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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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) × pi  × 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-GOmega 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
<IT>P</IT><SUB>A</SUB><IT>&cjs0823;  P</IT><SUB>B</SUB><IT>={z<SUP>2</SUP></IT><SUB>B</SUB><IT>×</IT>B<SUB>o</SUB><IT>×</IT>exp(&agr;<IT>z</IT><SUB>B</SUB><IT>E</IT><SUB>r</SUB>)

× [1−exp(<IT>&agr;z</IT><SUB>A</SUB><IT>E</IT><SUB>r</SUB>)]<IT>}&cjs0823;  {z<SUP>2</SUP></IT><SUB>A</SUB><IT>×</IT>A<SUB>i</SUB><IT>×</IT>[<IT>1−</IT>exp(<IT>&agr;z</IT><SUB>B</SUB><IT>E</IT><SUB>r</SUB>)]<IT>},</IT>
where alpha  = 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


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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).


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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 (down-triangle), and after addition of cocktail + DPC (400 µM, black-down-triangle ). 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.


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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 (down-triangle), and DPC (black-down-triangle ). 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 (down-triangle) 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 ATPgamma S permeated through the cAMP-activated anion pathway; however, the nucleotide GTP did not (Fig. 4).


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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. ATPgamma 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).


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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).


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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).


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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 (down-triangle) 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).


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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).



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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).


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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.



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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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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DISCUSSION
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