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Adenosine (Ado) is an important mediator of the endogenous defense against ischemia-induced injury in the heart. The action of Ado is mediated by activation of G protein-gated inwardly rectifying K+ (GIRK) channels. In turn, GIRK channels are inhibited by reducing phosphatidylinositol 4,5-bisphosphate (PIP2) through Gq protein-coupled receptors (GqPCRs). We previously found that GIRK channels activated by acetylcholine, a muscarinic M2 acetylcholine receptor agonist, are inhibited by GqPCRs in a receptor-specific manner. However, it is not known whether GIRK channels activated by Ado signaling are also regulated by GqPCRs. Presently, this was investigated in mouse atrial myocytes using the patch clamp technique. GIRK channels were activated by 100 µM Ado. When Ado was repetitively applied at intervals of 5~6 min, the amplitude of second Ado-activated GIRK currents (IK(Ado)) was 88.3±3.7% of the first IK(Ado) in the control. Pretreatment of atrial myocytes with phenylephrine, endothelin-1, or bradykinin prior to a second application of Ado reduced the amplitude of the second IK(Ado) to 25.5±11.6%, 30.5±5.6%, and 96.0±2.7%, respectively. The potency of IK(Ado) inhibition by GqPCRs was different with that observed in acetylcholine-activated GIRK currents (IK(ACh)) (endothelin-1>phenylephrine>bradykinin). IK(Ado) was almost completely inhibited by 500 µM of the PIP2 scavenger neomycin, suggesting low PIP2 affinity of IK(Ado). Taken together, these results suggest that the crosstalk between GqPCRs and the Ado-induced signaling pathway is receptor-specific. The differential change in PIP2 affinity of GIRK channels activated by Ado and ACh may underlie, at least in part, their differential responses to GqPCR agonists.
Adenosine (Ado) is released during cardiac ischemia and can mediate important protective functions in the heart [1-6]. Previous studies have shown that Ado or Ado receptor agonists can cause a reduction in the infarct size or an improvement in left ventricular function when given during reperfusion [1,6] or during both low-flow ischemia and reperfusion in the isolated and perfused heart [7,8]. The protective effect of Ado in the intact heart is mediated by Ado receptors. Transgenic mouse hearts overexpressing the Ado A1 receptor (A1AdoR) exhibit an enhanced resistance to the deleterious effect of ischemia , demonstrating that overexpression of the Ado receptor subtypes can lead to increased cardioprotection.
The mechanism of action of A1AdoR on cardiac myocytes has been intensively studied. It was demonstrated that A1AdoR hyperpolarizes the membrane potential and decreases the action potential duration [10,11]. These effects were known to be mediated by activation of G protein-gated inwardly rectifying K (GIRK) channels [12-14]. In atrial myocytes, the GIRK channel current also can be activated by the parasympathetic muscarinic M2 acetylcholine receptor (M2AChR) [15-17]. Thus, they are activated by Gi/o-coupled receptors and represent important mediators of vagally induced bradycardia and cardioprotection during myocardial ischemia [18,19].
Recently, it was shown that activation of GIRK channels by Gi/o protein depends on the presence of PIP2 [20-23]. Since PIP2 serves as a substrate for phospholipase C (PLC) that is activated by the Gq protein [24,25], we questioned whether PIP2 depletion induced by the stimulation of Gq protein-coupled receptors (GqPCRs) could inhibit GIRK currents in physiological conditions. This was previously investigated in acetylcholine (ACh)-activated GIRK channels; stimulation of the α1-adrenergic receptor resulted in the inhibition of ACh-activated GIRK currents (IK(ACh)) . Subsequent experiments on the specificity of action of GqPCRs demonstrated that prostaglandin F2α and endothelin-1 are potent inhibitors of IK(ACh), but that bradykinin has little or no effect . These results imply that crosstalk between GqPCRs and GIRK channels is specifically regulated and that the results obtained in ACh-activated GIRK channels cannot be extrapolated to other receptor and channel types. However, there has been limited research conducted on the regulation of Ado-activated GIRK channels, and the role of GqPCRs in Ado-induced signaling pathway in cardiac myocytes is poorly understood.
The present study investigated the effect of various GqPCR agonists, including phenylephrine, endothelin-1, and bradykinin, on Ado-induced GIRK currents (IK(Ado)). It is well-known that activation of these GqPCRs stimulates hydrolysis of PIP2 in cardiac myocytes . Presently, Ado-activated GIRK channels were inhibited by GqPCRs in a receptor-specific manner and the characteristic feature of GqPCR-induced IK(Ado) inhibition for each GqPCR agonist was different from that observed in IK(ACh) inhibition. The differential responses of Ado- and ACh-activated GIRK channels to the stimulation of GqPCRs might be mediated by the difference in PIP2 affinity.
Mouse atrial myocytes were isolated by perfusing a Ca2+ free normal Tyrode solution containing collagenase (0.14 mg/ml; Yakult Pharmaceutical, Tokyo) on a Langendorff column at 37 as previously described . Isolated atrial myocytes were kept in a high K+, low Cl- solution at 4 until used.
Membrane currents were recorded from single isolated myocytes in a perforated patch configuration by using nystatin (200 µg/ml; ICN Biomedical, Irvine, CA) or ruptured whole-cell patch clamp configuration at 35±1. Voltageclamp was performed using an EPS-8 amplifier (HEKA Instruments, Bellmore, NY) and filtered at 5 kHz. The patch pipettes (World Precision Instruments, Sarasota, FL) were made of a PP-830 Narishige puller (Narishige, Tokyo). The patch pipettes had a resistance of 2~3 MΩ when filled with the pipette solutions. Normal Tyrode solution contained 140 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2, 1.8 mM CaCl2, 10 mM glucose, and 5 mM HEPES, titrated to pH 7.4 with NaOH. The Ca2+-free solution contained 140 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2, 10 mM glucose, and 5 mM HEPES, titrated to pH 7.4 with NaOH. The high K+ and low Cl- solution contained 70 mM KOH, 40 mM KCl, 50 mM L-glutamic acid, 20 mM taurine, 20 mM KH2PO4, 3 mM MgCl2, 10 mM glucose, 10 mM HEPESs, and 0.5 mM EGTA. The pipette solution for perforated patches contained 140 mM KCl, 10 mM HEPES, 1 mM MgCl2, and 5 mM EGTA, titrated to pH 7.2 with KOH. To ensure a rapid solution turn-over, the rate of superfusion was kept >5 ml/min, which corresponded to 50 times bath volume (100 µl/min).
ACh (Sigma-Aldrich, St Louis, MO) was dissolved in deionized water to make a 10 mM stock solution and was stored at -20. On the day of the experiments, one aliquot was thawed and used. Phenylephrine, endothelin-1, and bradykinin were from Sigma-Aldrich.
Results in the text and the figures are presented as mean±SEM. (n=number of cells tested). Statistical analyses were performed by using Student's t test. The difference between two groups was considered to be significant when p<0.01.
IK(Ado) was well-characterized in rat and guinea-pig atrial myocytes [12-14]. However, in mouse preparations, IK(Ado) has not yet studied in great detail. To investigate the regulation of IK(Ado) by GqPCRs, IK(Ado) was first characterized in mouse atrial myocytes. IK(Ado) was activated by adding 100 µM Ado to the bath solution, while the cell was voltage-clamped at the holding potential of -40 mV (Fig. 1A). Upon the application of Ado, an increase in outward current was observed. To ensure that the Ado-induced outward currents were GIRK currents, I-V relationships were examined by applying voltage-ramp pulses from -120 mV to +60 mV (at a speed of ±0.6 Vs-1) before and during the application of Ado, as indicated in Fig. 1A. The I-V curves for net IK(Ado) were obtained by subtracting the control curve (a) from the I-V curves in the presence of Ado (b) (Fig. 1A, inset). This shows a typical inward rectification known for GIRK current with a reversal potential of around -70 mV, which is close to the equilibrium potential of the potassium ion as calculated using the Nernst equation . To compare IK(Ado) with IK(ACh), the I-V curves in the presence of ACh was also obtained. The shape of inward rectification and the reversal potential of two curves were not different, indicating that the current induced by both Ado and ACh were same GIRK currents. However, the current that was activated by Ado was significantly smaller than the total current activated upon stimulation of M2AChR if saturating concentrations of ACh (≥10 µM) and Ado (≥10 µM) were compared. This is illustrated by the representative current recording in Fig. 1B. On average, the amplitude of IK(Ado) was 30.9±7.0% of IK(ACh) (n=5, Fig. 1C). This was consistent with the previous finding that the current activated by saturating concentrations of Ado in atrial myocytes from rat and guinea-pig is about 30% of maximum current activated upon stimulation of M2AChR [12-14]. In addition, whereas IK(ACh) was characterized by rapid activation, activation of IK(Ado) was slow (Fig. 1B, inset).
Next, the effects of various GqPCR agonists on IK(Ado) were examined in mouse atrial myocytes. The protocol of the experiments for investigating the effects of GqPCR activation on IK(Ado) is shown in Fig. 2. As shown in Fig. 2A, the re-application of Ado at intervals of 5~6 min triggered an IK(Ado) (I2, the second IK(Ado)) with a similar amplitude to I1 (the first IK(Ado)). To enable quantitative analysis, the peak amplitude of I2 was normalized to the peak current amplitude of I1. Under control conditions, the peak value of I2 (I2, peak) was calculated to be 88.3±3.7% (n=4) of that of I1 (I1, peak). In subsequent experiments, such a paired application of Ado was used to investigate the effect of various GqPCR agonists on regulation of Ado-activated GIRK channel, regarding the IK(Ado) at the first response as the control. As shown in Fig. 2B, 100 µM of the α1 adrenergic receptor agonist phenyleprine was applied 4 min before the second application of Ado, and I2 in the presence of Ado was compared with I1. The amplitude of I2 was significantly reduced by pretreatment of phenylephrine. The peak current amplitude of I2 was 25.58±11.6% (n=4) of the I1, peak, which was significantly smaller than the I2, peak in the absence of GqPCR agonists (p<0.01) (Fig. 2E). Phenylephrine-induced inhibition of IK(Ado) was reversible after a recovery period, and the third exposure to Ado elicited an outward current with a similar peak amplitude to that of I1 (Fig. 2C).
GqPCRs regulate ACh-activated GIRK channels in a receptor-specific manner. Adrenergic agonists and angiotensin II (acting via the α1 and AT1 receptors, respectively) produce a moderate effect, and endothelin-1 and prostaglandin F2α induced strong inhibition of the channels . However, bradykinin was almost ineffective in inducing channel inhibition . So, we examined whether the potency of inhibition of Ado-activated GIRK channels by GqPCR agonists matched with their potency in inhibition of GIRK channels activated by ACh (endothelin-1>prostaglandin F2α>phenylephrine>angiotensin II>bradykinin). The effects of endothelin-1 (30 nM) and bradykinin (10 µM) on IK(Ado) were tested using the same experimental protocol (Fig. 2C and 2D). The peak current amplitude of I2 after pretreatment of endothelin-1 was 30.5±5.6% (n=4) of the I1, peak, (Fig. 2E, p<0.01 vs control). However, the effect of bradykinin on IK(Ado) was negligible. The peak current amplitude of I2 after pretreatment of bradykinin was 96.0±2.7% (n=4) of the I1, peak, (p<0.05) (Fig. 2E, p>0.05 vs control). These results indicate that phenylephrine, which exhibited the medium potency in inhibiting IK(ACh), induced strong inhibition of the channels with a similar extent of inhibition to that obtained following the application of endothelin-1. Thus, it can be suggested that crosstalk between GqPCRs adn Ado-activated GIRK channels occurs in a receptor-specific manner and the characteristic feature of GqPCR-induced IK(Ado) inhibition for each GqPCR agonist differs from that observed in IK(ACh) inhibition.
Adrenergic and endothelin-1 receptors are known to activate PLC  and inhibit ACh-activated GIRK channels via depletion of PIP2 in cardiac myocytes [26,27]. However, in hippocampal neurons, GIRK channel inhibition by Gq-coupled muscarinic M1 receptors is mediated by protein kinase (PKC)C activation, even though stimulation of muscarinic M1 receptors induces robust PIP2 hydrolysis . To investigate whether PKC activation is required for receptor-induced IK(Ado) inhibition, we tested if direct activation of PKC by phobol ester inhibited IK(Ado). Phorbol-12,13-dibutyrate (PDBu), a PKC activator, however, had no effects on IK(Ado) when pretreated for 4 min before second Ado application (Fig. 3A). The I2, peak in the presence of PDBu was 97.5±0.5% of the I1, peak (n=5), which was not significantly different from control condition (88.3±3.7%, n=4, P>0.05, Fig. 3B). This result was consistent with the notion that PKC activation did not affect IK(Ado) and that PKC was not a major mechanism in the inhibition of IK(Ado) by GqPCRs.
Differences in the affinity of inwardly rectifying K+ (Kir) channels for PIP2 may be responsible for differences among Kir channels in their specific regulation by a given modulator . Thus, we hypothesized that the differential sensitivity of IK(Ado) and IK(ACh) to GqPCR agonists might reflect their different affinities for PIP2. Given that βγ subunits of Gi/o proteins activate GIRK channels by directly binding to the GIRK channel, increasing the channels' affinity for PIP2 , it is plausible that A1AdoR and M2AChR increase the affinity of GIRK channels for PIP2 to different extents. The PIP2 affinity of ACh- and Ado-activated GIRK channels can be measured using neomycin. Neomycin is a polycation that binds specifically to PIP2 . In electrophysiological experiments, the neomycin sensitivity of a Kir channel has been used as a measure of its PIP2 affinity; channels with a high PIP2 affinity are less sensitive to neomycin than those with low PIP2 affinity . In previous studies, we showed that neomycin does not significantly affect the activation of IK(ACh) even at 500 µM . Presently, an experiment was devised to examine whether IK(Ado) was affected by 500 µM neomycin. As shown in Fig. 4A, neomycin pretreatment completely inhibited the IK(Ado). On average, 500 µM neomycin caused a reduction of IK(Ado) to 11.0±3.9% (n=3, Fig. 4C). The effect of neomycin on IK(Ado) was concentration-dependent. When 100 µM neomycin was pretreated before second Ado application, I2, peak was reduced to 60.5±3.5% (n=3, Fig. 4B and 4C). Taken together, these data indicate that IK(Ado) is more sensitive to neomycin than IK(ACh), suggesting that IK(Ado) has a lower PIP2 affinity.
This study demonstrates that A1AdoR-activated GIRK channels are inhibited by GqPCR agonists in mouse atrial myocytes, and that the potency of inhibition is GqPCR-specific. Adrenergic and endothelin receptor stimulation reduced the IK(Ado) by about 70%, whereas bradykinin receptor stimulation had little effect on IK(Ado). Such a specific regulation of IK(Ado) was not attributable to the difference in the potency of PIP2 hydrolysis by GqPCRs, since all GqPCRs tested in this study could induce PIP2 hydrolysis in cardiac myocytes . This discrepancy might support the view that there is coupling specificity of GqPCR and GIRK channels . The sensitivity of A1AdoR-activated GIRK channels to GqPCRs is different from that observed in ACh-activated GIRK channels (endothelin-1>phenylephrine>bradykinin, ). When PIP2 affinity in GIRK channels was estimated by neomycin inhibition, the PIP2 affinity of GIRK channels activated by Ado was lower than that of ACh-activated GIRK channels. Considering that the channels with low PIP2 affinity were more susceptible to PIP2 depletion, it can be suggested that differential change in PIP2 affinity of GIRK channels activated by Ado and ACh may underlie, at least in part, their different responses to the stimulation of GqPCRs.
It is well-known that the PIP2 affinity of Kir channels varies widely among different types of Kir channels [21,23], and that the differences in their affinity are responsible for differences among Kir channels in their specific regulation by a given modulator . For example, Kir 3.4 or Kir 2.3 channels, which have low affinity for PIP2, are inhibited by ACh or by the PKC activator phorbol 12-myristate 13-acetate (PMA), whereas Kir 2.1 or Kir 3.4 channels in the presence of Gβγ, which have high affinity for PIP2, are only marginally affected. Here, we showed that the GIRK channel-PIP2 interactions in the presence of Gi/o-coupled receptor agonists became different depending on the type of Gi/o-coupled receptors. Thus, the magnitude and kinetics of GIRK inhibition by GqPCRs might differ widely among different Gi/o-coupled receptor agonists. This hypothesis is supported by the previous finding that ACh-activated GIRK channels in native myocytes are not affected by PDBu  whereas baclofen-activated GIRK channels in hippocampal neurons are inhibited by PDBu . This discrepancy may reflect the fact that GIRK channel-PIP2 interactions in the presence of Gi/o-coupled receptor agonists in hippocampal neurons are not as strong as those in cardiac myocytes. The present study confirmed that the Ado-induced GIRK channels are not inhibited by PDBu in mouse atrial myocytes (Fig. 3).
The present data represents the first evidence that Ado-activated GIRK channels are regulated by GqPCRs. The functional consequence of inhibition of IK(Ado) by the α1-adrenergic and endothelin receptors may be an early cessation of the protective effect of Ado during cardiac ischemia. This discovery may be of particular importance, since it provides a novel pathway for the interaction of GqPCRs with Ado-induced signaling. The clinical importance of this interaction needs to be investigated in future studies.
This work was supported by the grant of the Korea Healthcare Technology R&D project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (A080604).