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Circulation Research. 1995;77:379-393

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*PHENYLEPHRINE
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(Circulation Research. 1995;77:379-393.)
© 1995 American Heart Association, Inc.


Articles

{alpha}-Adrenergic Control of Volume-Regulated Cl- Currents in Rabbit Atrial Myocytes

Characterization of a Novel Ionic Regulatory Mechanism

Dayue Duan, Bernard Fermini, Stanley Nattel

From the Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada; the Department of Medicine, University of Montreal; and the Department of Medicine and the Research Center, Montreal Heart Institute.

Correspondence to Stanley Nattel, Research Center, Montreal Heart Institute, Montreal, Quebec, Canada H1T 1C8.


*    Abstract
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*Abstract
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Abstract {alpha}-Adrenergic stimulation is known to play a role in cardiac arrhythmogenesis and to modulate a variety of cardiac K+ currents. The effects of {alpha}-adrenergic stimulation on Cl- currents are largely unknown. Many cardiac cell types show a volume-sensitive Cl- current induced by cell swelling (ICl.swell). The present experiments were designed to assess the potential {alpha}-adrenergic modulation of ICl.swell in rabbit atrial myocytes. ICl.swell was induced with the use of a hypotonic superfusate, under conditions designed to prevent currents carried by K+, Na+, and Ca2+ ions. A basal Cl- current (ICl.b) was observed under isotonic conditions in 128 of 150 cells (85%), had the same dependency on [Cl-]o as ICl.swell, and was reduced by cell shrinkage induced by hypertonic superfusion, suggesting that ICl.b is carried by the same volume-sensitive Cl- conductance as ICl.swell. Phenylephrine produced a concentration-dependent and near-complete inhibition of ICl.b and ICl.swell, with EC50 values of 86±5 and 72±7 (mean±SEM) µmol/L, respectively, at +20 mV. Norepinephrine (administered in the presence of 1 µmol/L propranolol) also inhibited ICl.b and ICl.swell, with EC50 values of 2.6±0.1 and 2.8±0.4 µmol/L, respectively. The concentration-response curve for phenylephrine was shifted significantly (P<.001) to the right by the {alpha}1-adrenoceptor antagonist prazosin and by the {alpha}1A-receptor antagonists (+)-niguldipine and 5-methylurapidil but was unaltered by the {alpha}1B-receptor antagonist chloroethylclonidine (100 µmol/L). Inhibition of protein kinase C (PKC) with staurosporine, H-7, or 18-hour preincubation with the phorbol ester 4ß-phorbol 12-myristate 13-acetate (PMA, 500 nmol/L) blocked the effects of phenylephrine on ICl.swell, and the highly selective PKC inhibitor bisindolylmaleimide blocked the effects of norepinephrine on ICl.swell and ICl.b. Both PMA and 1-oleoyl-2-acetylglycerol inhibited ICl.swell in a concentration-dependent fashion. In blinded studies, the phorbol ester phorbol 12,13-didecanoate (PDD) reduced ICl.swell by 91±3%; its inactive analogue 4{alpha}-PDD had no effect (mean change, 3±1%). Preincubation with pertussis toxin (PTX) prevented the actions of phenylephrine on ICl.swell, indicating a role for a PTX-sensitive guanine nucleotide–binding (G) protein. We conclude that {alpha}-adrenergic agonists inhibit volume-sensitive Cl- currents in rabbit atrial cells by interacting with an {alpha}1A-adrenoceptor mechanism that is coupled to PKC via a PTX-sensitive G protein. These results suggest a potentially novel mechanism of {alpha}-adrenergic control of cardiac electrical activity, the inhibition of volume-sensitive Cl- currents, and indicate that PKC, well known to elicit phosphorylation-dependent Cl- currents in cat and guinea pig ventricular myocytes, is also capable of potently inhibiting other forms of cardiac Cl- current.


Key Words: action potential • Cl- currents • ion channels • autonomic nervous system • phenylephrine


*    Introduction
up arrowTop
up arrowAbstract
*Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
In most mammalian species, the stimulation of cardiac {alpha}-adrenoceptors increases action potential duration and the force of cardiac contraction.1 2 A variety of arrhythmia mechanisms may be enhanced by {alpha}-adrenergic stimulation,3 and {alpha}-adrenoceptor blockade can reduce the severity of arrhythmias induced by myocardial ischemia and reperfusion.1 A number of actions of {alpha}-adrenergic receptor activation on ionic currents have been described. Consistent with its ability to prolong action potential duration, {alpha}1-adrenoceptor activation inhibits a variety of K+ currents, including Ito, in rat ventricular4 5 6 and rabbit atrial7 8 9 myocytes, IK1 in rabbit atrial and ventricular myocytes,10 11 and IKACh in rabbit atrial and ventricular cells.10 11 In guinea pig myocytes, {alpha}-adrenergic stimulation shortens action potential duration,12 apparently by enhancing IK.13

Since the discovery in 1989 of ICl.cAMP in guinea pig and rabbit ventricular myocytes,14 15 16 the properties of several types of cardiac Cl- currents have been described. These include ICl.cAMP,14 15 16 17 18 ICl.Ca,19 20 ICl.b,21 22 ICl.swell,23 24 25 26 ICl.purinergic,27 and ICl.PKC.28 29 30 31 At physiological Cl- concentrations, the reversal potential for Cl- currents has been estimated at values ranging from -65 to -30 mV.26 32 33 Thus, Cl- current can play a role both in the repolarization of the cell from plateau potentials and in phase 4 depolarization underlying spontaneous activity.

Although the actions of {alpha}-adrenoceptor stimulation on K+ currents have been widely studied, {alpha}-adrenergic effects on Cl- currents have not been extensively investigated. Preliminary data showing that {alpha}-adrenoceptor stimulation activates a Cl- current by stimulating PKC in cat ventricular myocytes have been presented previously.30 Cell swelling and cell stretch, both of which activate ICl.swell,23 24 25 26 may occur during a variety of clinical conditions, including acute myocardial ischemia34 and congestive heart failure. Increased catecholamine concentrations are a feature of both conditions,35 36 so that the interactions of adrenergic agonists with ICl.swell may be of physiological and clinical importance. Isoproterenol has been shown to enhance ICl.swell in canine atrial myocytes, presumably by stimulating ß-adrenoceptors.23 The ability of {alpha}-adrenoceptor stimulation to modify ICl.swell is presently unknown. The present experiments were designed to determine (1) the effects of {alpha}-adrenoceptor stimulation on ICl.swell in rabbit atrial myocytes, (2) the ability of subtype-selective {alpha}-adrenoceptor antagonists to block any response seen, and (3) the signal transduction mechanisms linking {alpha}-adrenergic stimulation to changes in Cl- current. The results to be presented show that in contrast to the effects of {alpha}-adrenergic and PKC-mediated enhancement of Cl- currents in cat and guinea pig ventricular myocytes, {alpha}-adrenoceptor stimulation and the resulting activation of PKC cause concentration-dependent inhibition of ICl.swell.


*    Materials and Methods
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*Materials and Methods
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Preparation of Single Cells
Single atrial cells were obtained from rabbit hearts by using a previously described dissociation technique.21 37 Briefly, rabbits (1.5 to 2.0 kg) were killed by a blow on the neck, and the hearts were quickly removed and perfused in the Langendorff mode, first with a modified HEPES-buffered Tyrode's solution at 37°C, then with a nominally Ca2+-free Tyrode's solution until the heart ceased to beat, and finally with the same solution containing 0.04% collagenase (CLS II, Worthington Biochemical) and 1.0% bovine serum albumin (Sigma Chemical Co) for 10 minutes. The left atrium was removed and further dissected into small pieces, and cell dissociation was achieved by gentle mechanical agitation. All cells studied were rod-shaped, had clear cross-striations, and lacked any visible blebs on their surfaces. Cell dimensions were determined with a calibrated graticule in the microscope, and cell volumes were estimated with assumed right cylindrical geometry according to the following equation:

where V, L, and D are cell volume, length, and diameter, respectively.

Electrophysiological Recording
The tight-seal whole-cell voltage-clamp configuration of the patch-clamp technique was used. Recordings were performed with an Axopatch 1D or 200 amplifier (Axon Instruments). Voltage-clamp pulses were generated by a 12-bit digital-to-analog (D/A) convertor. Membrane current data were acquired by an analog-to-digital (A/D) conversion board (Medical Systems) with a maximum sampling rate of 100 kHz and simultaneously digitized (model TM 125, Scientific Solutions) and stored on the hard disk of an IBM PC–compatible computer under the control of PCLAMP software (Axon Instruments). Recording pipettes were prepared from borosilicate glass electrodes (outer diameter, 1.5 mm) with tip resistances of 2 to 5 M{Omega} (3.4±0.2 M{Omega}, mean±SEM, n=118) when filled. Junction potentials were corrected before achieving the membrane seal. After a tight seal between the cell membrane and the pipette tip (seal resistance >10 G{Omega}) had been formed, the bath solution was changed from Tyrode's solution to the standard isotonic experimental solution. After seal formation, the membrane patch was ruptured with brief additional suction.

The capacitive transients elicited by symmetrical 5-mV steps from -40 mV were recorded at 100 kHz for subsequent calculation of capacitance and access resistance. The mean cellular capacitance was 75±2 pF, and the input resistance averaged 557±33 M{Omega}, a value of the same order as that obtained by Giles and van Ginneken38 in rabbit atrial cells from the crista terminalis. Series resistance was then compensated to minimize the duration of the capacitive surge on the current record during 5-mV hyperpolarizations from -40 mV, and over 70% compensation was usually obtained. The time constant of the capacitive transient averaged 205±7 microseconds (n=118), and series resistance averaged 2.9±0.1 M{Omega} (n=118) after compensation. In most experiments, the maximum outward current was in the range of 1.5 nA, but in occasional experiments, the current at very positive voltages (eg, +80 mV) was larger, exceeding 2 nA. All analyses of drug action were therefore based on currents measured at +20 mV, a voltage in the physiologically-relevant range at which currents were always <1 nA.

To obtain whole-cell I-V relations, 300-millisecond hyperpolarizing and depolarizing pulses were imposed at 0.1 Hz in +10-mV increments between -100 and +80 mV from a holding potential of -40 mV. Current amplitudes were measured relative to the 0 current level. Leak-subtraction procedures were not used, but cells with evidence of a significant leak were rejected from study. All experiments were performed at 30±1°C. Na+ current was inactivated before the voltage steps by holding the cell at -40 mV. BaCl2 (500 µmol/L), CdCl2 (100 µmol/L), ouabain (10 µmol/L), and propranolol (1 µmol/L) were added to all superfusion solutions to block IK1, ICa, Na+,K+-ATPase, and ß-adrenoceptors, respectively.

Solutions and Drugs
The modified Tyrode's solution for cell isolation contained (mmol/L) NaCl 126, KCl 5.4, CaCl2 2.0, MgCl2 1.0, NaH2PO4 0.33, glucose 10, and HEPES 10, with pH adjusted to 7.4 with NaOH. The high-K+ storage solution contained (mmol/L) KCl 20, KH2PO4 10, glucose 10, potassium glutamate 70, ß-hydroxybutyric acid 10, taurine 10, and EGTA 10, along with 1% bovine serum albumin, pH 7.4 (KOH). K+-free pipette solutions were used to avoid contamination by outward K+ currents. The pipette (internal) solution contained (mmol/L) NMDG chloride 100, HEPES 10, EGTA 5.0, and Mg2+-ATP 5.0, with pH adjusted to 7.4 with NMDG hydroxide and osmolarity adjusted to 270 to 290 mOsm/kg H2O by adding mannitol (mean final osmolarity, 284±2 mOsm/kg H2O). Solution osmolarities were measured by freezing-point depression (Osmomette A, Precision Systems Inc). In experiments analyzing the response of currents to extracellular Cl- replacement, the pipette solution contained (mmol/L) NMDG chloride 24, NMDG aspartate 100, HEPES 10, EGTA 5, and Mg2+-ATP 5, with pH adjusted to 7.4 with NMDG hydroxide. [Cl-]o was modified by equimolar replacement with aspartate. The standard isotonic bath solution contained (mmol/L) NaCl 126, CsCl 5.4, MgCl2 0.8, CaCl2 1.0, NaH2PO4 0.33, HEPES 10, and glucose 5.5, with pH adjusted to 7.4 with NaOH (mean osmolarity, 294±3 mOsm/kg H2O). In some experiments, a hypertonic bath solution was used, which was prepared by adding 75 mmol/L mannitol to the standard isotonic solution to create a final osmolarity of 361±3 mOsm/kg H2O. The standard hypotonic bath solution contained (mmol/L) NaCl 100, MgCl2 0.8, CaCl2 1.0, NaH2PO4 0.33, HEPES 10, and glucose 5.5, pH adjusted to 7.4 with NaOH (217±2 mOsm/kg H2O). In some experiments, cell swelling was induced by using a hypertonic pipette solution that contained (mmol/L) CsCl 160, CsOH 40, MgCl2 1.0, HEPES 10, EGTA 5.0, and Mg2+-ATP 5.0 (pH 7.4 with HCl, 400 to 420 mOsm/kg H2O) while cells were perfused with standard bath solution (270 to 285 mOsm/kg H2O) as described above. Similar results were obtained with either method of inducing cell swelling, and the experiments presented were performed with hypotonic superfusate-induced swelling.

Phenylephrine, norepinephrine, propranolol, prazosin, ouabain, and PTX were purchased from Sigma. H-7, staurosporine, PMA, and OAG were obtained from ICN Biochemicals. The highly selective PKC inhibitor bisindolylmaleimide, the phorbol ester PDD, and its inactive analogue 4{alpha}-PDD were purchased from Calbiochem/Novobiochem. CEC, 5MU, and S(+)-niguldipine were bought from Research Biochemicals Inc. The disulfonic stilbene Cl- transport blockers DIDS and SITS were purchased from Sigma and made up as fresh solutions on the day of each experiment. Staurosporine, PMA, 4{alpha}-PDD, and PDD were prepared as 1 or 2 mmol/L stock solutions in dimethyl sulfoxide. OAG was first dissolved in chloroform, then dispersed in standard bath solution by sonication after evaporation of chloroform with N2 gas, and finally diluted in the standard bath solution to obtain the desired concentration. Stock solutions of the other drugs were prepared in distilled water and added to known volumes of superfusion solution to produce the desired concentrations. In all experiments with staurosporine or H-7, staurosporine (0.1 µmol/L) or H-7 (20 µmol/L) was present in the pipette solution and was added to the superfusate at the times indicated in "Results."

Data Analysis
All analyses are based on comparisons of currents in the presence of a drug with those recorded before drug superfusion in the same cell. Dose-response experiments were performed by superfusing the same cell with control solutions and then with each concentration of the drug to be tested. All drug effects were assessed after a superfusion interval long enough to achieve steady state effects, which was 10 to 15 minutes unless otherwise indicated.

EC50 was calculated from the concentration-response relation with an Emax model,39 in which the measured effect (E) of an agent at a known concentration C was fitted to the relation E=EmaxC/(C+EC50), where the variables determined by curve fitting are Emax (maximum effect) and EC50 (concentration for 50% of maximum effect). All results are expressed as mean±SEM. Statistical comparisons were performed either by ANOVA with Scheffé contrasts for group data or by Student's t test when only two groups were compared. A two-tailed probability of <5% was taken to indicate statistical significance.


*    Results
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up arrowIntroduction
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*Results
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Properties of ICl.swell and ICl.b
Fig 1ADown shows the properties of swelling-induced current in a myocyte lacking any significant conductance under basal conditions (Fig 1ADown, a). After exposure to hypotonic conditions, a substantial current is seen (Fig 1ADown, b). The current shows outward rectification and has a reversal potential (-36 mV) that is close to the calculated Cl- equilibrium potential (-38.5 mV). Fig 1ADown, c and d, shows the effects of extracellular Cl- replacement with aspartate on swelling-induced currents in the same cell. Reduction of [Cl-]o shifted the reversal potential to more positive values and strongly reduced the amplitude of outward currents. Fig 1BDown shows the I-V relations for swelling-induced currents in three cells that lacked any significant basal Cl- conductance. Results (mean±SEM) before hypotonic cell swelling are shown by open symbols; the results after cell swelling are shown by filled symbols. Under basal conditions (open circles), the conductance is extremely small and reverses at 0 mV. Cell swelling induces a large outwardly rectifying conductance whose reversal potential shifts strongly with changes in [Cl-]o. Fig 1CDown shows the relation between mean reversal potential in these cells and the logarithm of [Cl-]o. The relation was highly linear (r2=.999) and had a slope of -57 mV per decade. Experimentally determined values were close to the relation predicted for a pure Cl--specific conductance, as calculated from the Nernst equation and shown by the line in the figure.



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Figure 1. A, ICl.swell in a cell lacking ICl.b. Results are shown from the same cell under control conditions (a) and then after hypotonic superfusate–induced cell swelling in the presence of [Cl-]o of 105 (b), 25 (c), and 5 (d) mmol/L. In this and other figures showing original recordings, the horizontal line to the right of the recordings indicates the zero current level. Similar results were obtained in two other cells lacking ICl.b. B, Current-voltage relations from cells lacking ICl.b under isotonic conditions ({circ}) and in the presence of cell swelling at three values of [Cl]o (filled symbols). Results are mean±SEM in three cells. C, Relation between reversal potential (Erev) of ICl.swell and [Cl]o in cells lacking ICl.b. Results are mean±SEM, but error bar falls within symbol for mean. The square of the correlation coefficient for the relation between Erev and [Cl]o is .999, and the slope is -57 mV per decade. The line shown is the relation predicted for a pure Cl- conductance.

A minority of cells had the basal conductance characteristics shown in Fig 1Up. Of a total of 150 cells studied, 128 (85.3%) showed a significant basal conductance as illustrated in Fig 2ADown. ICl.b was present immediately after membrane rupture and remained constant under isotonic conditions for observation periods of up to 40 minutes. ICl.bs were outwardly rectifying and reversed at a potential (-46 mV) very close to the calculated Cl- equilibrium potential (-45.4 mV) in the presence of 136 mmol/L [Cl-]o and 24 mmol/L [Cl-]i. Reductions in [Cl-]o to 25 (Fig 2BDown) and 5 (Fig 2CDown) mmol/L reduced the outward current amplitude and shifted the reversal potential, changes that were reversible upon returning to a [Cl-]o of 136 mmol/L (Fig 2DDown). Exposure to hypotonic conditions caused cell swelling and greatly increased the conductance (Fig 2EDown). Overall, cell size increased upon hypotonic swelling from 114±2x10.6±0.2 µm to 107±2x16.4±0.5 µm, corresponding to a calculated volume increase of 140±15%. Reducing [Cl-]o reduced the magnitude of swelling-induced currents and shifted their reversal potential in the positive direction, as illustrated in Figs 2FDown and 2GDown.



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Figure 2. Currents under isotonic conditions (A through D) and in the presence of hypotonic superfusate–induced cell swelling (E through G) at the [Cl]o values shown.

Fig 3Down shows a quantitative analysis of the effects of changes in [Cl-]o on currents recorded under both hypotonic and isotonic conditions in six cells exposed to various [Cl-] values before and after cell swelling. For a given value of [Cl-]o, the I-V relation has the same form and a similar reversal potential under both isotonic and hypotonic superfusate conditions (Fig 3ADown). The relations between reversal potentials and [Cl-]o under isotonic and hypotonic conditions are shown in Fig 3BDown. The relations were highly linear (r2=.997 and .999 for isotonic and hypotonic conditions, respectively), with a slope of 56 mV per decade under each condition. Since currents were recorded with both hypotonic and isotonic superfusate at corresponding values of [Cl-]o in each cell, we were able to determine the response of the swelling-induced component to changes in [Cl-]o by subtracting ICl.b (under isotonic conditions) from the current recorded in the presence of hypotonic cell swelling. The resulting values have the I-V relations shown by the open squares in Fig 3ADown. The reversal potential of the swelling-induced component follows the Cl- equilibrium potential (Fig 3BDown) and has a linear relation to log ([Cl-]o), with an r2 of .999 and a slope of 57 mV per decade. Values of the reversal potential of swelling-induced current in the absence of ICl.b, as illustrated in Fig 1CUp, are reproduced as open triangles in Fig 3BDown. Note that the reversal potentials of ICl.b (open circles), the total current in the presence of swelling in cells with ICl.b (open triangles), the swelling-induced component in cells with ICl.b (open squares), and the swelling-induced current in cells without ICl.b (open diamonds) virtually superimpose on each other at each [Cl-]o (Fig 3BDown).



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Figure 3. A, I-V relations before ({circ}) and after ({triangleup}) hypotonic-induced cell swelling at [Cl-]o values of 105 (a), 25 (b), and 5 (c) mmol/L in six cells with ICl.b exposed to various [Cl-] values under both control and swelling conditions. {square} indicates the current induced by swelling, obtained by subtracting ICl.b from total current in the presence of swelling. B, Relation between reversal potential (Erev) and [Cl]o for currents shown in panel A (graphs a through c). Values are also shown for swelling-induced currents in cells lacking ICl.b ({diamond}, same data as in Fig 1CUp) and for ICl.b at 136 mmol/L [Cl]o. All results are mean±SEM. Isot. indicates isotonic; Hypot., hypotonic; and Ib, ICl.b.

The results shown in Fig 3Up suggest that ICl.b may be carried by the same ionic current mechanism as the swelling-induced current. Therefore, we sought to determine whether ICl.b is volume sensitive and can be suppressed by reducing cell volume with hypertonic superfusates. These experiments (and all others shown subsequently) were performed with pipettes containing 100 mmol/L [Cl-] in the pipette, bringing the Cl- reversal potential toward 0 mV. Fig 4ADown, a, shows ICl.b recorded immediately after rupturing the membrane and compensating for capacitance and series resistance. After 30 minutes of continued superfusion with isotonic solution, ICl.b was unchanged (Fig 4ADown, b). Subsequent exposure to hypertonic superfusate caused a gradual reduction in current amplitude, with results after 30 minutes shown in Fig 4ADown, c. Similar results were obtained in a total of four cells studied in this fashion, for which the mean I-V relations before and after 30 minutes of exposure to hypertonic solution are shown in Fig 4BDown. Overall, exposure to hypertonic solution for 30 minutes reduced ICl.b by 52±8% (P<.001) at +20 mV. Calculated mean volume of these cells averaged 9056±1514 µm3 (length, 123±13 µm; width, 9.5±0.4 µm) under isotonic conditions and 4163±884 µm3 (length, 121±12 µm; width, 6.5±0.4 µm) after hypertonic superfusion (a mean reduction in cell volume of 53±6%).



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Figure 4. Response of ICl.b to superfusion with hypertonic solution. A, Results from a typical cell. Results with isotonic superfusate are shown immediately after membrane rupture, capacitance, and series resistance compensation (a), after an additional 30 minutes of superfusion with isotonic solution (b), and after 30 minutes of superfusion with hypertonic superfusate (c). B, Effects of 30 minutes of exposure to hypertonic superfusate on the mean I-V relation of four cells. Calculated mean volume of these cells averaged 9056±1514 µm3 under isotonic conditions and 4163±884 µm3 after hypertonic superfusion.

The data shown in Figs 1 through 3UpUpUp indicate that ICl.b and the current induced by swelling are anion currents that have a similar and substantial selectivity for Cl- ions over aspartate and a similar I-V relation. Fig 4Up shows that ICl.b is volume sensitive and can be decreased by hypertonic superfusate-induced cell shrinkage. Therefore, it is quite likely that ICl.b and the current induced by swelling are carried by the same underlying volume-sensitive anion conductance. Since relatively few cells lack ICl.b, making it difficult to study swelling-induced current in isolation, we studied the {alpha}-adrenergic regulation of the total current in the presence of cell swelling, which we will call ICl.swell in this article and which we believe to represent the total magnitude of volume-regulated Cl- current in the presence of cell swelling in each cell. Several additional series of experiments were performed with both ICl.b and ICl.swell to determine whether they respond similarly to drug interventions, as would be expected if they are carried by the same underlying volume-sensitive current mechanism.

Effects of {alpha}-Adrenoceptor Stimulation on ICl.b and ICl.swell
Fig 5Down shows the effects of varying concentrations of phenylephrine on ICl.b. ICl.b was present as soon as the first voltage-clamp steps could be made after membrane rupture, capacitance compensation, and measurement of series and input resistance (Fig 5ADown) and did not change during 20 minutes of observation (Fig 5BDown). Subsequent superfusion of the cells with the {alpha}-adrenoceptor agonist phenylephrine in the presence of the ß-adrenoceptor antagonist propranolol (1 µmol/L) caused a concentration-dependent inhibition of ICl.b (Fig 5CDown through 5E). At +20 mV in four experiments in which all drug concentrations could be tested in each cell, 5 µmol/L phenylephrine caused a 9.8±1.5% reduction (P<.05), and 100 µmol/L phenylephrine reduced the current by 48±1% (P<.001). At a greater concentration (800 µmol/L), phenylephrine suppressed ICl.b by 82±4%, an effect that disappeared in the example shown after 45 minutes of washout (Fig 5FDown). The concentration-response curve for phenylephrine inhibition of outward current at +20 mV is shown in Fig 5GDown. The EC50 for phenylephrine inhibition of ICl.b averaged 86±5 µmol/L in four cells in which all drug concentrations could be studied.



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Figure 5. Effects of phenylephrine (PE) on ICl.b. Ionic currents shown in panels A through F were recorded from the same cell by imposing 10-mV 300-millisecond voltage steps from the holding potential of -40 mV to voltages between -100 and +80 mV (voltage protocol shown in inset). A, Currents recorded in the presence of isotonic superfusate, 2 minutes after membrane rupture. B, Currents recorded 20 minutes after membrane rupture. C through E, Currents recorded in the presence of incrementally increasing PE concentrations. F, Currents 45 minutes after washout of PE. G, Concentration-response curve for inhibition by PE of ICl.b as measured upon steps to +20 mV in the cell shown in panels B through E. The best-fit curve (shown) to the Emax equation provided in the text had an EC50 of 101 µmol/L and an Emax of 94%. (In all current recordings, horizontal line indicates zero current level.)

Fig 6Down shows the response of ICl.swell to phenylephrine. Under isotonic conditions, ICl.b is present (Fig 6ADown). Membrane conductance started to increase 3 to 5 minutes after exposure to the hypotonic solution and approached steady state values within 20 minutes (Fig 6BDown). Subsequent exposure to phenylephrine caused a concentration-dependent inhibition of ICl.swell, which was partially reversible upon washout of the largest concentration (Fig 6FDown). At +20 mV, the EC50 for phenylephrine action on ICl.swell averaged 72±7 µmol/L (n=9), not significantly different from the EC50 for phenylephrine inhibition of ICl.b. Although phenylephrine was somewhat more potent at inhibiting ICl.swell at more positive potentials (EC50 of 90±13, 71±10, 72±7, 77±8, 64±4, and 64±5 µmol/L at test potentials of -100, -80, +20, +40, +60, and +80 mV, respectively; n=9 for each), these differences were not statistically significant (ANOVA).



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Figure 6. Response of ICl.swell to phenylephrine (PE) in a representative cell. A, Recordings under isotonic conditions. B, Recordings obtained 16 minutes after changing to standard hypotonic superfusate. C through E, Response to increasing concentrations of PE. F, Partial reversal of the effect of PE after 80 minutes of washout. G, Concentration-response curve for PE-induced inhibition of ICl.swell in the cell shown in panels B through E. The best-fit curve (shown) to the Emax equation provided in the text had an EC50 of 94 µmol/L and an Emax of 100%.

To determine whether the changes observed with phenylephrine are also produced by the endogenous neurotransmitter norepinephrine, we studied the concentration-dependent effects of norepinephrine (in the presence of 1 µmol/L propranolol) on ICl.b and ICl.swell, as shown in Fig 7Down. Norepinephrine produced a concentration-dependent and voltage-independent inhibition of ICl.b (Fig 7ADown) and ICl.swell (Fig 7BDown), with EC50 values of 2.6±0.1 µmol/L (n=4) and 2.8±0.4 µmol/L (n=4), respectively. The results shown in Figs 5 through 7UpUpDown indicate that {alpha}-adrenergic stimulation is highly effective in inhibiting the volume-sensitive Cl- currents (ICl.b and ICl.swell) in rabbit atrial myocytes. The similar sensitivity of ICl.b and ICl.swell to both phenylephrine and norepinephrine supports the contention that both currents are carried by the same underlying mechanism.



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Figure 7. Inhibition of ICl.b (A) and ICl.swell (B) by norepinephrine (NE) in the presence of 1 µmol/L propranolol. A, Currents under isotonic control conditions (a) and in the presence of NE at 5 (b) and 20 (c) µmol/L. I-V relations for ICl.b in the absence and presence of NE are shown (d). B, Currents under isotonic conditions (a), after superfusion with hypotonic superfusate (b), and in the presence of NE at 5 (c) and 20 (d) µmol/L. I-V relations for ICl.swell in the absence and presence of NE are shown (e). Results shown for I-V relations of ICl.b and ICl.swell are for four cells, each studied under control conditions and in the presence of all [NE] values.

Effects of Selective {alpha}-Receptor Antagonists on Phenylephrine Action
Fig 8Down illustrates the antagonism of the effects of phenylephrine on ICl.swell by the {alpha}1-adrenoceptor antagonist prazosin. Fig 8ADown, a and b, shows currents before and after the induction of cell swelling in a representative myocyte. Exposure to prazosin (2 µmol/L) in the absence of phenylephrine did not alter ICl.swell (Fig 8ADown, c). Exposure to 800 µmol/L phenylephrine in the continued presence of prazosin inhibited ICl.swell (Fig 8ADown, d) but to a much smaller extent than the same concentration of phenylephrine in cells not exposed to prazosin (eg, compare with Figs 5EUp and 6EUp). Subsequent exposure to a superfusate containing the same concentration of phenylephrine in the absence of prazosin resulted in much stronger inhibition of ICl.swell (Fig 8ADown, e), which was partially reversed by the reintroduction of prazosin (Fig 8ADown, f).



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Figure 8. Inhibition of the effect of phenylephrine (PE) on ICl.swell by 2 µmol/L prazosin (PZ). A, Sample recordings from a representative cell: a, recordings were obtained under isotonic conditions; b, currents were recorded after changing to standard hypotonic superfusate; c, recordings were obtained 5 minutes after changing to a hypotonic superfusate containing 2 µmol/L PZ (PZ did not alter the currents recorded); d, addition of 800 µmol/L PE in the presence of PZ caused a modest reduction in ICl.swell; e, currents were markedly reduced after a change to a hypotonic superfusate with PE in the absence of PZ; and f, currents increased when superfusion with a hypotonic solution containing both PE and PZ was resumed. B, I-V relations obtained under control conditions ({bullet}), in the presence of PZ ({square}), in the presence of both PE and PZ ({blacktriangledown}), and in the presence of PE alone ({blacktriangleup}) in four cells studied under all conditions. C, Concentration-response curve for PE inhibition of ICl.swell at +20 mV in the absence ({bullet}, nine cells) and presence ({square}, six cells) of 2 µmol/L PZ. The best-fit curve (shown) to the Emax equation provided in the text had an EC50 of 635 µmol/L and an Emax of 85% in the presence of PZ and an EC50 of 61 µmol/L and an Emax of 91% in the absence of PZ.

Fig 8BUp shows mean I-V relations for ICl.swell under control conditions, in the presence of 2 µmol/L prazosin alone, in the presence of 800 µmol/L phenylephrine and 2 µmol/L prazosin, and in the presence of 800 µmol/L phenylephrine alone in four cells exposed to all conditions. Prazosin alone did not alter the I-V curve, and phenylephrine strongly inhibited the current at all voltages. The effect of phenylephrine was greatly attenuated by coadministration with prazosin. Prazosin (2 µmol/L) shifted the phenylephrine concentration-response curve in a parallel fashion to the right (Fig 8CUp), with the phenylephrine EC50 at +20 mV increased by prazosin from 72±7 to 737±99 µmol/L (n=6, P<.001).

To gain insights into the {alpha}1-receptor subtype mediating the effect of phenylephrine on ICl.swell, we studied the effect of the {alpha}1A-receptor antagonists (+)-niguldipine and 5MU and the {alpha}1B-receptor antagonist CEC on the phenylephrine concentration-response curve. As shown in Fig 9Down, even large concentrations of CEC (100 µmol/L) did not perceptibly alter the response to phenylephrine. On the other hand, 0.1 µmol/L 5MU substantially inhibited the action of phenylephrine, with relatively little current inhibition occurring at a concentration of 800 µmol/L.



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Figure 9. Changes in response to phenylephrine (PE) in the presence of CEC (A) and 5MU (B) in representative cells. A, Currents recorded in the absence of CEC are shown under isotonic (a) and hypotonic (b) conditions, followed by currents in the presence of CEC alone (c) and phenylephrine (800 µmol/L) in the presence of CEC (d). Similar results were obtained in a total of eight cells. B, Currents recorded in the absence of 5MU are similarly shown under isotonic (a) and hypotonic (b) conditions, followed by currents in the presence of 5MU alone (c) and phenylephrine (800 µmol/L) in the presence of 5MU (d). Similar results were obtained in a total of five cells.

The actions of several antagonists were assessed quantitatively by studying their effects on the phenylephrine concentration-response curve (Fig 10Down). While CEC (100 µmol/L) did not significantly alter the phenylephrine EC50 (84±12 µmol/L in presence of CEC, n=8, P=NS vs phenylephrine alone), (+)-niguldipine, 5MU, and prazosin all significantly (P<.001 for each) increased the phenylephrine EC50 by shifting the concentration- response curve to the right in a parallel fashion (Fig 10Down). The action of (+)-niguldipine was concentration dependent [EC50 of 368±74 and 691±47 µmol/L for 0.1 and 1.0 µmol/L (+)-niguldipine, respectively; n=4 for each], and its potency was substantially less than that of 5MU. 5MU, at a concentration of 0.1 µmol/L, increased the phenylephrine EC50 in five cells to 2418±53 mmol/L, a greater increase than produced by a 10 times higher concentration of (+)-niguldipine. None of the antagonists significantly altered the Emax for phenylephrine, which averaged 93±4% under control conditions and 94±3%, 93±3%, 91±5%, 96±2%, and 99±1% in the presence of prazosin, CEC, niguldipine at 0.1 µmol/L, niguldipine at 1 µmol/L, and 5MU, respectively.



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Figure 10. Concentration-dependent effects of phenylephrine (PE) alone and in the presence of CEC, (+)-niguldipine (NIG), and 5MU on ICl.swell. The PE concentration-response curve was not altered by 100 µmol/L CEC ({blacktriangleup}). It was shifted to the right in a concentration-dependent way by 0.1 ({triangledown}) and 1.0 ({blacktriangledown}) µmol/L NIG. A still greater shift was caused by 0.1 µmol/L 5MU ({diamondsuit}). The best-fit curves (shown) to the Emax equation provided in the text had EC50 values of 61 (PE alone), 69 (PE+CEC), 367 (PE+NIG, 0.1 µmol/L), 840 (PE+NIG, 1 µmol/L), and 1869 µmol/L (PE+5MU) and Emax values of 91%, 88%, 95%, 98%, and 97%, respectively.

Effects of PKC Inhibition on the Response of ICl.swell and ICl.b to {alpha}-Adrenoceptor Agonists
To determine whether the {alpha}-adrenoceptor–induced decrease in ICl.swell is mediated by the activation of PKC, we applied phenylephrine in the presence of the PKC inhibitors staurosporine40 and H-7.41 Fig 11ADown shows the effect of staurosporine on the response of ICl.swell to phenylephrine. In contrast to the reproducible inhibition caused by phenylephrine in the absence of PKC inhibitors (eg, Figs 5Up and 6Up), 800 µmol/L phenylephrine had minimal effect on Cl- current in their presence. Fig 11BDown shows overall data for the concentration-dependent inhibitory effects of phenylephrine on ICl.swell under control conditions (n=9) and in the presence of 100 nmol/L staurosporine (n=6) and 20 µmol/L H-7 (n=5). In the presence of PKC inhibitors, no statistically significant effects of phenylephrine on ICl.swell could be demonstrated at concentrations up to 1600 µmol/L.



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Figure 11. A, Effects of staurosporine (STAUR) on the response of ICl.swell to phenylephrine (PE) in a representative cell. Currents were recorded in the presence of isotonic superfusate (a). ICl.swell was recorded after superfusate was changed to hypotonic solution (b). The addition of STAUR (0.1 µmol/L) to the hypotonic superfusate did not alter ICl.swell (c). When phenylephrine was added to the superfusate at 800 µmol/L in the presence of STAUR, ICl.swell was not affected (d). STAUR (0.1 µmol/L) was in the pipette solution throughout the experiment. B, Concentration-response relation for PE alone (n=9 cells) and in the presence of 0.1 µmol/L STAUR (n=6) and 20 µmol/L H-7 (n=5). C, Effects of PKC downregulation with PMA on action of PE on ICl.swell (ICl). Cells were incubated at room temperature in high-K+ storage solution in the absence (n=5) or presence (n=5) of PMA. The PE concentration-response relation for cells incubated without PMA was not significantly different from that of fresh cells (nonincubation, n=9). In contrast, preincubation with PMA prevented any response to PE at concentrations up to 1600 µmol/L.

Organic PKC inhibitors like H-7 and staurosporine are not completely specific in their actions. Therefore, additional experiments were performed to establish the effects of PKC inhibition on phenylephrine action. Prolonged stimulation (>6 hours) of PKC by phorbol esters such as PMA leads to a loss of PKC enzymatic activity and high-affinity phorbol ester binding.9 11 42 This allows for the role of PKC to be tested in a fashion independent of the use of organic PMA inhibitors. Therefore, we incubated cells in the high-K+ storage solution (for contents, see "Materials and Methods") overnight (>15 hours) at room temperature, with or without the addition of 500 nmol/L PMA. Experiments were done in a paired fashion, with the cells from each atrial isolate divided into two lots, one to be incubated with and the other without PMA. ICl.swell and its response to phenylephrine were then assessed in cells from both groups in random order the next day. Fig 11CUp shows the mean concentration-response curve for phenylephrine inhibition of ICl.swell at +20 mV in cells incubated with (n=5) or without (n=5) PMA, along with data from nine cells studied without prior overnight incubation. In cells incubated without PMA, phenylephrine caused a concentration-dependent inhibition of ICl.swell with an EC50 of 66±8 µmol/L (n=5), a value not significantly different from that in cells studied without preincubation. In contrast, exposure to phenylephrine at concentrations up to 2 mmol/L did not significantly alter ICl.swell in cells incubated overnight with PMA before study.

Finally, we used the recently developed and highly selective PKC inhibitor bisindolylmaleimide,43 to determine whether PKC inhibition alters the response to norepinephrine (coadministered with 1 µmol/L propranolol). Fig 12Down shows the effect of 30 nmol/L bisindolylmaleimide on the response of ICl.b (panel A) and ICl.swell (panel B) to norepinephrine at concentrations up to 20 µmol/L. In marked contrast to the strong inhibition of these currents caused by norepinephrine in the absence of bisindolylmaleimide (Fig 7Up), PKC inhibition completely prevented {alpha}-adrenergic actions on these currents. Furthermore, the similar effects of PKC inhibition on the response to norepinephrine of ICl.b and ICl.swell suggest that these volume-sensitive currents are both inhibited by {alpha}-adrenergic agonists via the activation of PKC.



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Figure 12. Response of ICl.b (A) and ICl.swell (B) in typical cells to bisindolylmaleimide (BIM, 30 nmol/L) alone and to increasing concentrations of norepinephrine (NE) in the presence of 30 nmol/L BIM. Similar results were obtained in three cells for ICl.b and in three cells for ICl.swell.

Effects of PKC Activators on ICl.swell
To further assess the ability of PKC activation to inhibit ICl.swell, we examined the effects of addition to the superfusate of PKC activators.44 Fig 13Down shows that PMA (50 to 800 nmol/L) induced a concentration-dependent decrease in ICl.swell. The EC50 for inhibition of ICl.swell at +20 mV by PMA was 210±23 nmol/L (n=5) (Fig 13EDown). Similar results were obtained with OAG but at higher concentrations (5 to 200 µmol/L). The EC50 for inhibition of ICl.swell by OAG was 47±14 µmol/L (n=5) at +20 mV (Fig 13FDown). To exclude a nonspecific action of phorbol esters unrelated to PKC activation, we performed blinded experiments in which coded stock solutions of either PDD or 4{alpha}-PDD (structurally very similar to PDD but ineffective in activating PKC) were used in a randomized fashion to study the changes in ICl.swell caused by 1 µmol/L of each in the superfusate. Fig 14Down shows results in one cell exposed to both compounds. 4{alpha}-PDD had no effect on ICl.swell, whereas PDD produced strong inhibition. Overall, PDD reduced ICl.swell by 91±3% (n=4, P<.001), whereas the mean change in ICl.swell occurring in the presence of 1 µmol/L 4{alpha}-PDD averaged 3±1% (n=6, P=NS). These results indicate that activators of PKC are capable of mimicking the effect of {alpha}-adrenoceptor stimulation on ICl.swell.



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Figure 13. Concentration-dependent inhibition of ICl.swell by PMA. Currents were recorded from one cell under isotonic (A) and hypotonic (B) superfusate conditions in the absence of PMA and then in the presence (C and D) of increasing PMA concentrations. E and F, Concentration-response curves for inhibition of ICl.swell (ICl) by PMA (E) and OAG (F).



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Figure 14. Effects of 1 µmol/L PDD and the same concentration of its inactive 4{alpha} analogue (4{alpha}-PDD) on ICl.swell upon blinded administration to a typical cell. A, Currents were recorded under isotonic conditions. B, Currents were recorded after hypotonic superfusate–induced swelling. C, No changes in currents were observed 10 minutes after the addition of 1 µmol/L 4{alpha}-PDD. D, Ten minutes of subsequent superfusion with 1 µmol/L PDD produced profound depression of ICl.swell. Similar results were obtained in six cells with 4{alpha}-PDD and in four with PDD.

Effects of PTX on Phenylephrine-Induced Inhibition of ICl.swell
Cardiac {alpha}1-adrenoceptors are functionally linked to heterotrimeric GTP-binding regulatory proteins (G proteins),45 which are thought to play an important role in mediating {alpha}1-adrenoceptor–induced increases in phospholipase C activity46 47 that result in PKC activation. PTX inactivates G proteins (Go and Gi) that are potentially coupled with {alpha}1-adrenoceptors,1 48 49 50 by catalyzing the ADP ribosylation of the {alpha} subunit at a C-terminal cysteine residue and thus blocking the interaction between activated receptors and the holo-G protein.51

To determine whether the {alpha}1-adrenergic inhibition of ICl.swell is coupled via PTX-sensitive G proteins, we incubated myocytes for over 18 hours at room temperature in storage solution containing 0.5 µg/mL of PTX. This procedure has been reported to cause the ADP ribosylation of up to 90% of the available PTX-sensitive G proteins in rabbit atrial cells.9 11 As shown in Fig 15Down, pretreatment of cells with PTX abolished the effect of phenylephrine (25 to 3200 µmol/L) on ICl.swell (n=5). Preincubation of cells in the storage solution alone did not alter the response to phenylephrine (Fig 11CUp). These results indicate that PTX-sensitive G proteins are essential for the inhibition of ICl.swell by {alpha}-adrenoceptor stimulation.



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Figure 15. Effect of >18-hour incubation in high-K+ storage solution containing 0.5 µg/mL PTX on the response to phenylephrine (PE). Currents in the absence of PE under isotonic (A) and hypotonic (B) conditions were similar to those recorded in fresh cells. The addition of increasing PE concentrations to the hypotonic superfusate (C through F) failed to alter ICl.swell in cells pretreated with PTX. Similar results were obtained in a total of five cells incubated with PTX. Incubation in the storage solution alone did not alter the response of ICl.swell to PE (squares in Fig 7Up).


*    Discussion
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
We have shown that ICl.b and ICl.swell in rabbit atrial myocytes share properties of outward rectification, Cl- selectivity, volume regulation, and concentration-dependent inhibition by {alpha}-adrenergic agonists, suggesting that they are carried by the same underlying volume-sensitive anion conductance. The inhibitory actions of phenylephrine on ICl.swell are antagonized by the {alpha}1-receptor antagonist prazosin, not altered by the {alpha}1B-receptor antagonist CEC, and prevented by the {alpha}1A-selective receptor antagonists (+)-niguldipine and 5MU, indicating mediation by an {alpha}1A-receptor subtype. The actions of {alpha}-adrenergic stimulation on volume-regulated Cl- currents are mimicked by the PKC-stimulating phorbol esters PDD, PMA, and OAG and are prevented when PKC is inhibited by staurosporine, H-7, bisindolylmaleimide, or downregulation by prolonged exposure to PMA. Exposure to PTX also blocks {alpha}-adrenergic inhibition of ICl.swell, indicating the participation of a PTX-sensitive G protein in the signal transduction pathway leading to PKC activation by {alpha}1A-receptor stimulation.

Properties of Cl- Currents Studied
The properties of ICl.b in the present experiments resemble those we have previously reported in the same preparation.21 22 Although ICl.swell has not previously been reported to exist in rabbit atrium, the properties of ICl.swell described in the present article resemble those described in other cardiac preparations23 24 26 and those of a stretch-induced Cl- current in rabbit atrial cells.25 The reversal potential of ICl.swell responded to changes in [Cl-] gradient in a fashion consistent with a Cl--selective current and with the same slope factor for [Cl-]o dependence as ICl.b. Exposure to hyperosmotic superfusate substantially reduced ICl.b. ICl.b and ICl.swell were inhibited by {alpha}-adrenoceptor agonists in a quantitatively similar fashion, and {alpha}-adrenergic inhibition of either was PKC dependent. These findings suggest that both currents are carried by the same underlying volume-sensitive Cl- channel.

Comparison With Previous Observations of {alpha}1-Adrenoceptor Modulation of Ion Currents
Recent reports have indicated that {alpha}1-adrenergic stimulation suppresses several K+ currents, including Ito,6 7 8 9 52 IK1, and IKACh.10 11 In their studies on rat ventricular myocytes, Ravens and colleagues6 52 reported that {alpha}1-adrenergic stimulation inhibited both the 4-AP–sensitive transient outward current and the 4-AP–insensitive sustained current.6 52 In rabbit atrium, the sustained current activated by depolarization is resistant to 4-AP and has properties suggesting a potentially important contribution from ICl.b.21 In the present study, we have found that {alpha}1-adrenoceptor stimulation inhibits both ICl.b and ICl.swell, at concentrations very similar to those found to inhibit K+ current in other studies.6 7 8 9 52 This is, to our knowledge, the first report of {alpha}-adrenergic inhibition of a cardiac Cl- current.

Subtype Selectivity of {alpha}1-Adrenoceptor Effects on ICl.swell
McGrath and Wilson53 suggested the existence of two subtypes of {alpha}1-adrenoceptors based on differences in agonist affinity. Han et al54 and Minneman55 classified {alpha}1-adrenoceptors into {alpha}1A and {alpha}1B subtypes, based on tissue responses to various agonists and antagonists and on competitive ligand-binding studies with WB 4101 and CEC. The {alpha}1A subtype has a high affinity for the competitive antagonist WB 4101 and is not inactivated by the alkylating agent CEC. The {alpha}1B subtype has a 20- to 1500-fold lower affinity for WB 4101 and is potently inhibited by CEC.54 55 56 Additional {alpha}1A-selective antagonists have been developed, including 5MU,57 58 a serotonin antagonist, and (+)-niguldipine,59 60 a dihydropyridine Ca2+ channel blocker. It has been suggested that 5MU is currently the best antagonist for identifying {alpha}1A-receptor–mediated responses.61 Recently, two additional subtypes of {alpha}-adrenoceptors, {alpha}1C and {alpha}1D, have been identified on the basis of molecular cloning.62 63 The {alpha}1D-receptor differs from the {alpha}1A-receptor clone by two amino acids and, in contrast to the latter, is sensitive to inhibition by CEC.62 The {alpha}1C-receptor shows exquisite and equal sensitivity to inhibition by (+)-niguldipine and 5MU, whereas the {alpha}1A-receptor is >10 times as sensitive to 5MU as it is to (+)-niguldipine.63

In the present study, phenylephrine-induced inhibition of ICl.swell was unaffected by CEC at high concentrations but was inhibited by (+)-niguldipine and 5MU, showing particular sensitivity [>10-fold greater than that to (+)-niguldipine] to the latter agent. These observations suggest the involvement of an {alpha}1A type of receptor. Both {alpha}1A- and {alpha}1B-receptors have been shown to exist in canine ventricle on the basis of radioligand and electrophysiological studies.64 The {alpha}1A-receptor–mediated system is involved in the induction of abnormal automaticity in canine cardiac Purkinje fibers exposed to ischemic conditions.65 66 67 This system is PTX sensitive66 67 and causes an increase in longitudinal resistance consistent with a decrease in membrane conductance.67 It would be interesting to explore the possible activation of volume-sensitive Cl- currents by ischemia in canine Purkinje fibers, which could be amenable to {alpha}-adrenergic inhibition by the system described in the present study.

Intracellular Signaling Mechanisms Underlying {alpha}-Adrenergic Modulation of ICl.swell
Stimulation of {alpha}1-adrenoceptors leads to various biochemical responses, including enhanced Ca2+ influx, phospholipase C and A2 activation, and changes in intracellular cyclic nucleotide levels.2 55 68 69 In the heart, the most extensively documented signaling responses to {alpha}1-adrenoceptor stimulation are mediated via phospholipase C–induced hydrolysis of phosphatidylinositol 4,5-diphosphate, giving rise to a variety of potential second messengers, including inositol 1,4,5-tris-phosphate and 1,2-diacylglycerol,49 69 70 71 which is thought to be an endogenous activator of PKC.68 69 PKC has been implicated in the modulation of ion channel function in numerous studies.44 Many of the signaling mechanisms mediating {alpha}-adrenoceptor–induced responses are coupled by G proteins, at least two of which are sensitive to inhibition by PTX.48

PTX-sensitive G proteins, which may appear with development,72 mediate the effects of {alpha}1-adrenergic stimulation on abnormal automaticity in canine Purkinje fibers66 67 and the positive chronotropic response to {alpha} agonists in rat hearts.72 PKC produces action potential changes similar to those caused by methoxamine in guinea pig papillary muscles,12 and phospholipase C produces positive chronotropic responses in canine Purkinje fibers similar to those resulting from {alpha}-adrenergic stimulation.73 In contrast, {alpha}-adrenergic inhibition of a variety of K+ currents in rabbit atrial myocytes is insensitive to PTX and agents that inhibit PKC activity.9 11 In the present study, {alpha}1-adrenergic inhibition of Cl- current was found to be sensitive to inhibition by PTX and interventions that suppress PKC function, making this system a candidate to account for some of the electrophysiological effects of {alpha}-adrenergic activation. Our findings are consistent with studies in which PKC activation caused a reduction in Cl- currents in a variety of noncardiac preparations.74 75 76

Limitations
Both ICl.b and ICl.swell are inhibited by {alpha}1-agonists, which raises a potential problem. Since in many cells ICl.swell is composed of both a basal (ICl.b) and swelling-induced component, the observed changes in total Cl- current represent the sum of {alpha}-adrenergic effects on each. To have studied each selectively in each cell would have been prohibitively difficult, requiring exposure to control solutions, multiple drug concentrations, hypotonic solutions, and then the same drug concentrations again. We have presented extensive evidence (in Figs 1 through 7UpUpUpUpUpUpUp and Fig 12Up), discussed above, suggesting that ICl.b and ICl.swell are due to the same volume-sensitive anion conductance and justifying the use of ICl.swell as an index of mechanisms of {alpha}-adrenergic regulation of volume-sensitive Cl- current.

The response of ICl.b and ICl.swell in rabbit atrial myocytes to {alpha}-adrenergic and PKC stimulation that we observed is opposite the response to the Cl- current stimulation noted in feline and guinea pig ventricular myocytes by Zhang and colleagues30 31 and Walsh and Long.28 29 Recently published work by Zhang et al31 suggests that PKC and protein kinase A may act on the same set of Cl- channels to elicit Cl- current in feline ventricular myocytes. Rabbit atrium lacks transcripts for the cystic fibrosis transmembrane conductance regulator77 and ICl.cAMP,78 which appears to be the target for PKC activation of Cl- current.31 This may explain the apparently simple inhibitory effect of {alpha}1-adrenergic activation on ICl.b and ICl.swell that we observed. Under conditions that cause cell swelling in tissues that express both ICl.swell and ICl.PKC, a more complex response to {alpha}1-adrenergic activation, including both stimulation of ICl.PKC and inhibition of ICl.swell, might occur. This question remains to be addressed in future studies. Walsh and Long29 cite unpublished results suggesting that in guinea pig ventricular myocytes, which manifest ICl.PKC, ICl.swell is absent.

The response of ICl.b to hypertonic superfusate raises the question of whether cell swelling or cell stretch occurred during cell isolation, resulting in the activation of ICl.swell, which then appeared as a background current at the onset of whole-cell recording. An alternative explanation of these findings is that rabbit atrial cells have a background Cl- current that is sensitive to cell volume (and possibly cell stretch), and can be enhanced by exposure to hypotonic media (which cause cell swelling) or suppressed by exposure to hypertonic media (which cause cell shrinkage). If this were the case, the volume-sensitive Cl- current could modulate cell function in response to either an increase or a decrease in cell volume.

We obtained concentration-response curves by exposing each cell to five or six drug concentrations over the steep portion of the concentration response curve. Since saturating concentrations were not studied in all cells, there is some uncertainty about the precise maximum inhibition caused by {alpha}-agonists, introducing some uncertainty in the calculation of EC50 values. These uncertainties are relatively small and do not alter the qualitative differences that were seen among various {alpha}-receptor antagonists.

Conclusions
We have found that {alpha}1-adrenergic activation inhibits the volume-regulated Cl- currents ICl.b and ICl.swell in rabbit atrial myocytes by a PTX-sensitive PKC-dependent mechanism. This is, to our knowledge, the first demonstration of the inhibition of Cl- current by an {alpha}-adrenergic mechanism in the heart. It contrasts with the stimulatory effect of PKC on Cl- current previously described in ventricular tissues from the cat and guinea pig and indicates the potential complexity of the {alpha}-adrenergic regulation of cardiac Cl- currents. The inhibition of ICl.b by {alpha}1-adrenergic activation could contribute to some of the electrophysiological effects of the latter, and {alpha}-adrenergic inhibition of ICl.swell may play a role in settings, such as acute myocardial ischemia and heart failure, in which catecholamine concentrations are increased and cell swelling and/or stretch can activate ICl.swell.


*    Selected Abbreviations and Acronyms
 
CEC = chloroethylclonidine
Emax = maximum effect of an agent
ICa = Ca2+ current
ICl.Ca = Ca2+-activated Cl- current
ICl.cAMP = cAMP-dependent Cl- current
ICl.b = basal Cl- current
ICl.PKC = PKC-activated Cl- current
ICl.swell = total Cl- current in presence of swelling
IK = delayed rectifier K+ current
IKACh = acetylcholine-activated K+ current
IK1 = inward rectifier K+ current
Ito = transient outward current
I-V = current-voltage
NMDG = N'-methyl-D-glucamine
OAG = 1-oleoyl 2-acetylglycerol
PDD = phorbol 12,13-didecanoate
PKC = protein kinase C
PMA = 4ß-phorbol 12-myristate 13-acetate
PTX = pertussis toxin
4-AP = 4-aminopyridine
5MU = 5-methylurapidil


*    Acknowledgments
 
This study was supported by operating grants from the Medical Research Council of Canada, the Quebec Heart Foundation, and the Fonds de Recherche de l'Institut de Cardiologie de Montréal. D. Duan holds an MRC graduate studentship. The authors thank Guylaine Nicol and Ling Yu Ye for technical assistance and Martine Dufort and Luce Bégin for typing the manuscript.

Received July 13, 1994; accepted May 2, 1995.


*    References
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
up arrowResults
up arrowDiscussion
*References
 
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