| BackgroundRecently, we reported that zacopride, a known potent5-HT3receptor antagonist and5-HT4receptor agonist which is experimentally used as a gastrointestinal prokinetic agent, is also a selective Ik1channel agonist in rat ventricular myocytes and can suppress triggered ventricular arrhythmia. Upregulation of Ik1in atrial myocytes and activation of5-HT4receptor in atria may lead to potential adverse effect on atrial electrophysiology. It is worth noting that zacopride actually does not induce any atrial arrhythmia when applied to treat triggered ventricular arrhythmia in the rat, although it is the selective agonists both for5-HT4receptor and Ik1channel. The mechanism underlying this "atrial waive" character is poorly understood and is the main focus of the present study.It is known that Kir2.1(KCNJ2), Kir2.2(KCNJ12) and Kir2.3(KCNJ4) channels which assembled as homotetramers or heterotetramers are the molecular basis of native pore-forming subunit of Ik1channels in the heart. Tissue and species-specific expression profiles of Kir2.x isoforms determine the biophysical and physiological characteristics of Ik1channel in the heart. Based on these understandings, we hypothesize that zacopride exerts differential effects on atrial and ventricular Ik1due to its selective actions on different Kir2.x channels.It is also known that all of the Kir2.x channels, including Kir2.1, Kir2.2and Kir2.3channels, are the substrates of PKA and PKC. Thus, it is expected that phosphorylations of these Kir2.x channel pore-forming proteins may play important regulatory roles in Ik1function. We surmise that zacopride modulates Ik1channel activities potentially via acting on the phosphorylations of certain Kir2.x proteins, rather than via the5-HT receptor. This signaling pathway may potentially serve as a mechanism by which zacopride suppresses ventricular arrhythmias without inducing atrial arrhythmias.ObjectiveIn support of this hypothesis, we studied the effects of zacopride on Ik1in rat atrial myocytes, and further observed the effects of zacopride on the homotetrameric or heterotetrameric channels of Kir2.1, Kir2.2, Kir2.3and a mutant Kir2.1channel (Kir2.1S425L) transfected in HEK293cells, aiming to determine which of these Kir2.x channels is the target of zacopride and what is the signaling pathway underlying the tissue-and channel protein-specific actions of the drug.MethodsRat atrial myocytes were isolated and routine whole cell recording was performed to measure the Ik1currents. Respective rat cardiac orthologs of Kir2.1, Kir2.2and Kir2.3were cloned by reverse transcriptase-PCR. S425L mutation of KCNJ2gene (encoding Kir2.1) was introduced by site-directed mutagenesis. cDNAs were subcloned into the eukaryotic expression vector pEGFP-Nl. HEK293cells were transfected with genes encoding Kir2.1, Kir2.2, Kir2.3and Kir2.1S425L, and then the respective Ikir2.x were recorded using the same patch clamp technique. Western blots were performed to estimate the relative expression levels of Kir2.x channels in rat ventricles and atria.Results1.1.0μmol/L zacopride did not affect the rat atrial IK1and action potential (P>0.05).2. Kir2.1, Kir2.2and Kir2.3proteins were all expressed in rat ventricles and atria. Similar levels of Kir2.3protein were expressed in atria and ventricles, but atrial Kir2.1protein level was only25%of that measured in the ventricle.3. Zacopride at100μmol/L significantly increased the outward component of IKir2.1 by40.7%±9.7%at-50mV (P<0.01), but did not affect the inward component of Ikir2.1(changed by9.6%±4.2%at-110mV, P>0.05). Zacopride (100μmol/L) did not affect the currents of both Kir2.2and Kir2.3channels (P>0.05).The heteromeric (Kir2.1plus Kir2.2, Kir2.2plus Kir2.3and Kir2.1plus Kir2.3) channels were insensitive to zacopride (100μmol/L).4. In HEK293cells which do dot express5-HT3receptor, the enhancement of Ikir2.1by zacopride(100μmol/L) could not be inhibited by a5-HT4receptor antagonist RS23597-190(10μmol/L)(P<0.05).2-1-(4-Piperonyl) piperazinylbenzothiazole (PBT), a known selective5-HT4receptor agonist, did not show an effect on the outward component of IKir2.1at-50mV (P>0.05), observably decreased (rather than increased) the inward IKir2.1by31.4%±8.6%at-110mV (P<0.05).5. The PKA inhibitor (KT5720,5μmol/L) diminished the Kir2.1-enhancing effect of zacopride alone on the outward component when coapplicated with and zacopride. In contrast, PKC inhibitor (GF109203X,5μmol/L) and the PKG inhibitor (KT5823,5μmol/L) did not significantly alter the Kir2.1activator effect of zacopride. At-50mV,50迷mol/L forskolin (adenylyl cyclase activator) increased the Kir2.1current by24.1%±5.9%(P<0.01) and50μmol/L8-bromo-cAMP (exogenous cAMP) increased the current by22.1%±1.9%(P<0.05). At-50mV, zacopride induced only a mild and statistically insignificant increase of Kir2.1(S425L) current (by13.3%±6.5%,P>0.05).Conclusion1. Zacopride failed to impact the Ik1in rat atrial myocytes in which Kir2.3is the predominant isoform of Ik1pore-forming subunits.2. Zacopride could increase the currents of Kir2.1homomeric channels but not the Kir2.2and Kir2.3homomeric channels and Kir2.1-Kir2.2, Kir2.1-Kir2.3and Kir2.2-Kir2.3heteromeric channels.3. Zacopride regulates Kir2.1channel via a5-HT receptor-independent pathway4. Zacopride increases Kir2.1current via a PKA-mediated signaling pathway In conclusion, the present pharmacological study demonstrates that zacopride selectively activates the homomeic Kir2.1channel via a PKA-mediated signaling pathway. This finding may suggest an important mechanism by which zacopride exerts an Ik1-agonizing effect in the ventricle but not in atria. This unique feature of zacopride suggests that the potential risk of atrial fibrillation induction by this agent may be overestimated when using it as a drug of anti-ventricular arrhythmia. The study may shed light on the development of Ik1channel agonists as anti-arrhythmia drugs. |
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