From the University Department of Pharmacology,
University of Oxford, Mansfield Road, Oxford, OX1 3QT and the
¶ University Laboratory of Physiology, University of Oxford, Parks
Road, Oxford, OX1 3PT, United Kingdom
Received for publication, June 5, 2000, and in revised form, December 13, 2000
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ABSTRACT |
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Hypoxic pulmonary vasoconstriction is unique to
pulmonary arteries and serves to match lung perfusion to ventilation.
However, in disease states this process can promote hypoxic pulmonary
hypertension. Hypoxic pulmonary vasoconstriction is associated with
increased NADH levels in pulmonary artery smooth muscle and with
intracellular Ca2+ release from ryanodine-sensitive
stores. Because cyclic ADP-ribose (cADPR) regulates ryanodine receptors
and is synthesized from Since it was first described over 50 years ago, hypoxic pulmonary
vasoconstriction (HPV)1 has
been recognized as the critical and distinguishing characteristic of
the blood vessels of the lung (1). Thus, in marked contrast to systemic
arteries, which dilate in response to hypoxia, pulmonary arteries
constrict. Physiologically, HPV contributes to the matching of lung
perfusion and ventilation. However, when alveolar hypoxia is global, as
it is in disease states such as cystic fibrosis, emphysema, and
mountain sickness, it results in pulmonary hypertension, which can
ultimately lead to right heart failure. Unfortunately, the precise
mechanisms that underpin HPV remain to be identified, and current
therapies for hypoxic pulmonary hypertension are poor.
Certain key characteristics of HPV have been described. In isolated
pulmonary arteries, HPV is biphasic. An initial transient constriction
(phase 1) is followed by a slowly developing, sustained phase of
constriction (phase 2). It is widely thought that the first phase of
constriction is initiated by a reduction in membrane K+
conductance in pulmonary artery smooth muscle cells (2-4), membrane depolarization, and Ca2+ influx through voltage-gated
Ca2+ channels (5-8). Phase 2 of the constriction is tonic
and may depend on the release of a vasoconstrictor from the
endothelium, which sensitizes the contractile apparatus to
Ca2+ (9, 10).
Our recent findings (11) do not support the above hypothesis. They
suggest that hypoxia may, by activating a mechanism intrinsic to
pulmonary artery smooth muscle cells, induce intracellular Ca2+ release from ryanodine-sensitive stores in the absence
of transmembrane Ca2+ influx. This proposal is also
supported by the findings of others (12, 13). Also, we have established
that the hypoxia-induced SR Ca2+ release initiates and
maintains acute HPV (11). Details of the signal transduction pathway
remain to be clarified. One possibility is that the
We have investigated the role of the We report that the enzyme activities for cADPR synthesis and metabolism
are particularly high in pulmonary artery smooth muscle, as opposed to
systemic artery smooth muscle. We show that hypoxia, by increasing
Male New Zealand White rabbits (1-2 Kg) were stunned and killed
by cervical dislocation. Heart and lungs were removed and placed in PSS
A (124 mM NaCl, 5 mM KCl, 15 mM NaH2CO3, 1.8 mM CaCl2, 1 mM MgCl2, 0.5 mM NaH2PO4, 0.5 mM
KH2PO4, 15 mM HEPES, and 10 mM glucose, pH 7.4, 95% air, 5% CO2).
Pulmonary arteries, mesenteric arteries, and aorta were dissected free
and cleaned.
Homogenates--
The endothelium was removed by rubbing the
luminal surface with a cotton bud, and 2-mm artery strips were cut and
placed in 1 ml of Ca2+-free sucrose-HEPES buffer (250 mM and 20 mM, respectively, pH 7.2). The
preparation was homogenized by 2× 5-s bursts using an ultra-turrex
homogenizer; 2× 5 strokes using a glass Dounce homogenizer (pestle D).
Homogenates were centrifuged for 12 min at 2,000 × g
and 4 °C to remove debris and nuclei. The supernatant was stored at
Smooth Muscle Cell Isolation--
Smooth muscle cells were
isolated as previously described (24). Prior to enzyme assays only,
cells were washed in sucrose-HEPES buffer, centrifuged at 1,000 × g for 5 min at 4 °C, resuspended, and washed once in
(a) phosphate-buffered saline (120 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer salts; pH 7.4),
(b) phosphate-buffered saline plus 1 mM EGTA, or
(c) 100-300 µl of sucrose-HEPES buffer. Buffers contained
protease inhibitors: 25 µg/ml leupeptin, 20 µg/ml aprotinin, and
100 µg/ml soy bean trypsin inhibitor. Cells were permeabilized
with saponin (50 µg/ml) for 30 min at 24 °C. Permeabilization was
confirmed by trypan blue exclusion.
Sea Urchin Egg Homogenate Ca2+ Release
Bioassay--
Homogenates were prepared and "calcium-loaded" in an
intracellular medium (250 mM potassium gluconate, 250 mM N-methyl-glucamine, 20 mM HEPES,
1 mM MgCl2, 1 mM ATP, 10 units/ml
creatine phosphokinase, and 10 mM phosphocreatine, pH 7.2)
as described previously (25, 26). Intracellular medium also contained
protease inhibitors (25 mg/ml leupeptin, 10 mg/ml aprotinin, and 100 µg/ml soy bean trypsin inhibitor) and mitochondrial inhibitors
(1 µg/ml oligomycin, 1 µg/ml antimycin, and 1 mM sodium
azide). Fluo-3 (3 mM) was used as a Ca2+
indicator (excitation = 490 nm, emission = 535 nm). Changes
in free Ca2+ induced by 5-µl test samples were measured
at 17 °C using 500 µl of sea urchin egg homogenate in a
PerkinElmer LS-50B fluorimeter. cADPR was identified by homologous
desensitization of ryanodine receptors (27) and quantified by
interpolation with standard concentration-response curves (28).
Continuous ADP-ribosyl Cyclase Assay--
The substrate was
NGD+ (250 µM). Autofluorescence of the
product, cyclic GDP-ribose, was measured with excitation and emission wavelengths of 300 and 410 nm, respectively (29). Reaction mixture (500 µl) contained test homogenate diluted 1 in 10 with sucrose-HEPES buffer.
Differential Centrifugation--
Homogenates were separated by
differential centrifugation: P1, nuclear and whole cell debris
(1,000 × g); P2, plasma membrane (8,000 × g); P3, microsomal membranes (SR and mitochondria;
100,000 × g); S3, cytosolic fraction (30).
Protein Concentration--
Estimated using the BCA protein assay (Sigma).
Electrophysiology--
Membrane potential recordings in isolated
pulmonary artery smooth muscle cells were made at 22 °C, under
current clamp (I = 0) and using the whole cell
configuration of the patch clamp technique, as described
previously (31). Data were acquired and analyzed using an Axopatch 200B
amplifier and pCLAMP 6.0 data acquisition and analysis software (Axon
Instruments). The pipette solution was 140 mM KCl, and 10 mM HEPES, pH 7.4. The bath solution was 130 mM
NaCl, 5 mM KCl, 2 mM glucose, 10 mM
HEPES, 1.7 mM CaCl2, and 2 mM
MgCl2, pH 7.45.
Small Vessel Myography--
Vessels were always obtained from
the same anatomical position within the lung, i.e. third
order branches of the pulmonary arterial tree (inner diameter, 300-400
µm). Vessels (2-3 mm in length) were mounted on to the jaws of an
automated myograph (AM10, Cambustion Biological, Cambridge, UK)
using 50-µm tungsten wire. Initial tension was set to be equivalent
to typical pulmonary arterial pressure. The detailed technique,
protocol, and theory have been described previously (32). Vessels were
bathed in PSS B (constituents as for PSS A, but
with 24 mM NaHCO3 and no HEPES) at 37 ± 1 °C and in a bath volume of 4 ml. The solution was bubbled with
75% N2, 20% O2, 5% CO2 to
maintain a pH of 7.4.
All arteries were first subjected to four exposures of high
K+ (75 mM) to test their responsiveness and
stability. Resting tension was taken to be zero. The bath chambers
containing PSS B were individually sealed and bubbled with
either normoxic (154-160 Torr; 75% N2, 20%
O2, 5% CO2) or hypoxic (16-21 Torr; 93%
N2, 2% O2, 5% CO2) gas, supplied
via a gas-mixing flowmeter (Cameron Instruments Ltd., Port Aransas,
TX). The desired PaO2 was confirmed using an Oxel
O2 electrode and meter (World Precision
Instruments). All drugs were applied to the bath directly. All
solutions were warmed to 37 °C before adding them to the bath.
When required, the endothelium of the arteries was removed by rubbing
the inner surface with braided silk surgical thread. Removal of the
endothelium was assessed by the ability of 100 µM
acetylcholine to relax constrictions induced by 1 µM
prostaglandin F2 Extraction and Measurement of Endogenous cADPR--
Second and
third order branches of the pulmonary artery were placed in chambers
containing PSS B that were individually sealed and bubbled
with either normoxic (154-160 Torr; 75% N2, 20%
O2, 5% CO2) or hypoxic (16-21 Torr; 93%
N2, 2% O2, 5% CO2) gas as described above (see small vessel myography). Vessels were quickly removed from the experimental chambers and snap frozen in liquid nitrogen. Acid extraction of nucleotides/cADPR was carried out using a
variation of a method described previously (33). Briefly, the frozen
tissue was powdered, added to ice-cold 3 M perchloric acid
(1:1 w/v) and sonicated for 20 s. After sonication the samples were left in an ice-salt bath ( [32P] cADPR Binding Assay--
Enzymatic synthesis
of [32P]cADPR and determination of specific
[32P]cADPR binding to sea urchin egg homogenate were
performed using methods described previously (35, 36).
Materials--
Lytechinus pictus sea urchins were
from Marinus Inc. (Long Beach, CA). Chemicals were from Sigma, except
Fluo-3, which was from Molecular Probes, and
[32P] Statistical Significance--
Statistical significance was
assessed by analysis of variance and assumed if p < 0.05. Data were expressed as the means ± S.E. for n
animals tested unless stated. In biochemical assays each sample was
assayed in triplicate.
Synthesis and Degradation of cADPR Occurs in Pulmonary Artery
Smooth Muscle Homogenates--
Fig.
1A illustrates the time course
of cADPR synthesis from 2.5 mM cADPR Synthesis Occurs at an Intracellular Site--
Because both
ecto-enzymes and intracellular enzymes with ADP-ribosyl cyclase
activities have been reported (37), it was important to show whether or
not cADPR could be synthesized intracellularly, because this would
probably be more relevant to an intracellular signaling role. To
achieve this we studied the enzyme activities in the cellular fractions
obtained from differential centrifugation of smooth muscle homogenates
from third order branches of the pulmonary artery. Fig.
2A shows that the synthesis of
cADPR was detected in each of the membrane fractions. However, the rate of synthesis was highest in the P3 (microsomal; SR and mitochondria) fraction at 9.5 ± 1.1 nmol/mg of protein/h (n = 3 animals), compared with 2.5 ± 0.7 nmol/mg of protein/h
(n = 3 animals) in the P2 fraction (plasma membrane),
0.25 ± 0.01 nmol/mg of protein/h (n = 3 animals)
in the P1 fraction (nuclear and whole cell debris), and 0.01 ± 0.1 nmol/mg of protein/h (n = 3 animals) in the S3 fraction (cytosolic). Fig. 2B shows a similar distribution
for cADPR metabolism. The rate of metabolism was highest in the P3 (microsomal; SR and mitochondria) fraction at 2.6 ± 0.3 nmol/mg of protein/h (n = 3 animals), compared with 0.92 ± 0.02 nmol/mg of protein/h (n = 3 animals) in the P2
fraction (plasma membrane). In contrast, the hydrolase activity in the
P1 fraction (nuclear and whole cell debris; n = 3 animals) and in the S3 fraction (cytosolic; n = 3 animals), respectively, was undetectable. Fig. 2C shows the
distribution of a 5' nucleotidase reaction used to estimate the
relative level of plasma membrane associated protein in all four
fractions. The highest level of 5' nucleotidase activity was 129 ± 19 units/liter for the P2 (plasma membrane) fraction (n = 3 animals), compared with 86 ± 5.5 U/liter
(n = 3 animals) for the P3 (microsomal) fraction and
56 ± 2.2 U/liter (n = 3 animals) for the P1
(nuclear and whole cell debris) fraction. Clearly, the P2 fraction had
a much lower level of ADP-ribosyl cyclase and cADPR hydrolase activity
than the P3 pellet. The ADP-ribosyl cyclase and cADPR hydrolase
activity did not, therefore, follow the plasma membrane
contamination.
Fig. 2D shows cADPR synthesis using whole and saponin (50 µg/ml) permeabilized pulmonary artery smooth muscle cells isolated from the large extrapulmonary artery. A significantly greater level of
synthesis was detected in permeabilized smooth muscle cells. Thus,
after a 120-min incubation with 2.5 mM ADP-ribosyl Cyclase and Hydrolase Activity Is Differentially
Distributed in Pulmonary and Systemic Artery Smooth Muscle--
Fig.
3A compares the level of cADPR
synthesis by homogenates of pulmonary and systemic arterial smooth
muscle following a 1-h incubation with 2.5 mM
Modulation by Hypoxia Increases cADPR Content in Pulmonary Artery Smooth
Muscle--
We used a sea urchin egg homogenate
[32P]cADPR receptor binding assay to compare the cADPR
content of pulmonary artery smooth muscle under normoxic (151-160
Torr) and hypoxic (16-21 Torr) conditions. Fig.
5 shows that the level of cADPR in second
order branches of the pulmonary artery increased ~2-fold in the
presence of hypoxia from 1.5 ± 0.4 pmol/mg protein to 3.6 ± 0.5 pmol/mg protein (n = 3 animals). Significantly, the
increase in cADPR induced by hypoxia was greater still in third order
branches of the pulmonary artery, in which we measured a 10-fold
increase in cADPR from 1.5 ± 0.6 pmol/mg protein to 19.7 ± 1.1 pmol/mg protein (n = 3 animals).
cADPR Releases Ca2+ from Ryanodine-sensitive SR Stores
in Isolated Pulmonary Artery Smooth Muscle Cells--
We examined the
sensitivity of pulmonary artery smooth muscle Ca2+ stores
to cADPR. This was achieved by applying cADPR intracellularly from a
patch pipette in the whole cell configuration and in current clamp mode
(I = 0). Changes in intracellular Ca2+ were
monitored by recording changes in the membrane potential mediated via
the activation of large conductance Ca2+-activated
potassium channels (BKCa) in smooth muscle cells isolated from second and third order branches of the pulmonary artery. In the
absence of cADPR the membrane potential was stable at approximately 8-Bromo-cADPR Inhibits Hypoxic Pulmonary Vasoconstriction in
Pulmonary Artery Rings in Vitro--
We next investigated the effect
of a membrane-permeant antagonist of cADPR, 8-bromo-cADPR (25), on HPV
in isolated rabbit pulmonary artery rings. Fig.
7A shows that hypoxia induced
a characteristic biphasic constriction in an intact pulmonary artery
ring. Fig. 7B shows the response obtained in the absence of
the pulmonary artery endothelium. The initial fast transient
constriction seen in Fig. 7A remains, but the second slowly
developing, tonic phase of constriction is lost. Instead the
constriction falls to a plateau level above base line that is
maintained for the duration of exposure to hypoxia. Previous studies
(11) have shown that the initial fast transient constriction and the
maintained plateau rely on Ca2+ release from
ryanodine-sensitive SR stores. Panels C and D of Fig. 7 show the effect on the hypoxic constriction of preincubating (10 min) intact (C) and de-endothelialized (D)
arteries with 300 µM 8-bromo-cADPR. Panels E
and F of Fig. 7 show the mean (1-min intervals) ± S.E.
(5-min intervals) for the constriction obtained in the presence and
absence of 300 µM 8-bromo-cADPR, both with (E)
and without (F) the endothelium. The peak of the fast
transient constriction remained unaffected in the presence of 300 µM 8-bromo-cADPR (n = 6 arteries). In
marked contrast, the second phase of the hypoxia-induced constriction
was abolished both in the presence and absence of the endothelium
(n = 6 arteries). These data suggest that prolonged,
cADPR-dependent SR Ca2+ release is required to
maintain hypoxia-induced constriction of pulmonary artery smooth
muscle. However, the full development of phase 2 of HPV also requires
the release of an endothelium-derived vasoconstrictor (10). Because
8-bromo-cADPR abolished phase 2 of HPV when the endothelium was
present, we cannot rule out that this was due, in part, to its
inhibition of vasoconstrictor release from the endothelium or the
blockade of the action of the vasoconstrictor in the smooth muscle. We
know that the endothelium-derived vasoconstrictor induces constriction
by sensitizing the smooth muscle myofilaments to Ca2+ and
that it does not appear to mobilize Ca2+ in its own right
(10). Thus, we would expect arteries that have been preconstricted by
Ca2+ derived from another source (e.g. the
extracellular fluid) to constrict further on release of the
endothelium-derived vasoconstrictor during hypoxia, even when
maintained SR Ca2+ release has been blocked by
8-bromo-cADPR. Because K+ induces pulmonary artery
constriction by depolarizing the smooth muscle cell membrane and
activating voltage-gated Ca2+ channels, we were able to
preconstrict pulmonary arteries with K+ even in the
presence of 8-bromo-cADPR. We used 20 mM K+ to
provide a plateau constriction similar in magnitude (2.4 ± 0.2 mN/mm, n = 6) to the endothelium-independent
constriction that was maintained by hypoxia and blocked by
8-bromo-cADPR (2.8 ± 1.1 mN/mm, n = 6; see also Fig. 7 D). Fig.
8A shows a control response to
hypoxia (16-21 Torr) as before. Fig. 8B shows the response
of the same vessel to hypoxia after the vessel had been preconstricted
with 20 mM K+. Fig. 8C shows the
effect of 300 µM 8-bromo-cADPR on the constriction to
hypoxia, after the vessel had been preconstricted with 20 mM K+. Under these conditions and when reported
as a percentage of the constriction to 75 mM
K+, hypoxia induced a typical biphasic constriction similar
in magnitude to control both in the presence and absence of
8-bromo-cADPR. The peak of phase 1 measured 62 ± 11% in the
absence of 8-bromo-cADPR and in the absence of K+ (20 mM)-induced preconstriction; 55 ± 10% in the absence
of 8-bromo-cADPR and in the presence of K+ (20 mM)-induced preconstriction; and 53 ± 7% in the
presence of 300 µM 8-bromo-cADPR and K+ (20 mM)-induced preconstriction (n = 5 arteries). The peak of phase 2 of HPV after 40 min of exposure to
hypoxia measured 32 ± 8% in the absence of 8-bromo-cADPR and in
the absence of K+ (20 mM)-induced
preconstriction; 36 ± 8% in the absence of 8-bromo-cADPR and in
the presence of K+ (20 mM)-induced
preconstriction; and 25 ± 9% in the presence of both 300 µM 8-bromo-cADPR and K+ (20 mM)-induced preconstriction (n = 5 arteries). This finding suggests that although 8-bromo-cADPR inhibits
SR Ca2+ release to hypoxia, it does not affect
hypoxia-induced release of the endothelium-derived vasoconstrictor(s),
subsequent myofilament sensitization, or the constriction to
Ca2+ per se.
Extracellular application of 300 µM cADPR, which is
membrane-impermeant, had no effect on either resting tone (after 60 min) or acute HPV (20 min of preincubation) in isolated pulmonary
artery rings (n = 4; not shown). This supports the view
that the cADPR antagonist 8-bromo-cADPR is acting intracellularly.
We have investigated the possible role of ADP-ribosyl cyclase,
cADPR hydrolase, and cADPR as a redox sensor in pulmonary artery smooth
muscle. Our findings suggest that increased cADPR synthesis may
mediate, in part, the hypoxia-induced increase in SR Ca2+
release in pulmonary artery smooth muscle and hence contribute to
HPV.
We measured a high level of cADPR production in smooth muscle
homogenates from pulmonary arteries, whereas little synthesis could be
detected in smooth muscle homogenates from aortic or mesenteric
arteries. A similar trend was observed with respect to the metabolism
of cADPR, although a small amount of metabolism was observed in
homogenates of both aortic and mesenteric artery smooth muscle.
Furthermore we have shown that synthesis and metabolism of cADPR occurs
intracellularly. These findings point to an important role for
ADP-ribosyl cyclase, cADPR hydrolase and cADPR in the regulation of
pulmonary artery function. Support for this proposal comes from the
fact that the level of cADPR synthesis in pulmonary artery homogenates
was inversely related to artery diameter, being 18-fold higher in
homogenates from the small third order branches than it was in the
homogenates of the main extrapulmonary artery. Because the magnitude of
the hypoxic constriction is also inversely related to artery diameter
(39, 40), the ability of hypoxia to constrict pulmonary arteries
increases with the increasing levels of smooth muscle ADP-ribosyl
cyclase and cADPR hydrolase activity. An intriguing possibility,
therefore, is that hypoxia may increase the rate of synthesis of cADPR
in pulmonary artery smooth muscle.
In a separate series of experiments, we found that Given the above findings and the fact that the hypoxia-induced increase
in NAD(P)H autofluorescence in carotid body type I cells occurs over
the same time scale as does the hypoxia-induced increase in
intracellular Ca2+ concentration (21), it seemed likely
that hypoxia may promote SR Ca2+ release by reducing the
NAD(P):NAD(P)H ratio and by subsequently increasing cADPR synthesis.
This proposal gained support from the finding that tissue cADPR levels
increased when pulmonary arteries were exposed to hypoxia under
physiological conditions. Furthermore, a 5-fold greater increase in
cADPR content was observed in third order branches than in second order
branches of the pulmonary artery. Thus, like the enzyme activities (see
above), the magnitude of the hypoxia-induced increase in cADPR content
was inversely related to artery diameter, as is the sensitivity of the
arteries to hypoxia (39, 40).
In functional studies we first showed that cADPR induced
Ca2+ release from ryanodine-sensitive SR stores in isolated
pulmonary artery smooth muscle cells, as does hypoxia (11, 12, 13). We
therefore investigated the role of cADPR in HPV, using a
membrane-permeant cADPR antagonist, 8-bromo-cADPR. Our findings
strongly suggest that hypoxia activates both
cADPR-dependent and cADPR-independent Ca2+
release from ryanodine-sensitive SR stores. The first transient phase
of constriction to hypoxia remained unaffected in the presence of
8-bromo-cADPR. Thus, a cADPR-independent O2-sensing
mechanism must initiate the ryanodine-sensitive SR Ca2+
release associated with this phase of HPV (11). Recent investigations have suggested that phase 1 of HPV may result from an initial fall in
ATP levels and inhibition of the SR Ca2+ ATPase, leading to
an increase in the net efflux of Ca2+ from the SR (Ref. 43
and Fig. 9). This may in turn promote calcium influx because of the subsequent activation of the store refilling current (43). These findings (43) and our own (11), do not
support the view (2-8) that phase 1 of HPV in isolated vessels is
mediated by Ca2+influx through voltage-gated
Ca2+ channels. In marked contrast, the subsequent sustained
phase of acute HPV in de-endothelialized pulmonary artery rings (Fig. 7B), which is also mediated by ryanodine-sensitive SR
Ca2+ release (11), was blocked by 8-bromo-cADPR. This
finding suggests that a sustained increase in SR Ca2+
release and hence constriction may depend on an increase in cADPR synthesis during HPV (Fig. 9). Perhaps the most important observation, however, was that the second tonic phase of HPV in intact isolated pulmonary artery rings was abolished by 8-bromo-cADPR and could be
reconstituted if the arteries were preconstricted with K+
(Figs. 7 and 8). These findings suggest that an increase in cADPR levels and subsequent SR Ca2+ release in the smooth muscle
is essential for the maintenance of acute HPV. Furthermore they show
that 8-bromo-cADPR did not block (a) constriction to
Ca2+ per se, (b) the release of the
endothelium-derived vasoconstrictor in response to hypoxia, or
(c) the increase in myofilament Ca2+
sensitivitiy promoted by the released vasoconstrictor. Thus, the
release of the endothelium-derived vasoconstrictor during the second
tonic phase of HPV (9, 10, 40) may be insufficient to promote pulmonary
artery constriction in the absence of maintained cADPR-dependent SR Ca2+ release, despite the
fact that the vasoconstrictor may activate a Rho associated
kinase and increase smooth muscle myofilament Ca2+
sensitivity (Ref. 44 and Fig. 9).
-NAD+, we investigated the
regulation by
-NADH of cADPR synthesis and metabolism and the role
of cADPR in hypoxic pulmonary vasoconstriction. Significantly higher
rates of cADPR synthesis occurred in smooth muscle homogenates of
pulmonary arteries, compared with homogenates of systemic arteries.
When the
-NAD+:
-NADH ratio was reduced, the net
amount of cADPR accumulated increased. This was due, at least in part,
to the inhibition of cADPR hydrolase by
-NADH. Furthermore, hypoxia
induced a 10-fold increase in cADPR levels in pulmonary artery smooth
muscle, and a membrane-permeant cADPR antagonist, 8-bromo-cADPR,
abolished hypoxic pulmonary vasoconstriction in pulmonary artery rings. We propose that the cellular redox state may be coupled via an increase
in
-NADH levels to enhanced cADPR synthesis, activation of ryanodine
receptors, and sarcoplasmic reticulum Ca2+ release. This
redox-sensing pathway may offer new therapeutic targets for hypoxic
pulmonary hypertension.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-NAD+ metabolite cyclic ADP-ribose (cADPR; Refs.
14-16), a messenger that regulates SR Ca2+ release via
ryanodine receptors (RyRs) in a variety of cell types (17-19), plays a
role in this process.
-NAD+:
-NADH
ratio in regulating cADPR synthesis in pulmonary artery smooth muscle
because of the fact that (a) the cellular redox couple
-NAD+ is the recognized substrate for cADPR synthesis;
(b) the redox state of O2-sensing cells is
uniquely sensitive to changes in the level of O2 (20);
(c) hypoxia has been shown to reduce the NAD(P): NAD(P)H
ratio in all O2-sensing cells studied to date, including
carotid body type I cells (20, 21), airway neuroepithelial cells (22),
and pulmonary artery smooth muscle cells (3, 23); and (d)
the hypoxia-induced fall in the NAD(P):NAD(P)H ratio in carotid body
type I cells occurs with a time course similar to the hypoxia-induced
increase in intracellular Ca2+ (21).
-NADH levels, inhibits cADPR hydrolase, which in turn promotes an
increase in cADPR levels in pulmonary artery smooth muscle.
Furthermore, we show that a membrane-permeant cADPR antagonist,
8-bromo-cADPR, abolishes the second, sustained phase of HPV. The role
of ADP-ribosyl cyclase and cADPR hydrolase as a redox sensor is discussed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. Production of cADPR from 2.5 mM
-NAD+ and metabolism of 5-10 µM cADPR,
respectively, was assessed in 10-50-µl samples of smooth muscle
homogenate at 37 °C. Test samples (5 µl) were assayed for cADPR
using a Ca2+ release bioassay (see below).
.
5 °C) for 30 min to allow for extraction of nucleotides. Precipitated protein was then removed by
centrifugation (15,000 × g for 10 min). The
supernatant was neutralized by the addition of 2 M
KHCO3. The potassium perchlorate precipitate was removed by
centrifugation at 15,000 × g for 10 min. To remove
contaminating nucleotides that weakly interfere with
[32P]cADPR binding, the neutralized acid extracts were
treated with NADase (0.25 unit/ml), nucleotide pyrophosphatase (1.75 units/ml), alkaline phosphatase (50 units/ml), and apyrase (5 units/ml)
for 4 h, as previously described (15). The concentration of cADPR in acid extracts was assessed by comparing inhibitory effects on the
[32P]cADPR binding assay, with a standard curve
constructed using authentic cADPR. As a control each sample was heat
treated at 85 °C for 45 min, to hydrolyze cADPR to ADPR. The
inhibitory effect on [32P]cADPR binding of all samples
was abolished by heat treatment. The recovery of cADPR, monitored by
recovery of [32P]cADPR added prior to acid extraction,
was 69.9 ± 5.2% (n = 3). Correction was
introduced for recovery of cADPR.
-NAD+, which was from Amersham
Pharmacia Biotech.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-NAD+ in
smooth muscle homogenates from third order branches of the pulmonary
arterial tree, as determined by the sea urchin homogenate bioassay. The
synthesis was complete after 45 min, when 18 ± 1.7 nmol of cADPR
were synthesized per mg of protein (n = 5 animals). Homogenates showed a similar time-dependent hydrolysis of 5 µM cADPR, assessed by bioassay. This was complete at 60 min, when 10 ± 0.6 nmol of cADPR had been metabolized per mg of
protein (n = 4 animals; Fig. 1B). The
inset in Fig. 1A shows that 250 µM
NGD+, an alternative substrate for ADP-ribosyl cyclase,
yielded a fluorecent cyclic product (cyclic GDP-ribose) when
NGD+ was added to smooth muscle homogenates of third order
branches of the pulmonary artery. When NGD+ was excluded,
no change in fluorescence was observed with time (trace 1).
Upon addition of NGD+ a significant increase in the
fluorescence was observed with time (trace 2). The increase
in fluorescence was clearly dependent on cyclic GDP-ribose formation
and therefore ADP-ribosyl cyclase, because it was inhibited by the
ADP-ribosyl cyclase antagonist nicotinamide (10 mM;
trace 3; see also Refs. 25 and 26).
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Fig. 1.
Time course for the synthesis and degradation
of cADPR in smooth muscle homogenates of third order branches of the
pulmonary artery. A, the production of cADPR from 2.5 mM -NAD+. The inset shows the
rate of change in fluorescence resulting from the production of cyclic
GDP-ribose in the absence of substrate (trace 1), in the
presence of 250 µM NGD+ (trace 2),
and in the presence of 250 µM NGD+ and 10 mM nicotinamide (trace 3); the excitation
wavelength was 300 nm, and the emission wavelength was 410 nm.
B, rate of degradation of 5 µM cADPR by smooth
muscle homogenates. The points and error bars
represent the means ± S.E. for
5 animals. All incubations were
carried out at 37 °C and in triplicate.
RFU, change in relative fluorescence
units.
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Fig. 2.
The enzymes for the synthesis of cADPR are
located at an intracellular site. A, the rate of cADPR
synthesis (nmol/mg protein/h) in the P1 (nuclear and whole cell
debris), P2 (plasma membrane), P3 (microsomal), and S3 (cytosolic)
fractions of smooth muscle homogenates of third order branches of the
pulmonary artery; each fraction was separated by differential
centrifugation. B, the rate of cADPR metabolism (nmol/mg
protein/h) in each fraction. C, the relative plasma membrane
contamination as indicated by the 5' nucleotidase (5'ND)
activity (units of activity/liter) in P1, P2, P3, and S3 fractions.
D, production of cADPR by whole (open bars) and
saponin permeabilized (black bars) pulmonary artery smooth
muscle cells acutely isolated from the extrapulmonary artery. The
inset shows a typical image of an isolated pulmonary artery
smooth muscle cell in sucrose-HEPES buffer. All incubations were
carried out at 37 °C and in triplicate. Bars represent
the means ± S.E. for 3 animals. *, significance of
p < 0.05.
-NAD+
the production of cADPR measured 0.51 ± 0.07 nmol/mg protein (n = 3 animals) in whole cells and 1.5 ± 0.4 nmol/mg protein (n = 3 animals) in paired and
time-matched permeabilized cells (Fig. 2D). A clear
separation in the level of cADPR synthesis in whole versus
permeabilized pulmonary artery smooth muscle cells, respectively, was
not, however, observed at the 60-min time point. This apparent nonlinearity may be a product of (a) the sensitivity of the
Ca2+ release bioassay to cADPR and (b) the time
required for sufficient quantities of the newly synthesized cADPR to
accumulate in the samples tested. Note that the saponin concentration
used was found to have no effect on the Ca2+ release
bio-assay (see methods). Fig. 2D (inset) shows a
picture of an isolated and saponin permeabilized pulmonary artery
smooth muscle cell in suspension.
-NAD+. The quantity of cADPR produced was high in smooth
muscle homogenates from all sections of the pulmonary arterial tree,
whereas no significant production could be detected in homogenates of
either the aorta or mesenteric artery. Moreover, the level of cADPR
synthesis was inversely related to pulmonary artery diameter, measuring
1.0 ± 0.4 nmol/mg of protein/h (n = 4 animals) in
homogenates of the large conduit or extrapulmonary artery, 9.1 ± 2.8 nmol/mg protein/h (n = 5 animals) in the second
order branches of the pulmonary artery (intrapulmonary artery) and
18 ± 1.7 nmol/mg protein/h (n = 6 animals) in the
third order branches. Fig. 3B shows that the rate of
hydrolysis of 5 µM cADPR in the same series of artery homogenates followed a similar pattern, measuring 11 ± 1.5 nmol/mg protein/h (n = 4 animals) in smooth muscle
homogenates of the third order branches, 5.7 ± 2.3 nmol/mg
protein/h (n = 5 animals) in second order branches, and
2.3 ± 2.2 nmol/mg protein/h (n = 4 animals) in
homogenates of the extrapulmonary artery. In contrast to cADPR
synthesis, a small amount of cADPR hydrolysis was detected in
homogenates of both the aorta (0.6 ± 0.3 nmol/mg protein/h; n = 3 animals) and the near-resistance sized mesenteric
arteries (200-1000 µm external diameter; 0.4 ± 0.2 nmol/mg
protein/h, n = 3 animals). These findings suggest that
ADP-ribosyl cyclase and cADPR may play an important role in the
regulation of pulmonary artery smooth muscle cell function.
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Fig. 3.
Enzyme activities for cADPR synthesis and
metabolism in rabbit pulmonary and systemic arteries.
A, the rate of cADPR production from 2.5 mM
-NAD+ in homogenates of smooth muscle from third order
branches of the pulmonary artery (RP), second order branches
of the pulmonary artery (IP), and the main or extrapulmonary
artery (EP). These are compared with the rate of cADPR
production in the thoracic aorta, third order branches of the
mesenteric artery (RM) and the main conduit mesenteric
artery (CM). B, rate of degradation of 5 µM cADPR in smooth muscle homogenates. All incubations
were carried out at 37 °C and in triplicate. The bars
represent the means ± S.E. for
3 animals.
-NADH of cADPR Synthesis and Metabolism--
Fig.
4A compares the rate of
synthesis of cADPR from a maximally effective concentration (2.5 mM) of
-NAD+ to the rate of synthesis of
cADPR from 25 mM
-NADH (n = 4 animals). From these data
-NADH can be seen to be a poor substrate for ADP-ribosyl cyclase in pulmonary artery smooth muscle. However, addition of
-NADH in combination with
-NAD+ could
produce up to a 3-fold increase in cADPR synthesis when compared with
the time-matched synthesis obtained in the presence of
-NAD+ alone. Fig. 4B shows the effect of
-NADH (1-10 mM) on the synthesis of cADPR from a
maximally effective concentration of
-NAD+ (2.5 mM) in smooth muscle homogenates from third order branches of the pulmonary artery.
-NADH increased the rate of synthesis of
cADPR from
-NAD+ in a
concentration-dependent manner. The rate of synthesis of cADPR increased from 13.52 ± 1.5 nmol/mg of protein/h
(n = 4 animals) in the absence of
-NADH to a maximum
of ~30 nmol/mg of protein/h (n = 4 animals) in the
presence of 4-10 mM
-NADH. The fact that
-NADH
increased cADPR synthesis from a maximally effective concentration of
-NAD+ raised the possibility that
-NADH may mediate
this effect via the inhibition of cADPR hydrolase. Fig. 4C,
shows that 4 mM
-NADH inhibited the hydrolysis of cADPR
(10 µM) in pulmonary artery smooth muscle homogenates. In
smooth muscle homogenates of third order branches of the pulmonary
artery, 3.63 ± 0.49 nmol of cADPR were metabolized/mg protein/h
(n = 3 animals) in the absence of
-NADH. This fell
to 0.61 ± 0.37 nmol/mg protein/h (n = 3 animals) in the presence of 2 mM
-NADH and was undetectable in
the presence of 4 mM
-NADH. Taken together these
findings suggest that net amount of cADPR accumulated from
-NAD+ may be increased by
-NADH in a nonadditive and
synergistic manner. This may be due, at least in part, to the
inhibition by
-NADH of cADPR hydrolase. Note that the small amount
of cADPR synthesis observed with 25 mM
-NADH may be due,
in part, to the 0.5%
-NAD+ contamination.
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Fig. 4.
Regulation by -NADH
of ADP-ribosyl cyclase and cADPR hydrolase in smooth muscle of third
order branches of the pulmonary artery. A, production
of cADPR from a maximally effective concentration (2.5 mM)
of
-NAD+ and from 25 mM
-NADH in the
absence of added
-NAD+ in smooth muscle homogenates of
third order branches of the pulmonary artery. B,
concentration-response curve describing the effect of increasing
concentrations of
-NADH (1-10 mM) on the production of
cADPR from a fixed and maximally effective concentration (2.5 mM) of
-NAD+ in smooth muscle homogenates of
third order branches of the pulmonary artery. The upper x
axis shows the equivalent
-NAD+:
-NADH ratio.
C, concentration-effect relationship for
-NADH (0.5-4
mM) versus cADPR (10 µM)
metabolism. Incubations were carried out in triplicate at 37 °C.
Bars represent the means ± S.E. from
2 animals. **,
significance of p < 0.001.
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Fig. 5.
Elevation of cADPR content in second and
third order branches of the pulmonary artery by hypoxia. Bar
chart shows the total cADPR content (pmol/mg protein) in second
order branches (IP) and in third order branches
(RP), respectively, of the pulmonary artery under normoxic
(154-160 Torr, open bars) and hypoxic (16-21 Torr,
filled bars) conditions. The error bars represent
the means ± S.E. of three determinations (n = 3 animals).
45 ± 5 mV (n = 3 cells), in agreement with the
findings of others (38). In marked contrast, Fig.
6A shows the effect of
intracellular dialysis of 30 µM cADPR from the patch
pipette; dialysis began immediately after breaking the membrane patch
under the pipette tip, i.e. at the start of the record
shown. Clearly, cADPR induced a pronounced hyperpolarization of the
membrane potential. The hyperpolarization reached a maximum after
~1-2 min, at which point pronounced oscillations in the membrane
potential were observed. When the oscillations in membrane potential
commenced the smooth muscle cells began to contract, and it proved
impossible to hold the smooth muscle cells in a tight whole cell
configuration for any length of time. On average (mean ± S.E.;
n = 5 cells), the membrane potential hyperpolarized
from
48.6 ± 5.3 mV (recorded immediately after entering the
whole cell configuration) to
73 ± 2.4 mV. The
hyperpolarization, oscillations in membrane potential, and cell
contraction were not observed after the ryanodine-sensitive SR stores
had been depleted by prior application of 10 µM ryanodine and 10 mM caffeine (Fig. 6B). Under these
conditions the membrane potential remained stable at
55 ± 3 mV
(n = 4 cells) for the duration of the experiment (
20
min). The activation of BKCa channels was confirmed by the
fact that the hyperpolarization was not observed in the presence of TEA
(10 mM; not shown) or iberiotoxin (100 nM; not
shown). These data suggest that cADPR can induce Ca2+
release from ryanodine-sensitive SR stores in pulmonary artery smooth
muscle cells.
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Fig. 6.
cADPR induces Ca2+ release from
ryanodine-sensitive SR stores in isolated pulmonary artery smooth
muscle cells. A, record of the effect on the membrane
potential of intracellular dialysis of 30 µM cADPR from
the patch pipette in a smooth muscle cell isolated from the second and
third order branches of the pulmonary artery. B, effect of
intracellular dialysis of 30 µM cADPR after the
ryanodine-sensitive SR stores had been depleted by prior application of
10 µM ryanodine and 10 mM caffeine. The
line break in A indicates where the seal between
the patch pipette and the cell was broken after the pulmonary artery
smooth muscle cell began to contract. Each experiment was carried out
at 22 °C.
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Fig. 7.
Hypoxic pulmonary vasoconstriction is
inhibited by 8-bromo-cADPR in the presence and absence of the pulmonary
artery endothelium. A, control constriction to 75 mM K+. The pulmonary artery ring was then
exposed to hypoxia (16-21 Torr) for 30 min. After the artery had
recovered from the exposure to hypoxia, the vessel was then exposed
once more to 75 mM K+. B, first
shows a control response of a de-endothelialized pulmonary artery ring
to 75 mM K+ and then the effect of exposing the
vessel to hypoxia (16-21 Torr). C, control response to
K+ (75 mM) and then the effect of preincubation
(10 min) with 300 µM 8-bromo-cADPR on the hypoxia (16-21
Torr)-induced constriction of a pulmonary artery ring in the presence
of the endothelium. D, control response to K+
(75 mM) and then the effect of preincubation (10 min) with
300 µM 8-bromo-cADPR on the hypoxia (16-21 Torr)-induced
constriction of a pulmonary artery ring in the absence of the
endothelium. The records in A and C and those in
B and D, respectively, were obtained from the
same artery at 37 °C. E and F, means (1-min
intervals) ± S.E. (5-min intervals) for six experiments carried
out in the presence (E) and absence (F) of the
endothelium. The closed and open circles
represent the constriction measured in the absence and presence of 300 µM 8-bromo-cADPR, respectively.
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Fig. 8.
Hypoxic pulmonary vasoconstriction is not
inhibited by 8-bromo-cADPR in pulmonary arteries preconstricted with
potassium. A, control constriction to 75 mM
K+. The pulmonary artery ring was then exposed to hypoxia
(16-21 Torr) for 30 min. After the artery had recovered from the
exposure to hypoxia, the vessel was then exposed once more to 75 mM K+. B, first shows a control
response to 75 mM K+. The vessel was then
preconstricted with 20 mM K+ and subsequently
exposed to hypoxia (16-21 Torr). C, shows a control
response to K+ (75 mM). The vessel was then
preincubated (10 min) with 300 µM 8-bromo-cADPR,
preconstricted with 20 mM K+ and subsequently
exposed to hypoxia (16-21 Torr). The results were obtained from the
same artery at 37 °C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-NADH induced a
concentration-dependent increase in the rate of cADPR synthesis from a fixed and maximally effective concentration of its
substrate
-NAD+. Because
-NADH was found to be a poor
substrate for cADPR synthesis, this effect was clearly nonadditive,
i.e. synergistic. We also found
-NADH to inhibit, in a
concentration-dependent manner, cADPR metabolism in
pulmonary artery smooth muscle homogenates. Thus,
-NADH may promote
an increase in cADPR accumulation from
-NAD+, at least
in part, by blocking the metabolism of cADPR by a cADPR hydrolase. The
effect of
-NADH on cADPR synthesis may be of importance to the
regulation of O2-sensing cells because the redox state of
such cells is uniquely sensitive to changes in O2 (3, 20, 21, 22), and hypoxia has been shown to increase
-NADH levels in all
O2-sensing cells studied to date (3, 20, 21, 22, 23).
Previous investigations of pulmonary artery smooth muscle suggest that
total tissue
-NAD+ levels may be in the mM
range during normoxia and hypoxia and that during hypoxia a small fall
in
-NAD+ levels yields a large increase in
-NADH
levels. Estimates suggest that
-NADH concentration in the smooth
muscle may increase at least 5-fold during hypoxia from ~0.03
mM (normoxia; Ref. 23) to 0.15 mM (hypoxia;
Ref. 23). However, it should be noted that any estimates of total
tissue
-NAD+ and
-NADH levels are likely
underestimates of the true levels that may be experienced in intact
cells. The estimates given here assume a total tissue H2O
content of 0.75 liter/Kg and assume that the extracellular fluid would
account for 20% of the total tissue H2O. They do not take
into account known subcellular compartmentalization, which may result
in significant concentration of
-NAD+ and
-NADH
levels and, indeed, cADPR. In fact
-NAD+/
-NADH and
the enzymes for cADPR synthesis and metabolism may be localized in
cellular compartments from the plasma membrane to the mitochondria
(41). In addition hypoxia may reduce the redox state of the cytoplasmic
compartment, by modulating glycolysis (42) and/or the mitochondrial
redox state (20). It is, however, clear that these data give equivalent
-NAD+:
-NADH ratios of ~17 and 5 (23),
respectively, and the latter is close to the range (5-0.7) of
-NAD+:
-NADH ratios, over which our data indicate that
we may see an increase in cADPR synthesis from
-NAD+
(Fig. 4B).
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Fig. 9.
Schematic representation of the proposed
redox sensing pathway. We propose that hypoxia may increase
-NADH levels and thereby increase the net amount of cADPR
accumulated from
-NAD+, at least in part, by inhibiting
cADPR metabolism by the cADPR hydrolase (CH). The increase
in cADPR synthesis in combination with the inhibition of the SR
Ca2+ ATPase by hypoxia may then promote SR Ca2+
release from ryanodine-sensitive SR stores and constriction via
Ca2+-dependent activation myosin light chain
kinase (MLCK). The release by hypoxia of a vasoconstrictor
from the pulmonary artery endothelium may then promote further
constriction by activating a Rho-associated kinase (ROCK),
leading to the inhibition of smooth muscle myosin phosphatase
(SMMP) and an increase in myofilament Ca2+
sensitivity. AC, ADP-ribosyl cyclase; ADPR,
ADP-ribose.
Given the above, it may be of some significance that the level of cADPR synthesis is at least 2 orders of magnitude higher in pulmonary artery smooth muscle than it is in systemic artery smooth muscle. Thus, in pulmonary artery smooth muscle hypoxia may induce an increase in cADPR levels at least 2 orders of magnitude greater than that observed in systemic artery smooth muscle. This may provide a platform for the evident pulmonary selective effects of hypoxia; systemic arteries dilate in response to hypoxia. This is a significant point, because all three ryanodine receptor subtypes (RyR1, RyR2, and RyR3) may be present in both systemic and pulmonary vascular smooth muscle (45), and all three RyR subtypes can be expressed in a cADPR-sensitive form (46-48).
In summary, our findings suggest that (a) the enzyme
activities for the metabolism of cADPR are significantly higher in
pulmonary artery smooth muscle than they are in systemic artery smooth
muscle; (b) the expression of these enzyme activities was
highest in the smaller pulmonary arteries, which exhibit the highest
sensitivity to hypoxia; (c) an increase in -NADH
concentration increased the net amount of cADPR synthesized from
-NAD+, and this was due, at least in part, to the
inhibition by
-NADH of cADPR metabolism; (d) hypoxia
increased cADPR levels in pulmonary arteries; (e) cADPR
induced Ca2+ release from ryanodine-sensitive SR stores in
pulmonary artery smooth muscle cells; and (f) 8-bromo-cADPR,
a cADPR antagonist, abolished the sustained phase of acute HPV in
intact arteries. We propose that ADP-ribosyl cyclase, cADPR hydrolase,
and cADPR act as a redox sensor that couples changes in the cellular
redox potential to SR Ca2+ release (Fig. 9) and that cADPR
is the primary mediator of acute HPV. This pathway may offer an
important new therapeutic target for the treatment of hypoxic pulmonary
hypertension. It is also possible that this pathway may play a role in
(a) ischemia/ischemic reperfusion injury and (b)
Ca2+ signaling in pancreatic
cells, in which cADPR may
regulate stimulus secretion coupling and in which glucose induces
concomitant oscillations in NAD(P)H autofluorescence and in cytoplasmic
Ca2+ (34, 49, 50).
![]() |
ACKNOWLEDGEMENT |
---|
We are grateful to Dr. Grant Churchill for helpful discussion.
![]() |
FOOTNOTES |
---|
* This work was supported by the Wellcome Trust and the BBSRC.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Special Biotechnology and Biological Sciences Research Council studentship. Present address: Inst. of Molecular Physiology, Sheffield University, Alfred Denny Bldg., Western Bank, Sheffield, S10 2TN, UK.
** Wellcome Trust Non-Clinical Lecturer. To whom correspondence should be addressed: Division of Biomedical Sciences, School of Biology, Buke Building, University of St. Andrew, St. Andrew, Fife, KY 169TS, UK. Tel: 44-1-334-463579; Fax: 44-1334-463600; E-mail: ame3@st-and.ac.uk.
Wellcome Trust Senior Research Fellow.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M004849200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HPV, hypoxic pulmonary vasoconstriction; SR, sarcoplasmic reticulum; cADPR, cyclic ADP-ribose; RyR, ryanodine receptor.
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REFERENCES |
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