1Department of Physiology, Loyola University Chicago, Maywood, Illinois 60153; and 2Arbeitsgruppe Muskelphysiologie, Fakultät für Biologie, Ruhr-Universität, D-44780 Bochum, Germany
Submitted 4 December 2003 ; accepted in final form 5 April 2004
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ABSTRACT |
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atrial myocytes; intracellular calcium
In the heart, palytoxin leads to myocardial ischemia, ventricular fibrillation, and cardiac failure. These effects have been attributed in part to intense constriction of the coronary vasculature (25, 50). In addition, palytoxin exerts direct effects on the heart, including depolarization of resting membrane potential, changes of action potential (AP) configuration, generation of afterpotentials and arrhythmias, reduction of phasic tension, and contracture (23, 24, 38, 41, 42, 51). Studies of the underlying mechanisms, however, are sparse and controversial (11, 12, 41, 42). Given the pronounced effects on ion homeostasis, membrane potential, and tension, palytoxin is expected to markedly alter cardiac excitation-contraction (E-C) coupling. However, palytoxin effects on Ca2+-induced Ca2+ release (CICR) and the resulting intracellular Ca2+ concentration ([Ca2+]i) transient, which underlies contraction, have not been investigated so far. Furthermore, most previous studies have focused on ventricular preparations, whereas palytoxin effects on the atrium have gained much less attention. Atrial physiology differs in several respects from ventricular physiology. For example, AP duration is much shorter in the atrium than in the ventricle. Atrial myocytes are smaller than ventricular cells. They lack a regular T-tubular system and contain peripheral junctional and central nonjunctional sarcoplasmic reticulum (SR) (30). Thus, unlike ventricular cells, atrial myocytes exhibit characteristic spatiotemporal inhomogeneities in AP-induced SR Ca2+ release (for review, see Ref. 7). Ca2+ release starts in the peripheral junctional SR, triggered by Ca2+ influx via L-type Ca2+ channels during the AP, and then spreads actively in a wavelike fashion to the central nonjunctional SR via CICR. In addition, Na+/Ca2+ exchange plays a prominent role in atrial Ca2+ homeostasis, particularly during diastole, resulting in a decrease of SR Ca2+ content after a rest period, which is in clear contrast to the postrest potentiation observed in the ventricle (32). Finally, the atrium also performs endocrine functions by secreting atrial natriuretic peptide (ANP) in response to stretch. ANP secretion has a Ca2+-dependent component and is likely to be affected by changes in Ca2+ homeostasis.
Because of the paucity of mechanistic insight into palytoxin action on cardiac tissue in general and the distinct differences between the atrium and the ventricle in particular with respect to E-C and excitation-secretion coupling, we set out to characterize palytoxin effects on the various steps of E-C coupling in atrial myocytes. We investigated the effect of palytoxin on membrane potential and AP configuration, voltage-activated Ca2+ current (ICa), SR function, and the AP-induced [Ca2+]i transient. The results revealed that palytoxin interferes with the sarcolemmal (SL) Na+-K+ pump and the SR Ca2+ pump (SERCA), two major P-type ion pumps in cardiac myocytes, and thereby disrupts atrial E-C coupling. A preliminary account of this work was published previously in abstract form (29).
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METHODS |
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Imaging of [Ca2+]i transients.
Cells were loaded with the fluorescent Ca2+ indicator fluo 3-acetoxymethyl ester (fluo 3-AM; Molecular Probes, Eugene, OR) in Tyrode solution (in mM: 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 D-glucose, pH 7.35 adjusted with NaOH) for 2030 min at room temperature. A coverslip with fluo 3-AM-loaded myocytes was placed on the stage of an inverted microscope (Axiovert 100; Carl Zeiss, Oberkochen, Germany). Cells were superfused continuously with Tyrode solution for 15 min to allow sufficient time for deesterification of the dye. [Ca2+]i transients were evoked by electrical field stimulation of the myocytes with 2- to 4-ms suprathreshold rectangular voltage pulses delivered through a pair of extracellular platinum electrodes at frequencies between 0.5 and 1.0 Hz. They were recorded with a confocal laser scanning system (LSM 410; Carl Zeiss) equipped with a x40 oil-immersion objective lens (Plan-Neofluar, NA 1.3; Carl Zeiss) as described previously (19). Fluo 3 was excited with the 488-nm line of an argon ion laser. The emitted fluorescence was collected at wavelengths >515 nm. [Ca2+]i transients were recorded in linescan mode. The line was positioned at a central axial depth perpendicular to the longitudinal axis of the cells (transverse linescan) and scanned repetitively at intervals between 4 and 10 ms. Background-corrected fluorescence images were converted to [Ca2+]i images according to the equation
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For recording of ICa, cells were superfused with a modified Tyrode solution composed of (in mM) 135 NaCl, 4 CsCl, 5 CaCl2, 1 MgCl2, 0.1 ouabain, 10 HEPES, and 10 D-glucose, pH 7.3 (adjusted with NaOH). The pipette solution contained (in mM) 130 cesium glutamate, 20 CsCl, 0.33 MgCl2, 4 Na2ATP, 5 EGTA, and 10 HEPES, pH 7.3 (adjusted with CsOH). Cs+ served to suppress K+ currents. Ouabain was used to prevent palytoxin effects on the Na+-K+ pump. Myocytes were voltage clamped at a holding potential of 40 mV to inactivate Na+ channels. ICa was elicited by 200-ms rectangular voltage steps to +10 mV every 5 s. Current amplitude was measured as the difference between the peak inward current and the steady-state current at the end of the depolarization step. Current-voltage (I-V) relationships were obtained by step depolarizations to potentials between 30 and +50 mV in 10-mV increments. To allow comparison between cells, ICa was normalized to cell membrane capacitance (Cm), estimated by integration of the capacitative current transient during hyperpolarizing voltage steps. The average Cm of the atrial myocytes used for ICa recordings was 48 ± 2 pF (n = 14).
AP were recorded in the current-clamp mode. Cells were superfused continuously with Tyrode solution containing (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 10 HEPES, and 10 D-glucose, pH 7.4 (adjusted with NaOH). The pipette solution was composed of (in mM) 100 potassium glutamate, 40 KCl, 1 MgCl2, 4 Na2ATP, 10 HEPES, and 2 EGTA, pH 7.2 (adjusted with KOH).
Planar lipid bilayer recordings of ryanodine receptor channel activity. Rat ventricular SR vesicles were obtained as described previously (54). Planar lipid bilayers were formed from a lipid mixture containing phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine (ratio 5:4:1) dissolved in n-decane at a final lipid concentration of 45 mg/ml. SR vesicles were added to the cis-chamber corresponding to the cytosolic side of the ryanodine receptor (RyR) channel. The trans-chamber (luminal side of the RyR) was connected to the virtual ground of the amplifier. During fusion, the cis- and trans-chambers contained the following solutions (in mM): 400 CsCH3SO3 (cis), 40 (trans); 0.1 CaCl2; and 20 HEPES, pH 7.3 (CsOH). After channel incorporation, the concentration of CsCH3SO3 in the trans-chamber was increased to 400 mM and free [Ca2+] in the cis-chamber was adjusted to 3 µM by addition of EGTA. Free [Ca2+] in the experimental solutions was verified with a Ca2+-sensitive minielectrode (4). Single-channel currents were recorded with the use of an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). All recordings were made at a holding potential of 15 mV. Currents were filtered at 1 kHz and digitized at 5 kHz.
Ca2+ uptake by SR microsomes. SR vesicles (2550 µg) prepared from rat ventricle were added to a cuvette containing 1 ml of buffered phosphate medium comprising (in mM) 100 KH2PO4, 3 MgCl2, 2 ATP, 0.01 ruthenium red, and 0.2 antipyrylazo III (APIII; Sigma), pH 7.0. Changes in Ca2+ were measured as changes in absorbance between 710 and 790 nm of the Ca2+-sensitive dye APIII by means of an ultraviolet-visible diode array spectrophotometer (Cory 50; Varian). Ca2+ uptake was initiated by the addition of Ca2+ aliquots (CaCl2, 10 µM) to the cuvette. The rapid rise in Ca2+-dependent APIII absorbance was followed by a slower absorbance decrease due to ATP-dependent Ca2+ uptake by the SR vesicles. The rate of Ca2+ loading of the vesicles, i.e., the net Ca2+ uptake, equals the SR pumping rate minus the Ca2+ leak rate. The latter reflects the activity of the RyRs, which were blocked by ruthenium red. Thus, under our experimental conditions, net Ca2+ uptake by the vesicles was equal to the SR Ca2+ pumping rate.
Drugs. Palytoxin was isolated (6) and generously provided by Dr. L. Béress (Universität Kiel, Germany) or was purchased from Sigma-RBI. Palytoxin from both sources was prepared as 105 M aqueous stock solution and stored in aliquots at 20°C. Solutions containing 110 nM palytoxin were used throughout this study. The respective solutions were prepared freshly immediately before the experiments by diluting the desired amount of stock solution in the experimental solution. The cardiac glycoside ouabain (Alexis Biochemicals, San Diego, CA) was used as a specific inhibitor of the Na+-K+-ATPase (43). Ouabain was directly dissolved in the extracellular solution at concentrations (10500 µM) indicated elsewhere in the text.
Statistics. Data from n cells are presented as means ± SE. In bilayer single-channel recordings and SR microsome Ca2+ uptake experiments, n refers to the number of individual measurements obtained from different SR preparations. Statistical differences between data sets were evaluated by performing Student's t-test and were considered significant at P < 0.05.
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RESULTS |
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Palytoxin effects on electrically evoked atrial [Ca2+]i transients.
Figure 2 shows examples of palytoxin-induced changes of electrically evoked [Ca2+]i transients in single atrial myocytes. When an atrial myocyte was challenged with palytoxin (Fig. 2A), there was a quickly developing, monotonic reduction in the amplitude of the [Ca2+]i transient. Peak systolic [Ca2+]i decreased from 650 nM under control conditions to 200 nM in the presence of the coral toxin (Fig. 2A, left). The reduction in the amplitude of the [Ca2+]i transient was not accompanied by a reduction of SR Ca2+ content. Application of 10 mM caffeine (to assess SR Ca2+ content) induced a large increase in [Ca2+]i, indicating that, although electrically evoked [Ca2+]i transients were small, a large amount of releasable Ca2+ was stored in the SR (Fig. 2A, middle). Moreover, propagating waves of [Ca2+]i were observed shortly after the caffeine application (Fig. 2A, right). Thus palytoxin reduced the amplitude of the electrically evoked [Ca2+]i transient and, at the same time, induced [Ca2+]i waves, most likely by causing SR Ca2+ overload.
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Figure 3B illustrates the subcellular Ca2+ release patterns of another cell treated with palytoxin. The atrial myocyte and the position of the scan line are illustrated in Fig. 3Bb, right. Figure 3Ba shows the transverse linescan image, and Fig. 3Bb demonstrates the corresponding [Ca2+]i signals from the right and left sides of the cell. Dashed vertical lines indicate the time of electrical stimulations [stimuli 15 (S1S5)]. Except for S3 and S5, SR Ca2+ release was spatially inhomogeneous and not directly correlated with the stimulus. During S1, for example, SR Ca2+ release was greatly reduced on the left side and completely absent on the right side of the myocyte. Shortly after the stimulus, however, a large [Ca2+]i signal was generated, apparently by spontaneous release of [Ca2+]i from the central nj-SR (asterisk). Before the next stimulus (S2), spontaneous propagating Ca2+ release occurred, first on the left side and then on the right side of the cell. The latter event is marked by a black arrow. It took place immediately (120 ms) before the electrical stimulation. Thus SR Ca2+ release is still refractory at this time, and no further release is triggered by S2. Instead, the spontaneous Ca2+ release propagated in a wavelike fashion from the right to the left side of the cell. Similarly, spontaneous Ca2+ release from the subsarcolemmal j-SR on the left side of the myocyte (arrow) preceded S4 (by
120 ms), rendering AP-induced release at this site refractory. However, S4 was able to trigger SR Ca2+ release on the right side of the myocyte, which then traveled back as a [Ca2+]i wave across the cell center to the left side. These results demonstrate that the palytoxin-induced alterations of E-C coupling can exhibit significant subcellular variations, including subcellular Ca2+ alternans (Fig. 3A), and illustrate how uncoordinated, spatially restricted Ca2+ release can generate local failure of AP-evoked Ca2+ release and arrhythmogenic [Ca2+]i waves.
Atrial [Ca2+]i transients in the presence of ouabain and palytoxin.
Because ouabain was able to completely block the palytoxin-induced inward current (Fig. 1), we tested whether it could also attenuate or abolish the palytoxin-mediated changes of E-C coupling. Exposure of an atrial myocyte to 10 µM ouabain resulted in a large increase of systolic [Ca2+]i from 400 to 730 nM (Fig. 4A), consistent with the well-known positive inotropic effect of the cardiac glycoside. In five myocytes, ouabain (1025 µM) increased systolic [Ca2+]i from 643 ± 178 to 807 ± 177 nM (n = 5; P < 0.05). When palytoxin was applied in the continued presence of ouabain (Fig. 4A), the amplitude of the [Ca2+]i transient started to decline immediately, and, after 30 s, Ca2+ alternans developed. The decrease in [Ca2+]i transient amplitude occurred despite high SR Ca2+ load, as assessed by caffeine. In additional experiments, palytoxin was also able to cause failures of Ca2+ release and [Ca2+]i waves in the presence of ouabain (data not shown). In all seven atrial myocytes pretreated with 1025 µM ouabain, palytoxin (2 nM) elicited a reduction of the [Ca2+]i transient amplitude [from 812 ± 128 to 431 ± 91 nM (n = 7; P < 0.01), or by 45 ± 9%], Ca2+ alternans in six cells (86%), failure of Ca2+ release in one cell (14%), and [Ca2+]i waves in two cells (29%). These changes qualitatively and quantitatively resemble the changes in E-C coupling induced by the coral toxin in the absence of ouabain and indicate that micromolar concentrations of ouabain were unable to prevent palytoxin effects on atrial SR Ca2+ release. We therefore set out to study palytoxin effects in the presence of higher concentrations of ouabain. Figure 4B shows electrically evoked [Ca2+]i transients of an atrial myocyte challenged with 500 µM ouabain. In sharp contrast to the effects of lower concentrations of the cardiac glycoside (Fig. 4A), this high concentration of ouabain elicited a decrease in [Ca2+]i transient amplitude. Furthermore, Ca2+ alternans developed within less than 15 s. Similar results were obtained in five more cells. At 500 µM, ouabain never led to an increase of the [Ca2+]i transient amplitude (0 of 6 cells) but instead induced Ca2+ alternans in four (67%) and [Ca2+]i waves in one of these cells (17%). Thus high concentrations of ouabain induced changes in SR Ca2+ release comparable to those evoked by palytoxin. This finding rendered irrelevant any further studies of palytoxin actions in the presence of high concentrations of ouabain. It suggested that serious impairment of Na+-K+ pump function by either palytoxin or high concentrations of ouabain may result in similar defects in E-C coupling.
An alternative explanation for the palytoxin-mediated disruption of E-C coupling might be a direct action of the coral toxin on the CICR mechanism. Therefore, we examined whether palytoxin modulated the activity of the RyR Ca2+ release channel, Ca2+ uptake by the SR, and/or voltage-dependent Ca2+ influx (i.e., ICa).
Palytoxin does not affect RyR channel activity. Activity of single RyR channels reconstituted into planar lipid bilayers was recorded before (control) and after the addition of 3 and 10 nM palytoxin (Fig. 5). Examples of original current traces are presented in Fig. 5A. Statistical analysis of single-channel currents from a total of six RyRs (Fig. 5B) revealed that open probability (Po), mean open time (top), and current amplitude (I) were unaffected by the toxin. Mean values for the three parameters under control conditions and after the addition of 3 and 10 nM palytoxin were Po, 0.076 ± 0.029, 0.078 ± 0.027, and 0.070 ± 0.020, respectively; top, 2.52 ± 0.69, 2.38 ± 0.74, and 2.27 ± 0.65 ms, respectively; and I, 7.78 ± 0.37, 7.84 ± 0.41, and 7.78 ± 0.33 pA, respectively (n = 6 channels for all data; all differences not statistically significant). Thus palytoxin did not affect cardiac RyR Ca2+ release channels.
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Palytoxin depolarizes the resting membrane potential and induces delayed afterdepolarizations. In a final step, palytoxin effects on atrial AP were studied. AP were elicited by extracellular field stimulation (1 Hz), and membrane potential was measured by means of whole cell current clamp. Figure 8A displays a recording of the membrane potential of an atrial myocyte before and during exposure to palytoxin (2 nM). The baseline indicates resting membrane potential (RP), and upward deflections are due to evoked AP. The parts of the recording marked ae (horizontal lines under the voltage trace) are shown on an expanded scale in Fig. 8B. Under control conditions, RP was fairly stable at about 68 mV (dashed line in Fig. 8A). Palytoxin caused an initial depolarization of RP to 50 mV (next to trace b) followed by a short repolarization to 75 mV (trace c). Finally, a large and sustained depolarization to values close to 0 mV occurred. During the transient repolarization (Fig. 8Bc), RP was stable, similar to control conditions (Fig. 8Ba). By contrast, during both the initial (Fig. 8Bb) and the final depolarizations (Fig. 8Bd), significant fluctuations of RP were observed, including first occasional (Fig. 8Bb) and later regular (Fig. 8Bd) delayed afterdepolarizations (DADs, arrows in b and d), presumably because of spontaneous SR Ca2+ release and [Ca2+]i waves. AP marked iiv are shown superimposed in Fig. 8C. Comparison of AP iiv revealed that, except for a broader shoulder in iv, AP configuration remained rather constant. At the end of the recording, when RP was close to 0 mV, AP no longer could be evoked and only a stimulus artifact remained [Fig. 8, v in Be and C]. In a total of three cells, RP was initially depolarized from 66.3 ± 2.7 to 40.3 ± 7.7 mV (P < 0.05); after transient repolarization to 64.7 ± 5.8 mV (P < 0.05), sustained depolarization to 17.0 ± 6.6 mV occurred (P = 0.05). In all cells, palytoxin induced frequent DADs with amplitudes up to 12 mV. Changes in AP configuration were minor, however, and no trend toward longer or shorter AP was evident.
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DISCUSSION |
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In line with the observations in ventricular myocytes (2, 20, 27, 35), we found that palytoxin generated a very similar current in atrial myocytes. In particular, the palytoxin-induced current displayed a linear I-V relationship that was reversed at 5 mV, was comparable in magnitude, and could be antagonized by cardiac glycosides. From these results, we conclude that palytoxin induced an inward current in cat atrial myocytes through conversion of Na+-K+ pumps into nonselective cation channels. Assuming a single-channel conductance of
10 pS (2, 17), a specific membrane capacitance of 1 µF/cm2, and a Na+-K+ pump density of 1,0002,500 µm2 (13), we estimate that far less than 1 of every 1,000 Na+-K+ pumps was converted into a channel by palytoxin.
One of the novel findings of this study is that in addition to its action on the Na+-K+ pump, palytoxin also inhibited the function of the SERCA in isolated SR vesicles. The fact that palytoxin interferes with both the Na+-K+ pump and the SERCA might be explained by structural similarities and the high degree of homology between the two ion pumps (48). In line with this notion, Scheiner-Bobis et al. (45) suggested in a recent report that palytoxin can convert the colonic H+-K+ pump, another closely related P-type ion pump, into a cation channel. While our experiments on isolated SR vesicles clearly establish an inhibitory action of palytoxin on the SERCA, they do not provide any insights into the mechanism of inhibition. Furthermore, it is not known whether the toxin is able to enter the cells and reach the SERCA. Thus a possible contribution of SERCA inhibition to the palytoxin effects in intact cells remains elusive. Nonetheless, the results regarding SERCA lend further support to the notion that, from a biochemical point of view, palytoxin acts on P-type ion pump structures. Likewise, it is not known whether the SL Ca2+ pump, another related P-type ion pump involved in Ca2+ regulation, could also be a target for palytoxin. While we cannot rule out this possibility, putative inhibition of the SL Ca2+ pump is unlikely to contribute significantly to palytoxin effects on atrial Ca2+ signaling, given that Na+/Ca2+ exchange (NCX) and SERCA are the dominating Ca2+ transport systems in cardiac myocytes, accounting for 98% of Ca2+ elimination during relaxation (37; see also Ref. 47). Most important, however, is that conversion of SL Na+-K+ pumps into nonselective cation channels is sufficient to explain the observed palytoxin actions and thus might be the major mechanism underlying palytoxin effects on atrial E-C coupling, as discussed below.
Secondary effects of palytoxin: disruption of atrial E-C coupling. To the best of our knowledge, this is the first report of palytoxin effects on atrial Ca2+ signaling. It indicates that the coral toxin elevated diastolic [Ca2+]i, decreased the amplitude of the AP-induced [Ca2+]i transient, and caused Ca2+ alternans and [Ca2+]i waves as well as failures of Ca2+ release (Figs. 2 4). These alterations of atrial [Ca2+]i signaling may well explain the negative inotropic and arrhythmogenic effects of palytoxin noted in earlier studies (23, 38, 41). Further examination of the subcellular patterns of Ca2+ signaling revealed spatial inhomogeneities in the palytoxin effects and identified spontaneous release of Ca2+, predominantly from the subsarcolemmal j-SR, as the underlying mechanism for the palytoxin-induced [Ca2+]i waves and failures of SR Ca2+ release.
Because palytoxin seriously impairs atrial Ca2+ signaling, it follows that the mechanism of CICR is somehow affected by the toxin, either directly or indirectly. Previous voltage clamp studies of atrial trabeculae and isolated ventricular myocytes found palytoxin-mediated changes of ICa (27, 41). In both cases, however, these changes were attributed to secondary effects caused mainly by a shift of the reversal potential of ICa due to intracellular Ca2+ accumulation. Consistent with these results, we did not observe any effect of the toxin on ICa in [Ca2+]i-buffered atrial myocytes. Furthermore, RyR activity remained completely unaffected by palytoxin, suggesting that the coral toxin does not exhibit any direct effects on CICR but rather impairs atrial E-C coupling via indirect mechanisms. Recordings of membrane potential indicated that AP configuration was not significantly affected by palytoxin, confirming the lack of a direct effect on ICa. Rather, palytoxin caused depolarization of the RP and generation of delayed afterdepolarizations (DADs). The depolarization is a direct consequence of the induction of cation channels by the toxin (27) and a general feature of its action (14). The DADs, on the other hand, were probably caused by SR Ca2+ overload (via reverse mode NCX) and spontaneous SR Ca2+ release as visualized in the Ca2+ imaging studies.
Involvement of Na+/Ca2+ and Na+/H+ exchange. Palytoxin-induced Na+ loading of the atrial myocytes affects the activities of Na+-dependent ion transporters. In this regard, NCX and Na+/H+ exchange (NHE) are of particular importance because of their involvement in the regulation of Ca2+ and H+ homeostasis and thus atrial contraction. Previous studies of palytoxin cardiotoxicity have implicated either NCX or NHE or both in mediating some of the toxin's effects (1012). Although we did not test the involvement of either transporter directly in our experiments, it is likely that altered activities of NCX and NHE contributed to the effects of palytoxin on atrial myocytes. Na+ loading of the cells in conjunction with depolarization inhibits forward and favors reverse mode NCX and thereby causes Ca2+ overload and Ca2+-induced arrhythmias. This mechanism might be particularly pronounced in atrial myocytes, in which NCX plays a major role in AP configuration and the development of DADs (5). Similarly, Na+ loading is expected to reduce H+ extrusion via the NHE and to decrease intracellular pH (pHi), which might affect Ca2+ handling at various levels, including Ca2+ release from the SR (53).
Unresolved questions: ouabain vs. palytoxin. Both ouabain and palytoxin promote Na+ loading of atrial myocytes. The major difference in the action of the two drugs is that palytoxin induces a large depolarization of RP, whereas ouabain, especially at moderate concentrations, does not. This difference might explain why ouabain, at moderate concentrations, increases [Ca2+]i transient amplitude, while palytoxin reduces it and causes arrhythmias. Furthermore, the lack of antagonistic effects of lower concentrations of ouabain (1025 µM) on palytoxin action could be explained by the much higher apparent affinity of palytoxin to the Na+-K+ pump. At present, however, it remains unclear why high and low concentrations of ouabain exerted opposite effects on the [Ca2+]i transient. It is noteworthy, though, that in rabbit ventricular myocytes, a high concentration of ouabain (100 µM) reduced the amplitude of the electrically evoked [Ca2+]i transient (3) and that ouabain might have effects other than inhibiting the Na+-K+ pump. For example, at higher concentrations, ouabain could increase the SR Ca2+ leak through the RyR (33, 40), thereby causing decreased SR Ca2+ content and reduced [Ca2+]i transients.
Proposed mechanism of palytoxin action in atrium. According to our results, we propose the following mechanism of palytoxin action in atrial myocytes. The primary molecular target of the toxin is the SL Na+-K+ pump. Palytoxin binding converts a small fraction of pumps into nonselective cation channels, resulting in K+ efflux, Na+ influx, and concomitant depolarization. Intracellular Na+ loading and depolarization reduce the driving force for Ca2+ extrusion by NCX and thus promote intracellular Ca2+ loading. Elevated diastolic [Ca2+]i and depolarization may decrease ICa and the AP-induced [Ca2+]i transient. Furthermore, sufficiently large depolarizations lead to failures of stimulation-induced Ca2+ release. The increased SR Ca2+ content, on the other hand, causes spontaneous Ca2+ release, [Ca2+]i waves, DADs, and, ultimately, arrhythmias and contracture.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present addresses: G. U. Ahmmed, Dept. of Pharmacology, University of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612-3748; J. Kockskämper, Abt. Kardiologie und Pneumologie, Universität Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen, Germany; K. A. Sheehan, Dept. of Physiology and Biophysics, Univ. of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612-3748.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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