cAMP activates an ATP-permeable
pathway in neonatal rat cardiac myocytes
Alan S.
Lader1,2,
Yong-Fu
Xiao1,2,
Catherine
R.
O'Riordan3,
Adriana G.
Prat1,2,
G. Robert
Jackson Jr.1, and
Horacio F.
Cantiello1,2
1 Renal Unit, Massachusetts General Hospital East,
Charlestown 02129; 2 Department of Medicine, Harvard
Medical School, Boston 02115; and 3 Genzyme Corporation,
Framingham, Massachusetts 01701
 |
ABSTRACT |
The molecular mechanisms associated with
intracellular ATP release by the heart are largely unknown. In this
study the luciferin-luciferase assay and patch-clamp techniques were
used to characterize the pathways responsible for ATP release in
neonatal rat cardiac myocytes (NRCM). Spontaneous ATP release by NRCM
was significantly increased after cAMP stimulation under physiological
conditions. cAMP stimulation also induced an anion-selective
electrodiffusional pathway that elicited linear,
diphenylamine-2-carboxylate (DPC)-inhibitable Cl
currents
in either symmetrical MgCl2 or NaCl. ATP, adenosine 5'-O-(3-thiotriphosphate), and the ATP derivatives ADP and
AMP, permeated this pathway; however, GTP did not. The cAMP-induced ATP
currents were inhibited by DPC and glibenclamide and by a monoclonal
antibody raised against the R domain of the cystic fibrosis
transmembrane conductance regulator (CFTR). The channel-like nature of
the cAMP-induced ATP-permeable pathway was also determined by assessing
protein kinase A-activated single channel Cl
and ATP
currents in excised inside-out patches of NRCM. Single channel currents
were inhibited by DPC and the anti-CFTR R domain antibody. Thus the
data in this report demonstrate the presence of a cAMP-inducible
electrodiffusional ATP transport mechanism in NRCM. Based on the
pharmacology, patch-clamping data, and luminometry studies, the data
are most consistent with the role of a functional CFTR as the anion
channel implicated in cAMP-activated ATP transport in NRCM.
ATP channels; ATP release; cystic fibrosis transmembrane
conductance regulator
 |
INTRODUCTION |
IN CARDIAC TISSUES,
ATP is released to the extracellular milieu in response to transient
anoxia (10, 49), exposure to adrenaline
(49), and activation of the cAMP pathway
(22). However, the molecular mechanisms associated with
ATP release in cardiac myocytes are largely unknown. Recent studies
have identified ATP transport pathways in various cell types.
P-glycoproteins, for example, have been shown to be responsible for ATP
release (1, 5, 37). ATP also
permeates through the related ATP-binding cassette (ABC)
transporters, human epithelial (6, 31,
36, 41) and shark cystic fibrosis
transmembrane conductance regulator (CFTR) (7,
8). Recent studies indicate that the cAMP-activated Cl
conductance of cardiac cells may be associated with
the expression of CFTR (17, 26,
29). CFTR is present in various mammalian heart
preparations including rabbit (47) and guinea pig
(29). However, adult rat and mouse cardiac myocytes were
previously reported to lack a cAMP-activated Cl
conductance, tantamount to a functional CFTR (11,
27). Similar findings have been reported in adult human
cardiac myocyte preparations (30).
Studies from our laboratory have recently demonstrated the presence of
a functional cardiac CFTR in primary cultures of neonatal mouse cardiac
myocytes (NMCM) (25). In that study, a cAMP-inducible, time-independent, and Cl
- and ATP-permeable conductance
was found in wild-type NMCM. This Cl
- and ATP-permeable
conductance was absent in NMCM obtained from homozygous CFTR knockout
mice, yet present in NMCM from mice heterozygous for the CFTR knockout.
Consistent with CFTR function in this cell preparation, the
cAMP-activated anion currents of NMCM were inhibited by
diphenylamine-2-carboxylate (DPC), glibenclamide, and an anti-CFTR antibody. Recent studies, however, have also provided evidence suggesting the presence of a functional CFTR in neonatal rat cardiac myocytes (NRCM) (48, 50, 51).
Thus the possibility exists for this cardiac preparation to also
express a cAMP-inducible ATP-permeable electrodiffusional pathway.
To date, no comprehensive study has addressed the issue of noncytolytic
ATP movement by cardiac myocytes in the rat heart.
Therefore, the purpose of the present study was to assess whether NRCM
release cellular ATP and to determine whether CFTR may play a role in
ATP transport in this cardiac myocyte model. The data indicate that
cultured NRCM released cellular ATP both spontaneously and following
cAMP stimulation. Activation of NRCM by cAMP also induced an
anion-selective conductance that was permeable both to Cl
and ATP. The cAMP-activated Cl
and ATP conductance was
linear (nonrectifying) and was inhibited by DPC, glibenclamide, and an
anti-CFTR R domain antibody, but was insensitive to the anion channel
blocker DIDS. The data are most consistent with the presence of a
functional CFTR, which is implicated in the cAMP-stimulated anion
conductance present in NRCM. Basal ATP movement, however, may represent
a distinct transport mechanism that will require further investigation.
 |
MATERIALS AND METHODS |
Primary culture of NRCM.
Primary cultures of NRCM were obtained with a commercial isolation kit
(Worthington Biochemical, Freehold, NJ) as previously reported
(20, 21). Cardiac cells, mostly of
ventricular origin, displayed a pattern of spontaneous beating and
contraction. Healthy (beating) batches of cells were seeded and grown
for up to 2 wk on glass coverslips for patch clamping as previously
indicated for other cells (1, 36).
Luciferin-luciferase assay.
ATP release from NRCM was assessed with the luciferin-luciferase assay
using a monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) and methods previously described
(1, 33). Briefly, extracellular and total
cellular ATP were measured from confluent NRCM grown on glass
coverslips. At the time of the experiment, the coverslips were placed
in 12 × 75 mm plastic cuvettes containing 0.1 ml of the
luciferin-luciferase assay mix and 0.5 ml of a Ca2+-free
solution containing (in mM) 140 NaCl 140, 5.0 KCl, 0.8 MgCl2, and 10 HEPES, pH 7.4. The purified
luciferin-luciferase solution (Analytical Luminescence Laboratory)
contained 3 µg/ml luciferase and 400 µM luciferin in 25 mM HEPES
buffer (pH 7.75; catalog no. 234671; Analytical Luminescence
Laboratory). The HEPES buffer also contained 10 mM MgCl2, a
cofactor of the luciferin-luciferase reaction. The final pH of the
mixture was 7.40. Photon release was continuously measured in a
luminometer (MonoLight 2010; Analytical Luminescence Laboratory).
Coverslips were placed in a plastic cuvette after washing the
"contaminating" culture medium (33) and were held
vertically with microclips (Roboz Surgical Instruments, Rockville, MD).
The ATP release was followed by the photon release of the
luciferin-luciferase assay for ~2 min before membrane
permeabilization was performed. Total intracellular ATP release was
accomplished by addition of alamethicin (10 µM; Sigma, St. Louis, MO)
and vigorous shaking to lyse the cell membrane. Photon release was
again followed for another 2 min. To determine the amount of ATP
released from cells, known concentrations of ATP in solution were also
measured to construct a calibration curve. To obtain values of
intracellular ATP concentration, the cell volume (Vc),
993 ± 237 µm3 (n = 6) was obtained
using microscopy by measuring the cell diameter (r × 2) and calculating Vc = (4/3) ×
× r3, since cultured NRCM are largely of round shape.
CFTR activation for the ATP release assay.
To activate the cAMP-dependent stimulatory pathway of NRCM, cells were
incubated for 6-12 h with cholera toxin (CT; 1 µg/ml; Sigma).
This maneuver was chosen because it does not interfere with the
luciferin-luciferase assay. CT has been extensively used to activate
Gs and, hence, adenylyl cyclase in cardiac preparations, further validating the technique in this preparation. Neither cell
volume nor cell count changed significantly after CT treatment. Both
control and CT-treated cells excluded Trypan blue, an indication that
cell permeability was not impaired by the experimental conditions imposed. This was further supported by the ability of CT-treated cells
to spontaneously beat. The best assessment of the integrity of the
CT-treated cells, however, relied on the intracellular ATP
concentration as it was assessed after cell permeabilization with
alamethicin (see RESULTS; for comparison see Ref. 33). In
some experiments, cAMP-activated cells grown on coverslips were used to
assess the effect of CFTR activation on ATP release. Coverslips were
incubated for 20-30 min in the presence of a cAMP-stimulatory cocktail containing 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 500 µM), IBMX (200 µM), and forskolin (10 µM), and then transferred to a cocktail-free medium and rinsed before
transferring to the cuvette.
To pharmacologically assess the ATP release mechanism, either the
Cl
channel blocker DPC (400 µM; Fluka Chemical,
Ronkonkoma, NY) or DIDS (400 µM; Sigma) was added to the incubation
medium. The coverslips were incubated with the blockers for 15-20
min before the experiments. Coverslips were then rinsed of the
contaminating culture medium before being placed in the cuvette. This
process took <30 s, thus accounting for the inhibitory effect of DPC
after withdrawal from the extracellular medium. The entire process took <3 min, validating the remaining effect, because DPC works from the
intracellular side of the cell.
Whole cell currents.
Currents and command voltages were obtained and driven, respectively,
with a Dagan 3900 amplifier (Dagan, Minneapolis, MN) using a 1-G
headstage, and further analyzed as previously described (1, 36). Whenever indicated, the patch
pipette was filled up to at least one third of its height with either
MgATP or TrisATP (100 mM, pH 7.4 adjusted with
N-methyl-D-glucamine), and the remainder of the
pipette was backfilled with the NaCl-containing solution as
previously reported (1, 36). This technique
allows a suitable liquid-liquid interface that separates the
Cl
-plated electrode, which was in contact with the top
NaCl-containing solution, from the ATP solution (tip and shank). Thus
this setup provided a gradient front with a safe margin for the ATP
solution (several thousand times the cell volume) in contact with the
intracellular compartment, and preventing Cl
contamination under any of the experimental conditions
(1). This has been previously determined experimentally
and also conforms with theoretical calculations of diffusional movement
of Cl
into the ATP solution, which may take several hours
to reach the tip of the pipette (19). Thus, in experiments
where ATP was present in the pipette, the cell was dialyzed in the
complete absence of Cl
. In some cases, ATP solutions (100 mM) were also used as bathing solutions. Otherwise, the pipette and
bathing solution contained (in mM) either 140 NaCl , 1.0 MgCl2, 5 KCl, and 10 HEPES, at pH 7.4, or 70 MgCl2, 10 HEPES, pH 7.4. The bathing solution also contained 1.0 mM CaCl2. Experiments were conducted at room
temperature. Conductances were determined by fitting the current vs.
voltage relationship between ±100 mV to the Goldman-Hodgkin-Katz (GHK) equation or to a linear equation.
Single channel studies.
The excised patch-clamp configuration was carried out as previously
described (1, 36). Data from excised
inside-out patches with upward and downward deflections indicating the
channel open state for positive and negative holding potentials,
respectively, were obtained between ±100 mV.
Calculation of perm-selectivity ratios under asymmetrical ATP
conditions.
To calculate the perm-selectivity ratio
PA/PB under either
asymmetrical intracellular ATP (ATPi)/extracellular
Cl
(Clo) conditions or asymmetrical ATP
salts, the following solution of the GHK equation was used
where
= F/RT, zA is
the valence of anion A, zB is the valence of
anion B, and A and B are the two different anion concentrations. The
subscripts i and o represent the intra- and extracellular compartments,
respectively. Er is the reversal potential under asymmetrical conditions; F, R, and T have their usual meaning.
Drugs and chemicals.
The cAMP stimulatory cocktail contained 8-BrcAMP, IBMX, and forskolin
and was used at a final concentration of 500, 200, and 10 µM,
respectively. The cAMP analog 8-BrcAMP (Sigma) was used from a 25 mM
stock in a 1:1 (vol/vol) ethanol/methyl sulfoxide (DMSO) solution.
Forskolin (Sigma) was used from a 100% ethanol, 10 mM stock solution.
The phosphodiesterase inhibitor IBMX (Sigma) was used from a 20 mM
solution in ethanol. In some cases, CT (1 µg/ml, Sigma) was added to
the medium to stimulate the cAMP pathway for 6-12 h before the
experiment. The catalytic subunit of the cAMP-dependent protein kinase
(PKA; Sigma) was used at a final concentration of 20 µg/ml. The
Cl
channel blocker DPC (Fluka Chemical) was kept in a
100-fold stock solution (20 mM) in 50% water/ethanol. The DIDS (Sigma)
was kept in a 10 mM stock solution in 100% DMSO. Glibenclamide (RBI,
Natick, MA) was kept in a 40 mM stock solution in 100% DMSO. The
monoclonal antibody (MAb) raised against amino acids 729-736 of
the R domain of CFTR (MAb no. 13-1; Genzyme, MA) was directly diluted
to 1:100 times in the intracellular or bathing solution from a stock
solution (292 µg/ml). An inactive antibody was obtained by preheating
the CFTR antibody for 30 min at 100°C. For the whole cell
experiments, the antibody was diluted into the intracellular solution
and loaded into the pipette, thus gaining access to the R domain by way
of dialysis from the pipette.
 |
RESULTS |
ATP release by NRCM: effect of cAMP stimulation.
The release of ATP by cultured NRCM, originally reported for cardiac
myocytes in 1959 (15), was determined in both control and
CT-treated cells using the luciferin-luciferase assay, as previously
described (1, 33). Cells were first
stimulated with CT (1 µg/ml) for 6-12 h to increase
intracellular cAMP. Similar intracellular ATP concentrations were
observed in controls (2.19 ± 0.21 mM, n = 15) and
CT-treated NRCM (2.13 ± 0.23 mM, n = 15, P < 0.8). The control values were in agreement with
previous reports on cardiac cells (13). The ATP released
by the NRCM, however, was 109% higher for the CT-treated cells
compared with controls (106 ± 15 nmol/g cells, n = 15, vs. 222 ± 48 nmol/g cells, n = 15, P < 0.05), indicating a significant increase in ATP
release after cAMP activation. In addition, the ratio (expressed in %) of released (extracellular) ATP to total ATP, measured after cell permeabilization (see MATERIALS AND METHODS) was 117%
higher in the CT-treated cells compared with controls. Individual
ratios for the CT-treated cells ranged between 4.9% and 32.6% with
most (>90%) cells in the range of 6-15%. Similar results were
obtained with a cAMP-stimulatory cocktail, which increased the ratio of released ATP to total ATP by 97% (6.45 ± 0.65%,
n = 25, vs. 12.7 ± 2.21%, n = 26, P < 0.05, Fig.
1A).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1.
ATP release by cultured neonatal rat cardiac myocytes
(NRCM). A: cells were studied under control conditions
(n = 25) and after being treated with a
cAMP-stimulatory cocktail (n = 26) for 15-20 min
before the measurement of ATP release. Also shown are the effects of
diphenylamine-2-carboxylate (DPC; n = 18) and DIDS
(n = 16) on cAMP-stimulated cells. The
luciferin-luciferase assay was used as indicated in MATERIALS AND
METHODS and as previously reported (1,
33). Values are means ± SE. * Statistical
significance (P < 0.05) between control and
cAMP-stimulated cells; # statistical significance (P < 0.05) between cAMP-stimulated cells with and without DPC treatment.
B: cells were studied under control conditions and following
DPC treatment in the absence of cAMP stimulation. Differences did not
reach statistical significance. Values are means ± SE for 25 and
8 experiments under control and DPC treatment, respectively.
|
|
ATP release by NRCM was not increased by cAMP-independent stimulation.
The released ATP-to-total ATP ratio following addition of the L-type
Ca2+ channel agonist, BAY K 8644 (Sigma), was 5.35 ± 0.42% (n = 5), thus similar to controls
(P < 0.5).
Addition of DPC, a known inhibitor of the cAMP-induced and
CFTR-mediated ATP release (33), completely inhibited the
CT-induced ATP release of NRCM. The released-to-total ATP ratio of
CT-treated cells decreased significantly after DPC treatment (4.3 ± 1.6%, n = 5; P < 0.05), thus
returning ATP release to control values (4.40 ± 1.20%,
n = 5, P < 0.9). Addition of DPC (400 µM) to NRCM stimulated with the cAMP-stimulatory cocktail also
significantly reduced the released-to-total ATP ratio (12.7 ± 2.21%, n = 26, vs. 5.16 ± 0.56%,
n = 18, P < 0.01, Fig. 1A);
however, DIDS (400 µM) was without effect (12.7 ± 2.21%,
n = 26, vs. 15.4 ± 4.82%, n = 16, P < 0.7, Fig. 1A). Interestingly, DPC
also reduced basal ATP release by 22% in the absence of CT or cAMP
stimulation (4.98 ± 0.98%, n = 8, Fig.
1B); however, this difference did not reach statistical
significance (P < 0.3).
Whole cell Cl
currents of NRCM.
To assess the presence of a functional CFTR in NRCM, whole cell
Cl
currents were first assessed, as this inorganic anion
permeates this channel protein after cAMP stimulation (3,
36). Whole cell Cl
currents in either
symmetrical NaCl or MgCl2 solutions were recorded with the
voltage-clamp technique (14). Addition of the
cAMP-stimulatory cocktail increased the nonrectifying whole cell
currents by 1,440% (0.86 ± 0.21 nS/cell vs. 15.5 ± 2.95 nS/cell, n = 7, P < 0.01, Fig.
2, A and B).
Currents were partially blocked (55.1 ± 10.2%, n = 7, Fig. 2, A and B) by the Cl
channel blocker DPC (400 µM), with individual inhibition values ranging from 20 to 82% (P < 0.005). No effect was
observed by addition of DIDS (400 µM, data not shown). cAMP also
stimulated Cl
currents of NRCM in symmetrical
MgCl2 (70 mM), thus indicating the anionic nature of the
conductance pathway, which was reversibly inhibited by addition of
glibenclamide (100 µM) to the bathing solution (Fig. 2C).
Thus the data are consistent with the presence of a cAMP-stimulated
Cl
conductance in NRCM, in agreement with the recently
reported presence of a functional CFTR in this cell model
(48).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of cAMP on Cl currents of
NRCM. A: whole cell currents (I) were obtained in
symmetrical NaCl (140 mM), before ( ), after addition of the
cAMP stimulatory cocktail ( ), and after addition of
cocktail + DPC (400 µM, ). Data are the average
of 7 experiments for basal and stimulated conditions, respectively.
Similar results were obtained in the presence of symmetrical
MgCl2 70 mM. B: tracings indicate whole cell
currents under either control conditions (top) or in the
presence of cAMP-stimulatory cocktail (middle) and DPC
(bottom). Dashed line indicates zero current. C:
whole cell currents were obtained in symmetrical MgCl2 (70 mM) before (top), after addition of the cAMP-stimulatory
cocktail (second), after addition of the cocktail + glibenclamide (third), and after washout of the bathing
solution (bottom). Vh, holding
potential.
|
|
Whole cell ATP currents of NRCM.
To assess the possibility that the cAMP-activated anion pathway of NRCM
was also permeable to cellular nucleotides, cells were dialyzed
intracellularly with various salts of ATP (100 mM) in the presence of
the Cl
bathing saline, as previously reported
(34, 36). In the presence of 100 mM MgATP in
the pipette, and 150 mM Cl
in the bathing solution,
addition of the cAMP-stimulatory cocktail induced a 795% stimulation
of the whole cell currents (1.05 ± 0.09 vs. 9.40 ± 0.51 nS/cell, n = 10, P < 0.001, Fig.
3, A and B). The ATP/Cl
electrodiffusional pathway
induced by cAMP stimulation was inhibited by DPC (48.0 ± 9.8%,
n = 10, with individual inhibition values ranging from
10 to 100%; Fig. 3, A and B) but not by DIDS
(9.82 ± 0.72 vs. 10.04 ± 0.53 nS/cell, n = 12, P < 0.8, Fig. 3C). A linear
current-voltage (I-V) relationship was found for the
cAMP-activated ATP/Cl
currents consistent with a
perm-selectivity ratio
(PATP/PCl) of 0.35 (for
zATP =
2) as calculated from the
cAMP-stimulated whole cell currents. cAMP stimulation was also
associated with a
39.4 mV (51.4 ± 8.7 vs. 12.2 ± 4.2 mV,
n = 10; P < 0.001, Fig. 3A)
depolarizing shift in the reversal potential, in agreement with
activation of an anionic conductive pathway. Treatment of NRCM with CT
(6-12 h) also resulted in a 544% increase in the ATP/Cl
whole cell currents (1.05 ± 0.09 nS/cell,
n = 10, vs. 6.76 ± 0.33 nS/cell,
n = 5; P < 0.001). The CT-induced
whole cell currents were inhibited by 66.3% with DPC (2.28 ± 0.23 nS/cell, n = 5, P < 0.001). A
strong linear correlation between the cAMP-stimulated Cl
and ATP currents was found with a slope of 1.22 ± 0.14 (Fig. 3D, n = 26), thus indistinguishable from
1.0, and consistent with the simultaneous activation of a
Cl
/ATP permeable pathway. With no cAMP stimulation (basal
currents), however, only ATP currents were observed, indicating that,
under these conditions, ATP movement is not accompanied by
Cl
transport (Fig. 3D). These data suggest
that, in the absence of cAMP stimulation, a CFTR-independent
electrodiffusional pathway may be present, in agreement with the
DPC-independent basal ATP release.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of cAMP on ATP currents of NRCM. A:
whole cell currents were obtained in the presence of intracellular
MgATP (100 mM) and bathing Cl (140 mM) before ( ),
after addition of the stimulatory cocktail ( ), and DPC
( ). Data are the average of 10 experiments for basal
and stimulated conditions, respectively. Solid lines indicate the curve
fit of the data to the Goldman-Hodgkin-Katz equation. B:
tracings indicate control (top), in the presence of
cAMP-stimulatory cocktail (middle), and DPC
(bottom). Dashed line indicates zero current. C:
effect of cAMP and DIDS on whole cell conductance. cAMP-stimulated
whole cell currents were obtained in the presence of intracellular ATP
(100 mM). The bathing solution contained 140 mM Cl . Whole
cell conductance was obtained before and after addition of the
cAMP-stimulatory cocktail and further addition of DIDS (400 µM). Bars
representing the various experimental conditions are means ± SE
of 4-10 experiments. D, top: whole cell
currents were obtained before ( ) and after cAMP
stimulation ( ) in the presence of intracellular MgATP and
bathing Cl (140 mM). Respective single cell values were
plotted as the whole cell currents at +100 mV (Cl ,
abcissa), and 100 mV (ATP, ordinate). A strong linear correlation was
found between Cl and ATP currents. The slope, 1.22 ± 0.14 (n = 26), indistinguishable from 1.0, indicates
simultaneous activation of movement for both ions. An expanded plot for
control values is shown below (bottom), indicating a lack of
correlation between the ATP and the Cl currents in the
absence of cAMP stimulation (n = 22).
|
|
Interestingly, the ATP analog ATP
S permeated through the
cAMP-activated anion pathway; however, the nucleotide GTP did not (Fig.
4).

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 4.
Whole cell conductance for different nucleotides and
analogs. Currents were obtained in the presence of 100 mM intracellular
nucleotides, as indicated in the respective bars, and bathing
Cl (140 mM). Whole cell conductance was obtained as the
difference between whole cell currents before and after addition of the
cAMP-stimulatory cocktail. Bars representing the various experimental
conditions are means ± SE of 4-10 experiments. ATP S,
adenosine 5'-O-(3-thiotriphosphate).
|
|
Effect of NRCM aging in culture on whole cell conductance.
Both the ATP and Cl
currents activated by cAMP were
maximal between 1-3 days in culture and simultaneously decreased
to another, lower level that was maintained between 7 and 13 days in
culture (Fig. 5). This phenomenon is
consistent with the possibility that aging and/or redifferentiation of
the cultured NRCM downregulated the anion pathway, in agreement with
previously unsuccessful attempts to observe cAMP-activated
Cl
currents in adult rat cardiac myocytes
(11, 44).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of time in culture on the cAMP-stimulated whole
cell conductances of NRCM. cAMP-dependent whole cell conductance was
obtained as the difference between controls (before) and
cAMP-stimulated whole cell currents as a function of NRCM time in
culture. Whole cell currents were obtained in the presence of
intracellular MgATP (100 mM), and bathing 140 mM NaCl. Values are
means ± SE of 3-11 experiments.
|
|
ATP currents of NRCM in the absence of Cl
.
cAMP also induced a 2,600% increase in ATP currents under symmetrical
(100 mM) MgATP conditions (0.25 ± 0.03 vs. 6.75 ± 0.59 nS/cell, n = 7; P < 0.001, Fig.
6). Under these conditions, ATP currents
were linear between ±100 mV and inhibited by DPC (53.9 ± 3.3%,
n = 4). The whole cell conductance in the presence of symmetrical MgATP (6.75 ± 0.59 nS/cell, n = 7;
Fig. 6) was similar to that in symmetrical TrisATP (6.23 ± 0.17 nS/cell, n = 4, P < 0.5; Fig. 6).
cAMP-induced whole cell currents originally obtained in symmetrical
MgATP were also subsequently tested with extracellular TrisATP to
further assess the role of the ATP counterion to the cAMP-stimulated
ATP currents. In the presence of asymmetrical ATP solutions
(MgATPi/TrisATPo, where i = intracellular
and o = extracellular), the whole cell conductance was 8.07 ± 0.40 nS/cell (n = 4, Fig. 6), also indistinguishable
from values under either symmetrical Mg2+ or Tris
conditions. The results are also consistent with similar reversal
potentials obtained under either conditions of
MgATPi/ TrisATPo (11 ± 7 mV,
n = 4), MgATPi/MgATPo (9 ± 6 mV, n = 7), or
TrisATPi/TrisATPo (16 ± 8 mV,
n = 4).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
Whole cell currents in symmetrical ATP. Whole cell
currents were obtained in symmetrical ATP salts (100 mM), before (open
symbols) and after (filled symbols) stimulation with cocktail. Whole
cell currents were obtained with either symmetrical MgATP
(n = 7, circles) or TrisATP (n = 5, triangles). cAMP-stimulated whole cell currents obtained with
intracellular MgATP and extracellular TrisATP are indicated by
filled squares (n = 4). The solid lines indicate the
curve fit of the data to the Goldman-Hodgkin-Katz equation. Data are
means ± SE.
|
|
Effect of anti-CFTR antibodies on the whole cell currents of NRCM.
To further characterize the cAMP-activated whole cell
ATP/Cl
currents in NRCM, the effect of a monoclonal
antibody raised against the R domain of human CFTR, known to block CFTR
function (25, 34), was assessed. In cells
intracellularly dialyzed with a 1:100 dilution of MAb no. 13-1 (Genzyme), the active antibody completely abolished the cAMP-stimulated
Cl
and ATP currents (Fig.
7, A and B), while
intracellular dialysis of heat-inactivated antibody was without effect
on the cAMP-stimulated whole cell currents (Fig. 7, C and
D).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of anti-cystic fibrosis transmembrane conductance
regulator (CFTR) R domain antibody on the whole cell currents of NRCM.
Whole cell currents were obtained in the presence of intracellular
MgATP (100 mM) and bathing Cl (140 mM), before
( ) and after ( ) addition of the
cAMP-stimulatory cocktail. The intracellular solution contained either
1:100 dilution of active (A, B) anti-R domain antibody (MAb
no. 13-1; Genzyme) or heat-inactivated antibody (C, D). The
experimental points are the average of 4 experiments for basal and
stimulated conditions, respectively. Tracings (B, D)
indicate control (top) and in the presence of
cAMP-stimulatory cocktail (bottom).
|
|
Single channel ATP currents of NRCM.
The ATP currents were further characterized in excised inside-out
patches of NRCM. Under asymmetrical conditions, in the presence of 100 mM ATP in the patch pipette and Cl
(140 mM) in the
bathing solution, addition of PKA (20 µg/ml) and ATP (1 mM) to
excised patches induced single channel currents both in negative (ATP;
Fig. 8A) and positive
(Cl
; Fig. 8A) holding potentials in 10 of 12 patches tested with a single channel conductance of 12.2 ± 1.4 pS
(n = 7; Fig. 8B). The PKA-activated single
channel currents were inhibited by anti-CFTR antibodies against the R
domain (MAb no. 13-1; Fig. 8C) and DPC (Fig.
9), both similar to findings in cells
expressing human epithelial CFTR (36) and purified CFTR
reconstituted into lipid bilayers (6).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 8.
Single channel ATP and Cl currents in
excised patches from NRCM. A: single channel ATP currents
(top traces) were activated by addition of cAMP-dependent
protein kinase (PKA; 20 µg/ml) and ATP (1 mM) to quiescent excised,
inside-out patches. The histogram for the ATP currents shows 2 distinct
conductance states with the low conductance state indicated by the
arrow. Addition of PKA (20 µg/ml) and ATP (1 mM) to quiescent
excised, inside-out patches also activated single channel
Cl currents (bottom traces). B:
single channel conductance for the Cl /ATP currents. Shown
is the linear fit (solid line) of the data along with the 95%
confidence interval. Mean conductance was 12.2 ± 1.4 pS
(n = 7). C: single channel ATP currents
(top trace) were readily inhibited by addition of anti-CFTR
R domain antibody (MAb no. 13-1) to the bathing solution (bottom
trace).
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of DPC on ATP currents in asymmetrical
Cl /ATP. Addition of bathing DPC (400 µM) decreased the
PKA-induced single channel activity under asymmetrical conditions
(compare traces in A and C). Histograms
(B and D) indicate the decrease in channel activity
that is evident in the fewer second open states. The presence of 2 different channel states (6 and 17 pS) are also indicated in both
histograms. The calculated probability of the main conductance state
decreased from 0.58 to 0.18 within 30 s of DPC addition,
consistent with the inhibition observed with DPC in the whole cell
experiments.
|
|
Single ATP channel currents were also observed in 52% of the patches
(11/21) activated by addition of PKA in the presence of symmetrical
MgATP conditions (100 mM). Under these conditions, linear single
channel currents with a single channel conductance of 5.04 ± 1.57 pS (n = 12) were observed (Fig.
10). A rectifying "large-conductance" channel also was observed, with a conductance (at positive potentials) of 57.9 ± 2.60 pS (n = 4; Fig. 11, A and B). The larger channel currents presented conductance
substates of the same magnitude as the small channel species (Fig.
11C). However, due to the fact that the relative frequency
of the two substates of the ATP channel currents could not be
quantitatively assessed, the data still cannot rule out that different
conductances may represent different channel entities. Nevertheless,
various conductance states are in agreement with multiple ATP channel conductance substates (both 50 and 5 pS) for CFTR, as previously reported for cells expressing CFTR (34, 36),
and more recently for the reconstituted protein (6).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 10.
Small conductance single channel ATP currents in
excised patches from NRCM. A: single channel ATP currents
(symmetrical MgATP, 100 mM) were activated by addition of PKA (20 µg/ml) to excised, inside-out patches. Bottom trace is the
expanded segment marked with the bold line in the top tracing.
B: current-to-voltage relationship for the small conductance
channel. The linear fit is shown (solid line) along with the 95%
confidence interval. Data are means ± SE from 12 experiments.
|
|

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 11.
Large conductance single channel ATP currents
in excised patches from NRCM. A: PKA-activated ATP single
channel currents with a large conductance state. B:
current-to-voltage relationship for the large conductance channel. Data
are expressed as means ± SE from 4 experiments. Also shown is the
linear fit (solid line) along with the 95% confidence interval.
C: single large conductance channel tracings also contained
"small conductance" currents. Left: expanded large
conductance ATP currents also showing the presence of the "small"
conductance substates (solid line and expanded trace,
bottom). The small conductance in this example was 14.5 pS.
Right: all point histograms for the tracings on the
left.
|
|
 |
DISCUSSION |
Effects of ATP and derivatives in the mammalian heart.
Both ATP and hydrolytic metabolites, chief among which is adenosine,
exert profound electrophysiological effects in the mammalian heart,
including a negative chronotropic effect on cardiac pacemakers and
dromotropic effects on the atrial-ventricular node (32). Extracellular nucleotides also affect coronary blood flow and, hence,
heart function by acting on coronary vasculature (16, 23, 24, 45). However,
extracellular nucleotides may also target purinergic receptors in
cardiac myocytes and/or cardiac endothelial cells. ATP-mediated
purinergic receptor regulation may affect cardiac electrical activity
by coupling to ion channel modulation associated with Ca2+
homeostasis, and excitation-contraction coupling (35,
54, 55). Extracellular ATP, for example,
decreases action potential duration in ferret ventricular myocytes
(35), a phenomenon that seemed to be mediated by a
decrease in L-type Ca2+ currents. This may be species
specific, however, as the opposite has been observed in rat ventricular
myocytes (38, 40). Dual effects have been
observed, on the other hand, in bullfrog cardiac myocytes
(2).
This implies that external ATP-regulated cardiac electrical activity
may be more complex than invoking direct changes in Ca2+
homeostasis. Extracellular ATP, for example, has been shown to regulate
other ion channel activity in cardiac cells, including Na+
(39), muscarinic acetylcholine K+
(42), and Cl
channel function
(18, 27). More recently, the effect of
extracellular ATP has been associated with the activation of
nonselective cation channels in the heart (42). Thus
extracellular ATP may be responsible for a variety of effects in
cardiac function, yet to be more clearly assessed. Extracellular ATP
has been associated with the induction of action potential
afterdepolarization in cardiac myocytes (43) and
alterations in gene expression associated with cardiac hypertrophy (52, 53). Interestingly, possible
explanations for these phenomena, such as alterations in the release
and pericellular concentration of ATP in the heart, have never been invoked.
Findings of the present study.
The data in this study demonstrate that cAMP stimulation of NRCM with
either CT or a cAMP-stimulatory cocktail increased the release of
cellular ATP. The cAMP-stimulated release of ATP was inhibitable with
DPC but was insensitive to DIDS, suggesting the involvement of CFTR as
previously reported (33). The data indicated, however,
that NRCM also displayed spontaneous ATP release, which was relatively
insensitive to DPC, suggesting that another, as yet undetermined ATP
pathway is likely involved. This is further supported by the lack of
correlation found between the basal Cl
and ATP currents,
suggesting that ATP transport in the absence of cAMP is uncoupled from
Cl
transport. Thus it is unlikely that CFTR was involved
in the basal ATP-permeable pathway.
Under asymmetrical conditions, with ATP in the pipette and
Cl
in the bathing solution, cAMP activation with either a
cAMP-stimulatory cocktail or treatment with CT, elicited an
electrodiffusional pathway permeable to both Cl
and ATP.
This anion conductance was inhibited by DPC, a known inhibitor of CFTR
(28). However, the ATP/Cl
conductance was
unaffected by DIDS. The pattern of cAMP activation and blocker
pharmacology was in agreement with the data observed with the ATP
release assay. The presence of a cAMP-stimulated linear whole cell ATP
conductance was further observed under symmetrical ATP conditions with
either Tris or Mg2+ as the counterion. The permeation of
ATP was confirmed by similar currents under asymmetrical ATP conditions
(MgATP in the pipette and TrisATP in the bathing solution), indicating
that ATP was indeed the charge carrier. One interesting finding was
that only adenine, but not guanine nucleotides, permeated the
cAMP-activated anion pathway. The nucleotide GTP failed to permeate
this pathway. The possibility that CFTR may be involved in the
cAMP-activated whole cell Cl
and ATP conductance was
supported by the simultaneous inhibition of both ATP and
Cl
conductances with an anti-CFTR R domain antibody,
known to block human epithelial (34) and mouse cardiac
CFTR (25).
Further characterization of the electrodiffusional pathway for ATP was
conducted at the single channel level. The PKA-activated single channel
currents displayed characteristics similar to those previously reported
for cells expressing human epithelial CFTR (34,
36), including the presence of multiple substates, and inhibition by DPC and anti-CFTR antibodies. Addition of DPC inhibited the single channel open probability to a degree consistent with the
inhibition observed in the whole cell data, whereas the anti-CFTR antibody completely blocked the ATP channel activity, also consistent with the lack of cAMP-stimulated whole cell currents in the presence of
the active but not the preheated antibody. Thus the most likely scenario is that cAMP stimulation activates CFTR in this cell model.
The possibility that more than one ion channel species is present in
this cell preparation cannot be ruled out, however, since more than one
single channel conductance was observed under symmetrical ATP
conditions. Nevertheless, both 50- and 5-pS single channel conductances
have been previously observed in CFTR expressing cells (9,
36), suggesting that the similar findings in NRCM may be
functionally related to CFTR. It is interesting to note, however, that,
under basal conditions in symmetrical ATP, excised patches of NRCM
showed no channel activity. This is in contrast to the ATP release and
to ATP currents (but not Cl
) under basal conditions. The
presence of PKA-insensitive ATP channels will have to be further
explored. All channel activity described in this report was activated
by PKA.
Our data suggest that the cAMP-activated electrodiffusional ATP
movement of NRCM may be largely responsible for the delivery of cardiac
ATP to the extracellular milieu, which can be validated by comparing
the cAMP-induced ATP release, measured under physiological conditions,
and the whole cell ATP currents, which for technical reasons were
conducted in the presence of high intracellular ATP (33).
Provided that the cAMP-induced ATP release of NRCM under physiological
conditions is considered electrodiffusional, the ATP current generated
by its movement was calculated: Icalc = zFJ, where J represents the single-cellular, cAMP-induced
release of ATP (1.75 × 10
16
mol·cell
1·s
1), F is
Faraday's constant, and z, the valence of ATP, was either
2 or
4. The Icalc obtained was either 3.38 or 6.76 pA/cell for z =
2 or
4, respectively. The
electrophysiological data, on the other hand, were calculated as
previously reported (36) using the GHK equation for
electrophysiological conditions, including an average membrane
potential of
45 mV and intracellular ATP = 2.2 mM. The
calculated whole cell currents expected for the observed ATP release
under physiological conditions were 5.79 or 5.66 pA/cell for z
=
2 or
4, respectively, thus in close agreement with the
values observed.
Mechanisms and implications of cardiac ATP release.
Various stimuli trigger the noncytolytic release of cellular ATP by
cardiac myocytes, including catecholamine stimulation (49), and an increase in intracellular cAMP
(22). However, the molecular mechanisms associated with
this ATP transport are as yet unknown. To date, only few molecular
structures have been identified in electrodiffusional ATP transport.
These include P-glycoproteins (1, 5,
37) and human epithelial (6, 31,
36, 41) and shark CFTR (7,
8). Cardiac muscle expresses several members of the ABC
family of transporters, including P-glycoprotein, the related lung
resistance protein (4, 12, 46),
and CFTR (25, 26, 29,
48). All are likely candidates for membrane-associated ATP transport.
Cardiac CFTR, a spliced version of its epithelial homolog, has been
observed in cardiac tissues, where it has been implicated in the
cAMP-activated Cl
currents of cardiomyocytes
(26, 29). Previous studies have determined
the presence of cAMP-induced Cl
currents, a paradigm
associated with the presence of CFTR in adult guinea pig
(29) but not adult rat (11, 44)
or human cardiomyocytes (30). However, recent evidence has
demonstrated the presence of CFTR in the neonatal rat heart by Western
blotting and demonstrated that CFTR is indeed functional
(48). The possibility exists, however, for as yet unknown
putative ATP channels to also be associated with ATP transport in this preparation.
Conclusion.
The data in this report indicate that cAMP stimulation of NRCM
activates an anion-permeable pathway that allows cellular ATP to move
out of the cells. The close agreement between the electrophysiological and ATP release data suggests the presence of a largely
electrodiffusional pathway that can be regulated by the electrical
activity of the activated cell. Based on the electrophysiological data
and pharmacology of inhibition, the data also imply the presence of a
functional CFTR to be at least in part responsible for the phenomenon.
The spontaneous, DPC-insensitive ATP release and ATP currents uncoupled to Cl
currents under basal conditions, however, suggest
the presence of at least another ATP transport pathway, which may or
may not be associated with CFTR. Only further experimentation will
determine the molecular identity of the different ATP transport
mechanisms in the neonatal heart.
 |
ACKNOWLEDGEMENTS |
We are extremely grateful to Dr. Alexander Leaf and his group from
the Department of Preventive Medicine at the Massachusetts General
Hospital for the supply of initial batches of primary cultures of rat
cardiac myocytes.
 |
FOOTNOTES |
This work was partially supported (H. F. Cantiello) by a
Grant-in-Aid from the American Heart Association (AHA) and with funds contributed in part by the AHA, Massachusetts Affiliate.
Address for reprint requests and other correspondence:
H. F. Cantiello, Renal Unit, Massachusetts General Hospital East,
149 13th St., Charlestown, MA 02129 (E-mail:
cantiello{at}helix.mgh.harvard.edu).
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. §1734 solely to indicate this fact.
Received 26 February 1999; accepted in final form 21 January 2000.
 |
REFERENCES |
1.
Abraham, EH,
Prat AG,
Gerweck L,
Seneveratne T,
Arceci RJ,
Kramer R,
Guidotti G,
and
Cantiello HF.
The multidrug resistance (mdr1) gene product functions as an ATP channel.
Proc Natl Acad Sci USA
90:
312-316,
1993[Abstract].
2.
Alvarez, JL,
Mongo K,
Scamps F,
and
Vassort G.
Effects of purinergic stimulation on the Ca2+ current in single frog cardiac cells.
Pflügers Arch
416:
189-195,
1990[ISI][Medline].
3.
Anderson, MP,
Rich DP,
Gregory RJ,
Smith AE,
and
Welsh MJ.
Generation of cAMP-activated chloride current by expression of CFTR.
Science
251:
679-682,
1991[ISI][Medline].
4.
Baas, F,
and
Borst P.
The tissue dependent expression of hamster P-glycoprotein genes.
FEBS Lett
229:
329-332,
1988[ISI][Medline].
5.
Bosch, I,
Jackson GR, Jr,
Croop J,
and
Cantiello HF.
Expression of Drosophila melanogaster P-glycoproteins is associated with ATP-channel activity.
Am J Physiol Cell Physiol
271:
C1527-C1538,
1996[Abstract/Free Full Text].
6.
Cantiello, HF,
Jackson GR, Jr,
Grosman CF,
Prat AG,
Borkan SC,
Wang Y-H,
Reisin IL,
O'Riordan CR,
and
Ausiello DA.
Electrodiffusional ATP movement through the cystic fibrosis transmembrane conductance regulator.
Am J Physiol Cell Physiol
274:
C799-C809,
1998[Abstract/Free Full Text].
7.
Cantiello, HF,
Jackson GR, Jr,
Prat AG,
Gazley JL,
Forrest JN, Jr,
and
Ausiello DA.
cAMP activates an ATP-conductive pathway in cultured shark rectal gland cells.
Am J Physiol Cell Physiol
272:
C466-C475,
1997[Abstract/Free Full Text].
8.
Cantiello, HF,
Jackson GR, Jr,
Prat AG,
Gazley JL,
Forrest JN, Jr,
and
Ausiello DA.
Evidence for an ATP conductive pathway induced by cyclic AMP in rectal gland cells from the shark Squalus acanthias.
Bull Mt Desert Isl Biol Lab
33:
47-48,
1994.
9.
Cantiello, HF,
Prat AG,
Reisin IL,
Abraham EH,
Ercole LB,
Amara JF,
Gregory RJ,
and
Ausiello DA.
External ATP activates the cystic fibrosis transmembrane conductance regulator.
J Biol Chem
269:
11224-11232,
1994[Abstract/Free Full Text].
10.
Clemens, MG,
and
Forrester T.
Appearance of adenosine triphosphate in the coronary sinus effluent from isolated working heart in response to hypoxia.
J Physiol (Lond)
312:
143-158,
1980[Abstract].
11.
Dukes, IT,
Cleeman L,
and
Morad M.
Tedesamil blocks the transient and delayed rectifier K+ currents in mammalian cardiac and glial cells.
J Pharmacol Exp Ther
254:
560-569,
1990[Abstract].
12.
Flens, M,
Zaman G,
van der Valk P,
Izquierdo M,
Schroeijers A,
Scheffer G,
van der Groep P,
de Haas M,
Meijer C,
and
Scheper R.
Tissue distribution of the multidrug resistance protein.
Am J Pathol
148:
1237-1247,
1996[Abstract].
13.
Forrester, T.
Release of ATP from heart. Presentation of a release model using human erythrocyte.
In: Biological Actions of Extracellular ATP, edited by Dubyak GR,
and Fedan JS.. New York: NY Acad Sci, 1990, p. 335-352.
14.
Hamill, OP,
Marty A,
Neher E,
Sakmann B,
and
Sigworth FJ.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch
391:
85-100,
1981[ISI][Medline].
15.
Holton, P.
The liberation of adenosine triphosphate on antidromic stimulation of sensory nerves.
J Physiol (Lond)
145:
494-504,
1959[ISI].
16.
Hopwood, AM,
and
Burnstock G.
ATP mediates coronary vasoconstriction via P2x-purinoceptors and coronary vasodilatation via P2y-purinoceptors in the isolated perfused rat heart.
Eur J Pharmacol
136:
49-54,
1987[ISI][Medline].
17.
Horowitz, B,
Tsung SS,
Hart P,
Levesque PC,
and
Hume JR.
Alternative splicing of CFTR Cl
channels in heart.
Am J Physiol Heart Circ Physiol
264:
H2214-H2220,
1993[Abstract/Free Full Text].
18.
Horowitz, B,
Tsung SS,
Levesque PC,
Hart P,
and
Hume JR.
The cardiac cAMP-dependent Cl
conductance is encoded by an alternatively spliced isoform of CFTR (Abstract).
Biophys J
64:
A17,
1993[ISI].
19.
Jacobs, MH
(Editor).
Diffusion Processes. New York: Springer-Verlag, 1967, p. 136-140.
20.
Kang, J,
and
Leaf A.
Effects of long-chain polyunsaturated fatty acids on the contraction of neonatal rat cardiac myocytes.
Proc Natl Acad Sci USA
91:
9886-9890,
1994[Abstract/Free Full Text].
21.
Kang, J,
and
Leaf A.
Prevention and termination of
-adrenergic agonist-induced arrhythmias by free polyunsaturated fatty acids in neonatal rat cardiac myocytes.
Biochem Biophys Res Commun
208:
629-636,
1995[ISI][Medline].
22.
Katsugari, T,
Tokunaga T,
Ohba M,
Sato C,
and
Furukawa T.
Implication of ATP released from atrial, but not papillary, muscle segments of guinea pig by isoproterenol and forskolin.
Life Sci
53:
961-967,
1993[ISI][Medline].
23.
Korchazhkina, O,
Wright G,
and
Exley C.
Action of Al-ATP on the isolated working rat heart.
J Inorg Biochem
69:
153-158,
1998[ISI][Medline].
24.
Kwan, Y,
and
Qi A.
Inhibition by extracellular ATP of L-type calcium channel currents in guinea-pig single sinoatrial nodal cells: involvement of protein kinase C.
Can J Cardiol
13:
1202-1211,
1997[ISI][Medline].
25.
Lader, AS,
Xiao Y-F,
Wang Y,
Jackson GRJ,
Borkan SC,
and
Cantiello HF.
cAMP-activated anion conductance is associated with expression of CFTR in neonatal mouse cardiac myocytes.
Am J Physiol Cell Physiol
278:
C436-C451,
2000[Abstract/Free Full Text].
26.
Levesque, PC,
Hart PJ,
Hume JR,
Kenyon JL,
and
Horowitz B.
Expression of cystic fibrosis transmembrane conductance regulator Cl
channels in heart.
Circ Res
71:
1002-1007,
1992[Abstract].
27.
Levesque, PC,
and
Hume JR.
ATPo but not cAMPi activates a chloride conductance in mouse vetricular myocytes.
Cardiovasc Res
29:
336-343,
1995[ISI][Medline].
28.
McCarty, NA,
McDonough S,
Cohen BN,
Riordan JR,
Davidson N,
and
Lester HA.
Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl
channel by two closely related arylaminobenzoates.
J Gen Physiol
102:
1-23,
1993[Abstract].
29.
Nagel, G,
Hwang T-C,
Nastiuk KL,
Nairn AC,
and
Gadsby DC.
The protein kinase A-regulated cardiac Cl
channel resembles the cystic fibrosis transmembrane conductance regulator.
Nature
360:
81-84,
1992[ISI][Medline].
30.
Oz, MC,
and
Sorota S.
Forskolin stimulates swelling-induced chloride current, not cardiac cystic fibrosis transmembrane-conductance regulator current, in human cardiac myocytes.
Circ Res
76:
1063-1070,
1995[Abstract/Free Full Text].
31.
Pasyk, EA,
and
Foskett JK.
Cystic fibrosis transmembrane conductance regulator-associated ATP and adenosine 3'-phosphate 5'-phosphosulfate channels in endoplasmic reticulum and plasma membranes.
J Biol Chem
272:
7746-7751,
1997[Abstract/Free Full Text].
32.
Pelleg, A,
Hurt CM,
and
Michelson EL.
Cardiac effects of adenosine and ATP.
In: Biological Actions of Extracellular ATP, edited by Dubyak GR,
and Fedan JS.. New York: NY Acad Sci, 1990, p. 19-30.
33.
Prat, AG,
Reisin IL,
Ausiello DA,
and
Cantiello HF.
Cellular ATP release by the cystic fibrosis transmembrane conductance regulator.
Am J Physiol Cell Physiol
270:
C538-C545,
1996[Abstract/Free Full Text].
34.
Prat, AG,
Xiao Y-F,
Ausiello DA,
and
Cantiello HF.
cAMP-independent regulation of CFTR by the actin cytoskeleton.
Am J Physiol Cell Physiol
268:
C1552-C1561,
1995[Abstract/Free Full Text].
35.
Qu, Y,
Himmel HM,
Campbell DL,
and
Strauss HC.
Effects of extracellular ATP on ICa, [Ca2+]i, and contraction in isolated ferret ventricular myocytes.
Am J Physiol Cell Physiol
264:
C702-C708,
1993[Abstract/Free Full Text].
36.
Reisin, IL,
Prat AG,
Abraham EH,
Amara JF,
Gregory RJ,
Ausiello DA,
and
Cantiello HF.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a dual ATP and chloride channel.
J Biol Chem
269:
20584-20591,
1994[Abstract/Free Full Text].
37.
Roman, RM,
Wang Y,
Lidofsky SD,
Feranchak AP,
Lomri N,
Scharschmidt BF,
and
Fitz JG.
Hepatocellular ATP-binding cassette protein expression enhances ATP release and autocrine regulation of cell volume.
J Biol Chem
272:
21970-21976,
1997[Abstract/Free Full Text].
38.
Scamps, F,
Legssyer A,
Mayoux E,
and
Vassort G.
The mechanism of positive inotropy induced by adenosine triphosphate in rat heart.
Circ Res
67:
1007-1016,
1990[Abstract].
39.
Scamps, F,
and
Vassort G.
Effect of extracellular ATP on the Na+ current in rat ventricular myocytes.
Circ Res
74:
710-717,
1994[Abstract].
40.
Scamps, F,
and
Vassort G.
Mechanism of extracellular ATP-induced depolarization in rat isolated ventricular cardiomyocytes.
Pflügers Arch
417:
309-316,
1990[ISI][Medline].
41.
Schwiebert, EM,
Egan ME,
Hwang T-H,
Fulmer SB,
Allen SS,
Cutting GR,
and
Guggino WB.
CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.
Cell
81:
1063-1073,
1995[ISI][Medline].
42.
Shoda, M,
Hagiwara N,
Kasanuki H,
and
Hosoda S.
ATP-activated cationic current in rabbit sino-atrial node cells.
J Mol Cell Cardiol
29:
689-695,
1997[ISI][Medline].
43.
Song, Y,
and
Belardinelli L.
ATP promotes development of afterdepolarizations and triggered activity in cardiac myocytes.
Am J Physiol Heart Circ Physiol
267:
H2005-H2011,
1994[Abstract/Free Full Text].
44.
Sorota, S,
Rose EA,
and
Oz MC.
Failure to detect a protein kinase A-regulated chloride current in human atrial myocytes (Abstract).
Biophys J
66:
A434,
1994[ISI].
45.
Stowe, DF,
Sullivan TE,
Dabney JM,
Scott JB,
and
Haddy FJ.
Role of ATP in coronary flow regulation in the isolated perfused guinea pig heart.
Physiologist
17:
339,
1979.
46.
Sugawara, I,
Akiyama S,
Scheper R,
and
Itoyama S.
Lung resistance protein (LRP) expression in human normal tissues in comparison with that of MDR1 and MRP.
Cancer Lett
112:
23-31,
1997[ISI][Medline].
47.
Takano, M,
and
Noma A.
Distribution of the isoprenaline-induced Cl
currents in rabbit heart.
Pflügers Arch
420:
223-226,
1992[ISI][Medline].
48.
Tilly, B,
Bezstarosti K,
Boomaars W,
Marino C,
and
Lamers J.
Expression and regulation of chloride channels in neonatal rat cardiomyocytes.
Mol Cell Biochem
157:
129-135,
1996[ISI][Medline].
49.
Vial, C,
Owen P,
Opie LH,
and
Posel D.
Significance of release of adenosine triphosphate and adenosine induced by hypoxia or adrenaline in perfused rat heart.
J Mol Cell Cardiol
19:
187-197,
1987[ISI][Medline].
50.
Xiao, Y-F,
and
Cantiello HF.
Autocoid role of extracellular ATP on the duration of the action potential in cultured rat neonate ventricular myocytes (Abstract).
Biophys J
68:
A109,
1995.
51.
Xiao, Y-F,
O'Riordan CR,
and
Cantiello HF.
Characteristics of the cardiac CFTR-mediated ATP currents in rat neonatal cardiac myocytes in culture (Abstract).
J Gen Physiol
104:
38A,
1994.
52.
Zheng, J,
Boluyt M,
Long X,
O'Neill L,
Lakatta E,
and
Crow M.
Extracellular ATP inhibits adrenergic agonist-induced hypertrophy of neonatal cardiac myocytes.
Circ Res
78:
525-535,
1996[Abstract/Free Full Text].
53.
Zheng, J,
Boluyt M,
O'Neill L,
Crow M,
and
Lakatta E.
Extracellular ATP induces immediate-early gene expression but not cellular hypertrophy in neonatal cardiac myocytes.
Circ Res
74:
1034-1041,
1994[Abstract].
54.
Zheng J-S, Christie A, De Young MB, Levy MN, and SA.
Synergism between cAMP and ATP in signal transduction in cardiac
myocytes. Am J Physiol Cell Physiol 262:
C128-C135, 1992.
55.
Zheng J-S, De Young MB, Wiener E, Levy MN, and SA. Extracellular
ATP-induced Ca2+ transients in cardiac myocytes are
potentiated by an increase in cellular cAMP. In: Biological
Actions of Extracellular ATP, edited by Dubyak GR and Fedan JS.
New York: NY Acad Sci, 1990, p. 448-451.
Am J Physiol Cell Physiol 279(1):C173-C187
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society