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Cardiovascular Research 2006 70(1):88-96; doi:10.1016/j.cardiores.2006.01.010
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Copyright © 2006, European Society of Cardiology

Sphingosine-1-phosphate effects on guinea pig atrial myocytes: Alterations in action potentials and K+ currents

Rikuo Ochia,b, Yasunori Momoseb, Kosuke Oyamab and Wayne R. Gilesa,*

aDepartments of Bioengineering and Medicine, University of California San Diego, La Jolla, California, USA
bDepartment of Clinical Pharmacy, School of Pharmaceutical Sciences, Toho University, Funabashi, Japan

* Corresponding author. Department of Bioengineering and Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0412, USA. Tel.: +1 858 822 4424; fax: +1 858 534 4535. Email address: wgiles{at}bioeng.ucsd.edu

Received 23 August 2005; revised 30 December 2005; accepted 10 January 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective Sphingosine-1-phosphate (S-1-P), a potent lysophospholipid mediator which is released from platelets during clotting, activates a G-protein-gated inwardly rectifying K+ current (GIRK) in atrial and sino-atrial node myocytes. We denote this current IK(S-1-P). A similar GIRK, which is activated by acetylcholine (ACh) and denoted IK(ACh), is expressed in atrium. It shortens the action potential duration (APD) and reduces the effective refractory period (ERP). We have examined the effect of S-1-P on APD in guinea pig atrial myocytes by characterizing the rectification properties of IK(S-1-P) and evaluating whether IK(S-1-P) and IK(ACh) exhibit convergence/occlusion.

Methods Membrane potential and K+ currents were recorded from guinea pig atrial myocytes. Inwardly rectifying K+ currents were recorded using a ramp voltage clamp waveform between +30 to – 130mV from a holding potential of – 7mV. Agonist-induced current changes were obtained by subtracting the control current.

Results S-1-P (1 and 10nM) altered both passive and active properties of atrial myocytes. S-1-P increased the threshold current for excitation and decreased the time constant of the subthreshold electrotonic potentials. In addition, both APD50 and APD90 were decreased substantially. Voltage clamp analysis showed that the outward conductance of IK(IR) (GK(IR)out) was 134.8±11.3pS pF– 1 (n=19) in S-1-P (100nM), and 207.0±19.6pS pF– 1 (n=18) in ACh (10µM). The ratio of GK(IR)out:GK(IR)in was about 0.7 for both S-1-P and ACh. The EC50 values for the activation of GK(IR)out and GK(IR)in by S-1-P were 1.6 and 1.3nM, respectively. Addition of S-1-P (100nM) after the effect of ACh (10µM) had developed fully caused very little additional change.

Conclusion IK(S-1-P) is carried by weakly inwardly-rectifying K+ channels that are the same as those activated by ACh. This K+ current can markedly shorten APD in guinea pig atrial myocytes. This effect would be expected to increase the incidence of atrial rhythm disturbances.

KEYWORDS Arrhythmia (mechanism); Atrial function; K-channel; Lipid signaling; Sphingosine-1-phosphate

This article is referred to in the Editorial by R. Schubert (pages 9–11) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Sphingosine-1-phosphate (S-1-P), is a membrane-derived lipid signaling substance. It can modulate cell proliferation, migration, and differentiation as well as promoting apoptosis and other pathophysiological phenomena in various tissues, including the heart and blood vessels [1,2]. S-1-P binds to well-defined plasmalemma receptor proteins (S-1-P/Edg receptors) which are linked to specific G proteins [3,4].

In guinea pig atrial myocytes, S-1-P, at nanomolar concentrations, produces an inwardly rectifying K+ current (IK(S-1-P)) via a pertussis toxin-sensitive G protein [5]. IK(S-1-P) increases the cycle length in rabbit sino-atrial node cells [6]. Intravenous administration of S-1-P causes significant negative chronotropic and inotropic effects in rat heart [7]. Human trials have demonstrated that FTY720, an agent which can alter immune status and is used in kidney transplant procedures [8,9], produces transient bradycardia and lymphopenia [10]. A recent study in mice [11] has shown that this FTY720-induced decrease in heart rate is also caused by activation of G-protein-gated K+ channels.

In atrial myocytes, a G protein coupled receptor (GPCR)-mediated K+ conductance has been shown to markedly accelerate repolarization and may also produce a small hyperpolarization [12,13]. This shortening of APD in atrial myocytes decreases the effective refractory period (ERP) and increases its spatial heterogeneity. These effects contribute to the initiation and maintenance of supraventricular arrhythmias, including atrial flutter and fibrillation (AF) [14,15]. Chronic AF is often accompanied by hypercoagulable state of the blood [16]. As S-1-P can be released from platelets by thrombin [17,18] or from blood clots [19,20], it could shorten the APD and thus may predispose to supraventricular arrhythmias.

S-1-P-induced K+ current and associated S-1-P receptors have been identified in guinea pig atrial myocytes in culture [5,21] as well as in freshly isolated atrial myocytes from mouse and human [22]. However, the effect of S-1-P on APD has not been studied in detail, and important biophysical principles which regulate the extent of the inward rectification of the IK(S-1-P) have not been determined (Ref. [1] is a comprehensive review of S-1-P effects in the cardiovascular system). In the present study using guinea pig atrial myocytes, the effects of S-1-P on APD were measured, and the ion transfer or rectifier properties of IK(S-1-P) were derived. In addition, we determined whether S-1-P activates K+ currents other than the acetylcholine-sensitive current, IK(ACh). Our results demonstrate that S-1-P can markedly shorten APD, and that the corresponding K+ current, IK(S-1-P), exhibits weak inward rectification, very similar to IK(ACh). Our analysis also confirms that IK(S-1-P) and IK(ACh) are carried by the same population of K+ channels.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
The experimental work in this paper was conducted at Toho University in accordance with the guiding principles for the care and use of animals in the field of Physiological Sciences of Japan. These procedures were also in accordance with the US National Institutes of Health (NIH Publication NO. 85-23, revised 1996).

2.1 Isolation of guinea pig atrial myocytes
Guinea pigs (300~450g) were heparinized (1200U kg– 1 i.p.) and then anesthetized with sodium pentobarbitone (50mg kg– 1 i.p.). After cervical dislocation, hearts were quickly removed and Langendorff-perfused at 37°C as follows: normal Tyrode's solution for 2min, Ca2+-free Tyrode's solution for 5min, and Ca2+-free Tyrode's solution with collagenase (1mg/ml, Sigma Type 1, Sigma Chemical, St Louis, MO, U.S.A.) for 15min. After this digestion with collagenase, the heart was perfused for 5min with a modified Kraftbrühe (KB) solution containing (mM): 100 potassium glutamate, 10 potassium aspartate, 25 KCl, 10 KH2PO4, 2 MgSO4, 20 taurine, 5 creatine base, 0.5 EGTA, 5 HEPES, 20 glucose, and 1mg ml– 1 BSA (pH adjusted to 7.3 with KOH). The atria were then removed, placed in KB solution and cut into small pieces. These atrial tissue segments were mechanically separated into single myocytes by gentle trituration at room temperature using a fire-polished Pasteur pipette with an inner diameter of 3mm. Populations of isolated myocytes in KB solution were kept in the refrigerator until they were introduced into the recording chamber, approximately 20min before the patch clamp experiments began.

2.2 Electrophysiological measurements
Normal Tyrode's solution contained (in mM): 135 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 5 HEPES, and 10 glucose. pH was adjusted to 7.4 with NaOH. Effects of S-1-P on membrane potential were examined by superfusing the myocytes with normal Tyrode's solution. The voltage clamp experiments were conducted in the presence of 0.2mM Cd2+ which reversibly blocked L-type Ca2+ current channels. The whole-cell pipette solution contained (in mM): 30 KCl, 100 potassium aspartate, 1 MgCl2, 5 HEPES, 10 EGTA, 3 Na2ATP and 0.3 GTP. pH was adjusted to 7.3 with KOH. Sphingosine-1-phosphate (S-1-P, Avanti, Alabaster, AL, U.S.A.) stock solution (0.1mM) was prepared by incubating S-1-P in 4mg/ml fatty acid-free bovine serum albumin solution for 30min at 37°C. Acetylcholine chloride was from Sigma Ltd. (St. Louis, MO, U.S.A.).

For electrophysiological recordings, an aliquot of atrial myocyte suspension was allowed to settle for several minutes in a shallow rectangular chamber (approximate volume of 0.2ml) that was mounted on the stage of inverted microscope (IX-70, Olympus, Tokyo, Japan). This chamber was continuously superfused with modified Tyrode's solution at a flow rate of 1~2ml min– 1. S-1-P and other pharmacological agents were applied using a three-way switch connected in series to the tube that superfused the chamber. Patch pipettes were made from borosilicate glass and their resistance was 4~8M{Omega} when filled with the pipette solution. Whole-cell currents were recorded by patch clamp (whole-cell mode) using Axopatch 1-D patch-clamp amplifier (Axon Instruments, Foster City, CA, U.S.A.). The junction potential between pipette solution and the Tyrode's solution was corrected by 10mV as outlined in our previous study [23]. Membrane potential and voltage clamp procedures were controlled by pClamp (v. 6, Axon Instruments). Membrane currents were filtered at 2kHz, digitized at 10kHz and stored. Both membrane potential and currents were digitized at 4kHz and recorded continuously using Powerlab Software (Chart v. 5, ADInstruments, Castle Hill, Australia). Off-line data analysis was done using Igor Pro 4 or 5 (Wavemetrics Inc., Lake Oswego, OR, U.S.A.). Membrane capacitance (Cm) was estimated by integrating the charging transients elicited by a 5mV hyperpolarizing voltage clamp step from – 70mV. In most voltage clamp experiments ramp waveforms (150ms in duration) were applied between +30 and – 130mV from a holding potential (HP) of – 60mV at 0.1Hz. The outward (GK(IR)out) and inward conductances (GK(IR)in) of the inwardly rectifying K+ currents were quantified by calculating the chord conductances at membrane potentials near the reversal potential (Erev), i.e. within voltage ranges ±40mV from Erev. APs were elicited in current clamp experiments by applying 2.5ms rectangular current steps. These stimuli were applied in 100pA increments at 0.2Hz. Action potential durations were measured in terms of APD30, APD50 and APD90. Each of these parameters represents the time interval between the time of the peak of AP and the time to a 30%, 50% or 90% decrease of the AP amplitude, respectively. All experiments were conducted at room temperature (22±1°C).

2.3 Data analysis
S-1-P-induced K+ currents were obtained by subtracting all background currents which were recorded prior to application of S-1-P. The fixed dV/dt of the ramp command waveforms used in voltage clamp experiments resulted in there being no measurable changes in the charging or discharging of the capacitance of the myocyte. Possible differences of parameters among multiple groups were evaluated statistically using one or two way ANOVA, followed by Bonferroni's multiple comparison test, except when otherwise described (Prism v. 4, GraphPad Software Inc., San Diego, CA, U.S.A.). Statistical data are presented as means±s.e. of the mean. P<0.05 was the criterion used to establish a statistically significant difference.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effects of S-1-P on atrial action potential repolarization
Superfusion of S-1-P (1 and 10nM) onto atrial myocytes freshly isolated from the hearts of adult guinea pigs resulted in a marked and dose-dependent shortening of APD (Fig. 1). S-1-P, even at a very low concentration (1nM), caused significant shortening of APD and this effect was completely reversible (Fig. 1A). A subsequent increase of S-1-P concentration, from 1 to 10nM, caused a more pronounced shortening of APD. These large changes were not completely reversible during a 3min washout (Fig. 1B). S-1-P consistently shortened APD when its effects were measured in terms of reductions in APD30, APD50 and APD90. The S-1-P effects on APD50 and APD90 were statistically significant (Fig. 1C). APD90, which corresponds to ERP, decreased from its control value, 98.0±11.4ms (n=25) to 42.5±5.2ms (n=11, P<0.001 compared to control) in the presence of 1nM S-1-P. APD90 was further reduced to 15.6±1.0pA (n=14, P<0.001 compared to control) by 10nM S-1-P. We noted also that S-1-P increased the stimulus current needed to elicit an AP (Fig. 2A~C). At a stimulus duration of 2.5ms, the threshold current increased from the control level 445±25pA (n=23) to 550±20pA (n=10, P<0.05) in the presence of 1nM S-1-P. The threshold current increased further to 930±65pA (n=13, P<0.001) when 10nM S-1-P was present in the superfusate. These changes were due mainly to activation of IK(S-1-P), which decreased the amplitude and time to peak of the electrotonic potentials during the stimulus current pulses (Fig. 2A~C). In the subthreshold range, the electrotonic changes in membrane potential decayed faster in the presence of S-1-P than in control conditions. The time constant of the decay phase of these electrotonic changes in membrane potential were decreased from control 5.6±0.4ms (n=25) to 3.3±0.5ms (n=11, P<0.001 compared with control) by 1nM S-1-P. In 10nM S-1-P this time constant decreased further to 1.5±0.1ms (n=14, P<0.001 compared with control and 1nM S-1-P). In contrast to these significant changes, the resting membrane potential (RP) changed only slightly. The control RP was – 82.5±0.5mV (n=25) and it was – 83.4±0.5 (n=11) and – 84.2±0.8mV (n=14), in the presence of 1 and10 nM S-1-P, respectively.


Figure 1
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  		Fig. 1 Effect of S-1-P on the duration of the membrane action potential (APD) in myocytes from guinea pig atrium. S-1-P (1nM) produced a reversible shortening of APD (A). Application of 10nM S-1-P shortened APD more markedly. This effect was not completely reversible during a 3min washout (B). (C) Effect of S-1-P (1 and 10nM) on APD. APD30, APD50 and APD90 during control, and following S-1-P (1 and 10nM). Data are plotted versus the normalized APD parameter. Results are presented as mean±s.e.; **P<0.01; ***P<0.001 compared with the control data.

 

Figure 2
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  		Fig. 2 Effect of S-1-P on threshold current and electrotonic membrane potentials in guinea pig atrial myocytes. Electrotonic potentials due to subthreshold stimulation and the initial part of first action potential elicited by suprathreshold stimulation are shown. These depolarizing stimuli (with a duration of 2.5ms) were applied in increments of 100pA, as shown in bottom traces. The largest membrane potential change in B was generated by a premature excitation. Note that the passive electrotonic potentials caused by the subthreshold current pulses decayed exponentially, and that the rate of their decay was accelerated by S-1-P.

 
3.2 Voltage-dependent properties of S-1-P-activated K+ current
The effects of S-1-P at levels between 1 and 100nM on inwardly rectifying K+ currents, IK(IR), were recorded from voltage clamped atrial myocytes in the presence of 5.4mM [K+]o. S-1-P resulted in rapid increases in both the holding current at – 60mV and the current changes elicited by a 150-ms ramp waveform from +30 to – 130mV. In each experiment, S-1-P (10nM) produced a marked increase in the outward current at membrane potentials positive to the resting potential, as well as increasing the inward current at hyperpolarized potentials (Fig. 3A). The S-1-P sensitive current (IK(S-1-P)) was isolated by subtracting the control or background current. It showed weak inward rectification and had an apparent reversal potential (Erev) near the expected K+ equilibrium potential (EK, – 88.5mV). In contrast, in all experiments the control I–V exhibited much stronger inward rectification. This pattern of results was expected since it is known that the endogenous inwardly rectifying background K+ current, IK1, is due to Kir2.1/2.2 transcripts.


Figure 3
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  		Fig. 3 Current–voltage (I–V) relations of S-1-P and ACh induced K+ currents. S-1-P- or ACh-induced changes in whole-cell currents in guinea pig atrial myocytes recorded in the presence of 5.4mM [K+]o. A, Typical effect of S-1-P on membrane current. Currents were elicited by a ramp voltage clamp waveform between +30 and – 130mV, starting from HP of – 60mV. Time calibration bar indicates that the duration of the ramp pulse was similar to APD in Fig. 1. Control current and current changes in the presence of S-1-P (10nM) are shown. IK(S-1-P) was obtained as the difference between the control current and the current after washout of S-1-P. B, Comparison of I–V relations of IK(S-1-P) and IK(ACh) denoted as averaged current densities (mean±s.e.). Note that these current densities are significantly different at several membrane potentials; ***P<0.001, **P<0.01, *P<0.05.

 
Fig. 3B compares the I–Vs of IK(S-1-P) in the presence of 100nM with IK(ACh) in the presence of 10µM ACh. Note that the I–Vs for both IK(S-1-P) and IK(ACh) show only moderate inward rectification. Specifically, these I–V relationships were approximately linear in the inward direction but in the outward direction the slope conductance decreased beginning at membrane potential at about 20mV positive than Erev. The voltage dependence of IK(S-1-P) was very similar to that of IK(ACh). Erev for IK(S-1-P) and for IK(ACh) were close to the EK (Table 1).


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  		Table 1 Comparison of parameters of S-1-P- and ACh-activated K+ currents

 
To provide a quantitative basis for comparing these two K+ currents chord conductances were calculated. The outward chord conductance (GK(IR)out) and its inward equivalent (GK(IR)in) of IK(S-1-P) and IK(ACh) were calculated in a range of membrane potentials (±40mV from Erev, see Table 1). Note that both GK(IR)out and GK(IR)in of IK(S-1-P) are smaller than those for IK(ACh). This difference was statistically significant. Our results showed that the GK(IR)out activated by S-1-P was only approximately 0.6 as large as that activated by ACh.

Additional analysis of the rectifier properties of these ligand-gated inwardly rectifying K+ channels was done in terms of an inward rectification factor, (Fir). We define this as the ratio: GK(IR)out/GK(IR)in [24]. This parameter was estimated from the raw data of each experiment in this series. Fir averaged approximately 0.7 for both IK(S-1-P) and IK(ACh) (Table 1). The much more pronounced inward rectification of the background IK1 current yielded an Fir value of 0.16±0.01 (n=36). The close agreement of the Erev values and Fir ratio between IK(S-1-P) and IK(ACh) strongly suggests that S-1-P and ACh can activate an identical population of K+ channels in the atrial myocardium.

3.3 Dose–response relationship of S-1-P-induced K+ currents
Progressive increases in S-1-P concentration produced corresponding increases in both GK(IR)out and GK(IR)in (Fig. 4). The resulting dose–response curves were well fitted by a Hill equation with EC50 of 1.6 and 1.3nM for GK(IR)out and GK(IR)in, respectively. The inward rectification factor (Fir), was not affected by changes in S-1-P concentration; 0.58±0.08 (n=20) at 1nM, 0.68±0.08 (n=18) at 10nM. See also Table 1.


Figure 4
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  		Fig. 4 Dose-dependence of GK(IR) activation by S-1-P. Mean±s.e. of GK(IR)out and GK(IR)in are plotted versus concentration of S-1-P. **P<0.01; ***P<0.001 denote significant differences compared with the GK(IR)out at the same S-1-P concentration. Fitted curves are based on the Hill equation: GK(IR)=GK(IR)max/(1+(EC50/[S-1-P]). GK(IR)max denotes maximal GK(IR) and Hill coefficient. For GK(IR)out; GK(IR)out max was 137.1pS pF– 1 and EC50 was 1.6nM, ±0.98. For GK(IR)in; GK(IR)in max was 224.9pS pF– 1 and EC50 1.3 nM, ±0.96.

 
3.4 Do S-1-P- and ACh-mediated increases in K+ current exhibit occlusion?
To more fully understand the molecular pharmacology of S-1-P effects in mammalian heart, it is important to determine: i) whether S-1-P regulates a different population of K+ channels than those modulated by ACh, or ii) whether S-1-P can regulate the same K+ channels that are opened by ACh. In principle, this can be tested by sequential application of saturating concentrations of each of these compounds, followed by measurement of maximal effects in terms of outward K+ currents or the corresponding conductances. Accordingly, S-1-P and ACh were applied at their maximally effective concentrations. Either S-1-P (100nM) or ACh (10µM) was applied for 20~30s. This maneuver was followed immediately by simultaneous application of both agonists for 30s at their maximal concentrations. The time of agonist exposure was kept very short to minimize the effects of receptor desensitization [5]. This protocol also allowed these agonists to be applied in the reverse order to the same myocytes (after an appropriate washout period). In this way, records of IK(S-1-P) or IK(ACh) were compared with the K+ current activated by both S-1-P and ACh (IK(S-1-P+ACh)). Both S-1-P and ACh activated K+ currents during ramp waveform voltage clamp (Fig. 5A,B). This analysis revealed that addition of ACh after S-1-P produced a significant increase in K+ current IK(IR) (Fig. 5C), suggesting that ACh could activate or recruit additional K+ channels. In contrast, when the combination of S-1-P and ACh was applied following an ACh challenge only a small increase in K+ was recorded (Fig. 5D). Additional, complementary analysis of these results in terms of chord conductances yielded a similar pattern of results. Specifically, GK(IR)out obtained from these I–Vs showed that ACh alone can produce almost the same increase in GK(IR)out as that produced by the combination of S-1-P and ACh (Fig. 5E). These findings can be expressed based on a normalization of the parameters for IK(S-1-P) and IK(ACh) to those for the combination, IK(S-1-P+ACh). This analysis showed that the S-1-P-induced GK(IR)out was 0.53±0.06 (n=9) of total GK(IR)out. In contrast, this ratio for the effect of ACh applied immediately before the S-1-P and ACh combination was 0.94±0.02 (n=9). Similar results were obtained when the data were examined at membrane potentials negative to the reversal potential, that is in terms of GK(IR)in.


Figure 5
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  		Fig. 5 Comparison of changes in IK(IR) or GK(IR)out activated by S-1-P, ACh or S-1-P plus ACh. Panels A and B show I–V relationships obtained before (control), and after application of S-1-P (100nM) (A) or ACh (10µM) (B) applied individually. This was followed by simultaneous application of S-1-P and ACh. Finally, both compounds were washed out. Note that the I–Vs of the control and washout were very similar (Con/Wo). Currents obtained in the presence of ACh are shown in Panel B. The symbol ACh2 denotes the second application of ACh (10µM) which produced the smaller response. Panels C and D show I–V relationships for the agonist-induced K+ currents. These were obtained by subtracting control current from the current in the presence of one agonist (IK(S-1-P) in C, and IK(ACh) in D) or both agonists (dotted line, IK(S-1-P+ACh)). Data summarized in Panel E are based on sequential changes of GK(IR)out during a series of applications of S-1-P or ACh, individually, or S-1-P plus ACh. GK(IR)out for the ACh was obtained from control IK(ACh) recorded just before the application of S-1-P. The letters (a) and (b) above the abscissa denote the trial which produced the membrane current changes shown in Panels A and B above. Note that ACh (10µM) activated IK(IR) or GK(IR)out to a similar extent as the simultaneous application of ACh (10µM) and S-1-P (100nM).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Our results demonstrate that S-1-P at nanomolar concentrations causes a marked shortening of APD in the guinea pig atrial myocytes. This effect is due to a G-protein-mediated activation of a weakly inwardly rectifying K+ current. A decrease in APD will shorten the effective refractory period (ERP). This may predispose the atria to reentry-induced arrhythmias [14]. Atrial myocytes from AF patients show marked shortening of APD [28]. This working hypothesis is based in part on the well-known propensity of the atria to fibrillate during vagal stimulation [26] as a result of activation of IK(ACh). Further, atrial rhythm disturbances are abolished in a murine model in which the K+ channels responsible for K(ACh) are knocked out [27].

4.1 APD shortening and threshold increase by S-1-P
Repolarization of the atrial AP is due to a net outward current which results from the algebraic sum of a number of different inward and outward currents [29]. The expression level of the classical inward rectifier K+ channel (IK1) is very low in atrial myocytes from adult mammalian hearts. However, in response to a number of drugs or naturally occurring ligands other inwardly rectifying K+ currents are activated. These are capable of generating quite substantial outward currents at membrane potentials corresponding to overshoot, plateau and terminal repolarization of AP (Fig. 3). Our results confirm that IK(S-1-P) can markedly accelerate the repolarization and decrease the APD50 and APD90 (Fig. 1). We acknowledge, however, that the marked shortening of APD shown in Fig. 1 would be unlikely to be this pronounced in the intact atrial syncytium. Two primary reasons for this are that S-1-P is unlikely to be liberated in a uniform fashion, thus its effects would be blunted through electrotonic interactions. In addition, under physiological conditions the intrinsic heart rate will be much higher than the stimulus rate of these experiments and this will produce a shorter control APD.

In principle, a S-1-P effect on L-type Ca2+ current could contribute to the observed change in action potential duration. Yasui and Palade [28] have reported that sphingolipid compounds, specifically sphingosine at 25µM can alter the kinetics and the IV curve of the Ca2+ current in rat ventricular myocytes. In contrast, our previous work on this current in rabbit sinoatrial node myocytes failed to identify any significant effect [6]. Under the experimental conditions in which the IV curves in this paper were obtained, any significant contribution of an inhibition of L-type Ca2+ current by S-1-P can be excluded since Cd2+ was present in the superfusate. Nevertheless, the remaining and important issue of whether S-1-P (at physiological levels) can modulate the Ca2+ current, i.e., under adrenergic tone should be fully evaluated.

Our results have identified an effect of S-1-P on atrial excitability and changes in the time-course of the electrotonic potential which precedes action potential firing (threshold). Nanomolar concentrations of S-1-P increased the stimulus current needed to elicit the AP, and also reduced the time constant of the decay of subthreshold electrotonic potentials. The increase of threshold current could be primarily due to the decrease of resting membrane resistance due to activation of IK(S-1-P). The size of this increase in the threshold current is related to, but not identical with, the decrease of the time constant. Nonlinearities in the small background K+ currents and the very short applied stimuli result in the subthreshold depolarizations being partly due to charge redistribution under control conditions and in the presence of S-1-P. Following action potential firing and conduction, this S-1-P-induced increase in the threshold current for AP initiation can be proarrhythmic since it results in a decrease of conduction velocity. This would be expected to contribute to a reduction of the wavelength of the propagating wave of excitation (ERP x conduction velocity) [14]. A reduction in this parameter is considered to be a classical indication of increased likelihood for reentrant rhythm disturbances.

4.2 Characterization of weak inward rectification of IK(S-1-P)
The weak inward rectification of G-protein-activated IK(IR) (GIRK) currents is an essential functional characteristic. This allows the generation of a substantial net outward current which contributes to the bradycardia. It also causes the shortening of APD and an increase in the firing threshold in atrial myocytes. We have analysed the inward rectifying properties of IK(IR) activated by S-1-P and ACh by calculating the ratio of outward to inward chord conductance (Fir) in normal [K+]o (Table 1).

G-protein-activated inwardly rectifying K+ channels (GIRK, Kir3.X) in mammalian heart are tetramers consisting of Kir3.1 and Kir3.4 subunits [30]. GIRK is activated by direct interaction of beta/gamma subunits of G protein with Kir3.1/Kir3.4 channels [25]. Our Fir values are similar to those for Kir3.1/Kir3.4 channels activated by G beta/gamma subunits in HEK293 cells in the presence of 20mM [K+]o [24]. Hommers et al. [24] reported that the extent of this inward rectification of Kir3.X channels can be reduced by increases in agonist concentration in feline atrial myocytes. A similar effect was observed in heterologous co-expression experiments designed to study G-protein subunit interactions with Kir3.1/3.4 expressed HEK293 cells. However, some of our observations differ from this pattern. Specifically: (i) the normalized I–V relations of IK(ACh) and IK(S-1-P) were similar, in spite of differences in their amplitude (Fig. 3B), (ii) the EC50 of GK(IR) was only weakly dependent on the direction of the current flow (Fig. 4), and (iii) Fir was independent on S-1-P concentration. In combination, our results suggest that the inward rectification of IK(S-1-P) is not solely modulated by G-protein beta/gamma subunit levels in guinea pig atrial myocytes. An alternative explanation for the observed weak inward rectification is that it may be due to weak binding of intracellular polyamines to Kir3.4 subunits. It is known that the inward rectification of K(IR) channels is produced by binding of Mg2+ and/or polyamines to these channels [25,31]. Polyamines bind to the alpha subunits of Kir3.1/Kir3.4 channels near the selectivity filter of this K+ channel, and this can cause inward rectification [32]. Furthermore, overexpression of Kir3.4 subunits can reduce this inward rectification in rat [33] and murine atrial myocytes [34].

4.3 Convergence of IK(S-1-P) and IK(ACh)
IK(S-1-P) activation is initiated by the binding of S-1-P to tissue and/or cell specific S-1-P/Edg receptors. The Edg-3 (S-1-P3) receptor has been suggested to be involved in S-1-P-induced activation of K+ current in human and murine atrial myocytes [22]. Consistent with this, S-1-P-induced bradycardia can be suppressed by genetically deleting S-1-P3 receptor in mice [35] and S-1-P binds to the human S-1-P3 receptors with high affinity, yielding an apparent IC50 of 0.2nM [36]. The EC50 in guinea pig [5,22], murine and human atrial myocytes [22] has been reported to be approximately 1nM. We obtained a similar EC50 for IK(S-1-P) activation based on the dose-dependence of GK(IR)out and GK(IR)in (Fig. 4). This is an interesting result given that the S-1-P receptor of guinea pig heart has a much higher sensitivity to sphingosylphosphorylcholine [21,22] than S-1-P3 receptor of human and mouse heart [22].

Previous experimental results have provided evidence that ligands which are linked to Kir3.1/3.4 can exhibit a nonadditive effect, which resembles saturation. This phenomenon has also been termed ‘occlusion’. A prime example is the adenosine (Ado)-activated IK(IR) with the maximal m2-muscarinic acetylcholine receptor (M2R)-mediated IK(ACh) in guinea pig atrial myocytes [37]. These results have been interpreted in terms of Ado-A1-activation converging on M2R-related biochemical signaling pathway at G beta/gamma or downstream IK(IR) channels. Indeed, previous work on cultured guinea pig atrial myocytes [5] has shown that the ACh-induced increase in holding current is significantly reduced by prior application of S-1-P. Thus, we conclude that S-1-P receptor signaling mechanism converges with the ACh–M2R activation system by activating the same population of weakly inwardly rectifying K+ channels. However, since in the same myocyte IK(S-1-P) is smaller than IK(ACh) (Fig. 3B) and application of ACh following a maximal concentration of S-1-P consistently induced an additional K+ current, other factors must also be involved. It is now known that GIRK can be activated by interaction of receptor-associated G proteins with Kir3.1/Kir3.4 channels, that is, via protein–protein interactions which do not require the disassociation of beta/gamma subunits from the receptors [38]. It therefore seems possible that S-1-P receptors are expressed at a smaller density than M2R receptors. This could contribute to altered levels of interaction with the Kir3.1/Kir3.4 channels that are in the immediate proximity of M2 muscarinic receptors in the sarcolemma of guinea pig atrial myocytes.

4.4 Possible role of S-1-P in atrial rhythm disturbances
S-1-P is produced from sphingosine by sphingosine kinase, and it is stored in platelets [17]. S-1-P can be rapidly released from platelets (in the absence of or during their aggregation) into the extracellular environment on stimulation with agonists such as thrombin [17,18]. An increase of S-1-P concentration in plasma from 0.2nM to 0.5µM has been estimated during clotting [19]. Chronic AF is accompanied by a hypercoagulable state including increases of the tissue factor, Factor III, an initiator of coagulation [16], and fibrin D-dimer, a marker of thrombogenesis [39]. Based on our findings, it seems plausible that S-1-P released from platelets could shorten the APD of atrial myocytes, and thus tend to induce atrial fibrillation/flutter. S-1-P3 receptors which mediate I(KS-1-P) activation are expressed in human atrium [22]. However, at present, neither the effect of S-1-P on the APD in human atrium nor the change of plasma S-1-P concentration in the setting of AF has been established. The multifactorial and very complex milieu from which supraventricular rhythm disturbances arise makes it necessary to insure that possible species differences (guinea pig to human) of receptor subtypes or relative contributions of different ion channels are properly accounted for (c.f. 22,40,41).


   Acknowledgements
 
We are grateful for the financial assistance from the Office of the Deans of Medicine and Engineering at UCSD which provided a Visiting Professorship for R. Ochi. In addition, this study was supported by the Canadian Institutes of Health Research and the Heart Stroke Foundation of Canada.


   Notes
 
Time for primary review 27 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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