Reiko Takikawa

Cardiac Ca current does not run down and is very sensitive to isoprenaline in the nystatin-method of whole cell recording

SummaryThe conventional l type Ca2+ channel current (ICa.L) was measured in single atrial and ventricular myocytes, with a new whole-cell recording technique using an ionophore, nystatin. The membrane of a cell-attached patch was gradually permeabilized by nystatin (100–200 gg/ml), added to the pipette solution, within 2–5 min after formation of a GΩ-seal. The electrical activity of the cells was measured through the pipette. ICa.L, measured with the nystatin-whole cell recording technique, did not exhibit the so-called “run-down” phenomenon for up to 90 min. The response of ICa.L to isoprenaline was also well preserved during the measurement. The half maximal concentration for the isoprenaline-induced increase of ICa.L was 8.2 × 10−9 M, which is a much smaller value than that reported previously. Thus, the nystatin-whole cell clamp recording is a useful technique to measure membrane currents of cardiac myocytes with preserving the physiological intracellular milieu.

Anti-cholinergic effect of verapamil on the muscarinic acetylcholine receptor-gated K+ channel in isolated guinea-pig atrial myocytes.

SummaryEffects of verapamil on the acetylcholine (ACh)-induced K+ current were examined in single atrial cells, using the tight-seal whole-cell clamp technique. The pipette solution contained guanosine-5′-triphosphate (GTP) or guanosine-5′-O-(3-thiotriphosphate) (GTP-γS, a non-hydrolysable GTP analogue). In GTP-loaded cells, ACh induced a specific K+ current, which is known to be mediated by pertussis toxin-sensitive GTP-binding (G) proteins. Verapamil (0.1–100 μM) depressed the ACh-induced K+ current in a concentration-dependent fashion. In GTP-γS-loaded cells, the K+ current remained persistently after wash-out of ACh, probably due to irreversible activation of G proteins by GTP-γS. Verapamil (0.1–100 μM) also depressed the intracellular GTP-γS-induced K+ current. However, the magnitude of verapamil-depression of the K+ current in GTP-γS-loaded cells was significantly smaller than that in GTP-loaded cells at concentrations between 1 and 10 μM of the drug. From these results, it is suggested that verapamil may block not only the function of muscarinic ACh receptors but also of G proteins and/or the K+ channel itself and thereby depress the ACh-induced K+ current in isolated atrial myocytes.

Heparin uncouples the muscarinic receptors from GK protein in the atrial cell membrane of the guinea-pig heart

The effects of heparin on activation of the G protein-gated muscarinic K+ channel were examined in atrial cells of guinea-pig heart. The inside-out patch clamp technique was used. The pipette solution contained 1.1 μM acetylcholine (ACh). In the inside-out patches, intracellular GTP activated the muscarinic K+ channel. When heparin (0.05–5 units/ml) was further added to the intracellular side of the patch membrane, the channel openings were depressed in a concentration-dependent fashion. The effects of heparin were reversible after wash-out. Heparin did not affect GTP-γS-induced activation of the K+ channel. Therefore, it is suggested that heparin may uncouple the muscarinic receptors from GK protein in the cardiac atrial cell membrane.

Adenosine-5'-triphosphate-induced sinus tachycardia mediated by prostaglandin synthesis via phospholipase C in the rabbit heart.

Effects of adenosine 5′-triphosphate (ATP) and adenosine on cardiac sinus pacemaker activity were examined in the rabbit heart. Electrocardiograms of hearts were recorded while using the Langendorff perfusion method. Both adenosine and ATP, added to the perfusate, slowed the sinus pacemaker activity in a concentration-dependent manner. But in about 40% of the cases, ATP higher than 300 μM initially accelerated and then slowed the heart. The sinus slowing caused by adenosine and ATP was blocked by theophylline (a p1 receptor antagonist) and disappeared in the hearts pre-treated with islet-activating protein (IAP). In contrast, the ATP-induced sinus acceleration was not affected by either theophylline or IAP. In about 75% of the IAP-treated hearts, ATP persistently accelerated the sinus pacemaker. In the remaining 25% of the hearts, ATP caused junctional tachycardia, which may have masked the ATP-induced sinus acceleration. Apamin specifically blocked the ATP-induced sinus acceleration, suggesting that P2 receptors are involved. Among various adenine nucleotide analogues, the order of potency in inducing tachycardia in IAP-treated hearts is adenosine-5′-[γ-thio]triphosphate > adenylyl imidodiphosphate > adenosine 5′-[α, β-methylene]triphosphate = ATP > adenosine diphosphate = adenosine 5′-[β, γ-methylene]triphosphate. ATP-induced acceleration was partially blocked by indomethacin and aspirin (cyclooxygenase inhibitors), but not by nordihydroguaiaretic acid (a lipoxygenase inhibitor). These results suggest that cyclooxygenase and not lipoxygenase metabolites of arachidonic acid, e.g. prostaglandins, may be involved in the generation of tachycardia. Consistent with this notion, exogenously applied cyclooxygenase metabolites, prostaglandin E2 and 6-keto-prostaglandin F1α, which are known to be produced by extracellular ATP in the rabbit heart [Schwartzman et al. (1981) Eur J Pharmacol 74: 167–173], accelerated the sinus rate. We also observed that the ATP-induced tachycardia was almost completely blocked by neomycin (a phospholipase C inhibitor). We suggest, therefore, that cardiac P2 receptors may be coupled to prostaglandin synthesis via an IAP-insenstive stimulation of phospholipase C.

Effects of calcitonin gene-related peptide on membrane currents in mammalian cardiac myocytes

We examined the effects of calcitonin gene-related peptide (CGRP) on the membrane currents of single atrial and ventricular cells of guinea pig heart. The tightseal whole-cell voltage-clamp technique was used. In atrial cells, like isoproterenol, CGRP increased the L-type Ca channel current (ICa.L) in a concentration-dependent manner. Human CGRP-(8-37), a putative CGRP receptor antagonist, completely abolished the CGRP-induced increase of ICa.L. Although the effects of CRGP were similar to those of isoproterenol, propranolol, a β-adrenergic receptor antagonist, did not affect the CGRP-induced increase of ICa.L. After ICa.L had been maximally activated by isoproterenol (2 μM) or intracellular cyclic adenosine 5′-monophosphate (100 μM), CGRP failed to increase ICa.L. Acetylcholine antagonized the effects of CGRP on ICa.L. Unlike the effects on atrial cells, CGRP had no significant effects on the membrane currents of ventricular myocytes. Thes results indicate that CGRP increases ICa.L via adenylate cyclase activation by binding to specific membrane receptors in cardiac atrial myocytes. Furthermore, CGRP receptors are expressed in atrial cells but probably not in ventricular cells.

Coenzyme Q10 attenuates cyanide-activation of the ATP-sensitive K+ channel current in single cardiac myocytes of the guinea-pig

SummaryThe effect of coenzyme Q10 (CoQ10) on the cyanide (CN−)-induced ATP-sensitive K+ channel current (KATP) was examined in single atrial myocytes, using the patch clamp technique. Superfusion of the cells with a CN−/low glucose bathing solution induced an outward current in the whole-cell clamp condition. Glibenclamide (1 μM) abolished this current, indicating that the current was carried through the KATP channel. After steady-state activation by CN−, pinacidil (a KATP channel opener, 300 μM) failed to further increase the current. In cell-attached patches, CN−, when applied to the bath, induced bursting openings of an 80 pS channel (the KATP channel). In cells preincubated for 30 min in a solution containing CoQ10 (100 μg/ml), CN−-activation of the KATP channel was markedly attenuated both at the whole cell and at the single channel level. At the steady-state effect of CN− in CoQ10-treated cells, pinacidil (300 μM) activated the current to the maximum level achieved by CN− in the control cells. These results suggest that CoQ10 reduces in the CN−-induced KATP current not by affecting the channel itself but by preventing depletion of intracellular ATP caused by CN−.

Noninvasive study of the presystolic component of the first heart sound in mitral stenosis.

Echophonocardiography and pulsed Doppler echocardiography were performed in 30 patients with mitral stenosis (19 with atrial fibrillation and 11 with sinus rhythm) to investigate the genesis of the presystolic component or small apical vibrations preceding the first heart sound in mitral stenosis. In 27 patients, mitral valve closure preceded or coincided with tricuspid valve closure regardless of the preceding RR interval. Of three patients whose tricuspid valve closed prematurely, two had a prolonged PR interval. The soft apical vibrations, which were recorded during the final rapid closing motion of the mitral valve echogram (B-C slope), began with the upstroke of the apexcardiogram. During this event the pulsed Doppler echocardiogram revealed a deceleration in the velocity of mitral inflow. In two exceptional patients with a prolonged PR interval, this apical sound was separated from a presystolic rumble that occurred during an accelerated phase of mitral inflow or at the A wave of mitral valve echograms. In conclusion, the tricuspid valve is not a factor contributing to the genesis of the small apical vibrations preceding the first heart sound in mitral stenosis. These vibrations are caused by acceleration of left ventricular contraction and deceleration of mitral inflow in the presence of a stenotic valve.

Echophonocardiographic study of the initial low frequency component of the first heart sound.

To investigate the genesis of the initial low frequency component of the first heart sound that precedes the high frequency vibrations associated with closure of the atrioventricular valves, echophonocardiograms of 36 persons were recorded. These included 10 normal subjects and 26 patients with various types of heart disease including mitral valve replacement. Electrocardiograms demonstrated normal sinus rhythm in 23 subjects, atrial fibrillation in 9, complete atrioventricular block in 2 and atrial flutter in 2. In the phonocardiogram, the low frequency component of the first heart sound followed the onset of the QRS complex and preceded the first high frequency component of this sound. The low frequency component occurred simultaneously with the beginning of the final fast closing movement of the mitral valve on the echocardiogram and was found both in normal rhythm and in arrhythmias. However, in arrhythmias its intensity varied on a beat to beat basis, being loudest after a short RR interval or when atrial systole occurred very close to the expected time of ventricular systole. In patients in whom apexcardiograms were recorded, the low frequency component was coincident with or very close to the onset of ventricular systole. It is concluded that the low frequency component of the first heart sound represents vibrations caused by contraction of the left ventricle and deceleration of antegrade blood flow across the mitral valve. Neither atrial contraction nor mitral valve tension is necessary for the production of this soft initial component.