Propranolol inhibits IK(Ado) by competitive A1-receptor interaction.

OBJECTIVES: Betablocking agents are known to exert anti-arrhythmic effects in ischemic myocardium due to blockade of myocardial beta-1-receptors. Since adenosine (Ado) induced muscarinic potassium current (IK(Ado)) and ATP sensitive potassium current (IK(ATP)) are discussed to be activated during ischemia we studied the effect of propranolol on IK(Ado) and IK(ATP). METHODS AND RESULTS: The effect of propranolol on muscarinic potassium current and IK(ATP) was studied in isolated rat atrial myocytes by means of the whole-cell voltage clamp tech- nique. Propranolol (50 microM) completely inhibited IK(Ado). IC50 was 7 microM. Inhibition of acetylcholine induced current (IK(ACh)) and GTP-gamma-S induced muscarinic potassium current was less potent (IC50 29 microM and 31 microM respectively). Propranolol was active from the outside only. Intracellular application did not significantly affect muscarinic potassium current. (+)-propranolol, an isomer without beta-blocking properties, was as effective as (+/-)-propranolol. The inwardly rectifying potassium current IK(ATP) showed minor sensitivity to the compound (10% current reduction, propranolol 50 microM). CONCLUSIONS: Propranolol inhibits IK(Ado). Inhibition is not due to beta-receptor blockade. Predominantly an interaction with A1-receptors seems to be involved. The observations in part might explain the anti-arrhythmic properties of the drug in ischemic/fibrillating myocardium based on the prolongation of refractoriness.

Inhibition of G protein-coupled and ATP-sensitive potassium currents by 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran (KB130015), an amiodarone derivative.

2-Methyl-3- (3,5-diiodo-4-carboxymethoxybenzyl) benzofuran (KB130015; KB), a novel compound derived from amiodarone, has been proposed to have antiarrhythmic properties. Its effect on the G protein-coupled inward rectifying K+ current [IK(ACh) or IK(Ado)], ATP-sensitive K+ current [IK(ATP)], and background inward rectifying current (I(K1)) were studied in guinea pig atrial and ventricular myocytes by the whole-cell voltage-clamp technique. Receptor-activated IK(ACh/Ado), induced in atrial myocytes by the stimulation of either muscarinic or Ado receptors was concentration dependently (IC50 value of approximately 0.6-0.8 microM) inhibited by KB. Receptor-independent guanosine 5'-O-(3-thio)triphosphate-induced and background IK(ACh), which contributes to the resting conductance of atrial myocytes, were equally sensitive to KB (IC50 value of approximately 0.9 microM). IK(ATP) induced in atrial myocytes during metabolic inhibition with 2,4-dinitrophenol (DNP) was also suppressed by KB, whereas IK1 measured in ventricular myocytes was insensitive to the drug (KB < or =50 microM). Although being effective when applied from the outside, intracellular application of KB via the patch pipette affected neither IK(ACh) nor IK(ATP). 3,3',5-triodo-L-thyronin, which shares structural groups with KB, did not have an effect on the K+ currents. Consistent with the effects on single myocytes, KB did not depolarize the resting potential but antagonized the shortening of action potential duration by carbamylcholine-chloride or by DNP in multicellular preparations and antagonized the shortening of action potential duration by acetylcholine in single myocytes. It is concluded that KB inhibits IK(ACh) and IK(ATP) by direct drug-channel interaction at a site more easily accessible from extracellular side of the membrane.

Action potential changes associated with a slowed inactivation of cardiac voltage-gated sodium channels by KB130015.

1. We have studied the acute cardiac electrophysiological effects of KB130015 (KB), a drug structurally related to amiodarone. Membrane currents and action potentials were measured at room temperature or at 37 degrees C during whole-cell patch-clamp recording in ventricular myocytes. Action potentials were also measured at 37 degrees C in multicellular ventricular preparations. 2. The effects of KB were compared with those of anemone toxin II (ATX-II). Both KB and ATX-II slowed the inactivation of the voltage-gated Na(+) current (I(Na)). While KB shifted the steady-state voltage-dependent inactivation to more negative potentials, ATX-II shifted it to more positive potentials. In addition, while inactivation proceeded to completion with KB, a noninactivating current was induced by ATX-II. 3. KB had no effect on I(K1) but decreased I(Ca-L) The drug also did not change I(to) in mouse myocytes. 4. The action potential duration (APD) in pig myocytes or multicellular preparations was not prolonged but often shortened by KB, while marked APD prolongation was obtained with ATX-II. Short APDs in mouse were markedly prolonged by KB, which frequently induced early afterdepolarizations. 5. A computer simulation confirmed that long action potentials with high plateau are relatively less sensitive to a mere slowing of I(Na) inactivation, not associated with a persisting, noninactivating current. In contrast, simulated short action potentials with marked phase-1 repolarization were markedly modified by slowing I(Na) inactivation. 6 It is suggested that a prolongation of short action potentials by drugs or mutations that only slow I(Na) inactivation does not necessarily imply identical changes in other species or in different myocardial regions.

Diadenosine-5-phosphate exerts A1-receptor-mediated proarrhythmic effects in rabbit atrial myocardium.

(1) Diadenosine polyphosphates have been described to be present in the myocardium and exert purinergic- and nonreceptor-mediated effects. Since the electrophysiological properties of atrial myocardium are effectively regulated by A(1) receptors, we investigated the effect of diadenosine pentaphosphate (Ap(5)A) in rabbit myocardium. (2) Parameters of supraventricular electrophysiology and atrial vulnerability were measured in Langendorff-perfused rabbit hearts. Muscarinic potassium current (I(K(ACh/Ado))) and ATP-sensitive potassium current (I(K(ATP))) were measured by using the whole-cell voltage clamp method. (3) Ap(5)A prolonged the cycle length of spontaneously beating Langendorff perfused hearts from 225+/-14 (control) to 1823+/-400 ms (Ap(5)A 50 micro M; n=6; P<0.05). This effect was paralleled by higher degree of atrio-ventricular block. Atrial effective refractory period (AERP) in control hearts was 84+/-14 ms (n=6). Ap(5)A>/=1 micro M reduced AERP (100 micro M, 58+/-11 ms; n=6). (4) Extrastimuli delivered to hearts perfused with Ap(5)A- or adenosine (>/= micro M)-induced atrial fibrillation, the incidence of which correlated to the concentration added to the perfusate. The selective A(1)-receptor antagonist CPX (20 micro M) inhibited the Ap(5)A- and adenosine-induced decrease of AERP. Atrial fibrillation was no longer observed in the presence of CPX. (5) The described Ap(5)A-induced effects in the multicellular preparation were enhanced by dipyridamole (10 micro M), which is a cellular adenosine uptake inhibitor. Dipyridamole-induced enhancement was inhibited by CPX. (6) Ap(5)A (

AV nodal pathways in the R-R interval histogram of the 24-hour monitoring ECG in patients with atrial fibrillation.

BACKGROUND: Patients with more than one AV nodal pathway show two and more peaks in the histogram of the R-R intervals of the Holter monitoring ECG during atrial fibrillation. It was the purpose of the present study to determine the number of patients showing more than one AV nodal pathway in a larger patient group with permanent atrial fibrillation by analyzing the Holter monitoring ECG. METHODS: 250 patients with permanent atrial fibrillation during Holter monitoring ECG were studied; 203 patients had structural heart disease. The number of peaks in the R-R interval histogram of each patient was determined. The distribution of the number of peaks in the R-R interval histogram in different patient groups was analyzed. RESULTS: 153 patients (61%) had one peak, 80 patients (32%) two peaks, 13 patients (5%) three peaks, and four patients (2%) four peaks, reflecting the number of different AV nodal pathways. In the different patient groups, in the patients with or without structural heart disease, with coronary heart disease, with a history of syncope, and in patients with a mean heart rate of more than 100/min, there was no significant difference in the distribution of the number of peaks in the R-R interval histogram. CONCLUSIONS: In more than one third of all patients with permanent atrial fibrillation there are two, three, or four AV nodal pathways. It is suggested that this number of different AV nodal pathways found in the studied group can be applied to all humans. 38.8% of all patients with permanent atrial fibrillation have more than one AV nodal pathway; 6.4% of all patients with atrial fibrillation would benefit from an ablation of AV nodal pathways with shorter refractory periods for reduction of the heart rate.

Sphingosylphosphocholine is a naturally occurring lipid mediator in blood plasma: a possible role in regulating cardiac function via sphingolipid receptors.

Blood plasma and serum contain factors that activate inwardly rectifying GIRK1/GIRK4 K+ channels in atrial myocytes via one or more non-atropine-sensitive receptors coupled to pertussis-toxin-sensitive G-proteins. This channel is also the target of muscarinic M(2) receptors activated by the physiological release of acetylcholine from parasympathetic nerve endings. By using a combination of HPLC and TLC techniques with matrix-assisted laser desorption ionization-time-of-flight MS, we purified and identified sphingosine 1-phosphate (SPP) and sphingosylphosphocholine (SPC) as the plasma and serum factors responsible for activating the inwardly rectifying K+ channel (I(K)). With the use of MS the concentration of SPC was estimated at 50 nM in plasma and 130 nM in serum; those concentrations exceeded the 1.5 nM EC(50) measured in guinea-pig atrial myocytes. With the use of reverse-transcriptase-mediated PCR and/or Western blot analysis, we detected Edg1, Edg3, Edg5 and Edg8 as well as OGR1 sphingolipid receptor transcripts and/or proteins. In perfused guinea-pig hearts, SPC exerted a negative chronotropic effect with a threshold concentration of 1 microM. SPC was completely removed after perfusion through the coronary circulation at a concentration of 10 microM. On the basis of their constitutive presence in plasma, the expression of specific receptors, and a mechanism of ligand inactivation, we propose that SPP and SPC might have a physiologically relevant role in the regulation of the heart.

Inhibition of muscarinic potassium current by the class III antiarrhythmic drug RP58866 in guinea-pig atrial myocytes.

RP58866 is a potent antiarrhythmic drug that maintains its antiarrhythmic properties during ischemia. Since interstitial concentrations of adenosine increase during ischemia, we examined the properties of the drug with respect to the muscarinic K+ current (IK(ACh)), with a main emphasis on the adenosine (Ado)-induced current (IK(Ado)). Using different Gi-coupled receptors (M2, A1, sphingolipid), we studied the effect of RP58866 in isolated guinea-pig atrial myocytes by the whole-cell voltage clamp technique. Application of 50 microM RP58866 resulted in complete inhibition of the muscarinic K+ current. Inhibition was observed during activation of IK(ACh) by each of the three receptors. IC50 was approximately 2.0 microM. GTP-gamma-S induced IK(ACh) was reduced by RP58866. The drug was active from the outside only, and its intracellular application via the patch pipet had no inhibitory effect. Despite the structural homologies between inward rectifying K+ channels, the adenosine triphosphate-sensitive K+ current (IK(ATP)) was not inhibited by the compound. It is concluded that muscarinic K+ current is inhibited by RP58866, an inhibition not limited to IK1, Ito, and IKr. High interstitial adenosine concentrations during ischemia are expected to increase the participation of IK(Ado) on repolarization. RP58866-induced inhibition of IK(Ado) would, therefore, be of particular relevance during ischemia. The high sensitivity of IK(Ado) to RP58866 may partially explain the unique properties of the drug toward arrhythmias developing in the ischemic myocardium.

Uncoupling of ATP-sensitive potassium current from cell metabolism due to antisense oligonucleotides against sulfonylurea receptor in guinea-pig atrial myocytes.

ATP-sensitive K+ current (IK(ATP)) plays an important role in the regulation of cardiac electrical activity. In the myocardium, IK(ATP) is regulated by the sulfonylurea receptor (SURIIA) which mediates the inhibition of IK(ATP) due to glibenclamide (Gli). The role played by SURIIA in the sensitivity of IK(ATP) to metabolic inhibition is unclear. We studied the effect of SURIIA antisense oligonucleotides (ODNs) on the properties of IK(ATP) in cultured guinea-pig atrial myocytes. IK(ATP) was measured by the whole-cell voltage clamp method and was activated with cromakalim (Cro; 200 microns) and dinitrophenole (DNP; 100 microns). Mean IK(ATP) density activated by DNP and Cro in nonincubated cells was 117 +/- 12 pA/pF (n = 17) and 17 +/- 9 pA/pF (n = 16) respectively. No significant difference was observed after incubation with nODN [DNP: 121 +/- 13 pA/pF (n = 20); Cro:19 +/- 4 pA/pF (n = 8)]. Cells incubated with ODNs showed a significant reduction of IK(ATP) due to DNP (19 +/- 13 pA/pF; P < 0.05, n = 6), whereas Cro-induced IK(ATP) was unaffected (16 +/- 8 pA/pF, n = 8). The effectiveness of DNP-induced metabolic inhibition was apparent in a concomitant reduction of the nucleotide-phosphate dependent muscarinic K+ current (inhibition of IK(ACh) in ODN incubated myocytes without activation of IK(ATP)). The ATP sensitivity of IK(ATP) appears mediated by SURIIA. Activation of this current by Cro seems to be SURIIA-independent. ODN-induced metabolic uncoupling of IK(ATP) may be a useful experimental tool. A reduced sensitivity of IK(ATP) to intracellular ATP concentrations may be of clinical interest.

Antisense oligonucleotides against receptor kinase GRK2 disrupt target selectivity of beta-adrenergic receptors in atrial myocytes.

K+ channels composed of GIRK subunits are predominantly expressed in the heart and various regions of the brain. They are activated by betagamma-subunits released from pertussis toxin-sensitive G-proteins coupled to different seven-helix receptors. In rat atrial myocytes, activation of K(ACh) channels is strictly limited to receptors coupled to pertussis toxin-sensitive G-proteins. Upon treatment of myocytes with antisense oligodesoxynucleotides against GRK2, a receptor kinase with Gbetagamma binding sites, in a fraction of cells, K(ACh) channels can be activated by beta-adrenergic receptors. Sensitivity to beta-agonist is insensitive to pertussis toxin treatment. These findings demonstrate a potential role of Gbetagamma binding proteins for target selectivity of G-protein-coupled receptors.

Spatial and temporal variations in the magnetic fields produced by human gastrointestinal activity.

Magnetoenterography (MENG) is a new, non-invasive technique that measures gastrointestinal magnetic signals near the body surface. This study was undertaken to evaluate the temporal and spatial characteristics of the magnetic signals generated by gastric and duodenal slow wave activity. The gastrointestinal magnetic fields of eight normal subjects were measured for 60 minutes in both the fasting and fed state using 36 magnetic sensors simultaneously. The results were displayed as a succession of maps over time showing the temporal evolution of the spatial distribution of the signal over the upper abdomen. In all subjects, slow wave activity of the stomach centred at 3.0 +/- 0.5 cycles min-1 in both the fasting and fed state was observed. The duodenal signal at 11.0 +/- 1.0 cycles min-1 was observed in four subjects. The spatial distribution of these two signals is distinctly different. The observed spatial and temporal variations are described in terms of a model used previously to explain the potentials observed in electrogastrography (EGG).

Non-receptor-mediated activation of IK(ATP) and inhibition of IK(ACh) by diadenosine polyphosphates in guinea-pig atrial myocytes.

1. The effects of diadenosine polyphosphates (APnA, where n = 4-6) were studied on beating frequency of perfused guinea-pig hearts and on muscarinic K+ current (IK(ACh)) and ATP-regulated K+ current (IK(ATP)) in atrial myocytes from guinea-pig hearts using whole-cell voltage clamp. 2. Bradycardia induced by APnA in perfused hearts was completely inhibited by 8-cyclopentyl- 1,3-dipropylxanthine (CPX, 20 microM), a selective antagonist at A1 adenosine receptors, and was augmented by dipyridamole (Dipy), an inhibitor of cellular adenosine (Ado) uptake. 3. Whereas exposure of atrial myocytes to Ado (100 microM) within about 1 s induced a significant whole-cell IK(ACh), APnA up to 1 mM applied for some tens of seconds failed to activate IK(ACh). If present for periods > 2 min, APnA caused inhibition of agonist-evoked IK(ACh) and activation of a weakly inward rectifying K+ current, which was identified as IK(ATP) by its sensitivity to glibenclamide and its current-voltage curve. 4. The actions of extracellular APnA on IK(ACh) and IK(ATP) were mimicked by intracellular loading of compounds via the patch clamp pipette and by intracellular loading of AMP. 5. The results from isolated myocytes exclude APnA acting as A1 agonists. It is suggested that myocytes can take up APnA, which are degraded to AMP. In the presence of ATP, AMP is converted to ADP, a physiological activator of ATP-regulated K+ channels, by adenylate kinase. A similar mechanism resulting in a reduction of the [GTP]/[GDP] ratio might be responsible for inhibition of IK(ACh). 6. In the perfused heart and other multicellular cardiac preparations the actions of APnA are mediated by Ado via A1 receptors. It is suggested that APnA in multicellular cardiac tissue are hydrolysed by an ectohydrolase to yield AMP which is converted to Ado by ectonucleotidases.

Inhibition of muscarinic K+ current in guinea-pig atrial myocytes by PD 81,723, an allosteric enhancer of adenosine binding to A1 receptors.

1. PD 81,723 has been shown to enhance binding of adenosine to A1 receptors by stabilizing G protein-receptor coupling ('allosteric enhancement'). Evidence has been provided that in the perfused hearts and isolated atria PD 81,723 causes a sensitization to adenosine via this mechanism. 2. We have studied the effect of PD 81,723 in guinea-pig isolated atrial myocytes by use of whole-cell measurement of the muscarinic K+ current (I[K(ACh)]) activated by different Gi-coupled receptors (A1, M2, sphingolipid). PD 81,273 caused inhibition of I[K(ACh)] (IC50 approximately 5 microM) activated by either of the three receptors. Receptor-independent I[K(ACh)] in cells loaded with GTP-gamma-S and background I[K(ACh)], which contributes to the resting conductance of atrial myocytes, were equally sensitive to PD 81,723. At no combination of concentrations of adenosine and PD 81,723 could an enhancing effect be detected. 3. The compound was active from the outside only. Loading of the cells with PD 81,723 (50 microM) via the patch pipette did not affect either I[K(ACh)] or its sensitivity to adenosine. We suggest that PD 81,723 acts as an inhibitor of inward rectifying K+ channels; this is supported by the finding that ventricular I(K1), which shares a large degree of homology with the proteins (GIRK1/GIRK4) forming I[K(ACh)] but is not G protein-gated, was also blocked by this compound. 4. It is concluded that the functional effects of PD 81,723 described in the literature are not mediated by the A1 adenosine receptor-Gi-I[K(ACh)] pathway.

In vivo downregulation of M2 receptors revealed by measurement of muscarinic K+ current in cultured guinea-pig atrial myocytes.

1. Muscarinic K+ current (IK(ACh)) elicited by acetylcholine (ACh) was measured in guinea-pig atrial myocytes, which were either freshly isolated or cultured for up to 8 days. 2. The half-time of activation of inward IK(ACh) by a saturating concentration (10 microM) of ACh decreased from approximately 400 ms (in freshly isolated cells) to 250 ms after 6 days in culture. This was paralleled by an increase in the fast desensitizing component of IK(ACh). The density of steady-state currents was not changed. Downregulation of M2 receptors by long-term treatment of isolated myocytes with carbachol in vitro had opposite effects. 3. The EC50 of ACh for the activation of steady-state IK(ACh) was reduced from 5 x 10(-7) M (day 0) to 8 x 10(-8) M (day 6). The shift in EC50 occurred with a half-time of about 2 days, similar to the recovery from downregulation induced by treating atrial myocytes with carbachol in vitro. 4. The increase in sensitivity to ACh can be accounted for by an approximately 6-fold increase in the density of M2 receptors. 5. It is concluded that sensitization in culture reflects recovery from downregulation of M2 receptors due to the tonic vagal input to the heart in the intact animal.

A novel membrane receptor with high affinity for lysosphingomyelin and sphingosine 1-phosphate in atrial myocytes.

Activation of IK(ACh) is the major effect of the vagal neutrotransmitter acetylcholine in the heart. We report that both lysosphingomyelin (D-erythro-sphingosyl-phosphorylcholine; SPC) and sphingosine 1-phosphate (SPP) activate IK(ACh) in guinea pig atrial myocytes through the same receptor with an EC50 of 1.5 and 1.2 nM, respectively. Pertussis toxin abolished the activation of IK(ACh) by either lipid. The putative receptor showed an exquisite stereoselectivity for the naturally occurring D-erythro-(2S,3R)-SPC stereoisomer, the structure of which was confirmed by mass spectroscopy and NMR. These lipids caused complete homologous and heterologous desensitization with each other but not with ACh, indicating that both act on the same receptor. This receptor displays a distinct structure-activity relationship: it requires an unsubstituted amino group because N-acetyl-SPC, lysophosphatidic acid and lysophosphatidylcholine were inactive. Because SPP and SPC are naturally occurring products of membrane lipid metabolism, it appears that these compounds might be important extracellular mediators acting on a family of bona fide G protein-coupled receptors. Expression of these receptors in the heart raises the possibility that sphingolipids may be a part of the physiological and/or pathophysiological regulation of the heart. Based on their ligand selectivity we propose a classification of the sphingolipid receptors.

Downregulation of muscarinic M2 receptors linked to K+ current in cultured guinea-pig atrial myocytes.

1. Desensitization of muscarinic K+ current (IK(ACh)) was studied in cultured atrial myocytes from guinea-pig hearts using whole-cell voltage clamp. 2. Three different types of desensitization could be identified. A fast component which upon rapid superfusion with ACh resulted in a partial relaxation of IK(ACh) within a few seconds to a plateau which was maintained in the presence of ACh. Recovery from this type of desensitization paralleled the decay of IK(ACh) after washout of the agonist. A second type of desensitization was observed within minutes. This was reversed around 10 min after washout of ACh. Both types were heterologous with regard to the A1 receptor and the novel phospholipid (Pl) receptor, both of which activate IK(ACh) via the same signalling pathway. 3. A third type of desensitization (downregulation) occurred upon exposure of the cultures for 24-48 h to the muscarinic agonist carbachol (CCh). The level of downregulation depended on the concentration of CCh (0.1 microM < or = [CCh] < or = 10 microM). No recovery was observed within 5 h after washout of CCh. Thereafter sensitivity to ACh slowly returned (half-time (t1/2), approximately 20 h). 4. Downregulation by CCh (0.1-5 microM) was characterized by an increase in EC50 for ACh with no reduction in maximum IK(ACh). With 5 microM CCh, EC50 was increased from 0.1 to 3.7 microM. At 10 microM CCh EC50 was increased to 15 microM and maximal current that could be evoked by ACh was reduced to 15%. 5. Downregulation by CCh was homologous with regard to A1 and Pl receptors. Maximum IK(ACh), assayed by a saturating concentration of Pl, was not reduced in downregulated cells, suggesting a mechanism localized at the M2 receptor. 6. The changes in the concentration-response curves can be accounted for by assuming an excess of M2 receptors relative to the subsequent component of the signalling pathway. 7. As the intact heart is under tonic vagal control, downregulation is likely to contribute to controlling the sensitivity of the heart to vagal activity in situ.

Activation of muscarinic K+ current in guinea-pig atrial myocytes by sphingosine-1-phosphate.

1. Activation of muscarinic K+ current (IK(ACh)) by sphingosine-1-phosphate (Sph-1-P) was studied in isolated cultured guinea-pig atrial myocytes using whole-cell voltage clamp. 2. Sph-1-P caused activation of IK(ACh) with an EC50 of 1.2 nM. The maximal current that could be activated by Sph-1-P amounted to about 90% of the IK(ACh) caused by a saturating concentration of acetylcholine (ACh, 10 microM). Sphingosine (1 microM), which can mimic the signalling effects of Sph-1-P in other cells, failed to cause measurable activation of IK(ACh). 3. IK(ACh) activation by Sph-1-P was completely suppressed in cells treated with pertussis toxin. 4. Desensitization of muscarinic receptors by pre-incubation of the cells with carbachol did not affect the response to Sph-1-P; likewise, pre-incubation of the cells with Sph-1-P resulted in a reduced sensitivity to the phospholipid but not to ACh. In contrast, pre-incubation with either Sph-1-P or a serum phospholipid previously described as activating atrial IK(ACh) resulted in reduced sensitivity to both phospholipids. 5. It is concluded that activation of IK(ACh) by Sph-1-P in atrial myocytes is induced by binding to a novel G protein-coupled phospholipid receptor.