12
45 Vega-Saenz de Miera E, Rudy B. Modulation of K+ channels
by hydrogen peroxide. Biochem Biophys Res Commun 1992;
186:1681-87
22S
Thomas L.
Sharon Rounds, MD; Michael Cutaia, MD, FCCP;
Amos
Charles, MD; Joseph Meharg, MD;
Oldmixon, PhD; and
Doloretta Dawicki, PhD; Eben
Charles Kuhn, MD
(CHEST 1998; 114:22S-24S)
\M aintenance of normal intracellular/extracellular Na+
*¦**. and K+
gradients is of vital importance to all
mammalian cells. The Na+ gradient provides energy for
Na+-coupled transport of nutrients and other sub¬
stances into cells, including transport of glucose, amino
*From the
Pulmonary/Critical
Care Section
(Drs. Rounds,
Cutaia, Charles, Meharg, and Dawicki), Providence Veterans
Affairs Medical Center, Providence, RI; and the Department of
Pathology (Drs. Oldmixon and Kuhn), Memorial Hospital of
Rhode Island, Pawtucket, and Brown University School of
Medicine, Providence, RI.
Petty 40th Annual Aspen Lung Conference: Biology & Pathobiology of the Lung Circulation
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acids, and serotonin. Gradients of Na+ and H+ govern
the movement of other ions, such as Ca2+ across cell
membranes. Osmotic balance and cell volume are
dependent on normal ion gradients. The sodium-potassium-adenosine-triphosphate pump (Na/K pump, E.C.
3.6.1.3) is primarily responsible for maintenance of Na+
and K+ gradients.1 Thus, function of the Na/K pump is
critically important in cell homeostasis in the face of
injury. Maintenance of cell homeostasis is a crucial
component of endogenous mechanisms of resistance to
cell injury. Endothelial cell injury from inflammatory
processes may be important in the development of some
types of pulmonary hypertension.
The Na/K pump is a glycosylated heterodimer that
traverses the plasma membrane of nearly all mammalian
cells.1 The pump functions in a cyclic manner, trans¬
porting two moles of K+ intracellularly for every three
moles of Na+ moved extracellularly, a process requiring
Mg++ as a cofactor and the hydrolysis of adenosine
triphosphate (ATP).1 During its cycle, the pump is
phosphorylated at aspartate 376 of the a subunit (cat¬
alytic site phosphorylation). The pump is a heterodimer
consisting of a and (3 subunits.2-3 The a subunit exists in
ax, a2, and a3 isoforms, and is the site of cation and
ouabain binding and of catalytic site phosphorylation
during activation.3 The glycosylated, smaller (3 subunit
may be important in stabilization of the pump in the cell
membrane.2 A y subunit of unknown function has also
been reported.3
The activity of Na/K pump can be controlled in both
long- and short-term fashions. Long-term control involves
regulation of tissue expression of subunit isoforms, such as
that occurring in Type II epithelial cells under hyperoxic
conditions.4 Short-term regulation (<30 min) may be
mediated by changes in intracellular Na+ concentration,5
changes in subcellular distribution of pump units, by
reversible noncatalytic site phosphorylation of a subunit,
or by changes in association of pump with cytoskeletal
Results
We found that ax subunit
antibody bound
to intact
increased in the perinumonolayers
clear area.7 This binding pattern indicates that intact
pump is not localized on lateral aspects of endothelial
cells, in contrast to basolateral binding reported on epi¬
thelium. Using confocal lasar scanning microscopy, we
found cq subunit on both apical and basilar cell surfaces,
suggesting that the role of pump in endothelial cells is
"housekeeping" and maintenance of homeostasis, rather
than transcapillary ion flux.7
We found that short-term exposure (30 min) to reagent
H202 and xanthine/xanthine oxidase stimulate pulmonary
arterial endothelial Na/K pump activity (Fig l).8
Hydrogen peroxide decreased intracellular ATP con¬
tent; thus, changes in ATP content did not account for
increased pump activity.8 Scatchard analysis indicated that
the number of endothelial cell 3H-ouabain binding sites
was decreased by H202.8 Cell membrane expression of ax
Na/K pump subunit, as assessed by Western blots, was not
altered by H202.7 Thus, increased numbers of membrane
pump sites did not account for oxidant-induced enhance¬
ment of Na/K pump activity.
To assess noncatalytic site phosphorylation of a2 subunit, pulmonaiy arteiy endothelial cell cultures were
prelabeled with 32P, followed by exposure to H202 for 30
min. Oxidant stress did not change phosphorylation of
immunoprecipitated a: subunit.7 Thus, noncatalytic site
did not account for oxidant-induced in¬
phosphorylation
creases in Na/K pump activity.
Finally, we found that 30 min of exposure of endothelial
cells to H202 increased the influx of 22Na into cells. This
was not associated with increased 51Cr release, indicating
that cell permeability was otherwise intact.8
in a punctate fashion,
Discussion
These results indicate that oxidant stress stimulates endo¬
thelial Na/K pump activity because of increased intracellular
structures.6
Materials
and
Methods
100
Little is known about the function or modulation of Na/K
pump activity in vascular endothelial cells. Because little is known
regarding the distribution of vascular endothelial Na/K pump, we
used ax subunit as an immunologic marker for Na/K pump to
characterize the distribution of the pump in cultured bovine
pulmonary arterial endothelial cells, using immunofluorescence
microscopy and confocal scanning laser microscopy.
To determine the effects of oxidant stress on short-term
modulation of Na/K pump activity, we assessed the effects of
xanthine/xanthine oxidase and reagent hydrogen peroxide
(H202) on ouabain-inhibitable uptake of 86Rb by intact endo¬
thelial monolayers. To determine the mechanism of oxidantinduced changes in Na/K pump activity, we assessed intracel¬
lular content of ATP, influx of 22Na, the number of binding
sites of 3H-ouabain to intact monolayers, cell membrane
expression of aY subunit (by comparing immunoblots of cell
membrane preparations), and phosphorylation of a1 subunit
immunoprecipitates from monolayers that had been incubated
lLH H-Ll
CONTROL
X/XO
Figure 1. Confluent cultures of pulmonary artery endothelial cells
incubated for 30 min in Eagle's minimum essential medium
were
and 86Rb in the presence or absence of ouabain (0.1 mM), xanthine
(100 |xM), and/or xanthine oxidase (0.0153 U/mL). Pump activity
(nmol K+/106 cells/30 min) was calculated as difference between
86Rb uptake in absence and presence of ouabain.
CHEST / 114 / 1 / JULY, 1998 SUPPLEMENT
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23S
influx of Na+. Others have reported similar results using the
tert-butyl hydroperoxide model of endothelial cell injury.9
Intracellular concentration of Na+ is an important determi¬
nant of cell replication in the face of injury. Changes in Na/K
pump activity are likely important in the maintenance of
endothelial cell homeostasis after oxidant stress, such as that
encountered with vascular inflammation.
1
References
Horisberger J-D, Lemas V, Kraehenbuhl J-P, et al. Structurefunction relationship of Na/K ATPase. Ann Rev Physiol 1991;
53:565-84
Schmalzing G, Gloor S. Na+/K+-pump beta subunits: struc¬
and functions. Cell Physiol Biochem 1994; 4:96-114
3 Vasilets LA, Schwarz W. The Na+/K+ pump: structure and
function of the alpha-subunit. Cell Physiol Biochem 1994;
4:81-95
4 Nici L, Dowin R, Gilmore-Hebert M, et al. Upregulation of
rat lung Na-K-ATPase during hyperoxic injury. Am J Physiol
1991; 26LL307-14
5 Haber RS, Pressley TA, Loeb JN, et al. Ionic dependence of
active Na-K transport: 'clamping" of cellular Na+ with monensin. Am J Physiol 1987; 253:F26-33
6 Bertorello AM, Katz AL Short-term regulation of renal
Na-K-ATPase activity: physiological relevance and cellular
mechanisms. Am J Physiol 1993; 265:F743-55
7 Charles A, Dawicki DD, Oldmixon E, et al. Studies on the
mechanism of short-term regulation of pulmonary artery
endothelial cell Na/K pump activity. J Lab Clin Med 1997;
130:157-68
8 Meharg JV, McGowan-Jordan J, Charles A, et al. Hydrogen
peroxide stimulates sodium-potassium pump activity in cul¬
tured pulmonary arterial endothelial cells. Am J Physiol 1993;
265:L613-21
9 Elliott SJ, Schilling WP. Oxidant stress alters Na+ pump and
Na+-K+-Cl~ cotransporter activities in vascular endothelial
cells. Am J Physiol 1992; 263:H96-102
2
ture
Table 1.BPAF Results
Incorp,
[3H]Thy
% Control*
Normoxic
Control
ET-1 (KT7 M)
PDGF (3 ng/mL)
100
100
612
623
4,227
5,615
But Not in Those From the
Mesenteric Circulation*
Andrew J. Peacock, MD; P. Scott; R. Plevin; R. Wadsworth;
and D. Welsh, BSc
(CHEST 1998; 114:24S)
TTypoxia,
whether due
remodelling
of all three
to
altitude
or
chronic
layers
of the
pulmonary arteries,
*From the Pulmonary Vascular Unit, Department of Respiratory
Medicine, Western Infirmary, and the Department of Physiol¬
ogy and Pharmacology, University of Strathclyde, Glasgow,
Scotland.
Supported by the British Lung Foundation.
24S
Thomas L
Normoxic
Hypoxic
100
100
100
150
150
225
which renders them unresponsive to vasodilators. We have
previously shown that hypoxia increases the rate of repli¬
cation of pulmonary arteiy fibroblasts.1 We therefore
wished to determine whether the same effect is seen with
cells from systemic arteries and whether the effects of
hypoxia on replication are mirrored by changes in intra¬
cellular signalling.
Methods
We harvested bovine pulmonary arteiy fibroblasts
(BPAF) and bovine mesenteric artery fibroblasts (BMAF)
and utilized them between passages 3 to 10. Cells were
quiesced for 48 h and then stimulated by hypoxia for
24 h (Po2=20 mm Hg) with or without endothelin-1
(ET-1) (10~7 M) or platelet-derived growth factor
(PDGF) (3 ng/mL). Fibroblast replication was measured
by [3H]thymidine uptake. Inositol r,4',5'-trisphosphate
(IP3) generation was determined using the method of
Palmer and colleagues2.
Results
[3H]Thymidine incorporation was increased by hypoxia
(results expressed as percent
of control) (p<0.05). Peak IP3 levels occurred 10 to 30 s
after stimulation. In BPAF cells, hypoxia alone caused a
rise in control IP3 and also enhanced the effect of PDGF
but not of ET-1. In the BMAF cells, hypoxia did not
change IP3 generation.
Conclusion
Hypoxia stimulated replication and IP3 generation in
BPAF cells but not in BMAF cells. Hypoxia causes
pulmonary vasoconstriction and systemic vasodilation. We
have now shown that hypoxia stimulates replication in
pulmonaiy arteiy but not mesenteric artery cells.
lung
¦*¦-¦¦ disease, causes pulmonary hypertension (PH) but has
no effect on systemic arteries. The PH is accompanied by
% Control
*[3H]Thy incorp=[3H]Thymidine incorporatition.
in BPAF but not BMAF cells
Hypoxia Enhances Proliferation
and Generation of IP3 in
Pulmonary Artery Fibroblasts
Hypoxic
IP3 Generation,
1
References
Welsh D. Effects of hypoxia on IP3 generation and DNA
synthesis in bovine pulmonary artery fibroblasts. Am J Respir
Crit Care Med 1996; -153:A576
2 Palmer S, Hughes KT, Lee DY, et al. Development of a novel,
Ins(l,4,5)P3-specific binding assay: its use to determine the
intracellular concentration of Ins(l,4,5)P3 in unstimulated and
vasopressin-stimulated
1:147-56
rat
hepatocytes.
Cell
Signal 1989;
Petty 40th Annual Aspen Lung Conference: Biology & Pathobiology of the Lung Circulation
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