From the Division of Biomedical Sciences, School of
Biology, Bute Building, University of St Andrews, St Andrews, Fife,
KY16 9TS, United Kingdom and the ¶ University Department of
Pharmacology, University of Oxford, Mansfield Road,
Oxford, OX1 3QT, United Kingdom
Received for publication, May 17, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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In artery smooth muscle, adenylyl cyclase-coupled
receptors such as A variety of transmitters relax smooth muscle by increasing cAMP
levels and thereby activating cAMP-dependent protein kinase A (PKA).1 In arteries,
trachea, human airway, and lymphatic vessels, PKA-dependent smooth muscle relaxation has been shown to be mediated, in part, by
opening of Ca2+-activated potassium (BKCa)
channels and membrane hyperpolarization (1-11). In artery smooth
muscle, adenylyl cyclase-coupled receptors, such as Previous studies have suggested that activation of PKA may promote
BKCa-dependent hyperpolarization by direct
phosphorylation of the BKCa channel protein (4, 6, 10).
However, the most compelling evidence suggests that BKCa
channel activation results from Ca2+ release from the
sarcoplasmic reticulum (SR) due to the opening of RyRs proximal to the
plasma membrane (1, 2, 4, 5, 8, 11-17). There is evidence to suggest
that this may result from direct phosphorylation of RyRs by PKA (4,
12). There is also support for a role for PKA-dependent
activation of the SR Ca2+ ATPase by phosphorylation of
phospholamban, increased SR Ca2+ load, and thereby
increased resting SR Ca2+ release via RyRs in the vicinity
of the plasma membrane (4, 13-17). As yet, however, the extent to
which each of these three mechanisms contributes to physiological
responses remains controversial (4, 5, 10, 12-18).
We have now investigated the role of cyclic adenosine
diphosphate-ribose (cADPR), a Cell Isolation--
Single smooth muscle cells were
enzymatically isolated from second order branches of the pulmonary
artery of male Wistar rats (150-300 g, sacrificed by cervical
dislocation). Briefly, arteries were incubated (1 h, 22 °C)
in a low Ca2+ solution of the following composition: 124 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 0.5 mM NaH2PO4,
0.5 mM KH2PO4, 15 mM
NaHCO3, 0.16 mM CaCl2, 0.5 mM EDTA, 10 mM glucose, 10 mM
Taurine, 10 mM Hepes, pH 7.4, 0.5 mg/ml papain (Fluka), and
1 mg/ml bovine serum albumin (Sigma). Then, 0.25 mg/ml
1,4-dithio-DL-threitol (Fluka) was added to the solution
followed by a further 30-min incubation. The tissue was placed in
enzyme-free low Ca2+ solution, and single smooth
muscle cells were isolated by gentle trituration. Cells were placed, as
required, onto a glass coverslip in the experimental chamber.
Electrophysiological Recordings--
Membrane potential in
single pulmonary artery smooth muscle cells was measured in the
whole-cell configuration of the patch clamp technique in current-clamp
mode (I = 0) using an Axopatch 200B amplifier (Axon instruments,
Foster City, CA). Cells were bathed in physiological salt solution of
the following composition (physiological salt solution A): 130 mM NaCl, 5.2 mM KCl, 1 mM MgCl2, 1.7 mM CaCl2, 10 mM glucose, 10 mM Hepes, pH 7.45. The pipette
solution contained 140 mM KCl, 10 mM Hepes, 1 mM MgCl2, pH 7.4. All experiments were carried
out at room temperature (22 °C). Seal resistance was Ca2+ Imaging--
Cells were incubated for 30 min in
low Ca2+ solution (see above) containing 5 µM
Fura-2 AM, washed, and allowed to equilibrate for 20 min. 5 µM Fura-2 (free acid) was also added to the pipette solution used for intracellular dialysis (see above). Changes in
intracellular Ca2+ were monitored by assessing Fura-2
fluorescence, using excitation wavelengths of 340 nm (F340) and 380 nm
(F380), respectively, and an emission wavelength of 510 nm. Emitted
fluorescence was monitored using a Hamamatsu 4880 image-intensifying
CCD camera and recorded and analyzed using Openlab imaging
software (Improvision) on an Apple Macintosh G4 personal
computer. Fluorescence intensity was measured at 0.25-5 Hz with
background subtraction being carried out on-line. Changes in Fura-2
fluorescence are reported as the F340/F380 ratio and as the estimated
intracellular Ca2+ concentration.
Small Vessel Myography--
Artery rings (2-3 mm in length)
were isolated from second order branches of rat pulmonary arteries and
mounted onto an automated myograph (AM10, Cambustion Biological,
Cambridge, UK) using 50-µm tungsten wire. Tension was set to be
equivalent to a pressure of 30 mm Hg. The methods have been described
in detail previously (34, 35). The myograph contained physiological
salt solution B: 118 mM NaCl, 4 mM KCl, 24 mM NaHCO3, 1 mM MgSO4,
1.2 mM NaH2PO4, 2 mM
CaCl2, 5.56 mM glucose. The solution was
maintained at 37 °C and bubbled with 75% N2, 20%
O2, and 5% CO2 to maintain a pH of 7.4. The
endothelium was removed by rubbing the intima of the arteries with
braided silk surgical thread. Endothelium removal was confirmed by the
failure of 100 µM acetylcholine to relax constrictions
induced by 1 µM (5Z, 9 Chemicals--
Isoprenaline,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA), cADPR, ADP-ribose, iberiotoxin, 8-amino-cyclic adenosine diphosphate-ribose (8-NH2-cADPR), 8-bromo-cyclic
adenosine diphosphate-ribose (8-Br-cADPR), heparin,
N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89), cAMP, ryanodine, PGF2 cADPR-dependent SR Ca2+ Release via
Ryanodine Receptors Activates BKCa Channels and
Hyperpolarization--
Using Fura-2 fluorescence imaging techniques
and the whole-cell configuration of the patch clamp technique, we
measured, in parallel, changes in the intracellular Ca2+
concentration and resting membrane potential evoked by intracellular dialysis of cADPR in isolated rat pulmonary artery smooth muscle cells.
In agreement with previous investigations in arterial smooth muscle,
resting membrane potentials were within the range Does Ryanodine Deplete SR Ca2+ Stores?--
In
contrast to hyperpolarization by cADPR, ryanodine had no significant
effect on the global Ca2+ wave induced by intracellular
dialysis of IP3. Thus, intracellular dialysis of 1 µM IP3 increased the Fura-2
fluorescence ratio from 0.60 ± 0.01 to a peak of 2.5 ± 0.1 in the absence of 20 µM ryanodine (n = 5)
and from 0.8 ± 0.1 to a peak of 1.8 ± 0.2 in the presence of 20 µM ryanodine (20-min preincubation,
n = 5). Conversely, the IP3R antagonist
xestospongin C blocked IP3-induced Ca2+ signals
(n = 4, not shown) but had no effect on
Ca2+ signals or hyperpolarization by cADPR
(n = 4, not shown). These findings suggest that
ryanodine blocks hyperpolarization by cADPR without significant effect
on the ability of the SR to release Ca2+ over the time
course of our experiments.
Does 8-Bromo-cADPR Inhibit SR Ca2+ ATPase
Activity?--
Preincubation of pulmonary artery smooth muscle cells
with 300 µM 8-bromo-cADPR was without effect on the
transient increase in Fura-2 fluorescence ratio triggered upon SR store
depletion by cyclopiazonic acid. The Fura-2 fluorescence ratio
was increased by 20 µM cyclopiazonic acid from 0.37 ± 0.02 to a peak of 0.56 ± 0.03 (n = 10) in the
absence of 8-bromo-cADPR and from 0.35 ± 0.03 to a peak of
0.58 ± 0.04 (n = 4) in the presence of
8-bromo-cADPR (300 µM). Thus, it seems unlikely that
8-bromo-cADPR blocks cADPR-dependent hyperpolarization by
inhibiting SR Ca2+ ATPase activity. When taken together,
the aforementioned findings are consistent with the view that cADPR
mediates hyperpolarization by triggering SR Ca2+ release
via RyRs by a mechanism independent of the SR Ca2+ ATPase
and IP3Rs, respectively, which ultimately leads to
BKCa channel activation and hyperpolarization.
cADPR Mediates SR Ca2+ Release and Hyperpolarization by
Isoprenaline--
Given our finding that cADPR mediates
hyperpolarization by BKCa channel activation, we
investigated the role of cADPR in mediating hyperpolarization induced
by the activation of cAMP and PKA Mediate cADPR-dependent Hyperpolarization
by Isoprenaline--
Since
Preincubation with H89 (1 µM), a selective PKA inhibitor,
abolished hyperpolarization by isoprenaline (n = 5, Fig. 4, A and D).
In marked contrast, hyperpolarization by cADPR (20 µM)
remained unaffected in the presence of H89 (1 µM),
measuring cADPR-dependent SR Ca2+ Release via
Ryanodine Receptors Mediates BKCa-dependent
Vasodilation by Isoprenaline--
The role of cADPR in mediating
isoprenaline-induced vasodilation was investigated in isolated
pulmonary artery rings by tension recording experiments. Isolated
pulmonary artery rings, without endothelium, were first constricted
with 50 µM PGF2
After block of RyRs by ryanodine (20 µM) and depletion of
SR stores by cylclopiazonic acid (10 µM), isoprenaline
(100 nM) relaxed PGF2
Following block of BKCa channels by preincubation of artery
rings with iberiotoxin (100 nM), isoprenaline (100 nM) relaxed PGF2 As mentioned previously, vasodilators that activate adenylyl
cyclase-coupled receptors relax arterial smooth muscle by increasing cAMP levels and activating PKA. Vasodilation by
PKA-dependent signaling is mediated, in part, by
BKCa channel activation and membrane hyperpolarization
(1-11). There is evidence to suggest that PKA may activate these
channels by phosphorylating the BKCa channel protein (4, 6,
10) and/or RyRs in the SR (4, 12) or by activating the SR
Ca2+ ATPase, increasing the SR Ca2+ load, and
thereby increasing resting SR Ca2+ release in the vicinity
of the plasma membrane (4, 13-17). However, the extent to which each
of these three mechanisms contributes to physiological responses
remains controversial (4, 5, 10, 12-18). The present investigation now
provides strong support for an alternative mechanism.
Intracellular infusion of low concentrations of cADPR (20 µM) increased cytoplasmic Ca2+ concentration
at the perimeter of isolated pulmonary artery smooth muscle cells and
induced concomitant membrane hyperpolarization. Hyperpolarization was
reversed by the highly selective BKCa channel antagonist
iberiotoxin, which demonstrates that BKCa channel
activation underpins cADPR-dependent hyperpolarization.
Furthermore, SR Ca2+ release via RyRs was shown to be a
prerequisite for this response because hyperpolarization by cADPR was
abolished by chelating intracellular Ca2+ with BAPTA and by
selective block of RyRs with ryanodine. Thus, it seem likely that
Ca2+ release via RyRs mediates cADPR-dependent
hyperpolarization. Further support for this conclusion may be derived
from our finding that ryanodine did not deplete SR Ca2+
stores over the time course of our experiments, as indicated by the
fact that ryanodine blocked hyperpolarization by cADPR but not SR
Ca2+ release triggered by IP3. It should be
noted, however, that there is evidence to suggest that IP3R
and RyR activation, respectively, may mobilize Ca2+ from
functionally segregated SR compartments (38-41).
A role for the SR was confirmed using the SR Ca2+ ATPase
antagonist cyclopiazonic acid. Significantly, cyclopiazonic acid
reversed hyperpolarization by cADPR slowly, indicating that block by
depletion of SR Ca2+ stores exhibits a degree of use
dependence, as one might expect of a process that depends on SR
Ca2+ release via RyRs.
Most importantly, however, hyperpolarization by cADPR was blocked by
the cADPR antagonists 8-NH2-cADPR and 8-bromo-cADPR, respectively, whereas hyperpolarization by caffeine remained
unaffected. Furthermore, 8-bromo-cADPR was shown to be without effect
on the increase in intracellular Ca2+ concentration
triggered by depletion of SR Ca2+ stores by cyclopiazonic
acid, and 8-bromo-cADPR did not trigger an increase in intracellular
Ca2+ in its own right.
The findings described above are consistent with the view that the
cADPR antagonists tested here block the action of cADPR without
blocking the activation of RyRs per se and without
inhibiting SR Ca2+ ATPase activity. Thus, cADPR likely
mediates hyperpolarization in artery smooth muscle by inducing SR
Ca2+ release via RyRs and consequent activation of
BKCa channels in the plasma membrane.
We next investigated the likely physiological role of hyperpolarization
by cADPR. Previous studies have demonstrated that BKCa
channels contribute little to the resting potential in isolated pulmonary artery smooth muscle cells, and spontaneous transient outward
currents are not observed under resting conditions (42-44). Taking
this into consideration, it was not so surprising that the cADPR
antagonists tested here had no effect on the resting membrane potential
in isolated pulmonary artery smooth muscle cells nor on resting tone in
isolated pulmonary arteries. It should be noted, however, that SR
Ca2+ release via RyRs has been shown to initiate
spontaneous transient outward currents in resting systemic artery
smooth muscle, and these BKCa channel currents have been
shown to determine the resting tone in smooth muscle of pressurized
systemic arteries (for review, see Ref. 4). It may not be surprising,
therefore, if future studies demonstrate that
cADPR-dependent SR Ca2+ release, by activation
of BKCa channels, regulates resting smooth muscle tone in
certain vascular beds. By contrast, in the pulmonary vasculature it
seemed most likely that receptor-dependent regulation of
cADPR synthesis would serve pulmonary artery dilation by
BKCa channel activation.
Given that cADPR synthesis is up-regulated by cAMP in cardiac muscle
(25), it appeared plausible that cADPR may mediate hyperpolarization by
adenylyl cyclase-coupled receptors, such as Consistent with the above, hyperpolarization by intracellular dialysis
of cAMP was also blocked by iberiotoxin, BAPTA, ryanodine, and
8-amino-cADPR, respectively. However, our findings with respect to the
selective PKA antagonist H89 were quite different. H89 blocked
hyperpolarization by both isoprenaline and cAMP but was without effect
on hyperpolarization by cADPR. Thus, it would appear that cADPR is a
downstream element in this signaling cascade. We propose, therefore,
that hyperpolarization by In isolated artery rings without endothelium, the
membrane-permeant cADPR antagonist 8-bromo-cADPR reversed by ~50%
vasodilation evoked by isoprenaline. This is consistent with the view
that cADPR mediates, in part, dilation by isoprenaline. Block of RyRs with ryanodine and depletion of SR Ca2+ stores by
cyclopiazonic acid inhibited dilation by isoprenaline by between
50 and 60%. As mentioned previously, neither 8-bromo-cADPR nor
ryanodine appeared to have a significant effect on SR Ca2+
load in isolated smooth muscle cells over the time course of our
experiments. Furthermore, it is unlikely that 8-bromo-cADPR inhibits SR
Ca2+ ATPase activity because: 1) 8-bromo-cADPR is without
effect on the Ca2+ transient evoked by inhibition of SR
Ca2+ ATPase activity by cylcopiazonic acid and does not
itself increase intracellular Ca2+ concentration and 2)
cyclopiazonic acid induces constriction of pulmonary arteries, whereas
8-bromo-cADPR does not. These findings therefore provide strong support
for our view that cADPR-dependent SR Ca2+
release via RyRs mediates, in part, dilation by Importantly, 8-bromo-cADPR was without effect on residual dilation by
isoprenaline after blocking BKCa channels with iberiotoxin. We can conclude, therefore, that 8-bromo-cADPR reverses dilation by
isoprenaline by inhibiting BKCa channel activation by SR
Ca2+ release because 8-bromo-cADPR does not block
BKCa channel activation per se. It is
interesting to note, however, that iberiotoxin inhibited dilation by
isoprenaline by ~78% as compared with 50-60% reversal by
8-bromo-cADPR, ryanodine, and cyclopiazonic acid, respectively. It
would appear, therefore, that cADPR-dependent signaling is responsible for ~80% of BKCa-dependent vasodilation by
isoprenaline in isolated pulmonary arteries. A further 20% may depend
on a mechanism independent of SR Ca2+ release, possibly
PKA-dependent phosphorylation of the BKCa
channel protein (4, 6, 10).
Given our findings, it is interesting to note that previous studies in
coronary artery smooth muscle have suggested that increased synthesis
of ADP-ribose, a cADPR metabolite, may mediate
BKCa-dependent vasodilation by
11,12-epoxyeicosatrienoic acid (45). In contrast to the effects of 20 µM cADPR, however, we found 20 µM
ADP-ribose to be without effect on membrane potential in pulmonary
artery smooth muscle cells. A further variation on the theme comes from the proposal that nitric oxide may mediate vasodilation by inhibiting cADPR formation (46). It is possible, therefore, that the mechanism by
which pyridine nucleotide signaling promotes relaxation of artery
smooth muscle may vary in a manner dependent on the nature of the
vasodilator and/or vascular bed.
A recent report has also demonstrated that concentrations of cADPR in
the mM range may inhibit BKCa channel
activation (47). The physiological significance of this observation is
open to question, as the concentrations used were 50 times greater than those found to mediate BKCa-dependent hyperpolarization in
the present investigation. Despite this fact, it is possible that inhibition of BKCa channel activity by cADPR may 1) serve
as a negative feedback loop with respect to hyperpolarization and/or 2)
contribute to vasoconstriction by cADPR.
In summary, we have shown that adenylyl cyclase-coupled receptors may
mediate vasodilation by cADPR-dependent Ca2+
release via RyRs in the SR, leading to subsequent
BKCa-dependent hyperpolarization and
vasodilation. When taken together with previous investigations, it
would appear that cADPR-dependent Ca2+ release via
RyRs could lead to stimulus-dependent relaxation or
contraction in arterial smooth muscle (26-29). Given that RyR subtypes
1, 2, and 3 are present in vascular smooth muscle (48-51), this
paradox may be explained if: 1) -adrenoceptors evoke Ca2+
signals, which open Ca2+-activated potassium
(BKCa) channels in the plasma membrane. Thus, blood
pressure may be lowered, in part, through vasodilation due to membrane
hyperpolarization. The Ca2+ signal is evoked via
ryanodine receptors (RyRs) in sarcoplasmic reticulum proximal to
the plasma membrane. We show here that cyclic adenosine
diphosphate-ribose (cADPR), by activating RyRs, mediates, in part,
hyperpolarization and vasodilation by
-adrenoceptors. Thus,
intracellular dialysis of cADPR increased the cytoplasmic Ca2+ concentration proximal to the plasma membrane in
isolated arterial smooth muscle cells and induced a concomitant
membrane hyperpolarization. Smooth muscle hyperpolarization mediated by
cADPR, by
-adrenoceptors, and by cAMP, respectively, was abolished
by chelating intracellular Ca2+ and by blocking RyRs,
cADPR, and BKCa channels with ryanodine, 8-amino-cADPR, and
iberiotoxin, respectively. The cAMP-dependent protein
kinase A antagonist
N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked hyperpolarization by isoprenaline and cAMP,
respectively, but not hyperpolarization by cADPR. Thus, cADPR acts as a
downstream element in this signaling cascade. Importantly, antagonists
of cADPR and BKCa channels, respectively, inhibited
-adrenoreceptor-induced artery dilation. We conclude, therefore,
that relaxation of arterial smooth muscle by adenylyl cyclase-coupled
receptors results, in part, from a cAMP-dependent and
protein kinase A-dependent increase in cADPR synthesis, and subsequent activation of sarcoplasmic reticulum Ca2+
release via RyRs, which leads to activation of BKCa
channels and membrane hyperpolarization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenoceptors,
open BKCa channels by evoking Ca2+ signals
proximal to the plasma membrane, leading to smooth muscle cell
hyperpolarization and a consequent reduction in blood pressure through
vasodilation (1-5, 7-9, 11). Identifying the precise mechanisms
involved is therefore essential to our understanding of how blood
pressure may be regulated by PKA-dependent signaling and
may provide fundamental insights into the causes of essential hypertension.
-NAD+ metabolite (19, 20),
in mediating vasodilation by BKCa channel activation in
pulmonary artery smooth muscle. Previous investigations have identified
cADPR as a primary regulator of RyR function in a variety of
preparations, including arterial smooth muscle cells (19-23), and have
demonstrated a role for cADPR-dependent SR Ca2+
release in mediating contraction in both cardiac (24, 25) and smooth
muscle (26-33). Despite the wealth of information linking RyR
activation to vasodilation by adenylyl cyclase-coupled receptors, however, little attention has been paid to the role of cADPR in this
process. We show here that hyperpolarization and dilation by adenylyl
cyclase-coupled receptors is mediated, in part, by cADPR-dependent SR Ca2+ release and consequent
activation of BKCa channels in pulmonary artery smooth muscle.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 gigaohms,
series resistance was
5 megaohms, and pipette resistance was 2-4
megaohms. Fetchex and Fetchan software (Axon instruments, Foster City,
CA) were used to perform data acquisition and analysis.
, 11
, 13E,
15S)-9,11,15-Trihydroxyprosta-5,13-dienoic acid
(PGF2
).
, myo-inositol 1,4,5 trisphosphate (IP3), and cyclopiazonic acid were from
Sigma. Fura-2 and Fura-2-AM were from Molecular Probes. Xestospongin C
was from Calbiochem. Fura-2, Ryanodine and cyclopiazonic acid were
dissolved in Me2SO, final dilution of 1:1000. At
this concentration, Me2SO was without effect on the
preparations studied here.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
40 to
60 mV, and
estimated resting Ca2+ concentrations were in the range
70-150 nM (4, 36). Intracellular application of cADPR (20 µM) evoked a sustained increase in intracellular Ca2+ concentration, as indicated by an increase in the
Fura-2 fluorescence ratio (F340/F380) from 0.5 ± 0.1 to
0.8 ± 0.1 (mean ± S.E.; Fig. 1A) and a concomitant
hyperpolarization. The increase in intracellular Ca2+ was
localized at the cell perimeter, i.e. in close apposition to
the plasma membrane. However, the capacity of Fura-2 for binding Ca2+ resulted in marked attenuation of
cADPR-dependent hyperpolarization. Thus, subsequent
investigations of the transduction pathway leading to hyperpolarization
utilized electrophysiological techniques alone. In the absence of
Fura-2, intracellular dialysis of cADPR (20 µM) evoked
hyperpolarization from
40 ± 2 to
75 ± 1 mV
(n = 28, Fig. 1B), which was reversed by the
selective BKCa channel antagonist iberiotoxin (100 nM, n = 7, Fig. 1B).
Importantly, no hyperpolarization was observed when intracellular
Ca2+ was chelated by intracellular infusion of BAPTA (1 mM, Fig. 1C). Furthermore, cADPR-induced
hyperpolarization was slowly reversed when SR Ca2+ stores
were depleted by cyclopiazonic acid (10 µM,
n = 4, Fig. 1D), blocked following
preincubation with 20 µM ryanodine (20 min,
n = 4, Fig. 1E), and blocked by the
selective cADPR antagonists 8-NH2-cADPR (100 µM, n = 4, Fig. 1F) and
8-bromo-cADPR (100 µM, n = 4, not shown
(37)). These findings are summarized in Fig. 1G.
Hyperpolarization induced by 1 mM caffeine, which triggers Ca2+ release via RyRs by a mechanism independent of cADPR
(22), was insensitive to the cADPR antagonist 8-NH2-cADPR
(100 µM; n = 4, not shown) and
8-bromo-cADPR, respectively, (100-300 µM; n = 4, not shown). This is consistent with the view
that the cADPR antagonists tested here selectively inhibit SR
Ca2+ release by cADPR in pulmonary artery smooth muscle
cells but do not block SR Ca2+ release via RyRs or
BKCa channel activation per se. Note that we
found no measurable contamination of cADPR-containing solutions with
ADP or ADP-ribose and, in contrast to the effects of cADPR, intracellular infusion of 20 µM ADP-ribose had no effect
on membrane potential (n = 4, not shown). Extracellular
application of 20 µM cADPR was without effect on membrane
potential in isolated pulmonary artery smooth muscle cells, and neither
intracellular nor extracellular application of the cADPR antagonists
8-NH2-cADPR (100 µM, n = 4)
and 8-bromo-cADPR (
300 µM, n = 4),
respectively, had any effect on membrane potential.
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Fig. 1.
cADPR hyperpolarises
pulmonary artery smooth muscle cells by increasing Ca2+
release from ryanodine-sensitive SR stores. A, effect
of intracellular infusion of 20 µM cADPR, via a patch
pipette, on the Fura-2 fluorescence ratio (F340/F380) in an isolated
rat pulmonary artery smooth muscle cell. B, effect of
intracellular infusion of 20 µM cADPR on the membrane
potential in an isolated pulmonary artery smooth muscle cell, in the
absence and then in the presence of 100 nM iberiotoxin.
C, effect of intracellular infusion of 20 µM
cADPR in the absence and in the presence of BAPTA (1 mM),
respectively. D, effect of intracellular infusion of 20 µM cADPR on the membrane potential in an isolated
pulmonary artery smooth muscle cell in the absence and then in the
presence of 10 µM cyclopiazonic acid. E,
effect of intracellular infusion of 20 µM cADPR in the
absence and in the presence of 20 µM ryanodine (20 µM), respectively. F, effect of intracellular
infusion of 20 µM cADPR in the absence and then in the
presence of the cADPR antagonist 8-NH2-cADPR (100 µM), respectively. G, bar chart showing the
control membrane potential (CMP) recorded prior to
the onset of the cADPR-induced hyperpolarization and the membrane
potential recorded after the hyperpolarization by cADPR (20 µM) had reached its maximum in the absence and in the
presence of each of the antagonists tested. In this and subsequent
figures, the number of cells tested is indicated above the
bars. The columns and vertical bars
show the mean ± S.E.
-adrenoceptors, a family of adenylyl
cyclase-coupled vasodilator receptors that are known to mediate smooth
muscle relaxation, in part, by activating BKCa channels and
membrane hyperpolarization (4-7, 9). Extracellular application of the
selective
-adrenoreceptor agonist isoprenaline (10 µM)
induced sustained hyperpolarization from
39 ± 2 to
75 ± 1 mV (n = 20, Fig.
2A), which was reversed by the
selective BKCa channel antagonist iberiotoxin (100 nM, n = 5, Fig. 2A). As would be
expected from previous studies (6, 7, 9), hyperpolarization by
isoprenaline was also blocked by a selective
-adrenoreceptor
antagonist, propanolol (10 µM, n = 4, not
shown). In common with cADPR, hyperpolarization by isoprenaline was
blocked when intracellular Ca2+ was chelated by BAPTA (1 mM, n = 4, Fig. 2B),
underscoring the role of an increase in intracellular Ca2+
concentration. Isoprenaline-evoked hyperpolarization was also blocked
when SR Ca2+ release was inhibited with ryanodine (20 µM, n = 6, Fig. 2C). In
contrast, intracellular dialysis of heparin, an IP3R
antagonist, failed to inhibit hyperpolarization by isoprenaline
(n = 3, not shown), which mitigates against a possible
role for IP3Rs in this process. As RyRs mediate
isoprenaline-evoked hyperpolarization and cADPR mimicked the effects of
isoprenaline, we examined the possible role of cADPR in the
-adrenoreceptor signaling pathway. Hyperpolarization by isoprenaline
was abolished by intracellular infusion of 8-NH2-cADPR (100 µM, n = 6, Fig. 2D) and of
8-Br-cADPR (100 µM, n = 6, not shown),
respectively. The powerful effects of these two selective cADPR
antagonists strongly suggest a key role for cADPR in mediating
isoprenaline-induced membrane hyperpolarization in arterial smooth
muscle cells. These findings are summarized in Fig. 2E.
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Fig. 2.
Hyperpolarization induced by isoprenaline is
mediated by cADPR-dependent Ca2+ release from
ryanodine-sensitive SR stores. A, effect of 10 µM isoprenaline on the membrane potential in an isolated
pulmonary artery smooth muscle cell in the absence and then in the
presence of 100 nM iberiotoxin. Effect of
isoprenaline (10 µM) in the absence and then in the
presence, respectively, of intracellular infusion of 1 mM
BAPTA (B); ryanodine (20 µM) (C);
and intracellular infusion of 100 µM
8-NH2-cADPR (D). E, mean
hyperpolarization induced by isoprenaline (10 µM) in the
absence and in the presence of the antagonists tested. CMP
is the membrane potential recorded under control conditions,
i.e. prior to the application of isoprenaline and in the
absence of the antagonists tested.
-adrenoceptors activate adenylyl cyclase
(6, 7, 9), we investigated the relationship between isoprenaline, cAMP,
PKA, and cADPR-mediated hyperpolarization. Intracellular infusion of
cAMP (10 µM) induced hyperpolarization in pulmonary artery smooth muscle cells from
43 ± 4 mV to
82 ± 5 mV
(n = 10), which was reversed by the selective
BKCa channel antagonist iberiotoxin (100 nM,
n = 6, Fig.
3A). In common with cADPR and isoprenaline, cAMP-dependent hyperpolarization was blocked
in cells that had been preincubated with 20 µM ryanodine
(n = 5, Fig. 3B) and was abolished by
co-infusion with 8-NH2-cADPR (100 µM,
n = 11, Fig. 3C) and by co-infusion with
8-Br-cADPR (100 µM, not shown). These findings are
summarized in Fig. 3D.
View larger version (36K):
[in a new window]
Fig. 3.
Hyperpolarization induced by cAMP is mediated
by cADPR-dependent Ca2+ release from
ryanodine-sensitive SR stores. A, effect of
intracellular infusion of 10 µM cAMP, via a patch
pipette, on the membrane potential in an isolated pulmonary artery
smooth muscle cell before and then after extracellular application of
100 nM iberiotoxin. B, effect of intracellular
infusion of 10 µM cAMP in the absence and in the presence
of ryanodine (20 µM), respectively. C, effect
of intracellular infusion of 10 µM cAMP in the absence
and in the presence of 100 µM 8-NH2-cADPR,
respectively. D, bar chart showing the control membrane
potential (CMP) recorded prior to the onset of the
cAMP-mediated hyperpolarization and the membrane potential recorded
after the hyperpolarization by cAMP (10 µM) had reached
its maximum in absence and in the presence of each of the antagonists
tested.
76 ± 2 mV (n = 28) in the absence of
H89 and
78 ± 6 mV in the presence of H89 (1 µM;
n = 3; Fig. 4, B and D). In
common with isoprenaline, but in contrast to cADPR, cAMP failed to
induce membrane hyperpolarization in the presence of 1 µM
H89 (n = 6, Fig. 4, C and D).
These findings are consistent with cADPR acting downstream of cAMP and
PKA activation during
-adrenoreceptor signaling.
View larger version (32K):
[in a new window]
Fig. 4.
cADPR acts downstream of cAMP and PKA.
A, effect of extracellular application of 1 µM
isoprenaline in the absence and in the presence of H89 (1 µM). B, effect of intracellular infusion of 20 µM cADPR in the presence of H89 (1 µM).
C, effect of intracellular infusion of 10 µM
cAMP in the absence and in the presence of H89 (1 µM).
D, membrane potential recorded after the hyperpolarization
by isoprenaline (1 µM), cADPR (20 µM), cAMP
(10 µM), respectively, had reached its maximum in the
absence and in the presence of H89 (1 µM).
. Subsequent application of
isoprenaline (100 nM; IC50) relaxed the
PGF2
(50 µM)-induced constriction by
68 ± 4% (Fig. 5A,
n = 4). Application of 8-Br-cADPR (300 µM), a membrane-permeant cADPR antagonist (37), reversed
the isoprenaline-induced relaxation to 36 ± 2% (Fig. 5A, n = 4). Clearly, these findings are
consistent with a role for cADPR in mediating dilation by
-adrenoreceptor activation. Note that in the presence of 50 µM PGF2
, isolated pulmonary artery smooth
muscle cells were depolarized relative to control, and under these
conditions, intracellular dialysis of 20 µM cADPR elicited hyperpolarization consistent with control, from
36 ± 2 mV to
61 ± 12 mV (n = 4, Fig. 5,
inset). In contrast to its inhibition of dilation by
isoprenaline, 300 µM 8-bromo-cADPR has no effect on
constriction by potassium (i.e. by voltage-gated Ca2+ influx) and PGF2
, respectively
(29).
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[in a new window]
Fig. 5.
cADPR mediates dilation induced by
isoprenaline in isolated pulmonary artery rings without the
endothelium. A, effect of isoprenaline (100 nM) on a rat pulmonary artery ring preconstricted with
PGF2 (50 µM), in the absence and then
presence of 8-Br-cADPR (300 µM). B, effect of
isoprenaline (100 nM) on a rat pulmonary artery ring
preconstricted with PGF2
(50 µM) after
blocking ryanodine receptors with ryanodine (20 µM).
C, effect of isoprenaline (100 nM) on a rat
pulmonary artery ring preconstricted with PGF2
(50
µM) after depletion of SR Ca2+ stores with
cyclopiazonic acid (CPA, 10 µM). D,
as in A, but after block of BKca channels with
iberiotoxin (IbTx, 100 nM). The inset
shows hyperpolarization by intracellular dialysis of 20 µM cADPR into an isolated pulmonary artery smooth muscle
cell preincubated (10 min) with PGF2
(50 µM).
(50 µM)-induced constriction by 21 ± 5%
(n = 4) and 37 ± 4% (Fig. 5 B and
C; n = 4), respectively. In each case, the
inhibition of dilation by isoprenaline was equivalent to that obtained
in the presence of 8-bromo-cADPR. These findings are therefore
consistent with the view that cADPR mediates the component of
vasodilation by isoprenaline that is dependent on SR Ca2+
release via RyRs. Support for this viewpoint comes from our finding that preincubation of isolated pulmonary artery smooth muscle cells
with ryanodine (20 µM) did not deplete SR
Ca2+ stores and that 8-bromo-cADPR was without effect on
the Ca2+ transient evoked following inhibition of SR
Ca2+ ATPase activity by cyclopiazonic acid (see above).
Furthermore, inhibition of SR Ca2+ ATPase activity by
cyclopiazonic acid induced transient constriction of isolated arteries
(Fig. 5C), whereas 8-bromo-cADPR has no effect on resting
tone (28, 29).
(50 µM)-induced constriction by only 15 ± 3%
(n = 4, Fig. 5D). Under these conditions,
8-Br-cADPR (300 µM) was without effect on the
isoprenaline-induced dilation (n = 4, Fig.
5D). This finding is again consistent with a role for cADPR
in mediating a significant proportion of
BKCa-dependent dilation by isoprenaline. Clearly, however, iberiotoxin inhibited a greater proportion of isoprenaline-induced dilation than did 8-bromo-cADPR, ryanodine, or
cylclopiazonic acid. Thus, there may be a component of
BKCa-dependent dilation by isoprenaline that is
mediated by mechanisms independent of SR Ca2+ release.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenoceptors. As shown
previously in smooth muscle from a variety of tissues (for review, see
Ref. 4), isoprenaline-induced hyperpolarization in isolated pulmonary
artery smooth muscle cells was abolished by selective block of
BKCa channel activation. Thus, iberiotoxin completely
reversed hyperpolarization by isoprenaline, as was found to be the case
with cADPR. Consistent with a role for SR Ca2+ release via
RyRs in this process, hyperpolarization by isoprenaline was blocked by
chelation of intracellular Ca2+ with BAPTA and by
inhibition of RyR function with ryanodine, respectively. Significantly,
hyperpolarization by isoprenaline was also blocked by the cADPR
antagonists 8-amino-cADPR and 8-bromo-cADPR. In marked contrast, block
of IP3R activation had no effect on hyperpolarization by
cADPR and isoprenaline, respectively. These findings provide strong
support for our proposal that cADPR-dependent SR
Ca2+ release via RyRs mediates BKCa channel
activation by isoprenaline and mitigates against a role for
IP3Rs in this process.
-adrenoceptors in pulmonary artery smooth
muscle cells results, at least in part, from activation of adenylyl
cyclase, increased cytoplasmic cAMP levels, and activation of PKA,
leading to activation of ADP-ribosyl cyclase, increased cADPR
synthesis, consequent SR Ca2+ release via RyRs, and
hyperpolarization by BKCa channel activation. Neither
isoprenaline nor cAMP induced hyperpolarization in the presence of
cADPR antagonists, and cADPR antagonists were without effect on RyRs,
SR Ca2+ ATPase activity, and/or BKCa channel
activation per se. Thus, we find little evidence of a role
for PKA-dependent phosphorylation of the BKCa
channel protein, RyRs, or phospholamban in mediating hyperpolarization
by adenylyl cyclase-coupled receptors in isolated pulmonary artery
smooth muscle cells under the conditions of our experiments.
-adrenoreceptor activation.
-adrenoreceptor signaling targets,
via protein kinase A anchoring proteins (AKAPs; Refs. 52-54),
PKA-dependent cADPR synthesis to a particular RyR subtype in the "peripheral" SR that is in close apposition to
BKCa channels in the plasma membrane; 2)
cADPR-dependent vasoconstriction results from the
activation of a discrete RyR subtype localized in the central SR
proximal to the contractile apparatus; and 3) the peripheral and
central SR represent functionally segregated compartments. Further
investigations will, however, be required to confirm these proposals.
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ACKNOWLEDGEMENT |
---|
We thank Professor Alison Brading for kind help and support.
![]() |
FOOTNOTES |
---|
* This work was funded by a Wellcome Trust Project Grant (reference number 056423).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.
§ Present address: Worcester College, University of Oxford, Walton Street, Oxford, OX1 2HB.
To whom correspondence should be addressed. Tel.:
44-1334-463579; Fax: 44-1334-463600; E-mail: ame3@st-and.ac.uk.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M204891200
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ABBREVIATIONS |
---|
The abbreviations used are:
PKA, cAMP-dependent protein kinase;
BKCa, Ca2+-activated potassium;
SR, sarcoplasmic reticulum;
cADPR, cyclic ADP-ribose;
8-Br-cADPR, 8-bromo-cADPR;
8-NH2-cADPR, 8-amino-cADPR;
RyR, ryanodine receptors;
PGF2, (5Z, 9
, 11
, 13E,
15S)-9,11,15-Trihydroxyprosta-5,13-dienoic acid;
IP3, myo-inositol 1,4,5 trisphosphate;
IP3R, IP3
receptor;
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid;
H89, N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide
hydrochloride.
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