Effects of hydrogen peroxide on the transient outward current in rabbit atrial myocytes.

1. In the present study, we investigated the effects of hydrogen peroxide (H2O2) on the 4-aminopyridine-sensitive transient outward current (I(TO)) in rabbit atrial myocytes using the amphotericin B-perforated patch voltage-clamp method. 2. Superfusion of myocytes with H2O2 at 100 micromol/L gradually slowed the time-course of inactivation of I(TO) and increased the peak by 9% (n = 9). The H2O2-induced slowing of I(TO) inactivation was concentration dependent (over the concentration range 10 micromol/L to 1 mmol/L). These effects were hardly reversed by washout of H2O2, but were quickly abolished by dithiothreitol (2 mmol/L). 3. Bisindolylmaleimide (100 nmol/L), an inhibitor of protein kinase C, significantly attenuated the H2O2-induced effects on I(TO). 4. These results suggest that rabbit atrial I(TO) is susceptible to oxidation by H2O2 at concentrations relevant to those encountered during ischaemia/reperfusion and that protein kinase C modulates the effects of H2O2.

Increased sensitivity of neonate atrial myocytes to adenosine A1 receptor stimulation in regulation of the L-type Ca2+ current.

Adenosine has cardioprotective effects against ischemia, and newborn hearts show high resistance to ischemia. The effects of purinoceptor stimulation by adenosine and ATP on the L-type Ca2+ current (ICa) were examined in atrial cells from neonate and adult rabbits. ICa was measured by the membrane-perforated patch method. Adenosine inhibited the isoproterenol-stimulated ICa more potently in neonate cells than in adult cells. The high sensitivity of neonate myocytes to adenosine was accompanied not only by an increased maximum response but also by a lower IC50 concentration. ATP also inhibited isoproterenol-stimulated ICa. The effect of ATP on neonate cells was stronger than that on adult cells at high concentrations (greater than or = 100 microM). The effect of adenosine was antagonized by an A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). DPCPX or an ecto-5'-nucleosidase inhibitor (alpha,beta-methylene-ADP) blocked most (approximately 60%) of the effect of ATP (30 microM), and co-addition of DPCPX and suramin (P2 receptor blocker) abolished the effect of ATP. Suramin alone did not reduce the effect of ATP significantly in neonate cells. Both the effects of adenosine and ATP were eliminated by pre-treatment with pertussis toxin or by superfusion with forskolin plus 3-isobutyl-1-methylxanthine (IBMX). Inhibitors of the nitric oxide-cyclic GMP pathway did not affect the adenosine inhibition of ICa. In summary, neonatal myocardial cells are highly sensitive to adenosine A1 receptor stimulation. ATP stimulates both the adenosine A1 and P2 receptors. Adenosine A1 receptor stimulation, as a result of hydrolysis of ATP, predominantly mediates the effect of ATP, and the role of P2 receptors in the ATP inhibition of ICa is relatively small in neonate cells. The high sensitivity to adenosine may contribute to the ischemic tolerance of newborn hearts.

Electrophysiological effects of ibutilide on the delayed rectifier K(+) current in rabbit sinoatrial and atrioventricular node cells.

Biophysical and pharmacological characteristics of the delayed rectifier K(+) current (I(K)) of rabbit sinoatrial (SA) node and atrioventricular (AV) node cells have been studied using the whole-cell patch clamp technique together with a recently developed antiarrhythmic agent, ibutilide. Ibutilide is a potent blocker of the rapid delayed rectifier K(+) current, I(Kr). Superfusion with ibutilide (10(-7) M) caused a decrease in the spontaneous firing frequency, depolarization of the maximal diastolic potential and prolongation of the action potential duration in both SA and AV node cells. In whole cell voltage clamp experiments done on myocytes from SA node, ibutilide (10(-7) M) blocked I(K) strongly (40%) and had smaller effects on Ca(2+) current (10%) and hyperpolarization-activated inward current, I(f) (11%). In AV node cells, the corresponding reductions were I(K) (68%), I(Ca) (13%) and I(f) (10%), respectively. A 10-fold increase in the concentration of ibutilide further decreased I(K) in SA node cells (67+/-8%), and blocked I(K) almost completely in AV node cells. These results are consistent with the hypothesis that the delayed rectifier K(+) current in SA node cell is generated by both I(Kr) and I(Ks), whereas I(Kr) predominates in AV node cells. Knowledge of the differences in the distribution of I(Kr), as well as the different sensitivity to blockers of I(Kr) in nodal cells, is important for understanding modifications of the automaticity, conduction velocity, and refractoriness by class III antiarrhythmic agents.

[A case of intra-right atrial thrombus adhering to an intravenous hyperalimentation catheter].

We reported a clinical case of intra-right atrial giant thrombus adhering to an intravenous hyperalimentation(IVH) catheter. The patient was a 63 years-aged male, and was indwelled the IVH catheter for one month from right subclavian vein to right atrium to improve a malnutrition due to his advanced gastric cancer. Intra-cardiac mass was found in the right atrium by routine transthoracic echocardiography for the preoperative inspection; the giant mass(22 x 20 mm) had a low-echo density with a portion of high-echo density. In transesophageal echocardiography, the portion of high-echo density was found to be an IVH catheter and intra-right atrial mass was pierced by that catheter; however, it was difficult to determine whether the mass was either a vegetation or thrombus. The mass was removed by operation, and was diagnosed as a giant thrombus by histopathological examination. Thrombus should be taken into account as a possible cause of intra-right atrial mass when an IVH catheter was indwelled in the right atrium.

EP receptor-mediated inhibition by prostaglandin E(1) of cardiac L-type Ca(2+) current of rabbits.

Prostaglandin E(1) (PGE(1)) has cardioprotective effects on the ischemic-reperfused heart. To clarify the mechanisms underlying the protective action of PGE(1) on myocardium, we examined the effect of PGE(1) on the L-type Ca(2+) current (I(Ca)) using single atrial cells from rabbits. PGE(1) did not show a significant effect on basal I(Ca) but inhibited the I(Ca) prestimulated by isoproterenol (Iso, 30 nM). This inhibition was concentration dependent (EC(50) = 0.027 microM). Both sulprostone, a specific PGE receptor subtype (EP(1) and EP(3)) agonist, and 11-deoxy-PGE(1), an EP(3) agonist, inhibited the Iso-stimulated I(Ca), similar to PGE(1). Pretreatment with pertussis toxin (PTX) abolished the PGE(1) inhibition of I(Ca). Both the application of forskolin plus IBMX and intracellular dialysis with 8-bromoadenosine 3',5'-cyclic monophosphate eliminated the effect of PGE(1). PGE(1) did not show any further inhibition of I(Ca) when the effect of Iso was almost fully antagonized by acetylcholine. Methylene blue (guanylate cyclase inhibitor), KT-5823 (cGMP-dependent protein kinase inhibitor), and erythro-9-(2-hydroxy-3-nonyl)adenine (type II phosphodiesterase inhibitor) did not significantly change the inhibitory effect of PGE(1). These findings suggest that 1) PGE(1) inhibits Iso-stimulated I(Ca) by binding to the EP(3) receptor and 2) the PTX-sensitive and cAMP-dependent pathway is involved in the PGE(1) inhibition of I(Ca), but the nitric oxide-cGMP-dependent pathway is not. The PGE(1)-induced antiadrenergic effect shown in this study may contribute to the PGE(1) protection of myocardium against ischemia.

P2 purinoceptors contribute to ATP-induced inhibition of L-type Ca2+ current in rabbit atrial myocytes.

OBJECTIVE: Adenine compounds, including adenosine-5'-triphosphate (ATP) and adenosine (Ado), exert inhibitory effects on myocardium via P1 (subtype A1) purinoceptors. However, ATP per se is a potent activator of P2 purinoceptors. Our aim was to elucidate the respective roles of P1 and P2 purinoceptors in the actions of ATP on L-type calcium current (ICa) in rabbit atrial cells. METHODS AND RESULTS: A whole cell clamp technique was used to record ICa in single atrial cells from the rabbit heart. ATP (0.1 mumol/1-3 mmol/l) produced an inhibitory effect on ICa prestimulated by isoproterenol (ISO, 30 nmol/l), even in the presence of Ado (1 mmol/l). Both 1,3-dipropyl-8-cyclopentylxanthine (A1 blocker) and suramin (P2 blocker) partially blocked the ATP-induced inhibition of ICa, while their co-application nearly completely abolished the effect of ATP. ATP-gamma S (30 mumol/l) inhibited ISO-stimulated ICa significantly, and this inhibition was completely blocked by suramin. alpha, beta-Methylene-ADP, an inhibitor of hydrolysis of AMP to Ado, eliminated the suramin-resistant component of ICa inhibition by ATP. Pretreatment with pertussis toxin (PTX) abolished the ATP inhibition of ICa. Both intracellular dialysis with 8Br cAMP and the application of forskolin plus 3-isobutyl-1-methylxanthine also eliminated the effect of ATP. CONCLUSIONS: Both P1 and P2 purinoceptors are involved in the ATP inhibition of ISO-stimulated ICa in rabbit atrial cells. The P1 stimulation by ATP results from hydrolysis of ATP to Ado. Both the P2- and the P1-mediated effects of ATP and Ado, respectively. involve a PTX-sensitive and cAMP-dependent pathway.

Tyrosine kinase-dependent modulation by interferon-alpha of the ATP-sensitive K+ current in rabbit ventricular myocytes.

We examined the effects of interferon-alpha on the ATP-sensitive K+ current (IK,ATP) in rabbit ventricular cells using the patch-clamp technique. IK,ATP was induced by NaCN. Whole-cell experiments indicated that interferon-alpha (5 x 10(2) - 2.4 x 10(4) U/ml) inhibited IK,ATP in a concentration-dependent manner (60.7+/-7.5% with 2.4 x 10(4) U/ml). In cell-attached configuration, interferon-alpha (2.4 x 10(4) U/ml) applied to the external solution also inhibited the activity of the single ATP-sensitive K+ (KATP) channel by 56.0+/-5.8% without affecting the single channel conductance. The inhibitory effect of IK,ATP by interferon-alpha was blocked by genistein and herbimycin A, tyrosine kinase inhibitors, but was not affected by N-(2-metylpiperazyl)-5-isoquinolinesulfoamide (H-7), an inhibitor of protein kinase C and cAMP-dependent protein kinase. These findings suggest that interferon-alpha inhibits the cardiac KATP channel through the activation of tyrosine kinase. The tyrosine kinase-mediated inhibition of IK,ATP by cytokines may aggravate cell damage during myocardial ischemia.

Modulation by chloramine-T of 4-aminopyridine-sensitive transient outward current in rabbit atrial cells.

The effects of an oxidizing agent, chloramine-T, on the 4-aminopyridine-sensitive transient outward current (ITO) were investigated in rabbit atrial myocytes by using patch-clamp techniques. Extracellular application of chloramine-T at 20 microM irreversibly slowed the time course of inactivation of the whole-cell ITO, and increased the peak by 19.3% (n = 19) at +40 mV. At 100 microM, chloramine-T decreased the peak by 22.5% (n = 9) of the control, and subsequently induced a glibenclamide-sensitive time-independent outward K+ current. Under superfusion with dithiothreitol (3 mM), chloramine-T (100 microM) produced no change in ITO. The chloramine-T-induced slowing of ITO inactivation was partially reversed by subsequent application of 3 mM dithiothreitol. In single-channel recordings with the cell-attached patch configuration, chloramine-T (20 microM) increased the open probability of the ITO channel from 0.15 to 0.46 at a potential 100 mV positive to the resting potential, and the mean open lifetime from 5.1 ms to 7.0 ms (n = 5). The unitary current amplitude was not affected. As a result, chloramine-T increased the ensemble current in amplitude and slowed its decay. These results indicated that: (1) inactivation of the native A-type channels of rabbit heart is susceptible to oxidation; and (2) oxidation of ITO channels may contribute to the genesis of arrhythmias.

Blockage by terfenadine of the adenosine triphosphate (ATP)-sensitive K+ current in rabbit ventricular myocytes.

We examined the blocking effects of terfenadine, an antihistaminic agent, on the ATP-sensitive K+ current (IK,ATP) in rabbit ventricular cells. IK,ATP was induced by cromakalim or NaCN. Terfenadine blocked the IK,ATP with an IC50 of 1.7 microM at -10 mV. This blockage was voltage dependent; depolarization induced a stronger blockage. According to the transmembrane electrical field model, terfenadine interacts with the site located 15 to 18% from the cytoplasmic membrane surface. In line with the assumption that the binding site is near the cytoplasmic surface, terfenadine applied to the cytoplasmic solution potently inhibited the single-channel activity for IK,ATP in the inside-out configuration (IC50 0.19 microM). In contrast, terfenadine applied to the external solution did not affect the channel activity in the cell-attached configuration, but inhibited it when applied into the pipette. The inhibition of the single channels by terfenadine was accompanied by flickering of the channels. These findings suggest that 1) terfenadine blocks the ATP-sensitive K+ channel in the open state, 2) the binding site is near the internal membrane surface and 3) terfenadine is poorly diffusible into the lipid biomembrane and accesses the binding site via the hydrophilic pathway. Terfenadine also inhibited the transient outward K+ current, inward rectifier K+ current and E4031-sensitive rectifier K+ current. However, the inhibition of these repolarization currents by terfenadine at 1 microM was not sufficient to prolong the action potential duration significantly. Whereas, terfenadine (1 microM) prolonged the action potential duration which had been shortened by cromakalim. Terfenadine may modify the ischemia-induced arrhythmias by blocking IK,ATP.

[Electrophysiological heterogeneity of rabbit atrioventricular node cells: possible relationship to fast and slow pathways].

Catheter ablation has been applied for the therapy of atrioventricular nodal re-entrant tachycardia for several years. Although this procedure is quite successful, the cellular electrophysiological mechanisms underlying fast and slow conductions remain unknown. Therefore, the characteristics of the action potential waveform of rabbit atrioventricular node (AVN) cells obtained from different regions within Koch's triangle were studied using nystatin-permeabilized patch methods, and 64 AVN cells were used for this study. Based on gross morphology, AVN cells were classified into three groups: ovoid cells, rod shaped cells, and cells with an intermediate shape. Results obtained by measuring the maximum velocity dV/dt of action potentials fell into four subgroups: Group I (dV/dt < 5 V/sec, n = 23), Group II (dV/dt > 5 but < 10 V/sec, n = 20), Group III (dV/dt > 10 but < 20 V/sec, n = 13), and Group IV (dV/dt > 20 V/sec, n = 8). Ovoid cells had the smallest dV/dt, whereas the rod shaped cells had the highest dV/dt. The maximum diastolic potential was more negative in Groups III and IV than in Groups I and II. A notch in the phase II of the action potential was observed in 23% of Group III cells and 56% of those in Group IV, but was not present in Groups I and II. These findings provide further evidence that AVN cells are heterogeneous both in morphology and electrophysiological characteristics. Provided that values of dV/dt can be related to conduction velocity, our findings suggest that cells in Groups III and IV may contribute to fast conduction pathways whereas those in Groups I and II are responsible for slow conduction pathways.

Endothelin-1 inhibits pacemaker currents in rabbit SA node cells.

Our recent study demonstrated that endothelin-1 (ET-1) inhibits the pacemaker activity of sinoatrial (SA) node cells via changes in the L-type Ca2+, delayed K+, and background K+ currents. Using the whole-cell patch-clamp technique in the same preparation, we found that ET-1 reduces other pacemaker currents, the T-type Ca2+ current (ICa,T) and the hyperpolarization-activated inward current (I(f)). The inhibitory actions of ET-1 on these currents were concentration-dependent, i.e., EC50 of 0.9 nM for ICa,T and 2.3 nM for I(f), with little reversal after washout of the peptide. In the presence of BQ485, both currents were not affected by ET-1. These results indicate additional mechanisms underlying negative chronotropic actions of ET-1 on the rabbit SA node.

Negative chronotropic actions of endothelin-1 on rabbit sinoatrial node pacemaker cells.

1. The effects of endothelin-1 (ET-1) on sinoatrial (SA) node preparations of the rabbit heart were studied by means of whole-cell clamp techniques. 2. ET-1 at 1 nM slowed the spontaneous beating activity and rendered half of the cells quiescent. At a higher concentration of 10 nM, the slowing and cessation of spontaneous activity were accompanied by hyperpolarization. 3. In voltage-clamp experiments, ET-1 decreased the basal L-type Ca2+ current (Ica(L)) dose-dependently with a half-maximal inhibitory concentration (EC50) of 0.42 nM and maximal inhibitory response (Emax) of 49.5%. The delayed rectifying K+ current (Ik) was also reduced by 33.2 +/- 11.1% at 1 nM. In addition an inwardly rectifying K+ current was activated by ET-1 at higher concentrations (EC50 = 4.8 nM). These ET-1-induced changes in membrane currents were abolished by BQ485 (0.3 microM), a highly selective ETA receptor antagonist. 4. When Ica(L) was inhibited by ET-1 (1 nM), subsequent application of 10 microM ACh showed no additional decrease in Ica(L), suggesting the involvement of cyclic AMP in the effects of ET-1 on Ica(L). In contrast, 1 nM ET-1 further decreased Ica(L) in the presence of 10 microM ACh, suggesting that ET-1 activates some additional mechanism(s) which inhibit Ica(L). The ET-1-induced Ica(L) inhibition was abolished by protein kinase A inhibitory peptide (PKI, 20 microM) or H-89 (5 microM). However, the Ica(L) inhibition was not affected by methylene blue (10 microM), suggesting a minor role for cyclic GMP in the effect of ET-1 under basal conditions. 5. ET-1 failed to inhibit Ica(L) when the pipette contained GDP beta S (200 microM). However, incubation of the 21.5 +/- 9.5%, whereas it abolished the inhibitory effect of ACh on Ica(L). 6. Intracellular perfusion of 8-bromo cyclicAMP (8-Br cyclicAMP, 500 microM) attenuated, but did not abolish the inhibitory effect of ET-1 on Ica(L). This 8-Br cyclicAMP-resistant component (17.5 +/- 14.4%, n = 20) was not affected by combined application of 8-Br cyclicAMP-bromo cyclicGMP (500 microM), ryanodine (1 microM) or phorbol-12-myristate-13-acetate (TPA; 50 nM). 7. In summary, ET-1 exerts negative chronotropic effects on the SA node via ETA-receptors. ET-1 inhibits both ICa(L) and Ik, and increases background K+ current. The inhibition of ICa(L) by ET-1 is mainly due to reduction of the cyclicAMP levels via PTX-sensitive G protein, but some other mechanism(s) also seems to be operative.

[Application of urinary free dopamine as a marker of renal function, and comparison with other renal marker].

Urinary free dopamine (U-f-DA) is derived from renal DA synthesized in the renal proximal tubules, and plays an important role for diuresis and natriuresis. We were previously reported that U-f-DA was the superior marker of renal function as compared with ordinary methods including alpha 1-microglobulin (U-alpha 1 MG), beta 2-microglobulin (U-beta 2 MG) and N-acetyl-beta-D-glucosaminidase (U-NAG) in spot urine samples. U-f-DA can be used as index for the evaluation of renal transplantation. In order to evaluate the clinical usefulness of U-f-DA as a marker of renal function, we investigated as follows; firstly, the age related changes of U-f-DA in healthy out-patients, secondly, the correlation between U-f-DA and creatinine clearance (CCr), serum creatinine (S-Cr) in in-patients, and thirdly, the chronological changes of U-f-DA, U-alpha 1 MG, U-beta 2 MG, CCr and S-Cr in patients with chronic renal failure before and after renal transplantation. There is no age-related changes in U-f-DA from patients with 3 years to 88 years old. U-f-DA was positively correlated with CCr and negatively correlated with S-Cr. There are parallel changes of U-f-DA and CCr in increasing direction, on the other hand, parallel changes of U-alpha 1 MG, U-beta 2 MG and S-Cr in decreasing direction after renal transplantation. In patients with post renal transplantation who were not well controlled, S-Cr increased gradually with the decreasing level of U-f-DA. These results suggest that the measurement of U-f-DA in spot urine samples is useful marker for evaluation of the renal function and can be used an index of viability of the transplanted kidney.

The mechanisms underlying heart stimulation by dopamine, with special reference to direct and indirect beta adrenoceptor stimulation.

1. The positive chronotropic and norepinephrine-releasing effects of dopamine were examined in the isolated guinea pig heart, using the Langendorff model. 2. The released norepinephrine was estimated from the norepinephrine concentration measured in the post-perfusion solution using HPLC. 3. The dose-response curve for dopamine to stimulate the heart rate (HR) closely resembled that for the norepinephrine release. A selective beta 1 antagonist bisoprolol completely abolished the positive chronotropic effect, but did not affect the norepinephrine release. 4. The HR increase in response to 3 mumol/L dopamine was 54 +/- 15% (n = 14) of the control in normal hearts. The response was decreased to 15 +/- 7% (n = 6) by pretreatment with reserpine. 5. A D1 antagonist, SKF83742, (3 mumol/L) shifted the dose-response curve for the dopamine-induced norepinephrine release toward the right, indicating the involvement of D1-like dopamine receptors. 6. Voltage clamp experiments using single cells isolated from the right atrium revealed that dopamine is a weak partial agonist for beta adrenoceptors. Dopamine stimulated the L-type Ca2+ current with a threshold concentration of 3 mumol/L. 7. These findings indicate the important role of the norepinephrine release in the stimulation of beta adrenoceptors by dopamine at clinically relevant concentrations.

Dopamine stimulation of cardiac beta-adrenoceptors: the involvement of sympathetic amine transporters and the effect of SKF38393.

1. Mechanisms underlying beta-adrenoceptor stimulation by dopamine were examined on guinea-pig Langendorff-perfused hearts and isolated cells from the right atrium, by using the chronotropic effects and the enhancement of L-type Ca2+ current (ICa,L) in the presence of prazosin as indicators of beta-adrenoceptor stimulation. Dopamine-induced overflow of noradrenaline (NA) concentrations was measured by high-performance liquid chromatography. 2. Dopamine caused positive chronotropic effects with an EC50 of 2.5 microM and induced NA overflow with a similar EC50 (1.3 microM). The chronotropic effect of dopamine was abolished by bisoprolol (1 microM). 3. The effects of dopamine were maintained during prolonged application, whereas the effects of tyramine faded with time. Dopamine (3 microM) restored the chronotropic effects and the NA release suppressed by pretreatment with tyramine, suggesting a de novo synthesis of NA during the exposure to dopamine. 4. Dopamine (3 microM)-induced NA release was not affected by tetrodotoxin, omega-conotoxin, rauwolscine, ICI118551 or sulpiride, but was inhibited by desipramine, a NA uptake inhibitor (IC50 approximately 1 microM). It was also not affected by GBR12909 and bupropion, dopamine uptake inhibitors in the central nervous system. 5. SKF38393, a D1 receptor partial agonist, potently inhibited the 3 microM dopamine-induced release of NA (IC50 approximately 0.1 microM). D1 receptors are not involved in the DA-induced release of NA, since SCH23390 (3 microM), a potent D1 antagonist, inhibited the NA release only slightly, and dihydrexidine (1 microM) and chloro-APB (1 microM), full D1 agonists, caused no significant NA release. 6. SKF38393 inhibited tyramine-induced overflow of NA, and potentiated the field stimulation-induced NA release. SKF38393 and desipramine retarded the decay of the stimulation-induced tachycardia in a similar manner. These results indicate that SKF38393 is a potent monoamine transport inhibitor and a useful tool for the functional evaluation of indirectly-acting sympathomimetic agonists in the heart. In the presence of SKF38393 (10 microM), dopamine at 1 microM showed no chronotropic effect. 7. Voltage clamp experiments with isolated atrial cells revealed that dopamine is a weak partial agonist. The EC50 for ICa,L stimulation by dopamine was high (13 microM). As a result, dopamine at 1 microM did not affect ICa,L. Bisoprolol abolished the stimulation of ICa,L by dopamine (30 microM), and dihydrexidine (1 microM) did not affect ICa,L. 8. It was concluded that the cardiac effects of dopamine at clinically relevant concentrations (< 1 microM) result almost exclusively from the indirect effect of beta adrenoceptor stimulation, involving the release of NA from sympathetic nerve terminals. The roles of the direct stimulation of beta adrenoceptors by dopamine at these concentrations and the stimulation of postjunctional D1 receptors seem negligible. The desipramine- and SKF38393-sensitive monoamine transporter mediates the release of NA.

Muscarinic inhibition of basal L-type calcium current in pacemaker cells from the rabbit atrioventricular node.

Effects of acetylcholine (ACh) on the L-type calcium current were examined in isolated atrioventricular nodal cells that exhibited spontaneous contractions. ACh (0.1 to 10 microM) inhibited basal calcium current dose-dependently. This inhibition was eliminated by dialysis with 8Br cAMP or cAMP-dependent kinase inhibitory peptide. Both extracellular N-ethylmaleimide 50 microM and intracellular GDPssS 0.2 mM abolished the ACh effect. Dialysis with cGMP or NG-monomethyl-L-arginine did not significantly affect ACh inhibition of basal calcium current. Similarly, cGMP-dependent protein kinase inhibitor KT5823 (1 microM) and the type II phosphodiesterase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (30 microM) did not attenuate the ACh effect. Therefore, ACh inhibits the basal calcium current in the atrioventricular node mainly by suppressing cAMP synthesis through the inhibitory GTP-binding protein.

Regulation by acetylcholine of Ca2+ current in rabbit atrioventricular node cells.

Effects of acetylcholine (ACh) on L-type Ca2+ current (ICa) were examined in isolated atrioventricular (AV) node cells exhibiting spontaneous contractions and pacemaker current (If). ACh at a saturating concentration of 10 microM reduced basal ICa by 48 +/- 6%. The ACh effect was abolished by dialysis with 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase inhibitor, or guanosine-5'-O-(2-thiodiphosphate). Dialysis with guanosine 3',5'-cyclic monophosphate (cGMP) or NG-monomethyl-L-arginine (L-NMMA) and application of the cGMP-dependent protein kinase inhibitor KT-5823 (1 microM) did not affect ACh inhibition of ICa. Nitric oxide donor 3-morpholinosydnonimine (100 microM) and type III phosphodiesterase (PDE) inhibitor trequinsin (10 nM) enhanced basal ICa by 10-20%, whereas type IV PDE inhibitor Ro-20-1724 (30 microM) together with trequinsin caused a large ICa stimulation comparable to that by 3-isobutyl-1-methylxanthine (IBMX). These findings indicate that ACh inhibits basal ICa primarily by suppressing cAMP synthesis and that these cells have a potent type III and IV PDE activity to determine the basal cAMP concentration. When ICa was stimulated by IBMX (100 microM), the inhibitory effect of ACh was slightly reduced by L-NMMA, cGMP, and methylene blue but not by KT-5823 or Ro-20-1724. ACh hardly inhibited, or even enhanced, IBMX-stimulated Ica when forskolin (3 microM) was coapplied or the IBMX concentration was increased to 500 microM. These findings suggest that cAMP is degraded in the presence of 100 microM IBMX to some extent. Type II PDE, for which IBMX has a relatively high inhibitor constant, seems to contribute partially to the cAMP degradation.

Delineation of premature P waves on four-dimensional electrocardiography, a new display of electrical forces by computer techniques.

This study investigated the feasibility of four-dimensional electrocardiography (4-D ECG), a new display in which the vector loop was rotated and scanned along a timed axis to overcome the shortcomings of vectorcardiography (VCG). The subjects consisted of 38 patients with premature atrial complexes and 30 controls. The orthogonal Frank electrocardiograms were rotated three-dimensionally according to the right-hand rectangular coordinate system and scanned along a timed axis. The P wave delineation score, signifying good agreement with the intraobserver and interobserver variability, was significantly higher in 4-D ECG than those in the orthogonal leads or those on the transverse and frontal projections (P < 0.001). The authors measured the premature P loop areas as viewed from 361 directions. P loop areas were best delineated when viewed from cranial directions of 42.6 +/- 34.0 degrees and from rightward directions of 11.3 +/- 30.7 degrees. Adequate cranial rotation followed by scanning along a timed axis will maximally delineate premature atrial signals and provide comprehensive visualization of electrical forces.