Keywords

A1 adenosine receptor; Pertussis toxin; G proteins; Catecholamines

Introduction

A cyclic adenosine 5’-monophosphate (cAMP)-activated Cl− current in rabbit and guinea pig heart was discovered in 1989 [1,2]. Since then, multiple chloride channels have been identified in cardiac cells [3]. These include CFTR, ClC-2, ClC-3, CLCA, Bestrophin and Ano1 (12). The β-adrenergic-agonists activated channel, which is regulated by cAMP-dependent protein kinase A (PKA) phosphorylation conducts Cl- dependent outward rectifying current (I_Cl) [4]. The cystic fibrosis transmembrane conductance regulator (CFTR), which is a member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily, may be responsible for some of the Cl− current activated by PKA [3,5]. Catecholamines, which stimulate adenylyl cyclase and thereby increase the intracellular level of cAMP, are important regulators of cardiac function; thus, it has been hypothesized that I_Cl plays an important role in cardiac electrophysiology, in particular during increased sympathetic input to the heart [6,7].

That extracellular adenosine (Ado) can regulate ionic currents in the mammalian heart is well established [8,9]. The effects of Ado on two ionic currents, i.e., the L type Ca²⁺ current (ICa) and hyperpolarization activated inward pacemaker current (If) are mediated, in part, by the anti-adrenergic action of the nucleoside [9]. Specifically, the enhancement of ICa and If by catecholamines is attenuated by Ado acting on A1Ado receptor (A1AdoR), and is mediated by a pertussis toxin (PTX)-sensitive GTP binding protein G_i and inhibitory effect on adenylyl cyclase [9]. More than two decades ago, a preliminary report (abstract) have shown for the first time that Ado can also inhibit isoproterenol-induced I_Cl in mammalian ventricular myocytes [10]. In addition, the antiadrenergic action of Ado can be the manifestation of inhibition of norepinephrine release from sympathetic nerve endings in the heart [11].

Thus, the present study was aimed at testing the hypothesis that adenosine can attenuate catecholamines/cAMP-dependent I_Cl in ventricular myocytes by activating A1AdoR and a PTX-sensitive G protein(s). The present data support this hypothesis and suggest that the attenuation of I_Cl by Ado is another important manifestation of the antiadrenergic action of the nucleoside on the guinea pig ventricular myocardium.

Material and Methods

The present studies have been approved by the institutional ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

Materials

The modified Krebs-Henseleit (K-H) solution contained (mM): 127 NaCl, 4.6 KCl, 2 CaCI₂, 1.1 MgSO₄, 2 sodium pyruvate, 10 glucose, 10 creatine, 20 taurine, 5 ribose, 0.01 adenine, 0.1 allopurinol, and 5 HEPES. The composition of the Ca²⁺- free solution was the same as that of the K-H solution except for the deletion of CaCl2 as extracellular solution. pH was adjusted to 7.4 with NaOH and the osmolarity was 310 mOsmol. The intracellular solution consisted of (mM): 120 CsCl, 20 NaCl, 2 EGTA, 1 Mg-ATP and 10 HEPES. pH was adjusted to 7.2 with CsOH and the osmolarity was 305 mOsmol. The following chemicals were used: Adenosine, N6- cyclopentyladenosine (CPA), a selective adenosine A₁ receptor agonist, 8-cyclopentyltheophylline (CPT), a potent and selective antagonist for the adenosine receptors, isoproterenol (ISO), forskolin (stock solution 23.3 mM in DMSO), Na-glutamate, a replacement of NaCl, nifedipine, to block Ca²⁺ currents, BaCl2, to block inward rectifying K+ current, TTX, to minimize rapid Na+ activation, bovine serum albumin, dispase (Boehringer Mannheim, Indianapolis, IN), collagenase (Worthington Biochemical Co., Freehold, N.J.) , trypsin, and PTX (Calbiochem Corp. San Diego, CA). Unless otherwise specified, all compounds were from Sigma-Aldrich, Inc. (St Louis, MO).

Myocyte isolation

Single guinea-pig cardiac myocytes were isolated using an established method [12,13]. Briefly, hearts were isolated from anesthetized (sodium pentobarbital, 30 mg/kg, i.v.) guinea-pigs pretreated with heparin (200 U/kg), and perfused retrogradely via the aorta with a warm (35°C) oxygenated buffer solution for 10 min followed by perfusion with Ca²⁺- free buffer solution for 20 min during the last 15 min of that period the following substances were added to the perfusate: collagenase (0.3 mg/ ml), dispase (0.04 mg/ml), trypsin (0.04 mg/ml) and albumin (2 mg/ml). Single cells were obtained by dispersion of small tissue specimens excised from the hearts.

Whole-cell recording

The whole cell voltage patch clamp technique was used to record Cl- currents [14,15] from guinea pig cardiac myocytes. An Axopatch 200A amplifier (Axon Instruments) and a PC running pClamp 8 were used to apply pulse protocols and record data. The holding potential was -30mV or -40 mV with identical results to inactivate Na+ channels and was consecutively stepped from –100 mV up to +50 mV in 10 mV increments for 220 ms. Patch micropipettes were fabricated from borosilicate glass (Kimex 51, KimbleGlass, Inc.) on a vertical pipet puller (Narishige, Amityville, NY) and fire-polished to a final bath resistance of 2-4 MΩ. The interior of the pipette is filled with an intracellular solution for whole-cell recording. A chlorided silver wire was placed in contact with this solution to connect to the amplifier. The micropipette was pressed against a cell membrane and suction is applied to form a high resistance seal between the glass and the cell membrane. After formation of a gigaseal, the cell membrane patch was ruptured to provide an access to the intracellular space of the cell. Stimuli were used via a multi-barrel pipet connected to a Pico-spritzer unit. Cells were continuously superfused with K-H solution containing different chemical compounds as mentioned above after mounting in a recording chamber. The recording chamber volume was 0.2 ml and it was superfused at a rate of 1.0 ml/min. Cl- current was induced by either isoproterenol (ISO; 0.1μM) or forskolin (1 μM). Ado (100 μM) and 5 μM CPA and 5μM CPT were used as agonists and antagonist at A1AdoR, respectively. Experiments were conducted at room temperature (23°C).

PTX treatment

To determine the role of PTX sensitive G protein(s) in the actions of Ado, isolated ventricular myocytes were treated with PTX (1 μg/ml) for three hours prior to experimentation. The efficacy of PTX in inactivating G_i/Go was determined using [³²P]NAD+ as a substrate for the PTX-catalyzed [³²P]ADP-ribosylation of the α-subunit of G_i and Go as described below.

PTX-induced G-protein inactivation

Control and PTX-treated cell membranes (180 μg protein) were incubated for 30 min at 30°C in a solution containing ATP (1 mM), GTP (0.1 mM), MgCl₂ (5 mM), EDTA (1 mM), DTT (1 mM), thymidine (10 mM), NAD⁺ (5 μM) including [32P]NAD⁺ to yield specific activity of 3x104 cpm/pmol, PTX (0.2 μg), HEPES (pH=8, 10 mM), BSA (5 μg) and SDS (0.025%). The assay mixture volume was 100 μl. Reactions were stopped by the addition of 2% SDS in 10 mM Tris, pH=7.5, EDTA (1 mM), NaCl (0.9%) (TEN), followed by boiling for two minutes and subsequent addition of an equal volume of TEN containing NP40 (5%). Samples were incubated overnight at 4°C with or without the anti-G_iα/Goα antibody 10C1 (2 μg/sample) a monoclonal antibody. Antibody-bound Gα subunits were precipitated using pansorbin and pellets were washed and resuspended by boiling in SDS-PAGE sample buffer. Samples were separated by SDS-PAGE on 12% minigels, which were dried and exposed to radiographic film. The 10C1 antibody is a derived monoclonal antibody against recombinant G_i2α, which reacts with G_i1-3α and G_oα subunits. In all cases, sample loads contained equal amounts of protein as well as equivalent levels of G_α immunoreactivity.

Data acquisition and analyses

Currents were measured during 220 msec voltage steps from either -30 mV or -40 mV holding potentials to -100 mV up to +50 mV using an amplifier with 100 MΩ headstage controlled by software (Axon Instruments) at a sampling rate of 10 kHz. Current vs. voltage plots were obtained using currents measured at each step. Data across all cells are presented as I_Cl at -100 mV membrane potential. Statistical significance at the level of p<0.05 was determined using ANOVA and Student’s t test corrected for multiple measurements.

Results

A typical example of current recordings under whole-cell voltage clamp in an isolated guinea-pig ventricular myocyte is shown in (Figure 1a-d). This conductance, which was a non-voltageactivated and without rectification, had a reversal potential near 0 mV when [Cl-]o was approximately equal to [Cl-]i. Both Ado (Figure 1c and 2a) and A1AdoR agonist CPA (Figure 2c) markedly attenuated the ISO-induced current; this inhibition was partially reversed by the A1AdoR antagonist CPT (Figure 1d and 2a). The induction of anion conductance by ISO was mimicked by forskolin, which activates adenylyl cyclase. Ado also inhibited the forskolin-induced current (Figure 1g and 2b). Forskolin carrier alone had no effect (data not shown). CPA, a selective adenosine A₁ receptor agonist, also can reverse the effects of ISO and forskolin (Figure 2c and 2d). The effects of Ado on ISO and forskolin are shown in the current vs. voltage curves in Figures 2a and 2b. Ado’s current inhibition had a relatively quick on-set occurring within one minute or less. Further characterization of ISO-induced conductance indicated that it was similar to cAMPactivated- conductance described previously [16]. Replacement of NaCl in the bath perfused with Na-glutamate, reduced [Cl-]o from 156.8 mM to 16.8 mM while maintaining the original cation concentrations. The reduction of [Cl-]i shifted the zero current potential of the I/V curve in the positive direction by ~30 mV indicating a significant Cl- selectivity of the channel (Figure 3). Similar results were obtained in additional four myocytes. Extracellular glutamate also decreased the slope inward current indicating reduced Cl- efflux (Figure 3). Similar reduction of cAMP-activated conductance has been reported when aspartate replaced extracellular Cl- [1,2,17]. Thus, the substitution of [Cl-]o with nonpermeant glutamate or aspartate seems also to produce a partial block of Cl- flux through the chloride channel, which is manifested as reduced inward current.

Figure 1: Adenosine (Ado) attenuated the isoproterenol (ISO)- induced and forskolin current. A typical example of current recordings under whole-cell voltage clamp in an isolated guinea-pig ventricular myocyte is shown (a and e). This conductance, which was a non-voltage-activated and without rectification, had a reversal potential near 0 mV under conditions [Cl-]o approximately equal to [Cl-]i. The ISO and forskolin, which activates adenylyl cyclase, induced current (b and f). Ado markedly attenuated the ISO- and forskolin-induced current (c and g). This inhibition by Ado could be partially reversed by the A1AdoR antagonist cyclopentyltheophylline (CPT) (d).

Figure 2: A typical example of the effects of adenosine (Ado) on isoproterenol- (ISO) and forskolin-induced current in the current vs. voltage curves. Ado markedly attenuated the ISO- and forskolin-induced current and 8-cyclopentyltheophylline (CPT) partially reversed the inhibition (a and b). N6-cyclopentyladenosine (CPA), selective Ado A1 receptor agonist, also can reverse the effects of ISO and forskolin (c and d). Ado’s current inhibition had a relatively quick on-set occurring within a one minute or less.

Figure 3: A typical example of the modification of the isoproterenol (ISO)-induced Cl- conductance by the replacement of NaCl in the bath-perfused with Na-glutamate, which reduced [Cl-]o from 156.8 mM to 16.8 mM while maintaining the original cation concentrations. The reduction of [Cl-]o shifted the zero current potential of the current vs. voltage curve in the positive direction by ~ + 30 mV indicating a significant Cl- selectivity of the channel. Extracellular glutamate also decreased the slope inward current indicating reduced Cl- efflux.

Although Iso-induced and forskolin-induced I_Cl they were qualitatively similar they differed quantitatively. Both ISO and forskolin had a pronounced effect on Cl- conductance (Figure 1-2), but the mean response to forskolin was approximately twice that of ISO. ISO and forskolin (each, 1 μM) enhanced transmembrane current from 120 ± 15 pA to 353 ± 50 pA (Figure 4a) and from 10 ± 15 pA to 606 ± 133 pA (Figure 4b) (each, p<0.05), respectively. A1AdoR stimulation by either Ado or CPA partially inhibited the effects of both ISO and forskolin. The A1AdoR agonist’s inhibition of the response to ISO was fully abolished by the A1AdoR antagonist CPT, and its inhibition of the response to forskolin was markedly reduced by CPT (Figure 4a and 4b).

Figure 4: N6-cyclopentyladenosine (CPA) attenuated the isoproterenol (ISO)-induced and forskolin current. Both ISO and forskolin had pronounced effect on Cl- conductance, but the mean response to forskolin (b) was approximately twice that of ISO (a). A1AdoR stimulation by CPA A1 agonist partially inhibited the effects of both ISO and forskolin; the effect of the former and the latter was fully (a) and partially (b) abolished by the A1AdoR antagonist 8-cyclopentyltheophylline (CPT), respectively. n=9-13; *p<0.002, +p<0.025, ‡p<0.05.

Pretreatment with PTX (1 μg/ml; for 3 hrs) did not modify the effect of ISO, but blocked the effect of Ado (Figure 5, filled symbols=PTX treated). Assays of membranes of PTX-treated myocytes indicated efficient ribosylation of candidate G proteins thereby, removing them from the pool of proteins available for radioactive ribosylation (Figure 6).

Figure 5: A typical example of the effect of pertussis toxin on the inhibitory effect of Ado on isoproterenol (ISO)-induced current. Ado significantly reduce the ISO-induced current without PTX. Pretreatment with PTX did not modify the effect of ISO, but blocked the effect of Ado. Open and closed symbols: Pre- and Post-PTX treatment, respectively.

Figure 6: Autoradiogram of a polyacrylamide gel showing the extent of [32P]-ADP ribosylation od αi1 and αo in homogenates of untreated cells (Control; left column) and PTX-treated cells (PTX; right column).

Discussion

The present study confirm the preliminary report regarding Ado’s inhibition of ISO-induced I_Cl [10]. In addition, the following new results were obtained in single isolated guinea-pig ventricular myocytes: (i) Ado also attenuated either also forskolin-induced I_Cl, (ii) CPA, an A1AdoR agonist had a similar effect, (iii) CPT, an A1AdoR antagonist attenuated the effects of both Ado and CPA on ISO-induced I_Cl and to a lesser extent their effects on forskolininduced I_Cl, and (iv) the effects on Ado and CPA were mediated by a PTX sensitive G protein(s).

These data demonstrate for the first time that the activation of A1AdoR and an associated PTX sensitive G protein mediate not only the negative dromotropic action of Ado [18], but also its inhibitory effect on catecholamine/cAMP-dependent I_Cl in the guinea-pig ventricular myocardium. Thus, the anti-adrenergic action of Ado is manifested not only in the attenuation of ISO-induced I_Ca and If [9] but also I_Cl. The present results are in congruence with the demonstration of ISO/cAMP dependent I_Cl in ventricular myocytes [6,7] and confirm Ado’s reversal of I_Cl activation by histamine and ISO [7,17].

In the mammalian heart, Ad exerts direct and indirect effects; the latter is manifested in the attenuation of β-adrenoceptordependent effects of catecholamines [19]. In the atria, sinus node and AV node, Ado exerts both types of actions, while in the ventricle only the indirect anti- β-adrenergic action, i.e., the attenuation of the biochemical, inotropic and electrophysiologic effects of catecholamines [20,21,22]. An exception is the direct action of Ado on basal ICa in the ferret ventricular myocardium [23]. The effect of Ado on ISO-induced I_Cl is in agreement with its effects on ISO-induced ICa and If and delayed afterdepolarizations [24,25]. Moreover, adenosine antagonized catecholamineinduced electrophysiologic effects in human His-Purkinje system in vivo [26].

Several mechanisms are involved in the anti- β-adrenergic action of Ado including (i) inhibition of adenylyl cyclase [27-29,30], (ii) modulation of β-adrenoceptor signal transduction [30,31] and (iii) dephosphorylation of intracellular proteins independent of cAMP [32]. The present observation regarding Ado’s equal effectiveness in reducing ISO- and forskolin-induced I_Cl argues against the involvement of the aforementioned second mechanism. Several studies have indicated that the inhibitory action of Ado on adenylyl cyclase and the anti- β-adrenergic actions of Ado are mediated by A1AdoR and a PTX-sensitive G protein [32-36]. The present data are in agreement with these previous observations and argue against the involvement of A2AdoR in the anti-β- adrenergic actions of Ado. In cardiac myocytes, acetylcholine (Ach) and carbachol decrease ISO- or forskolin-induced I_Cl via the activation of G_i [16]. In view of the similarity between the actions of Ado and Ach in the mammalian heart [9] and the present observation with PTX, it is highly likely that the inhibition of I_Cl by Ado was mediated by G_i.

It should be note that A1AdoR mediates multiple actions of Ado including negative chronotropic and dromotropic effects, reduction of atrial contractility, attenuation of the stimulatory actions of catecholamines on beta-adrenergic receptors, reduction of lipolysis in adipose tissue, reduction of urine formation, and inhibition of neurotransmitter release [37]. However, these actions are associated with a variable receptor reserve profiles, which determine the hierarchy of the manifestation of these effects caused by a given concentration of Ado [37]. Specifically, A1AdoR is coupled more efficiently to an inhibition of ISO-induced ICa than to the activation of IK [38].

In the present study, Ado inhibited the action of forskolin on I_Cl. This is in agreement with the well-established stimulatory effect of forskolin on adenylyl cyclase resulting in increased intracellular levels of cAMP [39]. In addition, these data are in agreement with the previously reported attenuation by Ado of forskolin-induced If [9]. CPT antagonized the depressant effect of Ado on forskolin-induced I_Cl, however, this antagonism was less pronounced than that observed with ISO-induced I_Cl. There is no immediate explanation for this difference; it could be speculated that forskolin interferes with the actions of CPT directly or indirectly. Indeed, interaction between forskolin and A2AdoR signal transduction has been reported [40].

In summary, the present data indicate the Ado suppresses catecholamine/cAMP-dependent chloride conductance in guinea-pig ventricular myocytes and that this action is mediated by A1AdoR and a PTX sensitive G protein(s). Multiple chloride channels modulated by various signals including stretch, calcium, and phosphorylation have been characterized in human myocardium [3,41-47]. However, cAMP-dependent chloride conductance has not been demonstrated in human myocardium heretofore. Thus, the clinical significance of the present characterization of another aspect of the anti-adrenergic action of Ado as another aspect If Ado’s cardio-protective mechanism remains to be determined [48,49].

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