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0022-3565/98/2841-0075$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics JPET 284:75–82, 1998 Vol. 284, No. 1 Printed in U.S.A. Amiodarone Inhibits the Na1-K1 Pump in Rabbit Cardiac Myocytes after Acute and Chronic Treatment1 DAVID F. GRAY,2 ANASTASIA S. MIHAILIDOU, PETER S. HANSEN,3 KERRIE A. BUHAGIAR, NERIDA L. BEWICK, HELGE H. RASMUSSEN and DAVID W. WHALLEY Cardiology Department, Royal North Shore Hospital, St. Leonards, 2065 NSW, and University of Sydney, 2006 NSW, Australia Accepted for publication September 16, 1997 This paper is available online at http://www.jpet.org The mechanism of action of the widely used antiarrhythmic agent amiodarone is thought to involve use-dependent block of Na1, Ca11, and K1 channels (see Nattel et al., 1992, for review). However, when studying electrophysiological effects of amiodarone, the Na1-K1 pump should also be considered. The pump directly, or indirectly through the operation of secondary active ion transport mechanisms, maintains the transmembrane ionic gradients. These gradients in turn drive the channel currents that are modulated by amiodarone. In addition, the electrogenic Na1-K1 pump current, arising from the 3Na1:2K1 exchange ratio, contributes to repolarization of the cardiac action potential (Gadsby et al., 1985). Treatment with amiodarone influences physicochemical properties of the cell membrane, including fluidity and cholesterol content (Chatelain et al., 1986). Because the Na1-K1 pump is affected by such changes in membrane properties Received for publication April 1, 1997. 1 This study was supported by the National Heart Foundation of Australia (Grant 94S4055) and by the North Shore Heart Research Foundation. 2 D.F.G. was the recipient of a National Health and Medical Research Council of Australia Medical Postgraduate Research Scholarship. 3 P.S.H. was the recipient of a Postgraduate Medical Research Scholarship from the National Heart Foundation of Australia. 10 mM Na1 but had no effect on Ip at 80 mM Na1. Amiodarone had no effect on the voltage dependence of the pump or the affinity of the pump for extracellular K1 either after chronic treatment or during acute exposure. We conclude that chronic amiodarone treatment reduces overall Na1-K1 pump capacity in cardiac ventricular myocytes. In contrast, acute exposure of myocytes to amiodarone reduces the apparent Na1 affinity of the Na1-K1 pump. An amiodarone-induced inhibition of the hyperpolarizing Na1-K1 pump current may contribute to the action potential prolongation observed during treatment with this drug. Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017 ABSTRACT Amiodarone has been shown to affect cell membrane physicochemical properties, and it may produce a state of cellular hypothyroidism. Because the sarcolemmal Na1-K1 pump is sensitive to changes in cell membrane properties and thyroid status, we examined whether amiodarone affected Na1-K1 pump function. We measured Na1-K1 pump current (Ip) using the whole-cell patch-clamp technique in single ventricular myocytes isolated from rabbits. Chronic treatment with oral amiodarone for 4 weeks reduced Ip when myocytes were dialyzed with patch-pipettes containing either 10 mM Na1 or 80 mM Na1. In myocytes from untreated rabbits, acute exposure to amiodarone in vitro reduced Ip when patch pipettes contained (Cornelius, 1991), it seems reasonable to speculate that amiodarone may alter pump function. Amiodarone might also alter pump function through an alternative mechanism. The drug is believed to induce a state of cellular hypothyroidism with chronic use (Patterson et al., 1986; Singh et al., 1983). Thyroid status in turn is an important determinant of synthesis of Na1-K1 pump units (Doohan et al., 1995; Kjeldsen et al., 1986). Several previous studies using isolated myocardial membrane or microsomal preparations (Broekhuysen et al., 1972; Dzimiri and Almotrefi, 1991) have suggested that acute exposure to amiodarone decreases Na1-K1/ATPase activity, the enzymatic equivalent of the Na1-K1 pump. Although demonstrating that amiodarone has the potential to affect pump activity, the physiological relevance of such studies is unclear. The drug concentrations used were much higher than those encountered clinically, and the studies were performed using K1 and Na1 concentrations expected to saturate binding sites rather than at physiologically relevant concentrations. In addition, Na1-K1/ATPase studies require the pump molecule to be isolated from the native lipid membrane and purified. As the membrane lipid environment ABBREVIATIONS: Ip, Na1-K1 pump current; QTc, corrected QT interval; T4, thyroxine; T3, triiodothyronine; HEPES, N-2-hydroxyethylpiperazineN9-2-ethanesulfonic acid; EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N9,N9-tetraacetic acid; TMA, tetramethylammonium; aiNa, intracellular Na1 activity; [Na]pip, pipette Na1 concentration; [K]o, extracellular K1 concentration; K0.5, concentration for half-maximal pump activation; Vm, membrane potential; Ip-Vm, pump current-voltage relationship. 75 76 Gray et al. Vol. 284 modulates pump activity (Cornelius, 1991), it is unclear how these results relate to the activity of in situ pumps. In the present study, we examined the effects of both acute and chronic amiodarone exposure on the Na1-K1 pump in intact cardiac myocytes. We used the whole-cell patch-clamp technique to measure Ip. This approach allows independent control of ligands for the Na1-K1 pump on both sides of the intact native membrane, as well as control of membrane potential, variables that are important determinants of pump function. Methods Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017 Treatment protocols. Male New Zealand White rabbits, weighing 2.5 to 3.0 kg, were used in the study. Amiodarone was supplied in its hydrochloride powder form. In experiments examining the effects of chronic treatment, gelatin capsules containing appropriate quantities of amiodarone were made up at two weekly intervals. A single daily amiodarone capsule was administered to rabbits orally in a dose of 80 mg/kg/day for 4 weeks. This protocol was based on a previous study in rabbits demonstrating clinically relevant changes in electrophysiological and electrocardiographic parameters with 50 to 100 mg/kg oral amiodarone daily (Kodama et al., 1992). Control rabbits received empty gelatin capsules in the same manner. A surface electrocardiogram was obtained before treatment and at the end of the 4-week treatment protocol to assess the effects of amiodarone on the QT interval. This parameter has been shown to correlate well with myocardial concentrations of amiodarone (Debbas et al., 1984). Bazett’s formula was used to correct the QT interval (QTc) for heart rate (QTc 5 QT/=RR interval). The ECG electrodes were placed on the shaved precordium of conscious rabbits in positions that provided ECG deflections of adequate amplitude to make accurate determinations of QT interval. Recordings were performed using a single-channel ECG machine (Nihon Kohden Cardiofax, model SC-513E) at a paper speed of 50 mm/sec. Recordings were obtained from at least two lead orientations. Blood was taken from a marginal ear vein at base line and after 4 weeks of treatment to assess the effects of amiodarone on thyroid function. Serum total T4 and T3 were measured quantitatively using the Ciba Corning Automated Chemiluminescence System (ACS:180 system, Ciba Corning Diagnostics Corp, Medfield, MA). The serum amiodarone level was determined in the blood sample collected after 4-week treatment. The level was measured using an HPLC method adapted from Law et al. (1984). In experiments examining the acute effects of amiodarone on the Na1-K1 pump, myocytes isolated from untreated rabbits were exposed to the drug in-vitro. A 1 mM stock solution of amiodarone in 100% ethanol was prepared and used within 2 weeks. On the day of experimentation, the stock solution was diluted 100-fold in solutions used to superfuse the myocytes; thus, the superfusates had a nominal amiodarone concentration of 10 mM in 1% ethanol, but these solutions appeared to be supersaturated. We therefore measured amiodarone content in the superfusate using HPLC. The mean final amiodarone concentration in the superfusate was 0.61 6 0.13 mM (n 5 6; range, 0.30 –1.20 mM). Control myocytes in these experiments were exposed to superfusates containing 1% ethanol only. Measurement of Ip. Single ventricular myocytes were isolated as described previously(Hool et al., 1995). After isolation, myocytes were stored at room temperature until used for experimentation. Myocytes were used on the day of isolation only, and pump currents were measured 2 to 10 hours after the heart was excised. Myocytes were voltage clamped with wide-tipped (4 –5 mm) borosilicate glass pipettes made as described previously (Whalley et al., 1993). In experiments measuring Ip at a fixed membrane potential of 240 mV, pipettes were filled with a solution containing (in mM) 70 potassium glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, 2 MgATP and sodium glutamate plus TMAzCl 90. The Na1 concentration in the solution was either 10 mM, which is near the physiological intracellular level, or 80 mM, a level expected to nearly saturate intracellular pump sites. The solution was titrated to pH 7.05 6 0.01 at 35°C with 1 M KOH. In experiments designed to examine the relationship between Ip and membrane voltage pipettes were filled with a solution containing (in mM) 10 sodium glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, 2 MgATP, 60 TMAzCl, 20 tetraethylammonium chloride, 70 CsCl and 50 aspartic acid. To examine the Ip-Vm relationship at a high intracellular Na1 level, the solution was similar except that sodium glutamate was 80 mM, CsCl was 65 mM, aspartic acid was 45 mM, and TMAzCl was omitted. The solution was titrated to pH 7.05 6 0.01 at 35°C with 1 M HCl. When filled with the above solutions, patch pipettes had resistances of 0.8 to 1.2 MV. Myocytes were suspended in a tissue bath mounted on an inverted microscope. They were superfused with modified Tyrode’s solution that contained (in mM) 140 NaCl, 5.6 KCl, 2.16 CaCl2, 1 MgCl2, 0.44 NaH2PO4, 10 glucose and 10 HEPES. It was titrated to pH 7.4 at 35°C with 1 M NaOH. This solution was used while the whole-cell configuration was established and cell membrane capacitance was measured. In most experiments, the superfusate was then changed to a solution that was similar except that it was nominally Ca11 free and in addition contained 2 mM BaCl2 and 0.2 mM CdCl2. Superfusates that were Ca11 free and contained CdCl2 were used to prevent Ca11 overloading of myocytes when cells were patch-clamped with pipettes containing high Na1 concentrations. In experiments examining the effect of extracellular K1 on pump activity, we varied the K1 concentration of this Ca11-free superfusate between 0 and 15 mM. TMAzCl was used to maintain constant osmolality in these experiments. Dose of ouabain. A previous study in noncardiac tissue suggested that amiodarone may competitively inhibit ouabain binding to the Na1-K1 pump (Prasada Rao et al., 1986). To ensure that the concentration of ouabain used in experimental protocols (100 mM) was sufficient to completely block the Na1-K1 pump in the presence of amiodarone, we performed a series of preliminary experiments on control myocytes and myocytes exposed to amiodarone in vitro. We found that after the pump had been inhibited by 100 mM ouabain, there was no additional shift in holding current on increasing the ouabain concentration to 500 mM, indicating that 100 mM ouabain caused complete pump inhibition. Unless otherwise indicated, Ip was identified by the shift in holding current induced by exposure of myocytes to 100 mM ouabain (Sigma Chemical, St. Louis, MO). Membrane currents were recorded using the continuous singleelectrode voltage-clamp mode of an Axoclamp-2A amplifier and Axotape or pCLAMP software (Axon Instruments, Foster City, CA). Voltage-clamp protocols were generated with pCLAMP. Details of the experimental protocols used to measure membrane capacitance and Ip and details of the electronic recording system have been described previously (Whalley et al., 1993). Ip is reported normalized for membrane capacitance unless otherwise indicated. Statistics. Results are expressed as mean 6 S.E.M. Statistical comparisons are made using Student’s t test for paired or unpaired observations. Dunnett’s test was used when the same control group was used for more than one comparison. P , .05 is regarded as significant in all comparisons. Nonlinear regression was used to fit the Hill equation to data. Results Effects of chronic amiodarone on ECG and thyroid function. Amiodarone is known to prolong the QT interval on the surface ECG and affects thyroid hormone levels by inhibiting peripheral conversion of T4 to T3. The effects of chronic treatment on thyroid function, heart rate, and QTc are summarized in table 1. There was no significant difference in any of these parameters between the control and treated groups of rabbits at base line. Amiodarone treatment Amiodarone Inhibits the Na1-K1 Pump 1998 77 TABLE 1 Effects of chronic amiodarone treatment on thyroid function, heart rate and QTc Placebo Amiodarone Pb T3 (nmol/liter) T4 (nmol/liter) Heart rate QTc (sec) a b a Base line 4 weeks P 1.9 6 0.2 61.4 6 4.4 226 6 10 0.28 6 0.003 2.3 6 0.2 66.8 6 5.7 227 6 15 0.27 6 0.003 NS NS NS .037 Base line 4 weeks Pa 2.2 6 .1 69.1 6 2.1 218 6 7 0.27 6 .003 2.2 6 .2 138.8 6 10.2 209 6 7 0.31 6 0.006 NS ,.001 NS ,.001 NS ,.001 NS ,.01 Level of significance for differences in variables at 4 weeks compared with base line in each group of rabbits. Level of significance for differences between control rabbits and amiodarone-treated rabbits after 4-week treatment. Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017 resulted in a significant increase in serum T4 and a significant prolongation of QTc. The treated rabbits had a serum amiodarone level of 1.14 6 0.15 mmol/liter, which is similar to the concentration found during chronic treatment in humans (Debbas et al, 1984; Ikeda et al., 1984; Weinberg et al., 1993). The treatment protocol was well tolerated. One rabbit lost weight during the treatment period and was found to have a toxic serum amiodarone level of 3.9 mmol/liter. Its surface ECG showed slow (80 bpm) broad QRS complexes. Results from this rabbit were not included in the analysis. Effect of chronic amiodarone treatment on Ip. Myocytes from 5 control rabbits and 5 rabbits treated with amiodarone were voltage-clamped at 240 mV, and Ip was measured using pipettes with Na1 concentrations ([Na]pip) of 10 or 80 mM. Results from these experiments are summarized in figure 1A. When [Na]pip was 10 mM, mean Ip of 14 myocytes from rabbits treated with amiodarone was 0.24 6 0.03 pA/pF, whereas mean Ip of 10 myocytes from rabbits given placebo was 0.36 6 0.03 pA/pF. This 33% difference was statistically significant. Mean Ip in myocytes from the rabbits given placebo was similar to mean Ip in myocytes from untreated rabbits reported previously (Gray et al., 1997). When [Na]pip was 80 mM, amiodarone treatment resulted in a significant, 25% reduction in mean Ip (1.46 6 0.07 pA/pF, n 5 7, vs. 1.94 6 0.05 pA/pF, n 5 6). We conclude that chronic amiodarone treatment causes a reduction in overall Na1-K1 pump capacity. Effect of chronic amiodarone on the affinity of the Na1-K1 pump for extracellular K1. Because it has been suggested that the effect of amiodarone on Na1-K1/ATPase is K1 dependent (Almotrefi and Dzimiri, 1991), we examined the effect of chronic amiodarone treatment on the apparent affinity of the pump for extracellular K1 concentrations ([K]o). We measured changes in Ip in response to changes in [K]o. To facilitate the detection of small pump currents at low [K]o we used [Na]pip of 80 mM to induce near-maximal activation of the pump at intracellular sites. After establishing the whole-cell configuration, we inactivated the pump by switching to a K1-free superfusate. This superfusate was nominally Ca11 free and contained 2 mM CdCl2 to eliminate Ca11 channel currents and Na1-Ca11 exchange current. Myocytes were voltage clamped at 240 mV, and Ip was identified as the membrane current resulting from reactivation of the pump on exposure to different [K]os. Each myocyte was exposed to a random sequence of at least three of the following [K]o concentrations (in mM): 0.5, 1, 2, 3, 5.6 and 15. The resulting currents were normalized relative to the current at 5.6 mM [K]o (Ip%). This concentration was therefore used in every series of exposures. Each exposure to a K1 concentration was bracketed by reexposure to the K1free superfusate until Ip had returned to its base-line level. Fig. 1. A, Effect of chronic amiodarone (amio) treatment on Na1-K1 pump current (Ip), measured when the Na1 concentration in the patch pipette ([Na]pip) was 10 mM (open bars) and 80 mM (solid bars). Numbers in parentheses indicate the number of myocytes studied in each group. Amiodarone treatment significantly reduced Ip when [Na]pip was 10 mM and when [Na]pip was 80 mM. B, Effect of extracellular K1 ([K]o) on Ip in myocytes from rabbits chronically treated with amiodarone (F, dashed line) and from control rabbits (‚, solid line). Ip has been normalized to the current measured when [K]o is 5.6 to eliminate variability of data arising from measurement of cell membrane capacitance. We previously published an illustration of the experimental protocol and representative traces of membrane currents (Gray et al., 1997). To examine whether pump run-down occurred, we concluded the protocol by reexposing a number of myocytes to the first [K]o concentration used. There was no 78 Gray et al. Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017 evidence of pump run-down in the time taken to conclude the experimental protocol ('18 –20 min). We previously determined that K1-induced shifts in holding currents attributed to the activation of the Na1-K1 pump were not contaminated by other K1-sensitive currents (Gray et al., 1997). Experiments were performed on 19 myocytes from 5 amiodarone-treated rabbits and 17 myocytes from 4 control rabbits. The [K]o/Ip% relationships are summarized in figure 1B. When the Hill equation was fitted to the data, we obtained a K1 concentration for half-maximal pump activation (K0.5) of 2.3 mM for myocytes from amiodarone-treated rabbits and 2.7 mM for control rabbits. The Hill coefficients were 1.58 and 1.59, respectively. To allow statistical comparisons, we repeatedly fitted the Hill equation to randomly selected series of values for Ip at all six [K]o concentrations tested. This allowed us to derive mean K0.5 values and mean Hill coefficients for 10 derived [K]o activation curves for myocytes from the amiodarone-treated rabbits and 11 derived [K]o activation curves for myocytes from control rabbits. There was no significant difference between the mean values. We conclude that chronic amiodarone treatment does not affect the apparent K1 affinity of the Na1-K1 pump. Effect of chronic amiodarone on the Ip-Vm relationship. Because amiodarone is an amphiphile and amphiphiles can alter the voltage dependence of the Na1-K1 pump (Läuger, 1991), we examined whether chronic amiodarone treatment affects the Ip-Vm relationship. After establishing the whole-cell configuration, we superfused myocytes with Ca11-free modified Tyrode’s solution, and we voltageclamped the myocytes at 240 mV. We then applied 320-msec voltage steps in 20-mV increments to test potentials ranging from 2100 to 160 mV. Averaged membrane currents at each test potential after superfusion of ouabain were subtracted from the respective averaged membrane currents before ouabain exposure to derive Ip at each test potential. Experiments were performed at [Na]pip of both 10 and 80 mM. Figure 2A illustrates the voltage step protocol and an example of the resulting membrane currents for a myocyte studied using [Na]pip of 10 mM. Details of the experimental protocol and data analysis have been published previously (Gray et al., 1997). The mean Ip-Vm relationships for 13 myocytes from 5 amiodarone-treated rabbits and 17 myocytes from 8 control rabbits at [Na]pip of 10 mM are illustrated in figure 2B. In this and all other Ip-Vm relationships, we normalized Ip to the current measured at 0 mV to facilitate comparisons between experiments. The Ip-Vm relationships in myocytes from both groups of rabbits are near-linear and have a positive slope over the range of voltages examined. There were no major differences in the relationships for myocytes from the two groups. The mean Ip-Vm relationships for 7 myocytes from 3 amiodarone-treated rabbits and 8 myocytes from 3 control rabbits at [Na]pip of 80 mM are shown in figure 2C. The relationships in myocytes from both groups of rabbits are virtually superimposable, exhibiting a positive slope at negative potentials and a negative slope at positive potentials. We conclude that chronic amiodarone treatment does not affect the Ip-Vm relationship, at either physiological levels of intracellular Na1 or levels of intracellular Na1 expected to cause near-saturation of Na1 binding sites. Effect of chronic amiodarone on intracellular Na1. If chronic amiodarone treatment inhibits Na1-K1 pump activ- Vol. 284 Fig. 2. A, Voltage-clamp protocol and holding currents before and after exposure of a myocyte to 100 mM ouabain. The myocyte was from a rabbit treated with amiodarone. The current traces shown are the averaged currents from three applications of the voltage-clamp protocol. [Na]pip was 10 mM. B, Normalized Ip-Vm relationships for 13 myocytes isolated from rabbits treated with amiodarone (F) and 8 myocytes from control rabbits (‚). [Na]pip was 10 mM. C, Ip-Vm relationships for 7 myocytes from amiodarone-treated rabbits and 8 myocytes from control rabbits when [Na]pip was 80 mM. Amiodarone had no significant effect on the Ip-Vm relationships. ity, it may be expected to alter steady-state intracellular Na1 activity (aiNa). However, the direction and magnitude of any such change in aiNa cannot be assumed because amiodarone may also influence Na1 influx. We therefore examined the 1998 79 Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017 effect of chronic amiodarone treatment on aiNa. We determined aiNa in papillary muscles from 11 control rabbits and 11 rabbits treated with amiodarone for 4 weeks. We used ion-sensitive microelectrodes to measure aiNa as described previously (Hool et al., 1995). One or two determinations of aiNa were made with separate microelectrode impalements in tissue from each rabbit. Where two measurements were made, the mean value was used for statistical comparisons. Determinations of aiNa were made only after microelectrode recordings had been stable for $20 minutes. The aiNa in control rabbits was 8.1 6 0.4 mM. In amiodarone-treated rabbits, aiNa was 9.8 6 0.8 mM. This difference was not statistically significant (P 5 .07). Amiodarone is expected to reduce Na1 influx via Na1 channels (Mason et al., 1984); because amiodarone reduces cardiac metabolic rate (Charlier et al., 1968), treatment may have reduced Na1 influx via the Na1-H1 exchanger. One may speculate that such decreases in Na1 influx partially offset the rise in aiNa expected from pump inhibition. Acute effect of amiodarone on Ip. We next examined whether acute exposure of myocytes to amiodarone affected the Na1-K1 pump. For these experiments, myocytes were isolated from untreated rabbits and exposed to amiodarone in the tissue bath. After establishing the whole-cell configuration, we superfused myocytes with Ca11-free Tyrode’s solution that contained 2 mM BaCl2 and either amiodarone in 1% ethanol or 1% ethanol alone. The cells were exposed to this superfusate for a minimum of 6 minutes before changing to a superfusate that was similar except that it contained 100 mM ouabain. The duration of exposure was adopted from previous studies on Na1 channels (Follmer et al., 1987) and Ca11 channels (Nishimura et al., 1989) in cardiac myocytes. These studies indicated that steady state effects were achieved after '6-min exposure to amiodarone in vitro. When [Na]pip was 10 mM, mean Ip of 10 myocytes exposed to (nominally) 10 mM amiodarone in 1% ethanol was 0.19 6 0.02 pA/pF, whereas mean Ip of 10 myocytes exposed to ethanol alone was 0.31 6 0.03 pA/pF. This 39% reduction was statistically significant. Mean Ip of myocytes exposed to 1% ethanol was similar to mean Ip of myocytes from untreated rabbits not exposed to ethanol (Gray et al., 1997; Whalley et al., 1993), indicating that 1% ethanol does not affect Ip when membrane potential is held constant at 240 mV. We performed an additional four experiments to determine that the duration of exposure was adequate to achieve steady state effects. In these experiments, myocytes were exposed to amiodarone for $20 min (range, 20 to 28 min) before Ip was measured. Mean Ip (0.21 6 0.01 pA/pF) was similar to the mean Ip of myocytes exposed to amiodarone for '6 min only (0.19 6 0.02 pA/pF). This indicates that steady state was achieved using 6-min exposure. To examine whether acute exposure of myocytes to amiodarone in vitro also causes pump inhibition when intracellular Na1 is at high levels, we measured Ip using a [Na]pip of 80 mM. Mean Ip of 6 myocytes was 1.90 6 0.13 pA/pF, which is not significantly different from mean Ip in 6 control myocytes (1.94 6 0.05 pA/pF). The mean Ip values measured using a [Na]pip of either 10 and 80 mM in myocytes exposed to amiodarone and control myocytes are summarized in figure 3A. We conclude from these experiments that acute exposure to amiodarone inhibits Na1-K1 pump function when intracellular Na1 is near physiological levels, whereas it has no Amiodarone Inhibits the Na1-K1 Pump Fig. 3. A, Effect of acute exposure to amiodarone on Ip, measured using [Na]pip of 10 mM (open bars) and 80 mM (solid bars). Numbers in parentheses indicate how many myocytes were studied in each group. Exposure of myocytes to amiodarone significantly reduced Ip when [Na]pip was 10 mM. 1% ethanol alone did not affect Ip. Amiodarone had no effect on Ip when [Na]pip was 80 mM. B, Effect of [K]o on Ip in myocytes exposed to amiodarone in 1% ethanol in vitro (F, dashed lines) and in myocytes exposed to 1% ethanol alone (‚, solid line). Ip has been normalized to the current measured when [K]o is 5.6 mM. C, Normalized Ip-Vm relationships for 9 myocytes exposed to amiodarone in 1% ethanol (F) and for 8 myocytes exposed to 1% ethanol alone (‚) in vitro. 80 Gray et al. Discussion The major finding of our study is that both chronic and acute exposure to amiodarone decreased Na1-K1 pump activity in intact cardiac myocytes. After chronic treatment, pump activity was reduced at both low and high [Na]pip. This pattern suggests a decrease in overall pump capacity. Acute exposure to amiodarone in vitro decreased pump activity only when [Na]pip was near the physiological level for intracellular Na1 and had no significant effect at levels expected to saturate intracellular pump sites. This pattern is consistent with a decrease in the apparent affinity of the pump for intracellular Na1. A difference in effects between acute and chronic exposure is also well recognized for the clinical use of the drug (Ikeda et al., 1984) and for its effects on ion channel function (Varró et al., 1996). The [Na]pip-dependent difference in effects between chronic and acute exposure suggests that amiodarone affects the pump via two different mechanisms. With chronic exposure, amiodarone might affect the pump via both mechanisms: one related to a decrease in the abundance of Na1-K1 pumps and one due to a direct effect of the drug in the membrane. However, amiodarone induced a similar degree of inhibition with chronic and acute exposure when [Na]pip was 10 mM. This similarity might be explained by wash-out of amiodarone while cells from treated animals were maintained in amiodarone-free solutions for 2 to 10 hr before Ip was measured, with a loss of a direct effect of the drug bound to the membrane. Two previous studies have examined the effect of chronic amiodarone treatment on the Na1-K1 pump. Prasada Rao et al. (1986) administered amiodarone parenterally to rats for 6 weeks. They found that amiodarone had no effect on activity of brain synaptosome Na1-K1/ATPase activity. To our knowledge, a study by Hensley et al. (1994) provides the only previous data on cardiac tissue. They examined the effects of 3- to 6-week parenteral amiodarone therapy on protein expression and mRNA levels for the alpha and beta subunits of the Na1-K1 pump in rat ventricular muscle. At 3 weeks, abundance of the alpha-1, alpha-2 and beta-1 subunits was significantly decreased. This was accompanied by a 30% to 33% decrease in serum T3 and T4 levels consistent with a systemic amiodarone-induced hypothyroid state. Because hypothyroidism reduces synthesis of Na1-K1 pump subunits in the heart (Horowitz et al., 1990, Kamitani et al., 1992), the decrease in subunit abundance at 3 weeks might be due to the effect of amiodarone on thyroid status. After 6-week therapy, T3 and T4 levels and alpha-1 and beta-1 subunit abundance had returned to control values, whereas the abundance of the alpha-2 subunit remained significantly depressed. Alpha-2 subunits do not appear to be quantitatively important in the rat, and Hensley et al. (1994) emphasized that the functional significance of altered alpha-2 expression was uncertain. The present study does not identify effects of amiodarone on specific subunits; however, it does indicate that chronic treatment has a functionally significant inhibitory effect on the sarcolemmal Na1-K1 pump. Our study indicates that chronic treatment with amiodarone induces a decrease in Ip when the pump is maximally stimulated. Maximal Ip is highly correlated with the number of Na1-K1 pump units expressed in the plasmalemma of voltage-clamped oocytes (Jaunin et al., 1992). If the same applies to pump units in the sarcolemmal membrane, our Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017 effect when intracellular Na1 is at levels expected to nearly saturate intracellular Na1 binding sites. This suggests that acute exposure to amiodarone reduces the apparent affinity of the pump for intracellular Na1. Effect of acute amiodarone on the affinity of the Na1-K1 pump for extracellular K1. To examine the effect of acute amiodarone exposure on K1 affinity, we measured Ip at different levels of [K]o. We used a [Na]pip of 80 mM. After establishing the whole-cell configuration, we inactivated the Na1-K1 pump by superfusing myocytes with K1-free Tyrode’s solution. This superfusate also contained amiodarone. Myocytes were then voltage-clamped at 240 mV, and after a minimum of 6-min exposure to the amiodarone-containing superfusate, the pump was reactivated by exposing myocytes to a superfusate that was similar except that it contained different [K]o values. In contrast to experiments examining the effects of chronic amiodarone treatment, each myocyte was exposed in a random sequence to all of the following [K]o values (in mM):1, 2, 3, 5.6 and 15. Results were normalized relative to 5.6 mM [K]o. Each exposure to a [K]o concentration was bracketed by return to the K1-free superfusate until Ip had returned to its base-line level. Control myocytes were maintained in the whole-cell configuration for the same duration as amiodarone-exposed myocytes before Ip was measured. Nine myocytes were acutely exposed to amiodarone in vitro. When the Hill equation was fitted to the data, we obtained a K0.5 value of 2.50 mM and a Hill coefficient of 1.84. To allow statistical comparisons, we fitted data from each experiment to the Hill equation to derive the respective mean values for K0.5 and Hill coefficient. There was no significant difference between the mean values of either for myocytes exposed to amiodarone or control myocytes. The [K]o/Ip% relationship for myocytes exposed to amiodarone has been plotted in figure 3B along with the relationship for control myocytes. We conclude from these experiments that acute exposure of myocytes to amiodarone has no effect on the apparent [K]o affinity of the Na1-K1 pump. Effect of acute amiodarone on the Ip-Vm relationship. We next examined whether acute exposure of myocytes to amiodarone affects the Ip-Vm relationship. After establishing the whole-cell configuration, the superfusate was changed to Ca11-free Tyrode’s solution that contained either amiodarone in 1% ethanol or 1% ethanol alone. The myocytes were voltage-clamped at 240 mV; after a minimum exposure time of 6 min to this superfusate, we applied the voltage step protocol illustrated in figure 2A before and after exposure to 100 mM ouabain as outlined previously. Because in vitro exposure had no effect on Ip when [Na]pip was 80 mM, these experiments were only performed at [Na]pip of 10 mM. The mean Ip-Vm relationships for 9 myocytes exposed to amiodarone and 8 myocytes exposed to ethanol have been plotted in figure 3C. The Ip-Vm relationships in myocytes exposed to amiodarone and to ethanol demonstrate a positive slope at negative potentials and a negative slope at positive potentials. The negative slope at positive potentials is not seen in control myocytes not exposed to amiodarone or ethanol (Gray et al., 1997). It would thus appear that the negative slope at positive potentials is due to an effect of the vehicle, 1% ethanol, rather than an effect of amiodarone. We conclude that acute exposure to amiodarone has no effect on the Ip-Vm relationship. Vol. 284 Amiodarone Inhibits the Na1-K1 Pump 1998 may speculate that the effect of amiodarone could be due to modification of the physicochemical properties of the lipid membrane. Amiodarone is known to insert into the hydrocarbon core of the lipid bilayer and decrease membrane lipid mobility and membrane fluidity (Chatelain et al., 1986). Changes in membrane fluidity, in turn, may affect pump activity by impairing lateral movement of the pump molecule and hence conformational changes during the pump cycle (see Cornelius, 1991, for review). An alternative explanation may be provided by the cationic amphiphilic nature of amiodarone. Cationic amphiphiles have been shown to decrease the apparent Na1 affinity of Na1-K1/ATPase reconstituted into liposomes (Cornelius, 1995). Chronic treatment with amiodarone reduced Ip by 33% when [Na]pip was 10 mM. This inhibitory effect is similar to that estimated for clinical use of digoxin (Rasmussen et al., 1990; Schmidt et al., 1991). This finding may have implications for our understanding of the effect of the drug when used clinically. It prolongs action potential duration and myocardial refractoriness, effects that are believed to be largely mediated via block of voltage-gated K1 channels (Balser et al., 1991). The Na1-K1 pump generates a hyperpolarizing membrane current that contributes to repolarization. This effect becomes more significant as intracellular Na1 increases, for example, during tachyarrhythmias (Gadsby, 1982). The inhibitory effect of amiodarone on the pump is therefore expected to contribute, at least in part, to the class III effects of the drug. Acknowledgments Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017 results suggest that amiodarone induces a decrease in the number of functional Na1-K1 pump units. The amiodaroneinduced decrease in pump function occurred in the absence of systemic hypothyroidism. However, this does not rule out the possibility that amiodarone exerted its effect on the pump via interference with thyroid hormone action at the intracellular level because amiodarone inhibits the binding of T3 to its nuclear receptors (Drvota et al., 1994; Latham et al., 1987). The pattern of pump inhibition is similar to that reported by Doohan et al. (1995) in rabbits with experimentally induced hypothyroidism. It is also of interest to note that some of the electrophysiological effects of amiodarone, including prolongation of action potential duration, QT interval and sinus cycle length, are at least partly dependent on thyroid hormone activity and are greatly diminished or abolished by concurrent hypothyroidism (Polikar et al., 1986; Talajic et al., 1989). Several previous studies have reported inhibition of purified cardiac Na1-K1/ATPase, the enzymatic equivalent of the Na1-K1 pump, during acute exposure to amiodarone in vitro (Broekhuysen et al., 1972; Dzimiri and Almotrefi., 1991). Although these studies suggest that acute amiodarone exposure may inhibit the pump, their significance is unclear for several reasons. Isolation of Na1-K1/ATPase from the sarcolemmal membrane necessarily involves removing the pump molecule from its native lipid environment. Because the lipid environment is an important regulator of pump activity (Cornelius, 1991) and may itself be influenced by amiodarone (Chatelain et al., 1985, 1986), the response of isolated Na1-K1/ATPase to amiodarone may not be representative of the behavior of the pump in the intact myocyte. In the studies by Broekhuysen et al. (1972) and Dzimiri and Almotrefi (1991), Na1-K1/ATPase was examined using saturating concentrations of Na1 and K1 to produce maximal pump activity. They therefore provide no information about the effect of amiodarone on the activity of the pump at physiological ligand concentrations. Finally, the concentrations of amiodarone necessary to induce significant ATPase inhibition were substantially higher than those encountered clinically. In the only published study on intact cardiac muscle, Aomine (1989) indirectly determined the acute effects of amiodarone on pump activity in guinea pig papillary muscles using conventional microelectrode techniques. He found that exposure to 44 mM amiodarone decreased the postoverdrive hyperpolarization attributed to pump activity. Somewhat paradoxically, no effect was observed at the higher concentration of 440 mM. Our in vitro study differs from previous studies in several aspects. We examined the effect of 0.3 to 1.2 mM amiodarone, a concentration range similar to that encountered clinically, and we examined the pump in its intact membrane while varying extracellular and intracellular ligand concentrations. In contrast to the studies on isolated ATPase, we found no effect on Ip measured under conditions expected to maximally stimulate the pump. However, pump inhibition could be demonstrated when we used a [Na]pip near physiological levels for intracellular Na1. This is consistent with a decrease in the apparent affinity of the pump for intracellular Na1 without an influence on overall pump capacity. Although the mechanism for modulation of the Na1 affinity of the pump cannot be determined from our study, one 81 Amiodarone was a gift from Sanofi Winthrop Australia. The assistance of Dr. Annette Gross in performing serum amiodarone measurements is gratefully acknowledged. 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