β 3 -adrenergic receptor (β 3 -AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of β 3 -AR in the human atrium. Unexpectedly, β 3 -AR activation increased human atrial tissue contractility and stimulated the L-type Ca 2+ channel current (I Ca,L ) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The I Ca,L and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective β 3 -AR agonists, SR58611 and BRL37344, and a β 3 -AR partial agonist, CGP12177, stimulated I Ca,L in HAMs with nanomolar potency and a 60%-90% efficacy compared with isoprenaline. The β 3 -AR agonists also increased contractility but with a much lower efficacy (~10%) than isoprenaline. The β 3 -AR antagonist L-748,337, β 1 -/β 2 -AR antagonist nadolol, and β 1 -/β 2 -/β 3 -AR antagonist bupranolol were used to confirm the involvement of β 3 -ARs (and not β 1 -/β 2 -ARs) in these effects. The β 3 -AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked I Ca,L stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, β 3 -ARs are positively coupled to L-type Ca 2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.
BackgroundTRPM7 (Transient Receptor Potential of the Melastatin subfamily) proteins are highly expressed in the heart, however, electrophysiological studies, demonstrating and characterizing these channels in human cardiomyocytes, are missing.MethodsWe have used the patch clamp technique to characterize the biophysical properties of TRPM7 channel in human myocytes isolated from right atria small chunks obtained from 116 patients in sinus rhythm during coronary artery and valvular surgery. Under whole-cell voltage-clamp, with Ca2+ and K+ channels blocked, currents were generated by symmetrical voltage ramp commands to potentials between -120 and +80 mV, from a holding potential of -80 mV.ResultsWe demonstrate that activated native current has dual control by intracellular Mg2+ (free-Mg2+ or ATP-bound form), and shows up- or down-regulation by its low or high levels, respectively, displaying outward rectification in physiological extracellular medium. High extracellular Mg2+ and Ca2+ block the outward current, while Gd3+, SpM4+, 2-APB, and carvacrol inhibit both (inward and outward) currents. Besides, divalents also permeate the channel, and the efficacy sequence, at 20 mM, was Mg2+>Ni2+>Ca2+>Ba2+>Cd2+ for decreasing outward and Ni2+>Mg2+>Ba2+≥Ca2+>Cd2+ for increasing inward currents. The defined current bears many characteristics of heterologously expressed or native TRPM7 current, and allowed us to propose that current under study is TRPM7-like. However, the time of beginning and time to peak as well steady state magnitude (range from 1.21 to 11.63 pA/pF, ncells/patients = 136/77) of induced TRPM7-like current in atrial myocytes from different patients showed a large variability, while from the same sample of human atria all these parameters were very homogenous. We present new information that TRPM7-like current in human myocytes is less sensitive to Mg2+. In addition, in some myocytes (from 24 out of 77 patients) that current was already up-regulated at membrane rupture.ConclusionsThis study provides the first electrophysiological description of TRPM7-like current in native human atrial myocytes. Less sensitivity to intracellular Mg2+ suggests for channel operation under physiological conditions. The TRPM7-like current up-regulation indicates the pathophysiological evidence of that current in human heart.
β3-Adrenergic receptor (β3-AR) is expressed in human atrial and ventricular tissues. Recently, we have demonstrated that it was involved in the activation of L-type Ca(2+) current (I(Ca,L)) in human atrial myocytes and the force of contraction of human atrial trabeculae. In the present study, we examined the effect of β3-AR agonist CGP12177 which also is a β1-AR/β2-AR antagonist on I(Ca,L) in human ventricular myocytes (HVMs) and the force of contraction of human ventricular trabeculae. CGP12177 stimulated I(Ca,L) in HVMs with high potency but much lower efficacy than isoprenaline. The β3-AR antagonist L-748,337 inhibited the effect of CGP12177. CGP12177 and L748,337 competed selectively on β3-ARs because L748,337 had no effect on isoprenaline-induced stimulation of I(Ca,L), while CGP12177 completely blocked the effect of isoprenaline. The activation of β3-ARs by CGP12177 does not involve the activation of Gi proteins because CGP12177 had no effect on forskolin-induced stimulation of I(Ca,L). CGP12177 had no effect on the force of contraction of human ventricular trabeculae. L-NMMA, an inhibitor of NO synthase, and IBMX, a nonselective inhibitor of phosphodiesterases, did not potentiate the effect of CGP12177 either on contraction of human ventricular trabeculae or on I(Ca,L) in HVMs. We conclude that in human ventricles β3-AR activation has no inotropic effect, while it slightly increases I(Ca,L). In contrast to human atrium, the activation of β3-ARs in human ventricle is not accompanied by increased activity of phosphodiesterases.
The aim of the study was to investigate an action of 2-aminopyridine and its new sulfonylcarbamide derivatives 2-AP21, 2-AP22, 2-AP26, and 2-AP27 (10–5–10–3 M) on carbachol-induced shortening of action potential duration and reduction of contraction force in guinea pig atrial muscles. Experiments were carried out using a standard method of myocardium electromechanical activity registration. Under control conditions (perfusion of atrial strips with Tyrode solution), an average of action potential duration, measured at 90% (AP90) and 50% (AP50) of repolarization, were 112.32±6.07 ms and 50.21±3.25 ms, (n=19), respectively, and contraction force was of 1.42±0.28 mN (n=20). Carbachol (10–6M), an agonist of muscarinic acetylcholine receptor and activator of KAch channels, markedly decreased AP90 to 35.31±4.21%, AP50 – to 26.42±2.66% (n=19) (P<0.001), and contraction force – to 24.23±2.0% (n=20) (P<0.001) vs. control. Modification of 2-aminopyridine structure by replacing 2-amino group by 4-toluolsulfonylcarbamide fragment and quaternization of nitrogen in pyridine ring increased anticholinergic effect on action potential duration and contraction force. According to their maximal prolongation of AP at 90% of repolarization, all new drugs ranked as follows: 2-AP27>>2-AP26>2-AP22³2-AP>2-AP21. 2-aminopyridine derivative 2-AP27, containing 4-toluolsulfonylcarbamide fragment and 4-nitrobenzyl radical at quaternized nitrogen of the pyridine, had the most potent anticholinergic effect on AP90 (936.60±178.23%). 2-AP22 and 2-AP26 (containing methyl or allyl radicals at quaternized nitrogen of the pyridine, respectively) showed a much weaker anticholinergic effect (231.39±28.48% and 318.25±63.81%, respectively). The weakest anticholinergic effect (63.59±34.38%) was induced by 2-aminopyridine derivative 2-AP21, which had non-quaternized nitrogen of the pyridine.
Aminopyridines are known to inhibit acetylcholine-activated K+ current (IKACh) in cardiac myocytes. The aim of this study was to examine the effect of 2-aminopyridine sulfonylcarbamide derivative 2-AP27 on isoprenaline-stimulated L-type Ca2+ current (ICaL) and to identify whether 2-AP27 acts via blocking of muscarinic M2-receptors in frog cardiomyocytes. The whole-cell configuration of the patch-clamp technique was used to record ICaL in enzymatically isolated cardiac myocytes. Isoprenaline (0.1 μM), an agonist of β1-β2-adrenoreceptors, stimulated the ICaL up to 475±61% (n=4) (P<0.05) vs. control. Then, in the first series of experiments, carbachol (0.01 μM), an agonist of M2 muscarinic receptors, reduced the stimulatory effect of isoprenaline to 42±15% vs. isoprenaline alone. 2- AP27 (100 μM) alone completely abolished the inhibitory effect of carbachol on isoprenaline-stimulated ICaL, which recovered to 95±5.8% of the effect of isoprenaline. In the second series of experiments, adenosine (1 μM), an agonist of A1-adenosine receptors, reduced the stimulatory effect of isoprenaline on ICaL to 56±10% (n=3) (P<0.05). Then 2-AP27 (100 μM) applied in the presence of adenosine, had no effect on ICaL, which remained at 51±7.9% (n=3) (P<0.05) of the effect of isoprenaline. These results suggest that 2-AP27, a new derivative of 2-AP, containing 4-toluolsulfonylcarbamide instead of amino group and quaternizated nitrogen by 4-nitrobenzylbromide in pyridine ring, is acting as an antagonist of muscarinic M2 receptors in frog ventricular myocytes.
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