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(Circulation Research. 1995;76:664-674.)
© 1995 American Heart Association, Inc.

Articles

Contribution of Na⁺-Ca²⁺ Exchange to Stimulation of Transient Inward Current by Isoproterenol in Rabbit Cardiac Purkinje Fibers

Xinqiang Han, Gregory R. Ferrier

From the Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada.

	Abstract

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Abstract Cellular mechanisms underlying ß-adrenergic stimulation of the arrhythmogenic transient inward current (TI) were investigated by using a two-microelectrode voltage-clamp technique in rabbit cardiac Purkinje fibers. TI induced by elevating [Ca²⁺]_o to 30 mmol/L and substituting [Na⁺]_o with N-methyl-D-glucamine (NMG) chloride had a distinct reversal potential (E_REV) of -25 mV, suggesting that Na⁺-Ca²⁺ exchange was not the charge carrier for TI. In the absence of [Na⁺]_o, isoproterenol (ISO, 0.01 to 5.0 µmol/L) had no effect on either inward or outward TI or on the current-voltage relation of TI. However, ISO (0.1 µmol/L) significantly increased both inward and outward TIs without affecting the E_REV of TI, if [Na⁺]_o was present. Pretreatment with propranolol (0.2 µmol/L) or atenolol (0.2 µmol/L) abolished the stimulatory effects of ISO. Addition of propranolol (0.2 to 0.5 µmol/L) after the effects of ISO had developed caused only partial reversal of TI stimulation. This indicates persistence of stimulatory effects downstream from the initial agonist-receptor interaction. Forskolin (1 µmol/L), a direct adenylate cyclase activator, also strongly increased both inward and outward TI in the presence of [Na⁺]_o. These effects also were abolished when [Na⁺]_o was substituted by NMG. Inward and outward TIs enhanced by either ISO or forskolin were reversed by two putative Na⁺-Ca²⁺ exchange blockers, dodecylamine (20 µmol/L) and quinacrine (20 µmol/L). These results suggest that ß-adrenergic stimulation of TI is mediated by the Na⁺-Ca²⁺ exchange; stimulation likely involves phosphorylation of the exchanger or some factor that modulates exchanger activity.

Key Words: ß-adrenoceptors • nonspecific cationic currents • Cl^- current • Na⁺-Ca²⁺ exchange • oscillatory afterpotentials

	Introduction

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Sympathetic regulation of cardiac contractility is mediated primarily by the release of noradrenaline and adrenaline, which act by binding to adrenergic receptors. Positive inotropic and chronotropic effects can be mediated by both - and ß-adrenoceptors, but the latter are more prominent.¹ It is generally accepted that ß-adrenergic stimulation leads to the formation of cAMP, an intracellular second messenger that activates cAMP-dependent protein kinase and thereby induces phosphorylation of proteins in the sarcolemma, sarcoplasmic reticulum, and thin filaments.² One of the most important consequences of activation of phosphorylation is the elevation of free intracellular Ca²⁺.

Although an increase in [Ca²⁺]_i is commonly discussed as the major mechanism for positive inotropism associated with ß-adrenergic stimulation, Ca²⁺-overloaded cardiac cells also manifest many abnormal electrophysiological alterations that can result in arrhythmias. For example, stimulation of ß-adrenoceptors has been shown to induce oscillatory afterpotentials (OAPs, also called delayed afterdepolarizations) in both multicellular and single-cell preparations from the heart^{3 4} and to increase the amplitude of OAPs induced by ouabain in isolated canine Purkinje fibers,⁵ spontaneous OAPs in isolated ventricular myocytes,⁶ and OAPs occurring in feline Purkinje fibers surviving in 2- to 4-month-old infarcts.⁷ OAPs represent a mechanism of abnormal automaticity that may be involved in the generation of triggered cardiac arrhythmias in a wide range of settings, including digitalis toxicity and reperfusion of previously ischemic or hypoxic tissues.^{8 9} The prerequisite for the generation of OAPs is intracellular Ca²⁺ overload.^{8 9}

The ionic current that generates OAPs is the transient inward current (TI). The TI was initially demonstrated in cardiac Purkinje fibers exposed to either toxic concentrations of digitalis,^{10 11 12} elevated [Ca²⁺]_o,¹¹ or K⁺-deficient solution.¹³ TI also has been induced in cardiac muscles and sinoatrial nodal preparations by similar treatments.^{14 15 16} ß-Adrenergic stimulation can increase the peak TI in atrial cells,¹⁷ sinoatrial nodal cells,¹⁸ and coronary sinus cells.¹⁹ We have demonstrated that in Purkinje fibers, the ß-adrenoceptor agonist isoproterenol (ISO) strongly enhances TI induced by toxic concentration of digitalis but cannot itself induce TI.²⁰

Although enhancement of TI by ß-adrenergic stimulation generally has been attributed to an increase in the L-type Ca²⁺ current I_Ca(L), the exact mechanism(s) is far from clear. In part, this is because the ionic basis underlying TI is still a subject of much debate.²¹ Earlier work has suggested that either a nonselective leak channel or electrogenic Na⁺-Ca²⁺ exchange may act as the ionic pathway carrying TI.¹² The former concept is strengthened by the discovery of nonspecific cationic currents, which are activated by increases in intracellular Ca²⁺.²² Activation of a nonspecific cationic conductance could generate TI. Studies at the single-channel level indicate that mammalian myocardium contains a Ca²⁺-activated cationic channel that is selective for monovalent ions.^{23 24} On the other hand, Na⁺-Ca²⁺ exchange, which plays an essential role in the Ca²⁺ extrusion following each contraction,²⁵ could also generate TI when intracellular Ca²⁺ is significantly elevated.^{12 15}

We have recently demonstrated that TI induced by elevated [Ca²⁺]_o in rabbit cardiac Purkinje fibers is conducted through two different types of TI channels: nonselective cationic channels, which mainly conduct inward TI, and a class of Ca²⁺-dependent Cl^- channels, which are mainly responsible for outward TI.^{26 27} Na⁺-Ca²⁺ exchange played little or no role as the charge carrier for TI in these preparations whether [Na⁺]_o was absent or present.²⁷

It is not clear whether ß-adrenergic stimulation of TI is mediated by the enhancement of Ca²⁺ loading (elevation of [Ca²⁺]_i) or by a stimulatory effect at the charge-carrying sites (the TI channels). Ca²⁺ loading can be achieved by an influx of Ca²⁺ through Ca²⁺ channels and/or Na⁺-Ca²⁺ exchange.^{28 29 30 31} In the present investigation, we addressed the following questions: (1) Is ß-adrenergic stimulation of TI secondary to the elevation of [Ca²⁺]_i or a direct effect on the TI charge carrier? (2) If elevation of [Ca²⁺]_i is involved, is this effect mediated by enhanced Na⁺-Ca²⁺ exchange? (3) Does stimulation of TI by ISO involve a phosphorylation process? Our results suggest that ß-adrenergic stimulation of TI is most likely caused by the elevation of [Ca²⁺]_i. This effect is mainly mediated by the enhancement of Na⁺-Ca²⁺ exchange (reverse mode), and ISO stimulation can be mimicked completely by forskolin, which directly activates adenylate cyclase and bypasses agonist–ß-receptor binding and G-protein activation.³²

	Materials and Methods

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All studies were conducted on isolated rabbit cardiac Purkinje fibers obtained as described previously.²⁶ After 30 minutes to 1 hour of perfusion with dissecting solution, the preparations were perfused with Tyrode's solution with the following composition (mmol/L): NaCl 145, KCl 4, MgCl₂ 0.5, CaCl₂ 2.5, glucose 10, HEPES 10, and Tris base 5, pH 7.35 at 37°C. Na⁺-containing high-Ca²⁺ solution was composed as follows unless otherwise indicated (mmol/L): CaCl₂ 30, NaCl 105, KCl 4, MgCl₂ 0.5, glucose 10, HEPES 10, and Tris base 5. Na⁺-free high-Ca²⁺ solution was made by replacing NaCl with equimolar N-methyl-D-glucamine (NMG) chloride. The pH of the high-Ca²⁺ solutions was 7.35 at 37°C (the temperature at which experiments were performed). All solutions were bubbled with 100% O₂, except for the dissecting solution, which was bubbled with 95% O₂/5% CO₂. In all experiments, at least 40 minutes of perfusion with Tyrode's solution was performed before exposure to high-Ca²⁺ solution.

The two-microelectrode voltage-clamp technique was used.³³ Beveled microelectrodes filled with 2.7 mol/L KCl and with resistances between 5 and 12 M were used for current passage and voltage recording. The reference electrode was a Ag/AgCl wire immersed in a glass pipette filled with 2.7 mol/L KCl. The tip of the pipette was plugged with 2.7 mol/L KCl-agar to minimize changes in liquid junction potentials that might occur when changing perfusing solutions and was placed downstream from the preparations. We measured a 2.5-mV difference in junction potentials between Tyrode's solution and the Cl^--poor solution (60 mmol/L Cl^-). This small change of liquid junction potential was so small that the raw measurements required no correction. Voltage steps were generated by PCLAMP software (Axon Instruments), which controlled the command voltage of the voltage-clamp feedback circuit (Axoclamp-2A, Axon Instruments). Recordings were displayed on a Tektronix 5111A storage oscilloscope. Data were acquired and stored simultaneously by a microcomputer (80286/287 processors) interfaced with an Axolab I analog/digital converting system (Axon Instruments). The sampling frequency of the Axoclamp 2A was set at 1 kHz by modifying the CLAMPEX parameters. The holding potential was -55 mV. TI was defined as the inward or outward deflection seen on repolarization to various test potentials following a previous depolarizing pulse (+20 mV, 1 s). Activating steps were repeated every 20 s except for the current-voltage (I-V) relations, for which they were repeated every 10 s. The magnitude of the peak TI was measured from the maximum inward or outward deflection to a line joining the immediate preceding and following minima.^{11 12} Since ISO affects the time to peak TI, digital current subtraction was not used to illustrate the effects of ISO. The ISO-sensitive current referred to in the present study was calculated by subtracting the peak TI measured under control conditions from the peak TI recorded in the presence of ISO.

The perfusion bath had a volume of 1.5 mL. A constant flow rate of 4.5 mL/min was used to enable quick changes of solutions. After the perfusing solution was changed, there usually was an equilibration period, during which the background current shifted in either the inward or outward direction depending on the compositions of the superfusates. This period took 3 minutes until the steady state was reached. Therefore, unless otherwise indicated, both TI magnitudes and reversal potentials referred to in the present study were obtained only after shifts in background current had stabilized.

All agents except dodecylamine used in the present study were purchased from Sigma Chemical Co. These include NMG, isoproterenol, propranolol, atenolol, forskolin, and quinacrine. Dodecylamine was from Aldrich Chemical Co. NMG chloride was made by titrating a stock solution with HCl to pH 7. Quinacrine was protected from light. Forskolin was dissolved in ethanol. Drugs were first prepared in stock solutions and then added to the perfusion solution to reach final concentrations.

Data were analyzed with Student's t test where appropriate. Differences were considered significant for values of P<.05.

	Results

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Stimulation of TI by ISO in the Presence of [Na⁺]_o
TI can be induced by 30 mmol/L Ca²⁺ either in the presence or absence of [Na⁺]_o in rabbit Purkinje fibers.^{26 27} Fig 1 shows the effects of ISO on TI induced by 30 mmol/L [Ca²⁺]_o in the presence of 105 mmol/L [Na⁺]_o. The membrane potential was clamped at a holding potential of -55 mV. TI was elicited by two sequential repolarizing steps from a 1-s depolarizing step to +20 mV. Our previous studies demonstrated that TI had a distinct reversal potential (E_REV) close to -20 mV with the ionic concentrations used here. Therefore, the two sequential repolarization steps, to -5 (for 1 s) and -55 mV (Fig 1B), were used to allow observation of effects of ß-adrenergic stimulation on outward and inward TIs simultaneously. Fig 1A shows superimposed current traces recorded in the absence (trace a) and presence (trace b) of ISO. Prominent outward and inward TIs were seen after repolarization to test potentials of -5 and -55 mV, respectively. Five minutes after the addition of 0.1 µmol/L ISO, both outward and inward TIs were markedly increased. Thus, under control conditions the outward and inward TIs were 6.5±1.5 and -19.5±2.7 nA, respectively (mean±SD, n=5). They were increased significantly (P<.05) to 11.5±2 and -37.5±4.5 nA, respectively.

View larger version (12K): [in this window] [in a new window]   Figure 1. Effects of isoproterenol (ISO) on the transient inward current (TI) induced by elevation of [Ca2+]o (30 mmol/L) in the presence of [Na+]o (105 mmol/L). A, Superimposed current traces recorded in the absence (trace a) and presence (trace b) of ISO. B, Voltage steps applied to the preparation. Prominent outward and inward TIs were seen after repolarizations to test potentials of -5 and -55 mV, respectively. Five minutes after addition of 0.1 µmol/L ISO, both outward and inward TIs were markedly increased.

Detailed analysis for the ISO-induced stimulation of TI will be illustrated in Figs 2 through 4. Fig 2 shows the original superimposed current traces recorded in the absence (Fig 2A) and presence (Fig 2B) of ISO in one Purkinje fiber. Voltage steps applied to the preparations are shown schematically on the bottom of Fig 2A. The time to peak current shortened progressively with depolarization; however, this did not obscure a distinct reversal of current polarity. Five minutes after the addition of 0.1 µmol/L ISO, both outward and inward TIs generated at all test potentials were greatly increased (Fig 2B). ISO also abbreviated the time to peak current but otherwise did not alter the relation between time to peak current and membrane potential. Maximum stimulation of TI was usually seen within 5 minutes; no significant changes in peak TI (both inward and outward) were observed with longer drug exposure (15 minutes). These effects were reversible on removal of the agonist.

View larger version (11K): [in this window] [in a new window]   Figure 2. Effects of isoproterenol (ISO) on the transient inward current (TI) generated at different test potentials. A, Superimposed current traces recorded in the absence of ISO. The voltage-step changes applied to the preparation are shown at the far right. B, Superimposed current traces recorded in the presence of ISO. Five minutes after addition of 0.1 µmol/L ISO, outward and inward TIs elicited at test potentials ranging from +5 to -55 mV showed marked increases.

View larger version (14K): [in this window] [in a new window]   Figure 3. Effects of isoproterenol (ISO) on the current-voltage (I-V) relations of the transient inward current (TI) in the presence of [Na+]o. Data were pooled from five Purkinje fibers. Voltage steps applied to the preparations were shown schematically in Fig 2. A, Graph showing I-V relations of TI in the absence () and presence () of ISO. B, Graph showing ISO-sensitive current. Peak inward and outward TIs were greater at test potentials away from the reversal potential (EREV). ISO increased peak TI at all test potentials without affecting its voltage dependence. EREV (-25 mV) was not significantly affected.

View larger version (30K): [in this window] [in a new window]   Figure 4. Effects of isoproterenol (ISO) on inward transient inward current (TI) generated at -55 mV in response to depolarization to +20 mV, followed by repolarizing steps (prepulses) to different potentials ranging from -55 to +5 mV. Voltage protocols were the same as illustrated in Fig 2. For simplicity, only the prepulse potentials that were followed by TI at -55 mV are shown schematically (A, top). Also illustrated are two sets of 13 superimposed current traces recorded in one preparation showing control TI amplitude (A, middle) and the effects of ISO (A, bottom). ISO markedly increased the amplitude of inward TI seen at -55 mV. The prepulse voltage-current relations obtained in the absence () and presence () of ISO are shown (B), and the ISO-sensitive current is also shown (C). These data were averaged from five preparations. ISO significantly increased the inward TI without changing its dependence on prepulse voltage.

Effects of ISO on the I-V relation of TI in the presence of [Na⁺]_o are shown in Fig 3. Results plotted in this figure were averaged from five preparations. The I-V relations obtained in the absence (open circle) and presence (filled circle) of ISO are shown in Fig 3A; the ISO-sensitive TI is shown in Fig 3B. The ISO-induced increases in peak TI were greatest at test potentials away from E_REV (-25 mV). E_REV was not significantly affected. A number of different mechanisms could explain this ISO-induced increase in TI: (1) increased Ca²⁺ overload through I_Ca(L) channels, (2) increased Ca²⁺ overload by Na⁺-Ca²⁺ exchange, and (3) direct phosphorylation of the charge carriers for TI, the TI channels. These possibilities were tested in the following experiments.

Fig 4 shows the effects of ISO on inward TI seen on repolarizing to -55 mV after a double prepulse; ie, the first prepulse was +20 mV, and the second prepulse varied between +5 and -55 mV. The voltage-step changes applied to the preparations are shown schematically on the top of Fig 4A. A set of 13 current traces recorded in the absence (middle) and presence (bottom) of ISO (0.1 µmol/L) from one Purkinje fiber are also shown. The prepulse-current relations, which were quantified from five preparations, are plotted in Fig 4B and 4C. As shown in Fig 4B, the most positive prepulse voltages are followed by the largest TI. ISO significantly increased this set of TIs without affecting its voltage (prepulse) dependence (Fig 4C). Increased Ca²⁺ loading through Na⁺-Ca²⁺ exchange is known to occur at depolarized potentials (ie, -30 to +5 mV); thus, it seems that the ISO stimulation of TI (at least for inward TI) is more consistent with a role of Na⁺-Ca²⁺ exchange, since I_Ca(L) should remain inactivated at these prepulse potentials.

Effects of ISO on TI Induced in the Absence of [Na⁺]_o
If stimulation of TI by ISO is mediated by the enhancement of Na⁺-Ca²⁺ exchange, one would expect stimulation of TI by ISO to be abolished when the Na⁺-Ca²⁺ exchange mechanism is rendered inoperative. Therefore, the effects of ISO on TI were determined in the absence of [Na⁺]_o to test this hypothesis. Fig 5 shows superimposed current traces recorded in one Purkinje fiber before (Fig 5A) and after (Fig 5B) the addition of 1 µmol/L ISO. The membrane potential was clamped at -55 mV. Depolarization to +20 mV for 1 s was followed by repolarization steps varied from +5 to -55 mV for 1 s (Fig 5C) to allow observation of effects of ß-adrenergic stimulation on outward and inward TIs. Marked outward and inward TIs were induced at these test potentials before exposure to ISO (Fig 5A). After the administration of 1 µmol/L ISO (Fig 5B), no significant effects on the magnitudes of either peak outward or inward TIs were seen after 5 to 10 minutes of drug exposures. The inward TIs seen on repolarization to -55 mV after various prepulse potentials also showed no increase. Importantly, however, the net outward current activated by the end of the depolarization step to +20 mV (I_step), which largely reflects delayed rectifier K⁺ current (I_K) activation, was increased significantly (by 25.7±3.4 nA, n=5, P<.05). Summarized data (n=5) showing the I-V relations for TIs recorded in the presence and absence of ISO are plotted in Fig 5D. ISO failed to stimulate TIs at all test potentials.

View larger version (27K): [in this window] [in a new window]   Figure 5. Isoproterenol (ISO) does not increase the magnitude of the transient inward current (TI) induced in the absence of [Na+]o. A and B, Superimposed current traces recorded before and after the addition of 1 µmol/L ISO, respectively. No significant changes in either outward or inward TIs were seen in response to 1 µmol/L ISO. However, the outward currents that were activated by the end of a 1-s depolarization pulse were greatly increased. C, Voltage steps applied to the preparation. D, Graph showing current-voltage relations of TI induced by high Ca2+ in the absence () and presence () of ISO. Each point represents four to seven determinations (mean±SD) made in five Purkinje fibers. The peak amplitudes of both inward and outward TIs were unaffected by ISO at all potentials tested. The reversal potential also was unaffected by addition of ISO.

Failure of ISO to stimulate TIs in the absence of [Na⁺]_o might reflect the absence of operative Na⁺-Ca²⁺ exchange or the interruption of ß-adrenoceptor–initiated phosphorylation. If phosphorylation is inhibited by exposure to 0 mmol/L [Na⁺]_o in our system, one would expect inhibition of stimulation of all membrane currents normally stimulated through phosphorylation. I_Ca(L) could not be measured quantitatively in rabbit Purkinje fibers under the conditions of our experiments because a large transient outward current (I_TO) overlaps I_Ca(L). However, it is well established that ß-adrenergic stimulation of I_K is mediated by phosphorylation of I_K channels.³⁴ In the present study, I_step, which roughly reflects I_K activation, was still significantly increased by ISO in the absence of [Na⁺]_o (Fig 5). When Purkinje fibers were pretreated with 0.2 µmol/L propranolol (n=2) or atenolol (n=2), stimulation of I_step was abolished (not illustrated). Stimulation of I_step by ISO provides evidence that ß-adrenoceptor–initiated phosphorylation was still functional in these preparations in the absence of extracellular Na⁺. ß-Receptor agonist–initiated phosphorylation also has been observed by other investigators in Na⁺-free solutions.^{35 36}

Since the phosphorylation process was apparently still functional, it is unlikely that ß-adrenergic stimulation of TI represents direct phosphorylation of TI channels. For the same reason, it is also unlikely that stimulation of TI is secondary to the phosphorylation of I_Ca(L). In addition, ISO can stimulate the digitalis-induced TI in the presence of two I_Ca(L) blockers, verapamil and Mn²⁺.²⁰ Thus, stimulation of TI by ISO is most likely mediated by an action on Na⁺-Ca²⁺ exchange. Stimulation of Na⁺-Ca²⁺ exchange would be expected to cause Ca²⁺ loading during depolarization^{28 29 30 31} and could thereby mediate ß-adrenergic enhancement of TI.

Evidence for a Phosphorylation Step: Effects of ß-Adrenergic Blockade and Forskolin
The stimulatory effects of ISO (1 or 5 µmol/L) in the presence of [Na⁺]_o on both inward and outward TIs were totally prevented by pretreating preparations with 0.2 µmol/L propranolol (n=2) or atenolol (n=2), indicating that the effects were mediated by ß-adrenoceptors (not illustrated). However, if preparations were stimulated first with ISO and then exposed to propranolol, the same or even higher concentrations of the ß-adrenoceptor antagonists only partially reversed stimulation. Fig 6 shows such an experiment. Superimposed current traces recorded in the absence (trace a) and presence of ISO (trace b) and after the addition of propranolol (trace c) are shown on the top. Voltage steps applied to the preparation are illustrated on the bottom. Outward and inward TIs were generated on repolarization to -5 and -55 mV, respectively. After the addition of 0.1 µmol/L ISO, marked stimulation of both outward and inward TIs was observed within 2 minutes (trace b). Propranolol (0.5 µmol/L) was added to the perfusion solution at this time. Five minutes after the addition of propranolol, both outward and inward TIs showed significant decreases (trace c). However, both outward and inward TIs were still greater than those generated in the absence of ISO (trace a). Thus, in the four preparations in which both ISO and propranolol were tested, the outward TIs were 5.7±1.1, 10.8±2.1 (P<.05), and 7.3±1.8 (P>.05) nA under control, ISO, and ISO plus propranolol conditions, respectively; the inward TIs were -17.4±4.7, -44.6±8.9 (P<.05), and -26.5±6.6 (P>.05) nA under control, ISO, and ISO plus propranolol conditions, respectively (mean±SD).

View larger version (17K): [in this window] [in a new window]   Figure 6. ß-Adrenoceptor antagonist propranolol incompletely reverses isoproterenol (ISO) stimulation of the transient inward current (TI). Superimposed current traces recorded in the absence (trace a) and presence (trace b) of ISO and after the addition of propranolol in the presence of ISO (trace c) are shown on the top. Voltage steps applied to the preparation are illustrated on the bottom. Outward and inward TIs were generated on repolarizations to -5 and -55 mV, respectively. After the addition of 0.1 µmol/L ISO, marked stimulation of both inward and outward TIs were seen (trace b). Five minutes after the addition of propranolol (0.5 µmol/L), both inward and outward TIs were decreased significantly (trace c).

Propranolol is a competitive ß-adrenergic antagonist. Its action is believed to prevent agonist-receptor binding or to displace the agonist from receptors. Therefore, incomplete reversal of ISO stimulation of TI suggests that agonist-receptor binding has already triggered a cascade process (activation of G protein, adenylate cyclase, cAMP, cAMP-dependent protein kinase, and protein phosphorylation) that is not readily reversed by blockade at the level of ß-adrenergic receptors. If phosphorylation plus these later events is required for stimulation, forskolin, a direct activator of adenylate cyclase, should also stimulate TI.

The effects of forskolin (1 µmol/L) on high-Ca²⁺–induced TI in the presence of [Na⁺]_o are shown in Fig 7. Experiments were performed and recorded under conditions identical to those described for Figs 2 and 3. The original current traces recorded in the absence and presence of ISO from one preparation are illustrated in Fig 7A and 7B, respectively. The effects of forskolin on TI were identical to those of ISO. Five minutes after exposure to forskolin, both inward and outward TIs were significantly increased at all test potentials, and the time to peak TI was decreased (Fig 7B). Quantified data showing the I-V relations for TI in control conditions and in the presence of forskolin, which were obtained from four preparations, are plotted in Fig 7C. The increases in TI were greater near the extremes of test potentials, and E_REV was not affected (Fig 7C). The forskolin-sensitive current is plotted in Fig 7D. Forskolin increased TI without significantly affecting its voltage dependence. Pretreatment of preparations with propranolol (0.2 µmol/L) did not prevent stimulation of TI by forskolin (data not shown), indicating that the effects of forskolin were independent of agonist binding at the ß-adrenergic receptor.

View larger version (28K): [in this window] [in a new window]   Figure 7. Effects of forskolin (Forsk, 1 µmol/L) on high-Ca2+–induced transient inward current (TI) in the presence of [Na+]o. Experiments were performed and recorded under conditions identical to those described in Fig 2. A and B, Superimposed current traces recorded in the same preparation showing control conditions and the effects of Forsk, respectively. C, Graph showing current-voltage relations of TI obtained in the absence () and presence () of Forsk. D, Graph showing Forsk-sensitive current. These data were quantified from results obtained in four preparations. Five minutes after exposure to Forsk, both inward and outward TIs were significantly increased at all test potentials (B). Forsk increased the peak TI without affecting its voltage dependence (C and D). Thus, the increases were greater near the extremes of test potentials. Forsk also increased a set of inward TIs seen at -55 mV (as described in Fig 4).

Effects of Forskolin in the Absence of [Na⁺]_o
If forskolin promotes [Ca²⁺] overload and induction of TI by inducing phosphorylation of the Na⁺-Ca²⁺ exchanger protein or some other factor essential to stimulation of exchanger activity, the removal of [Na⁺]_o should interrupt the stimulatory effects of forskolin. The effects of forskolin on TI in Na⁺-free solution are illustrated in Fig 8, which is representative of observations made in three Purkinje fibers. The experiments were performed and recorded under conditions identical to those described for Fig 5. Fig 8A shows superimposed current traces recorded before (trace a) and after (trace b) the addition of forskolin (1 µmol/L). Fig 8B illustrates the voltage steps applied to the preparation. Neither inward nor outward TI was changed after the administration of forskolin. However, I_step clearly was increased by forskolin in the absence of [Na⁺]_o.

View larger version (10K): [in this window] [in a new window]   Figure 8. Absence of stimulatory effects of forskolin (1 µmol/L) on the transient inward current (TI) in Na+-free solution. A, Superimposed current traces recorded before (trace a) and after (trace b) the addition of forskolin. B, Voltage steps applied to the preparation. Neither inward nor outward TIs showed significant change in response to forskolin. However, the outward current activated by the end of a 1-s depolarization pulse was markedly increased by forskolin in the absence of [Na+]o.

Effects of Inhibitors of Na⁺-Ca²⁺ Exchange on Stimulation of TI by ISO and Forskolin
Experiments presented so far suggest that the enhancement of Na⁺-Ca²⁺ exchange via a phosphorylation-dependent process is essential for the stimulation of TI by ISO or forskolin. Further evidence supporting this conclusion comes from a previous study in which we demonstrated that pretreatment with quinacrine, a blocker of Na⁺-Ca²⁺ exchange, prevents stimulation of TI by ISO.²⁰ If stimulation of Na⁺-Ca²⁺ exchange is an essential "downstream" link in the cascade of events leading to the stimulation of TI, Na⁺-Ca²⁺ exchange blockers might reverse or block stimulation of TI when added after stimulation is initiated by ISO, unlike ß-adrenergic blockers. Fig 9 shows the effects of dodecylamine (20 µmol/L) on ISO (0.1 µmol/L) stimulation of TI. Voltage steps applied to the preparations are illustrated on the top. In Fig 9A, the current traces from top to bottom were recorded in control, dodecylamine-containing, and dodecylamine plus ISO–containing solutions, respectively. Pretreatment with dodecylamine did not result in significant changes in both inward and outward TIs (middle) but simply blocked the stimulatory effect of ISO (bottom). Thus, in the two preparations that were pretreated with dodecylamine, outward TIs were 6.0±1.4, 6.4±2.1, and 6.9±1.7 nA (mean±SD) under conditions of control, dodecylamine, and dodecylamine plus ISO, respectively; inward TIs were -15.4±3.9, -14.6±3.4, and -16.7±4.2 nA under conditions of control, dodecylamine, and dodecylamine plus ISO, respectively. In Fig 9B, the current traces from top to bottom were recorded in control, ISO, and ISO plus dodecylamine–containing solutions, respectively. Three minutes after the addition of ISO (0.1 µmol/L), both inward and outward TIs were dramatically increased. At this time, dodecylamine was added to the perfusing solution. After 5 minutes of exposure to dodecylamine, stimulation of TI was completely reversed (bottom). Averaged data obtained in five preparations showed that outward TIs were 5.7±1.5, 11.4±2.0 (P<.05), and 6.2±1.7 (P>.1) nA under conditions of control, ISO, and ISO plus dodecylamine, respectively; inward TIs were -16.8±4.3, -35.6±6.7 (P<.05), and -18.2±3.6 (P>.05) nA under conditions of control, ISO, and ISO plus dodecylamine (mean±SD), respectively. Although dodecylamine blocked the ISO stimulation of TI, it did not block the ISO-induced increase in outward current, which was activated on depolarization to +20 mV, and the shortening of time to peak inward TI.

View larger version (16K): [in this window] [in a new window]   Figure 9. Effects of dodecylamine (20 µmol/L) on isoproterenol (ISO, 0.1 µmol/L) stimulation of the transient inward current (TI). Voltage steps applied to the preparations are illustrated on the top. A, Current traces from top to bottom were recorded in control, dodecylamine, and dodecylamine plus ISO–containing solutions, respectively. Pretreatment with dodecylamine by itself did not result in significant changes in either inward or outward TIs but prevented the stimulatory effect of ISO. B, Current traces from top to bottom were recorded in control, ISO, and ISO plus dodecylamine–containing solutions, respectively. Enhancement of TI by ISO was reversed by dodecylamine. Outward and inward TIs are denoted by o and i, respectively. Zero current level is indicated by the horizontal line.

Quinacrine (10 µmol/L), another Na⁺-Ca²⁺ exchange blocker, which is structurally different from dodecylamine, also reversed the ISO stimulation of TI. Fig 10A shows such an experiment, which is typical of observations made in three Purkinje fibers. The voltage steps applied to the preparation are shown on the top. The current traces from top to bottom were recorded in control, ISO-containing, and ISO plus quinacrine–containing solutions, respectively. Three minutes of exposure to quinacrine completely reversed the ISO-induced increases in both inward and outward TIs (bottom trace). In three Purkinje fibers, outward TIs were 5.3±0.8, 10.6±1.7 (P<.05), and 5.5±1.7 (P>.2) nA under conditions of control, ISO, and ISO plus quinacrine, respectively; inward TIs were -19.2±4.6, -36.7±5.8 (P<.05), and -17.8±4.1 (P>.1) nA under conditions of control, ISO, and ISO plus quinacrine (mean±SD), respectively.

View larger version (16K): [in this window] [in a new window]   Figure 10. Effects of quinacrine (10 µmol/L) on isoproterenol (ISO, 0.1 µmol/L)– and forskolin (1 µmol/L)–stimulated transient inward current (TI). Quinacrine reverses stimulation of TI by ISO or forskolin. A, Effect of quinacrine on ISO stimulation of TI. Voltage steps applied to the preparation are shown at the top. The current traces from top to bottom were recorded in control, ISO, and ISO plus quinacrine–containing solutions, respectively. B, Effect of quinacrine on forskolin-induced enhancement of TI. Voltage step changes are illustrated on the top. Current traces from top to bottom were recorded in control, forskolin, and forskolin plus quinacrine–containing solutions, respectively. Outward and inward TIs are denoted by o and i, respectively. Zero current level is indicated by the horizontal line.

Complete reversal of stimulation by quinacrine (n=3) was observed when TI was stimulated by forskolin. Fig 10B shows the effect of quinacrine (10 µmol/L) on forskolin (1 µmol/L)–induced enhancement of TI. Voltage-step changes are illustrated on the top. Current traces from top to bottom were recorded in control, forskolin-containing, and forskolin plus quinacrine–containing solutions, respectively. Quinacrine apparently reversed the forskolin stimulation of both inward and outward TIs (bottom). In the three Purkinje fibers tested, outward TIs were 5.4±1.5, 10.2±2.1 (P<.05), and 6.1±1.7 (P>.01) nA under conditions of control, ISO, and ISO plus quinacrine, respectively; inward TIs were -20.4±5.3, -41.6±7.7 (P<.05), and -18.4±4.4 (P>.05) nA under conditions of control, ISO, and ISO plus quinacrine (mean±SD), respectively. Similar results were obtained in two additional preparations when dodecylamine (20 µmol/L) was applied after forskolin. Taken together, these observations strongly suggest that stimulation of Na⁺-Ca²⁺ exchange is the central action mediating ß-adrenergic stimulation of TI and that stimulation involves the adenylyl cyclase and possibly a phosphorylation step.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, a mechanism of ß-adrenergic arrhythmogenicity has been investigated in voltage-clamped rabbit cardiac Purkinje fibers. The most important conclusion drawn from the present study is that stimulation of Na⁺-Ca²⁺ exchange is likely a major mechanism by which ß-adrenergic agonists stimulate the arrhythmogenic TI. Furthermore, our observations suggest that the stimulation of TI is secondary to the elevation of [Ca²⁺]_i rather than a direct effect on TI channels. Because all effects of ISO can be mimicked by forskolin, a direct activator of adenylate cyclase,³² it is likely that a cAMP-dependent protein kinase activation and phosphorylation process is involved in the ß-adrenergic stimulation of TI.

The ionic mechanism of TI has been a subject of many studies, since this current was first demonstrated.¹⁰ Until recently, it was generally believed that two charge-carrying systems might conduct TI: a nonselective cationic channel and electrogenic Na⁺-Ca²⁺ exchange.^{8 9} Our recent studies show that TI induced by the elevation of [Ca²⁺]_o in rabbit cardiac Purkinje fibers is conducted through TI channels, not Na⁺-Ca²⁺ exchange.^{26 27} Furthermore, TI channels consist of two different classes: nonselective cationic channels, which mainly conduct inward TI at negative membrane potentials, and an anionic (Cl^-) channel, which conducts outward TI at membrane potentials positive to the reversal potential of Cl^- current (E_Cl). The Cl^- conductance also contributes to inward TI at membrane potentials negative to E_Cl.²⁶ The two classes of TI channels can be modulated differentially by a number of agents.²⁷

Clarification of the ionic basis of the TI in Purkinje fibers allows us to attempt to determine the mechanism by which ß-adrenergic stimulation enhances TI and thereby promotes OAP-initiated arrhythmias. It is well known that ß-adrenergic stimulation can lead to sequential activation of the stimulatory guanine nucleotide–binding regulatory protein (G protein); adenylate cyclase, which synthesizes cAMP from ATP; and cAMP-dependent protein kinase, which phosphorylates a number of membrane proteins including L-type Ca²⁺ channels and delayed rectifier K⁺ channels. Phosphorylation of these channels results in increased magnitudes of the currents (I_Ca(L) and I_K). Therefore, it is conceivable that ß-adrenergic enhancement of TI could be mediated by a similar mechanism, ie, phosphorylation of TI channels. Our results clearly do not support this explanation, because when both inward and outward TIs were induced by high Ca²⁺ in the absence of Na⁺, no stimulation was observed with ISO (Fig 5). Under the same experimental conditions, I_step (which reflects I_K activation) was increased by ISO, indicating that ß-adrenergic–dependent phosphorylation had been activated. In addition, ß-adrenergic receptor–initiated phosphorylation also has been observed by other investigators in Na⁺-free solutions.^{35 36}

Although ISO failed to stimulate TI in the Na⁺-free solution, it increased the outward deflection seen on the initial depolarization to +20 mV (Fig 5). For some preparations, this deflection resulted in an oscillation that superficially resembled an outward TI. However, this deflection occurs during a period when at least three membrane currents are activated (I_Ca(L), I_K, and I_TO) and cannot be identified as an outward TI for a number of reasons: (1) Slight differences in activation time for I_Ca(L), I_K, and I_TO could easily generate an oscillation in total current. (2) The time to peak outward deflection during depolarization is slower than the time to peak outward TI. (3) The time to peak outward deflection during depolarizations to different levels shows marked variability, whereas the time to peak outward TI is relatively constant. (4) The outward deflection during depolarization is seen only in some preparations, but the outward TI is seen in all experiments. (5) I_TO and I_Ca(L) should inactivate and remain inactivated at test potentials around 0 (-20 to +5) mV following a previous depolarization to +20 mV,³⁷ whereas outward TI clearly can be elicited by repolarizing steps in this range of potentials. Thus, it is reasonable to believe that the increased outward deflection in response to ISO during depolarization to +20 mV (Fig 5) is likely due to effects of ISO in I_Ca(L), I_K, or I_TO rather than stimulation of outward TI. This interpretation is consistent with known effects of ISO on the three major currents activated on depolarization.^{34 38 39}

It has been reported that there is a ß-adrenoceptor–stimulated cAMP-dependent Cl^- current (I_Cl(cAMP)) in heart^{40 41} and that this current is sensitive to the removal of [Na⁺]_o. Failure of ISO to stimulate TI, especially the outward TI conducted through a Cl^- channel, may therefore be related to the inability of ISO to initiate phosphorylation of TI channels in the absence of Na⁺. Although we cannot totally eliminate this possibility, we believe that this is very unlikely, because of the fundamental differences between the Cl^--conducting TI channels found in our experiments and I_Cl(cAMP). First, the Cl^- channels that conduct outward TI under our experimental conditions do not require the presence of ß-adrenergic agonist or cAMP to be activated. In contrast, very little basal I_Cl(cAMP) can be found in the absence of ß-receptor stimulation.⁴² Second, outward TI is activated only by elevation of [Ca²⁺]_i, because ryanodine, which abolishes release of [Ca²⁺]_i from the sarcoplasmic reticulum, totally eliminates TI,²⁶ whereas I_Cl(cAMP) is not affected by buffering [Ca²⁺]_i.⁴¹ On the other hand, a class of Ca²⁺-dependent Cl^- channels (I_Cl(Ca)) has been described recently in single Purkinje cells.⁴³ Activation of I_Cl(Ca) does not require the presence of cAMP or cAMP-generating agents but is solely dependent on [Ca²⁺]_i. Therefore, it is likely that the outward TI is conducted through this type of Cl^- channel.

Since TI is a Ca²⁺-activated current, any intervention that increases [Ca²⁺]_i overload should also increase the TI.⁸ Two pathways are thought to be important in causing [Ca²⁺]_i overload: inward Ca²⁺ currents through both T- and L-type Ca²⁺ channels³⁸ and Na⁺-Ca²⁺ exchange.^{28 29 30 31} T-type Ca²⁺ current is relatively small in the heart and has been reported to be insensitive to ß-adrenergic stimulation³⁸ ; therefore, the most likely candidates are the L-type Ca²⁺ channel and Na⁺-Ca²⁺ exchanger.

Although I_Ca(L) can be stimulated strongly by either a G protein or by cAMP-dependent phosphorylation, experimental evidence presented in the present study does not support I_Ca(L) as a major factor in ß-adrenergic stimulation of TI. This could be inferred from the observations that (1) no significant increase in TI was observed with ISO in the absence of [Na⁺]_o (Fig 5) and (2) significant stimulation of inward TI was seen at -55 mV after I_Ca(L) had been inactivated (Fig 4). Caution has to be taken in interpreting these data, because we did not directly measure I_Ca(L). Measurement of I_Ca(L) would require blockade of other interfering membrane currents, such as I_TO and I_K, and doing this may affect the induction of TI in our experiments. However, the increase in I_Ca(L) may be minimal for a number of reasons. First, ß-adrenergic modulation of the L-type Ca²⁺ channel is thought mainly to increase the existing channel conductance, which has been already saturated in the presence of high [Ca²⁺]_o.³⁸ Therefore, any further increase in the channel conductance by phosphorylation of the channel may not significantly increase I_Ca(L). Second, in the presence of an already elevated [Ca²⁺]_i, Ca²⁺-dependent inactivation of I_Ca(L)³⁸ can counterbalance the ISO-stimulated increase in I_Ca(L) (if any). Third, we have previously shown that blockade of L-type Ca²⁺ channels with Mn²⁺ or verapamil failed to prevent stimulation of TI by ISO.²⁰

The absence of stimulation of TI in Na⁺-free solution (Figs 5 and 8) and the presence of strong stimulation in Na⁺-containing solution (Figs 1 through 4, 6, 7, 9, and 10) are consistent with a role of Na⁺-Ca²⁺ exchange. Ca²⁺ entry through this pathway has been well established under conditions of elevated [Na⁺]_i.⁴⁴ Convincing evidence also exists for the direct contribution of Na⁺-Ca²⁺ exchange to the elevation of cytosolic Ca²⁺ on membrane depolarization.^{28 29 30 31} A role for Na⁺-Ca²⁺ exchange in the stimulation of TI is supported by our observation that two putative Na⁺-Ca²⁺ exchange blockers, quinacrine and dodecylamine,^{28 31 45} could abolish or reverse ISO stimulation of TI induced by either digitalis toxicity²⁰ or high Ca²⁺ (the present study). TI was not significantly affected after 5 minutes of exposure to 20 µmol/L dodecylamine (Fig 9A). Also, five minutes of exposure to these blockers only reversed the ISO-stimulated TI instead of abolishing it (Fig 10). These observations indicate that TI may not be affected significantly by nonspecific effects of these blockers (eg, on I_Ca(L) and I_Cl(Ca)) within the time frame used in the present study (5 to 10 minutes).

Evidence for phosphorylation/dephosphorylation of the Na⁺-Ca²⁺ exchanger has been reported previously in squid axons⁴⁶ and isolated cardiac sarcolemma.⁴⁷ The phosphorylation process increased both the affinity of the exchanger for Ca²⁺ and the maximal velocity of Ca²⁺ transport. Although phosphorylation does not seem to be involved in the ATP-induced stimulation of the Na⁺-Ca²⁺ exchange current in isolated giant membrane patches, this does not rule out the possibility of Na⁺-Ca²⁺ exchange regulation in intact cells by the cAMP-dependent protein kinase.⁴⁸ Therefore, it is possible that ß-adrenergic stimulation potentiates Ca²⁺ entry during depolarization by regulating phosphorylation of the Na⁺-Ca²⁺ exchanger protein or some other factor necessary for the stimulation or increased expression of the exchanger. Several lines of evidence support this hypothesis: (1) ISO stimulation of TI was abolished by pretreating the preparations with ß-blockers, which suggests that this effect is mediated by ß-adrenoceptors. (2) ß-Blockade only partially reversed ISO stimulation of TI once the increase in TI was evident, which is compatible with a cascade process that is triggered by the agonist-receptor binding and which is not stopped immediately by dissociation of the agonist from the receptor. The incomplete reversal of ISO-stimulated TI is more likely due to the slow removal of Ca²⁺ from the sarcoplasmic reticulum by dephosphorylation of phospholamban^{2 49} than a partial ß-receptor blockade, especially when observations were made with propranolol for a short time (5 minutes). (3) Forskolin, a direct adenylate cyclase activator that generates cAMP and initiates protein phosphorylation, mimicked the effects of ISO, including the absence of stimulation of TI in Na⁺-free solution and the presence of stimulation in Na⁺-containing solution. (4) Propranolol had no effect on forskolin stimulation of TI (which also indicates that the effect of propranolol was not nonspecific). Under physiological conditions, phosphorylation of the Na⁺-Ca²⁺ exchanger may not cause significant Ca²⁺ loading. Indeed, TI could not be induced by ISO alone in either Purkinje fibers²⁰ or sinoatrial nodal cells¹⁹ perfused with Tyrode's solution. However, if transmembrane Na⁺ and Ca²⁺ gradients are imbalanced so as to favor Ca²⁺ influx via the Na⁺-Ca²⁺ exchanger, ß-adrenergic stimulation should cause greater [Ca²⁺]_i overload and therefore potentiate TI- and OAP-initiated cardiac arrhythmias.

	Acknowledgments

 
This study was supported in part by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Nova Scotia. Dr Han is a fellow of the Medical Research Council of Canada. The authors wish to thank Claire Guyette and Louise Deal for valuable technical assistance.

	Footnotes

 
Reprint requests to Gregory R. Ferrier, PhD, Department of Pharmacology, Sir Charles Tupper Medical Bldg, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.

Received October 3, 1994; accepted December 20, 1994.

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