© 1995 American Heart Association, Inc.
Articles |
Forskolin Stimulates Swelling-Induced Chloride Current, Not Cardiac Cystic Fibrosis Transmembrane-Conductance Regulator Current, in Human Cardiac Myocytes
From the Departments of Surgery (M.C.O.) and Pharmacology (S.S.), Columbia University, New York, NY.
Correspondence to Dr Steve Sorota, Department of Pharmacology, Columbia University, 630 W 168th St, New York, NY 10032.
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Abstract Whole-cell patch clamp was used to look for cystic fibrosis transmembrane-conductance regulator (CFTR)–like chloride currents in calcium-tolerant human cardiac myocytes. Potassium-containing solutions were used initially. Steady state currents were measured with hyperpolarizing ramps (-16.25 mV/s). Peak net inward currents during voltage steps from -50 to +5 mV were used as an index of L-type calcium current. Isoproterenol (1 µmol/L) or forskolin (10 µmol/L) were used in attempts to evoke CFTR-like chloride current. No forskolin- or isoproterenol-induced steady state current was found in any of 17 atrial cells from seven patients in the absence of cell swelling. Every cell exhibited a large increase in net inward current in response to forskolin, suggesting that cAMP-dependent stimulation of L-type calcium current was present. Swelling with osmotic stress induced an outwardly rectifying steady state current with a reversal potential close to the chloride equilibrium potential. Once this current was activated, exposure to forskolin caused a further increase that subsided on washout (four of four cells, two patients). The atrial swelling-induced current was studied in more detail by using cesium-containing solutions. The current was determined to be a chloride current because the reversal potential was sensitive to changes in intracellular chloride and outward currents were blocked by 150 µmol/L DIDS. Ventricular cells were isolated from five failing human hearts. No CFTR-like current was found in any of 17 cells. In eight of eight ventricular cells, a swelling-induced current was found. The amplitude of the swelling-induced current was enhanced by forskolin. In human ventricular cells, outward swelling-induced currents were inhibited by DIDS. The present results are in apparent conflict with mRNA measurements made by others. Our results suggest that significant amounts of functional channels do not accumulate despite the reported presence of cardiac CFTR mRNA in human heart. However, human myocardium does express a swelling-induced chloride current that can be stimulated by forskolin.
Key Words: chloride current • protein kinase A • swelling • cystic fibrosis transmembrane-conductance regulator
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Introduction |
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Rabbit and guinea pig ventricular cells have a protein kinase A–activated chloride current that is very similar to the current through the epithelial cystic fibrosis transmembrane-conductance regulator (CFTR).1 2 3 4 The primary amino acid sequence of CFTR is known (reviewed in Reference 55 ). The protein has a repeated motif that is predicted to consist of six transmembrane-spanning regions and a nucleotide-binding region. Two of these motifs are connected by a highly charged region that contains many consensus sites for phosphorylation. The cardiac homologue of CFTR is an alternative splice variant of epithelial CFTR with a deletion of 30 amino acids in the first cytoplasmic loop.3
The current that flows through the cardiac variant of CFTR
(ICFTR-cardiac) is selective for chloride, is time
independent, and exhibits outward rectification when intracellular and
extracellular chloride concentrations are in the physiological
range.1 2 6 7 8 Normal intracellular chloride concentration
is 
20 mmol/L, with measurements of the chloride equilibrium
potential (ECl) ranging from -40 to -60
mV.9 10 11 If intracellular chloride concentrations are
raised to 150 mmol/L, then current through ICFTR-cardiac
exhibits a linear current-voltage relation.2 6 8
Important electrophysiological manifestations of ICFTR-cardiac have been demonstrated. Activation of ICFTR-cardiac can accelerate repolarization of the action potential and under certain conditions depolarize the resting potential to the point where cells become abnormally automatic.6 12 13 14 The repolarizing current at potentials positive to ECl is likely to prevent excessive action potential prolongation when calcium current is stimulated.6 However, the effect of ICFTR-cardiac activation might also promote arrhythmias. Depolarizing current at potentials negative to ECl could contribute to the generation of arrhythmias15 ; enhanced repolarization at potentials positive to ECl could perpetuate arrhythmias if a reentrant circuit were present.
The cardiac variant of CFTR (CFTR-cardiac) exhibits a species-, tissue-, and region-dependent distribution. In rabbit and guinea pig, ICFTR-cardiac is present in ventricular cells but absent in atrial cells.1 2 3 16 Subepicardial cells from rabbit ventricle have a larger ICFTR-cardiac than do subendocardial cells.16 Dog atrium and ventricle both lack this current.3 12 17 Rat ventricular cells also lack ICFTR-cardiac.18 In experimental animals, the distribution of mRNA for CFTR-cardiac, evaluated by either reverse transcriptase–polymerase chain reaction (RT-PCR) or Northern blot, corresponds with the distribution of ICFTR-cardiac measured in voltage-clamp studies3 4 19 ; that is to say, mRNA is found in rabbit and guinea pig ventricle but not in atrium or in dog heart.
Cell swelling can activate a distinct type of cardiac chloride current in mammalian heart cells. A swelling-induced chloride current (ICl-swelling) has been found in dog atrium, dog ventricle, rabbit atrium, and rabbit sinoatrial node.17 20 21 Like ICFTR-cardiac, ICl-swelling is time independent and outwardly rectifying, with physiological levels of intracellular chloride, but unlike CFTR it does not require cAMP-dependent phosphorylation to be activated.20 21 This current also differs from ICFTR-cardiac in its permeability sequence for other halides and its pharmacological sensitivity.4 21 22 23 ICl-swelling in dog atrium is stimulated by 1 µmol/L isoproterenol after the current has been activated by an increase in cell volume.17 The resulting macroscopic isoproterenol-induced current can appear to be similar to ICFTR-cardiac. ICl-swelling has also been found in cultured embryonic chick heart cells. This current differs from mammalian ICl-swelling in two important ways. First, chick heart ICl-swelling is inhibited by 20 µmol/L isoproterenol,24 whereas the mammalian current is clearly stimulated by isoproterenol.17 Second, ICl-swelling in chick heart is dependent on the elevation of intracellular calcium, whereas the mammalian current appears to be calcium independent.17 20 21 25 26
Since ICFTR-cardiac is species and tissue dependent, it is important to learn whether this current is present in human heart. The first report regarding CFTR-cardiac in human heart presented evidence that mRNA was present in human atrium.19 This result was unexpected because CFTR-cardiac is not known to be present in the atrium of any other species.3 16 17 19 mRNA for cardiac CFTR channels has also been detected in human ventricle by RT-PCR.27 The presence of mRNA suggests that ICFTR-cardiac should be detectable in human cardiac myocytes. In the present study, we have attempted to find this current by examining the effect of forskolin and isoproterenol on the membrane currents of single human atrial cells. We have also examined human ventricular cells in an attempt to find ICFTR-cardiac.
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Pieces of human right atrium were obtained from patients undergoing coronary bypass surgery. The patients ranged in age from 38 to 77 years. Seven of the patients were male and five were female. Coronary artery bypass graft surgery was being performed on eight of these patients. Aortic valve disease was the impetus for surgical intervention in three patients. One patient was operated on to repair the mitral valve. ECGs revealed atrial fibrillation in one of the patients. The remaining patients were in normal sinus rhythm with normal P waves. The following medications were being taken by the patients before surgery (number of patients appears in parentheses): nitrates (9), angiotensin-converting enzyme inhibitor (2), ß-blocker (1), amiodarone (1), and calcium channel blockers (3). The procedure for obtaining these samples has been reviewed and approved by the Institutional Review Board at Columbia University.
Atrial tissue was collected in an ice-cold saline solution (mmol/L:
NaCl 144, KCl 4, NaHCO3 12, NaH2PO4
1.6, MgCl2 1, CaCl2 1, and dextrose 10) that
had been equilibrated with 95% O2/5%
CO2. Atrium was transferred to a solution at room
temperature and cut into 3x3-mm pieces. The atrial pieces were
washed at 36°C for four 2-minute cycles in calcium-free solution
containing 20 mmol/L taurine and 30 mmol/L mannitol. The tissue was
stirred by gas bubbles during this step and all subsequent steps.
Because of the possibility of creating aerosols containing human
pathogens, the cell isolation was performed in a biological safety
cabinet. After the wash with calcium-free solution, the tissue was
exposed for 30 minutes to a solution that contained taurine, mannitol,
50 µmol/L calcium, 1 mg/mL collagenase A
(Boehringer-Mannheim Corp), and 0.37 mg/mL protease XXIV (Sigma
Chemical Co). The supernatant was removed, and subsequent cycles of
digestion were performed with the same solution without the protease.
At the end of each digestion cycle, supernatants were removed and
centrifuged at 50g to pellet the cells. Cells were washed
twice and stored in a modified KB medium (mmol/L: KCl 70, EGTA 0.5,
taurine 20, glucose 20, succinic acid 5, creatine 5,
K2HPO4 30, pyruvic acid 5, MgSO4 5,
Na2ATP 5, and CaCl2 0.12, along with 20 µg/mL
gentamicin).
Human ventricular tissue was obtained from five patients undergoing cardiac transplant. Two of the patients had ischemic heart failure, two were diagnosed with dilated idiopathic cardiomyopathy, and one had accelerated atherosclerosis after a prior cardiac transplant. The age of the patients ranged from 49 to 60 years. All of the patients were male. Medications included the following (number of patients appears in parentheses): amiodarone (1), angiotensin-converting enzyme inhibitors (2), nitrates (2), bumetanide (1), calcium channel blockers (1), and digoxin (1).
Human ventricular cells were prepared by a modification of the method
of Näbauer et al.28 A wedge of ventricle with a
central artery was transported from the operating room in ice-cold
cardioplegic solution containing (mmol/L) potassium aspartate 100,
KH2PO4 25, MgSO4 5, adenosine 5,
glutathione 3, raffinose 30, and allopurinol 1, along with 0.2 mg/mL
bovine albumin. The tissue was cannulated and perfused for 20 minutes
with calcium-free Tyrode's solution (36°C, equilibrated with 95%
O2/5% CO2) containing (mmol/L) NaCl
144, NaHCO3 24, KCl 4, NaH2PO4 1.6,
MgCl2 1, dextrose 11, mannitol 10, taurine 20, and pyruvic
acid 5, along with 2 mg/mL albumin. Next, the tissue was perfused with
a similar solution with 1 mg/mL collagenase, 0.l mg/mL
protease, and 100 µmol/L CaCl2 for 15 to 35 minutes.
Approximately 2 mm of the epicardium and endocardium was removed, and
the remaining myocardial tissue was minced into 3-mm3
pieces. The tissue was placed in a 50-mL plastic centrifuge tube and
shaken briefly. The supernatant was filtered through 200-µm nylon
mesh, and cells were recovered by centrifugation at 50g
for 3 minutes. Cells were washed one time with KB medium and then
stored in KB medium until used. The remaining undigested tissue was
subjected to several more cycles of enzyme digestion by using a chunk
procedure.
The bath solution used for most of these studies was a potassium-containing HEPES-buffered saline (mmol/L: NaCl 144, HEPES-NaOH 10, KCl 5.4, CaCl2 1.8, MgCl2 1, and dextrose 5.5, pH 7.35). In some cases, the NaCl concentration was reduced to 115 mmol/L, and the osmolarity of the bath solution was varied by using mannitol. The osmolarities of solutions were measured by freezing-point depression (µOsmette, Precision Instruments). Hypotonic bath solution (220 mOsm/kg) was used to induce cell swelling. Hypertonic bath solution (360 mOsm/kg) was used to reverse cell swelling, because return to isotonic was often ineffective for this purpose.
Electrodes were prepared from borosilicate glass (outer diameter, 1.5
mm; inner diameter, 0.86 mm; Sutter Instruments). Tip resistances when
filled with pipette solution ranged from 2 to 3.5 M
. The pipette
solution that was used with the bath solutions described above
contained (mmol/L) potassium aspartate 125, KCl 15, EGTA 4, MgATP 3,
Na2-phosphocreatine 5, MgCl2 1, GTP 0.1, and
HEPES-KOH 10, pH 7.2.
In a subset of experiments, cesium-containing solutions were used. The bath solution for these experiments contained (mmol/L) NaCl 103, CsCl 20, HEPES-NaOH 10, CaCl2 1.8, MgCl2 1, and dextrose 5.5, pH 7.35. The osmolarity of this solution was varied with the addition of mannitol. Cesium-containing pipette solution was composed of (mmol/L) cesium aspartate 0 or 100, CsCl 40 or 140 (sum of cesium aspartate and CsCl=140), HEPES-CsOH 10, EGTA 4, MgATP 3, Na2-phosphocreatine 5, and MgCl2 1, pH 7.2.
The amplifier offset was adjusted to 0 mV with the electrode immersed in the bath solution. All voltages were later corrected for the junction potential between the pipette solution and the bath solution. This junction potential was measured by using a two-chambered bath, with a flowing 3.3 mol/L KCl salt bridge in the downstream chamber as the indifferent electrode. We measured the change in potential when the solution flowing through the bath was changed from our normal bathing solution to that used for filling electrodes. Voltages were corrected by -10 mV when potassium-containing solutions were used. When cesium-containing solutions were used, the voltages were corrected by -9 mV when the pipette chloride concentration was 42 mmol/L and by -3 mV when it was 142 mmol/L.
Cells were allowed to adhere to laminin-coated glass coverslips. All
experiments were performed within 2 hours of adhesion to the coverslips
at 36±0.5°C. The flow rate used was 4 mL/min. A stock solution of
0.2 mol/L forskolin (Sigma) in dimethyl sulfoxide (DMSO) that was
stored at 4°C for up to 3 months was used to prepare the final
solutions. The final concentration of DMSO was 0.005%. Isoproterenol
stock solutions (10 mmol/L in 10 mmol/L HCl) were prepared fresh each
day and stored at 4°C until the final dilutions were made. DIDS
solutions were prepared and used in a darkened room. DIDS powder was
dissolved directly in the bath solution.
Whole-cell patch-clamp recordings were performed as previously described17 29 by using an Axopatch 1D amplifier and PCLAMP 5.7.1 or 6.01 software running on a Gateway 2000 486 DX 33 computer. A Labmaster TL1-125 interface board was used. All currents were low pass–filtered by using the 10-kHz filter built into the amplifier. Cell membrane capacitance and series resistance were calculated from the capacitance current evoked by a -10-mV voltage step as previously described.17 29 Peak net inward currents were measured during a 312-ms step from -50 to +5 mV. The data were sampled at an interval of 20 µs for the first 10 ms, and the remaining data were sampled using 600-µs intervals. Pseudo–steady state currents were measured by using slow hyperpolarizing voltage ramps (-16.25 mV/s). The ramps were preceded by a 2-s step to +20 mV to allow inactivation of voltage- and time-dependent currents (voltage-gated transient outward potassium current [ITO1] and L-type calcium current [ICa-L]). We saw no evidence of a slow delayed rectifier current in our cells, possibly because of rapid rundown.30 The sampling interval used for steady state current measurements was 10 ms.
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Calcium-tolerant human atrial cells with clear striations were used for these studies. Experiments were performed in potassium-containing solutions so that we could measure resting potentials before proceeding with voltage-clamp studies. Resting potentials ranged from -80 to -30 mV. The average resting potential was -56±16 mV. The large range of resting potentials is probably due to the small outward currents and the high membrane resistance of the cells between -70 and -30 mV (see Fig 2
). Action potentials were
recorded from cells with sufficiently negative resting potentials. An
example is shown in Fig 1.
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We did not block calcium currents, because we wanted to use the
activation of calcium currents as a positive control if isoproterenol
or forskolin failed to affect steady state currents. If
ICFTR-cardiac were present in human atrial cells,
agents that enhance intracellular cAMP should induce an outwardly
rectifying current with a reversal potential close to -58 mV under our
recording conditions. To reduce problems with spontaneous cell swelling
that can occur in atrial myocytes during whole-cell patch-clamp
experiments with osmotically matched pipette and bath
solutions,17 we used patch electrodes with an initial
resistance between 2 and 3.5 M and exposed the cells to
isoproterenol or forskolin shortly after patch rupture. A 90-s exposure
to cAMP-elevating agents was initiated between 3 and 7 minutes after
patch rupture. Lack of swelling was confirmed visually. Cells were
magnified 600 times, and cell width was monitored with an ocular
reticule.
An example of the influence of 10 µmol/L forskolin on the steady
state current-voltage relation in one atrial cell is shown in Fig 2
. There was no effect of forskolin on the steady state
current-voltage relation in this cell. Our results were consistent
between cells and patients if we were careful to avoid cell swelling.
We found no evidence of a forskolin-induced chloride current in 15 of
15 cells examined from six different patients. We also looked for an
effect of 1 µmol/L isoproterenol on steady state currents from three
atrial cells (two patients). We found no effect of isoproterenol on
steady state currents in any of the three cells examined. One cell was
exposed sequentially to isoproterenol and forskolin. Therefore, in
potassium-containing solutions we failed to find evidence for
ICFTR-cardiac in 17 of 17 cells.
The failure to find electrophysiological evidence of the presence of
CFTR-cardiac in human atrial cells could be due to a deficit in
cAMP-dependent phosphorylation. Therefore, we needed a positive control
to ensure that protein kinase A was functional in the atrial cells that
we studied. To this end, peak net inward current during voltage-clamp
steps from -50 to +5 mV was examined. The time-dependent currents
during this step will consist of ICa-L and ITO1
under our recording conditions. The contamination by ITO1
did not interfere with our ability to observe an increase in net inward
current. The stimulation of peak net inward current in a human atrial
cell by 10 µmol/L forskolin is shown in Fig 3. Similar
enhancements of peak net inward current were observed in all 15 cells
exposed to forskolin and all three cells exposed to isoproterenol. In
no case did we have to exclude a cell from the study because it failed
to respond to cAMP-elevating agents with an increase in peak net inward
current.
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Dog and rabbit atrial cells exhibit
ICl-swelling.17 21 Once activated,
ICl-swelling in the dog can be modulated in a stimulatory
manner by 1 µmol/L isoproterenol.17 A modified bathing
solution was used to look for a comparable current in human atrial
cells. The NaCl in the bathing solution was reduced by 20%. Isotonic
solution was prepared with 70 mmol/L mannitol used as an osmotic
supplement. Swelling was induced by exposing cells to mannitol-free
solutions (220 mOsm/kg). The results of one experiment in which cell
swelling was induced are shown in Fig 4. In this cell,
exposure to 10 µmol/L forskolin in isotonic solution had no effect on
steady state currents. During exposure to hypotonic solution, a small
outwardly rectifying current with a reversal potential of -45 mV
was induced (ECl=-52 with 80% NaCl). Subsequent exposure
to hypotonic solution containing 10 µmol/L forskolin caused a
dramatic increase in this current without altering the reversal
potential. Forskolin increased both outward current at positive
potentials and inward current at negative potentials. The effect of
forskolin subsided during washout with drug-free hypotonic solution.
Demonstration of the swelling in this cell is presented in Fig 5. In four of four cells, exposure to hypotonic solution
activated an outwardly rectifying current with a reversal potential
close to ECl. In each case, 10 µmol/L forskolin caused a
dramatic increase in the amplitude of this current.
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indicates the control condition (hidden by solid
circle); 
, 10 µmol/L forskolin in 290 mOsm/kg; 
,
hypotonic solution; 
, hypotonic solution plus forskolin; and
, washout of forskolin. Consistent with prior results,
application of 10 µmol/L forskolin in the 290 mOsm/kg solution
shortly after patch rupture had no effect on the steady state
current-voltage relation. Superfusion with hypotonic solution (0
mannitol, 220 mOsm/kg) induced an outwardly rectifying steady state
current with a reversal potential of -45 mV (chloride equilibrium
potential, -52 mV). After this current had been induced, superfusion
with 10 µmol/L forskolin in hypotonic solution stimulated an
outwardly rectifying steady state current that also reversed at -45
mV. The effect of forskolin subsided on washout; however, the current
that was induced by hypotonic solution increased slightly over time.
Difference currents relative to the control steady state
current-voltage relation are shown in the bottom panel. Cell 40194c01
was used.
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The observation that human atrial cells express a swelling-induced
steady state current that is outwardly rectifying and has a reversal
potential close to ECl is consistent with the presence of
ICl-swelling.17 To examine this possibility
further, we studied human atrial cells by using cesium-containing
solutions in the bath and pipette in order to block potassium currents.
As was observed when potassium-containing solutions were used,
forskolin had no effect on steady state currents in unswollen cells
(three of three cells, three patients; data not shown).
ICl-swelling could still be detected by using
cesium-containing solutions (six of seven cells, four patients). The
activation of ICl-swelling was associated with the
formation of membrane blebs in some, but not all, cases. The reversal
potential with 42 mmol/L intracellular chloride (-30±1 mV, four
cells) was identical to the calculated ECl (-30 mV) under
these experimental conditions. An example of one measurement with 42
mmol/L chloride in the pipette is shown in the top of Fig 6. When intracellular chloride was increased to 142
mmol/L, the reversal potential shifted to -4±1 mV, which is close to
the calculated ECl (+2 mV) (three cells; eg, see Fig 6,
bottom). As was observed in potassium-containing solutions, forskolin
stimulated the swelling-induced current without altering the reversal
potential or the shape of the current-voltage relation (three of three
cells, two patients; eg, see Fig 7). The chloride
channel blocker DIDS (150 µmol/L) was found to inhibit the
forskolin-enhanced swelling-induced current in a voltage-dependent
manner (three of three cells, two patients; eg, see Fig 7). Outward
currents were inhibited to a greater extent than were inward currents.
The voltage dependence of block by DIDS is similar to that seen when
DIDS is used to block ICl-swelling of canine atrial
cells.26 The effect of DIDS subsided when the drug was
washed out (Fig 7).
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Since ICFTR-cardiac is found in the ventricle but not the
atrium of guinea pigs and rabbits,1 2 3 16 it is important
to examine whether this current is present in human ventricle. To
address this issue, we performed studies on cells isolated from the
left ventricular free wall of five patients that received a cardiac
transplant. Potassium-containing solutions were used for all of the
studies on human ventricular cells. We examined 18 cells from these
patients. Average resting potential was -82±3 mV. Eight cells were
studied for their response to 10 µmol/L forskolin in our 100%-NaCl
bathing solution, and 10 cells were studied in 80%-NaCl solution that
was made isotonic by the addition of mannitol. A representative
cell is shown in Fig 8. There was no change in steady
state current in response to forskolin, but there was a dramatic
increase in the peak net inward current during voltage steps from -50
to +5 mV. The lack of effect of forskolin on steady state currents and
the increase in peak net inward current were observed in 17 of 17
cells.
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, forskolin; and
To determine whether human ventricular cells express a swelling-induced
current, we examined the response of eight cells from four patients to
hypo-osmotic stress by using a bathing solution that contained 80% of
the normal NaCl concentration. The results were similar to those
observed in human atrial cells. Swelling induced an outwardly
rectifying steady state current with a reversal potential close to
ECl in eight of eight cells (eg, see Fig 9).
After the cell membrane conductance had increased over control levels,
10 µmol/L forskolin enhanced the amplitude of the swelling-induced
steady state current (four of four cells, three patients; eg, see Fig 9). The amplitude of outward swelling-induced currents was inhibited by
150 µmol/L DIDS (six of six cells, three patients; eg, see Fig 10).
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, hypotonic solution plus forskolin; and
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No evidence was found in the present study to support the presence of ICFTR-cardiac in human heart. Because digestive enzymes were used to prepare isolated cardiac myocytes, it is possible that ICFTR-cardiac channels were proteolytically destroyed during the cell preparation. To examine whether our collagenase had an unusual effect on ICFTR-cardiac channels, we prepared guinea pig ventricular myocytes by using the same lot of collagenase that was used for isolating human cardiac cells in this study. Cesium-containing solutions were used for these experiments. The isotonic bath solution that was used contained 129 mmol/L NaCl and no mannitol. Forskolin stimulated an outwardly rectifying steady state current with a reversal potential close to ECl in five of six guinea pig ventricular cells under our recording conditions (chloride concentrations of 42 mmol/L [pipette] and 155 mmol/L [bath]) (data not shown). In agreement with previous studies,4 23 the forskolin-induced current that we found in guinea pig ventricle could not be inhibited by DIDS (not shown). This observation is consistent with the notion that the forskolin-induced current that we measured in guinea pig cells is ICFTR-cardiac.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human atria have been reported to contain mRNA for CFTR-cardiac.3 19 The positive reports for CFTR-cardiac message implied that functional channels would be present in human atrial cells. Therefore, we persisted in trying to find evidence for this current in spite of our initial failures. Our inability to observe ICFTR-cardiac in 20 of 20 cells (17 in potassium-containing solutions and 3 in cesium-containing solutions) from 10 different patients suggests that CFTR-cardiac channels do not accumulate in human atrial cells. Forskolin or isoproterenol increased peak net inward current, a crude measure of ICa-L, in every cell examined. Therefore, it is unlikely that our inability to see ICFTR-cardiac was due to a deficit in regulation of the channel by protein kinase A. We cannot completely exclude the possibility that our enzyme digestion may have damaged the CFTR channel protein and prevented us from detecting the current. However, this is not likely because other channels seemed to be unaffected by the disaggregation and because it is unlikely that we would have totally destroyed the CFTR-cardiac channel protein in each of the cells from which we recorded. As a positive control, the lot of collagenase that was used to isolate human cells was also used to prepare guinea pig ventricular cells, which are known to express ICFTR-cardiac. This lot of collagenase did not have any unusual effect that prevented the detection of ICFTR-cardiac in guinea pig ventricular cells.
In agreement with a preliminary report from another laboratory,27 our results show that there is a forskolin-stimulated chloride current in human atrial cells. It has been implied that this current is ICFTR-cardiac.27 However, our data demonstrate that the forskolin-regulated current is ICl-swelling, not ICFTR-cardiac. This conclusion is based on several observations: Forskolin has no effect on steady state membrane currents in unswollen human atrial cells but does enhance the amplitude of a steady state current that is induced by cell swelling. The reversal potential of the swelling-induced current is sensitive to changes in chloride concentration in the recording electrode. We have considered the possibility that the forskolin-induced current seen in hypotonic solutions is ICFTR-cardiac that is unmasked by cell swelling in human atrial cells, perhaps through relocation of the channel protein within the cell. However, we view this as being unlikely for the following reasons: (1) There are no reports of cell swelling causing an enhancement of ICFTR-cardiac. (2) The forskolin-stimulated steady state current that was present in swollen cells was inhibited by 150 µmol/L DIDS. (3) On several occasions we looked for forskolin-induced currents after relieving cell swelling by returning the cell to a hypertonic solution. We did this to evaluate whether cell swelling could cause a slowly reversible redistribution of ICFTR-cardiac channels to the sarcolemma. Hypertonic solution was used because return to an isotonic solution did not result in the prompt relief of cell swelling. Once cell swelling had subsided, forskolin no longer affected steady state membrane currents (data not shown).
The results that we observed in cells isolated from the ventricles of five human patients paralleled those obtained in the atrium. We saw no effect of forskolin on steady state currents if cells were not swollen. Swelling induced an outwardly rectifying steady state current with a reversal potential close to ECl. Exposure to forskolin enhanced the amplitude of the swelling-induced steady state current. Outward swelling-induced currents were inhibited by 150 µmol/L DIDS. These results suggest that human ventricular cells also lack CFTR-cardiac but express an ICl-swelling that can be stimulated by forskolin. Because failing human ventricle was used for these studies, we cannot exclude the possibility that the CFTR-like current is present in human ventricular cells isolated from nonfailing hearts. Even if this turns out to be the case, it will still be important to recognize the disappearance of the current in heart failure.
There is an apparent disagreement between our electrophysiological results and the report finding mRNA for CFTR-cardiac in human atrium by Northern blot analysis.19 Possible resolutions of this discrepancy include the presence of the channel in a very small percentage of cells, poor translation of the message, rapid turnover of the channel protein, or a failure of protein trafficking that prevents the channel from reaching the cell membrane. Another possibility is that the CFTR-cardiac mRNA found in human atrium may have come from nonmuscle cells that are present within the atrial wall. However, this is not very likely because of results presented in a follow-up study by Horowitz et al.3 No CFTR-cardiac mRNA was detected when RT-PCR amplification was used on mRNA from atrium of dog, guinea pig ,or rabbit.3 These tissues are known not to express ICFTR-cardiac but do contain the same nonmuscle cells that would be present in a piece of human atrium. It would be of interest to use immunocytochemical methods to look for ICFTR-cardiac protein to determine whether channel protein is present in human atrium and, if so, to determine why it does not result in the expression of significant levels of functional channels.
The present study demonstrates that swelling-induced chloride currents are present in human atrium and ventricle. Once activated, the swelling-induced chloride current can be stimulated by forskolin and inhibited by DIDS. DIDS blocks outward ICl-swelling26 but has no effect on ICFTR-cardiac.23 We found no evidence to support the hypothesis that human cardiac myocytes express ICFTR-cardiac. The results stress the importance of preventing cell swelling and confirming channel identity pharmacologically when studying ICFTR-cardiac in whole-cell patch-clamp experiments.
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Acknowledgments |
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This study was supported by a Grant-in-Aid from the American Heart Association, New York City Affiliate, Inc. Dr Sorota is an Investigator of the American Heart Association, New York City affiliate. The study was also supported by a gift from American Cyanamid Company Medical Research Division. The excellent technical assistance of Josefina Martinez is gratefully acknowledged.
Received May 23, 1994; accepted February 11, 1995.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1. Harvey RD, Hume JR. Autonomic regulation of a chloride current in heart. Science. 1989;244:983-985.
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