Ion Fluxes through KCa2 (SK) and Cav1 (L-type) Channels Contribute to Chronoselectivity of Adenosine A1 Receptor-Mediated Actions in Spontaneously Beating Rat Atria

Abstract

Impulse generation in supraventricular tissue is inhibited by adenosine and acetylcholine via the activation of A1 and M2 receptors coupled to inwardly rectifying GIRK/KIR3.1/3.4 channels, respectively. Unlike M2 receptors, bradycardia produced by A1 receptors activation predominates over negative inotropy. Such difference suggests that other ion currents may contribute to adenosine chronoselectivity. In isolated spontaneously beating rat atria, blockade of KCa2/SK channels with apamin and Cav1 (L-type) channels with nifedipine or verapamil, sensitized atria to the negative inotropic action of the A1 agonist, R-PIA, without affecting the nucleoside negative chronotropy. Patch-clamp experiments in the whole-cell configuration mode demonstrate that adenosine, via A1 receptors, activates the inwardly-rectifying GIRK/KIR3.1/KIR3.4 current resulting in hyperpolarization of atrial cardiomyocytes, which may slow down heart rate. Conversely, the nucleoside inactivates a small conductance Ca2+-activated KCa2/SK outward current, which eventually reduces the repolarizing force and thereby prolong action potentials duration and Ca2+ influx into cardiomyocytes. Immunolocalization studies showed that differences in A1 receptors distribution between the sinoatrial node and surrounding cardiomyocytes do not afford a rationale for adenosine chronoselectivity. Immunolabelling of KIR3.1, KCa2.2, KCa2.3, and Cav1 was also observed throughout the right atrium. Functional data indicate that while both A1 and M2 receptors favor the opening of GIRK/KIR3.1/3.4 channels modulating atrial chronotropy, A1 receptors may additionally restrain KCa2/SK activation thereby compensating atrial inotropic depression by increasing the time available for Ca2+ influx through Cav1 (L-type) channels.

The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

“…Isolated spontaneously beating atria were prepared using a previously described method (Braganca et al, 2016), with some modifications. In brief, hearts were rapidly excised after decapitation followed by exsanguination (Rodent guillotine, Stoelting 51330), and placed in a physiological solution (Tyrode’s solution) composed of (mM): NaCl 137; KCl 4.7; CaCl 2 1.8; MgCl 2 1; NaH 2 PO 4 0.4; NaHCO 3 11.9; glucose 11.2 and gassed with 95% O 2 + 5% CO 2 (at pH 7.4).…”

“…Tissue fragments were placed over a small lung lobule fragment with the endocardial layer facing down, stretched to all directions, pinned flat onto cork slices and embedded in Shandon cryomatrix (Thermo Scientific) before frozen in a liquid nitrogen–isopentane; frozen samples were stored at −80ºC until use. Frozen sections with 8 µm thickness were cut perpendicular to the crista terminalis of the RA and parallel to the long axis in the case of RV (see Braganca et al, 2016). Following fixation, the preparations were washed three times for 10 min each using 0.1 M PBS and incubated with a blocking buffer, consisting in fetal bovine serum 10%, bovine serum albumin 1%, Triton X-100 0.3% in PBS, for 2 h. After blocking and permeabilization, samples were incubated with selected primary antibodies ( Table 1 ) diluted in incubation buffer (fetal bovine serum 5%, serum albumin 1%, Triton X-100 0.3% in PBS), overnight at 4ºC.…”

“…The SAN was characterized as described in a previous study from our group (Braganca et al, 2016). SAN identification was also facilitated by observation of the sinus node artery surrounded by small-size cardiomyocytes positive for the hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and negative against connexin-43 (Cx43), a gap junction protein ubiquitously expressed in the heart apart from in nodal tissue.…”

“…Intravenous ATP produces negative chronotropy, as well as several other acute cardiovascular effects that resembles adenosine application, almost neglecting ATP-sensitive receptors as active and functional regulators of the cardiac function (Pelleg and Belhassen, 2010). Apart from a massive presence of adenosine A 1 receptors (Braganca et al, 2016), the SAN from both rodents and humans expresses several P2 purinoceptor subtypes, including ionotropic P2X4, P2X7, and metabotropic P2Y 1 , P2Y 2 , and P2Y 14 (Musa et al, 2009), yet their role is still largely unknown. Besides species differences, the type and relative abundance of P2 purinoceptors in the heart varies with location and disease conditions (Musa et al, 2009).…”

ADP‐Induced Ca2+ Signaling and Proliferation of Rat Ventricular Myofibroblasts Depend on Phospholipase C‐Linked TRP Channels Activation Within Lipid Rafts

“…After removal of the fluorophore loading solution, cells were washed again twice and 150 μl of Tyrode's solution was added per culture well. In some of the experiments the concentration of KCl was raised from 2.7 to 4.7 mM (see e.g., De Biasi et al, ; Bragança et al, ). For the recordings, temperature was maintained at 32°C and readings were made with 5 sec of interval, during approximately 30 min, using a tungsten halogen lamp.…”