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CARDIOVASCULAR
Departments of Medicine (Cardiology) and Molecular Pharmacology and Biological Chemistry and the Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Illinois
Received January 21, 2003; accepted August 5, 2003.
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Abstract |
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; Lee and Dagostino, 1982; Gadsby et al., 1985; Steimers et al., 1990). Indeed, studies supporting the sodium pump inhibition hypothesis are so frequent in the literature that the implication is that the action of cardiac glycosides on the sodium pump is the only significant effect mediated by cardiac glycosides in the myocardium. However, there is also substantial evidence that cardiac glycosides may act at sites other than the sodium pump, and that these alternative sites may account for important differences between agents. Support for alternative mechanisms underlying inotropic and/or toxic effects of cardiac glycosides includes findings that cardiac glycoside analogs differ significantly in their effects on intracellular [Na+], action and resting membrane potentials (Wasserstrom et al., 1991), and toxic to therapeutic ratios in both isolated preparations (Karagueuzian and Katzung, 1981) and whole animals (Mendez et.al., 1974). In addition, studies directed at defining alternative cardiac glycoside mechanisms have revealed intracellular sites of action independent of the sodium pump, including the sarcoplasmic reticulum (Fujino and Fujino, 1982; Isenberg, 1984) and its calcium release channel (Rardon and Wasserstrom, 1990; McGarry and Williams, 1993; Sagawa et al., 2002). Despite growing evidence in support of alternative mechanisms and their role in determining differences in the effects of cardiac glycoside analogs, few studies have compared cardiac glycoside analog actions using isolated cardiac myocytes. In contrast to multicellular preparations, the use of single cardiac cells provides considerable advantages in evaluating cardiac glycoside effects. Of key importance is the elimination of extracellular diffusion barriers that confound measurement of membrane currents and other cellular processes dependent on ion gradients. In addition, nearly all previous studies have used a variety of multicellular preparations under an equally varied number of experimental conditions, making comparisons between agents extremely difficult. The purpose of this study was to characterize the electrophysiological and inotropic effects of different cardiac glycosides in isolated cardiac myocytes in a standardized manner to determine whether there are indeed differences in cellular actions of these agents. Six cardiac glycoside analogs were chosen for study based on specific differences in their molecular structures. The study was designed first to characterize differences between these six cardiac glycoside analogs on action potential (AP) configuration and contractility in isolated cat ventricular myocytes, and then to define the underlying mechanisms that account for the differences in their actions.
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Materials and Methods |
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Solutions and Drugs. (Modified) Tyrode's solution: 140 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2, 5 mM HEPES, 0.4 mM NaH2PO4, 11 mM glucose, 1.8 mM CaCl2, pH = 7.4 (NaOH). ICa extracellular solution: 0.2 mM BaCl2 added to Tyrode's solution to block IK1. Ramp protocol extracellular solution: 0.1 mM CdCl2 added to Tyrode's solution to block ICa. High resistance microelectrode solution: 500 mM KCl. Whole-cell pipette solution: 10 mM NaCl, 120 mM potassium-aspartate, 25 mM KCl, 20 mM HEPES, 0.5 mM MgCl2, 4 mM K2ATP, 0.056 or 1.0 mM EGTA. Cardiac glycosides were purchased from Sigma-Aldrich (St. Louis, MO) with the exception of resibufogenin, which was obtained from Tientsin First Central Pharmaceutical (Tientsin, China). Cardiac glycoside stock solutions of 1 to 10 mM were made in distilled water [dihydroouabain (DHO), resibufogenin], or 50% EtOH/water (ouabain, ouabagenin, actodigin, digoxin). KB-R7943 was a gift from Kanebo, Ltd. (Osaka, Japan). In the experiments in which ICa was measured, a low concentration of Ba2+ (0.2 mM) was sufficient to block background K+ current but had little effect on magnitude and kinetics of ICa in the presence of 1.8 mM CaCl2. We have used this approach previously (Nishio et al., 2002) and found it to be a useful means to measure ICa under conditions in which normal physiological conditions are otherwise maintained (physiological [Ca2+], temperature, K+ gradient).
Molecular Structures of the Six Cardiac Glycosides Studied. To more accurately define differences between cardiac glycoside agents and further evaluate cardiac glycoside structure-activity relationships, six cardiac glycosides (ouabain, ouabagenin, DHO, actodigin, digoxin, and resibufogenin) were chosen for electrophysiology and contractility studies in single cat ventricular myocytes. Figure 1 shows the molecular structure of these agents. Of particular note, DHO and ouabagenin differ from the prototypical analog ouabain by saturation of the lactone ring and omission of the carbohydrate moiety, respectively. Actodigin differs from ouabain in the site of attachment of the lactone ring at C-17 of the steroid nucleus. Resibufogenin, which unlike the other analogs is isolated from an animal source (frog skin), has a unique 6-membered lactone ring at C-17, and also lacks the sugar at C-3. Digoxin, chosen primarily for its importance in clinical medicine, shares many similarities with ouabain but differs in the type of carbohydrate at C-3, and the number and position of hydroxyl groups attached to the steroid nucleus, thereby making it the most hydrophobic of the group.
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Measurement of Voltage, Current, and Fractional Shortening in Single Ventricular Myocytes. Several drops of cell suspension were placed in an experimental chamber (0.5 ml volume) mounted on the stage of an inverted microscope (Nikon Diaphot; Nikon, Tokyo, Japan). After allowing 10 min for the cells to adhere to the chamber bottom, they were superfused (2–3 ml/min) with Tyrode's solution maintained at 36 ± 1°C using a Peltier device.
For experiments that included measurement of fractional shortening, the video image of the long axis of the cell was aligned with the rasters of the video edge detector (Crescent Electronics, Salt Lake City, UT), which continuously monitored cell length along a single line. Fractional shortening was defined as the ratio of cell shortening to resting cell length. Intracellular access was then obtained using either high-resistance microelectrodes or whole-cell patch pipettes.
High-Resistance Microelectrode Method. A microelectrode (15–30 M
resistance) was pressed gently against the cell surface forming a relatively low-resistance seal. Access was then obtained by briefly turning up the capacitance compensation, causing the amplifier circuitry to ring. Immediately following access, negative holding current (
1 nA) was applied to maintain a resting membrane potential (RMP) of –70 to –80 mV. A high-resistance seal spontaneously formed within a few minutes, and RMP became more negative, indicating successful impalement. Background holding current was then turned off. The primary advantage of this technique is that it allows electrical access of the cell without appreciable diffusion between the cytoplasm and the electrode solution. The result is greater stability in measurement of ICa and contractility.
Whole-Cell Suction Pipette Method. A 2 to 3 M pipette was pressed gently against the cell surface, and suction was applied (50–100 cm of H2O), allowing formation of a gigaohm seal. Application of a brief suction pulse then ruptured the membrane patch giving electrical and physical access to the cell interior. Advantages of this technique include low-resistance access, and the ability to control the ionic composition of the cytoplasm (diffusion through a relatively wide-bore pipette). Disadvantages include ICa "run-down" that interferes with stable measurement of ICa and contractility.
After obtaining access to the cell interior, voltage- and current-clamp protocols were directed by pCLAMP6 software (Axon Instruments Inc., Union City, CA). For voltage-clamp experiments, discontinuous single-electrode voltage-clamp mode of the Axo-clamp-2A was used, which allowed simultaneous measurement of both current and actual (not command) membrane potential. In addition, series resistance compensation is unnecessary because current passage does not occur during voltage measurement since each task is performed during alternate phases of every duty cycle. Voltage, current, and cell-length signals were digitized by a TL-1 DMA interface (Axon Instruments Inc.) at 7 to 10 kHz and channeled into the Clampex data acquisition program, for later analysis using the Clampfit program (pCLAMP6). Additional details of the use of the switch clamp with high-resistance electrodes can be found in previous publications (see Salata and Wasserstrom, 1988; Wasserstrom and Salata, 1988).
Statistical Analysis. Concentration-response curves were analyzed using one-way analysis of variance followed by the Student-Newman-Keuls test if criteria for significance were met. Data sets that did not fit parametric criteria were analyzed using nonparametric rank analysis, followed by Dunn's test where appropriate. All means represent the results of five to eleven experiments, unless stated otherwise. Data were considered significant when p < 0.05.
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Results |
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Concentration-effect curves for inotropy for the six cardiac glycoside analogs are illustrated in Fig. 4. Calculated values for effective concentration at 50% of maximal effect (EC50) are shown in the tables below. The only significant difference in maximal inotropic response lies between ouabain and ouabagenin. Comparison of maximal inotropy (before the onset of toxicity) indicates that ouabain produces a significantly greater increase in cell shortening than its aglycone ouabagenin (2.16 ± 0.14 versus 1.43 ± 0.05, respectively; n = 11 and 7, respectively, p < 0.05).
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Additional electrophysiology data are presented in Table 1. Control AP parameters (Table 1) did not differ between experimental groups. In general, 1 to 3 mV of RMP depolarization and a 5 to 10 mV decrease in AP amplitude was observed at maximal cardiac glycoside concentrations, with no significant differences noted between agents.
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Effect of Ouabain on ICa. Finding significant differences between cardiac glycosides on AP shortening prompted an investigation into membrane currents that might explain these observed differences. Voltage-clamp studies were conducted to evaluate effects of ouabain on ICa as a possible mechanism for AP shortening using high-resistance microelectrodes. As demonstrated in Fig. 5A, exposure to 3 µM ouabain has little effect on the magnitude of ICa, although in this example the rate of inactivation appears to be increased. With longer exposure times (10 min), there is a small decrease in ICa; however, this decrease was not statistically significant (Fig. 5B). DHO shows a similar effect on ICa (Fig. 5C), with a small time-dependent decrease in magnitude. Thus the difference in action potential changes induced by these agents cannot be explained on the basis of alterations in ICa. Further experiments evaluating the stability of ICa measurements under control conditions (without cardiac glycoside) showed no time-dependent diminution of ICa (Fig. 5D) demonstrating that the slight decrease in current magnitude is in fact a result of actions of both drugs.
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Effect of Ouabain on IK1. Further voltage-clamp studies were conducted to evaluate effects of ouabain on the inward rectifier current (IK1) as a possible mechanism for action potential shortening. Figure 6A shows representative IK1 data obtained with high-resistance electrodes. The voltage-clamp protocol is presented in the top trace. From a holding potential of –40 mV, hyperpolarizing and depolarizing voltage steps were used to assess both the inward and outward IK1 currents, measured at the end of the test voltage step. Although ICa was not blocked in these experiments, values for IK1 at voltages positive to –40 mV were assumed to be fairly accurate at the end of the voltage-clamp steps after ICa was mostly inactivated (300 ms). Current recordings at hyperpolarizing potentials show large inward currents; depolarizing pulses yielded progressively diminishing outward currents typical of cat ventricular IK1. Exposure to 3 µM ouabain caused no change in inward currents and a slowly developing outward current during depolarization This outward current component increased with exposure time and was investigated further using a ramp protocol, discussed below. Composite voltage-current data (Fig. 6B) again shows no change in steady-state IK1 over the voltage range of –140 to 30 mV, with a small increase in outward current at voltage steps above 40 mV, which did not achieve statistical significance.
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Effect of Ouabain and DHO on the Steady-State Outward Current (Iss). A ramp protocol was used to evaluate the late outward current component noted above using high-resistance electrodes. The voltage protocol and representative data are presented in Fig. 7. Cd2+ (0.1 mM) was added to block ICa. As illustrated in Fig. 7A, 10 min of exposure to 3 µM ouabain induced a pronounced increase in outward current (previously termed "outward steady-state current", Iss; Levi, 1993) that is observed at voltages above –20 mV. However, pretreatment of cells with 10 µM KB-R7943, a blocker of reverse mode Na+-Ca2+ exchange (NCX), markedly attenuated the increase in this current (Fig. 7B), suggesting that the observed current is the result of increased outward (reverse mode) NCX. Composite data show that this cardiac glycoside-sensitive current (Fig. 8; represented as a difference current between control and cardiac glycoside treated conditions) is increased during exposure to maximal inotropic concentrations of both ouabain and DHO but is suppressed by pretreatment with 10 µM KB-R7943. The fact that there is an apparent voltage dependence to this outward current, which increases with test potential, is also consistent with the notion that the electrogenic NCX current may be involved. The finding that both ouabain and DHO are capable of increasing outward NCX current, however, does not explain the divergent effects of ouabain and DHO on the AP.
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Effect of Ouabain and DHO on APD with Increased Intracellular Ca2+ Buffering. Consideration of the possibility that the observed differences between ouabain and DHO on APD were secondary to changes in intracellular Ca2+ prompted a series of experiments that minimized the cardiac glycoside-induced rise in internal Ca2+. First, a series of experiments was performed with patch electrodes using an internal [Na+] of 10 mM and near physiological Ca2+ buffering (0.056 mM EGTA, measured [Ca2+]i = 82 nM; Vites and Wasserstrom, 1996). Under these conditions (which permitted dialysis and equilibration of the intracellular space with the patch pipette internal solution), APD was observed to shorten upon exposure to 3 µM ouabain (Fig. 9A), but not 10 µM DHO (Fig. 9B), just as we had observed previously using high-resistance electrodes (Fig. 2). The experiment was then repeated with markedly increased internal Ca2+ buffering (1 mM EGTA). As shown in Fig. 9, C and D, separate cells were allowed to equilibrate for 20 min with internal solution containing the high EGTA concentration before addition of the cardiac glycoside. During this equilibration time the APD shortened and then stabilized. Addition of a maximal inotropic concentration of both ouabain (Fig. 9C) and DHO (Fig. 9D) was now able to cause further shortening of the AP. Under these conditions, the decreases in AP50 and AP90 observed upon exposure to both 3 µM ouabain or 10 µM DHO were statistically significant, as indicated in Fig. 9E.
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Inotropic Action of Ouabain, DHO, and Ouabagenin under AP Clamp. To further characterize the observed diminished inotropic action of ouabagenin compared with ouabain under conditions independent of changes in resting and action potentials, myocytes were voltage-clamped using a protocol that approximated normal RMP (–78 mV) and AP configuration. High-resistance microelectrodes were used to maintain contractility during the experiment. Under these conditions, ouabain, ouabagenin, and DHO did not differ from each other significantly in the extent of their maximal inotropic action; all three cardiac glycosides were able to achieve similar inotropic effects; ouabain increased cell shortening from 4.1 ± 0.77% to 10.4 ± 0.76% of resting cell length (n = 9); ouabagenin increased shortening from 4.9 ± 0.87% to 11.4 ± 0.27% (n = 5); DHO increased shortening from 4.7 ± 0.27% to 12.1 ± 0.61% (n = 4). Unlike the results obtained with the free-running action potential stimulation of contraction, there were no significant differences in the magnitude of the positive inotropic effects between these three agents. In addition, the magnitude of the current during the plateau phase of the AP clamp increased with exposure to all three of the cardiac glycosides but did not differ significantly between agents (data not shown) as would be expected for a general increase in NCX current. This result suggests that inotropic differences between agents, particularly ouabagenin and ouabain, are minimized under conditions that control resting and excitation potentials.
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Discussion |
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Cardiac Glycoside Effects on Action Potentials, Inotropy, and Toxicity. Differences in activity among cardiac glycosides have been described both in intact animals and isolated cardiac preparations. However, attempts to explain these differences in action based on specific features of cardiac glycoside molecular structure have produced limited results. Part of this limitation stems from the fact that the majority of cardiac glycoside structure-activity studies are focused on Na+/K+-ATPase binding (De Pover and Godfraind, 1982; Brown and Erdmann, 1984), and few address physiological actions in intact systems. Notable exceptions include studies in guinea pig atria showing stimulation of the sodium pump at low concentrations of ouabain and ouabagenin, but not DHO (Ghysel-Burton and Godfraind, 1979), and the demonstration of differences in toxic to therapeutic ratios of cardiac glycosides in intact dogs, dependent on the position of the attachment of the lactone ring (Mendez et.al., 1974). However, none of these studies (individually or collectively) provides a comprehensive foundation for understanding cardiac glycoside structure-activity relationships, and it is likely that multiple factors in structure determine the specific response elicited by a given agent. Information provided by our investigation indicates that subtle differences in structure (including a simple saturation of the lactone ring) cause significant differences in physiological activity. If intracellular sites are indeed important in determining differences in action between cardiac glycoside agents (Fujino and Fujino, 1982; Isenberg, 1984; Sagawa et al., 2002), including the threshold for Ca2+ overload, one could speculate that the relative ability of a given glycoside to cross the sarcolemma (either passively or actively; Nunez-Duran et al., 1988) and affinity for specific intracellular sites would be important additional determinants of action. In addition, if the ryanodine receptor is an important determinant of this intracellular mechanism as postulated by some investigators (Isenberg, 1984; Rardon and Wasserstrom, 1990; McGarry and Williams, 1993; Sagawa et al., 2002), then differences in binding affinity to this receptor between cardiac glycosides might contribute to the observed differences in action.
Mechanisms Underlying Shortening of APD. Many studies evaluating the effects of cardiac glycosides on isolated myocardial preparations or single cells have shown a shortening of APD (McDonald et al., 1975; Levi, 1993). Explanations for this finding are reflected in contrasting theories, which attempt to characterize a cardiac glycoside-induced outward current. The first theory (Luk and Carmeliet, 1990) invokes an outward current that is increased by 10 to 100 µM ouabain in guinea pig cardiac myocytes with similar properties to a single channel current found in inside-out patches. This current is proposed to be a Na+-activated K+ current by virtue of the fact that it is dependent on the presence of Na+i, displays rectification that is dependent on the K+ gradient, and has a reversal potential near the K+ equilibrium potential. The second theory (Levi, 1993) suggests the involvement of an outward current elicited by 50 µM strophanthidin also in guinea pig cardiac cells with reversal potential of –54 mV. This current was shown to be consistent with reverse mode NCX by being more pronounced at positive voltages, abolished by removing external Ca2+ or addition of Ni2+, and unaffected by increasing intracellular Ca2+ buffering (BAPTA) or addition of K+ current blockers (Ba2+, TEA, and 4-AP). Data from our experiments indicate the cardiac glycoside-induced outward current is most likely due to increased reverse mode NCX because it does not develop in the presence of KB-R7943, a reverse mode NCX blocker (Kimura et al., 1999). In addition, AP shortening appears to be an action of some but not all cardiac glycosides. This could be a result of other drug actions, including the possibility that certain glycosides might also block IKs, the slowly activating component of the delayed rectifier current (Rocchetti et al., 2003), which could explain why DHO (and not ouabain or actodigin) caused AP prolongation. The balance of opposing direct and indirect actions on net membrane current could then be responsible for the variety of glycoside effects on AP duration, especially in different species with varying dependencies of repolarization on IKs.
Role of Ca2+ Overload in Development of APD Shortening and Inotropy. Data from this study suggest that Ca2+ overload is a primary determinant both for maximal inotropic response and alterations in AP configuration and could account for differences in response between cardiac glycoside analogs. This is not a new concept, particularly with regard to inotropic action as addressed by other investigators (Capogrossi et al., 1988). The basic theory, supported by experimental data, contends that the myocardium is capable of increasing inotropy by Ca2+-dependent mechanisms until toxicity develops as indicated by spontaneous sarcoplasmic reticulum Ca2+ release and spontaneous contractions. This spontaneous, uncoordinated release of Ca2+ depletes the sarcoplasmic reticulum of Ca2+ for the next contraction, thereby reducing the inotropic state as well as any contributions of Ca2+-dependent conductances (including NCX current) to AP duration.
We found that Ca2+ overload in fact does influence whether or not cardiac glycosides induced AP abbreviation. Although direct evidence for sodium pump inhibition was not evaluated, the presence of a robust increase in reverse mode NCX current within the inotropic range of both ouabain and DHO would suggest that sodium pump inhibition is an important mechanism in the inotropic response of cardiac glycosides in vitro. In addition, the data suggest that a second cardiac glycoside mechanism (independent of sodium pump inhibition) influences the threshold for Ca2+ overload and thereby determines the maximal inotropic response observed. For example, the weaker inotropic response and lack of AP shortening observed with ouabagenin compared with ouabain is likely the result of earlier spontaneous SR Ca2+ release (lower toxicity threshold) elicited by ouabagenin prior to accumulation of equal levels of intracellular calcium.
How voltage alters spontaneous release from the SR and thereby alters the maximal inotropic response of ouabagenin (as demonstrated in the AP clamp experiments) can only be speculated. The most likely reason is that the positive feedback between SR Ca2+ release and membrane depolarization is prevented under voltage clamp. Thus, differences between drugs displaying maximal inotropic effects are likely to be blunted simply because steady-state drug effects are more closely approximated. However, it is also possible that this observation may be related to the idea linking membrane potential with SR release (Ferrier and Howlett, 1995). Because the threshold for spontaneous calcium release from the SR during diastole is a primary determinant of cardiac glycoside inotropy, it may be that voltage clamp prevents or delays diastolic calcium release. This implies that small voltage perturbations during diastole may contribute to spontaneous release or that spontaneous release during diastole is at least partially dependent on membrane potential.
Effects of Low Internal Free Mg2+ Concentration on Experimental Results. It is possible that certain of our experimental conditions might influence the results found in this study. One such issue is the low free [Mg2+] concentration in the internal solution (about 10–5 M). This is likely to have important effects on Mg2+-dependent process as in the cell. However, it should be noted that even with this low [Mg2+], there are still pronounced differences between different cardiac glycosides just as expected from data obtained using high-resistance microelectrodes in which the intracellular environment is closer to physiological. In addition, the rectifying characteristics of IK1 are largely unaffected by the buffering of internal Mg2+ (data not shown), suggesting that effective concentrations in critical regions of the cytoplasm remain at normal regulatory levels despite calculated changes in bulk concentration. This fact suggests that it is difficult to extrapolate bulk calculated [Mg2+] to true free concentration at regulatory sites.
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Footnotes |
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ABBREVIATIONS: AP, action potential; APD, action potential duration; KHB, Krebs-Henseleit buffer; DHO, dihydroouabain; NCX, sodium-calcium exchanger; RMP, resting membrane potential; Iss, outward steady-state current; ICa, calcium current (L-type); IK1, inward rectifying potassium current; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.
Address correspondence to: Dr. J. Andrew Wasserstrom, Division of Cardiology—S203, Ward 3-105, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. E-mail: ja-wasserstrom{at}northwestern.edu
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