Single Atrial Myocytes Research Articles

Inhibition of L-type Ca2+ current by C-type natriuretic peptide in bullfrog atrial myocytes: an NPR-C-mediated effect.

Single atrial myocytes were isolated from the bullfrog heart and studied under current and voltage clamp conditions to determine the electrophysiological effects of the C-type natriuretic peptide (CNP). CNP (10(-8) M) significantly shortened the action potential and reduced its peak amplitude after the application of isoproteronol (10(-7) M). In voltage clamp studies, CNP inhibited isoproteronol-stimulated L-type Ca2+ current (ICa) without any significant effect on the inward rectifier K+ current. The effects of cANF (10(-8) M), a selective agonist of the natriuretic peptide C receptor (NPR-C), were very similar to those of CNP. Moreover, HS-142-1, an antagonist of the guanylyl cyclase-linked NPR-A and NPR-B receptors did not alter the inhibitory effect of CNP on ICa. Inclusion of cAMP in the recording pipette to stimulate ICa at a point downstream from adenylyl cyclase increased ICa, but this effect was not inhibited by cANF. These results provide the first demonstration that CNP can inhibit ICa after binding to NPR-C, and suggest that this inhibition involves a decrease in adenylyl cyclase activity, which leads to reduced intracellular levels of cAMP.

Decreases in ANP secretion by lysophosphatidylcholine through protein kinase C.

Lysophosphatidylcholine (LPC) is an endogenous phospholipid released from the cell membrane during ischemia, and it has potent, local effects on cardiac tissues. LPC has been implicated in arrhythmogenesis during ischemia by increasing intracellular Ca2+. However, it is not known whether LPC influences atrial release of atrial natriuretic peptide (ANP). The aim of this study was to investigate the effect of LPC on ANP secretion from isolated, perfused, beating rat atria. LPC (10 and 30 micromol/L) caused decreases in ANP secretion in a dose-dependent manner, with slight increases in intra-atrial pressure and extracellular fluid (ECF) translocation. Therefore, the ANP secretion in terms of ECF translocation was markedly decreased by LPC. The order of the suppressive effect of ANP release was stearoyl-LPC>LPC>myristoyl-LPC=lauroyl-LPC. Staurosporine and wortmannin significantly attenuated suppression of the ANP release and an increase in intra-atrial pressure by LPC. High extracellular Mg2+ also attenuated the LPC-induced suppression of ANP release. However, other protein kinase C inhibitors such as chelerythrine, GF 109203X, and tamoxifen citrate did not affect LPC-induced suppression of ANP release. In single atrial myocytes, LPC caused increases in intracellular Ca2+ in a dose-dependent manner. The order of an increase in intracellular Ca2+ by LPC was stearoyl-LPC>LPC>myristoyl-LPC=lauroyl-LPC. An increase in intracellular Ca2+ by LPC was attenuated by staurosporine. These results suggest that LPC-induced suppression of ANP release through protein kinase C/Ca2+ and phosphoinositol-3-kinase might in part play an important role in the development of hypertension.

Potentiation of the negative dromotropic effect of adenosine by rapid heart rates: possible ionic mechanism.

Adenosine-induced slowing of atrioventricular nodal conduction is a rate-dependent process that is potentiated by the A(1)-adenosine receptor allosteric enhancer, PD 81,723. The ionic mechanisms underlying these phenomena were investigated in guinea pig isolated hearts and single atrial myocytes by measuring the atrium-to-His bundle (A-H) interval and using patch-clamp recordings, respectively.A decrease in atrial cycle length from 300 to 190 ms decreased the concentration of adenosine needed to cause atrioventricular nodal block from 7.8 +/- 1.0 to 2.6 +/- 0.7 micromol/L (P < 0.001). Ba(2+) (100 micromol/L), a selective blocker of the adenosine-activated inward rectifier K(+) current I(K,ADO) in the atrioventricular node, failed to abolish this rate-dependent effect of adenosine. PD 81,723 (5 micromol/L) potentiated the negative dromotropic effect of adenosine even after I(K,ADO) was blocked by Ba(2+) and after attenuation of I(Ca,L) by adenosine was prevented by 8-Br-cAMP (1.5 mmol/L). In atrial myocytes, adenosine augmented a time- and voltage-dependent K(+) current (Ado-I(K)). Ado-I(K) was more sensitive to adenosine than I(K,ADO) (EC(50) values, 0.8 versus 1.4 micromol/L, P < 0.01). PD 81,723 blocked I(K,ADO), but potentiated Ado-I(K). Ado-I(K) was insensitive to Ba(2+) (P = 0.98), whereas it was blocked by chromanol 293B (5 micromol/L, P < 0.001). Unlike I(K,ADO), Ado-I(K) increased during rapid stimulation of myocytes (P < 0.001). Adenosine augments a time- and voltage-dependent K(+) current, Ado-I(K). The pharmacological and kinetic properties of Ado-I(K) are consistent with it playing an important role in the negative dromotropic effect of adenosine at lower concentrations of the nucleoside, at fast heart rates and in the presence of PD 81,723.

Monitoring of ANP secretion from single atrial myocytes using densitometry.

Atrial myocytes secrete atrial natriuretic peptide (ANP) in response to mechanical stretch and can serve as a challenging model for studying stretch-secretion coupling. We have developed a technique for monitoring ANP secretion from single atrial myocytes, using neutral red and a CCD video camera. Atrial-specific granules (ASGs) containing ANP were stained with neutral red. The cells were illuminated with monochromatic light (550 mm) and the grey value monitored within the region of interest (ROI) surrounding the region in which ASGs were densely located. Assuming that neutral red is evenly distributed in ASGs, the change in optical density (OD) was considered to represent the total amount of secretion. Under control, non-stimulated conditions, the OD decreased spontaneously (19.7+/-1.4%/10 min, n=14). Direct mechanical stretch (cell length increased by 20%) with two micropipettes or hypotonic swelling (200 mOsm) accelerated the decrease in OD significantly (48.7+/-7.4%/10 min; n=3, 47.2+/-2.4%/10 min; n=7, respectively). In conclusion, this method allows monitoring of ANP secretion with a relatively high time resolution while mechanical stress is applied. Furthermore, patch-clamp or intracellular perfusion techniques can be combined with the present technique for studying cellular mechanisms of stretch-secretion coupling.

Profile of RGS expression in single rat atrial myocytes

‘Regulators of G protein signaling’ (RGS proteins) are members of a large family of GTPase-activating proteins that are differentially expressed in various cell types and accelerate the termination of heterotrimeric G protein signaling. To identify RGS proteins that may affect autonomic regulation of atrial excitability, we screened the expression of nineteen RGS genes (RGS subfamilies A, B, C, and D) in single spontaneously beating rat atrial myocytes maintained in primary culture. Expression profiling by single-cell reverse transcriptase–polymerase chain reaction (RT–PCR) analysis revealed that seven distinct RGS genes are endogenously expressed in atrial myocytes which were also identified in poly(A) + mRNA from rat atria (RGS2, RGS3, RGS4, RGS6, RGS10, GAIP, and RGSZ2). Other RGS transcripts were detected in atrial poly(A) + mRNA but not single atrial myocytes (RGS5, RGS12, RGS16, and RGS18), and therefore are likely to originate from non-myocyte sources in atrial tissue. The single-cell RT–PCR experiments also led to the identification of putative splice variants for RGS6 and GAIP. Immunocytochemistry using RGS-specific antibodies confirmed the presence of selected RGS proteins in the cultured atrial myocytes. These results demonstrate a rich diversity of RGS expression in atrial myocytes whose specific role in G-protein signaling is yet to be determined. The identification of endogenous RGS proteins in atrial myocytes will facilitate targeted suppression and/or deletion studies to determine how each RGS protein may affect atrial excitability and its short-term and long-term regulation by G-protein signaling events.

Effects of ketamine on voltage-dependent calcium currents and membrane potentials in single bullfrog atrial cells.

This study was undertaken to assess the effect of ketamine on L-type calcium channel current (I(Ca)) and membrane action potential in the bullfrog single atrial myocyte. Bullfrog single atrial myocytes were prepared by enzymatic dispersion. Whole-cell voltage-clamp technique and current clamp technique were used to monitor I(Ca), membrane resting potential, and action potential. Ketamine (10(-5)-10(-3) M) showed dose-dependent inhibition of I(Ca) in a reversible manner. The 50% inhibitory concentration (IC(50)) of ketamine on I(Ca) was estimated to be 0.92 x 10(-5) M. Use-dependent block of I(Ca) was not observed. The resting membrane potential was depolarized at a high concentration (10(-4) M) of ketamine. Reduction of the plateau phase and prolonged duration of the action potential were observed in the presence of a high concentration of ketamine (10(-4) M). Ketamine has an inhibitory effect on I(Ca) in the bullfrog single atrial myocyte, and a high dose (10(-4) M) of ketamine prolonges the duration of the action potential. The mechanism of inhibition of I(Ca) seems to be a direct effect on the L-type calcium channel, not like an open channel blocker.

The Characteristics of Spontaneous Action Potential of Cardiac Myocytes in Rabbit Pulmonary Veins

Background and Objectives:Atrial fibrillation is one of the most prevalent arrhythmia with clinical significance. Recently, some subset of paroxysmal atrial fibrillation was reported to be originated from a focal, rapidly firing source inside the large thoracic veins, such as pulmonary veins, superior vena cava and coronary sinus. The pulmonary veins are known to be the most frequent source of this type of atrial fibrillation. The proximal segment of pulmonary vein was reported to be made up with cardiac muscle cells. This study was performed 1) to define the characteristics of action potential of cardiac myocytes inside the rabbit pulmonary vein in single cell preparation, 2) to observe the changes in action potential and current activation to acetylcholine and isoproterenol, and 3) to compare these changes with those in atrial myocytes. Method and Results: In most of rabbit specimens, myocardial tissue extended over the pulmonary vein for a few millimeters (1-2.5 mm). Single atrial myocyte and myocyte in pulmonary vein were successfully isolated. With using whole cell patch clamp technique, spontaneous activities of action potentials (APs) with diastolic depolarization were observed in 75% of pulmonary vein myocytes, in contrast to the absence of spontaneous activity in atrial myocytes. During spontaneous APs of pulmonary vein myocytes, the maximal diastolic potential was -50.5± 6.5 mV and peak potential was 32.5±9.5 mV, and the frequency of APs was 1-2.5 Hz. During perfusion of isolated pulmonary vein myocytes with acetylcholine, resting membrane potential was hyperpolarized and spontaneous APs activity was markedly reduced or completely disappeared. These effects were observed in very low concentration of acetylcholine, even with 1-2 nM. The analysis of change of currents by applying step pulse revealed this response was mediated by activation of I K (ACh) and the current change was more prominent in pulmonary vein myocytes than atrial myocytes. The responses of these cells to isoproterenol were variable from increased spontaneous APs to inhibition of APs. Conclusion:This study revealed that pulmonary vein myocytes was another automatic pacemaking focus, same as sinoatrial nodal and Purkinje 논문접수일:2000년 2월 17일 심사완료일:2001년 1월 31일 교신저자:임채헌, 138-736 서울 송파구 풍납동 388-1 울산대학교 의과대학 서울중앙병원 생리학교실 전화:(02) 2224-4287·전송:(02) 2224-4220 E-mail:leemch@www.amc.seoul.kr

C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling.

The purpose of the present experiments was to define the role of C-type natriuretic peptide (CNP) in the regulation of atrial secretion of atrial natriuretic peptide (ANP) and atrial stroke volume. Experiments were performed in perfused beating and nonbeating quiescent atria, single atrial myocytes, and atrial membranes. CNP suppressed in a dose-related fashion the increase in atrial stroke volume and ANP secretion induced by atrial pacing. CNP caused a right shift in the positive relationships between changes in the secretion of ANP and atrial stroke volume or translocation of the extracellular fluid (ECF), which indicates the suppression of atrial myocytic release of ANP into the paracellular space. The effects of CNP on the secretion and contraction were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). CNP increased cGMP production in the perfused atria, and the effects of CNP on the secretion of ANP and atrial dynamics were accentuated by pretreatment with an inhibitor of cGMP phosphodiesterase, zaprinast. An inhibitor of the biological natriuretic peptide receptor (NPR), HS-142-1, attenuated the effects of CNP. The suppression of ANP secretion by CNP and 8-BrcGMP was abolished by a depletion of extracellular Ca(2+) in nonbeating atria. Natriuretic peptides increased cGMP production in atrial membranes with a rank order of potency of CNP > BNP > ANP, and the effect was inhibited by HS-142-1. CNP and 8-BrcGMP increased intracellular Ca(2+) concentration transients in single atrial myocytes, and mRNAs for CNP and NPR-B were expressed in the rabbit atrium. From these results we conclude that atrial ANP release and stroke volume are controlled by CNP via NPR-B-cGMP mediated signaling, which may in turn act via regulation of intracellular Ca(2+).

Ionic mechanisms of electrical remodeling in human atrial fibrillation

Atrial fibrillation (AF) is associated with a decrease in atrial ERP and ERP adaptation to rate as well as changes in atrial conduction velocity. The cellular changes in repolarization and the underlying ionic mechanisms in human AF are only poorly understood. Action potentials (AP) and ionic currents were studied with the patch clamp technique in single atrial myocytes from patients in chronic AF and compared to those from patients in stable sinus rhythm (SR). The presence of AF was associated with a marked shortening of the AP duration and a decreased rate response of atrial repolarization. L-type calcium current (ICa,L) and the transient outward current (Ito) were both reduced about 70% in AF, whereas an increased steady-state outward current was detectable at test potentials between -30 and 0 mV. The inward rectifier potassium current (IKI) and the acetylcholine-activated potassium current (IKACh) were increased in AF at hyperpolarizing potentials. Voltage-dependent inactivation of the fast sodium current (INa) was shifted to more positive voltages in AF. AF in humans leads to important changes in atrial potassium and calcium currents that likely contribute to the decrease in APD and APD rate adaptation. These changes contribute to electrical remodeling in AF and are therefore important factors for the perpetuation of the arrhythmia.

L-type Ca2+ current and excitation-contraction coupling in single atrial myocytes from rainbow trout.

We have examined the contribution of L-type Ca2+ current (ICa) to the activation of contraction in trout atrial myocytes under basal and phosphorylating conditions. The average myocyte length was 197 +/- 14 micrometer, width was 5.5 +/- 0.2 micrometer, and cell capacitance was 36.2 +/- 2.2 pF. With 25 microM EGTA in the patch pipette and a stimulation frequency of 0.125 Hz, ICa was 2.6 +/- 0.4 pA/pF and it carried a total charge of 0.10 +/- 0.01 pC/pF, giving rise to a contraction of 15.2 +/- 2.8% of the resting cell length. With a cell volume of 2.4 +/- 0.3 pl, the charge carried by ICa corresponded to 14.7 +/- 2.2 micromol Ca2+/l nonmitochondrial cell volume (microM). This can account for only 30-40% of the Ca2+ binding to the myofilaments during a contraction. Increasing the stimulation frequency from 0.25 to 2 Hz decreased ICa amplitude and charge by 66 +/- 5 and 80 +/- 3%, respectively. Elevating the pipette EGTA concentration from 25 microM to 5 mM increased ICa amplitude and charge by approximately 290%. Both isoproterenol and cAMP increased ICa by approximately 230%. The total charge carried by the isoproterenol- or cAMP-stimulated current was increased by 170%. We conclude that the use of high-EGTA concentration may overestimate the total Ca2+ carried by ICa under physiological conditions. Furthermore, the results suggest that, in contrast to previous reports from other lower vertebrates, Ca2+ flux through L-type Ca2+ channels alone is not sufficient to fully activate contraction in trout atrial myocytes at room temperature.

Histamine H1-receptor-mediated increase in the Ca2+ transient without a change in the Ca2+ current in electrically stimulated guinea-pig atrial myocytes.

The effects of histamine on the intracellular Ca2+ concentration ([Ca2+]i), action potential and membrane currents were assessed in single atrial myocytes prepared from guinea-pigs. Histamine caused a concentration-dependent increase in the [Ca2+]i transient in indol/AM loaded myocytes when stimulated electrically at 0.5 Hz. However, the maximum increase in [Ca2+]i transient produced by histamine was less than 50% of that elicited by isoprenaline. The histamine-induced increase in [Ca2+]i transient was significantly inhibited by chlorpheniramine, but not by cimetidine. Pretreatment with nifedipine nearly completely suppressed the histamine-induced increase in [Ca2+]i transient. Cyclopiazonic acid did not affect the histamine response. In the whole-cell current-clamp mode of the patch-clamp method, both histamine and isoprenaline prolonged action potential duration (APD) in atrial myocytes. In the presence of Co2+ or nifedipine, the isoprenaline-induced APD prolongation was abolished and an APD shortening effect was manifested, while histamine still increased APD. The APD prolongation elicited by histamine was reversed by chlorpheniramine. In the voltage-clamp mode, the histamine-sensitive membrane current was inwardly rectifying and reversed close to the calculated value of the K+ equilibrium potential. Histamine had no apparent effect on L-type Ca2+ current, in contrast to the pronounced effect of isoprenaline. These results indicate that in guinea-pig atrial myocytes stimulation of H1-receptors with histamine does not directly activate Ca2+ channels but causes an elevation of [Ca2+]i transient by increasing Ca2+ influx through the channels during the prolonged repolarization of action potentials resulting from inhibition of the outward K+ current.

Inhibition of the delayed rectifier K current in guinea-pig cardiomyocytes by thiamine tetrahydrofurfuryl disulfide.

We examined effect of thiamine tetrahydrofurfuryl disulfide on electrophysiological characteristics of single atrial myocytes, obtained by digestion of guinea-pig heart, using collagenase. Membrane potential and ion channel current in the atrial myocytes were recorded by the patch clamp method. Thiamine tetrahydrofurfuryl disulfide prolonged action potentials at cycle lengths from 250 to 10,000 ms. The degree of thiamine tetrahydrofurfuryl disulfide-induced prolongation was similar among these cycle lengths. Thiamine tetrahydrofurfuryl disulfide inhibited the delayed rectifier K+ current, without affecting Ca2+ current and inward-rectifier K+ current. Thiamine tetrahydrofurfuryl disulfide blocked the delayed rectifier K+ current in voltage- and time-independent manner, indicating that thiamine tetrahydrofurfuryl disulfide blocked both subtypes of the delayed rectifier K+ current (rapid and slow components). Thiamine, the parent molecule of thiamine tetrahydrofurfuryl disulfide, blocked the delayed rectifier K+ current only when thiamine was applied intracellularly. Thiamine tetrahydrofurfuryl disulfide may be converted to thiamine in the cytoplasm, and then may block the the delayed rectifier K+ channel from the intracellular side. Although thiamine tetrahydrofurfuryl disulfide (or thiamine) has some of the properties of class III antiarrhythmics agents, thiamine tetrahydrofurfuryl disulfide did not exhibit reverse use-dependent prolongation of action potential.

Effects of dopamine on L-type Ca2+ current in single atrial and ventricular myocytes of the rat.

1. The effects of dopamine on the L-type Ca2+ current (ICa,L) of both atrial and ventricular single myocytes and on the force of contraction of atrial trabeculae in rat heart were investigated. 2. Dopamine increased atrial ICa,L at concentrations higher than 1 microM, but had little or no effect on ICa,L at lower concentrations. The increase in ICa,L at high concentrations was reversed by propranolol and acetylcholine, but not by phentolamine. Activation and inactivation kinetics of ICa,L were not altered by dopamine. 3. In rat ventricular myocytes in which the D4 receptor mRNA does not express, dopamine (20-100 microM) also increased the ICa,L amplitude and propranolol reversed this effect. 4. Clozapine, a potent D4 receptor antagonist, blocked the augmenting effect of dopamine on ICa,L. However, this effect could be explained by beta-antagonism, since clozapine also inhibited the isoprenaline effect. 5. In the atrial trabeculae, the increase in contraction by dopamine (1 to 30 microM) was reversed by 1 microM propranolol, but not by 2 microM phentolamine. Low doses of dopamine (0.01 to 0.3 microM) did not affect the contraction in the controls or during a modest stimulation of the beta-adrenoceptor with 0.01 microM isoprenaline. 6. These results indicate that the positive inotropic action of dopamine is mediated through direct stimulation of the beta-adrenoceptor in both atrial and ventricular myocytes. Involvement of D4 receptor appears unlikely in the regulation of the atrial contraction.

Development of muscarinic potassium current in fetal and neonatal rat heart.

Single atrial myocytes were isolated from fetal, neonatal, and adult rat hearts. The muscarinic K+ current activated by rapid application of acetylcholine (ACh) and adenosine (Ado) was recorded under the whole cell voltage clamp. The current density (pA/pF) of ACh-induced K+ current increased from gestation day 12 to the maximum on neonatal day 20 and decreased in the adult myocytes due to greater increase of the membrane capacitance. The development of Ado-induced K+ current followed a similar time course except for a remarkable decrease after neonatal day 10. No significant change was found in single-channel properties during the development. Receptor subtypes were M2 and A1 receptors for ACh and Ado, respectively. In the dose-response relationship, the half-maximal concentration for ACh-induced current markedly decreased with age, from 1.44 (fetus) to 0.17 microM (adult), whereas that for Ado increased from 0.45 (fetus) to 0.99 microM (adult). These changes of the muscarinic K+ current were discussed in relation to the functional development of cardiac myocytes and underlying mechanisms.

Isoflurane and sevoflurane modulate inactivation kinetics of Ca2+ currents in single bullfrog atrial myocytes.

To clarify the mechanism(s) of anesthetic depression of myocardial Ca2+ currents, the effects of isoflurane and sevoflurane on the inactivation kinetics of Ca2+ current in single bullfrog atrial myocytes were studied. Freshly isolated bullfrog atrial myocytes were obtained with an enzymatic dispersion procedure. Ca2+ currents were recorded with a whole-cell voltage-clamp technique. Both isoflurane (1.25, 2.5, 5.0 vol%) and sevoflurane (2.5, 5.0 vol%) decreased the peak amplitude of Ca2+ current ICa with a minimal change in the time to peak and the reversal potential. The inactivation kinetics studies revealed that (1) isoflurane (2.5 vol%) and sevoflurane (5.0 vol%) markedly reduced the time constant of inactivation in ICa to 55% and 75% of control, respectively; (2) isoflurane (2.5 vol%) shifted the midpoint (V1/2) of steady-state inactivation curve of ICa toward negative by 2.3 mV; and (3) isoflurane (2.5 vol%) delayed the reactivation time constant of ICa to 119% of control. The further computer-simulation study demonstrated that the observed decrease of time constant by isoflurane (1.25, 2.5 vol%) and sevoflurane (2.5 vol%) can explain the reduction in amplitude of ICa. The depression of ICa by lower concentration of isoflurane (1.25, 2.5 vol%) and sevoflurane (2.5 vol%) mainly is due to the decrease of time constant and, at higher concentration, isoflurane and sevoflurane may affect the other membrane components.

Activation of the muscarinic K+ channel by P2-purinoceptors via pertussis toxin-sensitive G proteins in guinea-pig atrial cells.

1. Whole-cell voltage clamp and cell-attached patch-clamp techniques were applied to single atrial myocytes enzymatically dissociated from adult guinea-pig hearts. 2. In whole-cell clamp conditions, external applications, of ATP activated the muscarinic K+ (KACh) current, identified by its inward rectification, its reversal potential near the calculated K+ equilibrium potential (EK) and its relaxation properties during step changes of whole-cell membrane potential. Theophylline, an antagonist for Pi-purinoceptors, did not affect the action of ATP on the KACh current, indicating that the response was evoked through P2-purinoceptors. 3. The concentration-response relationship for ATP was well described by a Hill equation with a half-maximal concentration of 1.84 microM and a Hill coefficient of 0.94. ATP (100 microM) produced a maximal increase of the KACh current to 10.92 microA microF-1, which corresponds to 44.9 and 80.9% of the maximal increases evoked by ACh (10 microM) and adenosine (100 microM), respectively. 4. The activation of KACh current gradually declined to a steady level despite the continuous presence of ATP (desensitization). Recovery from the desensitization was relatively rapid with a half-time of approximately 1.5 min. 5. The activation of KACh current by ATP was completely abolished by pre-incubating myocytes with pertussis toxin (PTX, 5 micrograms ml-1), indicating that P2-purinoceptors are coupled to PTX-sensitive G proteins to activate the KACh channel. 6. In the cell-attached patch recording, ATP (5 microM) applied to the pipette solution enhanced the activity of a channel with single-channel conductance of 52.7 +/- 0.9 pS (mean +/- S.E.M., n = 10), reversal potential near EK and mean open time of 1.1 +/- 0.1 ms. These conductance and kinetic properties are identical to those of the KACh channel in the heart. In contrast, ATP applied to the bath solution did not significantly affect the basal activity of KACh channel openings. These observations suggest that the mechanism coupling the P2-purinoceptor to the activation of the KACh channel involves membrane-delimited component(s) rather than soluble second messenger(s). 7. These results strongly suggest a direct coupling of the P2-purinoceptor to the KACh channel through PTX-sensitive G proteins, analogous to the coupling mechanism of the muscarinic ACh receptor and Pi-purinoceptor to this channel.

Inhibition of metabolism abolishes transient outward current in rabbit atrial myocytes

Outward currents were measured in single rabbit atrial myocytes using the whole cell configuration of the patch-clamp technique in the presence of tetrodotoxin (5-10 microM) and MnCl2 (2 mM) to block inward currents. Depolarizing voltage-clamp steps from a holding potential of -80 mV elicited a predominant 4-aminopyridine (4-AP)-sensitive transient outward current (Ito). Inhibitors of oxidative metabolism, 2,4-dinitrophenol (DNP; 100 microM) and cyanide (3 mM) abolished Ito and caused a large increase in the steady-state outward current. This steady-state outward current was inhibited by glibenclamide (5 microM), a blocker of the ATP-regulated potassium current (IKATP). In the presence of DNP, glibenclamide (5 microM) not only inhibited IKATP but also partially restored Ito. Absence of ATP from the pipette produced effects on outward currents similar to those induced by DNP or cyanide. We conclude that metabolic inhibition abolishes Ito in rabbit atrial myocytes and suggest that ATP may be required for the activation of the channel.

Calcium transients caused by calcium entry are influenced by the sarcoplasmic reticulum in guinea-pig atrial myocytes.

1. Single atrial myocytes obtained by enzyme perfusion from hearts of adult guinea-pigs were investigated using whole-cell voltage clamp and Indo-1 micro-fluorometry. 2. In myocytes loaded with a solution containing citrate as a low-affinity, non-saturable Ca2+ chelator, two types of [Ca2+]i transients could be recorded during repetitive activation of L-type Ca2+ current. Both large and small [Ca2+]i transients occurred; large transients reached peak values of about 1 microM, and small transients were about 100 nM or less in amplitude. 3. In the case of the large transients, peak [Ca2+]i was usually reached with a variable delay after repolarization from a voltage step that activated calcium current (ICa). For the small transients the rise in [Ca2+]i paralleled ICa. Upon repolarization [Ca2+]i started to decay. 4. The small transients reflect entry of Ca2+ through Ca2+ channels (entry transients), whereas the large transients are due to entry and release from the sarcoplasmic reticulum (release transients). 5. The entry transients displayed a positive staircase pattern during trains of depolarizing voltage steps despite constant or even decreasing amplitude of ICa. The steepness of the staircase was increased by elevation of [Ca2+]o. Entry transients were always smallest immediately after a release transient. 6. After functional removal of the sarcoplasmic reticulum by caffeine (1-5 mM) the staircase pattern of the transients reflecting Ca2+ entry was abolished. 7. It is concluded that the staircase pattern is due to rapid uptake by the sarcoplasmic reticulum of Ca2+ entering the cell, resulting in an attenuation of the signal. The attenuation is strongest shortly after a release signal, when the rate of sequestration of Ca2+ by the SR should be highest. 8. Evidence is provided that a compartment of the SR is involved in attenuation of the entry transients. This compartment has been identified recently as a peripheral release compartment.

Effects of anions on the G protein-mediated activation of the muscarinic K+ channel in the cardiac atrial cell membrane. Intracellular chloride inhibition of the GTPase activity of GK.

The effects of various intracellular anions on the G protein (GK)-mediated activation of the muscarinic K+ (KACh) channel were examined in single atrial myocytes isolated from guinea pig hearts. The patch clamp technique was used in the inside-out patch configuration. With acetylcholine (ACh, 0.5 microM) in the pipette, 1 microM GTP caused different magnitudes of KACh channel activation in internal solutions containing different anions. The order of potency of anions to induce the KACh channel activity at 0.5 microM ACh and 1 microM GTP was Cl- greater than or equal to Br- greater than 1-. In the SO4(2-) or aspartic acid internal solution, no channel openings were induced by 1 microM GTP with 0.5 microM ACh. In both the Cl- and SO4(2-) internal solutions (with 0.5 microM ACh) the relationship between the concentration of GTP and the channel activity was fit by the Hill equation with a Hill coefficient of approximately 3-4. However, the concentration of GTP at the half-maximal activation (Kd) was 0.2 microM in the Cl- and 10 microM in the SO4(2-) solution. On the other hand, the quasi-steady-state relationship between the concentration of guanosine-5'-o-(3-thiotriphosphate) and the channel activity did not differ significantly between the Cl- and SO4(2-) solutions; i.e., the Hill coefficient was approximately 3-4 and the Kd was approximately 0.06-0.08 microM in both solutions. The decay of channel activity after washout of GTP in the Cl- solution was much slower than that in the SO4(2-) solution. These results suggest that intracellular Cl- does not affect the turn-on reaction but slows the turn-off reaction of GK, resulting in higher sensitivity of the KACh channel for GTP. In the Cl- solution, even in the absence of agonists, GTP (greater than 1 microM) or ATP (greater than 1 mM) alone caused activation of the KACh channel, while neither occurred in the SO4(2-) solution. These observations suggest that the activation of the KACh channel by the basal turn-on reaction of GK or by phosphate transfer to GK by nucleoside diphosphate-kinase may depend at least partly on the intracellular concentration of Cl-.

Mechanisms of the anticholinergic effect of SUN 1165 in comparison with flecainide, disopyramide and quinidine in single atrial myocytes isolated from guinea‐pig

1. The mechanism of the anticholinergic effect of SUN 1165 on the acetylcholine (ACh)-induced K+ current (IK.ACh) was examined and compared with those of flecainide, disopyramide and quinidine in single atrial myocytes, in a whole-cell configuration by use of the concentration-jump technique. This technique combines an intracellular perfusion and a rapid exchange of external solution surrounding the voltage-clamped single myocyte within 2 ms. 2. In the cells loaded with guanosine-5'-triphosphate (GTP), 100 microM, the muscarinic ACh response, (IK.ACh), was mediated by GTP-binding proteins. The concentrations of the test drugs that produced a half-maximal inhibition of ACh (1 microM)-induced IK.ACh (IC50) were 29 microM for SUN 1165, 3.6 microM for flecainide, 1.7 microM for disopyramide, and 1.6 microM for quinidine. The blockade of IK.ACh by SUN 1165 and its recovery from the inhibition occurred within a few seconds. Disopyramide had a similar rapid action, while the effects of flecainide and quinidine occurred much more slowly within a few tens of seconds. 3. In cells loaded with 100 microM guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S, a nonhydrolysable GTP analogue), the K+ channel was uncoupled from the muscarinic receptors and activated irreversibly due to direct activation of GTP-binding proteins by GTP gamma S. SUN 1165 and disopyramide had a weak inhibitory effect (IC50 greater than 100 microM for both), while flecainide and quinidine depressed the GTP gamma S-induced K+ current with similar potencies to the cases of ACh-induced currents; IC50 was 5.3 microM for flecainide and 4.4 microM for quinidine. 4 These results suggest that the mechanisms underlying the anticholinergic effects of these antiarrhythmic drugs are different; disopyramide and high concentrations of SUN 1165 mainly block muscarinic ACh receptors in atrial myocytes, while flecainide and quinidine inhibit the K+ channel itself and/or GTP-binding proteins.